bluuebunny/crossref_metadata_2025 huggingface dataset:
Removal of One Nonhomologous DNA End During Gene Conversion by a RAD1- and MSH2-Independent Pathway
Authors: Colaiácovo Mónica P , Pâques Frédéric , Haber James E
Published: April 2021

Repair of a double-strand break (DSB) by homologous recombination depends on the invasion of a 3′-ended strand into an intact template sequence to initiate new DNA synthesis. When the end of the invading DNA is not homologous to the donor, the nonhomologous sequences must be removed before new synthesis can begin. In Saccharomyces cerevisiae, the removal of these ends depends on both the nucleotide excision repair endonuclease Rad1p/Rad10p and the mismatch repair proteins Msh2p/Msh3p. In rad1 or msh2 mutants, when both ends of the DSB have nonhomologous ends, repair is reduced ∼90-fold compared to a plasmid with perfect ends; however, with only one nonhomologous end, repair is reduced on average only 5-fold. These results suggest that yeast has an alternative, but less efficient, way to remove a nonhomologous tail from the second end participating in gene conversion. When the removal of one nonhomologous end is impaired in rad1 and msh2 mutants, there is also a 1-hr delay in the appearance of crossover products of gene conversion, compared to noncrossovers. We interpret these results in terms of the formation and resolution of alternative intermediates of a synthesis-dependent strand annealing mechanism.
Distribution of Crossing Over on Mouse Synaptonemal Complexes Using Immunofluorescent Localization of MLH1 Protein
Authors: Anderson Lorinda K , Reeves Aaron , Webb Lisa M , Ashley Terry
Published: April 2021

We have used immunofluorescent localization to examine the distribution of MLH1 (MutL homolog) foci on synaptonemal complexes (SCs) from juvenile male mice. MLH1 is a mismatch repair protein necessary for meiotic recombination in mice, and MLH1 foci have been proposed to mark crossover sites. We present evidence that the number and distribution of MLH1 foci on SCs closely correspond to the number and distribution of chiasmata on diplotene-metaphase I chromosomes. MLH1 foci were typically excluded from SC in centromeric heterochromatin. For SCs with one MLH1 focus, most foci were located near the middle of long SCs, but near the distal end of short SCs. For SCs with two MLH1 foci, the distribution of foci was bimodal regardless of SC length, with most foci located near the proximal and distal ends. The distribution of MLH1 foci indicated interference between foci. We observed a consistent relative distance (percent of SC length in euchromatin) between two foci on SCs of different lengths, suggesting that positive interference between MLH1 foci is a function of relative SC length. The extended length of pachytene SCs, as compared to more condensed diplotene-metaphase I bivalents, makes mapping crossover events and interference distances using MLH1 foci more accurate than using chiasmata.
A New Ac-Like Transposon of Arabidopsis Is Associated With a Deletion of the RPS5 Disease Resistance Gene
Authors: Henk Adam D , Warren Randall F , Innes Roger W
Published: April 2021

The RPS5 and RFL1 disease resistance genes of Arabidopsis ecotype Col-0 are oriented in tandem and are separated by 1.4 kb. The Ler-0 ecotype contains RFL1, but lacks RPS5. Sequence analysis of the RPS5 deletion region in Ler-0 revealed the presence of an Ac-like transposable element, which we have designated Tag2. Southern hybridization analysis of six Arabidopsis ecotypes revealed 4–11 Tag2-homologous sequences in each, indicating that this element is ubiquitous in Arabidopsis and has been active in recent evolutionary time. The Tag2 insertion adjacent to RFL1 was unique to the Ler-0 ecotype, however, and was not present in two other ecotypes that lack RPS5. DNA sequence from the latter ecotypes lacked a transposon footprint, suggesting that insertion of Tag2 occurred after the initial deletion of RPS5. The deletion breakpoint contained a 192-bp insertion that displayed hallmarks of a nonhomologous DNA endjoining event. We conclude that loss of RPS5 was caused by a double-strand break and subsequent repair, and cannot be attributed to unequal crossing over between resistance gene homologs.
Allele-Specific Interactions Between ttg and gl1 During Trichome Development in Arabidopsis thaliana
Authors: Larkin John C , Walker Jason D , Bolognesi-Winfield Agnese C , Gray John C , Walker Amanda R
Published: April 2021

Trichome development in Arabidopsis thaliana is a well-characterized model for the study of plant cell differentiation. Two genes that play an essential role in the initiation of trichome development are GL1 and TTG. Mutations in either gene prevent the initiation of most trichomes. The GL1 gene encodes a myb-related transcription factor. Mutations in TTG are pleiotropic, affecting anthocyanins, root hairs, and seed coat mucilage in addition to trichomes. Six ttg alleles were examined and shown to form a hypomorphic series. The severity of all aspects of the ttg phenotype varied in parallel in this allelic series. The weakest allele, ttg-10, causes frequent clusters of adjacent trichomes, suggesting a role for TTG in inhibiting neighboring cells from choosing the trichome fate. This allele results from a mutation in the 5′-untranslated region of ttg and creates an out-of-frame upstream AUG codon. The ttg-10 allele shows several unusual genetic interactions with the weak hypomorphic gl1-2 allele, including intergenic noncomplementation and a synthetic glabrous phenotype. These interactions are specific for the gl1-2 allele. The implication of these results for current models of trichome development is discussed.
RAD50 and RAD51 Define Two Pathways That Collaborate to Maintain Telomeres in the Absence of Telomerase
Authors: Le Siyuan , Moore J Kent , Haber James E , Greider Carol W
Published: April 2021

