bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the S-Locus Region of Almond (Prunus dulcis): Analysis of a Somaclonal Mutant and a Cosmid Contig for an S Haplotype
Authors: Ushijima Koichiro , Sassa Hidenori , Tamura Mihoko , Kusaba Makoto , Tao Ryutaro , Gradziel Thomas M , Dandekar Abhaya M , Hirano Hisashi
Published: April 2021

Almond has a self-incompatibility system that is controlled by an S locus consisting of the S-RNase gene and an unidentified “pollen S gene.” An almond cultivar “Jeffries,” a somaclonal mutant of “Nonpareil” (ScSd), has a dysfunctional Sc haplotype both in pistil and pollen. Immunoblot and genomic Southern blot analyses detected no Sc haplotype-specific signal in Jeffries. Southern blot showed that Jeffries has an extra copy of the Sd haplotype. These results indicate that at least two mutations had occurred to generate Jeffries: (1) deletion of the Sc haplotype and (2) duplication of the Sd haplotype. To analyze the extent of the deletion in Jeffries and gain insight into the physical limit of the S locus region, ∼200 kbp of a cosmid contig for the Sc haplotype was constructed. Genomic Southern blot analyses showed that the deletion in Jeffries extends beyond the region covered by the contig. Most cosmid end probes, except those near the Sc-RNase gene, cross-hybridized with DNA fragments from different S haplotypes. This suggests that regions away from the Sc-RNase gene can recombine between different S haplotypes, implying that the cosmid contig extends to the borders of the S locus.
Comparative Gene Expression Profiles Following UV Exposure in Wild-Type and SOS-Deficient Escherichia coli
Authors: Courcelle Justin , Khodursky Arkady , Peter Brian , Brown Patrick O , Hanawalt Philip C
Published: April 2021

The SOS response in UV-irradiated Escherichia coli includes the upregulation of several dozen genes that are negatively regulated by the LexA repressor. Using DNA microarrays containing amplified DNA fragments from 95.5% of all open reading frames identified on the E. coli chromosome, we have examined the changes in gene expression following UV exposure in both wild-type cells and lexA1 mutants, which are unable to induce genes under LexA control. We report here the time courses of expression of the genes surrounding the 26 documented lexA-regulated regions on the E. coli chromosome. We observed 17 additional sites that responded in a lexA-dependent manner and a large number of genes that were upregulated in a lexA-independent manner although upregulation in this manner was generally not more than twofold. In addition, several transcripts were either downregulated or degraded following UV irradiation. These newly identified UV-responsive genes are discussed with respect to their possible roles in cellular recovery following exposure to UV irradiation.
Dissecting the Genetic Pathway to Extreme Fruit Size in Tomato Using a Cross Between the Small-Fruited Wild SpeciesLycopersicon pimpinellifoliumandL. esculentumvar. Giant Heirloom
Authors: Lippman Zachary , Tanksley Steven D
Published: April 2021

In an effort to determine the genetic basis of exceptionally large tomato fruits, QTL analysis was performed on a population derived from a cross between the wild species Lycopersicon pimpinellifolium (average fruit weight, 1 g) and the L. esculentum cultivar var. Giant Heirloom, which bears fruit in excess of 1000 g. QTL analysis revealed that the majority (67%) of phenotypic variation in fruit size could be attributed to six major loci localized on chromosomes 1-3 and 11. None of the QTL map to novel regions of the genome—all have been reported in previous studies involving moderately sized tomatoes. This result suggests that no major QTL beyond those already reported were involved in the evolution of extremely large fruit. However, this is the first time that all six QTL have emerged in a single population, suggesting that exceptionally large-fruited varieties, such as Giant Heirloom, are the result of a novel combination of preexisting QTL alleles. One of the detected QTL, fw2.2, has been cloned and exerts its effect on fruit size through global control of cell division early in carpel/fruit development. However, the most significant QTL detected in this study (fw11.3, lcn11.1) maps to the bottom of chromosome 11 and seems to exert its effect on fruit size through control of carpel/locule number. A second major locus, also affecting carpel number (and hence fruit size), was mapped to chromosome 2 (fw2.1, lcn2.1). We propose that these two carpel number QTL correspond to the loci described by early classical geneticists as fasciated (f) and locule number (lc), respectively.
Detection of Closely Linked Multiple Quantitative Trait Loci Using a Genetic Algorithm
Authors: Nakamichi Reiichiro , Ukai Yasuo , Kishino Hirohisa
Published: April 2021

The existence of a quantitative trait locus (QTL) is usually tested using the likelihood of the quantitative trait on the basis of phenotypic character data plus the recombination fraction between QTL and flanking markers. When doing this, the likelihood is calculated for all possible locations on the linkage map. When multiple QTL are suspected close by, it is impractical to calculate the likelihood for all possible combinations of numbers and locations of QTL. Here, we propose a genetic algorithm (GA) for the heuristic solution of this problem. GA can globally search the optimum by improving the “genotype” with alterations called “recombination” and “mutation.” The “genotype” of our GA is the number and location of QTL. The “fitness” is a function based on the likelihood plus Akaike’s information criterion (AIC), which helps avoid false-positive QTL. A simulation study comparing the new method with existing QTL mapping packages shows the advantage of the new GA. The GA reliably distinguishes multiple QTL located in a single marker interval.
A Frameshift Mutation in MC1R and a High Frequency of Somatic Reversions Cause Black Spotting in Pigs
Authors: Kijas J M H , Moller M , Plastow G , Andersson L
Published: April 2021

