bluuebunny/crossref_metadata_2025 huggingface dataset:
Positive Selection of Novel Peroxisome Biogenesis-Defective Mutants of the Yeast Pichia pastoris
Authors: Johnson Monique A , Waterham Hans R , Ksheminska Galyna P , Fayura Liubov R , Cereghino Joan Lin , Stasyk Oleh V , Veenhuis Marten , Kulachkovsky Aleksander R , Sibirny Andrei A , Cregg James M
Published: April 2021

We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.
The Role of Nucleotide Binding and Hydrolysis in the Function of the Fission Yeast cdc18+ Gene Product
Authors: DeRyckere Deborah , Smith Cheryl L , Martin G Steven
Published: April 2021

The fission yeast cdc18+ gene is required for both initiation of DNA replication and the mitotic checkpoint that normally inhibits mitosis in the absence of DNA replication. The cdc18+ gene product contains conserved Walker A and B box motifs. Studies of other ATPases have shown that these motifs are required for nucleotide binding and hydrolysis, respectively. We have observed that mutant strains in which either of these motifs is disrupted are inviable. The effects of these mutations were examined by determining the phenotypes of mutant strains following depletion of complementing wild-type Cdc18. In both synchronous and asynchronous cultures, the nucleotide-hydrolysis motif mutant (DE286AA) arrests with a 1C–2C DNA content, and thus exhibits no obvious defects in entry into S phase or in the mitotic checkpoint. In contrast, in cultures synchronized by hydroxyurea arrest and release, the nucleotide-binding motif mutant (K205A) exhibits the null phenotype, with 1C and <1C DNA content, indicating a block in entry into S phase and loss of checkpoint control. In asynchronous cultures this mutant exhibits a mixed phenotype: a percentage of the population displays the null phenotype, while the remaining fraction arrests with a 2C DNA content. Thus, the phenotype exhibited by the K205A mutant is dependent on the cell-cycle position at which wild-type Cdc18 is depleted. These data indicate that both nucleotide binding and hydrolysis are required for Cdc18 function. In addition, the difference in the phenotypes exhibited by the nucleotide-binding and hydrolysis motif mutants is consistent with a two-step model for Cdc18 function in which nucleotide binding and hydrolysis are required for distinct aspects of Cdc18 function that may be executed at different points in the cell cycle.
Coordination of the Initiation of Recombination and the Reductional Division in Meiosis in Saccharomyces cerevisiae
Authors: Jiao Kai , Bullard Steven A , Salem Laura , Malone Robert E
Published: April 2021

Early exchange (EE) genes are required for the initiation of meiotic recombination in Saccharomyces cerevisiae. Cells with mutations in several EE genes undergo an earlier reductional division (MI), which suggests that the initiation of meiotic recombination is involved in determining proper timing of the division. The different effects of null mutations on the timing of reductional division allow EE genes to be assorted into three classes: mutations in RAD50 or REC102 that confer a very early reductional division; mutations in REC104 or REC114 that confer a division earlier than that of wild-type (WT) cells, but later than that of mutants of the first class; and mutations in MEI4 that do not significantly alter the timing of MI. The very early mutations are epistatic to mutations in the other two classes. We propose a model that accounts for the epistatic relationships and the communication between recombination initiation and the first division. Data in this article indicate that double-strand breaks (DSBs) are not the signal for the normal delay of reductional division; these experiments also confirm that MEI4 is required for the formation of meiotic DSBs. Finally, if a DSB is provided by the HO endonuclease, recombination can occur in the absence of MEI4 and REC104.
The pro1+ Gene From Sordaria macrospora Encodes a C6 Zinc Finger Transcription Factor Required for Fruiting Body Development
Authors: Masloff Sandra , Pöggeler Stefanie , Kück Ulrich
Published: April 2021

During sexual morphogenesis, the filamentous ascomycete Sordaria macrospora differentiates into multicellular fruiting bodies called perithecia. Previously it has been shown that this developmental process is under polygenic control. To further understand the molecular mechanisms involved in fruiting body formation, we generated the protoperithecia forming mutant pro1, in which the normal development of protoperithecia into perithecia has been disrupted. We succeeded in isolating a cosmid clone from an indexed cosmid library, which was able to complement the pro1- mutation. Deletion analysis, followed by DNA sequencing, subsequently demonstrated that fertility was restored to the pro1 mutant by an open reading frame encoding a 689-amino-acid polypeptide, which we named PRO1. A region from this polypeptide shares significant homology with the DNA-binding domains found in fungal C6 zinc finger transcription factors, such as the GAL4 protein from yeast. However, other typical regions of C6 zinc finger proteins, such as dimerization elements, are absent in PRO1. The involvement of the pro1+ gene in fruiting body development was further confirmed by trying to complement the mutant phenotype with in vitro mutagenized and truncated versions of the pro1 open reading frame. Southern hybridization experiments also indicated that pro1+ homologues are present in other sexually propagating filamentous ascomycetes.
Introgression Through Rare Hybridization: A Genetic Study of a Hybrid Zone Between Red and Sika Deer (Genus Cervus) in Argyll, Scotland
Authors: Goodman Simon J , Barton Nick H , Swanson Graeme , Abernethy Kate , Pemberton Josephine M
Published: April 2021

