Final combat meet the mechanism of dna

final combat meet the mechanism of dna

When the level of ROS exceeds the defense mechanisms, a cell is said to be in in plants to combat oxidative stress induced by various environmental stresses. . It directly oxidizes protein, unsaturated fatty acids, and DNA [32]. . then, enters the Calvin cycle and reduces the final electron acceptor, CO2. The game makers of China, not content to let the Koreans corner the market on Team Fortress 2 clones, have created their own in Final Combat. The publisher's final edited version of this article is available at Front Biosci ( Landmark Ed) The major pathway tasked with removal of oxidative DNA damage, .. glycosylases has evolved to maintain genomic stability and combat the various base modifications. DNA repair: how Ku makes ends meet.

This high frequency might be increased further by using microinjection in conjunction with specially developed vectors, derived from the Ti plasmid or plant transposable elements see sections on these vectors. Because of the high transformation frequency possible with microinjection, a direct selection scheme e. Furthermore, specific hostrange requirements associated with the Ti plasmid or viral vectors are obviated.

Although at present the recipient plant species must be amenable to cell culture and regeneration from protoplasts, suspension cultures or pollen grains may be used in the future, which would bypass the problem of regeneration. Alternatively, DNA may be injected into the developing floral side-shoots of plants, where it can pass into germ cells. Microinjection of individual chromosomes or cellular organelles e.

Transfer of traits by microinjection would be more direct, precise, and faster than by breeding or cell fusion described in the next sectionbecause microinjection transfers a specific, limited amount of genetic information. There would be less need for selection or backcrossing, which are often time-consuming, difficult processes. Most agronomic traits are polygenic, that is, they are caused by the interplay of several different genes in the plant. Genetic studies often reveal that these genes are linked in blocks on specific segments of chromosomes.

Classical plant breeding can sometimes transfer such traits between species via interspecific crosses, but these crosses are not always successful. Transfer of individual chromosomes would permit researchers to introduce traits that result from the interaction of several genes linked on that chromosome. Chromosome microinjection would also enable the transfer of traits that are encoded by single genes that have not yet been identified and isolated.

Much of the sophisticated biochemistry and genetics of single-gene traits known for animals and used to isolate important genes is lacking for plants. Consequently, few plant genes of agronomic importance have been isolated.

Overview of DNA Repair in Trypanosoma cruzi, Trypanosoma brucei, and Leishmania major

Whole chromosome transfer may allow scientists to genetically engineer plants that would not be tractable at this time by more sophisticated gene-splicing recombinant DNA techniques. Attempts are being made to transform plant cells by microinjection of isolated chromosomes Greisbach, Cell Fusion Cell fusion combines the entire genetic contents of two cells, producing hybrid cells that often express certain traits from both parents.

The parent cells can be from different species or from different types of the same species. Fusion is usually mediated by chemicals such as polyethylene glycol or dimethylsulfoxide, although newer techniques use electrofusion. Animal Cells Cell fusion is the basis for the manufacture of monoclonal antibodies.

Monoclonal antibody-producing cell lines hybridomas are created by fusing antibody-producing B-cells from animals with myeloma cells, which grow indefinitely in culture. The pure, highly specific antibodies thus obtained are important reagents for research, medicine, and agriculture.

Diagnostic kits and vaccines for animal health based on monoclonal antibodies are already on the market Gamble, Diagnosis of plant pathogens such as viruses, bacteria, fungi, and nematodes can also be facilitated by tests based on monoclonal antibodies; commercial products should be available in the near future Gamble, Certain agricultural applications have been held back by lack of suitable myeloma lines for fusion with B-cells from farm animals, as opposed to standard laboratory animals such as the mouse.

However, this problem can be surmounted by creating hybridomas by direct DNA uptake. DNA from B-cells and myeloma cells is simultaneously introduced into recipient cells by calcium phosphate coprecipitation or by electroporation Gamble, This approach obviates the need to fuse interspecific cell lines, and thus solves the problem of finding suitable myeloma lines for different livestock species.

Fusion of animal cell lines in culture is also exploited to map genes to specific chromosomes, an important step in locating genes to use in transfer experiments and in breeding strategies. Gene maps for mice and men are quite advanced.

