bluuebunny/crossref_metadata_2025 huggingface dataset:
Pooling Analysis of Genetic Data: The Association of Leptin Receptor (LEPR) Polymorphisms With Variables Related to Human Adiposity
Authors: Heo M , Leibel R L , Boyer B B , Chung W K , Koulu M , Karvonen M K , Pesonen U , Rissanen A , Laakso M , Uusitupa M I J , Chagnon Y , Bouchard C , Donohoue P A , Burns T L , Shuldiner A R , Silver K , Andersen R E , Pedersen O , Echwald S , Sørensen T I A , Behn P , Permutt M A , Jacobs K B , Elston R C , Hoffman D J , Allison D B
Published: April 2021

Analysis of raw pooled data from distinct studies of a single question generates a single statistical conclusion with greater power and precision than conventional metaanalysis based on within-study estimates. However, conducting analyses with pooled genetic data, in particular, is a daunting task that raises important statistical issues. In the process of analyzing data pooled from nine studies on the human leptin receptor (LEPR) gene for the association of three alleles (K109R, Q223R, and K656N) of LEPR with body mass index (BMI; kilograms divided by the square of the height in meters) and waist circumference (WC), we encountered the following methodological challenges: data on relatives, missing data, multivariate analysis, multiallele analysis at multiple loci, heterogeneity, and epistasis. We propose herein statistical methods and procedures to deal with such issues. With a total of 3263 related and unrelated subjects from diverse ethnic backgrounds such as African-American, Caucasian, Danish, Finnish, French-Canadian, and Nigerian, we tested effects of individual alleles; joint effects of alleles at multiple loci; epistatic effects among alleles at different loci; effect modification by age, sex, diabetes, and ethnicity; and pleiotropic genotype effects on BMI and WC. The statistical methodologies were applied, before and after multiple imputation of missing observations, to pooled data as well as to individual data sets for estimates from each study, the latter leading to a metaanalysis. The results from the metaanalysis and the pooling analysis showed that none of the effects were significant at the 0.05 level of significance. Heterogeneity tests showed that the variations of the nonsignificant effects are within the range of sampling variation. Although certain genotypic effects could be population specific, there was no statistically compelling evidence that any of the three LEPR alleles is associated with BMI or waist circumference in the general population.
The Distribution of Early Recombination Nodules on Zygotene Bivalents From Plants
Authors: Anderson Lorinda K , Hooker Kristina D , Stack Stephen M
Published: April 2021

Early recombination nodules (ENs) are protein complexes ~100 nm in diameter that are associated with forming synaptonemal complexes (SCs) during leptotene and zygotene of meiosis. Although their functions are not yet clear, ENs may have roles in synapsis and recombination. Here we report on the frequency and distribution of ENs in zygotene SC spreads from six plant species that include one lower vascular plant, two dicots, and three monocots. For each species, the number of ENs per unit length is higher for SC segments than for (asynapsed) axial elements (AEs). In addition, EN number is strongly correlated with SC segment length. There are statistically significant differences in EN frequencies on SCs between species, but these differences are not related to genome size, number of chromosomes, or phylogenetic class. There is no difference in the frequency of ENs per unit length of SC from early to late zygotene. The distribution of distances between adjacent ENs on SC segments is random for all six species, but ENs are found at synaptic forks more often than expected for a random distribution of ENs on SCs. From these observations, we conclude that in plants: (1) some ENs bind to AEs prior to synapsis, (2) most ENs bind to forming SCs at synaptic forks, and (3) ENs do not bind to already formed SCs.
Bayesian Methods for Quantitative Trait Loci Mapping Based on Model Selection: Approximate Analysis Using the Bayesian Information Criterion
Authors: Ball Roderick D
Published: April 2021

We describe an approximate method for the analysis of quantitative trait loci (QTL) based on model selection from multiple regression models with trait values regressed on marker genotypes, using a modification of the easily calculated Bayesian information criterion to estimate the posterior probability of models with various subsets of markers as variables. The BIC-δ criterion, with the parameter δ increasing the penalty for additional variables in a model, is further modified to incorporate prior information, and missing values are handled by multiple imputation. Marginal probabilities for model sizes are calculated, and the posterior probability of nonzero model size is interpreted as the posterior probability of existence of a QTL linked to one or more markers. The method is demonstrated on analysis of associations between wood density and markers on two linkage groups in Pinus radiata. Selection bias, which is the bias that results from using the same data to both select the variables in a model and estimate the coefficients, is shown to be a problem for commonly used non-Bayesian methods for QTL mapping, which do not average over alternative possible models that are consistent with the data.
On themod rescModel and the Evolution of Wolbachia Compatibility Types
Authors: Charlat Sylvain , Calmet Claire , Merçot Hervé
Published: April 2021