Telomere length is maintained by the de novo addition of telomere repeats by telomerase, yet recombination can elongate telomeres in the absence of telomerase. When the yeast telomerase RNA component, TLC1, is deleted, telomeres shorten and most cells die. However, gene conversion mediated by the RAD52 pathway allows telomere lengthening in rare survivor cells. To further investigate the role of recombination in telomere maintenance, we assayed telomere length and the ability to generate survivors in several isogenic DNA recombination mutants, including rad50, rad51, rad52, rad54, rad57, xrs2, and mre11. The rad51, rad52, rad54, and rad57 mutations increased the rate of cell death in the absence of TLC1. In contrast, although the rad50, xrs2, and mre11 strains initially had short telomeres, double mutants with tlc1 did not affect the rate of cell death, and survivors were generated at later times than tlc1 alone. While none of the double mutants of recombination genes and tlc1 (except rad52 tlc1) blocked the ability to generate survivors, a rad50 rad51 tlc1 triple mutant did not allow the generation of survivors. Thus RAD50 and RAD51 define two separate pathways that collaborate to allow cells to survive in the absence of telomerase.
Interorganelle Signaling Is a Determinant of Longevity in Saccharomyces cerevisiae
Authors: Kirchman Paul A , Kim Sangkyu , Lai Chi-Yung , Jazwinski S Michal
Published: April 2021

Replicative capacity, which is the number of times an individual cell divides, is the measure of longevity in the yeast Saccharomyces cerevisiae. In this study, a process that involves signaling from the mitochondrion to the nucleus, called retrograde regulation, is shown to determine yeast longevity, and its induction resulted in postponed senescence. Activation of retrograde regulation, by genetic and environmental means, correlated with increased replicative capacity in four different S. cerevisiae strains. Deletion of a gene required for the retrograde response, RTG2, eliminated the increased replicative capacity. RAS2, a gene previously shown to influence longevity in yeast, interacts with retrograde regulation in setting yeast longevity. The molecular mechanism of aging elucidated here parallels the results of genetic studies of aging in nematodes and fruit flies, as well as the caloric restriction paradigm in mammals, and it underscores the importance of metabolic regulation in aging, suggesting a general applicability.
stumps, a Drosophila Gene Required for Fibroblast Growth Factor (FGF)-directed Migrations of Tracheal and Mesodermal Cells
Authors: Imam Farhad , Sutherland David , Huang Wilmer , Krasnow Mark A
Published: April 2021

Fibroblast growth factors (FGFs) bind to FGF receptors, transmembrane tyrosine kinases that activate mitogenic, motogenic, and differentiative responses in different tissues. While there has been substantial progress in elucidating the Ras-MAP kinase pathway that mediates the differentiative responses, the signal transduction pathways that lead to directed cell migrations are not well defined. Here we describe a Drosophila gene called stumps that is required for FGF-dependent migrations of tracheal and mesodermal cells. These migrations are controlled by different FGF ligands and receptors, and they occur by different cellular mechanisms: the tracheal migrations occur as part of an epithelium whereas the mesodermal migrations are fibroblast-like. In the stumps mutant, tracheal cells fail to move out from the epithelial sacs, and only rudimentary tracheal branches form. Mesodermal cells fail in their dorsal migrations after gastrulation. The stumps mutation does not block all FGF signaling effects in these tissues: both random cell migrations and Ras-MAP kinase-mediated induction of FGF-specific effector genes occurred upon ectopic expression of the ligand or upon expression of a constitutively activated Ras protein in the migrating cells. The results suggest that stumps function promotes FGF-directed cell migrations, either by potentiating the FGF signaling process or by coupling the signal to the cellular machinery required for directed cell movement.
N-Ethyl-N-Nitrosourea Mutagenesis of a 6- to 11-cM Subregion of the Fah–Hbb Interval of Mouse Chromosome 7: Completed Testing of 4557 Gametes and Deletion Mapping and Complementation Analysis of 31 Mutations
Authors: Rinchik Eugene M , Carpenter Donald A
Published: April 2021

An interval of mouse chromosome (Chr) 7 surrounding the albino (Tyr; c) locus, and corresponding to a long 6- to 11-cM Tyr deletion, has been the target of a large-scale mutagenesis screen with the chemical supermutagen N-ethyl-N-nitrosourea (ENU). A segment of Chr 7, from a mutagenized genome bred from ENU-treated males, was made hemizygous opposite the long deletion for recognition and recovery of new recessive mutations that map within the albino deletion complex. Over 6000 pedigrees were analyzed, and 4557 of these were completely tested for mutations specifying both lethal and gross visible phenotypes. Thirty-one nonclustered mutations were identified and assigned to 10 complementation groups by pairwise trans-complementation crosses. Deletion-mapping analyses, using the extensive series of radiation-induced Tyr deletions, placed the loci defined by each of these complementation groups into defined intervals of the Tyr-region deletion map, which facilitates the identification of each locus on physical and transcription maps of the region. These mutations identified seven new loci and provided new ENU-induced alleles at three previously defined loci. Interestingly, no mutations were recovered that recapitulated three phenotypes defined by analysis of homozygous or partially complementing albino deletions. On the basis of our experience with this screen, we discuss a number of issues (e.g., locus mutability, failure to saturate, number of gametes to screen, allelic series) of concern when application of chemical mutagenesis screens to megabase regions of the mouse genome is considered.
Trichome Cell Growth in Arabidopsis thaliana Can Be Derepressed by Mutations in at Least Five Genes
Authors: Perazza Daniel , Herzog Michel , Hülskamp Martin , Brown Spencer , Dorne Anne-Marie , Bonneville Jean-Marc
Published: April 2021

