StemCells Therapy » Genetic medicine

Functional Genomics Analysis of the Saccharomyces cerevisiae Iron Responsive Transcription Factor Aft1 Reveals Iron-Independent Functions [Genome and systems biology]

admin — Tue, 27 Jul 2010 08:19:51 +0000

The Saccharomyces cerevisiae transcription factor Aft1 is activated in iron-deficient cells to induce the expression of iron regulon genes, which coordinate the increase of iron uptake and remodel cellular metabolism to survive low-iron conditions. In addition, Aft1 has been implicated in numerous cellular processes including cell-cycle progression and chromosome stability; however, it is unclear if all cellular effects of Aft1 are mediated through iron homeostasis. To further investigate the cellular processes affected by Aft1, we identified >70 deletion mutants that are sensitive to perturbations in AFT1 levels using genome-wide synthetic lethal and synthetic dosage lethal screens. Our genetic network reveals that Aft1 affects a diverse range of cellular processes, including the RIM101 pH pathway, cell-wall stability, DNA damage, protein transport, chromosome stability, and mitochondrial function. Surprisingly, only a subset of mutants identified are sensitive to extracellular iron fluctuations or display genetic interactions with mutants of iron regulon genes AFT2 or FET3. We demonstrate that Aft1 works in parallel with the RIM101 pH pathway and the role of Aft1 in DNA damage repair is mediated by iron. In contrast, through both directed studies and microarray transcriptional profiling, we show that the role of Aft1 in chromosome maintenance and benomyl resistance is independent of its iron regulatory role, potentially through a nontranscriptional mechanism.

Sex-Linked Inheritance in Macaque Monkeys: Implications for Effective Population Size and Dispersal to Sulawesi [Population and evolutionary genetics]

admin — Tue, 27 Jul 2010 08:19:50 +0000

Sex-specific differences in dispersal, survival, reproductive success, and natural selection differentially affect the effective population size (N_e) of genomic regions with different modes of inheritance such as sex chromosomes and mitochondrial DNA. In papionin monkeys (macaques, baboons, geladas, mandrills, drills, and mangabeys), for example, these factors are expected to reduce N_e of paternally inherited portions of the genome compared to maternally inherited portions. To explore this further, we quantified relative N_e of autosomal DNA, X and Y chromosomes, and mitochondrial DNA using molecular polymorphism and divergence information from pigtail macaque monkeys (Macaca nemestrina). Consistent with demographic expectations, we found that N_e of the Y is lower than expected from a Wright–Fisher idealized population with an equal proportion of males and females, whereas N_e of mitochondrial DNA is higher. However, N_e of 11 loci on the X chromosome was lower than expected, a finding that could be explained by pervasive hitchhiking effects on this chromosome. We evaluated the fit of these data to various models involving natural selection or sex-biased demography. Significant support was recovered for natural selection acting on the Y chromosome. A demographic model with a skewed sex ratio was more likely than one with sex-biased migration and explained the data about as well as an ideal model without sex-biased demography. We then incorporated these results into an evaluation of macaque divergence and migration on Borneo and Sulawesi islands. One X-linked locus was not monophyletic on Sulawesi, but multilocus data analyzed in a coalescent framework failed to reject a model without migration between these islands after both were colonized.

Searching for Footprints of Positive Selection in Whole-Genome SNP Data From Nonequilibrium Populations [Population and evolutionary genetics]

admin — Tue, 27 Jul 2010 08:19:50 +0000

A major goal of population genomics is to reconstruct the history of natural populations and to infer the neutral and selective scenarios that can explain the present-day polymorphism patterns. However, the separation between neutral and selective hypotheses has proven hard, mainly because both may predict similar patterns in the genome. This study focuses on the development of methods that can be used to distinguish neutral from selective hypotheses in equilibrium and nonequilibrium populations. These methods utilize a combination of statistics on the basis of the site frequency spectrum (SFS) and linkage disequilibrium (LD). We investigate the patterns of genetic variation along recombining chromosomes using a multitude of comparisons between neutral and selective hypotheses, such as selection or neutrality in equilibrium and nonequilibrium populations and recurrent selection models. We perform hypothesis testing using the classical P-value approach, but we also introduce methods from the machine-learning field. We demonstrate that the combination of SFS- and LD-based statistics increases the power to detect recent positive selection in populations that have experienced past demographic changes.

