Somaclonal variations
Introduction
Somaclonal variation
It is the term used to describe the genetic variation seen in plants that have been produced by plant tissue culture. Chromosomal rearrangements are an important source of this variation. Somaclonal variation is not restricted to, but is particularly common in plants regenerated from callus. The variations can be genotypic or phenotypic, which in the later case can be either genetic or epigenetic in origin.
Typical genetic alterations are:
1. changes in chromosome numbers (polyploidy and aneuploidy),
2. chromosome structure (translocations, deletions, insertions and duplications)
3. DNA sequence (base mutations)
4. Typical epigenetic related events are: gene amplification and gene methylation.
History
Historically, plant cell culture has been viewed by most to be a method for rapid cloning. In essence, it was seen as a method of sophisticated asexual propagation, rather than a technique to add new variability to the existing population. For example, it was believed that all plants arising from such tissue culture were exact clones of the parent, such that terms like calliclone mericlone and protoclone were used to describe the regenrants from callus meristems, and protoplasts, respectively. Although phenotypic variants were observed among these regenrants, often they were considered as artifacts of tissue culture. Such variation was though to be due to epi genetic factor such as exposure to plant growth regulators (PGRs) and prolonged culture time.
ORIGINS AND MECHANISMS OF SOMACLONAL VARIABILITY:
Somaclonal variation can be of two sorts:
• Genetic (i.e. heritable) variability – caused by mutations or other changes in DNA.
• Epigenetic (i.e. non-heritable) variability – caused by temporary phenotypic changes.
GENETIC VARIABILITY
Various molecular mechanisms are responsible for genetic variability associated with somaclonal variation.
Changes in ploidy
One of the more frequently encountered types of somaclonal variation results from changes in chromosome number, that is, aneuploidy, polyploidy, or mixoploidy. Changes in ploidy originate from abnormalities that occur during mitosis. For example, extra chromosomal duplication during interphase, spindle fusion or lack of spindle formation and cytoplasmic division. A plant cell grows and ages, the frequency of changes in ploidy increases. Therefore, changes in ploidy observed in cultures and regenerated plants might have their origins in the source of tissue explants used. Another cause of variability due to changes in ploidy is the in vitro culture regime itself. The longer the cell remains in culture the greater is its chromosomal instability. In addition, the composition of the growth medium can trigger changes in ploidy. For example, both kinetin and 2, 4-D are implicated in ploidy changes and cultures grown under nutrient limitation can develop abnormalities. Selecting a suitable explant and an appropriate culture medium can therefore enhance the chromosomal stability of the culture. However, high variations of ploidy in cultures do not always lead to high frequencies of somaclonal variation in regenerated plants. This is because, in mixed cultures, diploid cells appear to be better fitted than aneuploid or polyploidy cells for regeneration, as they are more likely to form meristems.
Structural changes in nuclear DNA
Structural changes in nuclear DNA appear to be a major cause of somaclonal variation. The changes can modify large regions of a chromosome and so may affect one or several genes at a time. These modifications include the following gross structural rearrangements.
• Deletion: loss of genes
• Inversion: alteration in gene order
• Duplication: duplication of genes
• Translocation: segments of chromosomes moving to new locations.
Activation of transposons can be a cause of somaclonal variation. Transposons or transposable elements are mobile segments of DNA that can insert into coding regions and cause gene disruption. In addition to these larger modifications of nuclear DNA sequence, changes at the level of a single DNA nucleotide that occur in a coding region can lead to somacloanal variation. For example, point mutations that result from a change of base in a single nucleotide or the altered methylation of a base can lead to gene inactivation.
Chimeral rearrangement of tissue layers: any horticulture plants are periclinal chimaeras, that is, the genetic composition of each concentric cell layer (LІ, LІІ, LІІІ) of a meristem (e.g. the shoot tip meristem) is different. These layers can be rearranged during rapid cellular proliferation. Therefore, regenerated plants may contain a different chimeral composition or may no longer be chimaeric at all. Shoot tip transformation procedures are particularly likely to cause chimeral transgenic.
Epigenetic variability
Epigenetic changes somaclonal variation can be temporary and over time are reversible. However, sometimes they can persist through the life of the regenerated plant. One common phenotypic change seen in plants produced through tissue culture is rejuvenation. Rejuvenation causes changes in morphology such as earlier flowering and enhanced adventitious root formation. Epigenetic changes may be caused by DNA methylation and thus may be one of the important causes of somaclonal variation.
The importance of somaclonal variation
As plant tissues are composed of heterogeneous array of cells of various ages, different physiological states and degree of differentiation and cells with different ploidy level exist. By placing cells in tissue culture, the genome at different molecular states is suddenly placed under stress to cope with in vitro conditions. It has also been reported that changes in tissue culture conditions could influenced the frequency of variation. The end effect seems to be an array of genetic engineering changes.
Studies concerning different aspects of somaclonal variation are important for several reasons.
1. First is hailed as a novel source of genetic Variation. However successful utilization of somaclonal variation heavily depends upon its systematic evaluation and judicious utilization in breeding programmes. This necessitates appropriate experimentation.
2. Second, soma clonal variation is of interest as a basic genetic process, since it contradicts the concept of clonal uniformity. The cells and tissues which are expected to produce true to type plants through the processes of de-differentiation, division and re-differentiation, possibly perceive the whole process as stress, as a result of which the genome, known for its plasticity, restructures itself to modulate the expression of gene as demanded by the in vitro conditions.
3. Third soma clonal variation is unwanted when the objective is mricropropagation of elite genotypes or genetic transformation that partly involved tissue culture. Under such circumstances, prevention or at least minimization of variation is of utmost importance. To achieve this, the frequency, nature and magnitude of somaclonal variation in relation to manipulation of media components, explant source, culture conditions etc. should essentially be understood.
Majority of studies under take on somaclonal variation are confined to early generation of soma clones. Therefore information on the nature, inheritance pattern and stability of morphological and molecular changes expressed in the advanced generation of soma clones is lacking.
The different aspects of somaclonal variation investigated so far are as follows
1. Generation of variation
2. Characterization of variant for morphological traits
3. Analysis of biochemical and chromosomal basis of variation
4. Relating the variation to alteration in DNA
Why variation occurs
Variation may also arise as a result of more suitable changes due to single gene mutation in culture, which have cells apparently showing no karyological changes. Every possible factor that could result in a genetic change has been accounted for as a cause for soma clonal variation. Recessive mutations are not detected in plant regenerated in vitro from any cell or tissue, but expressed in progeny. This shows that variants are the mutants. Single gene mutation responsible for somaclonal variation relates to transposable elements. Transposed induced changes have been observed in maize, tobacco and wheat. Somaclonal variation may also be due to changes caused by crossing over in regenerated plants. Such changes may also occur due to changes in organelles, DNA, is enzymes and protein profile example in wheat, potato, maize, barley and flax. changes in cytoplasmic genome have also been observed in somaclones. Factors that contribute to soma clonal variation are of categories i.e. physiological genetic and biochemical.
Physiological causes of variation
Variations induced by physiological factors were identified quite earlier. Such variations are those induced habituation to PGR in culture and culture conditions and are epigenetic. They may not be inherited in Mendelian fashion. Prolonged exposure to explanted tissue to powerful auxin such as phenoxyacetic acid (e.g., 2, 4-D or 2, 4, 5-T) often results in variation among the regenerants. In oil palm (Elaeis guineensis Jacq.), plants generated from long0term callus cultures in the presence of 2, 4-D show significant amount of variability in the field. In grapevine (Vitis vinifera L.), embryogenic cells that have been maintained in culture for several years gradually lose their ability to differentiate and regenerate into plants over time.
Genetic causes of variation
Tissue culture reentrants show certain variations which are results of alteration at the chromosomal level. Although the explanted tissue may be phenotypically similar, plants often have tissue made of diverse cell type or cells. That is there are cytological variation among the cell types with in the explanted tissue such as pre existing conditions often result in plant regenerates from the tissue that are dissimilar. These species are referred as poly somatic species (result of spontaneous mutation due to pre-existing conditions).species such as barely (Hordeum vulgare L.) and tobacco (Nicotiana tobacum L.) have been documented to possess such polysomatic tissues. Chromosomal mutation (deletion, duplication, inversion, translocation) are the source of genetic variation which are expressed by soma clones. Lee and Philips (1988) have described the possible mechanisms these chromosomal changes. They pointed out that late replicating heterochromatin is the primary cause of somaclonal variation in maize (Zea mays L.) and broad beans (Vicia faba L.). Transposable elements are activated during culture in explants tissue and results in altered gene types among the regenerated plants the well known transposable elements complex of AC-DS in maize which activation in vitro culture.
Biochemical cause of variation
Biochemical variations are pre dominant type of radiation in tissue culture. They have been noticed in barely (unless a specific test is performed). In tissue culture, several bio chemical variations have been identified in various crop plants and some of these variants show Mendelian inheritance (many may be epigenetic and may be lost in the plant regenerated). Biochemical variations also include alteration in carbon metabolism leading to lack of photosynthetic ability (albinos in cereals such as rice), starch biosynthesis carotenoid pathway. Nitrogen metabolism and antibiotic resistance. Genomic DNA exhibits normal methylation patterns. Methylation is a process where a particular nucleotide- usually adenine (A) or cytosine (C)-has a methyl group attached to it. Prolonged exposure to plant tissue to in vitro culture has resulted in the alteration of normal methylation pattern (e.g., maize, potato and grapevine). However, at present we do not know why this process happens.
Genetic action and crop improvement
Genetic variation appears during or after culture in vitro. It may occur in undifferentiated cells isolated protoplast, calli, tissue and morphological traits of regenerated plants. Most of reported genetic variation used in breeding programmes has occurred naturally and exist in germplasm collection of new and old cultivar, land races and genotypes. This variation through crosses is recombined to produce new and desired gene combination variants selected in tissue culture have been referred as Calliclones (from callus culture). The changes occur because of variation in chromosome and number and structure. Cytological heterogeneity in culture develops due to the following reasons:
• The expression of genetic disorders in cell of the initial explants.
• New irregularities brought about by culture conditions
Somaclonal variations are considered to be a good supplement to conventional crop improvement. There is evidence in different crops that the variant characteristics obtained from culture of somatic tissue are transmitted successfully to the progeny in terms of these desirable characteristics. Somaclonal variation is one of the aspects of tissue culture technology and is widely recommended for crop improvement especially of desired traits for the salt, drought, temperature and disease tolerance. The method refers to heritable change that accumulates in the callus from the somatic explant and express in the progeny of in vitro regenerations obtained from callus.
Possible advantages of Somoclonal vs Induced mutagenesis
1. The frequency of variation seems to be far greater than the yield of induced mutations.
2. The changes are very subtle and may not involve drastic alterations in the genetic background.
3. Somaclonal variations occur for trait of both nuclear and cytoplasmic origin.
In wide crosses somalonal variations provide a mechanism of gene introgression. Immature embryos of the wide cross can be callused and plants with the introgressed desired gene (or gene complex) are selected among the regenerants of their progenies.
Induced (or directed) causes of variationPlant improvement techniques involve screening of a large number of plants in the greenhouse or field for selection of a particular trait of interest. If one has to develop a salt-tolerant line of particular species, a large number of individual plants that can withstand the screening process. For this purpose limited material, space and time will be available. In addition, environmental factors will also interfere with the selection process. Cell culture systems provide the breeder with the ability to select from a very large amount of genetically uniform material and to conduct the screening quickly in a few Petri dishes or flasks. This provides much greater control over the selection process.
In vitro mutation
Certain crop plants such as bananas and plantains (Musa spp.) do not have a large genetic base and have been propagated by asexual means for thousands of tedyears. Genetic improvement in these species is very difficult because the seldom produce fertile seeds. Therefore, one has to look either for natural somatic mutations (do occur at an extreme low frequency), or induce mutations. In such vegetative propagated crops, even inducing mutations is rather difficult as their propagules are large as in the case of Banana sukers. Attempt to avoid such large vegetative propagules result either mosaics or fatalities. Development of a cell regeneration system, such as somatic embryogenesis, provides an opportunity to expose a large number of regenerative cells to either gamma irradiation or chemical mutagen such as ethyl methyl sulfonate etc., in a very controlled manner, and thus widen the existing germplam base (Novak, 1992).
Applications of tissue culture-derived applications
Cell culture systems offer plant breeders a well-defined environment where selection pressure can be imposed on thousands of genetically uniform single cells, each capable of growing into a whole plant. The effect of environment variation is minimized, so that escapes or adaptations that can revert back to original genetic background are also reduced. This controlled growth atmosphere in a minimal space provides the plant breeder new option for introducing variation. In addition, a scientist can study a tropical species and a temperate region or vice versa because specialized environmental conditions can be provided anywhere. Some of the important applications for induced variation are discussed below with one or two classical form the literature.
Development of disease-resistant plant from tissue culture
Hammerschlar (1992) pointed out the effectiveness in vitro selection for disease resistance can proceed. An effective selection agent, that can be produced and utilized in an in vitro system, must be identified. The identified selection agent should act at the cellular level and should be an important factor in the disease process. There must be a reliable protocol for regenerating whole plant from single cells for the species in question. The protocol must allow the cells to withstand several cycles of selection in a stringent environment and still be able to regenerate whole plants. In addition to these important factors, effective tools to determine if selected cells are truly resistant to the pathogen at the level and whole plant level are necessary.
Photo toxins (in late 1970s and early 1980s) were employed as selection agents to impart disease resistance. How in vitro-derived resistance occur is not well known. One possible reason is that these phytotoxins are produced by pathogen in a very timely and specific manner and in very low quantities during the disease process. When plant cells are subjected to higher doses of these toxins, they not only affect the ability of the cells to resist the phytotoxin, but also cause some unwarranted genetic damage to the cells. Another problem is that sometimes regenerants to the phytotoxin were not resistant to the pathogen. These results exposed problems of phytotoxins to select for disease resistance.
One of the main finding is that there are compounds either than phytotoxins produced by the pathogen that are involved in the disease process. For instance, `harpin’ proteins produced by the pathogen have been shown to elevate plant resistance against a diverse group of pathogens. Using these compounds as a whole unit (as in a crude culture filtrate) in suspension culture could be a better approach to bring out the true genetic resistance of the plant.
Induction of salt heavy metal tolerance through tissue culture
Crop development for saline regions (salt such as sodium chloride or heavy metals like aluminium) is still la high priority for agriculturists. The availability of arable land is continuously shrinking. Tolerance to sodium chloride using in vitro selection has been achieved in several crop species, such as rice, potato, sugarcane, and tomato. However, in most cases the resistance was epigenetic. A few reports are available for heavy metal tolerant somaclonal variation. The identification and cloning of genes that could elevate resistance to salt and heavy metals is a more breakthrough for plant geneticists.
The mechanism of somaclonal variation
According to Bhaskaran (1985) variations in somaclones occur due to the following reasons:
1. The pre-existing genetic variations in the explant tissue,
2. The spontaneous mutations that can accumulate during the many division cycles that cell of the explant go through before differentiating into an in vitro plant. The recessive mutation will naturally require a method by which they can express even diploid cells. Somatic crossing over followed by segregation is a likely mechanism, for the homozygosity and thus phenotypic expression of the recessive (Chopra and Sharma, 1988).
3. Intracellular mutagenic agents produced during in vitro growth.
4. Numerical and structural changed in chromosomes during in vitro growth.
5. Activation of transposable elements or jumping genes, are genetic entities which have the locus at which they get integrated is matured.
Agriculturists are very hopeful about practical advantages of somaclonal variation and they are waiting when this technique is fully integrated with the conventional plant breeding procedures.
Source material and culture conditions
Plant cell, tissue and somatic embryos developed from various explants sources for generating somaclonal variation. Explants are generally taken from any tissue, namely leaves, internodes, ovaries, roots and inflorescence. The source of explant has often been considered a critical variable for somaclonal variation.
Determination of cell number
Take an aliquot of suspension and filter off the culture through a wire mesh (300mm). note the volume of the filtrate (F) containing single cells and small clumps and place the drop of this suspension to heamocytometer to determine the number of cells by the equation
N = P x 100 x F 0.1 mm
where, N = total number of cells and clumps, P = number of cells in the squares of the haemocytometer, f = volume of the filtrate.
Forms of somaclonal variation:Many different forms of somaclonal variation arise. The most common forms include point mutation, chromosomal aberrations. And increase or decrease in the number of nuclear chromosomes. It is important to realize that not all forms of variability that arise in vitro are heritable. Some morphological and biochemical variants are due to physiological effects and are not exhibited in subsequent generations.
DETECTION AND ISOLATION OF SOMACLONAL VARIANTS:
There are several different approaches to detecting and isolating somaclaonal variants from cultured plant cell populations.
1. Morphologically distinct cells such as nonphotosynthetic (nongreen) cells or cells that accumulate anthocyanin and other plant pigments are detected visually.
2. To isolate herbicide- and antibiotic-resistant variants, plant cells are simply grown on media containing of the wild type cells in a culture.
3. The surviving cells are then subcultured and retested for growth on herbicide or antibiotic supplemented medium.
4. Through this method, one can eliminate any remaining wild-type cells that may have inadvertently survived the first round of selection.
We can also use this direct selection technique for isolation of temperature- resistant variants because those cells which survive in an extended incubation period at abnormally high or low temperature- resistant.
Those somaclonal variants that can not be detected visually or selected directly are isolated by indirect means. Those auxotrophic plants cells which unable to survive in absence of specific nutrient supplements not required by sensitive cells, which will not survive at temperatures above or below a certain threshold. This threshold will not affect normal wild type cells. In absence of the necessary nutrient supplement or when grown at excessive temperatures, these cells become very weak and at this weakened state, these cells assimilate exogenously supplied cytotoxic compounds (arsenate, bromodeoxyuracil, or fluorodeoxyuridine) at lower rate then that of wild type cells, thus treating a cell culture under restrictive growth conditions with one of these substances will tend to favor short term survival of the weaker auxotrophic or temperature sensitive cells.
Mutagenesis and somaclonal variation
Mutagenic agents (chemical and physical) are used to produce particular heritable forms of somacloanl variation e.g., ultra violet (UV) radiation, ethyl-methane sulfonate (EMS), UV radiation induces dimmer formation between adjacent thymine residues in DNA. This produces lesions that cause frame shift mutation and base pair substitutions during DNA replication. EMS and nitrosoguanidine are alkylating agents that cross-link and sever DNA molecules, often resulting in gross DNA alterations. In contrast, sodium azide induces single base pair substitutions or deletions, resulting mostly in small point mutation.
