Genetik Ufku

Mutations in either TSC1 or TSC2 result in tuberous sclerosis, a rare genetic disease that causes benign tumors. In this issue of PLoS Genetics, Hsieh et al. report that TSC1 and TSC2 in Drosophila regulate dE2F1 protein expression and cooperate with RBF1 to control cellular survival and proliferation. This image is an eye-antennal imaginal disc that contains tsc2 mutant clones. In the tsc2 mutant clones (GFP negative), dE2F1 proteins (red) are expressed at a higher level compared to the neighboring wild type clones (GFP positive cells). The difference can be clearly seen in photoreceptors that are marked by ELAV (blue).

Image Credit: Ting-Chiu Hsieh and Nam-Sung Moon

Patients affected by autosomal dominant retinitis pigmentosa (ADRP) experience gradual loss of vision, and mutations in the visual pigment Rhodopsin—a G protein-coupled receptor that mediates phototransduction—are associated with ADRP. The most common ADRP mutation is the substitution of proline 23 by histidine (RhP23H), which causes Rh misfolding and aggregation. It is currently unclear how mutant RhP23H leads to photoreceptor neuron (PN) degeneration in ADRP. We used the fruitfly Drosophila melanogaster in which Rh1P37H (the equivalent of mammalian RhP23H) was overexpressed in PNs. We found that the presence of both mutant Rh1P37H and endogenous Rh1 is required for neurodegeneration in Rh1P37H flies. To understand the impact of Rh1 misfolding and clearance on PN degeneration, we inactivated the chaperone VCP/ter94, which escorts misfolded proteins out of the ER (a process termed retrotranslocation) and delivers them for proteasomal degradation. Rh1P37H flies with decreased VCP function displayed more misfolded Rh1P37H but, remarkably, showed a potent suppression of PN degeneration and blindness. Treatment of Rh1P37H flies with VCP or proteasome inhibitors also mitigated PN degeneration. Our results suggest that excessive retrotranslocation and/or degradation of visual pigment is deleterious for PN expressing misfolded RhP23H.

Almost all living organisms deteriorate with time through the process of aging or senescence. Because most studies on senescence examined organisms possessing a juvenile state, it was thought that bacteria, which reproduce by producing two apparently identical daughter cells, were immortal and not senescent. Recent studies have demonstrated that bacteria senesce because one daughter is allocated a larger share of the mother's load of non-genetic damage. Nonetheless, it is still equivocal whether bacterial senescence renders them mortal. I have developed a model that demonstrates that bacteria can be immortal if they experience damage below a threshold rate. A fit of the model to data shows that bacteria grown under standard laboratory conditions are immortal because they encounter a rate below the threshold. Because bacteria often experience higher damage rates in nature, it is likely that bacteria are generally mortal. The allocation of more damage to one daughter and the resulting mortality is the price bacteria pay to survive higher damage rates. These results suggest that senescence originated with the evolution of the first single-celled organisms and that it is ancestral in all multicellular organisms.

Calcium ions are essential for many physiological processes, including neurosecretion and neuronal and muscle excitation. Paradoxically, abnormal accumulation of calcium ions is associated with cell death and has been documented as an early event in muscle and neural degenerative diseases. One mechanism to avoid detrimental calcium accumulation is to link the calcium increase with activation of calcium-dependent potassium ion channels, thereby reducing cell excitability and preventing further calcium influx. This negative feedback requires these potassium channels to be localized in close proximity to sites of calcium entry. In a Caenorhabditis elegans genetic screen, we identified α-catulin, known as a cytoskeletal regulatory protein in mammals, important for the localization of calcium-dependent potassium channels in both muscles and neurons. In muscle, α-catulin controls the localization of the dystrophin complex, a multimeric protein complex implicated in muscular dystrophy. The dystrophin complex in turn tethers the calcium-dependent potassium channels near calcium channels. In neurons, the α-catulin-mediated localization of the potassium channels is independent of the dystrophin complex. Lack of α-catulin results in mislocalization of the potassium channels, and in turn causes defects in neuromuscular function. Our results support the idea that cytoskeletal proteins function as anchor molecules that localize ion channels to specific cellular domains.

Single rare causal alleles and/or collections of multiple rare alleles have been suggested to create “synthetic associations” with common variants in genome-wide association studies (GWAS). This model predicts that associations with common variants will not be consistent across populations. In this study, we examined 19 T2D variants for association with T2D risk in 6,142 cases and 7,403 controls from five racial/ethnic populations in the Multiethnic Cohort (European Americans, African Americans, Latinos, Japanese Americans, and Native Hawaiians). In racial/ethnic pooled analysis, all 19 variants were associated with T2D risk in the same direction as previous reports in Europeans, and the sum total of risk variants was significantly associated with T2D risk in each racial/ethnic group. The consistent associations across populations do not support the Goldstein hypothesis that rare causal alleles underlie GWAS signals. We also did not find evidence that these markers underlie racial/ethnic disparities in T2D prevalence. Large-scale GWAS and sequencing studies in these populations are necessary in order to both improve the current set of markers at these risk loci and identify new risk variants for T2D that may be difficult, or impossible, to detect in European populations.