Black spotting on a red or white background in pigs is determined by the EP allele at the MC1R/Extension locus. A previous comparison of partial MC1R sequences revealed that EP shares a missense mutation (D121N) with the ED2 allele for dominant black color. Sequence analysis of the entire coding region now reveals a second mutation in the form of a 2-bp insertion at codon 23 (nt67insCC). This mutation expands a tract of six C nucleotides to eight and introduces a premature stop codon at position 56. This frameshift mutation is expected to cause a recessive red color, which was in fact observed in some breeds with the EP allele present (Tamworth and Hereford). RT-PCR analyses were conducted using skin samples taken from both spotted and background areas of spotted pigs. The background red area had transcript only from the mutant nt67insCC MC1R allele, whereas the black spot also contained a transcript without the 2-bp insertion. This indicates that black spots are due to somatic reversion events that restore the frame and MC1R function. The phenotypic expression of the EP allele is highly variable and the associated coat color ranges from red, red with black spots, white with black spots, to almost completely solid black. In several breeds of pigs the phenotypic manifestation of this allele has been modified by selection for or against black spots.
Estimating the Time to the Most Recent Common Ancestor for the Y chromosome or Mitochondrial DNA for a Pair of Individuals
Authors: Walsh Bruce
Published: April 2021

Bayesian posterior distributions are obtained for the time to the most recent common ancestor (MRCA) for a nonrecombining segment of DNA (such as the nonpseudoautosomal arm of the Y chromosome or the mitochondrial genome) for two individuals given that they match at k out of n scored markers. We argue that the distribution of the time t to the MRCA is the most natural measure of relatedness for such nonrecombining regions. Both an infinite-alleles (no recurring mutants) and stepwise mutation model are examined, and these agree well when n is moderate to large and k/n is close to one. As expected, the infinite alleles model underestimates t relative to the stepwise model. Using a modest number (20) of microsatellite markers is sufficient to obtain reasonably precise estimates of t for individuals separated by 200 or less generations. Hence, the multilocus haplotypes of two individuals can be used not only to date very deep ancestry but also rather recent ancestry as well. Finally, our results have forensic implications in that a complete match at all markers between a suspect and a sample excludes only a modest subset of the population unless a very large number of markers (>500 microsatellites) are used.
Multiple Signaling Mechanisms of the UNC-6/netrin Receptors UNC-5 and UNC-40/DCC in Vivo
Authors: Merz David C , Zheng Hong , Killeen Marie T , Krizus Aldis , Culotti Joseph G
Published: April 2021

Cell and growth cone migrations along the dorsoventral axis of Caenorhabditis elegans are mediated by the UNC-5 and UNC-40 receptor subtypes for the secreted UNC-6 guidance cue. To characterize UNC-6 receptor function in vivo, we have examined genetic interactions between unc-5 and unc-40 in the migrations of the hermaphrodite distal tip cells. We report that cell migration defects as severe as those associated with a null mutation in unc-6 are produced only by null mutations in both unc-5 and unc-40, indicating that either receptor retains some partial function in the absence of the other. We show that hypomorphic unc-5 alleles exhibit two distinct types of interallelic genetic interactions. In an unc-40 wild-type genetic background, some pairs of hypomorphic unc-5 alleles exhibit a partial allelic complementation. In an unc-40 null background, however, we observed that unc-5 hypomorphs exhibit dominant negative effects. We propose that the UNC-5 and UNC-40 netrin receptors can function to mediate chemorepulsion in DTC migrations either independently or together, and the observed genetic interactions suggest that this flexibility in modes of signaling results from the formation of a variety of oligomeric receptor complexes.
An Interaction Type of Genetic Screen Reveals a Role of the Rab11 Gene in oskar mRNA Localization in the Developing Drosophila melanogaster Oocyte
Authors: Jankovics Ferenc , Sinka Rita , Erdélyi Miklós
Published: April 2021

Abdomen and germ cell development of Drosophila melanogaster embryo requires proper localization of oskar mRNA to the posterior pole of the developing oocyte. oskar mRNA localization depends on complex cell biological events like cell-cell communication, dynamic rearrangement of the microtubule network, and function of the actin cytoskeleton of the oocyte. To investigate the cellular mechanisms involved, we developed a novel interaction type of genetic screen by which we isolated 14 dominant enhancers of a sensitized genetic background composed of mutations in oskar and in TropomyosinII, an actin binding protein. Here we describe the detailed analysis of two allelic modifiers that identify Drosophila Rab11, a gene encoding small monomeric GTPase. We demonstrate that mutation of the Rab11 gene, involved in various vesicle transport processes, results in ectopic localization of oskar mRNA, whereas localization of gurken and bicoid mRNAs and signaling between the oocyte and the somatic follicle cells are unaffected. We show that the ectopic oskar mRNA localization in the Rab11 mutants is a consequence of an abnormally polarized oocyte microtubule cytoskeleton. Our results indicate that the internal membranous structures play an important role in the microtubule organization in the Drosophila oocyte and, thus, in oskar RNA localization.
Positive and Negative Selection on the Human Genome
Authors: Fay Justin C , Wyckoff Gerald J , Wu Chung-I
Published: April 2021