Leaf trichomes in Arabidopsis are unicellular epidermal hairs with a branched morphology. They undergo successive endoreduplication rounds early during cell morphogenesis. Mutations affecting trichome nuclear DNA content, such as triptychon or glabra3, alter trichome branching. We isolated new mutants with supernumerary trichome branches, which fall into three unlinked complementation groups: KAKTUS and the novel loci, POLYCHOME and RASTAFARI. They map to chromosomes IV, II, and V, respectively. The trichomes of these mutants presented an increased DNA content, although to a variable extent. The spindly-5 mutant, which displays a constitutive gibberellin response, also produces overbranched trichomes containing more nuclear DNA. We analyzed genetic interactions using double mutants and propose that two independent pathways, defined by SPINDLY and TRIPTYCHON, act to limit trichome growth. KAKTUS and POLYCHOME might have redundant actions mediating gibberellin control via SPINDLY. The overall leaf polysomaty was not notably affected by these mutations, suggesting that they affect the control of DNA synthesis in a tissue- or cell type-specific manner. Wild-type tetraploids also produce overbranched trichomes; they displayed a shifted polysomaty in trichomes and in the whole leaf, suggesting a developmental program controlling DNA increases via the counting of endoreduplication rounds.
MOD-D, a Gα Subunit of the Fungus Podospora anserina, Is Involved in Both Regulation of Development and Vegetative Incompatibility
Authors: Loubradou Gabriel , Bégueret Joël , Turcq Béatrice
Published: April 2021

Cell death via vegetative incompatibility is widespread in fungi but molecular mechanism and biological function of the process are poorly understood. One way to investigate this phenomenon was to study genes named mod that modified incompatibility reaction. In this study, we cloned the mod-D gene that encodes a Gα protein. The mod-D mutant strains present developmental defects. Previously, we showed that the mod-E gene encodes an HSP90. The mod-E1 mutation suppresses both vegetative incompatibility and developmental defects due to the mod-D mutation. Moreover, we isolated the PaAC gene, which encodes an adenylate cyclase, as a partial suppressor of the mod-D1 mutation. Our previous results showed that the molecular mechanisms involved in vegetative incompatibility and developmental pathways are connected, suggesting that vegetative incompatibility may result from disorders in some developmental steps. Our new result corroborates the involvement of mod genes in signal transduction pathways. As expected, we showed that an increase in the cAMP level is able to suppress the defects in vegetative growth due to the mod-D1 mutation. However, cAMP increase has no influence on the suppressor effect of the mod-D1 mutation on vegetative incompatibility, suggesting that this suppressor effect is independent of the cAMP pathway.
The Drosophila melanogaster Suppressor of deltex Gene, a Regulator of the Notch Receptor Signaling Pathway, Is an E3 Class Ubiquitin Ligase
Authors: Cornell M , Evans D A P , Mann R , Fostier M , Flasza M , Monthatong M , Artavanis-Tsakonas S , Baron M
Published: April 2021

During development, the Notch receptor regulates many cell fate decisions by a signaling pathway that has been conserved during evolution. One positive regulator of Notch is Deltex, a cytoplasmic, zinc finger domain protein, which binds to the intracellular domain of Notch. Phenotypes resulting from mutations in deltex resemble loss-of-function Notch phenotypes and are suppressed by the mutation Suppressor of deltex [Su(dx)]. Homozygous Su(dx) mutations result in wing-vein phenotypes and interact genetically with Notch pathway genes. We have previously defined Su(dx) genetically as a negative regulator of Notch signaling. Here we present the molecular identification of the Su(dx) gene product. Su(dx) belongs to a family of E3 ubiquitin ligase proteins containing membrane-targeting C2 domains and WW domains that mediate protein-protein interactions through recognition of proline-rich peptide sequences. We have identified a seven-codon deletion in a Su(dx) mutant allele and we show that expression of Su(dx) cDNA rescues Su(dx) mutant phenotypes. Overexpression of Su(dx) also results in ectopic vein differentiation, wing margin loss, and wing growth phenotypes and enhances the phenotypes of loss-of-function mutations in Notch, evidence that supports the conclusion that Su(dx) has a role in the downregulation of Notch signaling.
Genetic Analysis of the Bone Morphogenetic Protein-Related Gene, gbb, Identifies Multiple Requirements During Drosophila Development
Authors: Wharton Kristi A , Cook James M , Torres-Schumann Sonia , de Castro Katherine , Borod Emily , Phillips Deborah A
Published: April 2021