Arachnomelia is a defect in skeletal development of cattle. Affected calves are born dead with elongated limbs and facial deformities. The causative mutation for this recessive condition had previously been mapped to a ~7 Mb interval. We exploited the special structure of cattle families to identify the causative mutation by a purely genetic approach. The rich pedigree records in cattle breeding allowed us to identify the founder animal of arachnomelia, a Brown Swiss bull born in 1957. A few generations later several cattle received two copies of the same chromosome segment from the father of this bull due to inbreeding. One copy was passed through the founder animal and acquired the causative mutation, while the other copy was transmitted through a different line of animals and stayed in its ancestral state. Using next-generation sequencing, we sequenced the entire critical interval in one of these inbred animals. As expected, we found only one single heterozygous position, which consequently represents the causative mutation for arachnomelia. The mutation affects the gene for sulfite oxidase, thus indicating a previously unrecognized important role for this enzyme in bone development. Our findings can immediately be applied to remove this deleterious mutation from the cattle breeding population.

Genome size (the amount of nuclear DNA) varies tremendously across organisms but is not necessarily correlated with organismal complexity. For example, genome sizes just within the grasses vary nearly 20-fold, but large-genomed grass species are not obviously more complex in terms of morphology or physiology than are the small-genomed species. Recent explanations for genome size variation have instead been dominated by the idea that population size determines genome size: mutations that increase genome size are expected to drift to fixation in species with small populations, but such mutations would be eliminated in species with large populations where natural selection operates at higher efficiency. However, inferences from previous analyses are limited because they fail to recognize that species share evolutionary histories and thus are not necessarily statistically independent. Our analysis takes a phylogenetic perspective and, contrary to previous studies, finds no evidence that genome size or any of its components (e.g., transposon number, intron number) are related to population size. We suggest that genome size evolution is unlikely to be neatly explained by a single factor such as population size.

Sensory motor neuropathies are diseases of the peripheral nervous system, involving primarily the nerves which control our muscles. These can result from either genetic or non-genetic causes, with genetic causes usually referred to as Charcot-Marie-Tooth (CMT) disease after the three clinicians who first described the key diagnostic markers. CMT patients lose muscle function, mainly in their arms and legs, with increasing severity during their lives. There are almost two dozen known genes that can mutate to cause CMT, and these fall into a wide variety of biochemical cellular pathways. We identified a group of patients with CMT from a small rural community, with good reason to suspect a genetic basis for their disease. Using high-throughput mapping and DNA sequencing technologies developed as part of the Human Genome Project, we were able to find the likely mutated gene, which was not any of the previously known CMT genes. Based on its sequence, the gene, called LRSAM1, probably plays a role in the correct metabolism of other proteins in the cell. Among the known CMT genes, some are also involved in protein metabolism, suggesting that this is a generally important pathway in the neurons that control muscle activity.

Over the last years, it has become evident that chromatin plays a crucial role in a variety of cellular processes. Insights into the tight regulation of chromatin opening and closing via chromatin remodelers, as well as post-translational modifications of proteins making up chromatin, have revealed new facets on the mechanisms used by cells in order to replicate, transcribe, or repair their DNA. In this report, we describe the involvement of a transcription-linked histone modification, methylation of histone H3 on lysine 4, in the DNA damage repair process. We discovered that in addition to its presence at promoters of highly transcribed genes, H3K4me3 is recruited to sites of newly created double-stranded breaks. Moreover, the results show that this recruitment is dependent on a chromatin remodeler, namely the RSC complex. Cells lacking this histone modification display similar defects as those devoid of the RSC complex; i.e. a significant decrease in the repair of DNA breaks by the non-homologous end-joining repair pathway and a difficulty to survive in presence of replication stresses. All these observations highlight the importance of this conserved histone modification, given that it is involved in a variety of mechanisms affecting genome function.