The distinction between deleterious, neutral, and adaptive mutations is a fundamental problem in the study of molecular evolution. Two significant quantities are the fraction of DNA variation in natural populations that is deleterious and destined to be eliminated and the fraction of fixed differences between species driven by positive Darwinian selection. We estimate these quantities using the large number of human genes for which there are polymorphism and divergence data. The fraction of amino acid mutations that is neutral is estimated to be 0.20 from the ratio of common amino acid (A) to synonymous (S) single nucleotide polymorphisms (SNPs) at frequencies of ≥ 15%. Among the 80% of amino acid mutations that are deleterious at least 20% of them are only slightly deleterious and often attain frequencies of 1–10%. We estimate that these slightly deleterious mutations comprise at least 3% of amino acid SNPs in the average individual or at least 300 per diploid genome. This estimate is not sensitive to human population history. The A/S ratio of fixed differences is greater than that of common SNPs and suggests that a large fraction of protein divergence is adaptive and driven by positive Darwinian selection.
Human DNA Sequence Variation in a 6.6-kb Region Containing the Melanocortin 1 Receptor Promoter
Authors: Makova Kateryna D , Ramsay Michele , Jenkins Trefor , Li Wen-Hsiung
Published: April 2021

An ∼6.6-kb region located upstream from the melanocortin 1 receptor (MC1R) gene and containing its promoter was sequenced in 54 humans (18 Africans, 18 Asians, and 18 Europeans) and in one chimpanzee, gorilla, and orangutan. Seventy-six polymorphic sites were found among the human sequences and the average nucleotide diversity (π) was 0.141%, one of the highest among all studies of nuclear sequence variation in humans. Opposite to the pattern observed in the MC1R coding region, in the present region π is highest in Africans (0.136%) compared to Asians (0.116%) and Europeans (0.122%). The distributions of π, θ, and Fu and Li's F-statistic are nonuniform along the sequence and among continents. The pattern of genetic variation is consistent with a population expansion in Africans. We also suggest a possible phase of population size reduction in non-Africans and purifying selection acting in the middle subregion and parts of the 5′ subregion in Africans. We hypothesize diversifying selection acting on some sites in the 5′ and 3′ subregions or in the MC1R coding region in Asians and Europeans, though we cannot reject the possibility of relaxation of functional constraints in the MC1R gene in Asians and Europeans. The mutation rate in the sequenced region is 1.65 × 10—9 per site per year. The age of the most recent common ancestor for this region is similar to that for the other long noncoding regions studied to date, providing evidence for ancient gene genealogies. Our population screening and phylogenetic footprinting suggest potentially important sites for the MC1R promoter function.
Accurate Partition of Individuals Into Full-Sib Families From Genetic Data Without Parental Information
Authors: Smith Bruce R , Herbinger Christophe M , Merry Heather R
Published: April 2021

Two Markov chain Monte Carlo algorithms are proposed that allow the partitioning of individuals into full-sib groups using single-locus genetic marker data when no parental information is available. These algorithms present a method of moving through the sibship configuration space and locating the configuration that maximizes an overall score on the basis of pairwise likelihood ratios of being full-sib or unrelated or maximizes the full joint likelihood of the proposed family structure. Using these methods, up to 757 out of 759 Atlantic salmon were correctly classified into 12 full-sib families of unequal size using four microsatellite markers. Large-scale simulations were performed to assess the sensitivity of the procedures to the number of loci and number of alleles per locus, the allelic distribution type, the distribution of families, and the independent knowledge of population allelic frequencies. The number of loci and the number of alleles per locus had the most impact on accuracy. Very good accuracy can be obtained with as few as four loci when they have at least eight alleles. Accuracy decreases when using allelic frequencies estimated in small target samples with skewed family distributions with the pairwise likelihood approach. We present an iterative approach that partly corrects that problem. The full likelihood approach is less sensitive to the precision of allelic frequencies estimates but did not perform as well with the large data set or when little information was available (e.g., four loci with four alleles).
Saccharomyces cerevisiae rad51 Mutants Are Defective in DNA Damage-Associated Sister Chromatid Exchanges but Exhibit Increased Rates of Homology-Directed Translocations
Authors: Fasullo Michael , Giallanza Peter , Dong Zheng , Cera Cinzia , Bennett Thomas
Published: April 2021