In this article we describe the structure of a hybrid zone in Argyll, Scotland, between native red deer (Cervus elaphus) and introduced Japanese sika deer (Cervus nippon), on the basis of a genetic analysis using 11 microsatellite markers and mitochondrial DNA. In contrast to the findings of a previous study of the same population, we conclude that the deer fall into two distinct genetic classes, corresponding to either a sika-like or red-like phenotype. Introgression is rare at any one locus, but where the taxa overlap up to 40% of deer carry apparently introgressed alleles. While most putative hybrids are heterozygous at only one locus, there are rare multiple heterozygotes, reflecting significant linkage disequilibrium within both sika- and red-like populations. The rate of backcrossing into the sika population is estimated as H = 0.002 per generation and into red, H = 0.001 per generation. On the basis of historical evidence that red deer entered Kintyre only recently, a diffusion model evaluated by maximum likelihood shows that sika have increased at ∼9.2% yr-1 from low frequency and disperse at a rate of ∼3.7 km yr-1. Introgression into the red-like population is greater in the south, while introgression into sika varies little along the transect. For both sika- and red-like populations, the degree of introgression is 30–40% of that predicted from the rates of current hybridization inferred from linkage disequilibria; however, in neither case is this statistically significant evidence for selection against introgression.
Tandem Repeat Recombination Induced by Replication Fork Defects in Escherichia coli Requires a Novel Factor, RadC
Authors: Saveson Catherine J , Lovett Susan T
Published: April 2021

DnaB is the helicase associated with the DNA polymerase III replication fork in Escherichia coli. Previously we observed that the dnaB107(ts) mutation, at its permissive temperature, greatly stimulated deletion events at chromosomal tandem repeats. This stimulation required recA, which suggests a recombinational mechanism. In this article we examine the genetic dependence of recombination stimulated by the dnaB107 mutation. Gap repair genes recF, recO, and recR were not required. Mutations in recB, required for double-strand break repair, and in ruvC, the Holliday junction resolvase gene, were synthetically lethal with dnaB107, causing enhanced temperature sensitivity. The hyperdeletion phenotype of dnaB107 was semidominant, and in dnaB107/dnaB+ heterozygotes recB was partially required for enhanced deletion, whereas ruvC was not. We believe that dnaB107 causes the stalling of replication forks, which may become broken and require repair. Misalignment of repeated sequences during RecBCD-mediated repair may account for most, but not all, of deletion stimulated by dnaB107. To our surprise, the radC gene, like recA, was required for virtually all recombination stimulated by dnaB107. The biochemical function of RadC is unknown, but is reported to be required for growth-medium-dependent repair of DNA strand breaks. Our results suggest that RadC functions specifically in recombinational repair that is associated with the replication fork.
Mutators, Population Size, Adaptive Landscape and the Adaptation of Asexual Populations of Bacteria
Authors: Tenaillon Olivier , Toupance Bruno , Le Nagard Hervé , Taddei François , Godelle Bernard
Published: April 2021

Selection of mutator alleles, increasing the mutation rate up to 10,000-fold, has been observed during in vitro experimental evolution. This spread is ascribed to the hitchhiking of mutator alleles with favorable mutations, as demonstrated by a theoretical model using selective parameters corresponding to such experiments. Observations of unexpectedly high frequencies of mutators in natural isolates suggest that the same phemonemon could occur in the wild. But it remains questionable whether realistic in natura parameter values could also result in selection of mutators. In particular, the main parameters of adaptation, the size of the adapting population and the height and steepness of the adaptive peak characterizing adaptation, are very variable in nature. By simulation approach, we studied the effect of these parameters on the selection of mutators in asexual populations, assuming additive fitness. We show that the larger the population size, the more likely the fixation of mutator alleles. At a large population size, at least four adaptive mutations are needed for mutator fixation; moreover, under stronger selection stronger mutators are selected. We propose a model based on multiple mutations to illustrate how second-order selection can optimize population fitness when few favorable mutations are required for adaptation.
Lesions in Many Different Spindle Components Activate the Spindle Checkpoint in the Budding Yeast Saccharomyces cerevisiae
Authors: Hardwick Kevin G , Li Rong , Mistrot Cathy , Chen Rey-Huei , Dann Phoebe , Rudner Adam , Murray Andrew W
Published: April 2021

The spindle checkpoint arrests cells in mitosis in response to defects in the assembly of the mitotic spindle or errors in chromosome alignment. We determined which spindle defects the checkpoint can detect by examining the interaction of mutations that compromise the checkpoint (mad1, mad2, and mad3) with those that damage various structural components of the spindle. Defects in microtubule polymerization, spindle pole body duplication, microtubule motors, and kinetochore components all activate the MAD-dependent checkpoint. In contrast, the cell cycle arrest caused by mutations that induce DNA damage (cdc13), inactivate the cyclin proteolysis machinery (cdc16 and cdc23), or arrest cells in anaphase (cdc15) is independent of the spindle checkpoint.
Identification of Novel Drosophila Meiotic Genes Recovered in a P-Element Screen
Authors: Sekelsky Jeff J , McKim Kim S , Messina Lisa , French Rachael L , Hurley Wendy D , Arbel Tamar , Chin Gregory M , Deneen Benjamin , Force Shelley J , Hari Kumar L , Jang Janet Ko , Laurençon Anne C , Madden Laurence D , Matthies Heinrich J , Milliken Dawn B , Page Scott L , Ring Amy D , Wayson Sarah M , Zimmerman Carin C , Hawley R Scott
Published: April 2021