Somatic genetics of Nitrogen metabolism
Plant cell in culture medium can metabolize ammonium, nitrate, and nitrite sources of inorganic nitrogen. The cells use ammonium directly while the nitrate is first reduced to nitrite (enzyme nitrate reductase, NR) and then nitrite is reduced to ammonium (enzyme nitrite reductase NiR). NR needs a molybdenum- containing cofactor (MoCo) for proper for isolation of variants, which unable to use nitrate and will be reduced to chlorate supplemented media. Chlorate is a close analog of nitrate and will be reduced to chlorite; a patent cytotoxin that accumulates internally and eventually kills the cell. Certain metabolic mutants can survive chlorate treatment. Some of these mutant cells carry mutations affecting NR expression or assimilation. Other mutations may affect MoCo expression or chlorate assimilation.
Methods for isolation of desired variant cells for NaCl-tolerant from callus suspension culture
1. Semisolid liquid media is prepared and NaCl (500 mg/l) is added to the medium, pH is adjusted at 5.6. Distribute the medium in 30-ml aliquots into 150-ml flasks or 50-ml aliquots into 250-ml flasks and grow the callus on NaCl containing medium. Select the granular friable callus for further subculture.
2. 1 g of callus is inoculated to each 250 ml flask containing 50ml of liquid medium then incubates the cultures on a gyratory (100 rpm) for 2 days at 25+2˚C.
3. pass the cell suspension through a stainless steel wire mesh (300 µm) and small aggregates (5-20 cells). Centrifuge the filtrate at 100g for 5 min and discard the supernatant, add 5ml of the sterilized medium to the pellet to obtain a cell suspension, and maintain the cell density of the suspension about 0.5-2.5 x 10*5 cell/ml. add to the flasks containing with or without NaCl.
4. Incubate the culture at 100 rpm at 25+2 C and centrifuge the cultures grown control and NaCl (500 mg/l) at 100 rpm for 5 min.
5. Transfer the pellets to fresh medium of the same composition. Incubate at 25+2 C for21 days and repeat these steps for 2-3 times to get a good growth rate of the NaCl tolerant cell line.
6. Grow cultures on 1000 mg/l NaCl and follow the sample steps, as carried out at 500 mg/l NaCl until a salt concentration should be increased gradually by repeating steps.
7. Regenerate variants cells into whole plants. Transfer 0.5 ml of cell suspension onto the surface of the regeneration medium gelled with agar by a wide mouth graduated sterile pipette.
8. In case of callus, inoculate 500 mg piece onto the surface of the organ/shoot forming medium having 500 mg/l NaCl
9. In most of the plant species, the differentiation of shoots and roots occur on different media. Hence once the shoots are obtained, they are transferred to the rooting medium. Transfer the selected NaCl tolerant plants of alkali, high temperature, drought, disease; herbicide can be developed by using in vitro selection system.
10. Use NaCl solution to water the salt tolerant plants. the concentration of NaCl should be the same as used in the regeneration medium. This procedure can also be used to develop tolerant plant of alkali, high temperature, drought, disease; herbicide can be developed by using in vitro selection system.
Applications in plant breeding
Somaclonal variation and gemetoclonal variation are the important source of introducing genetic variation that could be of value to plant breeders. Single gene mutation in the nuclear or organelle genome usually provides the best available variety in vitro which has a specific improved character. Somaclonal variations are used to uncover new variant retaining all the favorable characters along with an additional useful trait, e.g., resistance to disease or an herbicide. These variants can then be field tested to ascertain their genetic stability. Gametoclonal variation is induced by meiotic recombination during the sexual cycle of the F1 hybrid results in transgressive segregation to uncover unique gene combinations. Various cell lines selected un vitro and plant regenerated through it prove potentially applicable to agriculture and industry specially resistance to herbicide, pathotoxin, salt or aluminium, useful in the synthesis of secondary metabolites on a commercial scale, etc. The techniques used for development of somaclonal and gametoclonal variation are relatively easier than recombinant DNA technology and is the appropriate technology for genetic manipulation of some crops.
EXAMPLES
• Developed Fusarium wilt resistant plants of Carnation, lilium and Robinia pseudoacacia , Rhizoctonia root rot resistant plants of strawberry and cauliflower, Alternaria alternata resistant plants of tomato cultivar Solan Vajr, Alternaria dianthi resistant plants of Carnation cv Tempo and flower colour variants of chrysanthemum through somaclonal variations. Water stress tolerant plants of tomato were also developed through cell selection.
• Fusarium wilt tolerance cell line of peas, Septoria olera tolerant cell lines of Chrysanthemum and salt stress tolerant cell line in tomato through cell selection were developed.
• Apple rootstocks MM106 & M7 Cell lines/ shoots in vitro have been selected which are tolerant to fungi collar rot (Phytophthora cactorum) and white root rot (Dematophora necatrix).
Methods of assessing somaclonal variation
Although cytological and phenotypic analyses can be used to evaluate somaclonal variation, recently molecular techniques have been used with increasing frequency.
Restriction fragment length polymorphism (RFLP)
RFLP was one of the first techniques to be applied to somaclonal variation and has been widely used for several species. RFLP is a hybridization based technique that detects variation in the DNA sequence level but require the use of probes that hybridize to known sequences. A number of amplification techniques based on PCR technology have now been developed that avoid the need for prior sequence information.
Random amplified polymorphic DNA-PCR (RAPD-PCR)
RAPD-PCR or arbitrarily primed PCR (AP-PCR) (William et al., 1990) is a technique that has proved useful in detecting somaclonal variation in a number of species (e.g. Saker et al., 2000).RAPD-PCR is based on the premise that, because o fits complexity, eukaryotic nuclear DNA may contain paired random segment that are complementary to single decanucleotides and further more these segments have the correct orientation and are located close enough to each other for PCR amplification. RAPD-PCR uses single primers of arbitrary nucleotide sequence to initiate DNA synthesis. The DNA fragment can be separated by gel electrophoresis and the DNA variation is detected by the pattern of DNA bands from individual plants.
Amplified fragment length polymorphism
More recently another PCR-based technique known as AFLP (Vos et al., 1995) has been used to study somaclonal variation (Polanco and Ruiz,2002)AFLP is a DNA-finger printing procedure based on a selective PCR amplification of fragments from restriction digestion of genomic DNA. AFLP analysis consists of the following steps
• Genomic DNA is designed with two restriction enzymes, one that cuts frequently, for example Msel (4-bp recognition sequence) and one that cuts less frequently, for example EcoR1(6-bp recognition sequence).
• The resulting fragments are ligated to double stranded adaptor molecules that consist of a core sequence and a sequence specific for either the EcoR1 site or the Msel site.
• Pre-selective amplification by PCR using primer designed to include a core sequence, an enzyme specific sequence and a single base extension at the 3’ end. the primary products are fragments containing one Msel cut, one EcoR1 cut and a matching internal nucleotide. This amplification step achieves a 16-fold reduction in complexity.
• Selective PCR amplification using the products of the pre-selective amplification as template and identical primers as the pre-selective step, but containing two further additional nucleotides at the 3’ end. The primers are either radio labeled or fluorescent labeled. Only fragment having the matching nucleotides in all three positions (50-200) will be amplified. This step reduces the complexity 250 fold.
• Gel electrophoresis separation reveals the pattern (fingerprint) of the labeled fragments that are analyzed with the aid of an appropriate software package.
• The molecular analyzed described are rapid and more precise than phenotypic analyses. However. both the RAPD and AFLP techniques have proved to be inconclusive in some studies.
Benefits
The major likely benefit of somaclonal variation is in plant improvement. Somaclonal variation leads to the creation of additional genetic variability. Characteristics for which somaclonal mutants can be enriched during in vitro culture include resistance to disease pathotoxins, herbicides and tolerance to environmental or chemical stress, as well as for increased production of secondary metabolites.Micropropagation can be carried out throughout the year independent of the seasons.
Disadvantages
A serious disadvantage of somaclonal variation occurs in operations which require clonal uniformity, as in the horticulture and forestry industries where tissue culture is employed for rapid propagation of elite genotypes. Ways of reducing somaclonal variation: Different steps can be used. It is well known that increasing numbers of subculture increases the likelihood of somaclonal variation, so the number of subcultures in micropropagation protocols should be kept to a minimum. Regular reinitiation of clones from new explants might reduce variability over time. Another way of reducing somaclonal variation is to avoid 2,4-D in the culture medium, as this hormone is known to introduce variation.Vitrification[hyperhydracity] may be a problem in some species. In case of forest trees, mature elite trees can be identified and rapidly cloned by this technique. High production cost has limited the application of this technique to more valuable ornamental crops and some fruit trees.
Conclusion
sInduced variation still is the best route in perennial crop improvement, although one can argue on favor of the currently untapped potential of genetic transformation. However, it must be noted that tissue-culture induced variation does not have the socio-ethical hurdle like GM crops. In addition, there are not have the any significant technology owner ship issue as has become problematic with genetic engineering. Further, gene transfer technique, though successful in herbaceous species, still have not been commercialized in perennial and woody species. Molecular techniques have greatly aided in understanding the plant cell response to biotic and abiotic stresses at the sub cellular level. The future lies in utilizing these techniques to induce the species’ own resources, such as disease resistance, to our benefit.
Thursday, June 24, 2010
regulation of carcinogenicity
Regulation of cellular growth- Development of carcinogenicity
Contents:
• What is cancer
• What is Carcinogen and carcinogenicity
• Regulation of cancer
• Cancer and cell cycle
• Cancer and programmed cell death
• Genetic basis for cancer
• Oncogenes
• Proto- oncogenes
• Mutant cellular oncogenes and cancer
• Tumor suppresser genes
• Knudson’s two hit hypothesis
• Conclusion
What is cancer?
Cancer is uncontrolled, abnormal proliferation of any cell type.
Without regulation, cancer cells divide ceaselessly, piling up on top of each other to form tumors.
When cells detach form tumor and invade surrounding tissues, the tumor is malignant. When the cells do not invade surrounding tissues, the tumor is benign.
What is carcinogen and carcinogenicity?
The agents that can irreversibly transform normal cells into cancerous cells are called carcinogens. E.g. radiation, mutagenic chemicals, and certain types of viruses
Carcinogenicity- Ability of carcinogen to induce cancer is called carcinogenicity.
The abiding characteristic of all cancer cells is that their growth is unregulated. When normal cells are cultured in vitro, they form a single layer- a monolayer- on the surface of the culture medium. Cancer cells, by contrast, overgrow each other, piling up on the surface of the culture medium to form masses. This unregulated pileup occurs because cancer cells do not respond to the chemical signals that inhibit cell division and because they can not form stable associations with their neighbors.
The external abnormalities that are apparent in a culture of cancer cells are correlated with profound intracellular abnormalities. Cancer cells often have a disorganized cytoskeleton, they may synthesize unusual proteins and display them on their surfaces and they frequently have abnormal chromosome numbers- that is they are aneuploid.
Regulation of carcinogenicity- carcinogenicity is regulated at several levels viz.
• Cancer and cell cycle
• Cancer and programmed cell death
• Genetic basis for cancer
• Oncogenes
• Proto- oncogenes
• Mutant cellular oncogenes and cancer
• Tumor suppresser genes
• Knudson’s two hit hypothesis
Cancer and cell cycle-
The cell cycle consists of periods of growth, DNA synthesis and division. The length of cell cycle and the duration of each of its components are controlled by external and internal chemical signals. The transition from each phase of the cycle requires the integration of specific signals and precise responses to these signals. If the signals are incorrectly sensed or if the cell is not properly respond, the cell could become cancerous.
The current view of cell cycle control is that transitions between different phases of cell cycle (G1, S, G2 and M) are regulated at “checkpoints.” A checkpoint is a mechanism that halts progression through the cycle until a critical process such as DNA synthesis is completed or until damaged DNA is repaired. The molecular machinery that operates each checkpoint is complex. Two types of proteins are known to play especially critical roles: the cyclins and the cyclin dependant kinases, often abbreviated as CDKs. Complexes formed between cyclins and CDKs cause the cell cycle to progress.
The CDKs are the catalytically active components of the cell cycling mechanisms. These proteins regulate the activities of other proteins by transferring phosphate groups to them. However, the phosphorylation activity of the CDKs depends on the presence of the cyclins. The cyclins enable the CDKs to carry out their function by forming cyclin / CDK complexes. When the cyclins are absent, these complexes can not form, and CDKs are inactive. Cell cycling therefore requires the alternate formation and degradation of cyclin/ CDK complexes.
One of the most important cell cycle checkpoints, called START, is in mid G1. The cell receives both external and internal signals at this checkpoints to determine when it is appropriate to move into S phase. This checkpoint is regulated by D type cyclins in conjunction with CDK4. If a cell driven past the START checkpoint, it becomes committed to another round of DNA replication. Inhibitory proteins with the capability of sensing problems in late G1 phase such as low level of nutrients or DNA damage, can put a brake on the cyclin/ CDK complex and prevent the cell from entering the S phase. In the absence of such problems, the cyclin D/ CDK4 complex drives the cell through the end of G1 phase and into S phase, there by initiating the DNA replication that is a prelude to cell division.
In tumor cells, the checkpoints in the cell cycle are typically deregulated. This deregulation is due to genetic defects in the machinery that alternately raise and lower the abundance of cyclin/ CDK complexes. E.g the genes encoding the cyclins or CDKs may be mutated, or the genes encoding the proteins that respond to specific cyclin/ CDK complexes or that regulate the abundance of these complexes may be mutated. Many different types of genetic defects can deregulate the cell cycle, with the ultimate consequence that the cells may become cancerous.
Cells in which START checkpoint is dysfunctional are especially prone to become cancerous. The START checkpoint controls entry into the S phase of cell cycle. If DNA within a cell is damaged, it is important that entry into S phase be delayed to allow for the damaged DNA to be repaired. Otherwise, the damaged DNA will be replicated and transmitted to all cell’s descendents. Normal cells are programmed to pause at the START checkpoint to ensure that repair is completed before DNA replication commences. By contrast, cells in which the START checkpoints is dysfunctional move into S phase without repairing damaged DNA. Over a series of cell cycles, mutations that result from the replication of unrepaired DNA may accumulate and cause further deregulation of cell cycle. A clone of cells with a dysfunctional START checkpoint may therefore become aggressively cancerous.
Cancer and programmed cell death-
Every cancer involves accumulation of unwanted cells. In many animals, superfluous cells can be disposed of by mechanisms that are programmed into cells them selves. This programmed cell death was originally discovered in studies wit nematode Caenorhabitidis elegans. This tiny roundworm loses some of the cells that accumulate during the 10 or so cycles of division that occur during its development from a fertilized egg. Genetic analyses by Robert Horvitz demonstrated that the loss of these cells does not occur in certain mutants of C. Elegans. Thus cell death is part of normal developmental program in this animal- and in others too, e.g during development of hands and feet of many vertebrates, the cells that lie between the developing digits must die; if they do not, the digits remain fused. Programmed cell death is therefore a fundamental phenomenon among animals. Without it, the formation and function of organs would be impaired by cells that simply ‘get in the way.’
Incomplete differentiation in two toes (syndactyly) due to lack of apoptosis
Programmed cell death is also important in preventing the occurrence of cancers. If a cell with an abnormal ability to replicate is killed, it can not multiply to form tumor. Thus, programmed cell death is an important check against renegade cells that could otherwise proliferate uncontrollably in an organism.
Programmed cell death is called apoptosis from greek roots meaning “falling away.” The events that trigger cell death are only partially understood. A family of proteolytic enzymes called caspases play a crucial role in the cell death phenomenon. The caspases remove small part of other proteins by cleaving peptide bonds. Through this enzymatic trimming, the target proteins are inactivated. The caspases attack many different kinds of proteins, including the lamins, which make up the inner lining of nuclear envelope, and several components of cytoskeleton. The collective impact of these proteolytic cleavage is that cells in which it occurs lose their integrity; their chromatin becomes fragmented, blebs of cytoplasm form at their surfaces, and they begin to shrink. Cells undergoing this kind if disintegration are usually engulfed by Phagocytosis, and are then destroyed. If the apoptotic mechanism has been impaired or inactivated, a cell that should otherwise be killed can survive and proliferate. Such a cell has the potential to form a clone that could become cancerous if it acquire the ability to divide uncontrollably.
Genetic basis for cancer- There are strong evidence that the underlying causes of cancer are genetic.
1. It was known that cancerous state is clonally inherited. When cancer cells are grown in culture, their descendents are all cancerous. Therefore, cancerous condition is transmitted from each cell to its daughter cells at the time of division.
2. It was known that certain types of viruses ca induce the formation of tumors in experimental animals. The induction of cancer by viruses implies that the protein encoded by viral genes are involved in production of the cancerous state.
3. Cancer can be induced by agents capable of causing mutations. Mutagenic chemicals and ionizing radiations had been shown to induce tumors in experimental animals.
4. It was known that certain types of cancer run in families. In particular susceptibility to retinoblastoma, a rare cancer of eye, is inherited as dominant conditions, albeit with incomplete penetrance and variable expressivity.
So all these shows that special types of cancer is inherited- it seemed plausible that all cancer types might have their genetic basis.
In 1980s when molecular techniques were first used to study cancer cells, researchers discovered that the cancerous state is indeed, traceable to genetic defects. Typically, however not one but several such defects are required to convert a normal cell to cancerous cell.
Researchers identified two broad classes of genes that when mutated, can contribute to the development of a cancerous state.
• In one of these class mutated genes actively promote cell division
• In other class mutant genes fail to express cell division.
Genes in first class are called oncogenes from greek word tumor
In second class are called tumor suppressor genes.
Oncogenes- An oncogene is a gene that, when mutated or expressed at high levels, helps turn a normal cell into a tumor cell.
Many abnormal cells normally undergo a programmed form of death (apoptosis). Activated oncogenes can cause those cells to survive and proliferate instead. Most oncogenes require an additional step, such as mutations in another gene, or environmental factors, such as viral infection, to cause cancer. Since the 1970s, dozens of oncogenes have been identified in human cancer. Many cancer drugs target those DNA sequences and their products.
History-The first oncogene was discovered in 1970 and was termed src (pronounced sarc as in sarcoma). Src was in fact first discovered as an oncogene in a chicken retrovirus. Experiments performed by Dr G. Steve Martin of the University of California, Berkeley demonstrated that the SRC was indeed the oncogene of the virus.
In 1976 Drs. J. Michael Bishop and Harold E. Varmus of the University of California, San Francisco demonstrated that oncogenes were defective proto-oncogenes, found in many organisms including humans. For this discovery Bishop and Varmus were awarded the Nobel Prize in 1989.