The Conserved miR-51 microRNA Family Is Redundantly Required for Embryonic Development and Pharynx Attachment in Caenorhabditis elegans [Developmental and behavioral genetics]

admin — Tue, 27 Jul 2010 08:19:50 +0000

microRNAs (miRNAs) are ~22-nucleotide small RNAs that act as endogenous regulators of gene expression by base-pairing with target mRNAs. Here we analyze the function of the six members of the Caenorhabditis elegans miR-51 family of miRNAs (miR-51, miR-52, miR-53, miR-54, miR-55, miR-56). miR-51 family miRNAs are broadly expressed from mid-embryogenesis onward. The miR-51 family is redundantly required for embryonic development. mir-51 family mutants display a highly penetrant pharynx unattached (Pun) phenotype, where the pharyngeal muscle, the food pump of C. elegans, is not attached to the mouth. Unusually, the Pun phenotype in mir-51 family mutants is not due to a failure to attach, but instead a failure to maintain attachment during late embryogenesis. Expression of the miR-51 family in the mouth is sufficient to maintain attachment. The Fat cadherin ortholog CDH-3 is expressed in the mouth and is a direct target of the miR-51 family miRNAs. Genetic analysis reveals that miR-51 family miRNAs might act in part through CDH-3 to regulate pharynx attachment. This study is the first to assign a function to the miR-51/miR-100 miRNA family in any organism.

Specific {alpha}- and {beta}-Tubulin Isotypes Optimize the Functions of Sensory Cilia in Caenorhabditis elegans [Cellular genetics]

admin — Tue, 27 Jul 2010 08:19:50 +0000

Primary cilia have essential roles in transducing signals in eukaryotes. At their core is the ciliary axoneme, a microtubule-based structure that defines cilium morphology and provides a substrate for intraflagellar transport. However, the extent to which axonemal microtubules are specialized for sensory cilium function is unknown. In the nematode Caenorhabditis elegans, primary cilia are present at the dendritic ends of most sensory neurons, where they provide a specialized environment for the transduction of particular stimuli. Here, we find that three tubulin isotypes—the -tubulins TBA-6 and TBA-9 and the β-tubulin TBB-4—are specifically expressed in overlapping sets of C. elegans sensory neurons and localize to the sensory cilia of these cells. Although cilia still form in mutants lacking tba-6, tba-9, and tbb-4, ciliary function is often compromised: these mutants exhibit a variety of sensory deficits as well as the mislocalization of signaling components. In at least one case, that of the CEM cephalic sensory neurons, cilium architecture is disrupted in mutants lacking specific ciliary tubulins. While there is likely to be some functional redundancy among C. elegans tubulin genes, our results indicate that specific tubulins optimize the functional properties of C. elegans sensory cilia.

Autophosphorylation Within the Atg1 Activation Loop Is Required for Both Kinase Activity and the Induction of Autophagy in Saccharomyces cerevisiae [Cellular genetics]

admin — Tue, 27 Jul 2010 08:19:50 +0000

Autophagy is an evolutionarily conserved degradative pathway that has been implicated in a number of physiological events important for human health. This process was originally identified as a response to nutrient deprivation and is thought to serve in a recycling capacity during periods of nutritional stress. Autophagy activity appears to be highly regulated and multiple signaling pathways are known to target a complex of proteins that contains the Atg1 protein kinase. The data here extend these observations and identify a particular phosphorylation event on Atg1 as a potential control point within the autophagy pathway in Saccharomyces cerevisiae. This phosphorylation occurs at a threonine residue, T226, within the Atg1 activation loop that is conserved in all Atg1 orthologs. Replacing this threonine with a nonphosphorylatable residue resulted in a loss of Atg1 protein kinase activity and a failure to induce autophagy. This phosphorylation required the presence of a functional Atg1 kinase domain and two known regulators of Atg1 activity, Atg13 and Atg17. Interestingly, the levels of this modification were found to increase dramatically upon exposure to conditions that induce autophagy. In addition, T226 phosphorylation was associated with an autophosphorylated form of Atg1 that was found specifically in cells undergoing the autophagy process. In all, these data suggest that autophosphorylation within the Atg1 activation loop may represent a point of regulatory control for this degradative process.