The segregation of homologous chromosomes from one another is the essence of meiosis. In many organisms, accurate segregation is ensured by the formation of chiasmata resulting from crossing over. Drosophila melanogaster females use this type of recombination-based system, but they also have mechanisms for segregating achiasmate chromosomes with high fidelity. We describe a P-element mutagenesis and screen in a sensitized genetic background to detect mutations that impair meiotic chromosome pairing, recombination, or segregation. Our screen identified two new recombination-deficient mutations: mei-P22, which fully eliminates meiotic recombination, and mei-P26, which decreases meiotic exchange by 70% in a polar fashion. We also recovered an unusual allele of the ncd gene, whose wild-type product is required for proper structure and function of the meiotic spindle. However, the screen yielded primarily mutants specifically defective in the segregation of achiasmate chromosomes. Although most of these are alleles of previously undescribed genes, five were in the known genes αTubulin67C, CycE, push, and Trl. The five mutations in known genes produce novel phenotypes for those genes.
Pattern of Nucleotide Substitution and Rate Heterogeneity in the Hypervariable Regions I and II of Human mtDNA
Authors: Meyer Sonja , Weiss Gunter , von Haeseler Arndt
Published: April 2021

This study provides a comprehensive survey of the complex pattern of nucleotide substitution in the control region of human mtDNA, which is of central importance to the studies of human evolution. A total of 1229 different hypervariable region I (HVRI) and 385 different hypervariable region II (HVRII) sequences were analyzed using a complex substitution model. Moreover, we suggest a new method to assign relative rates to each site in the sequence. Estimates are based on maximum-likelihood methods applied to randomly selected subsets of sequences. Our results indicate that the rate of substitution in HVRI is approximately twice as high as in HVRII and that this difference is mainly due to a higher frequency of pyrimidine transitions in HVRI. However, rate heterogeneity is more pronounced in HVRII.
Saccharomyces cerevisiae Putative G Protein, Gtr1p, Which Forms Complexes With Itself and a Novel Protein Designated as Gtr2p, Negatively Regulates the Ran/Gsp1p G Protein Cycle Through Gtr2p
Authors: Nakashima Nobutaka , Noguchi Eishi , Nishimoto Takeharu
Published: April 2021

Prp20p and Rna1p are GDP/GTP exchanging and GTPase-activating factors of Gsp1p, respectively, and their mutations, prp20-1 and rna1-1, can both be suppressed by Saccharomyces cerevisiae gtr1-11. We found that gtr1-11 caused a single amino acid substitution in Gtr1p, forming S20L, which is a putative GDP-bound mutant protein, while Gtr1p has been reported to bind to GTP alone. Consistently, gtr1-S20N, another putative GDP-bound mutant, suppressed both prp20-1 and rna1-1. On the other hand, gtr1-Q65L, a putative GTP-bound mutant, was inhibitory to prp20-1 and rna1-1. Thus, the role that Gtr1p plays in vivo appears to depend upon the nucleotide bound to it. Our data suggested that the GTP-bound Gtr1p, but not the GDP-bound Gtr1p, interacts with itself through its C-terminal tail. S. cerevisiae possesses a novel gene, GTR2, which is homologous to GTR1. Gtr2p interacts with itself in the presence of Gtr1p. The disruption of GTR2 suppressed prp20-1 and abolished the inhibitory effect of gtr1-Q65L on prp20-1. This finding, taken together with the fact that Gtr1p-S20L is a putative, inactive GDP-bound mutant, implies that Gtr1p negatively regulates the Ran/Gsp1p GTPase cycle through Gtr2p.
Control of Ribosomal Protein L1 Synthesis in Mesophilic and Thermophilic Archaea
Authors: Kraft Alexander , Lutz Christina , Lingenhel Arno , Gröbner Peter , Piendl Wolfgang
Published: April 2021