We have isolated mutations in the Drosophila melanogaster gene glass bottom boat (gbb), which encodes a TGF-β signaling molecule (formerly referred to as 60A) with highest sequence similarity to members of the bone morphogenetic protein (BMP) subgroup including vertebrate BMPs 5-8. Genetic analysis of both null and hypomorphic gbb alleles indicates that the gene is required in many developmental processes, including embryonic midgut morphogenesis, patterning of the larval cuticle, fat body morphology, and development and patterning of the imaginal discs. In the embryonic midgut, we show that gbb is required for the formation of the anterior constriction and for maintenance of the homeotic gene Antennapedia in the visceral mesoderm. In addition, we show a requirement for gbb in the anterior and posterior cells of the underlying endoderm and in the formation and extension of the gastric caecae. gbb is required in all the imaginal discs for proper disc growth and for specification of veins in the wing and of macrochaete in the notum. Significantly, some of these tissues have been shown to also require the Drosophila BMP2/4 homolog decapentaplegic (dpp), while others do not. These results indicate that signaling by both gbb and dpp may contribute to the development of some tissues, while in others, gbb may signal independently of dpp.
Mytilus Mitochondrial DNA Contains a Functional Gene for a tRNASer(UCN) With a Dihydrouridine Arm-Replacement Loop and a pseudo-tRNASer(UCN) Gene
Authors: Beagley C Timothy , Okimoto Ronald , Wolstenholme David R
Published: April 2021

A 2500-nucleotide pair (ntp) sequence of F-type mitochondrial (mt) DNA of the Pacific Rim mussel Mytilus californianus (class Bivalvia, phylum Mollusca) that contains two complete (ND2 and ND3) and two partial (COI and COIII) protein genes and nine tRNA genes is presented. Seven of the encoded tRNAs (Ala, Arg, His, Met(AUA), Pro, Ser(UCN), and Trp) have the potential to fold into the orthodox four-armed tRNA secondary structure, while two [tRNASer(AGN) and a second tRNASer(UCN)] will fold only into tRNAs with a dihydrouridine (DHU) arm-replacement loop. Comparison of these mt-tRNA gene sequences with previously published, corresponding M. edulis F-type mtDNA indicates that similarity between the four-armed tRNASer(UCN) genes is only 63.8% compared with an average of 92.1% (range 86.2-98.5%) for the remaining eight tRNA genes. Northern blot analysis indicated that mature tRNAs encoded by the DHU arm-replacement loop-containing tRNASer(UCN), tRNASer(AGN), tRNAMet(AUA), tRNATrp, and tRNAPro genes occur in M. californianus mitochondria, strengthening the view that all of these genes are functional. However, Northern blot and 5′ RACE (rapid amplification of cDNA ends) analyses indicated that the four-armed tRNASer(UCN) gene is transcribed into a stable RNA that includes the downstream COI sequence and is not processed into a mature tRNA. On the basis of these observations the M. californianus and M. edulis four-armed tRNASer(UCN) sequences are interpreted as pseudo-tRNASer(UCN) genes.
Among-Locus Variation in Fst: Fish, Allozymes and the Lewontin-Krakauer Test Revisited
Authors: Baer Charles F
Published: April 2021

Variation among loci in the distribution of allele frequencies among subpopulations is well known; how to tell when the variation exceeds that expected when all loci are subject to uniform evolutionary processes is not well known. If locus-specific effects are important, the ability to detect those effects should vary with the level of gene flow. Populations with low gene flow should exhibit greater variation among loci in Fst than populations with high gene flow, because gene flow acts to homogenize allele frequencies among subpopulations. Here I use Lewontin and Krakauer’s k statistic to describe the variance among allozyme loci in 102 published data sets from fishes. As originally proposed, k ⪢ 2 was considered evidence that the variation in Fst among loci is greater than expected from neutral evolution. Although that interpretation is invalid, large differences in k in different populations suggest that locus-specific forces may be important in shaping genetic diversity. In these data, k is not greater for populations with expected low levels of gene flow than for populations with expected high levels of gene flow. There is thus no evidence that locus-specific forces are of general importance in shaping the distribution of allele frequencies at enzyme loci among populations of fishes.
The Causes of Synonymous Rate Variation in the Rodent Genome: Can Substitution Rates Be Used to Estimate the Sex Bias in Mutation Rate?
Authors: Smith Nick G C , Hurst Laurence D
Published: April 2021

Miyata et al. have suggested that the male-to-female mutation rate ratio (α) can be estimated by comparing the neutral substitution rates of X-linked (X), Y-linked (Y), and autosomal (A) genes. Rodent silent site X/A comparisons provide very different estimates from X/Y comparisons. We examine three explanations for this discrepancy: (1) statistical biases and artifacts, (2) nonneutral evolution, and (3) differences in mutation rate per germline replication. By estimating errors and using a variety of methodologies, we tentatively reject explanation 1. Our analyses of patterns of codon usage, synonymous rates, and nonsynonymous rates suggest that silent sites in rodents are evolving neutrally, and we can therefore reject explanation 2. We find both base composition and methylation differences between the different sets of chromosomes, a result consistent with explanation 3, but these differences do not appear to explain the observed discrepancies in estimates of α. Our finding of significantly low synonymous substitution rates in genomically imprinted genes suggests a link between hemizygous expression and an adaptive reduction in the mutation rate, which is consistent with explanation 3. Therefore our results provide circumstantial evidence in favor of the hypothesis that the discrepancies in estimates of α are due to differences in the mutation rate per germline replication between different parts of the genome. This explanation violates a critical assumption of the method of Miyata et al., and hence we suggest that estimates of α, obtained using this method, need to be treated with caution.
Intrachromosomal Recombination Between Well-Separated, Homologous Sequences in Mammalian Cells
Authors: Baker Mark D , Read Leah R , Ng Philip , Beatty Barbara G
Published: April 2021