In meiosis, sex cells that become eggs or sperm undergo a single round of DNA replication followed by two consecutive chromosomal divisions. In most organisms, the segregation of chromosomes at the first meiotic division is dependent upon at least one genetic exchange, or crossover event, between homologous chromosome pairs. Matched chromosomes that do not receive a crossover frequently undergo non-disjunction at the first meiotic division, yielding gametes that lack chromosomes or contain additional copies. Such missegregation events have been linked to Down syndrome and human infertility. This paper focuses on Msh4-Msh5, a complex required for the proper segregation of homologous chromosomes during the Meiosis I division. We performed a mutational analysis of the baker's yeast Msh4-Msh5 complex to study its role in implementing the decision to make a crossover. We identified a class of mutants that are functional in meiosis despite significant reductions in crossing over that occurred primarily on larger chromosomes. In combination with mutations (pch2Δ, spo11-HA) that disrupted early steps in crossover placement, this msh4/5 class of mutants displayed poor spore viability. Together, these data support the presence in yeast of a robust crossover distribution mechanism.

In Parkinson's disease (PD), motor neurons that produce dopamine degenerate, leading to a characteristic syndrome including tremor, rigidity, and bradykinesia. The mechanisms leading to PD have been under intense investigation, identifying hereditary mutations responsible for about 8% of the cases. However, multiple environmental factors contribute to PD; and, amongst those, manganese (Mn) exposure from pesticides, industrial fumes, and gasoline additives has been robustly associated with PD. To gain insights into processes leading to the specific degeneration of dopaminergic neurons, we used a simple animal model, the nematode Caenorhabditis elegans, which, upon Mn exposure, recapitulates key molecular processes known to be involved in PD. Combining biochemistry and genetics, we demonstrate that dopamine secreted by the neurons and not intracellular dopamine is directly involved in the generation of toxic reactive oxygen species. We identify two essential mediators of this dopamine-dependent effect which are an extracellularly active enzyme called dual-oxidase and the dopamine re-uptake transporter. We also reveal that a transcription factor which is strongly expressed in two neurons involved in the regulation of aging is a powerful modulator of the dopamine-dependent toxicity. Our study establishes novel evidence of the link among PD, aging, and oxidative stress within the context of exposure to Mn.

Ionotropic glutamate receptors (iGluRs) are a family of cell surface proteins best known for their role in allowing neurons to communicate with each other in the brain. We recently discovered a variant class of iGluRs in the fruit fly (Drosophila melanogaster), named Ionotropic Receptors (IRs), which function as olfactory receptors in its “nose,” prompting us to ask whether iGluR/IRs might have a more general function in detection of environmental chemicals. Here, we have identified families of IRs in olfactory and taste sensory organs throughout protostomes, one of the principal branches of animal life that includes snails, worms, crustaceans, and insects. Our findings suggest that this receptor family has an evolutionary ancient function in detecting odors and tastants in the external world. By comparing the repertoires of these chemosensory IRs among both closely- and distantly-related species, we have observed dynamic patterns of expansion and divergence of these receptor families in organisms occupying very different ecological niches. Notably, many of the receptors we have identified are in insects that are of significant harm to human health, such as the malaria mosquito. These proteins represent attractive targets for novel types of insect repellents to control the host-seeking behaviors of such pest species.

Mammalian embryonic development requires precise control of gene expression in the right place at the right time. One level of control of gene expression is through cis-regulatory elements controlled by transcription factors. Deregulation of gene expression by mutations in such cis-regulatory elements has been described in developmental disorders. Heterozygous mutations in the transcription factor p63 are found in patients with limb malformations, cleft lip/palate, and defects in skin and other epidermal appendages, through disruption of normal ectodermal development during embryogenesis. We reasoned that the identification of target genes and cis-regulatory elements controlled by p63 would provide candidate genes for defects arising from abnormally regulated ectodermal development. To test our hypothesis, we carried out a genome-wide binding site analysis and identified a large number of target genes and regulatory elements regulated by p63. We further showed that one of these regulatory elements controls expression of DLX6 and possibly DLX5 in the apical ectodermal ridge in the developing limbs. Loss of this element through a micro-deletion was associated with split hand foot malformation (SHFM1). The list of p63 binding sites provides a resource for the identification of mutations that cause ectodermal dysplasias and malformations in humans.

Centromeres form at the same chromosomal position from generation to generation, yet in most species this inheritance occurs in a DNA sequence–independent manner that is not well understood. Here, we determine the timing of DNA replication across the genome of the human fungal pathogen Candida albicans and find that centromeric DNA is the first locus to replicate on each chromosome. Furthemore, this unique replication timing may be important for centromere inheritance, based on several observations. First, DNA sequence patterns at centromeres indicate that, despite high levels of primary sequence divergence, the region has served as a replication origin for millions of years; second, formation of a neocentromere (a new centromere formed at an ectopic locus following deletion of the native centromere DNA) results in the establishment of a new, early-firing origin of replication; and third, a centromere-specific protein, Cse4p, recruits origin replication complex proteins in a concentration-dependent manner. Thus, centromere position is inherited by an epigenetic mechanism that appears to be defined by a distinctively early firing DNA replication origin.