Proto- oncogenes- A proto-oncogene is a normal gene that can become an oncogene due to mutations or increased expression. The resultant protein may be termed an oncoprotein. Proto-oncogenes code for proteins that help to regulate cell growth and differentiation. Proto-oncogenes are often involved in signal transduction and execution of mitogenic signals, usually through their protein products. Upon activation, a proto-oncogene (or its product) becomes a tumor-inducing agent, an oncogene. Examples of proto-oncogenes include RAS, WNT, MYC, ERK, and TRK.
Activation-The proto-oncogene can become an oncogene by a relatively small modification of its original function. There are three basic activation types:
• A mutation within a proto-oncogene can cause a change in the protein structure, causing
o An increase in protein (enzyme) activity
o A loss of regulation
• An increase in protein concentration, caused by
o An increase of protein expression (through misregulation)
o An increase of protein (mRNA) stability, prolonging its existence and thus its activity in the cell
o A gene duplication (one type of chromosome abnormality), resulting in an increased amount of protein in the cell
• A chromosomal translocation (another type of chromosome abnormality), causing
o An increased gene expression in the wrong cell type or at wrong times
o The expression of a constitutively active hybrid protein. This type of aberration in a dividing stem cell in the bone marrow leads to adult leukemia
The expression of oncogenes can be regulated by microRNAs (miRNAs), small RNAs 21-25 nucleotides in length that control gene expression by downregulating them Mutations in such microRNAs (known as oncomirs) can lead to activation of oncogenes. Antisense messenger RNAs could theoretically be used to block the effects of oncogenes.
Conversion of proto-oncogenes-There are two mechanisms by which proto-oncogenes can be converted to cellular oncogenes:
Quantitative: Tumor formation is induced by an increase in the absolute number of proto-oncogene products or by its production in inappropriate cell types.
Qualitative: Conversion from proto-oncogene to transforming gene (c-onc) with changes in the nucleotide sequence which are responsible for the acquisition of the new properties
Tumor suppressor gene- Tumor suppressor gene, or anti-oncogene, is a gene that protects a cell from one step on the path to cancer. When this gene is mutated to cause a loss or reduction in its function, the cell can progress to cancer, usually in combination with other genetic changes.
Two-hit hypothesis-Unlike oncogenes, tumor suppressor genes generally follow the 'two-hit hypothesis', which implies that both alleles that code for a particular gene must be affected before an effect is manifested. This is due to the fact that if only one allele for the gene is damaged, the second can still produce the correct protein. In other words, mutant tumor suppressors alleles are usually recessive whereas mutant oncogene alleles are typically dominant. The two-hit hypothesis was first proposed by A.G. Knudson for cases of retinoblastoma. Knudson observed that the age of onset of retinoblastoma followed 2nd order kinetics, implying that two independent genetic events were necessary. He recognized that this was consistent with a recessive mutation involving a single gene, but requiring biallelic mutation. Oncogene mutations, in contrast, generally involve a single allele because they are gain of function mutations. There are notable exceptions to the 'two-hit' rule for tumor suppressors, such as certain mutations in the p53 gene product. P53 mutations can function as a 'dominant negative', meaning that a mutated p53 protein can prevent the function of normal protein from the un-mutated allele. Other tumor-suppressor genes that are exceptions to the 'two-hit' rule are those which exhibit haploinsufficiency. An example of this is the p27Kip1 cell-cycle inhibitor, in which mutation of a single allele causes increased carcinogen susceptibility.
Examples-The first tumor-suppressor protein discovered was the Retinoblastoma protein (prb) in human retinoblastoma; however, recent evidence has also implicated prb as a tumor-survival factor.
Another important tumor suppressor is the p53 tumor-suppressor protein encoded by the TP53 gene. Homozygous loss of p53 is found in 70% of colon cancers, 30–50% of breast cancers, and 50% of lung cancers. Mutated p53 is also involved in the pathophysiology of leukemias, lymphomas, sarcomas, and neurogenic tumors. Abnormalities of the p53 gene can be inherited in Li-Fraumeni syndrome (LFS), which increases the risk of developing various types of cancers.
PTEN acts by opposing the action of PI3K, which is essential for anti-apoptotic, pro-tumorogenic Akt activation.
Other examples of tumor suppressors include APC, CD95, ST5, ST7, and ST14.
Functions-
Tumor-suppressor genes, or more precisely, the proteins for which they code, either have a dampening or repressive effect on the regulation of the cell cycle or promote apoptosis, and sometimes do both. The functions of tumor-suppressor proteins fall into several categories including the following:
1. Repression of genes that are essential for the continuing of the cell cycle. If these genes are not expressed, the cell cycle will not continue, effectively inhibiting cell division.
2. Coupling the cell cycle to DNA damage. As long as there is damaged DNA in the cell, it should not divide. If the damage can be repaired, the cell cycle can continue.
3. If the damage cannot be repaired, the cell should initiate apoptosis (programmed cell death) to remove the threat it poses for the greater good of the organism.
Some proteins involved in cell adhesion prevent tumor cells from dispersing, block loss of contact inhibition, and inhibit metastasis. These proteins are known as metastasis suppressors.
Conclusion-
Cancer results from breakdown of the regulatory mechanisms that govern from normal cell behavior. The proliferation, differentiation and survival of individual cells in multicellular organisms are carefully regulated to meet the needs of the organism as a whole. This regulation is lost in cancer cells, which grow and divide in an uncontrolled manner, ultimately spreading throughout body and interfering with the function of normal tissues and organs.
Because cancer results from defects in fundamental cell regulatory mechanisms, it is a disease that ultimately has to be understood at the molecular and cellular levels.
Contents:
• What is cancer
• What is Carcinogen and carcinogenicity
• Regulation of cancer
• Cancer and cell cycle
• Cancer and programmed cell death
• Genetic basis for cancer
• Oncogenes
• Proto- oncogenes
• Mutant cellular oncogenes and cancer
• Tumor suppresser genes
• Knudson’s two hit hypothesis
• Conclusion
What is cancer?
Cancer is uncontrolled, abnormal proliferation of any cell type.
Without regulation, cancer cells divide ceaselessly, piling up on top of each other to form tumors.
When cells detach form tumor and invade surrounding tissues, the tumor is malignant. When the cells do not invade surrounding tissues, the tumor is benign.
What is carcinogen and carcinogenicity?
The agents that can irreversibly transform normal cells into cancerous cells are called carcinogens. E.g. radiation, mutagenic chemicals, and certain types of viruses
Carcinogenicity- Ability of carcinogen to induce cancer is called carcinogenicity.
The abiding characteristic of all cancer cells is that their growth is unregulated. When normal cells are cultured in vitro, they form a single layer- a monolayer- on the surface of the culture medium. Cancer cells, by contrast, overgrow each other, piling up on the surface of the culture medium to form masses. This unregulated pileup occurs because cancer cells do not respond to the chemical signals that inhibit cell division and because they can not form stable associations with their neighbors.
The external abnormalities that are apparent in a culture of cancer cells are correlated with profound intracellular abnormalities. Cancer cells often have a disorganized cytoskeleton, they may synthesize unusual proteins and display them on their surfaces and they frequently have abnormal chromosome numbers- that is they are aneuploid.
Regulation of carcinogenicity- carcinogenicity is regulated at several levels viz.
• Cancer and cell cycle
• Cancer and programmed cell death
• Genetic basis for cancer
• Oncogenes
• Proto- oncogenes
• Mutant cellular oncogenes and cancer
• Tumor suppresser genes
• Knudson’s two hit hypothesis
Cancer and cell cycle-
The cell cycle consists of periods of growth, DNA synthesis and division. The length of cell cycle and the duration of each of its components are controlled by external and internal chemical signals. The transition from each phase of the cycle requires the integration of specific signals and precise responses to these signals. If the signals are incorrectly sensed or if the cell is not properly respond, the cell could become cancerous.
The current view of cell cycle control is that transitions between different phases of cell cycle (G1, S, G2 and M) are regulated at “checkpoints.” A checkpoint is a mechanism that halts progression through the cycle until a critical process such as DNA synthesis is completed or until damaged DNA is repaired. The molecular machinery that operates each checkpoint is complex. Two types of proteins are known to play especially critical roles: the cyclins and the cyclin dependant kinases, often abbreviated as CDKs. Complexes formed between cyclins and CDKs cause the cell cycle to progress.
The CDKs are the catalytically active components of the cell cycling mechanisms. These proteins regulate the activities of other proteins by transferring phosphate groups to them. However, the phosphorylation activity of the CDKs depends on the presence of the cyclins. The cyclins enable the CDKs to carry out their function by forming cyclin / CDK complexes. When the cyclins are absent, these complexes can not form, and CDKs are inactive. Cell cycling therefore requires the alternate formation and degradation of cyclin/ CDK complexes.
One of the most important cell cycle checkpoints, called START, is in mid G1. The cell receives both external and internal signals at this checkpoints to determine when it is appropriate to move into S phase. This checkpoint is regulated by D type cyclins in conjunction with CDK4. If a cell driven past the START checkpoint, it becomes committed to another round of DNA replication. Inhibitory proteins with the capability of sensing problems in late G1 phase such as low level of nutrients or DNA damage, can put a brake on the cyclin/ CDK complex and prevent the cell from entering the S phase. In the absence of such problems, the cyclin D/ CDK4 complex drives the cell through the end of G1 phase and into S phase, there by initiating the DNA replication that is a prelude to cell division.
In tumor cells, the checkpoints in the cell cycle are typically deregulated. This deregulation is due to genetic defects in the machinery that alternately raise and lower the abundance of cyclin/ CDK complexes. E.g the genes encoding the cyclins or CDKs may be mutated, or the genes encoding the proteins that respond to specific cyclin/ CDK complexes or that regulate the abundance of these complexes may be mutated. Many different types of genetic defects can deregulate the cell cycle, with the ultimate consequence that the cells may become cancerous.
Cells in which START checkpoint is dysfunctional are especially prone to become cancerous. The START checkpoint controls entry into the S phase of cell cycle. If DNA within a cell is damaged, it is important that entry into S phase be delayed to allow for the damaged DNA to be repaired. Otherwise, the damaged DNA will be replicated and transmitted to all cell’s descendents. Normal cells are programmed to pause at the START checkpoint to ensure that repair is completed before DNA replication commences. By contrast, cells in which the START checkpoints is dysfunctional move into S phase without repairing damaged DNA. Over a series of cell cycles, mutations that result from the replication of unrepaired DNA may accumulate and cause further deregulation of cell cycle. A clone of cells with a dysfunctional START checkpoint may therefore become aggressively cancerous.
Cancer and programmed cell death-
Every cancer involves accumulation of unwanted cells. In many animals, superfluous cells can be disposed of by mechanisms that are programmed into cells them selves. This programmed cell death was originally discovered in studies wit nematode Caenorhabitidis elegans. This tiny roundworm loses some of the cells that accumulate during the 10 or so cycles of division that occur during its development from a fertilized egg. Genetic analyses by Robert Horvitz demonstrated that the loss of these cells does not occur in certain mutants of C. Elegans. Thus cell death is part of normal developmental program in this animal- and in others too, e.g during development of hands and feet of many vertebrates, the cells that lie between the developing digits must die; if they do not, the digits remain fused. Programmed cell death is therefore a fundamental phenomenon among animals. Without it, the formation and function of organs would be impaired by cells that simply ‘get in the way.’
Incomplete differentiation in two toes (syndactyly) due to lack of apoptosis
Programmed cell death is also important in preventing the occurrence of cancers. If a cell with an abnormal ability to replicate is killed, it can not multiply to form tumor. Thus, programmed cell death is an important check against renegade cells that could otherwise proliferate uncontrollably in an organism.
Programmed cell death is called apoptosis from greek roots meaning “falling away.” The events that trigger cell death are only partially understood. A family of proteolytic enzymes called caspases play a crucial role in the cell death phenomenon. The caspases remove small part of other proteins by cleaving peptide bonds. Through this enzymatic trimming, the target proteins are inactivated. The caspases attack many different kinds of proteins, including the lamins, which make up the inner lining of nuclear envelope, and several components of cytoskeleton. The collective impact of these proteolytic cleavage is that cells in which it occurs lose their integrity; their chromatin becomes fragmented, blebs of cytoplasm form at their surfaces, and they begin to shrink. Cells undergoing this kind if disintegration are usually engulfed by Phagocytosis, and are then destroyed. If the apoptotic mechanism has been impaired or inactivated, a cell that should otherwise be killed can survive and proliferate. Such a cell has the potential to form a clone that could become cancerous if it acquire the ability to divide uncontrollably.
Genetic basis for cancer- There are strong evidence that the underlying causes of cancer are genetic.
1. It was known that cancerous state is clonally inherited. When cancer cells are grown in culture, their descendents are all cancerous. Therefore, cancerous condition is transmitted from each cell to its daughter cells at the time of division.
2. It was known that certain types of viruses ca induce the formation of tumors in experimental animals. The induction of cancer by viruses implies that the protein encoded by viral genes are involved in production of the cancerous state.
3. Cancer can be induced by agents capable of causing mutations. Mutagenic chemicals and ionizing radiations had been shown to induce tumors in experimental animals.
4. It was known that certain types of cancer run in families. In particular susceptibility to retinoblastoma, a rare cancer of eye, is inherited as dominant conditions, albeit with incomplete penetrance and variable expressivity.
So all these shows that special types of cancer is inherited- it seemed plausible that all cancer types might have their genetic basis.
In 1980s when molecular techniques were first used to study cancer cells, researchers discovered that the cancerous state is indeed, traceable to genetic defects. Typically, however not one but several such defects are required to convert a normal cell to cancerous cell.
Researchers identified two broad classes of genes that when mutated, can contribute to the development of a cancerous state.
• In one of these class mutated genes actively promote cell division
• In other class mutant genes fail to express cell division.
Genes in first class are called oncogenes from greek word tumor
In second class are called tumor suppressor genes.
Oncogenes- An oncogene is a gene that, when mutated or expressed at high levels, helps turn a normal cell into a tumor cell.
Many abnormal cells normally undergo a programmed form of death (apoptosis). Activated oncogenes can cause those cells to survive and proliferate instead. Most oncogenes require an additional step, such as mutations in another gene, or environmental factors, such as viral infection, to cause cancer. Since the 1970s, dozens of oncogenes have been identified in human cancer. Many cancer drugs target those DNA sequences and their products.
History-The first oncogene was discovered in 1970 and was termed src (pronounced sarc as in sarcoma). Src was in fact first discovered as an oncogene in a chicken retrovirus. Experiments performed by Dr G. Steve Martin of the University of California, Berkeley demonstrated that the SRC was indeed the oncogene of the virus.
In 1976 Drs. J. Michael Bishop and Harold E. Varmus of the University of California, San Francisco demonstrated that oncogenes were defective proto-oncogenes, found in many organisms including humans. For this discovery Bishop and Varmus were awarded the Nobel Prize in 1989.
Proto- oncogenes- A proto-oncogene is a normal gene that can become an oncogene due to mutations or increased expression. The resultant protein may be termed an oncoprotein. Proto-oncogenes code for proteins that help to regulate cell growth and differentiation. Proto-oncogenes are often involved in signal transduction and execution of mitogenic signals, usually through their protein products. Upon activation, a proto-oncogene (or its product) becomes a tumor-inducing agent, an oncogene. Examples of proto-oncogenes include RAS, WNT, MYC, ERK, and TRK.
Activation-The proto-oncogene can become an oncogene by a relatively small modification of its original function. There are three basic activation types:
• A mutation within a proto-oncogene can cause a change in the protein structure, causing
o An increase in protein (enzyme) activity
o A loss of regulation
• An increase in protein concentration, caused by
o An increase of protein expression (through misregulation)
o An increase of protein (mRNA) stability, prolonging its existence and thus its activity in the cell
o A gene duplication (one type of chromosome abnormality), resulting in an increased amount of protein in the cell
• A chromosomal translocation (another type of chromosome abnormality), causing
o An increased gene expression in the wrong cell type or at wrong times
o The expression of a constitutively active hybrid protein. This type of aberration in a dividing stem cell in the bone marrow leads to adult leukemia
The expression of oncogenes can be regulated by microRNAs (miRNAs), small RNAs 21-25 nucleotides in length that control gene expression by downregulating them Mutations in such microRNAs (known as oncomirs) can lead to activation of oncogenes. Antisense messenger RNAs could theoretically be used to block the effects of oncogenes.
Conversion of proto-oncogenes-There are two mechanisms by which proto-oncogenes can be converted to cellular oncogenes:
Quantitative: Tumor formation is induced by an increase in the absolute number of proto-oncogene products or by its production in inappropriate cell types.
Qualitative: Conversion from proto-oncogene to transforming gene (c-onc) with changes in the nucleotide sequence which are responsible for the acquisition of the new properties
Tumor suppressor gene- Tumor suppressor gene, or anti-oncogene, is a gene that protects a cell from one step on the path to cancer. When this gene is mutated to cause a loss or reduction in its function, the cell can progress to cancer, usually in combination with other genetic changes.
Two-hit hypothesis-Unlike oncogenes, tumor suppressor genes generally follow the 'two-hit hypothesis', which implies that both alleles that code for a particular gene must be affected before an effect is manifested. This is due to the fact that if only one allele for the gene is damaged, the second can still produce the correct protein. In other words, mutant tumor suppressors alleles are usually recessive whereas mutant oncogene alleles are typically dominant. The two-hit hypothesis was first proposed by A.G. Knudson for cases of retinoblastoma. Knudson observed that the age of onset of retinoblastoma followed 2nd order kinetics, implying that two independent genetic events were necessary. He recognized that this was consistent with a recessive mutation involving a single gene, but requiring biallelic mutation. Oncogene mutations, in contrast, generally involve a single allele because they are gain of function mutations. There are notable exceptions to the 'two-hit' rule for tumor suppressors, such as certain mutations in the p53 gene product. P53 mutations can function as a 'dominant negative', meaning that a mutated p53 protein can prevent the function of normal protein from the un-mutated allele. Other tumor-suppressor genes that are exceptions to the 'two-hit' rule are those which exhibit haploinsufficiency. An example of this is the p27Kip1 cell-cycle inhibitor, in which mutation of a single allele causes increased carcinogen susceptibility.
Examples-The first tumor-suppressor protein discovered was the Retinoblastoma protein (prb) in human retinoblastoma; however, recent evidence has also implicated prb as a tumor-survival factor.
Another important tumor suppressor is the p53 tumor-suppressor protein encoded by the TP53 gene. Homozygous loss of p53 is found in 70% of colon cancers, 30–50% of breast cancers, and 50% of lung cancers. Mutated p53 is also involved in the pathophysiology of leukemias, lymphomas, sarcomas, and neurogenic tumors. Abnormalities of the p53 gene can be inherited in Li-Fraumeni syndrome (LFS), which increases the risk of developing various types of cancers.