Cytoplasmic incompatibility (CI) is induced by the endocellular bacterium Wolbachia. It results in an embryonic mortality occurring when infected males mate with uninfected females. The mechanism involved is currently unknown, but the mod resc model allows interpretation of all observations made so far. It postulates the existence of two bacterial functions: modification (mod) and rescue (resc). The mod function acts in the males' germline, before Wolbachia are shed from maturing sperm. If sperm is affected by mod, zygote development will fail unless resc is expressed in the egg. Interestingly, CI is also observed in crosses between infected males and infected females when the two partners bear different Wolbachia strains, demonstrating that mod and resc interact in a specific manner: Two Wolbachia strains are compatible with each other only if they harbor the same compatibility type. Here we focus on the evolutionary process involved in the emergence of new compatibility types from ancestral ones. We argue that new compatibility types are likely to evolve under a wider range of conditions than previously thought, through a two-step process. First, new mod variants can arise by mutation and spread by drift. This is possible because mod is expressed in males and Wolbachia is transmitted by females. Second, once such a mod variant achieves a certain frequency, it can create the conditions for the deterministic invasion of a new resc variant, allowing the invasion of a new mod resc pair. Furthermore, we show that a stable polymorphism might be maintained in natural populations, allowing the long-term existence of “suicidal” Wolbachia strains.
Counteracting Regulation of Chromatin Remodeling at a Fission Yeast cAMP Responsive Element-Related Recombination Hotspot by Stress-Activated Protein Kinase, cAMP-Dependent Kinase and Meiosis Regulators
Authors: Mizuno Ken-ichi , Hasemi Tomoko , Ubukata Toshiharu , Yamada Takatomi , Lehmann Elisabeth , Kohli Jürg , Watanabe Yoshinori , Iino Yuichi , Yamamoto Masayuki , Fox Mary E , Smith Gerald R , Murofushi Hiromu , Shibata Takehiko , Ohta Kunihiro
Published: April 2021

In fission yeast, an ATF/CREB-family transcription factor Atf1-Pcr1 plays important roles in the activation of early meiotic processes via the stress-activated protein kinase (SAPK) and the cAMP-dependent protein kinase (PKA) pathways. In addition, Atf1-Pcr1 binds to a cAMP responsive element (CRE)-like sequence at the site of the ade6-M26 mutation, which results in local enhancement of meiotic recombination and chromatin remodeling. Here we studied the roles of meiosis-inducing signal transduction pathways in M26 chromatin remodeling. Chromatin analysis revealed that persistent activation of PKA in meiosis inhibited M26 chromatin remodeling, suggesting that the PKA pathway represses M26 chromatin remodeling. The SAPK pathway activated M26 chromatin remodeling, since mutants lacking a component of this pathway, the Wis1 or Spc1/Sty1 kinases, had no M26 chromatin remodeling. M26 chromatin remodeling also required the meiosis regulators Mei2 and Mei3 but not the subsequently acting regulators Sme2 and Mei4, suggesting that induction of M26 chromatin remodeling needs meiosis-inducing signals before premeiotic DNA replication. Similar meiotic chromatin remodeling occurred meiotically around natural M26 heptamer sequences. These results demonstrate the coordinated action of genetic and physiological factors required to remodel chromatin in preparation for high levels of meiotic recombination and eukaryotic cellular differentiation.
Phosphate Transport and Sensing in Saccharomyces cerevisiae
Authors: Wykoff Dennis D , O'Shea Erin K
Published: April 2021

Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate; however, little is known about how phosphate concentrations are sensed. The similarity of Pho84p, a high-affinity phosphate transporter in Saccharomyces cerevisiae, to the glucose sensors Snf3p and Rgt2p has led to the hypothesis that Pho84p is an inorganic phosphate sensor. Furthermore, pho84Δ strains have defects in phosphate signaling; they constitutively express PHO5, a phosphate starvation-inducible gene. We began these studies to determine the role of phosphate transporters in signaling phosphate starvation. Previous experiments demonstrated a defect in phosphate uptake in phosphate-starved pho84Δ cells; however, the pho84Δ strain expresses PHO5 constitutively when grown in phosphate-replete media. We determined that pho84Δ cells have a significant defect in phosphate uptake even when grown in high phosphate media. Overexpression of unrelated phosphate transporters or a glycerophosphoinositol transporter in the pho84Δ strain suppresses the PHO5 constitutive phenotype. These data suggest that PHO84 is not required for sensing phosphate. We further characterized putative phosphate transporters, identifying two new phosphate transporters, PHO90 and PHO91. A synthetic lethal phenotype was observed when five phosphate transporters were inactivated, and the contribution of each transporter to uptake in high phosphate conditions was determined. Finally, a PHO84-dependent compensation response was identified; the abundance of Pho84p at the plasma membrane increases in cells that are defective in other phosphate transporters.
Spontaneous Chromosome Loss in Saccharomyces cerevisiae Is Suppressed by DNA Damage Checkpoint Functions
Authors: Klein Hannah L
Published: April 2021