Systematic Epistasis Analysis of the Contributions of Protein Kinase A- and Mitogen-Activated Protein Kinase-Dependent Signaling to Nutrient Limitation-Evoked Responses in the Yeast Saccharomyces cerevisiae [Cellular genetics]

admin — Tue, 27 Jul 2010 08:19:50 +0000

Cellular responses to environmental stimuli require conserved signal transduction pathways. In budding yeast (Saccharomyces cerevisiae), nutrient limitation induces morphological changes that depend on the protein kinase A (PKA) pathway and the Kss1 mitogen-activated protein kinase (MAPK) pathway. It was unclear to what extent and at what level there is synergy between these two distinct signaling modalities. We took a systematic genetic approach to clarify the relationship between these inputs. We performed comprehensive epistasis analysis of mutants lacking different combinations of all relevant pathway components. We found that these two pathways contribute additively to nutrient limitation-induced haploid invasive growth. Moreover, full derepression of either pathway rendered it individually sufficient for invasive growth and thus, normally, both are required only because neither is maximally active. Furthermore, in haploids, the MAPK pathway contributes more strongly than the PKA pathway to cell elongation and adhesion, whereas nutrient limitation-induced unipolar budding is independent of both pathways. In contrast, in diploids, upon nutrient limitation the MAPK pathway regulates cell elongation, the PKA pathway regulates unipolar budding, and both regulate cell adhesion. Thus, although there are similarities between haploids and diploids, cell type-specific differences clearly alter the balance of the signaling inputs required to elicit the various nutrient limitation-evoked cellular behaviors.

Mitotic Expression of Spo13 Alters M-Phase Progression and Nucleolar Localization of Cdc14 in Budding Yeast [Cellular genetics]

admin — Tue, 27 Jul 2010 08:19:50 +0000

Spo13 is a key meiosis-specific regulator required for centromere cohesion and coorientation, and for progression through two nuclear divisions. We previously reported that it causes a G2/M arrest and may delay the transition from late anaphase to G1, when overexpressed in mitosis. Yet its mechanism of action has remained elusive. Here we show that Spo13, which is phosphorylated and stabilized at G2/M in a Cdk/Clb-dependent manner, acts at two stages during mitotic cell division. Spo13 provokes a G2/M arrest that is reversible and largely independent of the Mad2 spindle checkpoint. Since mRNAs whose induction requires Cdc14 activation are reduced, we propose that its anaphase delay results from inhibition of Cdc14 function. Indeed, the Spo13-induced anaphase delay correlates with Cdc14 phosphatase retention in the nucleolus and with cyclin B accumulation, which both impede anaphase exit. At the onset of arrest, Spo13 is primarily associated with the nucleolus, where Cdc14 accumulates. Significantly, overexpression of separase (Esp1), which promotes G2/M and anaphase progression, suppresses Spo13 effects in mitosis, arguing that Spo13 acts upstream or parallel to Esp1. Given that Spo13 overexpression reduces Pds1 and cyclin B degradation, our findings are consistent with a role for Spo13 in regulating APC, which controls both G2/M and anaphase. Similar effects of Spo13 during meiotic MI may prevent cell cycle exit and initiation of DNA replication prior to MII, thereby ensuring two successive chromosome segregation events without an intervening S phase.

The Drosophila Claudin Kune-kune Is Required for Septate Junction Organization and Tracheal Tube Size Control [Cellular genetics]