Saccharomyces cerevisiae Rad51 is structurally similar to Escherichia coli RecA. We investigated the role of S. cerevisiae RAD51 in DNA damage-associated unequal sister chromatid exchanges (SCEs), translocations, and inversions. The frequency of these rearrangements was measured by monitoring mitotic recombination between two his3 fragments, his3-Δ5′ and his3-Δ3′::HOcs, when positioned on different chromosomes or in tandem and oriented in direct or inverted orientation. Recombination was measured after cells were exposed to chemical agents and radiation and after HO endonuclease digestion at his3-Δ3′::HOcs. Wild-type and rad51 mutant strains showed no difference in the rate of spontaneous SCEs; however, the rate of spontaneous inversions was decreased threefold in the rad51 mutant. The rad51 null mutant was defective in DNA damage-associated SCE when cells were exposed to either radiation or chemical DNA-damaging agents or when HO endonuclease-induced double-strand breaks (DSBs) were directly targeted at his3-Δ3′::HOcs. The defect in DNA damage-associated SCEs in rad51 mutants correlated with an eightfold higher spontaneous level of directed translocations in diploid strains and with a higher level of radiation-associated translocations. We suggest that S. cerevisiae RAD51 facilitates genomic stability by reducing nonreciprocal translocations generated by RAD51-independent break-induced replication (BIR) mechanisms.
Correct Regulation of the Septation Initiation Network in Schizosaccharomyces pombe Requires the Activities of par1 and par2
Authors: Jiang Wei , Hallberg Richard L
Published: April 2021

In Schizosaccharomyces pombe, the initiation of cytokinesis is regulated by a septation initiation network (SIN). We previously reported that deletion of par1 and par2, two S. pombe genes encoding B′ regulatory subunits of protein phosphatase 2A, causes a multiseptation phenotype, very similar to that seen in hyperactive SIN mutants. In this study, we examined the genetic interactions between par deletions and mutations in the genes encoding components of SIN and found that deletion of par1 and par2 suppressed the morphological and viability defects caused by overproduction of Byr4p and rescued a loss-of-function allele of spg1. However, par deletions could not suppress any mutations in genes downstream of spg1 in the SIN pathway. We showed further that, in suppressing the lethality of a spg1 loss-of-function allele, the correct localization of Cdc7p to the spindle pole body (SPB), which is normally lost in spg1 mutant cells, was restored. The fact that par mutant cells themselves exhibited a symmetric localization of Cdc7p to SPBs indicated a hyperactivity of SIN in such cells. On the basis of our epistasis analyses and cytological studies, we concluded that par genes normally negatively regulate SIN at or upstream of cdc7, ensuring that multiple rounds of septation do not occur.
Regulation of Physiological Rates in Caenorhabditis elegans by a tRNA-Modifying Enzyme in the Mitochondria
Authors: Lemieux Jason , Lakowski Bernard , Webb Ashley , Meng Yan , Ubach Antonio , Bussière Frédéric , Barnes Thomas , Hekimi Siegfried
Published: April 2021

We show that the phenotype associated with gro-1(e2400) comprises the whole suite of features that characterize the phenotype of the clk mutants in Caenorhabditis elegans, including deregulated developmental, behavioral, and reproductive rates, as well as increased life span and a maternal effect. We cloned gro-1 and found that it encodes a highly conserved cellular enzyme, isopentenylpyrophosphate:tRNA transferase (IPT), which modifies a subset of tRNAs. In yeast, two forms of the enzyme are produced by alternative translation initiation, one of which is mitochondrial. In the gro-1 transcript there are also two possible initiator ATGs, between which there is a sequence predicted to encode a mitochondrial localization signal. A functional GRO-1::GFP fusion protein is localized diffusely throughout the cytoplasm and nucleus. A GRO-1::GFP initiated from the first methionine is localized exclusively to the mitochondria and rescues the mutant phenotype. In contrast, a protein initiated from the second methionine is localized diffusely throughout the cell and does not rescue the mutant phenotype. As oxygen consumption and ATP concentration have been reported to be unaffected in gro-1 mutants, our observations suggest that GRO-1 acts in mitochondria and regulates global physiology by unknown mechanisms.
High Genetic Load in the Pacific Oyster Crassostrea gigas
Authors: Launey Sophie , Hedgecock Dennis
Published: April 2021

The causes of inbreeding depression and the converse phenomenon of heterosis or hybrid vigor remain poorly understood despite their scientific and agricultural importance. In bivalve molluscs, related phenomena, marker-associated heterosis and distortion of marker segregation ratios, have been widely reported over the past 25 years. A large load of deleterious recessive mutations could explain both phenomena, according to the dominance hypothesis of heterosis. Using inbred lines derived from a natural population of Pacific oysters and classical crossbreeding experiments, we compare the segregation ratios of microsatellite DNA markers at 6 hr and 2–3 months postfertilization in F2 or F3 hybrid families. We find evidence for strong and widespread selection against identical-by-descent marker homozygotes. The marker segregation data, when fit to models of selection against linked deleterious recessive mutations and extrapolated to the whole genome, suggest that the wild founders of inbred lines carried a minimum of 8–14 highly deleterious recessive mutations. This evidence for a high genetic load strongly supports the dominance theory of heterosis and inbreeding depression and establishes the oyster as an animal model for understanding the genetic and physiological causes of these economically important phenomena.
Interaction Between Maternal Effect and Zygotic Effect Mutations During Maize Seed Development
Authors: Evans Matthew M S , Kermicle Jerry L
Published: April 2021