In the present study, we investigated intrachromosomal homologous recombination in a murine hybridoma in which the recipient for recombination, the haploid, endogenous chromosomal immunoglobulin μ-gene bearing a mutation in the constant (Cμ) region, was separated from the integrated single copy wild-type donor Cμ region by ∼1 Mb along the hybridoma chromosome. Homologous recombination between the donor and recipient Cμ region occurred with high frequency, correcting the mutant chromosomal μ-gene in the hybridoma. This enabled recombinant hybridomas to synthesize normal IgM and to be detected as plaque-forming cells (PFC). Characterization of the recombinants revealed that they could be placed into three distinct classes. The generation of the class I recombinants was consistent with a simple unequal sister chromatid exchange (USCE) between the donor and recipient Cμ region, as they contained the three Cμ-bearing fragments expected from this recombination, the original donor Cμ region along with both products of the single reciprocal crossover. However, a simple mechanism of homologous recombination was not sufficient in explaining the more complex Cμ region structures characterizing the class II and class III recombinants. To explain these recombinants, a model is proposed in which unequal pairing between the donor and recipient Cμ regions located on sister chromatids resulted in two crossover events. One crossover resulted in the deletion of sequences from one chromatid forming a DNA circle, which then integrated into the sister chromatid by a second reciprocal crossover.
Origin of Gene Overlap: The Case of TCP1 and ACAT2
Authors: Shintani Seikou , O’hUigin Colm , Toyosawa Satoru , Michalová Věra , Klein Jan
Published: April 2021

The human acetyl-CoA acetyltransferase 2 gene, ACAT2, codes for a thiolase, an enzyme involved in lipid metabolism. The human T-complex protein 1 gene, TCP1, encodes a molecular chaperone of the chaperonin family. The two genes overlap by their 3′-untranslated regions, their coding sequences being located on opposite DNA strands in a tail-to-tail orientation. To find out how the overlap might have arisen in evolution, the homologous genes of the zebrafish, the African toad, caiman, platypus, opossum, and wallaby were identified. In each species, standard or long polymerase chain reactions were used to determine whether the ACAT2 and TCP1 homologs are closely linked and, if so, whether they overlap. The results reveal that the overlap apparently arose during the transition from therapsid reptiles to mammals and has been retained for >200 million years. Part of the overlapping untranslated region shows remarkable sequence conservation. The overlap presumably arose during the chromosomal rearrangement that brought the two unrelated and previously separated genes together. One or both of the transposed genes found by chance signals that are necessary for the processing of their transcripts to be present on the noncoding strand of the partner gene.
Molecular Cloning and Tissue-Specific Expression of the mutator2 Gene (mu2) in Drosophila melanogaster
Authors: Kasravi Armin , Walter Marika F , Brand Stephanie , Mason James M , Biessmann Harald
Published: April 2021

We present here the molecular cloning and characterization of the mutator2 (mu2) gene of Drosophila melanogaster together with further genetic analyses of its mutant phenotype. mu2 functions in oogenesis during meiotic recombination, during repair of radiation damage in mature oocytes, and in proliferating somatic cells, where mu2 mutations cause an increase in somatic recombination. Our data show that mu2 represents a novel component in the processing of double strand breaks (DSBs) in female meiosis. mu2 does not code for a DNA repair enzyme because mu2 mutants are not hypersensitive to DSB-inducing agents. We have mapped and cloned the mu2 gene and rescued the mu2 phenotype by germ-line transformation with genomic DNA fragments containing the mu2 gene. Sequencing its cDNA demonstrates that mu2 encodes a novel 139-kD protein, which is highly basic in the carboxy half and carries three nuclear localization signals and a helix-loop-helix domain. Consistent with the sex-specific mutant phenotype, the gene is expressed in ovaries but not in testes. During oogenesis its RNA is rapidly transported from the nurse cells into the oocyte where it accumulates specifically at the anterior margin. Expression is also prominent in diploid proliferating cells of larval somatic tissues. Our genetic and molecular data are consistent with the model that mu2 encodes a structural component of the oocyte nucleus. The MU2 protein may be involved in controlling chromatin structure and thus may influence the processing of DNA DSBs.
Clustered Microsatellite Mutations in the Pipefish Syngnathus typhle
Authors: Jones Adam G , Rosenqvist Gunilla , Berglund Anders , Avise John C
Published: April 2021

Clustered mutations are copies of a mutant allele that enter a population's gene pool together due to replication from a premeiotic germline mutation and distribution to multiple successful gametes of an individual. Although the phenomenon has been studied in Drosophila and noted in a few other species, the topic has received scant attention despite claims of being of major importance to population genetics theory. Here we capitalize upon the reproductive biology of male-pregnant pipefishes to document the occurrence of clustered microsatellite mutations and to estimate their rates and patterns from family data. Among a total of 3195 embryos genetically screened from 110 families, 40% of the 35 detected de novo mutant alleles resided in documented mutational clusters. Most of the microsatellite mutations appeared to involve small-integer changes in repeat copy number, and they arose in approximately equal frequency in paternal and maternal germlines. These findings extend observations on clustered mutations to another organismal group and motivate a broader critique of the mutation cluster phenomenon. They also carry implications for the evolution of microsatellites with respect to mutational models and homoplasy among alleles.
Estimation of Past Demographic Parameters From the Distribution of Pairwise Differences When the Mutation Rates Vary Among Sites: Application to Human Mitochondrial DNA
Authors: Schneider Stefan , Excoffier Laurent
Published: April 2021