Those for livestock lag behind, but efforts are starting, notably for swine Fries and Ruddle, To map these genes, swine cells are fused to mouse cells in culture. The interspecies cell hybrids reject most of the swine chromosomes. Ideally, a set of cell lines, each harboring a single different swine chromosome, is made. Known DNA sequences are used as probes for particular genes with those sequences.

These probes bind to defined lengths of DNA from the fused cells. Because swine and mouse chromosomes can be distinguished by small differences in DNA sequences known as restriction fragment length polymorphismsdifferences in the lengths of DNA containing the gene detected by the probe indicate whether that gene is on a swine or a mouse chromosome of the hybrid cell.

Location on a swine chromosome pinpoints the gene to that single particular swine chromosome, which is the only swine chromosome in the hybrid cell. Gene mapping is expected to play an important role in finding genes for transfer of complex traits in livestock, such as lactation, fertility, growth, and disease resistance. Plant Cells In eucaryotic cells the cytoplasm—that part of the cell surrounding the nucleus—contains organelles that have their own separate DNA.

In plants, protoplast fusion is used to transfer genes from both the nucleus and the cytoplasm. Fusion combines the genomes of two parents, as in traditional breeding, but results can sometimes be obtained faster, even though the fusion product must be backcrossed to the recipient line for several generations to create a new, stable line possessing the one trait desired from the donor. Protoplast fusion can be used for transferring genes that are hard to identify, isolate, and clone or for polygenic traits.

Furthermore, protoplast fusion can be used for plants that cannot be crossed sexually although plants regenerated from such fused hybrids may sometimes be sterile. Most commonly, cells from closely related plants are fused in order to transfer one particular trait from the donor plant into the recipient. For example, a single dominant nuclear gene for resistance to tobacco mosaic virus Evans et al. Traits from a wild species can be introduced into a related cultivated species.

Cells of wild and cultivated potato plants were fused to transfer the wild species' resistance to potato leaf roll virus Austin et al. The hybrids were fertile, bore tubers like those of the cultivated species, and were resistant to the virus. Cytoplasmic mitochondrial and chloroplast traits can be transferred by fusing a donor cell whose nucleus has been inactivated, usually by irradiation, with an intact recipient cell to form a ''cybrid.

However, progeny cells that contain mitochondrial or chloroplast genotypes from one parent only quickly segregate. Plants are then regenerated from cells that harbor the desired donor cytoplasmic genotypes. Both cytoplasmic male sterility mitochondria and resistance to the triazine class of herbicides chloroplast have been transferred into a single Brassica line via cybrid formation Pelletier et al.

Vector-Mediated Gene Transfer A vector is a molecule of DNA that is attached to a foreign gene to facilitate its transfer, maintenance, and expression within the target cell.

final combat meet the mechanism of dna

Vectors offer many advantages: Vectors can, therefore, greatly improve gene transfer. However, different species and cell types may require different types of vectors, and often much work must go into creating an appropriate vector system before genes can be transferred into a specific organism.

Extra DNA, coding for foreign genes and for special markers "tags" to track their progress, are inserted into the virus's chromosome. These passenger genes can be expressed via their own regulatory sequences or, sometimes more efficiently, via those of the virus. The first animal virus used was SV40 simian virus 40; Hamer et al.

Fundamental studies on SV40 by Paul Berg and his coworkers laid the groundwork for their and other groups' subsequent development of it and other viruses as vectors for gene transfer, and earned Berg a Nobel Prize in SV40 can exist within the host cell both as an independent circular molecule or as a segment integrated in the host's DNA. This versatility, along with its well-characterized life cycle and gene regulation, have given researchers great flexibility in designing vector systems based on SV SV40's drawbacks are that it normally infects only cells of certain species notably primates and is severely limited in the amount of DNA it can carry.

Only about 2, base pairs the size of one small animal gene can be added to this virus, and even this addition must be compensated for by deleting some of its own DNA. Adenoviruses infect a wider variety of mammalian species than does SV Their DNA is a very long, linear molecule, which like SV40 can either replicate to give a high copy number of independent molecules or insert itself into the host's DNA in a low copy number.

Journal of Nucleic Acids

The molecular biology of adenoviruses has been well studied and like that of SV40, has provided fundamental insights into eucaryotic gene regulation. Adenovirus vectors have several advantages over SV40 and retroviruses which are discussed later.

Adenovirus can accommodate large, complete passenger genes with their own control sequences. Furthermore, two different genes at widely separated locations can be accommodated on the same vector molecule, permitting separate and distinct control of the two passenger genes within one cell. In addition, hybrid viruses composed of both adenovirus and SV40 can give even greater flexibility in control of gene expression and extend the host range for gene transfer van Doren and Gluzman, Several developments with SV40 and adenoviruses are of particular interest.

These viruses have been used to transfer genes into cells of diverse origin, notably mouse and human bone marrow cells Karlsson et al. However, the recombinant SV40 vector did not integrate into the cells' chromosomes.

With adenovirus-mediated transfer, one to three copies of foreign genes were transferred intact at very high frequency and maintained stably in the host cells' chromosomes. This low-copy number, stable integration is desirable for certain studies of gene regulation and for permanent genetic modification of animals. Viral vectors can also be used for large-scale production of specific proteins in cultured animal cells.

Although proteins can sometimes be efficiently manufactured in bacterial or yeast cells, many animal proteins are not correctly processed and assembled by cells of simpler organisms. In these cases it may be more efficient to manufacture proteins in cultured animal cells. To be economically feasible, protein manufacture by recombinant DNA technology must yield large amounts of the desired product. Researchers have developed SV40 and adenovirus vectors that meet this requirement by expressing any inserted gene at a high level Reddy et al.

The researchers made these "expression vectors" by connecting viral regulatory sequences that normally cause high-level production of proteins needed in huge quantities by the virus e. The expression vectors exploit the facts that many copies of viral DNA accumulate inside the cell and that each of these copies produces great quantities of the desired protein.

This virus does not integrate its DNA into the host cell's chromosome. Instead, the vector with its passenger DNA is maintained as an extrachromosomal DNA molecule, which usually replicates to give about copies of the transferred gene in every cell. The extrachromosomal maintenance and high copy number are advantageous for "transient expression" assays, detailed studies on gene expression, and production of proteins in quantity.

The circular shape of BPV's DNA and its ability to maintain itself as an independent chromosome have enabled scientists to further engineer BPV as well as SV40 vectors to replicate in both mammalian and bacterial cells. Researchers use these "shuttle vectors" to move cloned genes back and forth between mammalian and bacterial cells for ease of study and manipulation.

Drawbacks to BPV vectors are that the engineered DNA molecules are sometimes unstable, only a few types of cells usually epithelial can serve as hosts, and applications may be limited to cultured cells. Thus, researchers need to pursue fundamental studies on BPV's life cycle and regulatory mechanisms before optimal BPV vectors can be designed. It is famous for its role as the vaccine used to eradicate the deadly human disease smallpox in this century. Although vaccinia is similar enough to the smallpox variola virus to immunize against it, vaccinia itself does not cause disease.

Poxviruses are unique in that they set up shop in the cell's cytoplasm, unlike other viruses, which head for the cell's nucleus. Vaccinia expresses its genes in the cytoplasm using its own enzymes, which respond to vaccinia's regulatory sequences but cannot recognize those of the host cell. Therefore, when vaccinia is used as a vector for foreign genes, these genes are expressed only if they are hooked up to vaccinia's own regulatory sequences.

Among its advantages is vaccinia's ability to grow easily in cell culture. By inoculation into the skin, it can also infect a wide range of animal hosts, making it a versatile vector. Moreover, similar poxviruses could be used as vectors for additional species.

Because vaccinia is so large, it can accommodate more inserted DNA than any other virus—amounts greater than 25, base pairs are stable Smith and Moss, This is more than 10 times the carrying capacity of SV40, and covers the size of several genes. Vaccinia has two natural safety features: In addition, the virus can be attenuated further by genetic engineering. Scientists can insert passenger genes into the virus's gene for the enzyme thymidine kinase, thereby inactivating it.

Because this enzyme is needed for optimal growth of the virus, vaccinia recombinants cannot spread as easily as the normal virus Buller et al. In addition, viruses without thymidine kinase can survive treatment with a drug that kills the normal virus, enabling rapid laboratory detection of the desired recombinants. Because the large vaccinia DNA molecule is too cumbersome to handle in vitro, foreign genes must be transferred onto the vaccinia vector by a two-step process.