admin — Tue, 27 Jul 2010 08:19:50 +0000

The vertebrate tight junction is a critical claudin-based cell–cell junction that functions to prevent free paracellular diffusion between epithelial cells. In Drosophila, this barrier is provided by the septate junction, which, despite being ultrastructurally distinct from the vertebrate tight junction, also contains the claudin-family proteins Megatrachea and Sinuous. Here we identify a third Drosophila claudin, Kune-kune, that localizes to septate junctions and is required for junction organization and paracellular barrier function, but not for apical-basal polarity. In the tracheal system, septate junctions have a barrier-independent function that promotes lumenal secretion of Vermiform and Serpentine, extracellular matrix modifier proteins that are required to restrict tube length. As with Sinuous and Megatrachea, loss of Kune-kune prevents this secretion and results in overly elongated tubes. Embryos lacking all three characterized claudins have tracheal phenotypes similar to any single mutant, indicating that these claudins act in the same pathway controlling tracheal tube length. However, we find that there are distinct requirements for these claudins in epithelial septate junction formation. Megatrachea is predominantly required for correct localization of septate junction components, while Sinuous is predominantly required for maintaining normal levels of septate junction proteins. Kune-kune is required for both localization and levels. Double- and triple-mutant combinations of Sinuous and Megatrachea with Kune-kune resemble the Kune-kune single mutant, suggesting that Kune-kune has a more central role in septate junction formation than either Sinuous or Megatrachea.

IscR Regulates RNase LS Activity by Repressing rnlA Transcription [Gene expression]

admin — Tue, 27 Jul 2010 08:19:50 +0000

The Escherichia coli endoribonuclease LS was originally identified as a potential antagonist of bacteriophage T4. When the T4 dmd gene is defective, RNase LS cleaves T4 mRNAs and antagonizes T4 reproduction. This RNase also plays an important role in RNA metabolisms in E. coli. rnlA is an essential gene for RNase LS activity, but the transcriptional regulation of this gene remains to be elucidated. An Fe-S cluster protein, IscR, acts as a transcription factor and controls the expression of genes that are necessary for Fe-S cluster biogenesis. Here, we report that overexpression of IscR suppressed RNase LS activity, causing the loss of antagonist activity against phage T4. This suppressive effect did not require the ligation of Fe-S cluster into IscR. β-Galactosidase reporter assays showed that transcription from an rnlA promoter increased in iscR-deleted cells compared to wild-type cells, and gel-mobility shift assays revealed specific binding of IscR to the rnlA promoter region. RT–PCR analysis demonstrated that endogenous rnlA mRNA was reduced by overexpression of IscR and increased by deletion of iscR. From these results, we conclude that IscR negatively regulates transcription of rnlA and represses RNase LS activity.

Molecular Genetic Analysis of Chd3 and Polytene Chromosome Region 76B-D in Drosophila melanogaster [Gene expression]

admin — Tue, 27 Jul 2010 08:19:50 +0000

The Drosophila melanogaster Chd3 gene encodes a member of the CHD group of SNF2/RAD54 ATPases. CHD proteins are conserved from yeast to man and many are subunits of chromatin-remodeling complexes that facilitate transcription. Drosophila CHD3 proteins are not found in protein complexes, but as monomers that remodel chromatin in vitro. CHD3 colocalize with elongating RNA polymerase II on salivary gland polytene chromosomes. Since the role of Chd3 in development was unknown, we isolated and characterized the essential genes within the 640-kb region of the third chromosome (polytene chromosome region 76B-D) that includes Chd3. We recovered mutations in 24 genes that are essential for zygotic viability. We found that transposon-insertion mutants for 46% of the essential genes are included in the Drosophila Gene Disruption Project collection. None of the essential genes that we identified are in a 200-kb region that includes Chd3. We generated a deletion of Chd3 by targeted gene replacement. This deletion had no effect on either viability or fertility.

Stb3 Plays a Role in the Glucose-Induced Transition from Quiescence to Growth in Saccharomyces cerevisiae [Gene expression]

admin — Tue, 27 Jul 2010 08:19:50 +0000

Addition of glucose to quiescent Saccharomyces cerevisiae cells causes the immediate induction of ~1000 genes. These genes include ribosomal proteins (RP) and non-RP genes needed for ribosome production and other growth processes. RRPE sequence elements are commonly found 5′ of non-RP growth gene ORFs, and Stb3 has recently been identified as an RRPE binding protein. Stb3 overexpression (Stb3OE) produces a slow growth phenotype that is associated with reduced expression of non-RP genes and a drop in the rate of amino acid incorporation. Genes affected by Stb3 are associated with a TGAAAAA motif. Stb3 is restricted to the nucleus in quiescent cells and is immediately released into the cytoplasm after glucose repletion. The Stb3OE slow growth phenotype is reversed by loss of Hos2 histone deactylase activity, consistent with the idea that repression involves histone deacetylation. SCH9 overexpression or PPH22 deletion, mutations that activate target of rapamycin (Tor) nutrient sensing pathways, also reverse the Stb3OE phenotype. Inhibition of Tor signaling makes the phenotype more severe and restricts Stb3 to the nucleus. The results support a model in which Stb3 is one of the components that repress a large set of growth genes as nutrients are depleted. This repression is ended by glucose.