PTEN acts by opposing the action of PI3K, which is essential for anti-apoptotic, pro-tumorogenic Akt activation.
Other examples of tumor suppressors include APC, CD95, ST5, ST7, and ST14.
Functions-
Tumor-suppressor genes, or more precisely, the proteins for which they code, either have a dampening or repressive effect on the regulation of the cell cycle or promote apoptosis, and sometimes do both. The functions of tumor-suppressor proteins fall into several categories including the following:
1. Repression of genes that are essential for the continuing of the cell cycle. If these genes are not expressed, the cell cycle will not continue, effectively inhibiting cell division.
2. Coupling the cell cycle to DNA damage. As long as there is damaged DNA in the cell, it should not divide. If the damage can be repaired, the cell cycle can continue.
3. If the damage cannot be repaired, the cell should initiate apoptosis (programmed cell death) to remove the threat it poses for the greater good of the organism.
Some proteins involved in cell adhesion prevent tumor cells from dispersing, block loss of contact inhibition, and inhibit metastasis. These proteins are known as metastasis suppressors.
Conclusion-
Cancer results from breakdown of the regulatory mechanisms that govern from normal cell behavior. The proliferation, differentiation and survival of individual cells in multicellular organisms are carefully regulated to meet the needs of the organism as a whole. This regulation is lost in cancer cells, which grow and divide in an uncontrolled manner, ultimately spreading throughout body and interfering with the function of normal tissues and organs.
Because cancer results from defects in fundamental cell regulatory mechanisms, it is a disease that ultimately has to be understood at the molecular and cellular levels.
organisation and uses of chloroplast genome
Contents:
• What is chloroplast
• Ultrastructure of chloroplast
• Genome
• Physical properties
• Gene content
• Ct genes and encoded proteins
• Ultrastructure of ct genome
• Chloroplast transformation
• Recent uses of ct genome
Introduction-
Chloroplasts, as name suggests, are the plastids containing green pigment chlorophyll and is responsible for the green colour of photosynthetic organs of plants and algae. These are membrane bound, photosynthetic, eukaryotic organelle responsible for photosynthesis and observable as flat discs usually 2 to 10 µm in diameter and 1 µm thick. In land plants, they are, in general, 5 μm in diameter and 2.3 μm thick.
Ultrastructure of chloroplast-
The chloroplast is contained by an envelope that consists of an inner and an outer phospholipid membrane. Between these two layers is the intermembrane space. A typical parenchyma cell contains about 10 to 100 chloroplasts.
The material within the chloroplast is called the stroma, corresponding to the cytosol of the original bacterium, and contains one or more molecules of small circular DNA. It also contains ribosomes; however most of its proteins are encoded by genes contained in the host cell nucleus, with the protein products transported to the chloroplast Within the stroma are stacks of thylakoids, the sub-organelles, which are the site of photosynthesis. The thylakoids are arranged in stacks called grana (singular: granum). A thylakoid has a flattened disk shape. Inside it is an empty area called the thylakoid space or lumen. Photosynthesis takes place on the thylakoid membrane; as in mitochondrial oxidative phosphorylation, it involves the coupling of cross-membrane fluxes with biosynthesis via the dissipation of a proton electrochemical gradient.
1. outer membrane
2. intermembrane space
3. inner membrane (1+2+3: envelope)
4. stroma (aqueous fluid)
5. thylakoid lumen (inside of thylakoid)
6. thylakoid membrane
7. granum (stack of thylakoids)
8. thylakoid (lamella)
9. starch
10. ribosome
11. plastidial DNA
12. plastoglobule (drop of lipids
Chloroplast genome organization-
Chloroplasts posses a degree of autonomy within the cell that is in many ways similar to that of mitochondria. They do contain in the stroma a DNA that is unique to the organelle. With this genome a no. of chloroplast specific proteins are made using ribosomes that are also located in the stroma. Like mitochondria, chloroplasts replicate and thereby demonstrate a measure of reproductive autonomy.
Chloroplast contain their own genetic system, reflecting their evolutionary origins from photosynthetic bacteria. The 6 to 9 Mb genomes of present day free living photosynthetic cyanobacteria code for between 5400 and 7200 proteins. Like those of mitochondria, the genome of chloroplast consist of circular DNA molecules present in multiple copies per organelle. However, chloroplast genome are larger and more complex than those of mitochondria, ranging from 120 to 160 kb and containing approx. 150 genes.
Physical properties of chloroplast DNA-
• The entire chloroplast Genome resides within a single circular chloroplast DNA (ct DNA) molecule
• However, the DNA is generally present in multiple copies with as many as 20 to 60 ct DNA per chloroplast
• Depending on the species of organism, molecular weights of ct DNA commonly range from 85 to 140 ×106 daltons.
• The contour length is around 45µm, but may range from about 40 to 60 µm depending upon the species
• Isolated ct DNA exists in a variety of forms and conformations. Some are unicircular and other appeared as interlocked dimers.
• Two types of dimer are found:
-circular dimers which are formed by recombination between monomers, and
-catenated dimers in which two monomers interlink in a chain.
• Circular dimers may constitute upto 10% of ct DNA and catenated dimers about 2.5 %. The monomers often appear as relaxed circular duplexes in vitro, but in situ the closed supercoiled form is predominant.
• Ct DNA from higher plants and most algae have characteristic composition of 37+ 1 % (G+C).
• Density of 1.695 to 1.697 g cm-3. a notable exception is ct DNA from alga Euglena has G+C content of 28.2% and density of 1.685 g cm-3.
Gene Content of Chloroplast DNA-
The chloroplast genome of several plants have been completely sequenced, leading to the identification of many of the genes contained in the organelle DNAs.
First chloroplast genome sequenced was the liverwort (Marchantia polymorpha) genome which contains 121024 bp which contains 1024 inverted base repeats, separated by a small single copy region and a large single copy region.
These chloroplast genes code both RNAs and proteins involved in gene expression as well as variety of proteins that function in photosynthesis. Both the ribosomal and transfer RNAs used for translation of chloroplast mRNAs are encoded by the organelle genome
Genes encoded by the chloroplast DNA-
Function No. of genes
Genes for the genetic apparatus
rRNAs 4
tRNAs 30
Ribosomal proteins 21
RNA polymerase subunits 4
Genes for photosynthesis
photosystemI 5
photosystemII 12
Cytocrome bf complex 4
ATP synthase 6
Ribulose bis phosphate carboxylase 1
These include four rRNAs ( 23S, 16S, 5S and 4.5S). and 30 tRNA species. In contrast to the smaller no. of tRNAs encoded by mitochondrial genome, the chloroplast tRNAs are sufficient to translate all the mRNA codons according to the universal genetic code. In addition to these RNA components of the translation system, the chloroplast genome encodes about 20 ribosomal proteins, which represent approx. one third of the proteins of chloroplast ribosomes. Some subunits of RNA polymerase are also encoded by chloroplast, although additional RNA polymerase subunits and other factors needed for chloroplast gene expression are encoded in the nucleus.
The chloroplast genome also encodes approx. 30 proteins that are involved in photosynthesis, including the components of photosystem I and II, and of the cytochrome bf complex,, and of ATP synthase. In addition one of the subunits of ribulose bis phosphate carboxylase (rubisco) is encoded by chloroplast DNA. Rubisco is the critical enzyme that catalyzes the addition of CO2 to ribulose-1,5-bisphosphate during calvin cycle not only it is the major component of the chloroplast stroma, but it is also thought to be the single most abundant protein on earth, so it is noteworthy that one of its subunits is encode by the chloroplast genome.
Chloroplast Genes and protein encoded-
Ribosomal RNAs: Ribosomal RNA operons are designated as:
rrnA, B,C. Each operon normally includes genes for 16S rRNA, 23S rRNA, 5S rRNA, and 4.55 rRNA.
Transfer RNAs: tRNA genes are designated "trn' to indicate transfer RNA, followed by the single letter amino acid code indicating the amino acid accepted by the tRNA encoded by the gene. Where there is more than one gene for a particular amino acid, the isoaccepting species can be indicated either with sequential numbers or by giving the anticodon. About 40 tRNA genes are known to exist in the chloroplast genome.
Examples:
trnF-gene for tRNA(Phe)
trnC-gene for tRNA(Cys)
trn L1 (or trnL-UAA)--gene for tRNA(Leu)1
trn L2 (or trnL-CAA)--gene for tRNA(Leu)2
Ribosomal Proteins-
rps 4-ribosomal protein homologous to E. coli ribosomal protein S4.
rps 19---ribosomal protein homologous to E. coli ribosomal protein S 19
rpl 2-ribosomal protein homologous to E. coli ribosomal protein L2.
Photosystem I Proteins-
psaA 1-P700 chlorophyll a apoprotein
psaA2-P700 chlorophyll a apoprotein
Photosystem II Proteins-
psbA-"32 kilodalton' quinone-binding polypeptide. Also known as "photogene 32" and "Qb protein"; it contains the binding site for atrazine type herbicides.
psB-51 kilodalton chlorophyll a-binding polypeptide or p680 apoprotein.
pbC-44 kilodalton chlorophyll a-binding polypeptide.
pbD-"D2" protein.
psbE-cytochrome b559.
Photosynthetic Electron Proteins
pet-cytochrome f
petS-cytochrome b6
petD-subunit 4 of the cytochrome b6/f complex
Proteins of the ATP Synthase Complex-
atpA-CF1 alpha subunit
atpS-CF1 beta subunit
atpE-CF1 epsilon subunit
atpH-CFO subunit III, DCCD-binding proteolipid for the ATPase complex (proton translocating subunit)
Carbon Fixation Enzymes-
rbcL-ribulose bisphosphate carboxylase, large subunit
Other Stromal Polypeptides
tufA-translational elongation factor Tu
Ultra structure of chloroplast genome-
Chloroplast DNA contains long repetitive sequences making up 20 to 30 % of the contour length of the monomer. Shorter repetitive sequences that are inverted are also found in ct DNA.
The repetitive and non repetitive sequences are organized in segments in all ct DNA. The genome is thus divided into shorter regions, two of which contain repetitive sequences that are inverted with respect to each other and two that are made of nonrepetitive sequences . physical map of this sort have revealed that rRNA genes appear in the order 16, 23 and 5 S RNAs similar to that found in E. coli. Transcription is fron 16 to 23 S these two genomes are separated by approx. 2100 bp (in Zea mays). A 4.5S RNA, characteristic of chloroplast Ribosomes, is coded for by a gene in the vicinity of the 5S RNA genes.
Gene coding for chloroplast RNAs are scattered over the genome and are found both in inverted repeat regions and in non repetitive regions.
Chloroplast transformation (Transplastomic plant)-A transplastomic plant is a genetically modified plant in which the new genes have not been inserted in the nuclear DNA but in the DNA of the chloroplasts. The major advantage of this technology is that in many plant species plastid DNA is not transmitted through pollen, which prevents gene flow from the genetically modified plant to other plants.
Transformation technology-
The most common method to transform plastids is particle bombardment: Small gold or tungsten particles are coated with DNA and shot into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. The transformation efficiency is lower than in agrobacterial mediated transformation, which is also common in plant genetic engineering, but particle bombardment is especially suitable for plastid transformation.
In order to persist and be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the chromosome. Because transformation usually produces a mixture of rare transformed cells and abundant non-transformed cells, a method is needed to identify the cells that have acquired the plasmid. Plasmids used in transformation experiments will usually also contain a gene giving resistance to an antibiotic that the intended recipient strain of bacteria is sensitive to. Selection for cells able to grow on media containing this antibiotic can then select the cells that have acquired the plasmid by transformation, as cells lacking the plasmid will be unable to grow.
Transplastomic tobacco-However, plastid transformation is suitable only for certain crop species, and the reliability of this method has only been proven for tobacco. Led by Ralph Bock from the Max Planck Institute of Molecular Plant Physiology in Germany, researchers studied genetically modified tobacco in which the transgene was integrated in chloroplasts. The researchers analysed more than two million seedlings and found that less than 20 in 1,000,000 inherited the transgene. In the pollen of adult plants, the rate was even lower, remaining below 3 in 1,000,000. This reduction is because some parts of the seedlings are lost during their development into mature plants.
Because tobacco has a strong tendency towards self-fertilisation, the reliability of transplastomic plants is assumed to be even higher under field conditions. Therefore, the researchers believe that only one in 100,000,000 GM tobacco plants actually would transmit the transgene via pollen. Such values are more than satisfactory to ensure coexistence. However, for GM crops used in the production of pharmaceuticals, or in other cases in which absolutely no outcrossing is permitted, the researchers recommend the combination of chloroplast transformation with other biological containment methods, such as cytoplasmic male sterility or transgene mitigation strategies.
Recent applications of plastid genome-
• Expression of bar in the Plastid Genome Confers Herbicide Resistance-
Phosphinothricin (PPT) is the active component of a family of environmentally safe, nonselective herbicides. Resistance to PPT in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinothricin acetyltransferase, a detoxifying enzyme. We report here expression of a bacterial bar gene (b-bar1) in tobacco (Nicotiana tabacum cv Petit Havana) plastids that confers field-level tolerance to Liberty, an herbicide containing PPT.
• Polymorphic simple sequence repeat regions in chloroplast genomes: applications to the population genetics of pines-
Analysis of 305 individuals from seven populations of Pinus leucodermis Ant. revealed the presence of four variants with intrapopulational diversities ranging from 0.000 to 0.629 and an average of 0.320. Restriction fragment length polymorphism analysis of cpDNA on the same populations previously failed to detect any variation. Population subdivision based on cpSSR was higher (Gst = 0.22, where Gst is coefficient of gene differentiation) than that revealed in a previous isozyme study (Gst = 0.05). We anticipate that SSR loci within the chloroplast genome should provide a highly informative assay for the analysis of the genetic structure of plant populations.
• Use as plastid vector-The present invention provides a method to circumvent the problem of using antibiotic resistant selection markers. In particular, the target plants are transformed using a plastid vectorwhich contains a heterologous DNA sequences coding for a phytotoxindetoxifying enzyme or protein. the selection process involves converting a antibiotic free phytotoxic agent by the expresed phytotoxin detoxifying enzyme or protein to yield a nontoxic compound. The invention provides various methods to use antibiotic free selection in chloroplast transformation.
• What is chloroplast
• Ultrastructure of chloroplast
• Genome
• Physical properties
• Gene content
• Ct genes and encoded proteins
• Ultrastructure of ct genome
• Chloroplast transformation
• Recent uses of ct genome
Introduction-
Chloroplasts, as name suggests, are the plastids containing green pigment chlorophyll and is responsible for the green colour of photosynthetic organs of plants and algae. These are membrane bound, photosynthetic, eukaryotic organelle responsible for photosynthesis and observable as flat discs usually 2 to 10 µm in diameter and 1 µm thick. In land plants, they are, in general, 5 μm in diameter and 2.3 μm thick.
Ultrastructure of chloroplast-
The chloroplast is contained by an envelope that consists of an inner and an outer phospholipid membrane. Between these two layers is the intermembrane space. A typical parenchyma cell contains about 10 to 100 chloroplasts.
The material within the chloroplast is called the stroma, corresponding to the cytosol of the original bacterium, and contains one or more molecules of small circular DNA. It also contains ribosomes; however most of its proteins are encoded by genes contained in the host cell nucleus, with the protein products transported to the chloroplast Within the stroma are stacks of thylakoids, the sub-organelles, which are the site of photosynthesis. The thylakoids are arranged in stacks called grana (singular: granum). A thylakoid has a flattened disk shape. Inside it is an empty area called the thylakoid space or lumen. Photosynthesis takes place on the thylakoid membrane; as in mitochondrial oxidative phosphorylation, it involves the coupling of cross-membrane fluxes with biosynthesis via the dissipation of a proton electrochemical gradient.
1. outer membrane
2. intermembrane space
3. inner membrane (1+2+3: envelope)
4. stroma (aqueous fluid)
5. thylakoid lumen (inside of thylakoid)
6. thylakoid membrane
7. granum (stack of thylakoids)
8. thylakoid (lamella)
9. starch
10. ribosome
11. plastidial DNA
12. plastoglobule (drop of lipids
Chloroplast genome organization-
Chloroplasts posses a degree of autonomy within the cell that is in many ways similar to that of mitochondria. They do contain in the stroma a DNA that is unique to the organelle. With this genome a no. of chloroplast specific proteins are made using ribosomes that are also located in the stroma. Like mitochondria, chloroplasts replicate and thereby demonstrate a measure of reproductive autonomy.
Chloroplast contain their own genetic system, reflecting their evolutionary origins from photosynthetic bacteria. The 6 to 9 Mb genomes of present day free living photosynthetic cyanobacteria code for between 5400 and 7200 proteins. Like those of mitochondria, the genome of chloroplast consist of circular DNA molecules present in multiple copies per organelle. However, chloroplast genome are larger and more complex than those of mitochondria, ranging from 120 to 160 kb and containing approx. 150 genes.
Physical properties of chloroplast DNA-
• The entire chloroplast Genome resides within a single circular chloroplast DNA (ct DNA) molecule
• However, the DNA is generally present in multiple copies with as many as 20 to 60 ct DNA per chloroplast
• Depending on the species of organism, molecular weights of ct DNA commonly range from 85 to 140 ×106 daltons.
• The contour length is around 45µm, but may range from about 40 to 60 µm depending upon the species
• Isolated ct DNA exists in a variety of forms and conformations. Some are unicircular and other appeared as interlocked dimers.
• Two types of dimer are found:
-circular dimers which are formed by recombination between monomers, and
-catenated dimers in which two monomers interlink in a chain.
• Circular dimers may constitute upto 10% of ct DNA and catenated dimers about 2.5 %. The monomers often appear as relaxed circular duplexes in vitro, but in situ the closed supercoiled form is predominant.
• Ct DNA from higher plants and most algae have characteristic composition of 37+ 1 % (G+C).
• Density of 1.695 to 1.697 g cm-3. a notable exception is ct DNA from alga Euglena has G+C content of 28.2% and density of 1.685 g cm-3.
Gene Content of Chloroplast DNA-
The chloroplast genome of several plants have been completely sequenced, leading to the identification of many of the genes contained in the organelle DNAs.
First chloroplast genome sequenced was the liverwort (Marchantia polymorpha) genome which contains 121024 bp which contains 1024 inverted base repeats, separated by a small single copy region and a large single copy region.