Genomic instability is one of the hallmarks of cancer cells and is often the causative factor in revealing recessive gene mutations that progress cells along the pathway to unregulated growth. Genomic instability can take many forms, including aneuploidy and changes in chromosome structure. Chromosome loss, loss and reduplication, and deletions are the majority events that result in loss of heterozygosity (LOH). Defective DNA replication, repair, and recombination can significantly increase the frequency of spontaneous genomic instability. Recently, DNA damage checkpoint functions that operate during the S-phase checkpoint have been shown to suppress spontaneous chromosome rearrangements in haploid yeast strains. To further study the role of DNA damage checkpoint functions in genomic stability, we have determined chromosome loss in DNA damage checkpoint-deficient yeast strains. We have found that the DNA damage checkpoints are essential for preserving the normal chromosome number and act synergistically with homologous recombination functions to ensure that chromosomes are segregated correctly to daughter cells. Failure of either of these processes increases LOH events. However, loss of the G2/M checkpoint does not result in an increase in chromosome loss, suggesting that it is the various S-phase DNA damage checkpoints that suppress chromosome loss. The mec1 checkpoint function mutant, defective in the yeast ATR homolog, results in increased recombination through a process that is distinct from that operative in wild-type cells.
(CA/TG) Microsatellite Sequences Escape the Inhibition of Recombination by Mismatch Repair in Saccharomyces cerevisiae
Authors: Gendrel Christiane-Gabrielle , Dutreix Marie
Published: April 2021

Sequence divergence reduces the frequency of recombination, a process that is dependent on the activity of the mismatch repair system. In the yeast Saccharomyces cerevisiae, repair of mismatches results in gene conversion or restoration, whereas failure to repair mismatches results in postmeiotic segregation (PMS). By examining the conversion and PMS in yeast strains deficient in various MMR genes and heterozygous for large inserts (107 bp) with either a mixed sequence or a 39 (CA/TG) repetitive microsatellite sequence, we demonstrate that: (1) the inhibition of conversion by large inserts depends upon a complex containing both Msh2 and Pms1 proteins; (2) conversion is not inhibited if the single-stranded DNA loop in the heteroduplex is the microsatellite sequence; and (3) large heteroduplex loops with random sequence or repetitive sequence might be repaired by two complexes, containing either Msh2 or Pms1. Our results suggest that inhibition of recombination by heterologous inserts and large loop repair are not processed by the same MMR complexes. We propose that the inhibition of conversion by large inserts is due to recognition by the Msh2/Pms1 complex of mismatches created by intrastrand interactions in the heteroduplex loop.
Interactions of the Tribolium Sex combs reduced and proboscipedia Orthologs in Embryonic Labial Development
Authors: DeCamillis Mark A , Lewis David L , Brown Susan J , Beeman Richard W , Denell Robin E
Published: April 2021

The role of Hox genes in the development of insect gnathal appendages has been examined in three insects: the fruitfly, Drosophila melanogaster; the milkweed bug, Oncopeltus fasciatus; and the red flour beetle, Tribolium castaneum. In each of these organisms, the identity of the labium depends on the homeotic genes Sex combs reduced (Scr) and proboscipedia (pb). Loss of pb function in each of the three insects results in homeotic transformation of the labial appendages to legs. In contrast, loss of Scr function yields a different transformation in each species. Interestingly, mutations in Cephalothorax (Cx), the Tribolium ortholog of Scr, transform the labial appendages to antennae, a result seen in the other insects only when both pb and Scr are removed. We show here that the Tribolium labial appendages also develop as antennae in double mutants. Further, we demonstrate that expression of the Tribolium proboscipedia ortholog maxillopedia (mxp) is greatly reduced or absent in the labium of Cx mutant larvae. Thus, in the wild-type labial segment, Cx function is required (directly or indirectly) for mxp transcription. A similar interaction between Scr and pb during Drosophila embryogenesis has been described recently. Thus, this regulatory paradigm appears to be conserved at least within the Holometabola.
small bristles Is Required for the Morphogenesis of Multiple Tissues During Drosophila Development
Authors: Korey Christopher A , Wilkie Gavin , Davis Ilan , Van Vactor David
Published: April 2021

We found that mutations in small bristles (sbr) affect several tissues during the development of the fruit fly. In sbr embryos, neurons have defects in pathfinding and the body wall muscles have defective morphology. As adults, sbr flies have smaller and thinner bristles with a reduced diameter, suggesting a defective cytoskeleton within. The phenotypes we observe are consistent with defects in cell morphogenesis. We identified DmNXF1, the Drosophila homolog of a mRNA export protein that has been characterized in human (NXF1/TAP) and yeast (Mex67p) as the protein encoded by the small bristles locus. Given that a global decrease in mRNA export in these mutants is likely, the phenotypes we observe suggest that certain tissues are acutely sensitive to lower levels of cytoplasmic mRNA and the resultant decrease in protein synthesis during key stages of cellular morphogenesis.
Negative Crossover Interference in Maize Translocation Heterozygotes
Authors: Auger Donald L , Sheridan William F
Published: April 2021