Effect of Translesion DNA Polymerases, Endonucleases and RpoS on Mutation Rates in Salmonella typhimurium [Genome integrity and transmission]

admin — Tue, 27 Jul 2010 08:19:50 +0000

It has been suggested that bacteria have evolved mechanisms to increase their mutation rate in response to various stresses and that the translesion DNA polymerase Pol IV under control of the LexA regulon and the alternative sigma factor RpoS are involved in regulating this mutagenesis. Here we examined in Salmonella enterica serovar Typhimurium LT2 the rates for four different types of mutations (rifampicin, nalidixic acid, and chlorate resistance and Lac⁺ reversion) during various growth conditions and with different levels of four translesion DNA polymerases (Pol II, Pol IV, Pol V, and SamAB) and RpoS. Constitutive derepression of the LexA regulon by a lexA(def) mutation had no effect on Lac⁺ reversion rates but increased the other three mutation rates up to 11-fold, and the contribution of the translesion DNA polymerases to this mutagenesis varied with the type of mutation examined. The increase in mutation rates in the lexA(def) mutant required the presence of the LexA-controlled UvrB protein and endonucleases UvrC and Cho. With regard to the potential involvement of RpoS in mutagenesis, neither an increase in RpoS levels conferred by artificial overexpression from a plasmid nor long-term stationary phase incubation or slow growth caused an increase in any of the four mutation rates measured, alone or in combination with overexpression of the translesion DNA polymerases. In conclusion, mutation rates are remarkably robust and no combination of growth conditions, induction of translesion DNA polymerases by inactivation of LexA, or increased RpoS expression could confer an increase in mutation rates higher than the moderate increase caused by derepression of the LexA regulon alone.

Mek1 Suppression of Meiotic Double-Strand Break Repair Is Specific to Sister Chromatids, Chromosome Autonomous and Independent of Rec8 Cohesin Complexes [Genome integrity and transmission]

admin — Tue, 27 Jul 2010 08:19:50 +0000

During meiosis, recombination is directed to occur between homologous chromosomes to create connections necessary for proper segregation at meiosis I. Partner choice is determined at the time of strand invasion and is mediated by two recombinases: Rad51 and the meiosis-specific Dmc1. In budding yeast, interhomolog bias is created in part by the activity of a meiosis-specific kinase, Mek1, which is localized to the protein cores of condensed sister chromatids. Analysis of meiotic double-strand break (DSB) repair in haploid and disomic haploid strains reveals that Mek1 suppresses meiotic intersister DSB repair by working directly on sister chromatids. Rec8 cohesin complexes are not required, however, either for suppression of intersister DSB repair or for the repair itself. Regulation of DSB repair in meiosis is chromosome autonomous such that unrepaired breaks on haploid chromosomes do not prevent interhomolog repair between disomic homologs. The pattern of DSB repair in haploids containing Dmc1 and/or Rad51 indicates that Mek1 acts on Rad51-specific recombination processes.

An mre11 Mutation That Promotes Telomere Recombination and an Efficient Bypass of Senescence [Genome integrity and transmission]

admin — Tue, 27 Jul 2010 08:19:50 +0000

Preventing the formation of dysfunctional telomeres is essential for genomic stability. In most organisms, the ribo-nucleoprotein reverse transcriptase telomerase is responsible for telomere GT-strand elongation. However, in telomerase-negative cells, low-frequency recombination mechanisms can avert lethality by elongating critically short telomeres. This study focuses on the involvement of the budding yeast Mre11 in telomere recombination and homeostasis. We have identified a novel allele of MRE11, mre11-A470T, that, in telomerase-positive cells, confers a semidominant decrease in telomere size and a recessive defect in telomere healing. In addition, mutant cells lack normal telomere size homeostasis. Telomerase-negative mre11-A470T cells display a Rad51-dependent bypass of replicative senescence via induction of a highly efficient type I-related recombination pathway termed type IA. The type IA pathway involves an amplification of subtelomeric Y’ elements, coupled with elongated and more heterogeneous telomere tracts relative to the short telomere size of type I survivors. The data have led us to propose the involvement of break-induced replication in telomere expansion. The differing phenotypes elicited by the mre11-A470T mutants in telomerase-positive and telomerase-negative cells have also led us to speculate that the telomere end structure may be modified differentially in mre11-A470T cells, directing the telomere into specific pathways.