The mechanisms for the control of ribosomal protein synthesis have been characterized in detail in Eukarya and in Bacteria. In Archaea, only the regulation of the MvaL1 operon (encoding ribosomal proteins MvaL1, MvaL10, and MvaL12) of the mesophilic Methanococcus vannielii has been extensively investigated. As in Bacteria, regulation takes place at the level of translation. The regulator protein MvaL1 binds preferentially to its binding site on the 23S rRNA, and, when in excess, binds to the regulatory target site on its mRNA and thus inhibits translation of all three cistrons of the operon. The regulatory binding site on the mRNA, a structural mimic of the respective binding site on the 23S rRNA, is located within the structural gene about 30 nucleotides downstream of the ATG start codon. MvaL1 blocks a step before or at the formation of the first peptide bond of MvaL1. Here we demonstrate that a similar regulatory mechanism exists in the thermophilic M. thermolithotrophicus and M. jannaschii. The L1 gene is cotranscribed together with the L10 and L11 gene, in all genera of the Euryarchaeota branch of the Archaea studied so far. A potential regulatory L1 binding site located within the structural gene, as in Methanococcus, was found in Methanobacterium thermoautotrophicum and in Pyrococcus horikoshii. In contrast, in Archaeoglobus fulgidus a typical L1 binding site is located in the untranslated leader of the L1 gene as described for the halophilic Archaea. In Sulfolobus, a member of the Crenarchaeota, the L1 gene is part of a long transcript (encoding SecE, NusG, L11, L1, L10, L12). A previously suggested regulatory L1 target site located within the L11 structural gene could not be confirmed as an L1 binding site.
UV Stimulation of Chromosomal Marker Exchange in Sulfolobus acidocaldarius: Implications for DNA Repair, Conjugation and Homologous Recombination at Extremely High Temperatures
Authors: Schmidt Katherine J , Beck Kristen E , Grogan Dennis W
Published: April 2021

The hyperthermophilic archaeon Sulfolobus acidocaldarius exchanges and recombines chromosomal markers by a conjugational mechanism, and the overall yield of recombinants is greatly increased by previous exposure to UV light. This stimulation was studied in an effort to clarify its mechanism and that of marker exchange itself. A variety of experiments failed to identify a significant effect of UV irradiation on the frequency of cell pairing, indicating that subsequent steps are primarily affected, i.e., transfer of DNA between cells or homologous recombination. The UV-induced stimulation decayed rather quickly in parental cells during preincubation at 75°, and the rate of decay depended on the incubation temperature. Preincubation at 75° decreased the yield of recombinants neither from unirradiated parental cells nor from parental suspensions subsequently irradiated. We interpret these results as evidence that marker exchange is stimulated by recombinogenic DNA lesions formed as intermediates in the process of repairing UV photoproducts in the S. acidocaldarius chromosome.
Genetic Analysis of the Bacteriophage T4-Encoded Cochaperonin Gp31
Authors: Richardson Alexandra , Georgopoulos Costa
Published: April 2021

Previous genetic and biochemical analyses have established that the bacteriophage T4-encoded Gp31 is a cochaperonin that interacts with Escherichia coli’s GroEL to ensure the timely and accurate folding of Gp23, the bacteriophage-encoded major capsid protein. The heptameric Gp31 cochaperonin, like the E. coli GroES cochaperonin, interacts with GroEL primarily through its unstructured mobile loop segment. Upon binding to GroEL, the mobile loop adopts a structured, β-hairpin turn. In this article, we present extensive genetic data that strongly substantiate and extend these biochemical studies. These studies begin with the isolation of mutations in gene 31 based on the ability to plaque on groEL44 mutant bacteria, whose mutant product interacts weakly with Gp31. Our genetic system is unique because it also allows for the direct selection of revertants of such gene 31 mutations, based on their ability to plaque on groEL515 mutant bacteria. Interestingly, all of these revertants are pseudorevertants because the original 31 mutation is maintained. In addition, we show that the classical tsA70 mutation in gene 31 changes a conserved hydrophobic residue in the mobile loop to a hydrophilic one. Pseudorevertants of tsA70, which enable growth at the restrictive temperatures, acquire the same mutation previously shown to allow plaque formation on groEL44 mutant bacteria. Our genetic analyses highlight the crucial importance of all three highly conserved hydrophobic residues of the mobile loop of Gp31 in the productive interaction with GroEL.
Chromosome Size-Dependent Control of Meiotic Reciprocal Recombination in Saccharomyces cerevisiae: The Role of Crossover Interference
Authors: Kaback David B , Barber Dianna , Mahon Jim , Lamb Jacque , You Jerome
Published: April 2021

In the yeast Saccharomyces cerevisiae, small chromosomes undergo meiotic reciprocal recombination (crossing over) at rates (centimorgans per kilobases) greater than those of large chromosomes, and recombination rates respond directly to changes in the total size of a chromosomal DNA molecule. This phenomenon, termed chromosome size-dependent control of meiotic reciprocal recombination, has been suggested to be important for ensuring that homologous chromosomes cross over during meiosis. The mechanism of this regulation was investigated by analyzing recombination in identical genetic intervals present on different size chromosomes. The results indicate that chromosome size-dependent control is due to different amounts of crossover interference. Large chromosomes have high levels of interference while small chromosomes have much lower levels of interference. A model for how crossover interference directly responds to chromosome size is presented. In addition, chromosome size-dependent control was shown to lower the frequency of homologous chromosomes that failed to undergo crossovers, suggesting that this control is an integral part of the mechanism for ensuring meiotic crossing over between homologous chromosomes.
Mapping Quantitative Trait Loci by Genotyping Haploid Tissues
Authors: Wu R L
Published: April 2021