Negative interference describes a situation where two genetic regions have more double crossovers than would be expected considering the crossover rate of each region. We detected negative crossover interference while attempting to genetically map translocation breakpoints in maize. In an attempt to find precedent examples we determined there was negative interference among previously published translocation breakpoint mapping data in maize. It appears that negative interference was greater when the combined map length of the adjacent regions was smaller. Even positive interference appears to have been reduced when the combined lengths of adjacent regions were below 40 cM. Both phenomena can be explained by a reduction in crossovers near the breakpoints or, more specifically, by a failure of regions near breakpoints to become competent for crossovers. A mathematical explanation is provided.
Arabidopsis Genes Essential for Seedling Viability: Isolation of Insertional Mutants and Molecular Cloning
Authors: Budziszewski Gregory J , Lewis Sharon Potter , Glover Lyn Wegrich , Reineke Jennifer , Jones Gary , Ziemnik Lisa Schlater , Lonowski Jennifer , Nyfeler Beat , Aux George , Zhou Qing , McElver John , Patton David A , Martienssen Robert , Grossniklaus Ueli , Ma Hong , Law Marcus , Levin Joshua Z
Published: April 2021

We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening ~38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.
Functional Interactions Between SPO11 and REC102 During Initiation of Meiotic Recombination in Saccharomyces cerevisiae
Authors: Kee Kehkooi , Keeney Scott
Published: April 2021

In Saccharomyces cerevisiae, formation of the DNA double-strand breaks (DSBs) that initiate meiotic recombination requires the products of at least 10 genes. Spo11p is thought to be the catalytic subunit of the DNA cleaving activity, but the roles of the other proteins, and the interactions among them, are not well understood. This study demonstrates genetic and physical interactions between the products of SPO11 and another early meiotic gene required for DSB formation, REC102. We found that epitope-tagged versions of SPO11 and REC102 that by themselves were capable of supporting normal or nearly normal levels of meiotic recombination conferred a severe synthetic cold-sensitive phenotype when combined in the same cells. DSB formation, meiotic gene conversion, and spore viability were drastically reduced in the doubly tagged strain at a nonpermissive temperature. This conditional defect could be partially rescued by expression of untagged SPO11, but not by expression of untagged REC102, indicating that tagged REC102 is fully dominant for this synthetic phenotype. Both tagged and wild-type Spo11p co-immunoprecipitated with tagged Rec102p from meiotic cell extracts, indicating that these proteins are present in a common complex in vivo. Tagged Rec102p localized to the nucleus in whole cells and to chromatin on spread meiotic chromosomes. Our results are consistent with the idea that a multiprotein complex that includes Spo11p and Rec102p promotes meiotic DSB formation.
Precocious S-Phase Entry in Budding Yeast Prolongs Replicative State and Increases Dependence Upon Rad53 for Viability
Authors: Sidorova Julia M , Breeden Linda L
Published: April 2021

Precocious entry into S phase due to overproduction of G1 regulators can cause genomic instability. The mechanisms of this phenomenon are largely unknown. We explored the consequences of precocious S phase in yeast by overproducing a deregulated form of Swi4 (Swi4-t). Swi4 is a late G1-specific transcriptional activator that, in complex with Swi6, binds to SCB elements and activates late G1-specific genes, including G1 cyclins. We find that wild-type cells tolerate Swi4-t, whereas checkpoint-deficient rad53-11 cells lose viability within several divisions when Swi4-t is overproduced. Rad53 kinase activity is increased in cells overproducing Swi4-t, indicating activation of the checkpoint. We monitored the transition from G1 to S in cells with Swi4-t and found that there is precocious S-phase entry and that the length of S phase is extended. Moreover, there were more replication intermediates, and firing of at least a subset of origins may have been more extensive in the cells expressing Swi4-t. Our working hypothesis is that Rad53 modulates origin firing based upon growth conditions to optimize the rate of S-phase progression without adversely affecting fidelity. This regulation becomes essential when S phase is influenced by Swi4-t.
The ham-2 Locus, Encoding a Putative Transmembrane Protein, Is Required for Hyphal Fusion in Neurospora crassa
Authors: Xiang Qijun , Rasmussen Carolyn , Glass N Louise
Published: April 2021

Somatic cell fusion is common during organogenesis in multicellular eukaryotes, although the molecular mechanism of cell fusion is poorly understood. In filamentous fungi, somatic cell fusion occurs during vegetative growth. Filamentous fungi grow as multinucleate hyphal tubes that undergo frequent hyphal fusion (anastomosis) during colony expansion, resulting in the formation of a hyphal network. The molecular mechanism of the hyphal fusion process and the role of networked hyphae in the growth and development of these organisms are unexplored questions. We use the filamentous fungus Neurospora crassa as a model to study the molecular mechanism of hyphal fusion. In this study, we identified a deletion mutant that was restricted in its ability to undergo both self-hyphal fusion and fusion with a different individual to form a heterokaryon. This deletion mutant displayed pleiotropic defects, including shortened aerial hyphae, altered conidiation pattern, female sterility, slow growth rate, lack of hyphal fusion, and suppression of vegetative incompatibility. Complementation with a single open reading frame (ORF) within the deletion region in this mutant restored near wild-type growth rates, female fertility, aerial hyphae formation, and hyphal fusion, but not vegetative incompatibility and wild-type conidiation pattern. This ORF, which we named ham-2 (for hyphal anastomosis), encodes a putative transmembrane protein that is highly conserved, but of unknown function among eukaryotes.
Genetic Structure of the Mating-Type Locus of Chlamydomonas reinhardtii
Authors: Ferris Patrick J , Armbrust E Virginia , Goodenough Ursula W
Published: April 2021