Distributions of pairwise differences often called “mismatch distributions” have been extensively used to estimate the demographic parameters of past population expansions. However, these estimations relied on the assumption that all mutations occurring in the ancestry of a pair of genes lead to observable differences (the infinite-sites model). This mutation model may not be very realistic, especially in the case of the control region of mitochondrial DNA, where this methodology has been mostly applied. In this article, we show how to infer past demographic parameters by explicitly taking into account a finite-sites model with heterogeneity of mutation rates. We also propose an alternative way to derive confidence intervals around the estimated parameters, based on a bootstrap approach. By checking the validity of these confidence intervals by simulations, we find that only those associated with the timing of the expansion are approximately correctly estimated, while those around the population sizes are overly large. We also propose a test of the validity of the estimated demographic expansion scenario, whose proper behavior is verified by simulation. We illustrate our method with human mitochondrial DNA, where estimates of expansion times are found to be 10–20% larger when taking into account heterogeneity of mutation rates than under the infinite-sites model.
Spatial and Temporal Distribution of the Neutral Polymorphisms in the Last ZFX Intron: Analysis of the Haplotype Structure and Genealogy
Authors: Jaruzelska Jadwiga , Zietkiewicz Ewa , Batzer Mark , Cole David E C , Moisan Jean-Paul , Scozzari Rosaria , Tavaré Simon , Labuda Damian
Published: April 2021

With 10 segregating sites (simple nucleotide polymorphisms) in the last intron (1089 bp) of the ZFX gene we have observed 11 haplotypes in 336 chromosomes representing a worldwide array of 15 human populations. Two haplotypes representing 77% of all chromosomes were distributed almost evenly among four continents. Five of the remaining haplotypes were detected in Africa and 4 others were restricted to Eurasia and the Americas. Using the information about the ancestral state of the segregating positions (inferred from human-great ape comparisons), we applied coalescent analysis to estimate the age of the polymorphisms and the resulting haplotypes. The oldest haplotype, with the ancestral alleles at all the sites, was observed at low frequency only in two groups of African origin. Its estimated age of 740 to 1100 kyr corresponded to the time to the most recent common ancestor. The two most frequent worldwide distributed haplotypes were estimated at 550 to 840 and 260 to 400 kyr, respectively, while the age of the continentally restricted polymorphisms was 120 to 180 kyr and smaller. Comparison of spatial and temporal distribution of the ZFX haplotypes suggests that modern humans diverged from the common ancestral stock in the Middle Paleolithic era. Subsequent range expansion prevented substantial gene flow among continents, separating African groups from populations that colonized Eurasia and the New World.
A Molecular Description of Mutations Affecting the Pollen Component of the Nicotiana alata S locus
Authors: Golz J F , Su V , Clarke A E , Newbigin E
Published: April 2021

Mutations affecting the self-incompatibility response of Nicotiana alata were generated by irradiation. Mutants in the M1 generation were selected on the basis of pollen tube growth through an otherwise incompatible pistil. Twelve of the 18 M1 plants obtained from the mutagenesis screen were self-compatible. Eleven self-compatible plants had mutations affecting only the pollen function of the S locus (pollen-part mutants). The remaining self-compatible plant had a mutation affecting only the style function of the S locus (style-part mutant). Cytological examination of the pollen-part mutant plants revealed that 8 had an extra chromosome (2n + 1) and 3 did not. The pollen-part mutation in 7 M1 plants was followed in a series of crosses. DNA blot analysis using probes for S-RNase genes (encoding the style function of the S locus) indicated that the pollen-part mutation was associated with an extra S allele in 4 M1 plants. In 3 of these plants, the extra S allele was located on the additional chromosome. There was no evidence of an extra S allele in the 3 remaining M1 plants. The breakdown of self-incompatibility in plants with an extra S allele is discussed with reference to current models of the molecular basis of self-incompatibility.
Probability of Identity by Descent in Metapopulations
Authors: Kaj Ingemar , Lascoux Martin
Published: April 2021

Equilibrium probabilities of identity by descent (IBD), for pairs of genes within individuals, for genes between individuals within subpopulations, and for genes between subpopulations are calculated in metapopulation models with fixed or varying colony sizes. A continuous-time analog to the Moran model was used in either case. For fixed-colony size both propagule and migrant pool models were considered. The varying population size model is based on a birth-death-immigration (BDI) process, to which migration between colonies is added. Wright's F statistics are calculated and compared to previous results. Adding between-island migration to the BDI model can have an important effect on the equilibrium probabilities of IBD and on Wright's index.
In Vivo Analysis of the Domains of Yeast Rvs167p Suggests Rvs167p Function Is Mediated Through Multiple Protein Interactions
Authors: Colwill Karen , Field Deborah , Moore Lynda , Friesen James , Andrews Brenda
Published: April 2021