Mapping strategies based on a half- or full-sib family design have been developed to map quantitative trait loci (QTL) for outcrossing species. However, these strategies are dependent on controlled crosses where marker-allelic frequency and linkage disequilibrium between the marker and QTL may limit their application. In this article, a maximum-likelihood method is developed to map QTL segregating in an open-pollinated progeny population using dominant markers derived from haploid tissues from single meiotic events. Results from the haploid-based mapping strategy are not influenced by the allelic frequencies of markers and their linkage disequilibria with QTL, because the probabilities of QTL genotypes conditional on marker genotypes of haploid tissues are independent of these population parameters. Parameter estimation and hypothesis testing are implemented via expectation/conditional maximization algorithm. Parameters estimated include the additive effect, the dominant effect, the population mean, the chromosomal location of the QTL in the interval, and the residual variance within the QTL genotypes, plus two population parameters, outcrossing rate and QTL-allelic frequency. Simulation experiments show that the accuracy and power of parameter estimates are affected by the magnitude of QTL effects, heritability levels of a trait, and sample sizes used. The application and limitation of the method are discussed.
Reduced Sequence Variability on the NeoY Chromosome of Drosophila americana americana
Authors: McAllister Bryant F , Charlesworth Brian
Published: April 2021

Sex chromosomes are generally morphologically and functionally distinct, but the evolutionary forces that cause this differentiation are poorly understood. Drosophila americana americana was used in this study to examine one aspect of sex chromosome evolution, the degeneration of nonrecombining Y chromosomes. The primary X chromosome of D. a. americana is fused with a chromosomal element that was ancestrally an autosome, causing this homologous chromosomal pair to segregate with the sex chromosomes. Sequence variation at the Alcohol Dehydrogenase (Adh) gene was used to determine the pattern of nucleotide variation on the neo-sex chromosomes in natural populations. Sequences of Adh were obtained for neo-X and neo-Y chromosomes of D. a. americana, and for Adh of D. a. texana, in which it is autosomal. No significant sequence differentiation is present between the neo-X and neo-Y chromosomes of D. a. americana or the autosomes of D. a. texana. There is a significantly lower level of sequence diversity on the neo-Y chromosome relative to the neo-X in D. a. americana. This reduction in variability on the neo-Y does not appear to have resulted from a selective sweep. Coalescent simulations of the evolutionary transition of an autosome into a Y chromosome indicate there may be a low level of recombination between the neo-X and neo-Y alleles of Adh and that the effective population size of this chromosome may have been reduced below the expected value of 25% of the autosomal effective size, possibly because of the effects of background selection or sexual selection.
Temperature-Sensitive Mutations in the Saccharomyces cerevisiae MRT4, GRC5, SLA2 and THS1 Genes Result in Defects in mRNA Turnover
Authors: Zuk Dorit , Belk Jonathan P , Jacobson Allan
Published: April 2021

In a screen for factors involved in mRNA turnover, four temperature-sensitive yeast strains (ts1189, ts942, ts817, and ts1100) exhibited defects in the decay of several mRNAs. Complementation of the growth and mRNA decay defects, and genetic experiments, revealed that ts1189 is mutated in the previously unknown MRT4 gene, ts942 is mutated in GRC5 (encoding the L9 ribosomal protein), ts817 contains a mutation in SLA2 (encoding a membrane protein), and ts1100 contains a mutation in THS1 (encoding the threonyl-tRNA synthetase). Three of the four mutants (mrt4, grc5, and sla2) were not defective in protein synthesis, suggesting that these strains contain mutations in factors that may play a specific role in mRNA decay. The mRNA stabilization observed in the ths1 strain, however, could be due to the significant drop in translation observed in this mutant at 37°. While the three interesting mutants appear to encode novel mRNA decay factors, at least one could be linked to a previously characterized mRNA decay pathway. The growth and mRNA decay defects of ts942 (grc5) cells were suppressed by overexpression of the NMD3 gene, encoding a protein shown to participate in a two-hybrid interaction with the nonsense-mediated decay protein Upf1p.
Genetic Dissection of X-Linked Interspecific Hybrid Placental Dysplasia in Congenic Mouse Strains
Authors: Hemberger Myriam C , Pearsall Robert S , Zechner Ulrich , Orth Annie , Otto Sabine , Rüschendorf Franz , Fundele Reinald , Elliott Rosemary
Published: April 2021

Interspecific hybridization in the genus Mus results in male sterility and X-linked placental dysplasia. We have generated several congenic laboratory mouse lines (Mus musculus) in which different parts of the maternal X chromosome were derived from M. spretus. A strict positive correlation between placental weight and length of the M. spretus-derived part of the X chromosome was shown. Detailed analysis was carried out with one congenic strain that retained a M. spretus interval between 12.0 and 30.74 cM. This strain consistently produced hyperplastic placentas that exhibited an average weight increase of 180% over the weight of control placentas. In derived subcongenic strains, however, increased placental weight could no longer be observed. Morphometric analysis of these placentas revealed persistence of abnormal morphology. Fully developed placental hyperplasia could be reconstituted by recombination of proximal and central M. spretus intervals with an intervening M. musculus region. These results may suggest that placental dysplasia of interspecific mouse hybrids is caused by multiple loci clustered on the X chromosome that act synergistically. Alternatively, it is possible that changes in chromatin structure in interspecific hybrids that influence gene expression are dependent on the length of the alien chromosome.
The Age of Nonsynonymous and Synonymous Mutations in Animal mtDNA and Implications for the Mildly Deleterious Theory
Authors: Nielsen Rasmus , Weinreich Daniel M
Published: April 2021