Portions of the cloned mating-type (MT) loci (mt+ and mt−) of Chlamydomonas reinhardtii, defined as the ~1-Mb domains of linkage group VI that are under recombinational suppression, were subjected to Northern analysis to elucidate their coding capacity. The four central rearranged segments of the loci were found to contain both housekeeping genes (expressed during several life-cycle stages) and mating-related genes, while the sequences unique to mt+ or mt− carried genes expressed only in the gametic or zygotic phases of the life cycle. One of these genes, Mtd1, is a candidate participant in gametic cell fusion; two others, Mta1 and Ezy2, are candidate participants in the uniparental inheritance of chloroplast DNA. The identified housekeeping genes include Pdk, encoding pyruvate dehydrogenase kinase, and GdcH, encoding glycine decarboxylase complex subunit H. Unusual genetic configurations include three genes whose sequences overlap, one gene that has inserted into the coding region of another, several genes that have been inactivated by rearrangements in the region, and genes that have undergone tandem duplication. This report extends our original conclusion that the MT locus has incurred high levels of mutational change.
Unexpectedly High Allelic Diversity at the KIT Locus Causing Dominant White Color in the Domestic Pig
Authors: Pielberg G , Olsson C , Syvänen A-C , Andersson L
Published: April 2021

Mutations in KIT encoding the mast/stem cell growth factor receptor (MGF) are responsible for coat color variation in domestic pigs. The dominant white phenotype is caused by two mutations, a gene duplication and a splice mutation in one of the copies leading to skipping of exon 17. Here we applied minisequencing and pyrosequencing for quantitative analysis of the number of copies with the splice form. An unexpectedly high genetic diversity was revealed in white pigs. We found four different KIT alleles in a small sample of eight Large White females used as founder animals in a wild boar intercross. A similar number of KIT alleles was found in commercial populations of white Landrace and Large White pigs. We provide evidence for at least two new KIT alleles in pigs, both with a triplication of the gene. The results imply that KIT alleles with the duplication are genetically unstable and new alleles are most likely generated by unequal crossing over. This study provides an improved method for genotyping the complicated Dominant white/KIT locus in pigs. The results also suggest that some alleles may be associated with negative pleiotropic effects on other traits.
Genetic Variation for Phenotypically Invariant Traits Detected in Teosinte: Implications for the Evolution of Novel Forms
Authors: Lauter Nick , Doebley John
Published: April 2021

How new discrete states of morphological traits evolve is poorly understood. One possibility is that single-gene changes underlie the evolution of new discrete character states and that evolution is dependent on the occurrence of new single-gene mutations. Another possibility is that multiple-gene changes are required to elevate an individual or population above a threshold required to produce the new character state. A prediction of the latter model is that genetic variation for the traits should exist in natural populations in the absence of phenotypic variation. To test this idea, we studied traits that are phenotypically invariant within teosinte and for which teosinte is discretely different from its near relative, maize. By employing a QTL mapping strategy to analyze the progeny of a testcross between an F1 of two teosintes and a maize inbred line, we identified cryptic genetic variation in teosinte for traits that are invariant in teosinte. We argue that such cryptic genetic variation can contribute to the evolution of novelty when reconfigured to exceed the threshold necessary for phenotypic expression or by acting to modify or stabilize the effects of major mutations.
Release From Post-transcriptional Gene Silencing by Cell Proliferation in Transgenic Tobacco Plants: Possible Mechanism for Noninheritance of the Silencing
Authors: Mitsuhara Ichiro , Shirasawa-Seo Naomi , Iwai Takayoshi , Nakamura Shigeo , Honkura Ryoso , Ohashi Yuko
Published: April 2021

Transgenic tobacco plants that overproduce luciferase (Luc) frequently exhibit post-transcriptional gene silencing (PTGS) of luc. The silencing was observed over five generations and found not to be inherited but acquired by the next generation at a certain frequency. Luc imaging analysis of silenced plants revealed Luc activity only in proliferating tissues such as shoot meristem and developing flower. The luc gene expression has been recovered from silencing before development of germ cells, excluding a possible recovery from the PTGS at meiosis. A systemic silencing signal transferred from older tissue likely induces gene silencing of younger tissues in which cell proliferation has been completed. Only seeds maintained Luc activity, probably because of isolation from the silencing signal by a possible partition from the parent placenta. Calli newly induced from the leaf pieces of silenced plants recovered from the silencing and exhibited strong Luc activity similar to nonsilenced leaves, further indicating that the silencing cannot be maintained in proliferating cells. Thus release from PTGS in proliferating cells is a possible mechanism for noninheritance of silencing.
Differential Suppression of DNA Repair Deficiencies of Yeast rad50, mre11 and xrs2 Mutants by EXO1 and TLC1 (the RNA Component of Telomerase)
Authors: Lewis L Kevin , Karthikeyan G , Westmoreland James W , Resnick Michael A
Published: April 2021