Double fertilization of the embryo sac by the two sperm cells of a pollen grain initiates seed development. Proper development of the seed depends not only on the action of genes from the resulting embryo and endosperm, but also on maternal genes acting at two stages. Mutations with both sporophytic maternal effects and gametophytic maternal effects have been identified. A new maternal effect mutation in maize, maternal effect lethal1 (mel1), causes the production of defective seed from mutant female gametophytes. It shows reduced pollen transmission, suggesting a requirement in the male gametophyte, but has no paternal effect on seed development. Interestingly, the defective kernel phenotype of mel1 is conditioned only in seeds that inherit mel1 maternally and are homozygous for the recessive allele (endogenous to the W22 inbred line) of either of two genes, sporophyte enhancer of mel1 (snm1) or snm2, suggesting redundancy between maternally and zygotically required genes. Both mel1 and snm1 map to the short arm of chromosome 2, and snm2 maps to the long arm of chromosome 10. The mode of action of mel1 and the relationship between mel1 and snm1 and snm2 are discussed.
Roles for Internal and Flanking Sequences in Regulating the Activity of Mating-Type-Silencer-Associated Replication Origins in Saccharomyces cerevisiae
Authors: Sharma Karuna , Weinberger Martin , Huberman Joel A
Published: April 2021

ARS301 and ARS302 are inactive replication origins located at the left end of budding yeast (Saccharomyces cerevisiae) chromosome III, where they are associated with the HML-E and -I silencers of the HML mating type cassette. Although they function as replication origins in plasmids, they do not serve as origins in their normal chromosomal locations, because they are programmed to fire so late in S phase that they are passively replicated by the replication fork from neighboring early-firing ARS305 before they have a chance to fire on their own. We asked whether the nucleotide sequences required for plasmid origin function of these silencer-associated chromosomally inactive origins differ from the sequences needed for plasmid origin function by nonsilencer-associated chromosomally active origins. We could not detect consistent differences in sequence requirements for the two types of origins. Next, we asked whether sequences within or flanking these origins are responsible for their chromosomal inactivity. Our results demonstrate that both flanking and internal sequences contribute to chromosomal inactivity, presumably by programming these origins to fire late in S phase. In ARS301, the function of the internal sequences determining chromosomal inactivity is dependent on the checkpoint proteins Mec1p and Rad53p.
Selection Intensity Against Deleterious Mutations in RNA Secondary Structures and Rate of Compensatory Nucleotide Substitutions
Authors: Innan Hideki , Stephan Wolfgang
Published: April 2021

A two-locus model of reversible mutations with compensatory fitness interactions is presented; single mutations are assumed to be deleterious but neutral in appropriate combinations. The expectation of the time of compensatory nucleotide substitutions is calculated analytically for the case of tight linkage between sites. It is shown that selection increases the substitution time dramatically when selection intensity Ns > 1, where N is the diploid population size and s the selection coefficient. Computer simulations demonstrate that recombination increases the substitution time, but the effect of recombination is small when selection is weak. The amount of linkage disequilibrium generated in the process of compensatory substitution is also investigated. It is shown that significant linkage disequilibrium is expected to be rare in natural populations. The model is applied to the mRNA secondary structure of the bicoid 3′ untranslated region of Drosophila. It is concluded that average selection intensity Ns against single deleterious mutations is not likely to be much larger than 1.
Ras1 Interacts With Multiple New Signaling and Cytoskeletal Loci in Drosophila Eggshell Patterning and Morphogenesis
Authors: Schnorr Jon D , Holdcraft Robert , Chevalier Brett , Berg Celeste A
Published: April 2021

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61β, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a “neurospecific” receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (λtop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the λtop eggshell defect whereas smt3 and dock alleles significantly suppressed the λtop phenotype.
The Relationship Between Third-Codon Position Nucleotide Content, Codon Bias, mRNA Secondary Structure and Gene Expression in the Drosophilid Alcohol Dehydrogenase Genes Adh and Adhr
Authors: Carlini David B , Chen Ying , Stephan Wolfgang
Published: April 2021

To gain insights into the relationship between codon bias, mRNA secondary structure, third-codon position nucleotide distribution, and gene expression, we predicted secondary structures in two related drosophilid genes, Adh and Adhr, which differ in degree of codon bias and level of gene expression. Individual structural elements (helices) were inferred using the comparative method. For each gene, four types of randomization simulations were performed to maintain/remove codon bias and/or to maintain or alter third-codon position nucleotide composition (N3). In the weakly expressed, weakly biased gene Adhr, the potential for secondary structure formation was found to be much stronger than in the highly expressed, highly biased gene Adh. This is consistent with the observation of approximately equal G and C percentages in Adhr (~31% across species), whereas in Adh the N3 distribution is shifted toward C (42% across species). Perturbing the N3 distribution to approximately equal amounts of A, G, C, and T increases the potential for secondary structure formation in Adh, but decreases it in Adhr. On the other hand, simulations that reduce codon bias without changing N3 content indicate that codon bias per se has only a weak effect on the formation of secondary structures. These results suggest that, for these two drosophilid genes, secondary structure is a relatively independent, negative regulator of gene expression. Whereas the degree of codon bias is positively correlated with level of gene expression, strong individual secondary structural elements may be selected for to retard mRNA translation and to decrease gene expression.
Interallelic Complementation at theDrosophila melanogaster gastrulation defectiveLocus Defines Discrete Functional Domains of the Protein
Authors: Ponomareff Gregory , Giordano Heidi , DeLotto Yvonne , DeLotto Robert
Published: April 2021