McDonald/Kreitman tests performed on animal mtDNA consistently reveal significant deviations from strict neutrality in the direction of an excess number of polymorphic nonsynonymous sites, which is consistent with purifying selection acting on nonsynonymous sites. We show that under models of recurrent neutral and deleterious mutations, the mean age of segregating neutral mutations is greater than the mean age of segregating selected mutations, even in the absence of recombination. We develop a test of the hypothesis that the mean age of segregating synonymous mutations equals the mean age of segregating nonsynonymous mutations in a sample of DNA sequences. The power of this age-of-mutation test and the power of the McDonald/Kreitman test are explored by computer simulations. We apply the new test to 25 previously published mitochondrial data sets and find weak evidence for selection against nonsynonymous mutations.
A Random Model Approach to Mapping Quantitative Trait Loci for Complex Binary Traits in Outbred Populations
Authors: Yi Nengjun , Xu Shizhong
Published: April 2021

Mapping quantitative trait loci (QTL) for complex binary traits is more challenging than for normally distributed traits due to the nonlinear relationship between the observed phenotype and unobservable genetic effects, especially when the mapping population contains multiple outbred families. Because the number of alleles of a QTL depends on the number of founders in an outbred population, it is more appropriate to treat the effect of each allele as a random variable so that a single variance rather than individual allelic effects is estimated and tested. Such a method is called the random model approach. In this study, we develop the random model approach of QTL mapping for binary traits in outbred populations. An EM-algorithm with a Fisher-scoring algorithm embedded in each E-step is adopted here to estimate the genetic variances. A simple Monte Carlo integration technique is used here to calculate the likelihood-ratio test statistic. For the first time we show that QTL of complex binary traits in an outbred population can be scanned along a chromosome for their positions, estimated for their explained variances, and tested for their statistical significance. Application of the method is illustrated using a set of simulated data.
Multiple Chromosomes in Bacteria: The Yin and Yang of trp Gene Localization in Rhodobacter sphaeroides 2.4.1
Authors: Mackenzie Chris , Simmons Adrian E , Kaplan Samuel
Published: April 2021

The existence of multiple chromosomes in bacteria has been known for some time. Yet the extent of functional solidarity between different chromosomes remains unknown. To examine this question, we have surveyed the well-described genes of the tryptophan biosynthetic pathway in the multichromosomal photosynthetic eubacterium Rhodobacter sphaeroides 2.4.1. The genome of this organism was mutagenized using Tn5, and strains that were auxotrophic for tryptophan (Trp-) were isolated. Pulsed-field gel mapping indicated that Tn5 insertions in both the large (3 Mb CI) and the small (0.9 Mb CII) chromosomes created a Trp- phenotype. Sequencing the DNA flanking the sites of the Tn5 insertions indicated that the genes trpE-yibQ-trpGDC were at a locus on CI, while genes trpF-aroR-trpB were at locus on CII. Unexpectedly, trpA was not found downstream of trpB. Instead, it was placed on the CI physical map at a locus 1.23 Mb away from trpE-yibQ-trpGDC. To relate the context of the R. sphaeroides trp genes to those of other bacteria, the DNA regions surrounding the trp genes on both chromosomes were sequenced. Of particular significance was the finding that rpsA1, which encodes ribosomal protein S1, and cmkA, which encodes cytidylate monophosphate kinase, were on CII. These genes are considered essential for translation and chromosome replication, respectively. Southern blotting suggested that the trp genes and rpsA1 exist in single copy within the genome. To date, this topological organization of the trp “operon” is unique within a bacterial genome. When taken with the finding that CII encodes essential housekeeping functions, the overall impression is one of close regulatory and functional integration between these chromosomes.
A Core Activity Associated with the N Terminus of the Yeast RAD52 Protein Is Revealed by RAD51 Overexpression Suppression of C-Terminal rad52 Truncation Alleles
Authors: Asleson Erin N , Okagaki Ron J , Livingston Dennis M
Published: April 2021

C-terminal rad52 truncation and internal deletion mutants were characterized for their ability to repair MMS-induced double-strand breaks and to produce viable spores during meiosis. The rad52-Δ251 allele, encoding the N-terminal 251 amino acids of the predicted 504-amino-acid polypeptide, supports partial activity for both functions. Furthermore, RAD51 overexpression completely suppresses the MMS sensitivity of a rad52-Δ251 mutant. The absence of the C terminus in the truncated protein makes it likely that suppression occurs by bypassing the C-terminal functions of Rad52p. RAD51 overexpression does not suppress the low level of spore viability that the rad52-Δ251 allele causes and only partially suppresses the defect in rad52 alleles encoding the N-terminal 292 or 327 amino acids. The results of this study also show that intragenic complementation between rad52 alleles is governed by a complex relationship that depends heavily on the two alleles involved and their relative dosage. In heteroallelic rad52 diploids, the rad52-Δ251 allele does not complement rad52 missense mutations altering residues 61 or 64 in the N terminus. However, complementation is achieved with each of these missense alleles when the rad52-Δ251 allele is overexpressed. Complementation also occurs between rad52-Δ327 and an internal deletion allele missing residues 210 through 327. We suggest that the first 251 amino acids of Rad52p constitute a core domain that provides critical RAD52 activities.
Characterization of Seven Genes Affecting Caenorhabditis elegans Hindgut Development
Authors: Chamberlin Helen M , Brown Keith B , Sternberg Paul W , Thomas James H
Published: April 2021