Complete Switchgrass Genetic Maps Reveal Subgenome Collinearity, Preferential Pairing and Multilocus Interactions [Genome integrity and transmission]

admin — Tue, 27 Jul 2010 08:19:50 +0000

Polyploidy is an important aspect of the evolution of flowering plants. The potential of gene copies to diverge and evolve new functions is influenced by meiotic behavior of chromosomes leading to segregation as a single locus or duplicated loci. Switchgrass (Panicum virgatum) linkage maps were constructed using a full-sib population of 238 plants and SSR and STS markers to access the degree of preferential pairing and the structure of the tetraploid genome and as a step toward identification of loci underlying biomass feedstock quality and yield. The male and female framework map lengths were 1645 and 1376 cM with 97% of the genome estimated to be within 10 cM of a mapped marker in both maps. Each map coalesced into 18 linkage groups arranged into nine homeologous pairs. Comparative analysis of each homology group to the diploid sorghum genome identified clear syntenic relationships and collinear tracts. The number of markers with PCR amplicons that mapped across subgenomes was significantly fewer than expected, suggesting substantial subgenome divergence, while both the ratio of coupling to repulsion phase linkages and pattern of marker segregation indicated complete or near complete disomic inheritance. The proportion of transmission ratio distorted markers was relatively low, but the male map was more extensively affected by distorted transmission ratios and multilocus interactions, associated with spurious linkages.

A Fluorescence in Situ Hybridization System for Karyotyping Soybean [Methods, technology, and resources]

admin — Tue, 27 Jul 2010 08:19:50 +0000

The development of a universal soybean (Glycine max [L.] Merr.) cytogenetic map that associates classical genetic linkage groups, molecular linkage groups, and a sequence-based physical map with the karyotype has been impeded due to the soybean chromosomes themselves, which are small and morphologically homogeneous. To overcome this obstacle, we screened soybean repetitive DNA to develop a cocktail of fluorescent in situ hybridization (FISH) probes that could differentially label mitotic chromosomes in root tip preparations. We used genetically anchored BAC clones both to identify individual chromosomes in metaphase spreads and to complete a FISH-based karyotyping cocktail that permitted simultaneous identification of all 20 chromosome pairs. We applied these karyotyping tools to wild soybean, G. soja Sieb. and Zucc., which represents a large gene pool of potentially agronomically valuable traits. These studies led to the identification and characterization of a reciprocal chromosome translocation between chromosomes 11 and 13 in two accessions of wild soybean. The data confirm that this translocation is widespread in G. soja accessions and likely accounts for the semi-sterility found in some G. soja by G. max crosses.

Dictyostelium Finds New Roles to Model [Review]

admin — Tue, 27 Jul 2010 08:19:50 +0000

Any established or aspiring model organism must justify itself using two criteria: does the model organism offer experimental advantages not offered by competing systems? And will any discoveries made using the model be of wider relevance? This review addresses these issues for the social amoeba Dictyostelium and highlights some of the organisms more recent applications. These cover a remarkably wide gamut, ranging from sociobiological to medical research with much else in between.

The 1948 International Congress of Genetics in Sweden: People and Politics [Perspectives]

admin — Tue, 27 Jul 2010 08:19:50 +0000

The International Congresses have played an important role in the history of genetics. The Eighth International Congress, which in 1948 was held in Sweden, celebrated the conclusion of the war against Nazism and many new decisive scientific advances. It also signaled a hardening of the fight against Lysenkoism, which was growing in strength in the Soviet Union. A rare document is available from the Congress—an amateur film made by a young delegate, Nils Nybom. With its help a living description can be given of the scientific and political melees in which the delegates were involved.

ISSUE HIGHLIGHTS [Issue Highlights]

admin — Tue, 27 Jul 2010 08:19:50 +0000