The gastrulation defective (gd) locus encodes a novel serine protease that is involved in specifying the dorsal-ventral axis during embryonic development. Mutant alleles of gd have been classified into three complementation groups, two of which exhibit strong interallelic (intragenic) complementation. To understand the molecular basis of this interallelic complementation, we examined the complementation behavior of additional mutant alleles and sequenced alleles in all complementation groups. The data suggest that there are two discrete functional domains of Gd. A two-domain model of Gd suggesting that it is structurally similar to mammalian complement factors C2 and B has been previously proposed. To test this model we performed SP6 RNA microinjection to assay for activities associated with various domains of Gd. The microinjection data are consistent with the complement factor C2/B-like model. Site-directed mutagenesis suggests that Gd functions as a serine protease. An allele-specific interaction between an autoactivating form of Snake (Snk) and a gd allele altered in the protease domain suggests that Gd directly activates Snk in a protease activation cascade. We propose a model in which Gd is expressed during late oogenesis and bound within the perivitelline space but only becomes catalytically active during embryogenesis.
Patterns of DNA Sequence Variation Suggest the Recent Action of Positive Selection in the janus-ocnus Region of Drosophila simulans
Authors: Parsch John , Meiklejohn Colin D , Hartl Daniel L
Published: April 2021

Levels of nucleotide polymorphism in three paralogous Drosophila simulans genes, janusA (janA), janusB (janB), and ocnus (ocn), were surveyed by DNA sequencing. The three genes lie in tandem within a 2.5-kb region of chromosome arm 3R. In a sample of eight alleles from a worldwide distribution we found a significant departure from neutrality by several statistical tests. The most striking feature of this sample was that in a 1.7-kb region containing the janA and janB genes, 30 out of 31 segregating sites contained variants present only once in the sample, and 29 of these unique variants were found in the same allele. A restriction survey of an additional 28 lines of D. simulans revealed strong linkage disequilibrium over the janA-janB region and identified six more alleles matching the rare haplotype. Among the rare alleles, the level of DNA sequence variation was typical for D. simulans autosomal genes and showed no departure from neutrality. In addition, the rare haplotype was more similar to the D. melanogaster sequence, indicating that it was the ancestral form. These results suggest that the derived haplotype has risen to high worldwide frequency relatively recently, most likely as a result of natural selection.
Protein Variation in ADH and ADH-RELATED in Drosophila pseudoobscura: Linkage Disequilibrium Between Single Nucleotide Polymorphisms and Protein Alleles
Authors: Schaeffer Stephen W , Walthour C Scott , Toleno Donna M , Olek Anna T , Miller Ellen L
Published: April 2021

A 3.5-kb segment of the alcohol dehydrogenase (Adh) region that includes the Adh and Adh-related genes was sequenced in 139 Drosophila pseudoobscura strains collected from 13 populations. The Adh gene encodes four protein alleles and rejects a neutral model of protein evolution with the McDonald-Kreitman test, although the number of segregating synonymous sites is too high to conclude that adaptive selection has operated. The Adh-related gene encodes 18 protein haplotypes and fails to reject an equilibrium neutral model. The populations fail to show significant geographic differentiation of the Adh-related haplotypes. Eight of 404 single nucleotide polymorphisms (SNPs) in the Adh region were in significant linkage disequilibrium with three ADHR protein alleles. Coalescent simulations with and without recombination were used to derive the expected levels of significant linkage disequilibrium between SNPs and 18 protein haplotypes. Maximum levels of linkage disequilibrium are expected for protein alleles at moderate frequencies. In coalescent models without recombination, linkage disequilibrium decays between SNPs and high frequency haplotypes because common alleles mutate to haplotypes that are rare or that reach moderate frequency. The implication of this study is that linkage disequilibrium mapping has the highest probability of success with disease-causing alleles at frequencies of 10%.
Dynamics of Microsatellite Divergence Under Stepwise Mutation and Proportional Slippage/Point Mutation Models
Authors: Calabrese Peter P , Durrett Richard T , Aquadro Charles F
Published: April 2021

Recently Kruglyak, Durrett, Schug, and Aquadro showed that microsatellite equilibrium distributions can result from a balance between polymerase slippage and point mutations. Here, we introduce an elaboration of their model that keeps track of all parts of a perfect repeat and a simplification that ignores point mutations. We develop a detailed mathematical theory for these models that exhibits properties of microsatellite distributions, such as positive skewness of allele lengths, that are consistent with data but are inconsistent with the predictions of the stepwise mutation model. We use our theoretical results to analyze the successes and failures of the genetic distances (δμ)2 and DSW when used to date four divergences: African vs. non-African human populations, humans vs. chimpanzees, Drosophila melanogaster vs. D. simulans, and sheep vs. cattle. The influence of point mutations explains some of the problems with the last two examples, as does the fact that these genetic distances have large stochastic variance. However, we find that these two features are not enough to explain the problems of dating the human-chimpanzee split. One possible explanation of this phenomenon is that long microsatellites have a mutational bias that favors contractions over expansions.
Codon Usage Bias Covaries With Expression Breadth and the Rate of Synonymous Evolution in Humans, but This Is Not Evidence for Selection
Authors: Urrutia Araxi O , Hurst Laurence D
Published: April 2021