We have identified and characterized 12 mutations in seven genes that affect the development of the Caenorhabditis elegans hindgut. We find that the mutations can disrupt the postembryonic development of the male-specific blast cells within the hindgut, the hindgut morphology in both males and hermaphrodites, and in some cases, the expression of a hindgut marker in hermaphrodite animals. Mutations in several of the genes also affect viability. On the basis of their mutant phenotypes, we propose that the genes fall into four distinct classes: (1) egl-5 is required for regional identity of the tail; (2) sem-4 is required for a variety of ectodermal and mesodermal cell types, including cells in the hindgut; (3) two genes, lin-49 and lin-59, affect development of many cells, including hindgut; and (4) three genes, mab-9, egl-38, and lin-48, are required for patterning fates within the hindgut, making certain hindgut cells different from others. We also describe a new allele of the Pax gene egl-38 that is temperature sensitive and affects the conserved β-hairpin of the EGL-38 paired domain. Our results suggest that a combination of different factors contribute to normal C. elegans hindgut development.
Structure and Regulation of the Salivary Gland Secretion Protein Gene Sgs-1 of Drosophila melanogaster
Authors: Roth Günther E , Wattler Sigrid , Bornschein Hartmut , Lehmann Michael , Korge Günter
Published: April 2021

The Drosophila melanogaster gene Sgs-1 belongs to the secretion protein genes, which are coordinately expressed in salivary glands of third instar larvae. Earlier analysis had implied that Sgs-1 is located at the 25B2-3 puff. We cloned Sgs-1 from a YAC covering 25B2-3. Despite using a variety of vectors and Escherichia coli strains, subcloning from the YAC led to deletions within the Sgs-1 coding region. Analysis of clonable and unclonable sequences revealed that Sgs-1 mainly consists of 48-bp tandem repeats encoding a threonine-rich protein. The Sgs-1 inserts from single λ clones are heterogeneous in length, indicating that repeats are eliminated. By analyzing the expression of Sgs-1/lacZ fusions in transgenic flies, cis-regulatory elements of Sgs-1 were mapped to lie within 1 kb upstream of the transcriptional start site. Band shift assays revealed binding sites for the transcription factor fork head (FKH) and the factor secretion enhancer binding protein 3 (SEBP3) at positions that are functionally relevant. FKH and SEBP3 have been shown previously to be involved in the regulation of Sgs-3 and Sgs-4. Comparison of the levels of steady state RNA and of the transcription rates for Sgs-1 and Sgs-1/lacZ reporter genes indicates that Sgs-1 RNA is 100-fold more stable than Sgs-1/lacZ RNA. This has implications for the model of how Sgs transcripts accumulate in late third instar larvae.
The Genetic Analysis of Age-Dependent Traits: Modeling the Character Process
Authors: Pletcher Scott D , Geyer Charles J
Published: April 2021

The extension of classical quantitative genetics to deal with function-valued characters (also called infinite-dimensional characters) such as growth curves, mortality curves, and reaction norms, was begun by Kirkpatrick and co-workers. In this theory, the analogs of variance components for single traits are covariance functions for function-valued traits. In the approach presented here, we employ a variety of parametric models for covariance functions that have a number of desirable properties: the functions (1) are positive definite, (2) can be estimated using procedures like those currently used for single traits, (3) have a small number of parameters, and (4) allow simple hypotheses to be easily tested. The methods are illustrated using data from a large experiment that examined the effects of spontaneous mutations on age-specific mortality rates in Drosophila melanogaster. Our methods are shown to work better than a standard multivariate analysis, which assumes the character value at each age is a distinct character. Advantages over existing methods that model covariance functions as a series of orthogonal polynomials are discussed.
Uterine and Postnatal Maternal Effects in Mice Selected for Differential Rate of Early Development
Authors: Rhees Brian K , Ernst Cynthia A , Miao Christina H , Atchley William R
Published: April 2021

A series of mouse lines was produced by long-term restricted index selection for divergent rate of growth during early and late postnatal development. The selection program was based on the following treatments: E+ and E- lines were selected to alter birth to 10-day weight gain while holding late gain for both lines constant and a control line was established via random selection. Using embryo transfer and crossfostering methodology, we partitioned postnatal growth for E+, E-, and C lines into progeny genetic, uterine maternal, and nurse maternal components. Selection for differential early growth resulted in correlated response in uterine and nurse maternal effects on body weights, with significant genetic-by-environment interactions. Significant uterine effects were also observed in tail length measurements. Direct uterine effects on body weight were relatively small and resulted in growth rate differences early in development. Nurse effects were large, resulting in modification of progeny growth trajectory especially during early postnatal development. Genetic-by-uterine interactions were large and demonstrate progeny-specific effects of the prenatal uterine environment.
Fission Yeast Mutants That Alleviate Transcriptional Silencing in Centromeric Flanking Repeats and Disrupt Chromosome Segregation
Authors: Ekwall Karl , Cranston Gwen , Allshire Robin C
Published: April 2021