Most humans are infected with Helicobacter pylori. The H. pylori cag pathogenicity island (cagPAI) encodes a secretion apparatus that can translocate the CagA protein into host cells. Humans infected with cagPAI–carrying H. pylori are at increased risk of severe disease, including gastric cancer. We analyzed the nucleotide sequences and functional diversity of the cagPAI in a globally representative collection of isolates. Complete cagPAI sequences were obtained for 29 strains from all known H. pylori biogeographic populations. The gene content and arrangement of the cagPAI and its function were highly conserved. Diversity in most cag genes consisted in large part of synonymous polymorphisms. However some genes—in particular those that encode proteins predicted to be secreted or located on the outside of the bacterial cell—had particularly high frequencies of non-synonymous polymorphisms, suggesting that they were under diversifying selection. Our study provides evidence that the cagPAI was only acquired once and provides an important resource that can guide future research on the biological roles and host interactions of cagPAI proteins, including several whose function is still unknown.

The differentiation of cells into distinct cell-types, each of which is “remembered” for many generations, underlies the development of both healthy and cancerous tissues. Such differentiation, however, is not restricted to multi-cellular organisms: “white” and “opaque” cells of the unicellular fungal pathogen Candida albicans are two heritable cell-types, each thought to be adapted to unique niches within their human host. Here we examine the differences between these two cell-types by sequencing their RNA contents and subsequently reconstructing and comparing their gene expression profiles. We know that the transcription factor Wor1 plays a central role in mediating these expression differences. As with many other transcriptional regulators, however, a major unresolved issue is the apparent discordance between the genomic locations to which Wor1 binds and whether neighboring genes are differentially expressed. Here we resolve this discordance, showing that hundreds of Wor1 binding sites, previously without apparent function, actually flank differentially-expressed genes that were undiscovered, or not measured accurately, before. Additionally, we find that transcripts regulated by Wor1 have many unusual properties, several of which we also observe for transcripts regulated during the development of mammalian embryonic stem cells, suggesting they may be general hallmarks of cell differentiation.

Tuberous Sclerosis Complex genes 1 (TSC1) is a downstream component of the Insulin Receptor signaling pathway that is often deregulated in many tumors. In this study, we discovered that the fruit fly homolog of TSC1 regulates E2F transcription factor by controlling protein expression. E2F family proteins are key regulators of cellular division, and other tumor promoting events are previously shown to regulate E2F activity. Our findings demonstrate the importance of altering the E2F activity during tumorigenesis and provide new insights into the crosstalk between tumor promoting events.

The telomeric DNA of most eukaryotes consists of G-rich repetitive DNA with a 3′ single stranded DNA (ssDNA) overhang. In human and budding yeast (Saccharomyces cerevisiae) cells, the 3′ ssDNA overhang is bound by essential telomere capping proteins, POT1 and Cdc13 respectively. Maintenance of telomere capping is essential for the survival of cells. The RecQ helicases are a family of highly conserved proteins involved in the maintenance of telomere and genome stability. Loss of function of three RecQ helicases in humans results in cancer predisposition disorders Bloom's syndrome (BS), Werner's syndrome (WS), and Rothmund Thomson syndrome (RTS). Here we found that the RecQ helicase in budding yeast, Sgs1, plays a critical role in the resection of uncapped telomeres. Strikingly, simultaneous inactivation of Sgs1, the exonuclease Exo1, and checkpoint protein Rad9 allows budding yeast cells to divide in the absence of Cdc13, indicating that the essential role of the telomere cap is to counteract specific components of DNA damage response pathways. We speculate that, in certain genetic contexts, mammalian RecQ helicase also inhibit growth of cells with telomere capping defects, and a defect in this role could contribute to increased levels of tumorigenesis in BS, WS, and RTS patients.

How the axes of the embryo are established is an important question in developmental biology. In many organisms, the axes of the embryo are established during oogenesis through the generation of a polarized egg. Very little is known regarding the mechanisms of polarity establishment and maintenance in vertebrate oocytes and eggs. We have identified a zebrafish mutant called magellan, which displays a defect in egg polarity. The gene disrupted in the magellan mutant encodes the cytoskeletal linker protein microtubule actin crosslinking factor 1 (macf1). In vertebrates, it can take years to identify the molecular nature of a mutation. We used a new technique to identify the magellan mutation, which allowed us to rapidly isolate genomic DNA linked to the mutation and sequence it. Our results describe an important new function for macf1 in polarizing the oocyte and egg and demonstrate the feasibility of this new technique for the efficient identification of mutations.