In numerous species, from bacteria to Drosophila, evidence suggests that selection acts even on synonymous codon usage: codon bias is greater in more abundantly expressed genes, the rate of synonymous evolution is lower in genes with greater codon bias, and there is consistency between genes in the same species in which codons are preferred. In contrast, in mammals, while nonequal use of alternative codons is observed, the bias is attributed to the background variance in nucleotide concentrations, reflected in the similar nucleotide composition of flanking noncoding and exonic third sites. However, a systematic examination of the covariants of codon usage controlling for background nucleotide content has yet to be performed. Here we present a new method to measure codon bias that corrects for background nucleotide content and apply this to 2396 human genes. Nearly all (99%) exhibit a higher amount of codon bias than expected by chance. The patterns associated with selectively driven codon bias are weakly recovered: Broadly expressed genes have a higher level of bias than do tissue-specific genes, the bias is higher for genes with lower rates of synonymous substitutions, and certain codons are repeatedly preferred. However, while these patterns are suggestive, the first two patterns appear to be methodological artifacts. The last pattern reflects in part biases in usage of nucleotide pairs. We conclude that we find no evidence for selection on codon usage in humans.
A Filamentous Growth Response Mediated by the Yeast Mating Pathway
Authors: Erdman Scott , Snyder Michael
Published: April 2021

Haploid cells of the budding yeast Saccharomyces cerevisiae respond to mating pheromones by arresting their cell-division cycle in G1 and differentiating into a cell type capable of locating and fusing with mating partners. Yeast cells undergo chemotactic cell surface growth when pheromones are present above a threshold level for morphogenesis; however, the morphogenetic responses of cells to levels of pheromone below this threshold have not been systematically explored. Here we show that MATa haploid cells exposed to low levels of the α-factor mating pheromone undergo a novel cellular response: cells modulate their division patterns and cell shape, forming colonies composed of filamentous chains of cells. Time-lapse analysis of filament formation shows that its dynamics are distinct from that of pseudohyphal growth; during pheromone-induced filament formation, daughter cells are delayed relative to mother cells with respect to the timing of bud emergence. Filament formation requires the RSR1(BUD1), BUD8, SLK1/BCK1, and SPA2 genes and many elements of the STE11/STE7 MAP kinase pathway; this response is also independent of FAR1, a gene involved in orienting cell polarization during the mating response. We suggest that mating yeast cells undergo a complex response to low levels of pheromone that may enhance the ability of cells to search for mating partners through the modification of cell shape and alteration of cell-division patterns.
Dual Mutations Reveal Interactions Between Components of Oxidative Phosphorylation in Kluyveromyces lactis
Authors: Clark-Walker G D , Chen X J
Published: April 2021

Loss of mtDNA or mitochondrial protein synthesis cannot be tolerated by wild-type Kluyveromyces lactis. The mitochondrial function responsible for ρ0-lethality has been identified by disruption of nuclear genes encoding electron transport and F0-ATP synthase components of oxidative phosphorylation. Sporulation of diploid strains heterozygous for disruptions in genes for the two components of oxidative phosphorylation results in the formation of nonviable spores inferred to contain both disruptions. Lethality of spores is thought to result from absence of a transmembrane potential, ΔΨ, across the mitochondrial inner membrane due to lack of proton pumping by the electron transport chain or reversal of F1F0-ATP synthase. Synergistic lethality, caused by disruption of nuclear genes, or ρ0-lethality can be suppressed by the atp2.1 mutation in the β-subunit of F1-ATPase. Suppression is viewed as occurring by an increased hydrolysis of ATP by mutant F1, allowing sufficient electrogenic exchange by the translocase of ADP in the matrix for ATP in the cytosol to maintain ΔΨ. In addition, lethality of haploid strains with a disruption of AAC encoding the ADP/ATP translocase can be suppressed by atp2.1. In this case suppression is considered to occur by mutant F1 acting in the forward direction to partially uncouple ATP production, thereby stimulating respiration and relieving detrimental hyperpolarization of the inner membrane. Participation of the ADP/ATP translocase in suppression of ρ0-lethality is supported by the observation that disruption of AAC abolishes suppressor activity of atp2.1.
Overlapping Functions of the Saccharomyces cerevisiae Mre11, Exo1 and Rad27 Nucleases in DNA Metabolism
Authors: Moreau Sylvie , Morgan Elizabeth A , Symington Lorraine S
Published: April 2021

MRE11 functions in several aspects of DNA metabolism, including meiotic recombination, double-strand break repair, and telomere maintenance. Although the purified protein exhibits 3′ to 5′ exonuclease and endonuclease activities in vitro, Mre11 is implicated in the 5′ to 3′ resection of duplex ends in vivo. The mre11-H125N mutation, which eliminates the nuclease activities of Mre11, causes an accumulation of unprocessed double-strand breaks (DSBs) in meiosis, but no defect in processing HO-induced DSBs in mitotic cells, suggesting the existence of redundant activities. Mutation of EXO1, which encodes a 5′ to 3′ exonuclease, was found to increase the ionizing radiation sensitivity of both mre11Δ and mre11-H125N strains, but the exo1 mre11-H125N strain showed normal kinetics of mating-type switching and was more radiation resistant than the mre11Δ strain. This suggests that other nucleases can compensate for loss of the Exo1 and Mre11 nucleases, but not of the Mre11-Rad50-Xrs2 complex. Deletion of RAD27, which encodes a flap endonuclease, causes inviability in mre11 strains. When mre11-H125N was combined with the leaky rad27-6, the double mutants were viable and no more γ-ray sensitive than the mre11-H125N strain. This suggests that the double mutant defect is unlikely to be due to defective DSB processing.
Interval Mapping of Quantitative Trait Loci in Autotetraploid Species
Authors: Hackett C A , Bradshaw J E , McNicol J W
Published: April 2021

This article presents a method for QTL interval mapping in autotetraploid species for a full-sib family derived by crossing two parents. For each offspring, the marker information on each chromosome is used to identify possible configurations of chromosomes inherited from the two parents and the locations of crossovers on these chromosomes. A branch and bound algorithm is used to identify configurations with the minimum number of crossovers. From these configurations, the conditional probability of each possible QTL genotype for a series of positions along the chromosome can be estimated. An iterative weighted regression is then used to relate the trait values to the QTL genotype probabilities. A simulation study is performed to assess this approach and to investigate the effects of the proportion of codominant to dominant markers, the heritability, and the population size. We conclude that the method successfully locates QTL and estimates their parameters accurately, and we discuss different modes of action of the QTL that may be modeled.
Meiotic Alterations in CAG Repeat Tracts
Authors: Schweitzer Jill Kuglin , Reinke Shanda S , Livingston Dennis M
Published: April 2021

We have investigated meiotic changes in CAG repeat tracts embedded in a yeast chromosome. Repeat tracts undergo either conversion events between homologs or expansion and contraction events that appear to be confined to a single chromatid. We did not find evidence for conversion of tract interruptions or excess exchange of flanking markers.
Light and Clock Expression of the Neurospora Clock Gene frequency Is Differentially Driven by but Dependent on WHITE COLLAR-2
Authors: Collett Michael A , Garceau Norm , Dunlap Jay C , Loros Jennifer J
Published: April 2021

Visible light is thought to reset the Neurospora circadian clock by acting through heterodimers of the WHITE COLLAR-1 and WHITE COLLAR-2 proteins to induce transcription of the frequency gene. To characterize this photic entrainment we examined frq expression in constant light, under which condition the mRNA and protein of this clock gene were strongly induced. In continuous illumination FRQ accumulated in a highly phosphorylated state similar to that seen at subjective dusk, the time at which a step from constant light to darkness sets the clock. Examination of frq expression in several wc-2 mutant alleles surprisingly revealed differential regulation when frq expression was compared between constant light, following a light pulse, and darkness (clock-driven expression). Construction of a wc-2 null strain then demonstrated that WC-2 is absolutely required for both light and clock-driven frq expression, in contrast to previous expectations based on presumptive nulls containing altered Zn-finger function. Additionally, we found that frq light signal transduction differs from that of other light-regulated genes. Thus clock and light-driven frq expression is differentially regulated by, but dependent on, WC-2.
Cloning and Characterization of the Tribolium castaneum Eye-Color Genes Encoding Tryptophan Oxygenase and Kynurenine 3-Monooxygenase
Authors: Lorenzen Marcé D , Brown Susan J , Denell Robin E , Beeman Richard W
Published: April 2021

The use of eye-color mutants and their corresponding genes as scorable marker systems has facilitated the development of transformation technology in Drosophila and other insects. In the red flour beetle, Tribolium castaneum, the only currently available system for germline transformation employs the exogenous marker gene, EGFP, driven by an eye-specific promoter. To exploit the advantages offered by eye-pigmentation markers, we decided to develop a transformant selection system for Tribolium on the basis of mutant rescue. The Tribolium orthologs of the Drosophila eye-color genes vermilion (tryptophan oxygenase) and cinnabar (kynurenine 3-monooxygenase) were cloned and characterized. Conceptual translations of Tc vermilion (Tcv) and Tc cinnabar (Tccn) are 71 and 51% identical to their respective Drosophila orthologs. We used RNA interference (RNAi) to show that T. castaneum larvae lacking functional Tcv or Tccn gene products also lack the pigmented eyespots observed in wild-type larvae. Five available eye-color mutations were tested for linkage to Tcv or Tccn via recombinational mapping. No linkage was found between candidate mutations and Tccn. However, tight linkage was found between Tcv and the white-eye mutation white, here renamed vermilionwhite (vw). Molecular analysis indicates that 80% of the Tcv coding region is deleted in vw beetles. These observations suggest that the Tribolium eye is pigmented only by ommochromes, not pteridines, and indicate that Tcv is potentially useful as a germline transformation marker.