These chloroplast genes code both RNAs and proteins involved in gene expression as well as variety of proteins that function in photosynthesis. Both the ribosomal and transfer RNAs used for translation of chloroplast mRNAs are encoded by the organelle genome
Genes encoded by the chloroplast DNA-
Function No. of genes
Genes for the genetic apparatus
rRNAs 4
tRNAs 30
Ribosomal proteins 21
RNA polymerase subunits 4
Genes for photosynthesis
photosystemI 5
photosystemII 12
Cytocrome bf complex 4
ATP synthase 6
Ribulose bis phosphate carboxylase 1
These include four rRNAs ( 23S, 16S, 5S and 4.5S). and 30 tRNA species. In contrast to the smaller no. of tRNAs encoded by mitochondrial genome, the chloroplast tRNAs are sufficient to translate all the mRNA codons according to the universal genetic code. In addition to these RNA components of the translation system, the chloroplast genome encodes about 20 ribosomal proteins, which represent approx. one third of the proteins of chloroplast ribosomes. Some subunits of RNA polymerase are also encoded by chloroplast, although additional RNA polymerase subunits and other factors needed for chloroplast gene expression are encoded in the nucleus.
The chloroplast genome also encodes approx. 30 proteins that are involved in photosynthesis, including the components of photosystem I and II, and of the cytochrome bf complex,, and of ATP synthase. In addition one of the subunits of ribulose bis phosphate carboxylase (rubisco) is encoded by chloroplast DNA. Rubisco is the critical enzyme that catalyzes the addition of CO2 to ribulose-1,5-bisphosphate during calvin cycle not only it is the major component of the chloroplast stroma, but it is also thought to be the single most abundant protein on earth, so it is noteworthy that one of its subunits is encode by the chloroplast genome.
Chloroplast Genes and protein encoded-
Ribosomal RNAs: Ribosomal RNA operons are designated as:
rrnA, B,C. Each operon normally includes genes for 16S rRNA, 23S rRNA, 5S rRNA, and 4.55 rRNA.
Transfer RNAs: tRNA genes are designated "trn' to indicate transfer RNA, followed by the single letter amino acid code indicating the amino acid accepted by the tRNA encoded by the gene. Where there is more than one gene for a particular amino acid, the isoaccepting species can be indicated either with sequential numbers or by giving the anticodon. About 40 tRNA genes are known to exist in the chloroplast genome.
Examples:
trnF-gene for tRNA(Phe)
trnC-gene for tRNA(Cys)
trn L1 (or trnL-UAA)--gene for tRNA(Leu)1
trn L2 (or trnL-CAA)--gene for tRNA(Leu)2
Ribosomal Proteins-
rps 4-ribosomal protein homologous to E. coli ribosomal protein S4.
rps 19---ribosomal protein homologous to E. coli ribosomal protein S 19
rpl 2-ribosomal protein homologous to E. coli ribosomal protein L2.
Photosystem I Proteins-
psaA 1-P700 chlorophyll a apoprotein
psaA2-P700 chlorophyll a apoprotein
Photosystem II Proteins-
psbA-"32 kilodalton' quinone-binding polypeptide. Also known as "photogene 32" and "Qb protein"; it contains the binding site for atrazine type herbicides.
psB-51 kilodalton chlorophyll a-binding polypeptide or p680 apoprotein.
pbC-44 kilodalton chlorophyll a-binding polypeptide.
pbD-"D2" protein.
psbE-cytochrome b559.
Photosynthetic Electron Proteins
pet-cytochrome f
petS-cytochrome b6
petD-subunit 4 of the cytochrome b6/f complex
Proteins of the ATP Synthase Complex-
atpA-CF1 alpha subunit
atpS-CF1 beta subunit
atpE-CF1 epsilon subunit
atpH-CFO subunit III, DCCD-binding proteolipid for the ATPase complex (proton translocating subunit)
Carbon Fixation Enzymes-
rbcL-ribulose bisphosphate carboxylase, large subunit
Other Stromal Polypeptides
tufA-translational elongation factor Tu
Ultra structure of chloroplast genome-
Chloroplast DNA contains long repetitive sequences making up 20 to 30 % of the contour length of the monomer. Shorter repetitive sequences that are inverted are also found in ct DNA.
The repetitive and non repetitive sequences are organized in segments in all ct DNA. The genome is thus divided into shorter regions, two of which contain repetitive sequences that are inverted with respect to each other and two that are made of nonrepetitive sequences . physical map of this sort have revealed that rRNA genes appear in the order 16, 23 and 5 S RNAs similar to that found in E. coli. Transcription is fron 16 to 23 S these two genomes are separated by approx. 2100 bp (in Zea mays). A 4.5S RNA, characteristic of chloroplast Ribosomes, is coded for by a gene in the vicinity of the 5S RNA genes.
Gene coding for chloroplast RNAs are scattered over the genome and are found both in inverted repeat regions and in non repetitive regions.
Chloroplast transformation (Transplastomic plant)-A transplastomic plant is a genetically modified plant in which the new genes have not been inserted in the nuclear DNA but in the DNA of the chloroplasts. The major advantage of this technology is that in many plant species plastid DNA is not transmitted through pollen, which prevents gene flow from the genetically modified plant to other plants.
Transformation technology-
The most common method to transform plastids is particle bombardment: Small gold or tungsten particles are coated with DNA and shot into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. The transformation efficiency is lower than in agrobacterial mediated transformation, which is also common in plant genetic engineering, but particle bombardment is especially suitable for plastid transformation.
In order to persist and be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the chromosome. Because transformation usually produces a mixture of rare transformed cells and abundant non-transformed cells, a method is needed to identify the cells that have acquired the plasmid. Plasmids used in transformation experiments will usually also contain a gene giving resistance to an antibiotic that the intended recipient strain of bacteria is sensitive to. Selection for cells able to grow on media containing this antibiotic can then select the cells that have acquired the plasmid by transformation, as cells lacking the plasmid will be unable to grow.
Transplastomic tobacco-However, plastid transformation is suitable only for certain crop species, and the reliability of this method has only been proven for tobacco. Led by Ralph Bock from the Max Planck Institute of Molecular Plant Physiology in Germany, researchers studied genetically modified tobacco in which the transgene was integrated in chloroplasts. The researchers analysed more than two million seedlings and found that less than 20 in 1,000,000 inherited the transgene. In the pollen of adult plants, the rate was even lower, remaining below 3 in 1,000,000. This reduction is because some parts of the seedlings are lost during their development into mature plants.
Because tobacco has a strong tendency towards self-fertilisation, the reliability of transplastomic plants is assumed to be even higher under field conditions. Therefore, the researchers believe that only one in 100,000,000 GM tobacco plants actually would transmit the transgene via pollen. Such values are more than satisfactory to ensure coexistence. However, for GM crops used in the production of pharmaceuticals, or in other cases in which absolutely no outcrossing is permitted, the researchers recommend the combination of chloroplast transformation with other biological containment methods, such as cytoplasmic male sterility or transgene mitigation strategies.
Recent applications of plastid genome-
• Expression of bar in the Plastid Genome Confers Herbicide Resistance-
Phosphinothricin (PPT) is the active component of a family of environmentally safe, nonselective herbicides. Resistance to PPT in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinothricin acetyltransferase, a detoxifying enzyme. We report here expression of a bacterial bar gene (b-bar1) in tobacco (Nicotiana tabacum cv Petit Havana) plastids that confers field-level tolerance to Liberty, an herbicide containing PPT.
• Polymorphic simple sequence repeat regions in chloroplast genomes: applications to the population genetics of pines-
Analysis of 305 individuals from seven populations of Pinus leucodermis Ant. revealed the presence of four variants with intrapopulational diversities ranging from 0.000 to 0.629 and an average of 0.320. Restriction fragment length polymorphism analysis of cpDNA on the same populations previously failed to detect any variation. Population subdivision based on cpSSR was higher (Gst = 0.22, where Gst is coefficient of gene differentiation) than that revealed in a previous isozyme study (Gst = 0.05). We anticipate that SSR loci within the chloroplast genome should provide a highly informative assay for the analysis of the genetic structure of plant populations.
• Use as plastid vector-The present invention provides a method to circumvent the problem of using antibiotic resistant selection markers. In particular, the target plants are transformed using a plastid vectorwhich contains a heterologous DNA sequences coding for a phytotoxindetoxifying enzyme or protein. the selection process involves converting a antibiotic free phytotoxic agent by the expresed phytotoxin detoxifying enzyme or protein to yield a nontoxic compound. The invention provides various methods to use antibiotic free selection in chloroplast transformation.
biotechnology not possible without phages
Biotechnology Not possible Without phages
CONTENTS:
• What is biotechnology?
• What are phages?
• Applications of phages-
• As alternatives to antibiotics
• Phage display
• Phage display libraries
• Vaccine delivery vehicles
• Gene therapy delivery vehicles
• Detection of pathogenic bacteria
• Phage regulatory elements in gene expression technology
• Applications of phage lysis enzymes
• Clinical applications
• Conclusions
• Future directions
WHAT IS BIOTECHNOLOGY?
Biotechnology is a field of biology that involves the use of living things in engineering, technology, medicine, etc. Modern use of the term refers to genetic engineering as well as cell- and tissue culture technologies.
United Nations Convention on Biological Diversity defines biotechnology as:
"Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use."
Biotechnology is of following types:
• Blue biotechnology is a term that has been used to describe the marine and aquatic applications of biotechnology.
• Green biotechnology is biotechnology applied to agricultural processes. E.g Bt corn
• Red biotechnology is applied to medical processes. E.g. Designing of organisms to produce antibiotics, and the engineering of genetic cures through genetic manipulation.
• White biotechnology, also known as industrial biotechnology, is biotechnology applied to industrial processes.. Another example is the using of enzymes as industrial catalysts to either produce valuable chemicals or destroy hazardous/polluting chemicals.
The investment and economic output of all of these types of applied biotechnologies is termed as bioeconomy.
WHAT ARE BACTERIOPHAGES?
Bacteriophages (or phages) are viruses consisting of a DNA or RNA genome contained within a protein coat. They infect bacteria and either incorporate viral DNA into the host genome, replicating as part of the host (lysogeny), or multiply inside the host cell before releasing phage particles either by budding from the membrane or by actively lysing the cell.
APPLICATIONS OF PHAGES:Field of biotechnology does not seem possible without bacteriophages as bacteriophages have several potential applications in the modern biotechnology industry:
• Alternative to antibiotics
• Phage display
• Phage display libraries
• Vaccine delivery vehicles
• Gene therapy delivery vehicles;
• Detection of pathogenic bacteria
• Phage regulatory elements in gene expression technology
• Applications of phage lysis enzymes
• Clinical applications
1. ALTERNATIVE TO ANTIBIOTICS OR PHAGE THERAPYPhage therapy is the therapeutic use of bacteriophages to treat pathogenic bacterial infections. Phage therapy has many potential applications in human medicine as well as dentistry, veterinary science, and agriculture.
A Georgian, George Eliava, alongwith d'Hérelle, and in 1923 he founded the Eliava Institute in Tbilisi, Georgia, devoted to the development of phage therapy.
Bacteriophage treatment offers a possible alternative to conventional antibiotic treatments for bacterial infection. It is conceivable that, although bacteria can develop resistance to phage, the resistance might be easier to overcome than resistance to antibiotics. Bacteriophages are very specific, targeting only one or a few strains of bacteria. Traditional antibiotics have more wide-ranging effect, killing both harmful bacteria and useful bacteria such as those facilitating food digestion. The specificity of bacteriophages might reduce the chance that useful bacteria are killed when fighting an infection. Some evidence shows the ability of phages to travel to a required site—including the brain, where the blood brain barrier can be crossed—and multiply in the presence of an appropriate bacterial host, to combat infections such as meningitis.
• Enzobiotics are a new development at Rockefeller University that create enzymes from phage. These show potential for preventing secondary bacterial infections, e.g. pneumonia developing in patients suffering from flu, otitis etc.
• The first approval granted by the FDA and UDSA for a phage-based food additive is approval for ListShield targeted against Listeria monocytogenes) created by Intralytix. in August 2006, spraying meat with phages.
• The Southwest Regional Wound Care Centre in Texas (www.woundcarecenter.net) has been using phages (in combination with other methods) to treat antibiotic-resistant infections. To enable residents of the USA to access phage therapy more readily, the company Phage International (http://www.phageinternational.com) recently merged with the Georgian Phage Therapy Center, and opened a phage therapy centre in Tijuana, Mexico.
Example of a successful phage therapy treatment:
The patient was a 50-year-old female with venous leg ulcers. The wound was infected with a multidrug-resistant Pseudomonas, and had failed to heal after one year of conservative wound care management. It responded rapidly after the application of phage, forming a healthy, red granulating base within two weeks and later showing epithelial islands inside the wound, a characteristic of phage therapy.
2. PHAGE DISPLAY-
In phage display, a heterologous peptide or protein is displayed on the surface of the phage through transcriptional fusion with a coat-protein gene, producing novel phage particles that have a variety of potential uses. The most widely used phage display methods are based on the use of M13 and related filamentous phages of Escherichia coli but others, including the E. Coli phages lambda and T7, have also been used Because particulate phages are relatively easy and inexpensive to purify, phage display can also provide a means of purifying a particular protein or antibody.
Examples of some methods that have been used to fuse foreign proteins/peptides to the surface of bacteriophage. Foreign proteins can usually be displayed on more than one phage coat protein and in varying amounts. Generally, the smaller the foreign protein or peptide the more copies can be displayed, although this also depends on the phage used, the coat protein and the antigen displayed.
(a) The gene for a foreign peptide is fused directly to the coat-protein gene. All minor coat-proteins display the foreign antigen, which can limit the size of the peptide displayed. In this example, the minor coat-protein is used because it results in fewer copies of the foreign protein being displayed.
(b) Two copies of a coat protein gene can be present in the phage – one native and one fusion. As such, not all proteins display the foreign molecule, therefore larger proteins can often be displayed.
(c) Unmodified ‘helper’ phage infect cells containing a phagemid, which then expresses the coat protein fused to a foreign peptide or protein.
In a similar way to (b), this results in phage particles in which not all coat proteins display foreign antigens. Generally, a phagemid-type system is preferred because there is some control over how many copies of the foreign antigen are displayed, and manipulations can be carried out in a plasmid-like system. The exact methods used for phage display will vary, depending on the type of phage used and the antigen being displayed.
3. PHAGE-DISPLAY LIBRARIES-
can be screened in several ways to isolate displayed peptides or proteins with practical applications. For example, it is possible to isolate displayed peptides that bind target proteins with affinities similar to those of antibodies. These can then be used as therapeutics that act either as agonists or through the inhibition of receptor–ligand interactions. These high-affinity, displayed peptides also have the potential to be used for the detection of pathogens and agents posing a biological threat in the environment . In another example, directed evolution of proteins can be used to enhance enzymatic activity or binding properties. Here, the active site of an enzyme is randomly altered, and the library screened for increased activity.
A variation of phage display, which is also worthy of note, is the use of phages to display a library of Fab antibody fragments on the surface of filamentous phages . Although these libraries have generally been used for research purposes, one particularly novel use for phage-displayed antibodies was in the development of a nasally delivered treatment against cocaine addiction whole phage particles delivered nasally can enter the central nervous system where the specific phage-displayed antibody can bind to cocaine molecules and prevent their action on the brain.
4. PHAGES AS VACCINE DELIVERY VEHICLES-
Phages have been used as potential vaccine delivery vehicles in two different ways:
• by directly vaccinating with phages carrying vaccine antigens on their surface or
• by using the phage particle to deliver a DNA vaccine expression cassette that has been incorporated into the phage genome.
(i) In phage-display vaccination,- phages can be designed to display a specific antigenic peptide or protein on their surface. Alternatively, phages displaying peptide libraries can be screened with a specific antiserum to isolate novel protective antigens or mimetopes – peptides that mimic the secondary structure and antigenic properties of a protective carbohydrate, protein or lipid, despite having a different primary structure. The serum of convalescents can also be used to screen phage-display libraries to identify potential vaccines against a specific disease, without prior knowledge of protective antigens. In a few cases, whole phage particles displaying antigenic proteins have been used as vaccines in animal models. Because phage particles are naturally immunostimulatory an antigen presented on the phage coat would come ‘ready conjugated’ with a natural adjuvant activity, without the need for separate protein purification and subsequent conjugation to a carrier molecule before immunization.
(ii) DNA vaccine expression cassette- More recently, it has also been shown that unmodified phages can be used to deliver DNA vaccines more efficiently than standard plasmid DNA vaccination. The vaccine gene, under the control of a eukaryotic expression cassette, is cloned into a standard lambda bacteriophage, and purified whole phage particles are injected into the host. The phage coat protects the DNA from degradation and, because it is a virus-like particle, it should target the vaccine to the antigen-presenting cells. When compared with standard DNA vaccination, superior antibody responses have been shown in mice and rabbits. We have also recently shown that, similar to phage display, genome libraries in phage (such as Lambda ZAP expresstm containing both a prokaryotic and eukaryotic promoter) can be expressed in E. Coli, screened with convalescent serum and potential vaccines identified. These can then be used, directly, for vaccination.
5. PHAGES FOR TARGETED GENE-DELIVERY-
Phages have also been proposed as potential therapeutic-gene delivery vectors. Although conceptually different, the rationale for using phages for this purpose is similar to that for using phages for DNA vaccine delivery – the phage coat protects the DNA from degradation after injection, and the ability to display foreign molecules on the phage coat also enables targeting of specific cell types, a prerequisite for effective gene therapy.
Both artificial covalent conjugation and phage display have been used to display targeting and/or processing molecules on the surface of the phages. Targeting sequences, such as fibroblast growth factor, have been used to deliver phages to cells bearing the appropriate receptors and, whereas protein sequences such as the penton base of adenovirus, which mediates viral attachment, entry and endosomal release, have been used to enhance phage uptake and endosomal release. Similarly, the protein transduction domain of the HIV tat protein and the nuclear localization signal derived from the SV40 T antigen have been used to enhance uptake and nuclear targeting of modified phage lambda. Other examples of displayed peptides, which might facilitate phage-mediated gene delivery, include integrin-binding peptides (to enhance binding and uptake) and dnase II inhibitors (to reduce DNA degradation).
6. PHAGES FOR THE DETECTION AND TYPING OF BACTERIA-
For many years the specificity of phages for their bacterial hosts has enabled them to be used for the typing of bacterial strains and for the detection of low numbers of pathogenic bacteria. Phages bound to bacteria can be detected by specific, labeled antibodies, thereby increasing the sensitivity of detection. For specific typing, different species of phage can be plated out onto a lawn comprising an unidentified bacterial strain, and the presence of clear areas (plaques) where an individual phage particle has grown and lysed the surrounding cells enables identification of the specific bacteria. Other methods that have been used to detect pathogenic bacteria include: using phages specifically to deliver reporter genes (e.g. Lux or green fluorescent protein), which are expressed after infection of target bacteria; using phages that have a fluorescent dye covalently attached to the phage coat, and detecting the specific adsorption; the detection of released cellular components, such as adenylate kinase, after specific lysis; and using phages displaying peptides or antibody fragments that will bind specific bacterial pathogens or toxins.
7. PHAGE REGULATORY ELEMENTS IN GENE EXPRESSION TECHNOLOGY-
Several cloning vectors are either derivatives of bacteriophages (e.g., phages λ and M13 of E. Coli) or contain some elements of phage origin (e.g., cosmids and phagemid vectors). Site specific integration elements (attachment sites) in the genomes of temperate phages and host bacterium (attp and attb, respectively) can be used for stable insertion of cloned genes into the bacterial genome. Such integration vectors have been developed for a variety of organisms. Phages are a potential source of genetic elements for vector construction.
8. APPLICABILITY OF LYSIS GENES FOR LYSIS OF BACTERIA-
Recent advances in the purification technologies (like fluidisized beds and expanded bed adsorption methodology) permit the adsorption of proteins directly from the culture medium (Chase 1994). Such purification techniques are especially suitable for proteins secreted into culture medium. In order to make the intracellular or periplasmic located biomolecules accessible for purification, the bacterial cells must be disrupted.
The first suggestion for the use of phage genes to obtain lysates containing intracellular enzymes was from Sher and Mallette (1952), who purified L-lysine decarboxylase and L-arginine decarboxylase from a phage lysate after infection of E. Coli with phage T2. Auerbach and Rosenberg (1987) have patented the use of an E. Coli strain containing defective temperature sensitive lambda lysogens as a method for cell disruption. The prophage lacks the genes for replication or structural protein assembly, and functional phages can not therefore be produced. The lysis genes are under temperature sensitive control through the use of the lambda pl promoter and the ci857 repressor. The lysis could be induced in mid-log phase by a temperature shift to 42-44C. The phage lambda lysis genes S (holin), R (transglycosylase), and Rz cloned under control of the lac promoter cause rapid lysis within 40 minutes after induction of the gene expression with IPTG. In the absence of the functional holin gene S, lysis does not occur. E. Coli can tolerate relatively high amount of intracellularly accumulated phage lysin (up to 2 % in the case of the phage T4 lysin) without lysis (Perry et al. 1985). Injuries in the cytoplasmic membrane by phage holin, freeze-thawing, osmotic shock or chemicals (like chloroform or toluene) yield rapid degradation of the cell wall and lysis of bacteria. Phage T7 lysin (amidase) has been used for construction of E. Coli strains with increased susceptibility for lysis. Such strains can be conveniently used for externalization of intracellular gene products by osmotic shock treatment . The T7 lysin has a dual function. Beside its cell wall hydrolyzing activity it downregulates phage T7 RNA polymerase (and thus expression of the genes cloned under control of T7 promoter)
Recently, de Ruyter et al. (1997) cloned the holin and lysin genes of phage into Lactococcus lactis under control of a nisin inducible promoter. They were able to obtain nisin-inducible lysis of bacteria and acceleration of ripening of experimental cheeses. These very promising results wait for exploitation for larger scale cheese manufacturing.
9. CLINICAL APPLICATIONS –
Because S. sanguis is the first colonizer of newly cleaned teeth and because other bacteria then attach to it, the formation of dental plaque is reduced on newly cleaned teeth by introducing into the mouth bacteriophages which are parasitic to S. sanguis. Because S. sanguis is the means of attachment of plaque forming bacterial colonies to tooth surfaces and forms 10-15% of the organisms in plaque, destruction of S. Sanguis by introduction of its parasitic bacteriophages will remove plaque from teeth surfaces. And removal of plaque containing acid forming bacteria and other harmful bacteria reduces the incidence of dental caries and other disease.
10. OTHER APPLICATIONS:
• used as model viruses in a number of contexts:
• as indicators of the presence of human viruses in natural Waters and wastewater,
• in particle adsorption studies,
• as model viruses in treatment studies i.e. coagulation, filtration, chlorination,
• Ultraviolet [UV] disinfection studies
CONCLUSION-
These are an indication of the large range of biotechnology and/or medical applications of phages, ranging from disease prevention (phage vaccines) through diagnosis (detection and typing of bacteria) to actual treatment (anti-bacterial phage therapy, phage-display antibodies or phage-delivered gene therapy).
This versatility raises two interesting points:
• The possibility of engineering a ‘jack-of-all-trades’ bacteriophage, or phage mixture, to treat different stages of disease (i.e. Both prophylactic and therapeutic),
• The commonality of procedures and techniques across a wide range of potential applications.
FUTURE DIRECTIONS-
Conferences attempting to bridge the gap, such as the American Society for Microbiology Conference on New Phage Biology in Florida, USA in 2004, a Bacteriophage Group Session at the 156th Society for General Microbiology Meeting in production and operation Washington State, USA, which deserves a special mention for successfully bringing together practical and applied phage biologists. Although biotechnologists at these meetings are still the exception rather than the rule, this situation can hopefully only improve as the remarkable versatility and potential applications of these organisms becomes more widely known and developed.
CONTENTS:
• What is biotechnology?
• What are phages?
• Applications of phages-
• As alternatives to antibiotics
• Phage display
• Phage display libraries
• Vaccine delivery vehicles
• Gene therapy delivery vehicles
• Detection of pathogenic bacteria
• Phage regulatory elements in gene expression technology
• Applications of phage lysis enzymes
• Clinical applications
• Conclusions
• Future directions
WHAT IS BIOTECHNOLOGY?
Biotechnology is a field of biology that involves the use of living things in engineering, technology, medicine, etc. Modern use of the term refers to genetic engineering as well as cell- and tissue culture technologies.
United Nations Convention on Biological Diversity defines biotechnology as:
"Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use."
Biotechnology is of following types:
• Blue biotechnology is a term that has been used to describe the marine and aquatic applications of biotechnology.
• Green biotechnology is biotechnology applied to agricultural processes. E.g Bt corn
• Red biotechnology is applied to medical processes. E.g. Designing of organisms to produce antibiotics, and the engineering of genetic cures through genetic manipulation.
• White biotechnology, also known as industrial biotechnology, is biotechnology applied to industrial processes.. Another example is the using of enzymes as industrial catalysts to either produce valuable chemicals or destroy hazardous/polluting chemicals.
The investment and economic output of all of these types of applied biotechnologies is termed as bioeconomy.
WHAT ARE BACTERIOPHAGES?
Bacteriophages (or phages) are viruses consisting of a DNA or RNA genome contained within a protein coat. They infect bacteria and either incorporate viral DNA into the host genome, replicating as part of the host (lysogeny), or multiply inside the host cell before releasing phage particles either by budding from the membrane or by actively lysing the cell.
APPLICATIONS OF PHAGES:Field of biotechnology does not seem possible without bacteriophages as bacteriophages have several potential applications in the modern biotechnology industry:
• Alternative to antibiotics
• Phage display
• Phage display libraries
• Vaccine delivery vehicles
• Gene therapy delivery vehicles;
• Detection of pathogenic bacteria
• Phage regulatory elements in gene expression technology
• Applications of phage lysis enzymes
• Clinical applications
1. ALTERNATIVE TO ANTIBIOTICS OR PHAGE THERAPYPhage therapy is the therapeutic use of bacteriophages to treat pathogenic bacterial infections. Phage therapy has many potential applications in human medicine as well as dentistry, veterinary science, and agriculture.
A Georgian, George Eliava, alongwith d'Hérelle, and in 1923 he founded the Eliava Institute in Tbilisi, Georgia, devoted to the development of phage therapy.
Bacteriophage treatment offers a possible alternative to conventional antibiotic treatments for bacterial infection. It is conceivable that, although bacteria can develop resistance to phage, the resistance might be easier to overcome than resistance to antibiotics. Bacteriophages are very specific, targeting only one or a few strains of bacteria. Traditional antibiotics have more wide-ranging effect, killing both harmful bacteria and useful bacteria such as those facilitating food digestion. The specificity of bacteriophages might reduce the chance that useful bacteria are killed when fighting an infection. Some evidence shows the ability of phages to travel to a required site—including the brain, where the blood brain barrier can be crossed—and multiply in the presence of an appropriate bacterial host, to combat infections such as meningitis.
• Enzobiotics are a new development at Rockefeller University that create enzymes from phage. These show potential for preventing secondary bacterial infections, e.g. pneumonia developing in patients suffering from flu, otitis etc.
• The first approval granted by the FDA and UDSA for a phage-based food additive is approval for ListShield targeted against Listeria monocytogenes) created by Intralytix. in August 2006, spraying meat with phages.
• The Southwest Regional Wound Care Centre in Texas (www.woundcarecenter.net) has been using phages (in combination with other methods) to treat antibiotic-resistant infections. To enable residents of the USA to access phage therapy more readily, the company Phage International (http://www.phageinternational.com) recently merged with the Georgian Phage Therapy Center, and opened a phage therapy centre in Tijuana, Mexico.
Example of a successful phage therapy treatment:
The patient was a 50-year-old female with venous leg ulcers. The wound was infected with a multidrug-resistant Pseudomonas, and had failed to heal after one year of conservative wound care management. It responded rapidly after the application of phage, forming a healthy, red granulating base within two weeks and later showing epithelial islands inside the wound, a characteristic of phage therapy.
2. PHAGE DISPLAY-
In phage display, a heterologous peptide or protein is displayed on the surface of the phage through transcriptional fusion with a coat-protein gene, producing novel phage particles that have a variety of potential uses. The most widely used phage display methods are based on the use of M13 and related filamentous phages of Escherichia coli but others, including the E. Coli phages lambda and T7, have also been used Because particulate phages are relatively easy and inexpensive to purify, phage display can also provide a means of purifying a particular protein or antibody.
Examples of some methods that have been used to fuse foreign proteins/peptides to the surface of bacteriophage. Foreign proteins can usually be displayed on more than one phage coat protein and in varying amounts. Generally, the smaller the foreign protein or peptide the more copies can be displayed, although this also depends on the phage used, the coat protein and the antigen displayed.
(a) The gene for a foreign peptide is fused directly to the coat-protein gene. All minor coat-proteins display the foreign antigen, which can limit the size of the peptide displayed. In this example, the minor coat-protein is used because it results in fewer copies of the foreign protein being displayed.
(b) Two copies of a coat protein gene can be present in the phage – one native and one fusion. As such, not all proteins display the foreign molecule, therefore larger proteins can often be displayed.
(c) Unmodified ‘helper’ phage infect cells containing a phagemid, which then expresses the coat protein fused to a foreign peptide or protein.
In a similar way to (b), this results in phage particles in which not all coat proteins display foreign antigens. Generally, a phagemid-type system is preferred because there is some control over how many copies of the foreign antigen are displayed, and manipulations can be carried out in a plasmid-like system. The exact methods used for phage display will vary, depending on the type of phage used and the antigen being displayed.
3. PHAGE-DISPLAY LIBRARIES-
can be screened in several ways to isolate displayed peptides or proteins with practical applications. For example, it is possible to isolate displayed peptides that bind target proteins with affinities similar to those of antibodies. These can then be used as therapeutics that act either as agonists or through the inhibition of receptor–ligand interactions. These high-affinity, displayed peptides also have the potential to be used for the detection of pathogens and agents posing a biological threat in the environment . In another example, directed evolution of proteins can be used to enhance enzymatic activity or binding properties. Here, the active site of an enzyme is randomly altered, and the library screened for increased activity.
A variation of phage display, which is also worthy of note, is the use of phages to display a library of Fab antibody fragments on the surface of filamentous phages . Although these libraries have generally been used for research purposes, one particularly novel use for phage-displayed antibodies was in the development of a nasally delivered treatment against cocaine addiction whole phage particles delivered nasally can enter the central nervous system where the specific phage-displayed antibody can bind to cocaine molecules and prevent their action on the brain.
4. PHAGES AS VACCINE DELIVERY VEHICLES-
Phages have been used as potential vaccine delivery vehicles in two different ways:
• by directly vaccinating with phages carrying vaccine antigens on their surface or
• by using the phage particle to deliver a DNA vaccine expression cassette that has been incorporated into the phage genome.
(i) In phage-display vaccination,- phages can be designed to display a specific antigenic peptide or protein on their surface. Alternatively, phages displaying peptide libraries can be screened with a specific antiserum to isolate novel protective antigens or mimetopes – peptides that mimic the secondary structure and antigenic properties of a protective carbohydrate, protein or lipid, despite having a different primary structure. The serum of convalescents can also be used to screen phage-display libraries to identify potential vaccines against a specific disease, without prior knowledge of protective antigens. In a few cases, whole phage particles displaying antigenic proteins have been used as vaccines in animal models. Because phage particles are naturally immunostimulatory an antigen presented on the phage coat would come ‘ready conjugated’ with a natural adjuvant activity, without the need for separate protein purification and subsequent conjugation to a carrier molecule before immunization.
(ii) DNA vaccine expression cassette- More recently, it has also been shown that unmodified phages can be used to deliver DNA vaccines more efficiently than standard plasmid DNA vaccination. The vaccine gene, under the control of a eukaryotic expression cassette, is cloned into a standard lambda bacteriophage, and purified whole phage particles are injected into the host. The phage coat protects the DNA from degradation and, because it is a virus-like particle, it should target the vaccine to the antigen-presenting cells. When compared with standard DNA vaccination, superior antibody responses have been shown in mice and rabbits. We have also recently shown that, similar to phage display, genome libraries in phage (such as Lambda ZAP expresstm containing both a prokaryotic and eukaryotic promoter) can be expressed in E. Coli, screened with convalescent serum and potential vaccines identified. These can then be used, directly, for vaccination.
5. PHAGES FOR TARGETED GENE-DELIVERY-
Phages have also been proposed as potential therapeutic-gene delivery vectors. Although conceptually different, the rationale for using phages for this purpose is similar to that for using phages for DNA vaccine delivery – the phage coat protects the DNA from degradation after injection, and the ability to display foreign molecules on the phage coat also enables targeting of specific cell types, a prerequisite for effective gene therapy.
Both artificial covalent conjugation and phage display have been used to display targeting and/or processing molecules on the surface of the phages. Targeting sequences, such as fibroblast growth factor, have been used to deliver phages to cells bearing the appropriate receptors and, whereas protein sequences such as the penton base of adenovirus, which mediates viral attachment, entry and endosomal release, have been used to enhance phage uptake and endosomal release. Similarly, the protein transduction domain of the HIV tat protein and the nuclear localization signal derived from the SV40 T antigen have been used to enhance uptake and nuclear targeting of modified phage lambda. Other examples of displayed peptides, which might facilitate phage-mediated gene delivery, include integrin-binding peptides (to enhance binding and uptake) and dnase II inhibitors (to reduce DNA degradation).
6. PHAGES FOR THE DETECTION AND TYPING OF BACTERIA-
For many years the specificity of phages for their bacterial hosts has enabled them to be used for the typing of bacterial strains and for the detection of low numbers of pathogenic bacteria. Phages bound to bacteria can be detected by specific, labeled antibodies, thereby increasing the sensitivity of detection. For specific typing, different species of phage can be plated out onto a lawn comprising an unidentified bacterial strain, and the presence of clear areas (plaques) where an individual phage particle has grown and lysed the surrounding cells enables identification of the specific bacteria. Other methods that have been used to detect pathogenic bacteria include: using phages specifically to deliver reporter genes (e.g. Lux or green fluorescent protein), which are expressed after infection of target bacteria; using phages that have a fluorescent dye covalently attached to the phage coat, and detecting the specific adsorption; the detection of released cellular components, such as adenylate kinase, after specific lysis; and using phages displaying peptides or antibody fragments that will bind specific bacterial pathogens or toxins.
7. PHAGE REGULATORY ELEMENTS IN GENE EXPRESSION TECHNOLOGY-
Several cloning vectors are either derivatives of bacteriophages (e.g., phages λ and M13 of E. Coli) or contain some elements of phage origin (e.g., cosmids and phagemid vectors). Site specific integration elements (attachment sites) in the genomes of temperate phages and host bacterium (attp and attb, respectively) can be used for stable insertion of cloned genes into the bacterial genome. Such integration vectors have been developed for a variety of organisms. Phages are a potential source of genetic elements for vector construction.
8. APPLICABILITY OF LYSIS GENES FOR LYSIS OF BACTERIA-
Recent advances in the purification technologies (like fluidisized beds and expanded bed adsorption methodology) permit the adsorption of proteins directly from the culture medium (Chase 1994). Such purification techniques are especially suitable for proteins secreted into culture medium. In order to make the intracellular or periplasmic located biomolecules accessible for purification, the bacterial cells must be disrupted.
The first suggestion for the use of phage genes to obtain lysates containing intracellular enzymes was from Sher and Mallette (1952), who purified L-lysine decarboxylase and L-arginine decarboxylase from a phage lysate after infection of E. Coli with phage T2. Auerbach and Rosenberg (1987) have patented the use of an E. Coli strain containing defective temperature sensitive lambda lysogens as a method for cell disruption. The prophage lacks the genes for replication or structural protein assembly, and functional phages can not therefore be produced. The lysis genes are under temperature sensitive control through the use of the lambda pl promoter and the ci857 repressor. The lysis could be induced in mid-log phase by a temperature shift to 42-44C. The phage lambda lysis genes S (holin), R (transglycosylase), and Rz cloned under control of the lac promoter cause rapid lysis within 40 minutes after induction of the gene expression with IPTG. In the absence of the functional holin gene S, lysis does not occur. E. Coli can tolerate relatively high amount of intracellularly accumulated phage lysin (up to 2 % in the case of the phage T4 lysin) without lysis (Perry et al. 1985). Injuries in the cytoplasmic membrane by phage holin, freeze-thawing, osmotic shock or chemicals (like chloroform or toluene) yield rapid degradation of the cell wall and lysis of bacteria. Phage T7 lysin (amidase) has been used for construction of E. Coli strains with increased susceptibility for lysis. Such strains can be conveniently used for externalization of intracellular gene products by osmotic shock treatment . The T7 lysin has a dual function. Beside its cell wall hydrolyzing activity it downregulates phage T7 RNA polymerase (and thus expression of the genes cloned under control of T7 promoter)
Recently, de Ruyter et al. (1997) cloned the holin and lysin genes of phage into Lactococcus lactis under control of a nisin inducible promoter. They were able to obtain nisin-inducible lysis of bacteria and acceleration of ripening of experimental cheeses. These very promising results wait for exploitation for larger scale cheese manufacturing.
9. CLINICAL APPLICATIONS –
Because S. sanguis is the first colonizer of newly cleaned teeth and because other bacteria then attach to it, the formation of dental plaque is reduced on newly cleaned teeth by introducing into the mouth bacteriophages which are parasitic to S. sanguis. Because S. sanguis is the means of attachment of plaque forming bacterial colonies to tooth surfaces and forms 10-15% of the organisms in plaque, destruction of S. Sanguis by introduction of its parasitic bacteriophages will remove plaque from teeth surfaces. And removal of plaque containing acid forming bacteria and other harmful bacteria reduces the incidence of dental caries and other disease.
10. OTHER APPLICATIONS:
• used as model viruses in a number of contexts:
• as indicators of the presence of human viruses in natural Waters and wastewater,
• in particle adsorption studies,
• as model viruses in treatment studies i.e. coagulation, filtration, chlorination,
• Ultraviolet [UV] disinfection studies
CONCLUSION-
These are an indication of the large range of biotechnology and/or medical applications of phages, ranging from disease prevention (phage vaccines) through diagnosis (detection and typing of bacteria) to actual treatment (anti-bacterial phage therapy, phage-display antibodies or phage-delivered gene therapy).
This versatility raises two interesting points:
• The possibility of engineering a ‘jack-of-all-trades’ bacteriophage, or phage mixture, to treat different stages of disease (i.e. Both prophylactic and therapeutic),
• The commonality of procedures and techniques across a wide range of potential applications.
FUTURE DIRECTIONS-
Conferences attempting to bridge the gap, such as the American Society for Microbiology Conference on New Phage Biology in Florida, USA in 2004, a Bacteriophage Group Session at the 156th Society for General Microbiology Meeting in production and operation Washington State, USA, which deserves a special mention for successfully bringing together practical and applied phage biologists. Although biotechnologists at these meetings are still the exception rather than the rule, this situation can hopefully only improve as the remarkable versatility and potential applications of these organisms becomes more widely known and developed.
interaction of Agrobacterium with plants and non plant species
PLANT- AGROBACTERIUM INTERACTIONS
Contents:
• Agrobacterium tumefacians
• Characteristics
• Scientific classification
• Properties of crown gall cells
• The Ti plasmid
• Organization of T-DNA- (transferred DNA)
• Organization of vir region
• Molecular biology of Agrobacterium infection
• Attachment & Penetration
• Induction of vir genes
• Generation of T-DNA transfer complex
• Formation of the T-pilus
• Transfer and integration of T-DNA into plant cell
• Conclusion
Scientific classification
Kingdom: Bacteria
Phylum: Proteobacteria
Class: Alpha Proteobacteria
Order: Rhizobiales
Family: Rhizobiaceae
Genus: Agrobacterium
Species
• Agrobacterium radiobacter
• Agrobacterium rhizogenes
• Agrobacterium rubi
• Agrobacterium tumefaciens
A. tumefaciens a member of family Rhizobiaceae but Unlike the nitrogen fixing symbionts, tumor producing Agrobacterium are pathogenic.
A. tumefacians causes crown gall disease.
A. rhizogenes causes hairy roots which may often show negative geotropism.
Agrobacterium tumefacians-
Characteristics-
• Rod shaped
• Gram negative
• Soil bacterium
• Plant pathogen
• Causes crown gall disease in over 140 species of dicot.
Crown- part of plant which is present at junction of root and shoot; Gall- tumors.
• Gall forming genes are present on plasmid referred to as Ti plasmid
‘T’ stands for ‘tumor’ and ‘i’ stands for ‘inducing’
Means tumor inducing plasmid.
• Transmissible- When pTi is introduced into Rhizobium trifolii, it gains the ability to produce galls and to utilize opines and reverse is also true i.e. if nodulating genes from R. trifolii are transferred to Agrobacterium it can form nodules on specific host plant.
• The first evidences indicating this bacterium as the causative agent of the crown gall goes back to more than ninety years (Smith and Townsend, 1907).
Properties of crown gall and hairy root cells-
• Both hairy roots and crown gall cells are capable of growing in culture on a growth regulator (GR) free medium, while normal plant cells need exogenous auxin and or cytokinin.
• These plant cells have undergone cancerous or oncogenic alteration: they generally induce tumor when grafted onto a healthy plant.
• The hairy roots and crown gall cells synthesize unique nitrogenous compounds called opines, which are neither produced by normal plant cells nor utilized by them. Agrobacterium Cells use opines as carbon and nitrogen source; the bacteria are usually present in intercellular spaces of crown galls. Opines are of different types and type of opine produced by a crown gall depends on the bacterial strains.
A. tumefacians produce- octopine or nopaline
A. rhizogenes produce- agropine or mannopine.
A bacterial strain produces only one type of opine and it also catabolizes only that opine; the concerned genes are present in its pTi or pRi. These plasmids also carry genes for IAA and cytokinin production, which is the reason for indefinite growth of crown gall cells on a GR free culture medium.
The Ti plasmid-
The tumor inducing plasmid is a large conjugative plasmid or mega plasmid of about 200 kb (150-250kb). pTi is lost when Agrobacterium Grows above 28C; such cured bacteria do not induce crown galls i.e avirulent. pTi is unique in following respects:
It contain some genes (the genes located within their T-DNA), which have regulatory sequences recognized by plant cells, while their remaining genes have prokaryotic regulatory sequences. As a result former are expressed only in plant cells and not in Agrobacterium, while the latter expressed only in bacterium.
These plasmids naturally transfer their T-DNA into the host plant genome, which makes Agrobacterium Natural genetic engineer. The plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206,479 nucleotides long, the GC content is 56% and 81% of the material is coding genes. There are no pseudogenes.
The Ti plasmids are classified into different types based on type of opine produced by their genes. The different opines are- octopine, nopaline, succinamopine and leucinopine. The different Ti plasmids grouped in two categories:
• Octopine type and
• Nopaline type
Differ mainly in the organization of their T-DNAs.
Both contain following functional groups:
• T-DNA contains oncogenes and opine synthesis genes and is transferred into the host plant genome.
• vir region regulates the transfer of T-DNA into plant cells.
• Opine catabolism regions produce enzymes necessary for the utilization of opines by Agrobacterium.
• tra region for conjugative transfer of the plasmid.
• Origin of replication for propagation in Agrobacterium
Organization of T-DNA- (transferred DNA)-
T-DNA is 23 kb segment of Ti plasmids which is transferred into plant genome during agrobacterium Infection. T-DNA is defined on its both sides by a 24 bp repeats border sequences and contains the genes for tumor and opine synthesis. pTi has 3 genes, which are involved in crown gall formation. Two of these genes (iaaM and iaaH) encode enzymes that together convert tryptophan into indole acetic acid called auxin synthesizing genes. A deletion of these two genes produces shooty crowngalls. The third gene ipt encodes an enzyme which produces cytokinin isopentenyl adenine. In between ipt gene and RB there is nos gene codes for nopaline synthase enzyme involved in synthesis of nopaline.
All the genes present in T-DNA contain eukaryotic regulatory sequences. As a result these genes are expressed only in plant cells, and not in agrobacterium The gene ipt determines whether a given agrobacterium Strain has narrow or broad host range: all broad host range strains have a functional ipt gene, while those lacking functional ipt have a narrow host range.
Organization of vir region-
Vir region consists of 8 operons which together spans about 40 kb of DNA and have 25 genes this region mediates transfer of T-DNA into plant genome and hence is essential for virulence and therefore it is called as vir region. These genes are not transferred themselves; they only induce transfer of T-DNA.
Vir Genes and their Function
Vir Gene Function
Vir A, Vir G Sense phenolic compounds from wounded plant cells and induce expression
of other virulence genes
VirD2 Endonuclease; cuts T-DNA at right border to initiate T-strand synthesis
Vir D1 Topiosomerase; Helps Vir D2 to recognise and cleave within the 25bp
border sequence
Vir C Helicase; Binds to the 'overdrive' region to promote high efficiency T-strand
Synthesis
Vir E2 Binds to T-strand protecting it from nuclease attack, and intercalates
with lipids to form channels in the plant membranes through which the
T-complex passes
Vir E1 Acts as a chaperone which stabilises Vir E2 in the Agrobacterium
Vir B & Vir D4 Assemble into a secretion system which spans the inner and outer bacterial 0membranes. Required for Export of the T-complex and Vir E2 into the
plant cell
Vir G DNA binding proteins; formed dimer after phosphorylation by vir A
Molecular biology of Agrobacterium infection-
The molecular mechanism involved in Agrobacterium infection of plant cells became known only recently during 1980s. The process of infection involves transfer of small part of pTi into the plant cell genome; this DNA sequence is called as T-DNA. The infection process is governed by both chromosomal and plasmid borne genes of A. tumefaciens.
Attachment & Penetration-Infection begins when Agrobacterium cells become attached to plant cells: this step determine the host range of bacterium, is a function of host parasite interaction, and is governed by bacterial chromosomal genes, generally the chv (chromosomal virulence) genes. A. tumefaciens have flagella that allow them to swim through the soil towards photoassimilates that accumulate in the rhizosphere around roots. Chemotaxis: reaction of orientation and locomotion to chemical attractants. Without chemotaxis there will be no cell-cell contact. Some strains may chemotactically move towards chemical exudates coming out from wounded plant such as acetosyringone and sugars. Acetosyringone is recognised by the VirA protein, a transmembrane protein encoded in the virA gene on the Ti plasmid. Sugars are recognised by the chvE protein, a chromosomal gene-encoded protein located in the periplasmic space.
Most of the genes, e.g. chvB, exo, cel genes are involved in biosynthesis of cell attachment polysaccharides due to which the bacterial cells become adhered to plant cells. But at least two genes viz. chvD and chv E are needed for an optimal expression of pTi vir genes. These chromosomal genes are expressed constitutively i.e. expressed in all bacterial cells at all the times.
Attachment to the plant is a two stage process, firstly involving a weak initial adhesion, then the bacteria synthesise cellulose fibrils which anchor them to the wounded plant cell surface. Some of the bacterial genes required for this process have been identified, namely chvA, chvB, pscA and att, as a mutation in any of these genes leaves the bacterium unable to attach to the plant. There are also molecules within the plant which are thought to be involved in the attachment process. One such molecule is vitronectin; an adhesive glycoprotein which is a component of the plant extracellular matrix (ECM). Vitronectin is more commonly associated with the cohesion of plant cells, thus having a role in plant structure and rigidity.
After production of cellulose fibrils a Ca2+ dependent outer membrane protein called rhicadhesin is produced, which also aids in sticking the bacteria to the cell wall. Homologues of this protein can be found in other Rhizobia species.
Chromosomal genes Function
chvA Encodes an inner membrane protein essential for the transport of ß-1,2-glucan from cytoplasm to periplasm
chvB Encodes an inner membrane protein most likely involved in the synthesis of ß-1,2-glucan
chvD and chvE Needed for optimal expression of vir genes of pTi
exo locus genes Biosynthesis of attachment polysaccharides
exo C Encodes an enzyme directly involved in the biosynthesis of ß-1,2 glucan
cel Cellulose fibril synthesis especially during early phase of infection so that the bacterial cells become firmly adhered to plant cells.
Induction of vir genes
At least 25 vir genes on Ti plasmid are necessary for tumor induction.In addition to their perception role, virA and chvE induce other vir genes. The VirA protein has a kinase activity, it phosphorylates it self on a histidine residue. Then the VirA protein phosphorylates the VirG protein on its aspartate residue. The VirG protein is a cytoplasmic protein traduced from the virG Ti plasmid gene, it's a transcription factor. It induces the transcription of the vir operons. ChvE protein regulates the second mechanism of vir genes activation. It increases VirA protein sensibility to phenolic compounds.
Possible plant compounds, that initiate Agrobacterium to infect plant cells:
• Acetosyringone: Phenolic compound
• alpha-Hydroxyacetosyringone
• Catechol
• Ferulic acid
• Gallic acid
• p-Hydroxybenzoic acid
• Protocatechuic acid
• Pyrogallic acid
• Resorcylic acid
• Sinapinic acid
• Syringic acid
• Vanillin
Generation of T-DNA transfer complexAfter activation by phosphorylated vir G, first Vir C, a helicase, unwinds DNA then Vir D1, a topoisomerase, binds to RB sequence and relaxes supercoiling which facilitates the action of Vir D2. Vir D2, an endonuclease, nicks at RB and covalently binds to 5’ end so generated. The 3’ end so produced at the site of nick serves as primer for DNA synthesis in 5’ to 3’ direction. As a result ss of T-DNA is displaced from DNA duplex. T- strand is again nicked at LB to generate a ss copy of T-DNA. Vir E2 is SSBP stabilizes the ss copy of T-DNA. About 600 copies of it bind to ss T-DNA and protect from nuclease action.
Extensive mutation or deletion of the right T-DNA border is followed by almost completely loss of T-DNA transfer capacity, while at the left border results in lower transfer efficiency. This fact indicates that T-strand synthesis is initiated at the right border, it proceeds in the 5' to 3' direction the termination process takes place even when the left border is mutated or completely absent, although with lower efficiency. Left border may act as a starting site for ss T-strand synthesis but the efficiency is much lower. The difference may be a consequence of the presence of an enhancer or "overdrive" sequence next to the right border. This enhancer has been found to be specifically recognized by VirC1 protein. Deletion of virC operon is followed by attenuation of virulence of the Agrobacterium strains
Formation of the T-pilusIn order to transfer the T-DNA into the plant cell A. tumefaciens uses a Type IV secretion mechanism, involving the production of a T-pilus.
The VirA/VirG two component sensor system is able to detect phenolic signals released by wounded plant cells, in particular acetosyringone. This leads to a signal transduction event activating the expression of 11 genes within the VirB operon which are responsible for the formation of the T-pilus.
First, the VirB" pro-pilin is formed. This is a polypeptide of 121 amino acids which requires processing by the removal of 47 residues to form a T-pilus subunit. The subunit is circularized by the formation of a peptide bond between the two ends of the polypeptide.
Products of the other VirB genes are used to transfer the subunits across the plasma membrane. Yeast two-hybrid studies provide evidence that VirB6, VirB7, VirB8, VirB9 and VirB10 may all encode components of the transporter. An ATPase for the active transport of the subunits would also be required.
(Step 1) VirB1, VirB2, and VirB5 are translocated to the inner membrane via the GSP. Signal peptides (SPI) domains are represented by black wavy lines at the left N-terminal end of each protein.
(Step 2) Signal peptides are now removed from VirB1, VirB2, and VirB5. VirB1 is processed to release VirB1* from lytic transglycosylase domain.
(Step 3A) Lytic transglycosylase domain degrades peptidoglycan (PG) to create space for assembly of the vir-T4SS trans-envelope core.
(Step 3B) VirB1* binds to VirB2 and VirB5 during their transit from the GSP to the cell exterior.
(Step 4) At the site of T-pilus assembly,VirB2 and VirB5 are mobilized to the exterior of the cell. Mobilization of VirB1* to cell exterior does not require any additional Vir proteins.
A fraction of VirB1* remains associated with T pili, while some VirB1* is free in the medium. VirB2 is drawn as a shaded oval to represent the cyclized form of the mature peptide. VirB5 is drawn as shaded stacked trapezoids to represent the helix bundles of the solved structure of VirB5 . The VirB5 trapezoid is shown localized at the base of the T pilus; however, its precise localization within the T-pilus structure remains to be determined. VirB1 is shown with two domain structures. The N terminus of VirB1 in the periplasm is shown as a "Pac-Man" due to the function of this lytic transglycosylase-homologous domain to "chew" the peptidoglycan. C-terminal VirB1* is drawn as a white ellipsoid. *, VirB1*; ?, unknown exporter for VirB1*; IM, inner membrane; OM, outer membrane.
Transfer of T-DNA into plant cellIn the cytoplasm of the recipient cell, Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. VIP1 also appears to be an important protein in the process, possibly acting as an adapter to bring the VirE2 to the importin. Once inside the nucleus, VIP2 may target the T-DNA to areas of chromatin that are being actively transcribed, so that the T-DNA can integrate into the host genome.
Uses in plant improvement-• The ability of Agrobacterium to transfer genes to plants and fungi is used in biotechnology, in particular, genetic engineering for plant improvement.
• A modified Ti or Ri plasmid can be used.
• The plasmid is 'disarmed' by deletion of the tumor inducing genes; the only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation.
• The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the disarmed plasmid, together with a selectable marker (such as antibiotic resistance) to enable selection for plants that have been successfully transformed. Plants are grown on media containing antibiotic following transformation, and those that do not have the T-DNA integrated into their genome will die.
• Several vectors have been designed based in T plasmid like binary and cointegrate vectors.
• Several crops have been modified like Bt cotton, Bt brinjal etc.
Recent advancements in Agrobacterium interactionAlthough none of the Bryophytae and Pteridophytae species could be transformed by Agrobacterium upto 56% of gymnosperms and 58% of angiosperms (only 8% of monocotyledons) were susceptible to infection by wild type Agrobacterium. Functional similarity between T4SSs of Agrobacterium and other bacteria, such as intacellular pathogens of mammals Brucella spp and Legionella pnuemophila suggests that Agrobacterium can potentially exchange genetic material with non plant species. Under lab conditions, the host range of Agrobacterium can be extended to non plant eukaryotic organisms. These include yeast, filamentous fungi, cultivated mushrooms and human cultures cells.
First report of Agrobacterium mediated gene transfer (AGMT) of a non plant host involved cells of the budding yeast Saccaromyces cerevisieae whereby a ura- yeast strain was transformed to ura+ following introduction of the Ura3- encoding gene by T-DNA transfer and integration. Using specially designed Agrobacterium binary vectors, several unique features of AGMT of yeast cells were identified-
• Circularization and autonomous replication of T-DNA molecules in host cells could be achieved by introduction of the yeast 2 µ replication origin into T-DNA region.
• T-DNA integration into host genome by the homologous recombination was possible if T-DNA contained specific sequences that share homology with yeast genome.
By contrast no autonomous replication of T-DNA molecules has ever been observed in plant cells and integration in plant cells is mostly, if not solely, mediated by illegitimate recombination.
Some years later, Aspergillus awamori became first filamentous fungus to be genetically transformed by Agrobacterium.
Kunik et. al showed that cultured human cell lines (HeLa, HEK293 and neuronal PC12 cells were transformed by Agrobacterium using neomycin resistance as the selection marker. In their report, the T-DNA transfer and integration into human cells shared most of the features of plant AGMT
• Bacterial attachment to the host cell, an essential step in plant transformation by Agrobacterium was similar in human cells and plant protoplasts.
• Agrobacterium mutants in the chvA and chvB loci wre unable to bind to human cells.
• The mode of T-DNA integration into human genome was essentially similar to integration of DNA into plant genome suggesting bona fide T-DNA transfer rather than conjugative transfer of the Ti plasmid.
Moreover this mechanism of DNA transfer process, which relies on Agrobacterium Vir proteins, is essentially the same in plant and non plant hosts. However, the mechanism by which the T-DNA molecule integrates into host genome is most likely to be dictated by the nature of the host organism and nucleotide sequence of the T-DNA.
Conclusion-
Agrobacterium tumefaciens is more than the causative agent of crown gall disease affecting dicotyledonous plants. It is also the natural instance for the introduction of foreign genes in plants allowing its genetic manipulation. Similarities have been found between T-DNA and conjugal transfer systems. They are evolutionally related and apparently evolved from a common ancestor.
Undoubtedly, the development of transformation procedures based on A. tumefaciens-mediated gene transfer for new economically important species are advisable and the results obtained in recent years evidence a promising future.
Contents:
• Agrobacterium tumefacians
• Characteristics
• Scientific classification
• Properties of crown gall cells
• The Ti plasmid
• Organization of T-DNA- (transferred DNA)
• Organization of vir region
• Molecular biology of Agrobacterium infection
• Attachment & Penetration
• Induction of vir genes
• Generation of T-DNA transfer complex
• Formation of the T-pilus
• Transfer and integration of T-DNA into plant cell
• Conclusion
Scientific classification
Kingdom: Bacteria
Phylum: Proteobacteria
Class: Alpha Proteobacteria
Order: Rhizobiales
Family: Rhizobiaceae
Genus: Agrobacterium
Species
• Agrobacterium radiobacter
• Agrobacterium rhizogenes
• Agrobacterium rubi
• Agrobacterium tumefaciens
A. tumefaciens a member of family Rhizobiaceae but Unlike the nitrogen fixing symbionts, tumor producing Agrobacterium are pathogenic.
A. tumefacians causes crown gall disease.
A. rhizogenes causes hairy roots which may often show negative geotropism.
Agrobacterium tumefacians-
Characteristics-
• Rod shaped
• Gram negative
• Soil bacterium
• Plant pathogen
• Causes crown gall disease in over 140 species of dicot.
Crown- part of plant which is present at junction of root and shoot; Gall- tumors.
• Gall forming genes are present on plasmid referred to as Ti plasmid
‘T’ stands for ‘tumor’ and ‘i’ stands for ‘inducing’
Means tumor inducing plasmid.
• Transmissible- When pTi is introduced into Rhizobium trifolii, it gains the ability to produce galls and to utilize opines and reverse is also true i.e. if nodulating genes from R. trifolii are transferred to Agrobacterium it can form nodules on specific host plant.
• The first evidences indicating this bacterium as the causative agent of the crown gall goes back to more than ninety years (Smith and Townsend, 1907).
Properties of crown gall and hairy root cells-
• Both hairy roots and crown gall cells are capable of growing in culture on a growth regulator (GR) free medium, while normal plant cells need exogenous auxin and or cytokinin.
• These plant cells have undergone cancerous or oncogenic alteration: they generally induce tumor when grafted onto a healthy plant.
• The hairy roots and crown gall cells synthesize unique nitrogenous compounds called opines, which are neither produced by normal plant cells nor utilized by them. Agrobacterium Cells use opines as carbon and nitrogen source; the bacteria are usually present in intercellular spaces of crown galls. Opines are of different types and type of opine produced by a crown gall depends on the bacterial strains.
A. tumefacians produce- octopine or nopaline
A. rhizogenes produce- agropine or mannopine.
A bacterial strain produces only one type of opine and it also catabolizes only that opine; the concerned genes are present in its pTi or pRi. These plasmids also carry genes for IAA and cytokinin production, which is the reason for indefinite growth of crown gall cells on a GR free culture medium.
The Ti plasmid-
The tumor inducing plasmid is a large conjugative plasmid or mega plasmid of about 200 kb (150-250kb). pTi is lost when Agrobacterium Grows above 28C; such cured bacteria do not induce crown galls i.e avirulent. pTi is unique in following respects:
It contain some genes (the genes located within their T-DNA), which have regulatory sequences recognized by plant cells, while their remaining genes have prokaryotic regulatory sequences. As a result former are expressed only in plant cells and not in Agrobacterium, while the latter expressed only in bacterium.
These plasmids naturally transfer their T-DNA into the host plant genome, which makes Agrobacterium Natural genetic engineer. The plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206,479 nucleotides long, the GC content is 56% and 81% of the material is coding genes. There are no pseudogenes.
The Ti plasmids are classified into different types based on type of opine produced by their genes. The different opines are- octopine, nopaline, succinamopine and leucinopine. The different Ti plasmids grouped in two categories:
• Octopine type and
• Nopaline type
Differ mainly in the organization of their T-DNAs.
Both contain following functional groups:
• T-DNA contains oncogenes and opine synthesis genes and is transferred into the host plant genome.
• vir region regulates the transfer of T-DNA into plant cells.
• Opine catabolism regions produce enzymes necessary for the utilization of opines by Agrobacterium.
• tra region for conjugative transfer of the plasmid.
• Origin of replication for propagation in Agrobacterium
Organization of T-DNA- (transferred DNA)-
T-DNA is 23 kb segment of Ti plasmids which is transferred into plant genome during agrobacterium Infection. T-DNA is defined on its both sides by a 24 bp repeats border sequences and contains the genes for tumor and opine synthesis. pTi has 3 genes, which are involved in crown gall formation. Two of these genes (iaaM and iaaH) encode enzymes that together convert tryptophan into indole acetic acid called auxin synthesizing genes. A deletion of these two genes produces shooty crowngalls. The third gene ipt encodes an enzyme which produces cytokinin isopentenyl adenine. In between ipt gene and RB there is nos gene codes for nopaline synthase enzyme involved in synthesis of nopaline.
All the genes present in T-DNA contain eukaryotic regulatory sequences. As a result these genes are expressed only in plant cells, and not in agrobacterium The gene ipt determines whether a given agrobacterium Strain has narrow or broad host range: all broad host range strains have a functional ipt gene, while those lacking functional ipt have a narrow host range.
Organization of vir region-
Vir region consists of 8 operons which together spans about 40 kb of DNA and have 25 genes this region mediates transfer of T-DNA into plant genome and hence is essential for virulence and therefore it is called as vir region. These genes are not transferred themselves; they only induce transfer of T-DNA.
Vir Genes and their Function
Vir Gene Function
Vir A, Vir G Sense phenolic compounds from wounded plant cells and induce expression
of other virulence genes
VirD2 Endonuclease; cuts T-DNA at right border to initiate T-strand synthesis
Vir D1 Topiosomerase; Helps Vir D2 to recognise and cleave within the 25bp
border sequence
Vir C Helicase; Binds to the 'overdrive' region to promote high efficiency T-strand
Synthesis
Vir E2 Binds to T-strand protecting it from nuclease attack, and intercalates
with lipids to form channels in the plant membranes through which the
T-complex passes
Vir E1 Acts as a chaperone which stabilises Vir E2 in the Agrobacterium
Vir B & Vir D4 Assemble into a secretion system which spans the inner and outer bacterial 0membranes. Required for Export of the T-complex and Vir E2 into the
plant cell
Vir G DNA binding proteins; formed dimer after phosphorylation by vir A
Molecular biology of Agrobacterium infection-
The molecular mechanism involved in Agrobacterium infection of plant cells became known only recently during 1980s. The process of infection involves transfer of small part of pTi into the plant cell genome; this DNA sequence is called as T-DNA. The infection process is governed by both chromosomal and plasmid borne genes of A. tumefaciens.
Attachment & Penetration-Infection begins when Agrobacterium cells become attached to plant cells: this step determine the host range of bacterium, is a function of host parasite interaction, and is governed by bacterial chromosomal genes, generally the chv (chromosomal virulence) genes. A. tumefaciens have flagella that allow them to swim through the soil towards photoassimilates that accumulate in the rhizosphere around roots. Chemotaxis: reaction of orientation and locomotion to chemical attractants. Without chemotaxis there will be no cell-cell contact. Some strains may chemotactically move towards chemical exudates coming out from wounded plant such as acetosyringone and sugars. Acetosyringone is recognised by the VirA protein, a transmembrane protein encoded in the virA gene on the Ti plasmid. Sugars are recognised by the chvE protein, a chromosomal gene-encoded protein located in the periplasmic space.
Most of the genes, e.g. chvB, exo, cel genes are involved in biosynthesis of cell attachment polysaccharides due to which the bacterial cells become adhered to plant cells. But at least two genes viz. chvD and chv E are needed for an optimal expression of pTi vir genes. These chromosomal genes are expressed constitutively i.e. expressed in all bacterial cells at all the times.
Attachment to the plant is a two stage process, firstly involving a weak initial adhesion, then the bacteria synthesise cellulose fibrils which anchor them to the wounded plant cell surface. Some of the bacterial genes required for this process have been identified, namely chvA, chvB, pscA and att, as a mutation in any of these genes leaves the bacterium unable to attach to the plant. There are also molecules within the plant which are thought to be involved in the attachment process. One such molecule is vitronectin; an adhesive glycoprotein which is a component of the plant extracellular matrix (ECM). Vitronectin is more commonly associated with the cohesion of plant cells, thus having a role in plant structure and rigidity.
After production of cellulose fibrils a Ca2+ dependent outer membrane protein called rhicadhesin is produced, which also aids in sticking the bacteria to the cell wall. Homologues of this protein can be found in other Rhizobia species.
Chromosomal genes Function
chvA Encodes an inner membrane protein essential for the transport of ß-1,2-glucan from cytoplasm to periplasm
chvB Encodes an inner membrane protein most likely involved in the synthesis of ß-1,2-glucan
chvD and chvE Needed for optimal expression of vir genes of pTi
exo locus genes Biosynthesis of attachment polysaccharides
exo C Encodes an enzyme directly involved in the biosynthesis of ß-1,2 glucan
cel Cellulose fibril synthesis especially during early phase of infection so that the bacterial cells become firmly adhered to plant cells.
Induction of vir genes
At least 25 vir genes on Ti plasmid are necessary for tumor induction.In addition to their perception role, virA and chvE induce other vir genes. The VirA protein has a kinase activity, it phosphorylates it self on a histidine residue. Then the VirA protein phosphorylates the VirG protein on its aspartate residue. The VirG protein is a cytoplasmic protein traduced from the virG Ti plasmid gene, it's a transcription factor. It induces the transcription of the vir operons. ChvE protein regulates the second mechanism of vir genes activation. It increases VirA protein sensibility to phenolic compounds.
Possible plant compounds, that initiate Agrobacterium to infect plant cells:
• Acetosyringone: Phenolic compound
• alpha-Hydroxyacetosyringone
• Catechol
• Ferulic acid
• Gallic acid
• p-Hydroxybenzoic acid
• Protocatechuic acid
• Pyrogallic acid
• Resorcylic acid
• Sinapinic acid
• Syringic acid
• Vanillin
Generation of T-DNA transfer complexAfter activation by phosphorylated vir G, first Vir C, a helicase, unwinds DNA then Vir D1, a topoisomerase, binds to RB sequence and relaxes supercoiling which facilitates the action of Vir D2. Vir D2, an endonuclease, nicks at RB and covalently binds to 5’ end so generated. The 3’ end so produced at the site of nick serves as primer for DNA synthesis in 5’ to 3’ direction. As a result ss of T-DNA is displaced from DNA duplex. T- strand is again nicked at LB to generate a ss copy of T-DNA. Vir E2 is SSBP stabilizes the ss copy of T-DNA. About 600 copies of it bind to ss T-DNA and protect from nuclease action.
Extensive mutation or deletion of the right T-DNA border is followed by almost completely loss of T-DNA transfer capacity, while at the left border results in lower transfer efficiency. This fact indicates that T-strand synthesis is initiated at the right border, it proceeds in the 5' to 3' direction the termination process takes place even when the left border is mutated or completely absent, although with lower efficiency. Left border may act as a starting site for ss T-strand synthesis but the efficiency is much lower. The difference may be a consequence of the presence of an enhancer or "overdrive" sequence next to the right border. This enhancer has been found to be specifically recognized by VirC1 protein. Deletion of virC operon is followed by attenuation of virulence of the Agrobacterium strains
Formation of the T-pilusIn order to transfer the T-DNA into the plant cell A. tumefaciens uses a Type IV secretion mechanism, involving the production of a T-pilus.
The VirA/VirG two component sensor system is able to detect phenolic signals released by wounded plant cells, in particular acetosyringone. This leads to a signal transduction event activating the expression of 11 genes within the VirB operon which are responsible for the formation of the T-pilus.
First, the VirB" pro-pilin is formed. This is a polypeptide of 121 amino acids which requires processing by the removal of 47 residues to form a T-pilus subunit. The subunit is circularized by the formation of a peptide bond between the two ends of the polypeptide.
Products of the other VirB genes are used to transfer the subunits across the plasma membrane. Yeast two-hybrid studies provide evidence that VirB6, VirB7, VirB8, VirB9 and VirB10 may all encode components of the transporter. An ATPase for the active transport of the subunits would also be required.
(Step 1) VirB1, VirB2, and VirB5 are translocated to the inner membrane via the GSP. Signal peptides (SPI) domains are represented by black wavy lines at the left N-terminal end of each protein.
(Step 2) Signal peptides are now removed from VirB1, VirB2, and VirB5. VirB1 is processed to release VirB1* from lytic transglycosylase domain.
(Step 3A) Lytic transglycosylase domain degrades peptidoglycan (PG) to create space for assembly of the vir-T4SS trans-envelope core.
(Step 3B) VirB1* binds to VirB2 and VirB5 during their transit from the GSP to the cell exterior.
(Step 4) At the site of T-pilus assembly,VirB2 and VirB5 are mobilized to the exterior of the cell. Mobilization of VirB1* to cell exterior does not require any additional Vir proteins.
A fraction of VirB1* remains associated with T pili, while some VirB1* is free in the medium. VirB2 is drawn as a shaded oval to represent the cyclized form of the mature peptide. VirB5 is drawn as shaded stacked trapezoids to represent the helix bundles of the solved structure of VirB5 . The VirB5 trapezoid is shown localized at the base of the T pilus; however, its precise localization within the T-pilus structure remains to be determined. VirB1 is shown with two domain structures. The N terminus of VirB1 in the periplasm is shown as a "Pac-Man" due to the function of this lytic transglycosylase-homologous domain to "chew" the peptidoglycan. C-terminal VirB1* is drawn as a white ellipsoid. *, VirB1*; ?, unknown exporter for VirB1*; IM, inner membrane; OM, outer membrane.
Transfer of T-DNA into plant cellIn the cytoplasm of the recipient cell, Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. VIP1 also appears to be an important protein in the process, possibly acting as an adapter to bring the VirE2 to the importin. Once inside the nucleus, VIP2 may target the T-DNA to areas of chromatin that are being actively transcribed, so that the T-DNA can integrate into the host genome.
Uses in plant improvement-• The ability of Agrobacterium to transfer genes to plants and fungi is used in biotechnology, in particular, genetic engineering for plant improvement.
• A modified Ti or Ri plasmid can be used.
• The plasmid is 'disarmed' by deletion of the tumor inducing genes; the only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation.
• The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the disarmed plasmid, together with a selectable marker (such as antibiotic resistance) to enable selection for plants that have been successfully transformed. Plants are grown on media containing antibiotic following transformation, and those that do not have the T-DNA integrated into their genome will die.
• Several vectors have been designed based in T plasmid like binary and cointegrate vectors.
• Several crops have been modified like Bt cotton, Bt brinjal etc.
Recent advancements in Agrobacterium interactionAlthough none of the Bryophytae and Pteridophytae species could be transformed by Agrobacterium upto 56% of gymnosperms and 58% of angiosperms (only 8% of monocotyledons) were susceptible to infection by wild type Agrobacterium. Functional similarity between T4SSs of Agrobacterium and other bacteria, such as intacellular pathogens of mammals Brucella spp and Legionella pnuemophila suggests that Agrobacterium can potentially exchange genetic material with non plant species. Under lab conditions, the host range of Agrobacterium can be extended to non plant eukaryotic organisms. These include yeast, filamentous fungi, cultivated mushrooms and human cultures cells.
First report of Agrobacterium mediated gene transfer (AGMT) of a non plant host involved cells of the budding yeast Saccaromyces cerevisieae whereby a ura- yeast strain was transformed to ura+ following introduction of the Ura3- encoding gene by T-DNA transfer and integration. Using specially designed Agrobacterium binary vectors, several unique features of AGMT of yeast cells were identified-
• Circularization and autonomous replication of T-DNA molecules in host cells could be achieved by introduction of the yeast 2 µ replication origin into T-DNA region.
• T-DNA integration into host genome by the homologous recombination was possible if T-DNA contained specific sequences that share homology with yeast genome.
By contrast no autonomous replication of T-DNA molecules has ever been observed in plant cells and integration in plant cells is mostly, if not solely, mediated by illegitimate recombination.
Some years later, Aspergillus awamori became first filamentous fungus to be genetically transformed by Agrobacterium.
Kunik et. al showed that cultured human cell lines (HeLa, HEK293 and neuronal PC12 cells were transformed by Agrobacterium using neomycin resistance as the selection marker. In their report, the T-DNA transfer and integration into human cells shared most of the features of plant AGMT
• Bacterial attachment to the host cell, an essential step in plant transformation by Agrobacterium was similar in human cells and plant protoplasts.
• Agrobacterium mutants in the chvA and chvB loci wre unable to bind to human cells.
• The mode of T-DNA integration into human genome was essentially similar to integration of DNA into plant genome suggesting bona fide T-DNA transfer rather than conjugative transfer of the Ti plasmid.
Moreover this mechanism of DNA transfer process, which relies on Agrobacterium Vir proteins, is essentially the same in plant and non plant hosts. However, the mechanism by which the T-DNA molecule integrates into host genome is most likely to be dictated by the nature of the host organism and nucleotide sequence of the T-DNA.
Conclusion-
Agrobacterium tumefaciens is more than the causative agent of crown gall disease affecting dicotyledonous plants. It is also the natural instance for the introduction of foreign genes in plants allowing its genetic manipulation. Similarities have been found between T-DNA and conjugal transfer systems. They are evolutionally related and apparently evolved from a common ancestor.
Undoubtedly, the development of transformation procedures based on A. tumefaciens-mediated gene transfer for new economically important species are advisable and the results obtained in recent years evidence a promising future.
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