Rad50, Mre11, and Xrs2 form a nuclease complex that functions in both nonhomologous end-joining (NHEJ) and recombinational repair of DNA double-strand breaks (DSBs). A search for highly expressed cDNAs that suppress the DNA repair deficiency of rad50 mutants yielded multiple isolates of two genes: EXO1 and TLC1. Overexpression of EXO1 or TLC1 increased the resistance of rad50, mre11, and xrs2 mutants to ionizing radiation and MMS, but did not increase resistance in strains defective in recombination (rad51, rad52, rad54, rad59) or NHEJ only (yku70, sir4). Increased Exo1 or TLC1 RNA did not alter checkpoint responses or restore NHEJ proficiency, but DNA repair defects of yku70 and rad27 (fen) mutants were differentially suppressed by the two genes. Overexpression of Exo1, but not mutant proteins containing substitutions in the conserved nuclease domain, increased recombination and suppressed HO and EcoRI endonuclease-induced killing of rad50 strains. exo1 rad50 mutants lacking both nuclease activities exhibited a high proportion of enlarged, G2-arrested cells and displayed a synergistic decrease in DSB-induced plasmid:chromosome recombination. These results support a model in which the nuclease activity of the Rad50/Mre11/Xrs2 complex is required for recombinational repair, but not NHEJ. We suggest that the 5′–3′ exo activity of Exo1 is able to substitute for Rad50/Mre11/Xrs2 in rescission of specific classes of DSB end structures. Gene-specific suppression by TLC1, which encodes the RNA subunit of the yeast telomerase complex, demonstrates that components of telomerase can also impact on DSB repair pathways.
Dynamics of Telomeric DNA Turnover in Yeast
Authors: McEachern Michael J , Underwood Dana Hager , Blackburn Elizabeth H
Published: April 2021

Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template. We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template. Each initially produced normally regulated telomeres. One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats. This mutant differs from previously studied delayed elongation mutants in a number of properties. The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity. TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions. Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres. These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover.
Sls1p Is a Membrane-Bound Regulator of Transcription-Coupled Processes Involved in Saccharomyces cerevisiae Mitochondrial Gene Expression
Authors: Bryan Anthony C , Rodeheffer Matthew S , Wearn Christopher M , Shadel Gerald S
Published: April 2021

1Mitochondrial translation is largely membrane-associated in S. cerevisiae. Recently, we discovered that the matrix protein Nam1p binds the amino-terminal domain of yeast mtRNA polymerase to couple translation and/or RNA-processing events to transcription. To gain additional insight into these transcription-coupled processes, we performed a genetic screen for genes that suppress the petite phenotype of a point mutation in mtRNA polymerase (rpo41-R129D) when overexpressed. One suppressor identified in this screen was SLS1, which encodes a mitochondrial membrane protein required for assembly of respiratory-chain enzyme complexes III and IV. The mtRNA-processing defects associated with the rpo41-R129D mutation were corrected in the suppressed strain, linking Sls1p to a pathway that includes mtRNA polymerase and Nam1p. This was supported by the observation that SLS1 overexpression rescued the petite phenotype of a NAM1 null mutation. In contrast, overexpression of Nam1p did not rescue the petite phenotype of a SLS1 null mutation, indicating that Nam1p and Sls1p are not functionally redundant but rather exist in an ordered pathway. On the basis of these data, a model in which Nam1p coordinates the delivery of newly synthesized transcripts to the membrane, where Sls1p directs or regulates their subsequent handling by membrane-bound factors involved in translation, is proposed.
Structure/Function Analysis of the Saccharomyces cerevisiae Trf4/Pol σ DNA Polymerase
Authors: Wang Zhenghe , Castaño Irene B , Adams Carrie , Vu Clemence , Fitzhugh David , Christman Michael F
Published: April 2021

The Trf4p/Pol σ DNA polymerase (formerly Trf4p/Pol κ) couples DNA replication to the establishment of sister chromatid cohesion. The polymerase is encoded by two redundant homologs in Saccharomyces cerevisiae, TRF4 and TRF5, that together define a fourth essential nuclear DNA polymerase in yeast and probably in all eukaryotes. Here we present a thorough genetic analysis of the founding member of this novel family of DNA polymerases, TRF4. Analyses of mutants carrying 1 of 34 “surface-targeted” alanine scanning mutations in TRF4 have identified those regions required for Pol σ's essential function, for its role in DNA double-strand break repair, and for its association with chromosomes. The data strongly support the importance of the regions of predicted structural similarity with the Pol β superfamily as critical for Trf4p/Pol σ's essential and repair functions. Surprisingly, five lethal mutations lie outside all polymerase homology in a C-terminal region. The protein possesses Mg2+-dependent 3′ to 5′ exonuclease activity. Cell cycle analysis reveals that Trf4p/Pol σ associates with chromosomes in G1, S, and G2 phases, but that association is abolished coincident with dissolution of cohesion at the metaphase-to-anaphase transition.
Schizosaccharomyces pombe Bir1p, a Nuclear Protein That Localizes to Kinetochores and the Spindle Midzone, Is Essential for Chromosome Condensation and Spindle Elongation During Mitosis
Authors: Rajagopalan Srividya , Balasubramanian Mohan K
Published: April 2021

The inhibitor of apoptosis (IAP) family of proteins contains a subset of members characterized by the presence of highly conserved baculoviral IAP repeat (BIR) domains. Recent work has shown that some of these BIR-domain proteins play a prominent role in the regulation of cell division, in particular at the stage of chromosome segregation and cytokinesis. We and others have shown that the Schizosaccharomyces pombe BIR-domain protein, Bir1p/Pbh1p/Cut17p, is important for the regulation of mitosis. Here we further characterize S. pombe Bir1p using methods of cell biology and genetics. We show that Bir1p is dispersed throughout the nucleus during the cell cycle. In addition, a significant part of Bir1p is also detected at the kinetochores and the spindle midzone during mitosis and meiosis. Time-lapse microscopy studies suggest that Bir1p relocates from the kinetochores to the spindle at the end of anaphase A. Bir1p colocalizes with the S. pombe Aurora kinase homolog Aim1p, a protein essential for mitosis, at the kinetochores as well as the spindle midzone during mitosis, and functional Bir1p is essential for localization of Aim1p to the kinetochores and the spindle midzone. Analyses of bir1 conditional mutants revealed that Bir1p is essential for chromosome condensation during mitosis. In addition, anaphase cells show the presence of lagging chromosomes and a defect in spindle elongation. We conclude that Bir1p is important for multiple processes that occur during mitosis in S. pombe.
Dimorphic Mating-Type Chromosomes in the Fungus Microbotryum violaceum
Authors: Hood Michael E
Published: April 2021

Fungi often mate as haploids, and sex chromosomes (i.e., mating-type chromosomes) that are dimorphic for their size or overall DNA content have never been reported in this kingdom. Using electrophoretic techniques for karyotype analysis, a highly dimorphic chromosome pair that determines mating compatibility is shown to occur in populations of the fungus Microbotryum violaceum. This substantiates the evolution of such dimorphism as a general feature associated with haploid determination of mating compatibility, which previously had been known only in haplodioecious plants (mosses and liverworts). Size-dimorphic sex chromosomes are present in a lineage of M. violaceum native to Europe, as well as a lineage native to North America. However, they are very different in size between these lineages, indicating either independent evolution of the dimorphism or a large degree of divergence since their isolation. Several DNA sequences that show sequence similarity to transposons were isolated from these sex chromosomes.
Roles for Caenorhabditis elegans rad-51 in Meiosis and in Resistance to Ionizing Radiation During Development
Authors: Rinaldo Cinzia , Bazzicalupo Paolo , Ederle Sara , Hilliard Massimo , La Volpe Adriana
Published: April 2021

We have investigated the role of Caenorhabditis elegans RAD-51 during meiotic prophase and embryogenesis, making use of the silencing effect of RNA interference (RNAi). rad-51 RNAi leads to severe defects in chromosome morphology in diakinesis oocytes. We have explored the effect of rad-51 RNAi in mutants lacking fundamental components of the recombination machinery. If double-strand breaks are prevented by spo-11 mutation, rad-51 RNAi does not affect chromosome appearance. This is consistent with a role for RAD-51 downstream of the initiation of recombination. In the absence of MRE-11, as in the absence of SPO-11, RAD-51 depletion has no effect on the chromosomes, which appear intact, thus indicating a role for MRE-11 in DSB induction. Intriguingly, rad-51 silencing in oocytes that lack MSH-5 leads to chromosome fragmentation, a novel trait that is distinct from that seen in msh-5 mutants and in rad-51 RNAi oocytes, suggesting new potential roles for the msh-5 gene. Silencing of the rad-51 gene also causes a reduction in fecundity, which is suppressed by mutation in the DNA damage checkpoint gene rad-5, but not in the cell death effector gene ced-3. Finally, RAD-51 depletion is also seen to affect the soma, resulting in hypersensitivity to ionizing radiation in late embryogenesis.
Unexpected Stability of mariner Transgenes in Drosophila
Authors: Lozovsky Elena R , Nurminsky Dmitry , Wimmer Ernst A , Hartl Daniel L
Published: April 2021

A number of mariner transformation vectors based on the mauritiana subfamily of transposable elements were introduced into the genome of Drosophila melanogaster and examined for their ability to be mobilized by the mariner transposase. Simple insertion vectors were constructed from single mariner elements into which exogenous DNA ranging in size from 1.3 to 4.5 kb had been inserted; composite vectors were constructed with partial or complete duplications of mariner flanking the exogenous DNA. All of the simple insertion vectors showed levels of somatic and germline excision that were at least 100-fold lower than the baseline level of uninterrupted mariner elements. Although composite vectors with inverted duplications were unable to be mobilized at detectable frequencies, vectors with large direct duplications of mariner could be mobilized. A vector consisting of two virtually complete elements flanking exogenous DNA yielded a frequency of somatic eye-color mosaicism of ~10% and a frequency of germline excision of 0.04%. These values are far smaller than those observed for uninterrupted elements. The results imply that efficient mobilization of mariner in vivo requires the presence and proper spacing of sequences internal to the element as well as the inverted repeats.
The Drosophila Gene taranis Encodes a Novel Trithorax Group Member Potentially Linked to the Cell Cycle Regulatory Apparatus
Authors: Calgaro Stéphane , Boube Muriel , Cribbs David L , Bourbon Henri-Marc
Published: April 2021