Morphological changes during cell division in the yeast Saccharomyces cerevisiae are controlled by cell-cycle regulators. The Pcl-Pho85p kinase complex has been implicated in the regulation of the actin cytoskeleton at least in part through Rvs167p. Rvs167p consists of three domains called BAR, GPA, and SH3. Using a two-hybrid assay, we demonstrated that each region of Rvs167p participates in protein-protein interactions: the BAR domain bound the BAR domain of another Rvs167p protein and that of Rvs161p, the GPA region bound Pcl2p, and the SH3 domain bound Abp1p. We identified Rvs167p as a Las17p/Bee1p-interacting protein in a two-hybrid screen and showed that Las17p/Bee1p bound the SH3 domain of Rvs167p. We tested the extent to which the Rvs167p protein domains rescued phenotypes associated with deletion of RVS167: salt sensitivity, random budding, and endocytosis and sporulation defects. The BAR domain was sufficient for full or partial rescue of all rvs167 mutant phenotypes tested but not required for the sporulation defect for which the SH3 domain was also sufficient. Overexpression of Rvs167p inhibits cell growth. The BAR domain was essential for this inhibition and the SH3 domain had only a minor effect. Rvs167p may link the cell cycle regulator Pcl-Pho85p kinase and the actin cytoskeleton. We propose that Rvs167p is activated by phosphorylation in its GPA region by the Pcl-Pho85p kinase. Upon activation, Rvs167p enters a multiprotein complex, making critical contacts in its BAR domain and redundant or minor contacts with its SH3 domain.
A General Requirement for the Sin3-Rpd3 Histone Deacetylase Complex in Regulating Silencing in Saccharomyces cerevisiae
Authors: Sun Zu-Wen , Hampsey Michael
Published: April 2021

The Sin3-Rpd3 histone deacetylase complex, conserved between human and yeast, represses transcription when targeted by promoter-specific transcription factors. SIN3 and RPD3 also affect transcriptional silencing at the HM mating loci and at telomeres in yeast. Interestingly, however, deletion of the SIN3 and RPD3 genes enhances silencing, implying that the Sin3-Rpd3 complex functions to counteract, rather than to establish or maintain, silencing. Here we demonstrate that Sin3, Rpd3, and Sap30, a novel component of the Sin3-Rpd3 complex, affect silencing not only at the HMR and telomeric loci, but also at the rDNA locus. The effects on silencing at all three loci are dependent upon the histone deacetylase activity of Rpd3. Enhanced silencing associated with sin3Δ, rpd3Δ, and sap30Δ is differentially dependent upon Sir2 and Sir4 at the telomeric and rDNA loci and is also dependent upon the ubiquitin-conjugating enzyme Rad6 (Ubc2). We also show that the Cac3 subunit of the CAF-I chromatin assembly factor and Sin3-Rpd3 exert antagonistic effects on silencing. Strikingly, deletion of GCN5, which encodes a histone acetyltransferase, enhances silencing in a manner similar to deletion of RPD3. A model that integrates the effects of rpd3Δ, gcn5Δ, and cac3Δ on silencing is proposed.
DNA Sequence and Functional Analysis of Homologous ARS Elements of Saccharomyces cerevisiae and S. carlsbergensis
Authors: Theis James F , Yang Chen , Schaefer Christopher B , Newlon Carol S
Published: April 2021

ARS elements of Saccharomyces cerevisiae are the cis-acting sequences required for the initiation of chromosomal DNA replication. Comparisons of the DNA sequences of unrelated ARS elements from different regions of the genome have revealed no significant DNA sequence conservation. We have compared the sequences of seven pairs of homologous ARS elements from two Saccharomyces species, S. cerevisiae and S. carlsbergensis. In all but one case, the ARS308-ARS308carl pair, significant blocks of homology were detected. In the cases of ARS305, ARS307, and ARS309, previously identified functional elements were found to be conserved in their S. carlsbergensis homologs. Mutation of the conserved sequences in the S. carlsbergensis ARS elements revealed that the homologous sequences are required for function. These observations suggested that the sequences important for ARS function would be conserved in other ARS elements. Sequence comparisons aided in the identification of the essential matches to the ARS consensus sequence (ACS) of ARS304, ARS306, and ARS310carl, though not of ARS310.
Dictyostelium Myosin II G680V Suppressors Exhibit Overlapping Spectra of Biochemical Phenotypes Including Facilitated Phosphate Release
Authors: Wu Yuan , Nejad Mike , Patterson Bruce
Published: April 2021

We have biochemically characterized 13 intragenic suppressors of the G680V mutation of Dictyostelium myosin II. In the absence of the G680V mutation, the suppressors result in a number of deviant behaviors, most commonly an increase in the basal (actin-independent) ATPase of the motor. This phenotype is complementary to that of the G680V mutant and supports our proposal that the latter impairs phosphate release. Different subsets of the mutants also suffer from poor ATPase enhancement by 1 mg/ml actin, failure to release from actin in the presence of ATPγS (or ADP and salt), and excessive release from actin in the presence of ADP. The patterns of suppressor behaviors suggest that, in general, they are facilitating Pi-releasing state(s) of the motor, but that different individual suppressors may secondarily perturb other states or actions of the motor.
Roles of the C Terminus of Armadillo in Wingless Signaling in Drosophila
Authors: Cox Rachel T , Pai Li-Mei , Kirkpatrick Catherine , Stein Joel , Peifer Mark
Published: April 2021

