Genetik Ufku

A long-standing enigma in the fields of DNA repair and cancer chemotherapy is why it is that cells starved of the base thymine die rapidly. This process, called thymineless death (TLD), is conserved in bacterial, yeast, and human cells and is the mode of action of important cancer chemotherapeutic drugs. Tumors that become resistant to those drugs have ceased to die from TLD. Despite its ubiquity, importance, and having been studied for more than 50 years, the mechanism(s) of TLD remained elusive. Here we show that a large fraction of TLD requires RecA, the central protein in homologous recombinational (HR) DNA repair, and activation of the bacterial DNA–damage (or SOS) response, which RecA controls. We find that of the 40 or so proteins upregulated during an SOS response, SulA, an inhibitor of cell division, accounts for most of how SOS–activation causes TLD. In cells undergoing TLD, we observe blocked replication of the E. coli chromosome followed by loss of DNA near the replication origin then terminus. This implies that much of TLD results from an irreversible cell-cycle checkpoint that blocks cell division when single-stranded DNA (the SOS–inducing signal) accumulates and that the rest results from DNA destruction, models for which are presented.

Many association studies have been published looking for genetic variants contributing to a variety of human traits such as obesity, diabetes, and height. Because the frequency of genetic variants can differ across populations, it is important to have estimates of genetic ancestry in the individuals being studied. In this study, we were able to measure genetic ancestry in populations of mixed ancestry by genotyping pooled, rather than individual, DNA samples. This represents a rapid and inexpensive means for modeling genetic ancestry and thus could facilitate future association or population-genetic studies in populations of unknown ancestry for which whole-genome data do not already exist.

Our knowledge of the genetic basis of normal pigmentation variation in human populations is quite incomplete. Recent studies have identified some of the genes responsible for the reduction in melanin content in European populations, but this is not the case for other population groups, such as East Asians. Here, we report that a genetic variant located within the gene OCA2 (rs1800414) is associated with skin pigmentation in two samples of East Asian ancestry. The allele associated with lower melanin levels is found at high frequencies in East Asian populations, but is absent or at very low frequencies in other population groups. This is one of the first reports of association of genetic markers with quantitative measures of pigmentation in East Asian populations and it confirms previous evidence indicating that evolution towards light skin occurred, at least in part, independently in Europe and East Asia. The OCA2 gene has been under positive selection in Europe and East Asia, but different alleles have been selected in each region.

In contrast to animals, where germline differentiation initiates early in embryogenesis, germline differentiation in plants starts in the adult phase during reproductive development. Transpositions of transposable elements in both somatic and gametic cells can be transmitted to the next generation. As a result, plant genomes may contain transposable elements exhibiting a variety of tissue-specific activities. Thus far, the spatio-temporal activity of LTR retrotransposons, the most abundant class of transposable elements in plants, has not been well characterized. Here, we report a detailed analysis of the spatio-temporal transposition pattern of a plant LTR retrotransposon in the endogenous system. Using the model legume Lotus japonicus, we found that LORE1a, a member of the chromovirus LORE1 family that belongs to the Gypsy superfamily, was epigenetically de-repressed via tissue culture. Activation was stochastic and derepression was maintained in regenerated plants. This feature made it possible to trace the original spatio-temporal activity of the retrotransposon in the intact plants. We determined that the plant chromovirus retrotransposes mainly in the male germline, without obvious insertional preferences for chromosomal regions. This finding suggests that the tissue specificity of transposable elements should be taken into account when considering their impact on the host genome dynamics and evolution.

The regulation of gene activity by transcription factors is crucial to the function of all cells. Here, we studied the mechanisms of action of the largest family of gene regulators encoded by the human genome, the so-called KRAB–containing zinc finger proteins (KRAB–ZFPs), which in concert with their universal cofactor KAP1 act as transcriptional repressors. For this, we used two parallel approaches. First, by targeting an ectopic KRAB domain to hundreds of different genes, we found that KRAB/KAP1 can repress promoters located several tens of kilobases from the repressor DNA docking site. We further could show that KRAB induces such long-range effects by mediating the spread of repressive chromatin marks along the body of the gene, resulting in a block of transcriptional initiation at the promoter. In a second set of experiments, we analyzed an endogenous KRAB–ZFP gene cluster, where we could also document KAP1–dependent heterochromatin spreading and transcriptional repression. Together, these results support a model whereby KRAB–ZFPs and KAP1 can mediate long-range transcriptional repression through the spread of silencing chromatin marks. This study thus provides insight into KRAB/KAP1–induced gene regulation at KRAB–ZFP gene clusters, and will further help interpret genome-wide studies of KRAB–ZFPs and KAP1 DNA binding patterns.

Congenital ocular malformations affecting the optic nerve are an important cause of childhood blindness. The papillorenal syndrome (PRS) is an autosomal dominant disorder that causes congenital optic nerve and kidney abnormalities, which may result in legal blindness and renal failure, respectively. Many cases of PRS are caused by mutations in the paired-box transcription factor PAX2. In this paper, we describe a novel mouse model of this human disease caused by a missense mutation in the Pax2 gene at the same position of one of the few disease-causing missense mutations in humans. We characterize the ocular and non-ocular phenotypes of this mouse and model the effect that murine and human Pax2/PAX2 mutations have on protein structure. We also experimentally test the effect these missense mutations have on protein localization, transactivation, and DNA binding, concluding that all three reduce steady-state levels of protein in vitro and (in p.T74A) in vivo by reducing protein stability. This work will help us better understand the pathophysiology of PRS and to dissect the molecular interactions important in normal PAX2 function.

XPF-ERCC1 is a nuclease that plays a critical role in DNA repair. Mutations in XPF are linked to xeroderma pigmentosum, characterized by sun sensitivity, high incidence of skin cancer, and neurodegeneration, or XFE progeroid syndrome, a disease of accelerated aging. Herein we report the unexpected finding that mutations in XPF cause mislocalization of XPF-ERCC1 to the cytoplasm. Recombinant mutant XPF-ERCC1 derived from XP– and XFE–causing alleles are catalytically active and if delivered to the nucleus of cells restore DNA repair. This demonstrates that protein mislocalization contributes to defective DNA repair and disease arising as a consequence of mutations in XPF. It also illustrates a novel mechanism of regulating a cell's capacity for DNA repair: by manipulating nuclear localization of XPF-ERCC1 to enhance or inhibit repair and to prevent cancer or tumor resistance to chemotherapy, respectively.

In Drosophila the sex-specific ON/OFF regulation of Sex-lethal (Sxl) is controlled by an autoregulatory splicing mechanism that depends on the SXL protein interacting with general splicing factors. Here we identify PPS as a novel component of the machinery required for Sxl splicing autoregulation by showing that the lack of pps function interferes with Sxl expression and that the PPS protein is physically linked to the Sxl pre–mRNA, the SXL protein and components of the general splicing machinery. PPS, however, stands apart from all other proteins known to control Sxl splicing because it is not a general splicing factor. Furthermore, PPS has a distinct pattern of accumulation along the Sxl transcription unit that suggests PPS is loaded onto the RNA at the promoter. Together with the observation that the PPS protein contains four signature motifs typically found in proteins that function in transcriptional regulation, our data suggest that linking transcription to splicing regulation is important for controlling Sxl expression. This idea is especially intriguing because it indicates that the coupling of transcription and splicing seen in vitro and in cell culture studies is likely to be pertinent to developmentally controlled patterns of gene expression in the living animal.

In this issue of PLoS Genetics, Hohenlohe et al. report the first high-density genome scan of stickleback fish to identify signatures of selection. Oceanic stickleback have repeatedly formed freshwater populations that have evolved rapidly in many characters, including the head and jaws. Previous work demonstrated that the same genes were behind parallel phenotypic changes in independent populations. Hohenlohe et al. show that this is a general pattern in stickleback evolution, with many genomic regions exhibiting parallel signatures in independently derived freshwater populations. These results show that parallel phenotypic evolution may often be caused by extensive parallel genomic changes.

Image Credit: Mark Currey (Center for Ecology and Evolutionary Biology, University of Oregon, USA)

Bacterial lineages have repeatedly evolved intimate symbioses with eukaryotic hosts, the most famous cases being those of the cell organelles, mitochondria, and plastids. Symbiont genomes typically lose many ancestral genes, raising the question of how they function with so few genes. In organelles, part of the answer involves gene transfer to the host genome, allowing maintenance of essential functions. So far, the extent of gene transfer to hosts has not been assessed for other cases of intimate, obligate symbiosis. Aphids harbor an ancient coevolved intracellular symbiont, called Buchnera. We used the newly available sequence of the pea aphid genome to conduct an exhaustive computational search for genes of bacterial ancestry. We found that no functional genes have been transferred from Buchnera, ruling out such transfer as a driving force in genome reduction in this symbiont. However, the aphid genome does contain eight transcribed genes of apparent bacterial origin, some of which have been duplicated after transfer. Based on their expression patterns, most of these appear to function specifically in the aphid-Buchnera symbiosis, presenting the possibility that the maintenance of obligate intracellular symbioses can be affected by the acquisition and duplication of genes transferred from unrelated bacterial lineages.