Genes of the Drosophila Polycomb and trithorax groups (PcG and trxG, respectively) influence gene expression by modulating chromatin structure. Segmental expression of homeotic loci (HOM) initiated in early embryogenesis is maintained by a balance of antagonistic PcG (repressor) and trxG (activator) activities. Here we identify a novel trxG family member, taranis (tara), on the basis of the following criteria: (i) tara loss-of-function mutations act as genetic antagonists of the PcG genes Polycomb and polyhomeotic and (ii) they enhance the phenotypic effects of mutations in the trxG genes trithorax (trx), brahma (brm), and osa. In addition, reduced tara activity can mimic homeotic loss-of-function phenotypes, as is often the case for trxG genes. tara encodes two closely related 96-kD protein isoforms (TARA-α/-β) derived from broadly expressed alternative promoters. Genetic and phenotypic rescue experiments indicate that the TARA-α/-β proteins are functionally redundant. The TARA proteins share evolutionarily conserved motifs with several recently characterized mammalian nuclear proteins, including the cyclin-dependent kinase regulator TRIP-Br1/p34SEI-1, the related protein TRIP-Br2/Y127, and RBT1, a partner of replication protein A. These data raise the possibility that TARA-α/-β play a role in integrating chromatin structure with cell cycle regulation.
Rough eye Is a Gain-of-Function Allele of amos That Disrupts Regulation of the Proneural Gene atonal During Drosophila Retinal Differentiation
Authors: Chanut Françoise , Woo Katherine , Pereira Shalini , Donohoe Terrence J , Chang Shang-Yu , Laverty Todd R , Jarman Andrew P , Heberlein Ulrike
Published: April 2021

The regular organization of the ommatidial lattice in the Drosophila eye originates in the precise regulation of the proneural gene atonal (ato), which is responsible for the specification of the ommatidial founder cells R8. Here we show that Rough eye (Roi), a dominant mutation manifested by severe roughening of the adult eye surface, causes defects in ommatidial assembly and ommatidial spacing. The ommatidial spacing defect can be ascribed to the irregular distribution of R8 cells caused by a disruption of the patterning of ato expression. Disruptions in the recruitment of other photoreceptors and excess Hedgehog production in differentiating cells may further contribute to the defects in ommatidial assembly. Our molecular characterization of the Roi locus demonstrates that it is a gain-of-function mutation of the bHLH gene amos that results from a chromosomal inversion. We show that Roi can rescue the retinal developmental defect of ato1 mutants and speculate that amos substitutes for some of ato's function in the eye or activates a residual function of the ato1 allele.
Assembly of Two Transgenes in an Artificial Chromatin Domain Gives Highly Coordinated Expression in Tobacco
Authors: Mlynárová Ludmila , Loonen Annelies , Mietkiewska Elzbieta , Jansen Ritsert C , Nap Jan-Peter
Published: April 2021

The chromatin loop model predicts that genes within the same chromatin domain exhibit coordinated regulation. We here present the first direct experimental support for this model in plants. Two reporter genes, the E. coli β-glucuronidase gene and the firefly luciferase gene, driven by different promoters, were placed between copies of the chicken lysozyme A element, a member of the matrix-associated region (MAR) group of chromatin boundary elements, and introduced in tobacco (Nicotiana tabacum). In plants carrying A elements, quantitative enzyme activities and mRNA levels of both genes show high correlations compared to control plants. The A element thus creates an artificial chromatin domain that yields coordinated expression. Surprisingly, enzyme activities correlated poorly with their respective mRNA levels. We hypothesize that this indicates the occurrence of “error pipelines” in data generation: systematic errors of a given analytical method will point in the same direction and cancel out in correlation analysis, resulting in better correlations. In combining different methods of analysis, however, such errors do not cancel out and as a result relevant correlations can be masked. Such error pipelines will have to be taken into account when different types of (e.g., whole-genome) data sets are combined in quantitative analyses.
Likelihood-Based Estimation of the Effective Population Size Using Temporal Changes in Allele Frequencies: A Genealogical Approach
Authors: Berthier Pierre , Beaumont Mark A , Cornuet Jean-Marie , Luikart Gordon
Published: April 2021

A new genetic estimator of the effective population size (Ne) is introduced. This likelihood-based (LB) estimator uses two temporally spaced genetic samples of individuals from a population. We compared its performance to that of the classical F-statistic-based Ne estimator () by using data from simulated populations with known Ne and real populations. The new likelihood-based estimator () showed narrower credible intervals and greater accuracy than () when genetic drift was strong, but performed only slightly better when genetic drift was relatively weak. When drift was strong (e.g., Ne = 20 for five generations), as few as ~10 loci (heterozygosity of 0.6; samples of 30 individuals) are sufficient to consistently achieve credible intervals with an upper limit <50 using the LB method. In contrast, ~20 loci are required for the same precision when using the classical F-statistic approach. The estimator is much improved over the classical method when there are many rare alleles. It will be especially useful in conservation biology because it less often overestimates Ne than does and thus is less likely to erroneously suggest that a population is large and has a low extinction risk.