Drosophila melanogaster Armadillo and its vertebrate homolog β-catenin play multiple roles during development. Both are components of cell-cell adherens junctions and both transduce Wingless (Wg)/Wnt intercellular signals. The current model for Wingless signaling proposes that Armadillo binds the DNA-binding protein dTCF, forming a bipartite transcription factor that activates Wingless-responsive genes. In this model, Armadillo's C-terminal domain is proposed to serve an essential role as a transcriptional activation domain. In Xenopus, however, overexpression of C-terminally truncated β-catenin activates Wnt signaling, suggesting that the C-terminal domain might not be essential. We reexamined the function of Armadillo's C terminus in Wingless signaling. We found that C-terminally truncated mutant Armadillo has a deficit in Wg-signaling activity, even when corrected for reduced protein levels. However, we also found that Armadillo proteins lacking all or part of the C terminus retain some signaling ability if overexpressed, and that mutants lacking different portions of the C-terminal domain differ in their level of signaling ability. Finally, we found that the C terminus plays a role in Armadillo protein stability in response to Wingless signal and that the C-terminal domain can physically interact with the Arm repeat region. These data suggest that the C-terminal domain plays a complex role in Wingless signaling and that Armadillo recruits the transcriptional machinery via multiple contact sites, which act in an additive fashion.
RAPD-Based Genetic Linkage Maps of Tribolium castaneum
Authors: Beeman Richard W , Brown Susan J
Published: April 2021

A genetic map of the red flour beetle (Tribolium castaneum) integrating molecular with morphological markers was constructed using a backcross population of 147 siblings. The map defines 10 linkage groups (LGs), presumably corresponding to the 10 chromosomes, and consists of 122 randomly amplified polymorphic DNA (RAPD) markers, six molecular markers representing identified genes, and five morphological markers. The total map length is 570 cM, giving an average marker resolution of 4.3 cM. The average physical distance per genetic distance was estimated at 350 kb/cM. A cluster of loci showing distorted segregation was detected on LG9. The process of converting RAPD markers to sequence-tagged site markers was initiated: 18 RAPD markers were cloned and sequenced, and single-strand conformational polymorphisms were identified for 4 of the 18. The map positions of all 4 coincided with those of the parent RAPD markers.
Saccharomyces cerevisiae Checkpoint Genes MEC1, RAD17 and RAD24 Are Required for Normal Meiotic Recombination Partner Choice
Authors: Grushcow Jeremy M , Holzen Teresa M , Park Ken J , Weinert Ted , Lichten Michael , Bishop Douglas K
Published: April 2021

Checkpoint gene function prevents meiotic progression when recombination is blocked by mutations in the recA homologue DMC1. Bypass of dmc1 arrest by mutation of the DNA damage checkpoint genes MEC1, RAD17, or RAD24 results in a dramatic loss of spore viability, suggesting that these genes play an important role in monitoring the progression of recombination. We show here that the role of mitotic checkpoint genes in meiosis is not limited to maintaining arrest in abnormal meioses; mec1-1, rad24, and rad17 single mutants have additional meiotic defects. All three mutants display Zip1 polycomplexes in two- to threefold more nuclei than observed in wild-type controls, suggesting that synapsis may be aberrant. Additionally, all three mutants exhibit elevated levels of ectopic recombination in a novel physical assay. rad17 mutants also alter the fraction of recombination events that are accompanied by an exchange of flanking markers. Crossovers are associated with up to 90% of recombination events for one pair of alleles in rad17, as compared with 65% in wild type. Meiotic progression is not required to allow ectopic recombination in rad17 mutants, as it still occurs at elevated levels in ndt80 mutants that arrest in prophase regardless of checkpoint signaling. These observations support the suggestion that MEC1, RAD17, and RAD24, in addition to their proposed monitoring function, act to promote normal meiotic recombination.
Action of Repeat-Induced Point Mutation on Both Strands of a Duplex and on Tandem Duplications of Various Sizes in Neurospora
Authors: Watters Michael K , Randall Thomas A , Margolin Brian S , Selker Eric U , Stadler David R
Published: April 2021

In Neurospora crassa, DNA sequence duplications are detected and altered efficiently during the sexual cycle by a process known as RIP (repeat-induced point mutation). Affected sequences are subjected to multiple GC-to-AT mutations. To explore the pattern in which base changes are laid down by RIP we examined two sets of strains. First, we examined the products of a presumptive spontaneous RIP event at the mtr locus. Results of sequencing suggested that a single RIP event produces two distinct patterns of change, descended from the two strands of an affected DNA duplex. Equivalent results were obtained using an exceptional tetrad from a cross with a known duplication flanking the zeta-eta (ζ-η) locus. The mtr sequence data were also used to further examine the basis for the differential severity of C-to-T mutations on the coding and noncoding strands in genes. The known bias of RIP toward CpA/TpG sites in conjunction with the sequence bias of Neurospora accounts for the differential effect. Finally, we used a collection of tandem repeats (from 16 to 935 bp in length) within the mtr gene to examine the length requirement for RIP. No evidence of RIP was found with duplications shorter than 400 bp while all longer tandem duplications were frequently affected. A comparison of these results with vegetative reversion data for the same duplications is consistent with the idea that reversion of long tandem duplications and RIP share a common step.
Eyespot-Assembly Mutants in Chlamydomonas reinhardtii
Authors: Lamb Mary Rose , Dutcher Susan K , Worley Cathy K , Dieckmann Carol L
Published: April 2021

Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca++-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell.