Generation Of Genetic Diversity Research Articles

Meiotic instability of chicken ultra-long telomeres and mapping of a 2.8 megabase array to the W-sex chromosome

The objective of this research was to study the meiotic stability of a subset of chicken telomere arrays, which are the largest reported for any vertebrate species. Inheritance of these ultra-long telomere arrays (200 kb to 3 mb) was studied in a highly homozygous inbred line, UCD 003 (F >or= 99.9). Analysis of array transmission in four families indicated unexpected heterogeneity and non-Mendelian segregation including high-frequency-generation of novel arrays. Additionally, the largest array detected (2.8 Mb) was female-specific and correlated to the most intense telomeric DNA signal on the W-sex chromosome by fluorescence in situ hybridization (FISH). These results are discussed in regard to the potential functions of the ultra-long telomere arrays in the chicken genome including generation of genetic variation through enhanced recombination, protection against erosion by providing a buffer for gene-dense regions, and sex-chromosome organization.

DNA metabolism and genetic diversity in Trypanosomes

Trypanosomes are protozoan parasites that cause major diseases in humans and other animals. Trypanosoma brucei and Trypanosoma cruzi are the etiologic agents of African and American Trypanosomiasis, respectively. In spite of large amounts of information regarding various aspects of their biology, including the essentially complete sequences of their genomes, studies directed towards an understanding of mechanisms related to DNA metabolism have been very limited. Recent reports, however, describing genes involved with DNA recombination and repair in T. brucei and T. cruzi, indicated the importance of these processes in the generation of genetic variability, which is crucial to the success of these parasites. Here, we review these data and discuss how the DNA repair and recombination machineries may contribute to strikingly different strategies evolved by the two Trypanosomes to create genetic variability that is needed for survival in their hosts. In T. brucei, two genetic components are critical to the success of antigenic variation, a strategy that allows the parasite to evade the host immune system by periodically changing the expression of a group of variant surface glycoproteins (VSGs). One component is a mechanism that provides for the exclusive expression of a single VSG at any one time, and the second is a large repository of antigenically distinct VSGs. Work from various groups showing the importance of recombination reactions in T. brucei, primarily to move a silent VSG into an active VSG expression site, is discussed. T. cruzi does not use the strategy of antigenic variation for host immune evasion but counts on the extreme heterogeneity of their population for parasite adaptation to different hosts. We discuss recent evidence indicating the existence of major differences in the levels of genomic heterogeneity among T. cruzi strains, and suggest that metabolic changes in the mismatch repair pathway could be an important source of antigenic diversity found within the T. cruzi population.

Conformational model of the Holliday junction transition deduced from molecular dynamics simulations.

Homologous recombination plays a key role in the restart of stalled replication forks and in the generation of genetic diversity. During this process, two homologous DNA molecules undergo strand exchange to form a four-way DNA (Holliday) junction. In the presence of metal ions, the Holliday junction folds into the stacked-X structure that has two alternative conformers. Experiments have revealed the spontaneous transitions between these conformers, but their detailed pathways are not known. Here, we report a series of molecular dynamics simulations of the Holliday junction at physiological and elevated (400 K) temperatures. The simulations reveal new tetrahedral intermediates and suggest a schematic framework for conformer transitions. The tetrahedral intermediates bear resemblance to the junction conformation in complex with a junction-resolving enzyme, T7 endonuclease I, and indeed, one intermediate forms a stable complex with the enzyme as demonstrated in one simulation. We also describe free energy minima for various states of the Holliday junction system, which arise during conformer transitions. The results show that magnesium ions stabilize the stacked-X form and destabilize the open and tetrahedral intermediates. Overall, our study provides a detailed dynamic model of the Holliday junction undergoing a conformer transition.

Conidial anastomosis fusion between Colletotrichum species

Colletotrichum lindemuthianum is a pathogen of the common bean plant (Phaseolus vulgaris) causing anthracnose. Large numbers of isolates can rapidly arise with different genetic and chromosomal compositions but their origin is unknown since sexual fruit bodies have only been found in the laboratory. We have recently described the occurrence of special kinds of hyphae that create anastomoses directly between conidia. In this work we show that conidial anastomoses can occur between two different Colletotrichum species. The implications of this observation on the generation of genetic diversity in these species are discussed.

The Role of Prophage-like Elements in the Diversity of Salmonella enterica Serovars

The Salmonella enterica serovar Typhi CT18 ( S. Typhi) chromosome harbours seven distinct prophage-like elements, some of which may encode functional bacteriophages. In silico analyses were used to investigate these regions in S. Typhi CT18, and ultimately compare these integrated bacteriophages against 40 other Salmonella isolates using DNA microarray technology. S. Typhi CT18 contains prophages that show similarity to the lambda, Mu, P2 and P4 bacteriophage families. When compared to other S. Typhi isolates, these elements were generally conserved, supporting a clonal origin of this serovar. However, distinct variation was detected within a broad range of Salmonella serovars; many of the prophage regions are predicted to be specific to S. Typhi. Some of the P2 family prophage analysed have the potential to carry non-essential “cargo” genes within the hyper-variable tail region, an observation that suggests that these bacteriophage may confer a level of specialisation on their host. Lysogenic bacteriophages therefore play a crucial role in the generation of genetic diversity within S. enterica .

Molecular characterisation of rough strains of Vibrio cholerae isolated from diarrhoeal cases in India and their comparison to smooth strains

Sixteen of the 18 Vibrio cholerae rough strains isolated from hospitalised diarrhoea patients were found to contain O1 serotype-specific ( wbe) genes and all currently known virulence genes. Expression of the regulatory element ToxR was evident in these strains. Cholera toxin production ability of the rough strains was found to be higher (c. three- to five-fold) as compared to the smooth counterparts and this was transcriptionally regulated. Strains exhibiting the rough phenotype were more amenable to the uptake of CTXφ, which led us to consider that the rough phenotype could play a role in the generation of genetic diversity among V. cholerae strains.

Determination of intrachromosomal recombination rates in cultured mammalian cells.

Recombination is involved in many important biological processes including DNA repair, gene expression, and generation of genetic diversity. Recombination must be carefully regulated so as to prevent the deleterious consequences that may result from rearrangements between dissimilar sequences in a genome. It is of considerable interest to study the mechanisms by which genetic rearrangements in mammalian chromosomes are regulated in order to understand better how genomic integrity is normally maintained and to gain insight into the types of genetic mutations that may destabilize the genome. To explore such issues in mammalian chromosomes, a suitable experimental system must be developed. In this chapter, we describe a model system for studying intrachromosomal recombination in cultured mammalian cells. We discuss two model recombination substrates, a method for stably introducing the substrates into cultured Chinese hamster ovary cells, and a method for determining rates of intrachromosomal recombination between sequences contained within the integrated substrates. The general approach described here should be applicable to the study of a variety of aspects of recombination in virtually any cultured mammalian cell line.

New directions in testicular cancer; molecular determinants of oncogenesis and treatment success

Metastatic testicular cancer is highly curable with conventional cytotoxic drugs. This is in contrast to most other metastatic solid tumours which can only be palliated with chemotherapy achieving only a modest impact on overall survival. If we could understand at the molecular level why chemotherapy is so effective in the treatment of testicular cancer, we may be better able to move other forms of metastatic cancer into the curable bracket. Most cytotoxic drugs appear to induce cell death by activating intracellular apoptotic mechanisms. Thus, the ability of a cancer to activate and execute such mechanisms in response to treatment is paramount in determining the effectiveness of chemotherapy. The basic study of cancer molecular biology is providing some insight into the proteins involved in this process and the ability to apply this information to actual human tumours is essential to rationalise clinical treatment failures at a molecular level. Testicular cancer provides an excellent model system in this analysis. Whereas there are large numbers of patients that are cured by chemotherapy, there are some whose cancers become resistant to treatment. An understanding of testicular cancer molecular biology may allow the identification of the genes regulating such a crucial behavioural switch. It may then be possible to manipulate specific signalling pathways to overcome drug resistance. This review focuses on recent developments in our understanding of the molecular biology of testicular cancer. A number of key players have been implicated including p53, pRb, cyclin D2, p INK proteins, c-kit and the bcl-2 family of proteins. The exact manner by which cellular transformation occurs has still not been established, but it is clear that many of the above proteins also have important roles in normal spermatogenesis. This is a developmental phase when the generation of genetic diversity is at a premium, but when selective apoptotic mechanisms are paramount. We discuss why this may be relevant to the behaviour of germ cell tumours and address possible reasons why they can become resistant to conventional therapy.

Immunological disorders and DNA repair

Cellular DNA continuously incurs damage and a range of damage response mechanisms function to maintain genomic integrity in the face of this onslaught. During the development of the immune response, the cell utilises three defined processes, V(D)J recombination, class switch recombination and somatic hypermutation, to create genetic diversity in developing T and B cells. Curiously, the damage response mechanisms employed to maintain genomic stability in somatic cells have been exploited and adapted to help generate diversity during immune development. As a consequence of this overlap, there is mounting evidence that disorders attributable to impaired damage response mechanisms display associated immunodeficiency. Since double strand breaks (DSB) are created during at least two of the mechanisms used to create immunoglobulin diversity, namely V(D)J recombination and class switch recombination, it is not surprising that disorders associated with defects in the response to double strand breaks are those most associated with immunodeficiency. Here, we review the steps involved in the generation of genetic diversity during immune development with a focus on the damage response mechanisms employed and then consider human immunodeficiency disorders associated with impaired damage response mechanisms.

Nucleosome sliding: facts and fiction.

Nucleosome sliding is a frequent result of energy-dependent nucleosome remodelling in vitro. This review discusses the possible roles for nucleosome sliding in the assembly and maintenance of dynamic chromatin and for the regulation of diverse functions in eukaryotic nuclei.

Base excision repair in a network of defence and tolerance.

Survival of a species depends on balanced generation of genetic variation but at the same time on the protection of the genome from changes that cause disease and fitness reduction. DNA repair pathways limit mutations but do not totally eliminate them. In fact, some DNA repair pathways are error prone. DNA repair thus has a central function not only in protecting the genome, but also in the generation of genetic diversity. Expression of DNA repair proteins is subject to a delicate balance where both too few and too many of a type may result in increased cytotoxicity and/or mutation (1–4). DNA repair is integrated with transcription, replication, cell cycle control and apoptosis in complex networks (5), a full discussion of which is beyond the scope of this article, and our abilities. While DNA repair is an ancient and conserved defence mechanism, various pathways, and additions to basic pathways, have evolved during different time periods (6). Furthermore, the pathways have overlapping specificities and function as back-up systems for each other. Thus, cytotoxic and mutagenic abasic sites (AP sites) may be dealt with by the relatively accurate mechanisms nucleotide excision repair (NER) (7,8), base excision repair (BER) and recombination repair, as well as by highly error-prone translesion DNA synthesis (TLS) (Figure 1; 9). In addition, different enzymes may substitute for each other in a specific pathway, or variants of a pathway. Conditions that govern the selection of a pathway in each case are not well understood. Furthermore, components of DNA repair systems, such as error-prone DNA polymerases, may also cause untargeted mutations at sites where no apparent damage is present. In this commentary we will address functional aspects of BER and different complementary functions in the light of recent discoveries with regard to mutations, cancer, evolution and ageing. In summary, recent results from different DNA glycosylasedeficient mice have failed to demonstrate strongly increased mutation rates, increased cancer frequencies or other severely altered phenotypes. This may be due to overlap in functions between DNA glycosylases, as well as repair by alternative

Recombination is proportional to the number of chromosome arms in mammals.

Recombination is proportional to the number of chromosome arms in mammals.

Transgênicos e evolução dirigida

Mutation events are responsible for the generation of genetic variability in the populations enabling the occurrence of natural selection which favors the better-adapted types. The exploitation of this variability, though carried out empirically, dates from ten thousand years ago with the domestication of the first cultivated crops. With the advent of genetics, rational selection procedures were adopted with a view of the genetic breeding of plants, animals and microorganisms which might be of interest to men. Recently, new DNA manipulation techniques came up enabling the transference of genes between organisms, cutting across barriers which hindered crossing between the vegetable, animal, protist and fungus kingdoms. The generation of genetically modified organisms, or transgenics, has aroused a heated and controversial debate in various sectors of our society. Yet we must be cautious before generalizing the use of transgenics since each one should be analyzed at a time for its particular advantages and drawbacks, and for its contribution to the improvement of life quality. This paper also considers recent methods of mutation and in vitro genic recombination.

Genetic variation: molecular mechanisms and impact on microbial evolution.

On the basis of established knowledge of microbial genetics one can distinguish three major natural strategies in the spontaneous generation of genetic variations in bacteria. These strategies are: (1) small local changes in the nucleotide sequence of the genome, (2) intragenomic reshuffling of segments of genomic sequences and (3) the acquisition of DNA sequences from another organism. The three general strategies differ in the quality of their contribution to microbial evolution. Besides a number of non-genetic factors, various specific gene products are involved in the generation of genetic variation and in the modulation of the frequency of genetic variation. The underlying genes are called evolution genes. They act for the benefit of the biological evolution of populations as opposed to the action of housekeeping genes and accessory genes which are for the benefit of individuals. Examples of evolution genes acting as variation generators are found in the transposition of mobile genetic elements and in so-called site-specific recombination systems. DNA repair systems and restriction-modification systems are examples of modulators of the frequency of genetic variation. The involvement of bacterial viruses and of plasmids in DNA reshuffling and in horizontal gene transfer is a hint for their evolutionary functions. Evolution genes are thought to undergo biological evolution themselves, but natural selection for their functions is indirect, at the level of populations, and is called second-order selection. In spite of an involvement of gene products in the generation of genetic variations, evolution genes do not programmatically direct evolution towards a specific goal. Rather, a steady interplay between natural selection and mixed populations of genetic variants gives microbial evolution its direction.

Comparison of Mycobacterium tuberculosis Genomes Reveals Frequent Deletions in a 20 kb Variable Region in Clinical Isolates

The Mycobacterium tuberculosis complex is associated with a remarkably low level of structural gene polymorphism. As part of a search for alternative forms of genetic variation that may act as a source of biological diversity in M. tuberculosis, we have identified a region of the genome that is highly variable amongst a panel of unrelated clinical isolates. Fifteen of 24 isolates examined contained one or more copies of the M. tuberculosis-specific IS6110 insertion element within this 20 kb variable region. In nine of the isolates, including the laboratory-passaged strain H37Rv, genomic deletions were identified, resulting in loss of between two and 13 genes. In each case, deletions were associated with the presence of a copy of the IS6110 element. Absence of flanking tri- or tetra-nucleotide repeats identified homologous recombination between adjacent IS6110 elements as the most likely mechanism of the deletion events. IS6110 insertion into hot-spots within the genome of M. tuberculosis provides a mechanism for generation of genetic diversity involving a high frequency of insertions and deletions.

Cloning, Characterization, and Localization of Mouse and Human SPO11

Spo11 is a meiosis-specific protein in yeast that has been found covalently bound to DNA double-strand breaks (DSBs) during the early stages of meiosis. These DSBs initiate homologous recombination, which is required for proper segregation of chromosomes and the generation of genetic diversity during meiosis. Here we report the cloning, characterization, tissue expression, and chromosomal localization of both mouse and human homologues of Spo11. The putative mouse and human proteins are 82% identical and share approximately 25% identity with other family members. Northern blot analysis revealed testis-specific expression for both genes, but RT-PCR results showed ubiquitous expression of at least a portion of Spo11 in mouse. Human SPO11 was also detected in several somatic tissues. Mouse Spo11 was localized to chromosome 2H4, and human SPO11 was localized to chromosome 20q13.2–q13.3, a region amplified in some breast and ovarian tumors.

Identification of a new gene family expressed during the onset of sexual reproduction in the centric diatom Thalassiosira weissflogii.

An intriguing feature of the diatom life cycle is that sexual reproduction and the generation of genetic diversity are coupled to the control of cell size. A PCR-based cDNA subtraction technique was used to identify genes that are expressed as small cells of the centric diatom Thalassiosira weissflogii initiate gametogenesis. Ten genes that are up-regulated during the early stages of sexual reproduction have been identified thus far. Three of the sexually induced genes, Sig1, Sig2, and Sig3, were sequenced to completion and are members of a novel gene family. The three polypeptides encoded by these genes possess different molecular masses and charges but display many features in common: they share five highly conserved domains; they each contain three or more cysteine-rich epithelial growth factor (EGF)-like repeats; and they each display homology to the EGF-like region of the vertebrate extracellular matrix glycoprotein tenascin X. Interestingly, the five conserved domains appear in the same order in each polypeptide but are separated by variable numbers of nonconserved amino acids. SIG1 and SIG2 display putative regulatory domains within the nonconserved regions. A calcium-binding, EF-hand motif is found in SIG1, and an ATP/GTP binding motif is present in SIG2. The striking similarity between the SIG polypeptides and extracellular matrix components commonly involved in cell-cell interactions suggests that the SIG polypeptides may play a role in sperm-egg recognition. The SIG polypeptides are thus important molecular targets for determining when and where sexual reproduction occurs in the field.

Control of Meiotic Recombination in Schizosaccharomyces pombe

Homologous recombination occurs at high frequency during meiosis and is essential for the proper segregation of chromosomes and the generation of genetic diversity. Meiotic recombination is controlled in numerous ways. In the fission yeast Schizosaccharomyces pombe nutritional starvation induces meiosis and high-level expression of many genes, including numerous recombination (rec) genes, whose products are required for recombination. Accompanying the two meiotic divisions are profound changes in nuclear and chromosomal structure and movement, which may play an important role in meiotic recombination. Although recombination occurs throughout the genome, it occurs at high frequency in some intervals (hotspots) and at low frequency in others (coldspots). The well-characterized hotspot M26 is activated by the Mts1/Mts2 protein; this site and its binding proteins interact with the local chromosomal structure to enhance recombination. A coldspot between the silent mating-type loci is repressed by identified proteins, which may also alter local chromatin. We discuss in detail the rec genes and the possible functions of their products, some but not all of which share homology with other identified proteins. Although some of the rec gene products are required for recombination throughout the genome, others demonstrate regional specificity and are required in certain genomic regions but not in others. Throughout the review contrasts are made with meiotic recombination in the more thoroughly studied budding yeast Saccharomyces cerevisiae.

Molecular Variation in Isolated Plant Populations

Abstract Isolated plant populations face a series of demographic and genetic challenges. They potentially may suffer from a loss of allelic and genotypic diversity or from inbreeding depression. The demographic stability associated with migration between components of a metapopulation is lost as fragmentation increasingly isolates populations. Such isolated populations are then increasingly subject to demographic stocasticity and to more frequent population bottlenecks. Some plant species clearly endure this challenge better than others. In studies of native plant species whose populations have long endured isolation, population numbers very often appear stable and populations often contain ample genetic variation. Of course, such observations are biased, since those species which did not accommodate isolation are presumed extinct and thus not studied.When one considers species which have recently undergone habitat fragmentation and population isolation, the ability to accommodate such change is clearly not present in many species. Such populations often show a dramatic decline in numbers as well as erosion of genetic variability. In these populations the role of somatic processes in the generation of genetic variation may assume an increased importance. Since only a few isolated studies have documented such somatic genomic changes in plants, the importance of such changes is subject to speculation.

Generation of genetic diversity by DNA rearrangements in resting bacteria

Transpositional DNA rearrangements importantly contribute to the genomic plasticity of bacteria, their viruses and plasmids. Interestingly, enzymatically mediated transposition is not limited to propagating bacteria, but it also occurs in prolonged periods of rest. As revealed with RFLP analysis, genetic polymorphism increases steadily upon storage of bacteria in the resting phase. These results are discussed here in the wider context of accumulating knowledge on molecular mechanisms contributing to overall spontaneous mutagenesis, which is the result of a multitude of specific, often enzyme-mediated processes of variation generation.