First a small circular "insertion vector" is built in vitro. This vector contains the foreign gene, surrounded by cloned DNA from vaccinia's thymidine kinase gene. Second, animal cells are infected with normal vaccinia virus, and then insertion vector DNA is added to the infected cells by direct DNA uptake. Inside the cells an exchange occurs between the thymidine kinase sequences on the insertion vector and the identical homologous sequences on the viral DNA, placing the foreign gene into the viral DNA.

The foreign gene interrupts the thymidine kinase gene, inactivating it as described in the preceding paragraph Mackett et al.

final combat meet the mechanism of dna

The most important use of the vaccinia vector will be for the production of vaccines against viruses and parasites that have resisted conventional vaccines. Furthermore, a single recombinant vaccinia virus can carry antigenic genes from several disease agents or several strains of a virus like influenza.

Thus vaccinia can immunize against several diseases in one shot Perkus et al. Importantly, vaccinia vaccines not only stimulate antibody protection but also confer long-lasting cellular immunity Bennink et al.

Recombinant vaccinia vaccines for major diseases of livestock e. Because of its wide host range, vaccinia can immunize a large variety of animal species. Like the original smallpox vaccine, the vaccines would be cheap, easy to manufacture, dispense, and administer, and stable without refrigeration as freeze-dried preparations—ideal for field use. Retroviruses Retroviruses are a family of viruses that contain RNA as their primary genetic material.

On infection of a host cell, the RNA is copied into DNA, which then inserts itself into the host cell's chromosome, becoming a stable part of the host's genetic information. Retroviruses have been found in association with many animals, including humans, and probably exist for all agriculturally important animal species.

There are several particular advantages to retroviral vectors Anderson, They can infect a high percentage of the target cells, integrate in one copy at a single site in the cell's genome, and reliably express the foreign gene. Other methods often lead to the transfer of multiple copies of the gene, which may interfere with its correct expression.

Retroviruses are currently the focus of intense research on both their basic biology and their use as vectors. For example, engineered retroviruses can infect bone marrow cells in culture. These transformed cells can then be transplanted back into the animal. A gene introduced in this way may be able to correct a genetic defect in an animal or human, although it would not be inherited by the individual's progeny.

However, infection of germ line cells of early embryos of animals should allow heritable traits to be transferred for breeding purposes in agriculture.

The first key experiments in the use of retroviral vectors concentrated on the transfer of genes for drug resistance into blood-producing cells of the mouse Joyner et al. The HPRT gene functioned in both mouse and human cells in culture, as well as in live mice. Further experiments demonstrated efficient transfer of a rat growth hormone gene into mouse cells by retroviruses and correct expression of the gene by its own regulatory sequences Miller et al.

This demonstrates that retroviruses can deliver genes into the germ cells of early embryos so that the genes are inherited normally and function in intact animals.

The engineering of safe retroviral vectors involves some genetic tricks to ensure that the virus will not be able to reinfect other cells or spread to other organisms after the desired transfer of genes. In constructing the vector some of the retrovirus's own genes are replaced with foreign passenger genes, depriving the virus of the ability to replicate itself.

To overcome this handicap, a so-called ''helper virus" is used, which provides gene products that the engineered retrovirus can no longer make. These essential products are the enzyme for replication and the proteins for the virus coat. For the purpose of safety—and efficiency—the helper virus is debilitated by the removal of a small portion of the genetic material necessary to its reproduction. The helper is maintained only as an integrated "provirus" in a cell line; it is a permanent part of the cell's DNA and cannot become infectious.

The handicapped vector retroviruses that carry foreign genes are propagated in this cell line, aided by the replication and coat proteins manufactured by the helper provirus.

Vector viruses are then purified away from the cells containing the helper provirus. These purified vectors now can enter other target cells and integrate the foreign gene into the target cells' genome, but that is all they can do—without the helper provirus they cannot replicate in the target cells to produce more infectious viruses. Thus the retroviral vector is a gene delivery system, not an infectious agent. The vector can be further disabled by engineering a defective regulatory sequence at one end of its genome.

Such vectors integrate into the host's chromosome, and then become stuck. Even in the presence of the helper virus, they cannot express their viral genes, replicate further, or move out of the cell's chromosome.

Foreign genes transferred in by these vectors are expressed from their own regulatory sequences. Retroviral gene transfer vectors applicable to agricultural animals have been developed.

One system based on a turkey retrovirus efficiently delivers genes into avian and some mammalian cells Watanabe and Temin, Another retrovirus system can introduce genes into a broad range of mammalian species, including farm animals Cone and Mulligan, Thus, just a few retroviral vectors may serve for genetic engineering of many livestock species. Baculoviruses Baculoviruses, which infect lepidopteran insects, should have uses in agriculture for manipulation of both beneficial and harmful species.

Baculoviruses have some similarities to vaccinia virus in the way they are engineered for gene transfer Miller et al. Their large, double-stranded DNA genome may accommodate up toextra base pairs of DNA, due to the virus's extendable rod-shaped structure. Insertion of genes into such a large DNA molecule is accomplished via small insertion vectors, as described previously for vaccinia.

Viral and insertion vector DNA are simultaneously introduced into insect cells by direct uptake using calcium phosphate. Homologous recombination in vivo then places the foreign genes from the insertion vector into the baculovirus genome. Foreign genes are most conveniently inserted into the virus's gene for polyhedrin.

final combat meet the mechanism of dna

This strategy has several benefits. First, insertional inactivation of the polyhedrin gene gives an easily detected recombinant virus phenotype, because these viruses form areas of infected cells that look different from those made by the normal virus.

Second, viruses with a defective polyhedrin gene cannot be transmitted between host insects; they can move only from cell to cell within a single insect or cell culture. Thus the recombinant baculoviruses have a built-in safety feature. Third, the regulatory sequence promoter of the polyhedrin gene can express foreign proteins at high levels, as over 20 percent of the infected cell's messenger RNA and protein are normally made from this gene.

Foreign genes cloned in baculoviruses can also be expressed from their own promoters. A baculovirus, high-level expression system could be used to manufacture commercially useful proteins, as baculoviruses can be mass-produced in insect cell cultures. Baculoviruses might be particularly advantageous for the manufacture of insect-derived substances such as pheromones, which can be used for biological control of insect pests. Baculoviruses infect many lepidopteran insect species and can themselves be used as insecticides.

Their effectiveness as biological insecticides may be augmented by genetic engineering, for example, by introduction of insect-specific toxin genes.

Because baculoviruses infect only invertebrates, with different baculoviruses being relatively specific for certain lepidopteran insect hosts only, they should not spread indiscriminantly to other insects, animals, or plants. Plant Viruses Cauliflower Mosaic Virus Only small steps have been taken with viral vectors for plants, in contrast to the great strides in virally mediated gene transfer into animals.

There are no known plant retroviruses and only a few, small DNA viruses. The best-studied virus is cauliflower mosaic virus CaMVa small double-stranded DNA virus that infects cruciferous plants, such as cabbage and mustard. CaMV is transmitted in nature by aphids, but its DNA can infect plants if simply rubbed onto their leaves.

CaMV causes systemic infection and replicates abundantly throughout the plant. It thus should transfer many copies of a gene per cell into all tissues of a mature plant.

Furthermore, powerful CaMV gene regulation sequences can promote high-level expression of foreign genes. In fact, CaMV promoters are being used to augment the expression of plant genes transferred via other systems, as most plants recognize these promoters even when they are detached from the rest of CaMV.

The biggest obstacles to the development of a CaMV vector have been the severe limitation on the virus's size and thus on the quantity of DNA that can be inserted, and the instability of the genetically engineered virus.

This instability may be caused both by the packaging limitation on extra DNA and by the way the virus replicates. Furthermore, CaMV does not integrate into plant genomes under normal conditions of infection. Some success in introducing foreign genes into plants using CaMV has been reported, however.

Bacterial drug resistance genes were expressed and stably propagated in CaMV-infected turnip plants Brisson et al. Geminiviruses Geminiviruses are single-stranded DNA viruses of plants that are transmitted by insects, such as leafhoppers. Viruses in this group infect many crops, including the monocots wheat and corn and the dicots beans, tobacco, and tomatoes. Work on developing a vector system based on these viruses is in progress Kridl and Goodman, ; Lazarowitz, However, others such as XPA could not be identified in Tritryps.