In the fission yeast Schizosaccharomyces pombe genes are transcriptionally silenced when placed within centromeres, within or close to the silent mating-type loci or adjacent to telomeres. Factors required to maintain mating-type silencing also affect centromeric silencing and chromosome segregation. We isolated mutations that alleviate repression of marker genes in the inverted repeats flanking the central core of centromere I. Mutations csp1 to 13 (centromere: suppressor of position effect) defined 12 loci. Ten of the csp mutants have no effect on mat2/3 or telomere silencing. All csp mutants allow some expression of genes in the centromeric flanking repeat, but expression in the central core is undetectable. Consistent with defective centromere structure and function, chromosome loss rates are elevated in all csp mutants. Mutants csp1 to 6 are temperature-sensitive lethal and csp3 and csp6 cells are defective in mitosis at 36°. csp7 to 13 display a high incidence of lagging chromosomes on late anaphase spindles. Thus, by screening for mutations that disrupt silencing in the flanking region of a fission yeast centromere a novel collection of mutants affecting centromere architecture and chromosome segregation has been isolated.
The Saccharomyces cerevisiae RanGTP-Binding Protein Msn5p Is Involved in Different Signal Transduction Pathways
Authors: Alepuz Paula M , Matheos Dina , Cunningham Kyle W , Estruch Francisco
Published: April 2021

In eukaryotes, control of transcription by extracellular signals involves the translocation to the nucleus of at least one component of the signal transduction pathway. Transport through the nuclear envelope requires the activity of an import or export receptor that interacts with the small GTPase Ran. We have cloned the MSN5 gene of the yeast Saccharomyces cerevisiae that is postulated to encode one of these receptors. Msn5p belongs to a family of proteins with a conserved N-terminal sequence that acts as a RanGTP-binding domain. The results presented here provide genetic data supporting Msn5p involvement in several different signal transduction pathways. All of these pathways include changes in gene expression, and regulated nucleocytoplasmic redistribution of a component in response to external conditions has already been described in some of them. We have cloned MSN5 following two different strategies. Msn5p was constitutively localized in the nucleus. Phenotypic analysis of the msn5 mutant demonstrated that this protein participates in processes such as catabolite repression, calcium signaling, mating, and cell proliferation, as well as being involved in previously characterized phosphate utilization. Therefore, Msn5p could be a receptor for several proteins involved in different signaling pathways.
RNA Interference Can Target Pre-mRNA: Consequences for Gene Expression in a Caenorhabditis elegans Operon
Authors: Bosher Julia M , Dufourcq Pascale , Sookhareea Satis , Labouesse Michel
Published: April 2021

In nematodes, flies, trypanosomes, and planarians, introduction of double-stranded RNA results in sequence-specific inactivation of gene function, a process termed RNA interference (RNAi). We demonstrate that RNAi against the Caenorhabditis elegans gene lir-1, which is part of the lir-1/lin-26 operon, induced phenotypes very different from a newly isolated lir-1 null mutation. Specifically, lir-1(RNAi) induced embryonic lethality reminiscent of moderately strong lin-26 alleles, whereas the lir-1 null mutant was viable. We show that the lir-1(RNAi) phenotypes resulted from a severe loss of lin-26 gene expression. In addition, we found that RNAi directed against lir-1 or lin-26 introns induced similar phenotypes, so we conclude that lir-1(RNAi) targets the lir-1/lin-26 pre-mRNA. This provides direct evidence that RNA interference can prevent gene expression by targeting nuclear transcripts. Our results highlight that caution may be necessary when interpreting RNA interference without the benefit of mutant alleles.
Crossing Over During Caenorhabditis elegans Meiosis Requires a Conserved MutS-Based Pathway That Is Partially Dispensable in Budding Yeast
Authors: Zalevsky Jonathan , MacQueen Amy J , Duffy Joseph B , Kemphues Kenneth J , Villeneuve Anne M
Published: April 2021

Formation of crossovers between homologous chromosomes during Caenorhabditis elegans meiosis requires the him-14 gene. Loss of him-14 function severely reduces crossing over, resulting in lack of chiasmata between homologs and consequent missegregation. Cytological analysis showing that homologs are paired and aligned in him-14 pachytene nuclei, together with temperature-shift experiments showing that him-14 functions during the pachytene stage, indicate that him-14 is not needed to establish pairing or synapsis and likely has a more direct role in crossover formation. him-14 encodes a germline-specific member of the MutS family of DNA mismatch repair (MMR) proteins. him-14 has no apparent role in MMR, but like its Saccharomyces cerevisiae ortholog MSH4, has a specialized role in promoting crossing over during meiosis. Despite this conservation, worms and yeast differ significantly in their reliance on this pathway: whereas worms use this pathway to generate most, if not all, crossovers, yeast still form 30–50% of their normal number of crossovers when this pathway is absent. This differential reliance may reflect differential stability of crossover-competent recombination intermediates, or alternatively, the presence of two different pathways for crossover formation in yeast, only one of which predominates during nematode meiosis. We discuss a model in which HIM-14 promotes crossing over by interfering with Holliday junction branch migration.