Cooperative systems are susceptible to exploitation by cheaters who enjoy the benefits of cooperation without paying the costs. Such conflict is seen in biological systems at every level from individual genes within a cell to individuals within societies. The social amoebae Dictyostelium discoideum have a unique cooperative system in which large numbers of individual cells aggregate to form fruiting bodies with reproductive spores, and dead stalk cells that may help the survival and dispersal of the spores. Fruiting bodies can contain several genotypes, and hence can be exploited by cheater cells that preferentially form spores without contributing fairly to the stalk. We have studied a mutant, cheater C (chtC), which is defective in forming certain stalk cells, but is still able to form fruiting bodies on its own. However, when wild-type cells are mixed with chtC cells, the wild-type cells compensate for the stalk-forming defect of chtC and form more of the stalk cells. In that way, chtC cells cheat by taking advantage of developmental processes that normally regulate cell-type proportions. This study shows that existing mechanisms of developmental regulation can be exploited by cheater mutants, and the social amoebae offer a good system to study such mechanisms.

Humans utilize at least two major pathways to repair DNA double-strand breaks (DSBs): homologous recombination (HR) and non-homologous end joining (NHEJ), and there are at least two genetically discrete sub-pathways of NHEJ: classical-NHEJ (C-NHEJ) and alternative-NHEJ (A-NHEJ). Since the products generated by each of these three repair (sub)pathways differ substantially from one another, it is biologically critical that certain DSBs are repaired by certain DSB repair pathways. How this pathway choice is made in human cells was unclear. In this study, knockout human cell lines that are defective in core C-NHEJ factors were generated. These cell lines are by-and-large extremely deficient in DSB repair, proving that C-NHEJ is the major DSB repair pathway in human cells. Unexpectedly, cell lines reduced for the C-NHEJ factors Ku70 or Ku86, carried out proficient DSB repair because of hyperactive A-NHEJ. In published work we have also demonstrated that Ku suppresses HR throughout the genome and at telomeres. Collectively, these data imply that Ku ensures that C-NHEJ is the major DSB repair pathway by two mechanisms: i) enabling C-NHEJ and ii) by actively suppressing HR and A-NHEJ. Thus, Ku is the critical regulator of pathway choice in human somatic cells.

Genome-wide association studies have been used to identify genetic variants conferring susceptibility to diseases, intermediate phenotypes, and physiological traits such as height, hair color, and age at menarche. Here we analyze the NFBC1966 and ALSPAC birth cohorts to investigate the genetic determinants of a key developmental process: primary tooth development. The prospective nature of our studies allows us to exploit accurate measurements of age at first tooth eruption and number of teeth at one year, and also provides the opportunity to assess whether genetic variants affecting these traits are associated with dental problems later in the life course. Of the genes that we find to be associated with primary tooth development, several have established roles in tooth development and growth, and almost half have proposed links with the development of cancer. We find that one of the variants is also associated with occlusion defects requiring orthodontic treatment later in life. Our findings should provide a strong foundation for the study of the genetic architecture of tooth development, which as well as its relevance to medicine and dentistry, may have implications in evolutionary biology since teeth represent important markers of evolution.

The insulin/IGF signalling (IIS) pathway plays key roles in growth, metabolism, reproduction, and longevity in animals as diverse as flies and mammals. Most multicellular animals contain multiple IIS ligands, including 7 in the fruit fly Drosophila melanogaster (DILP1-7), implying that the diverse functions of IIS could in part be mediated by the functional diversification of the ligands. Although Drosophila is a prime model organism to study IIS, knowledge about the function of individual DILPs is still very limited due to the lack of gene-specific mutants. Therefore, we generated mutants for all 7 dilp genes and systematically analyzed their phenotypes. We show that loss of DILP2 extends lifespan and describe a novel role for DILP6 in growth control. Furthermore, we show that DILPs are evolutionary conserved and can act redundantly, supporting the hypothesis that functional redundancy itself can be of evolutionary advantage. We also describe a specific interaction between IIS and the endosymbiontic bacterium Wolbachia in lifespan regulation. This finding has implications for all longevity studies using IIS mutants in flies and offers the opportunity to study IIS as a mechanism involved in host/symbiont interactions. Finally, we show that DILPs mediate the response of lifespan and fecundity to dietary restriction.

Broken DNA molecules can be repaired by copying a homologous DNA sequence located elsewhere in the genome. This process, called homologous recombination, needs to be carefully regulated, because unwanted DNA exchanges can lead to genome rearrangements and cell death. Cdk1 kinase is required for cell cycle progression and phosphorylates DNA repair factors, such as Srs2, a protein that can both translocate on single-stranded DNA and open the two strands of DNA double helix. DNA translocation activity of Srs2 is crucial to prevent unwanted recombination, while DNA unwinding activity might be important to promote recombination. In this study, we used two srs2 mutants that constitutively express the unphosphorylated or Cdk1-dependent phosphorylated Srs2 protein isoforms. We found that Srs2 performs genetically distinct functions in preventing or promoting homologous recombination. Cdk1 targets Srs2 to promote accurate repair of double-stranded DNA breaks, but is not essential for the removal of toxic recombination intermediates assembled at single-stranded DNA breaks. Further, Cdk1 counteracts sumoylation of Srs2, which is responsible for recombination defects due to the lack of Srs2 phosphorylation. In summary, Cdk1-dependent Srs2 phosphorylation prevents its unscheduled sumoylation and targets the helicase to promote accurate homologous recombinational repair.

Recent advances in the field of arsenic microbial metabolism have revealed that bacteria colonize a large panel of highly contaminated environments. Belonging to the order of Burkholderiales, Thiomonas strains are ubiquitous in arsenic-contaminated environments. The genome of one of them, i.e. Thiomonas sp. 3As, was deciphered and compared to the genome of several other Thiomonas strains. We found that their flexible gene pool evolved to allow both the surviving and growth in their peculiar environment. In particular, the acquisition by strains of the same species of different genomic islands conferred heavy metal resistance and metabolic idiosyncrasies. Our comparative genomic analyses suggest that the natural environment influences the genomic evolution of these bacteria. Importantly, these results highlight the genomic variability that may exist inside a taxonomic group, enlarging the concept of bacterial species.

All organisms that undergo sexual reproduction carry out specific mechanisms to establish different sexes. In fungi, sexual identity is typically determined by components housed within specialized regions of chromosomes known as mating-type (MAT) loci. MAT–encoded genes function to define sexes via two distinct paradigms: 1) by controlling transcription of components common to both sexes, or 2) by expressing specially encoded factors (pheromones and their receptors) that differ between mating types. The two mating types of Cryptococcus neoformans (a and α) are specified by an extremely unusual MAT locus. The unique architecture of this locus makes it impossible to predict which paradigm governs mating type. To identify the mechanism by which the C. neoformans sexes are determined, we created an α strain where the pheromones and pheromone receptor were replaced with the analogous genes from an a strain. We discovered that the resulting strain (αa) now behaves as if it is an a. It senses and responds to α cells, mates with α cells, and no longer exhibits other α-specific behaviors. Our data show that replacement of two and only two genes completely alters the sexual identity of α cells, establishing pheromones and their receptors as the determinants of sexual identity in C. neoformans.

Oceanic threespine stickleback have invaded and adapted to freshwater habitats countless times across the northern hemisphere. These freshwater populations have often evolved in similar ways from the ancestral marine stock from which they independently derived. With the exception of a few identified genes, the genetic basis of this remarkable parallel adaptation is unclear. Here we show that the parallel phenotypic evolution is matched by parallel patterns of nucleotide diversity and population differentiation across the genome. We used a novel high-throughput sequence-based genotyping approach to produce the first high density genome-wide scans of threespine stickleback populations and identified several genomic regions indicative of both divergent and balancing selection. Some of these regions have been associated previously with traits important for freshwater adaptation, but others were previously unidentified. Within these genomic regions we identified candidate genes, laying the foundation for further genetic and functional study of key pathways. This research illustrates the complementary nature of laboratory mapping, functional genetics, and population genomics.