It is also possible that the Tritryps ligation step is different from the ligation step from higher eukaryotes. However, because their genomes encode ligase I, it might be possible that the ligation step is performed exclusively by this protein in those parasites.

In addition to that, a recent study showed that T.

These subunits are also present in the genomes of T. Protein-coding genes are constitutively transcribed in trypanosomatids [ 73 ]. This peculiarity implies that TCR could be one of the most crucial mechanisms involved in repairing DNA damage in those parasites.

Surprisingly, the Tritryps genomes apparently lack the gene that encodes CSA. The absence of an evident CSA in Tritryps implies that the trypanosomatid TCR differs from the standard TCR mechanism, either by the lack or divergence of this component, or by the presence of an alternative protein to perform this step.

This could be related to the peculiar constitutive transcription of Tritryps. In fact, overexpression of T. In addition, results obtained by our group show that T. Taken together, these results led us to hypothesize that, in T. Whether the CSA absence or the presence of an alternative CSA is an adaptation to this distinctive repair is a topic for future investigation. When compared to other taxons, GGR-NER in trypanosomatids seems to be similar to the GGR pathway encountered in plants, as both groups of organisms share peculiarities regarding the presence and absence of some NER genes.

In addition, the plant genome also carries two copies of XPB [ 72 ]. Interestingly, these DNA repair similarities found in Tritryps and plants can also be observed in the MMR pathway, which could suggest that both groups might share some commonalities in their DNA repair mechanisms.

The fundamental aspects of the pathway have been highly conserved throughout evolution. Strand discontinuities associated with DNA replication can serve as entry points for strand excision, conferring strand specificity to MMR [ 75 ].

Each trypanosomatid encodes a set of MMR proteins, which suggests they are fully competent for mismatch recognition and repair [ 715 ]. Components of the MMR pathway are major players in processes known to generate genetic diversity, such as mutagenesis and DNA recombination.

Evidences suggest that differences in MMR efficiency could be an important source of genetic diversity in organisms [ 76 — 79 ]. Despite its broad genetic diversity, three major lineages, named T. Studies with a number of molecular markers revealed that parasites belonging to the T. The great genetic diversity observed in T. It is conceivable that components of DNA repair pathways participate in processes that resulted in increasing genetic variability within the parasite population [ 85 ]. MSH2, the core eukaryotic mismatch repair gene, has been characterized in T.

It is possible that these isoforms have distinct protein activity, leading to variations in the efficiency of MMR. Further studies are needed to determine if these variations in MMR efficiency have a broader impact on genetic variation and behavior in T.

Mutations in both genes give rise to increased microsatellite instability and lead to increased tolerance to the alkylating agent MNNG [ 27 ]. Both phenotypes are consistent with an impairment of nuclear MMR activity [ 75 ]. These results indicate that MMR in trypanosomatids is active in repairing errors that arise during replication and in response to chemical damage. MMR also plays a regulatory role in homologous recombination in T.

However, MMR has little influence on antigenic variation in this parasite [ 28 ]. Interestingly, the heterologous expression of MSH2 from T. In addition, Helicobacter pylori, which is suggested to be MMR-defective due to the lack of MutH and MutL homologs, presents a MutS homolog that is involved in repairing oxidative damage [ 88 ].

Four additional MSH-like genes can be found in the trypanosomatids: The predicted MSH6 polypeptides in Tritryps have N-terminal truncations relative to eukaryotic orthologues [ 27 ]. In comparison, MSH7, unique to plants, bears similar truncations in the N-terminus along with the conserved mismatch interaction residues indicative of the MSH6 subgrouping [ 89 ].

MSH4 and MSH5 predicted proteins that appear to lack an N-terminal mismatch interaction, indicating an absence of function in the mismatch repair and a possible role in meiotic recombination [ 27 ].

DSBs can arise when replication forks encounter blocking lesions, which leads to fork collapse, or can be induced by ionizing radiation and radiomimetic chemicals. Failure to accurately repair such damage can result in cell death or large-scale chromosome changes, including deletions, translocations, and chromosome fusions that enhance genome instability.

Two distinct and evolutionarily conserved pathways for DSB repair exist: The two ends of the DSB are held together and religated, often following the loss of some sequence by nucleolytic degradation or addition by polymerization [ 90 ].

NHEJ seems to be absent in trypanosomatids. KU70 and KU80 have been identified in T. These absences in Tritryps suggest one of two possibilities: These possibilities should be further investigated. In addition, HR is involved in the repair of incomplete telomeres and in the correct segregation of homologous chromosomes during meiosis.

The broad reaction scheme [ 9394 ] can be considered in three steps: Homologous recombination is the major pathway of DSB repair in lower eukaryotes [ 95 ]. Essential components of this mechanism have been identified in the genome of T. HR can contribute to different strategies evolved by trypanosomatids to create genetic variability that is needed for survival in their hosts. Antigenic variation is used by T.

This mechanism is regulated by HR, allowing the switch of one VSG at time to the expression site [ 85 ]. Recent works have been suggesting that HR is responsible for creating mosaic genes of surface molecules through segmental gene conversion and for decreasing the divergence between duplicated regions such as surface multigenic families [ 8396 ].

Journal of Botany

In addition, experiments with genetic manipulation have shown that homologous recombination is the main mechanism for integration of transformed DNA in these organisms [ 97 — ]. However, only MRE11 from T. Mutation of MRE11 causes impairment in T. MRE11 does not contribute to recombination during antigenic variation, an important mechanism used by T. Both enzymes are present in Tritryps. DMC1, a putative meiosis-specific recombinase, has only been studied in T.

The presence of genes involved in meiosis is an intriguing feature of Tritryps because they reproduce primarily through clonal reproduction [ ]. Even though the population structure of each parasite is largely clonal [ ], evidence of genetic exchange in the wild-type populations of T. However, it is unclear whether or not the existence of meiotic recombination genes implies that the trypanosomatids use meiosis.

RAD51 has been characterized in the three trypanosomatids. The expression of RAD51 in T. Moreover, the overexpression of RAD51 in T. Using mutants deficient in key ROS-scavenging enzymes, Miller and coworkers [ 74 ] identified a signaling pathway that is activated in cells in response to ROS accumulation. Interestingly, many of the key players in this pathway, including different zinc finger proteins and WRKY transcription factors, are also central regulators of abiotic stress responses involved in temperature, salinity and osmotic stresses.

Reactive oxygen species ROS as second messengers in several plant hormone responses, including stomatal closure, root gravitropism, seed germination, lignin biosynthesis, programmed cell death, hypersensitive responses, and osmotic stress.

ROS are considered second messengers in the abscisic acid ABA transduction pathway in guard cells [ 1920 ]. ABA induced H2O2 is an essential signal in mediating stomatal closure to reduce water loss through the activation of calcium-permeable channels in the plasma membrane [ 77 ].

Jannat and coworkers [ 78 ] observed that ABA-inducible cytosolic H2O2 elevation functions in ABA-induced stomatal closure, while constitutive increase of H2O2 does not cause stomatal closure. Role of ROS as second messenger in root gravitropism has been demonstrated. Based on their work, Joo and coworkers [ 73 ] proposed that gravity induces asymmetric movement of auxin within 60 min, and, then, the auxin stimulates ROS generation to mediate gravitropism. Further, scavenging of ROS by antioxidants N-acetylcysteine, ascorbic acid, and Trolox inhibited root gravitropism [ 73 ].

ROS appear to be involved in dormancy alleviation. In dormant barley grains under control condition, gibberellic acid GA signaling and ROS content are low, while ABA signaling is high, resulting in dormancy. Exogenous H2O2 does not appear to alter ABA biosynthesis and signaling, but has a more pronounced effect on GA signaling, inducing a change in hormonal balance that results in germination [ 79 ]. Bethke and Jones [ 72 ] observed that GA-treated aleurone protoplasts are less tolerant to internally generated or exogenously applied H2O than ABA-treated protoplasts and suggested that ROS are components of the hormonally regulated cell death pathway in barley aleurone cells.

Plants have evolved a complex regulatory network to mediate biotic and abiotic stress responses based on ROS synthesis, scavenging, and signaling. Transient production of ROS is detected in the early events of plant-pathogen interactions and plays an important signaling role in pathogenesis signal transduction regulators.

This production-called oxidative burst could be considered as a specific signal during the interaction process [ 80 ]. ROS are shown to act as a second messenger for the induction of defense genes in tomato plants in response to wounding [ 82 ].