bluuebunny/crossref_metadata_2025 huggingface dataset:
Induced Chromosomal Exchange Directs the Segregation of Recombinant Chromatids in Mitosis of Drosophila
Authors: Beumer Kelly J , Pimpinelli Sergio , Golic Kent G
Published: April 2021

In meiosis, the segregation of chromosomes at the reductional division is accomplished by first linking homologs together. Genetic exchange generates the bivalents that direct regular chromosome segregation. We show that genetic exchange in mitosis also generates bivalents and that these bivalents direct mitotic chromosome segregation. After FLP-mediated homologous recombination in G2 of the cell cycle, recombinant chromatids consistently segregate away from each other (x segregation). This pattern of segregation also applies to exchange between heterologs. Most, or all, cases of non-x segregation are the result of exchange in G1. Cytological evidence is presented that confirms the existence of the bivalents that direct this pattern of segregation. Our results implicate sister chromatid cohesion in maintenance of the bivalent. The pattern of chromatid segregation can be altered by providing an additional FRT at a more proximal site on one chromosome. We propose that sister chromatid exchange occurs at the more proximal site, allowing the recombinant chromatids to segregate together. This also allowed the recovery of reciprocal translocations following FLP-mediated heterologous recombination. The observation that exchange can generate a bivalent in mitotic divisions provides support for a simple evolutionary relationship between mitosis and meiosis.
Detection of the Ongoing Sorting of Ancestrally Polymorphic SINEs Toward Fixation or Loss in Populations of Two Species of Charr During Speciation
Authors: Hamada Mitsuhiro , Takasaki Nobuyoshi , Reist James D , DeCicco Alfred L , Goto Akira , Okada Norihiro
Published: April 2021

The FokI family of short interspersed repetitive elements (SINEs) has been found only in the genomes of charr fishes (genus Salvelinus). In an analysis of the insertion of FokI SINEs using PCR, we characterized six loci at which FokI SINEs have been inserted into the genomes of Salvelinus alpinus (Arctic charr) and/or S. malma (Dolly Varden). An analysis of one locus (Fok-223) suggested that a sister relationship exists between S. alpinus and S. malma and the SINE at this locus might have been inserted in a common ancestor of these two species, being fixed in all extant populations examined. By contrast, SINEs at two other loci (Fok-211 and Fok-206) were present specifically in the genome of S. alpinus, with polymorphism among populations of this species. Moreover, the presence or absence of the SINEs of the other three loci (Fok-214, Fok-217, and Fok-600) varied among populations of these two species. The most plausible interpretation of this result is that SINEs, which were ancestrally polymorphic in the genome of a common ancestor of these two species, are involved in an ongoing process of differential sorting and subsequent fixation in the various populations of each species.
A Subset of Conserved tRNA Genes in Plastid DNA of Nongreen Plants
Authors: Lohan Amanda J , Wolfe Kenneth H
Published: April 2021

The plastid genome of the nonphotosynthetic parasitic plant Epifagus virginiana contains only 17 of the 30 tRNA genes normally found in angiosperm plastid DNA. Although this is insufficient for translation, the genome is functional, so import of cytosolic tRNAs into plastids has been suggested. This raises the question of whether the tRNA genes that remain in E. virginiana plastid DNA are active or have just fortuitously escaped deletion. We report the sequences of 20 plastid tRNA loci from Orobanche minor, which shares a nonphotosynthetic ancestor with E. virginiana. The two species have 9 intact tRNA genes in common, the others being defunct in one or both species. The intron-containing trnLUAA gene is absent from E. virginiana, but it is intact, transcribed, and spliced in O. minor. The shared intact genes are better conserved than intergenic sequences, which indicates that these genes are being maintained by natural selection and, therefore, must be functional. For the most part, the tRNA species conserved in nonphotosynthetic plastids are also those that have never been found to be imported in plant mitochondria, which suggests that the same rules may govern tRNA import in the two organelles. A small photosynthesis gene, psbI, is still intact in O. minor, and computer simulations show that some small nonessential genes have an appreciable chance of escaping deletion.
The Population Genetics of Synthetic Lethals
Authors: Phillips Patrick C , Johnson Norman A
Published: April 2021

Synthetic lethals are variants at different loci that have little or no effect on viability singly but cause lethality in combination. The importance of synthetic lethals and, more generally, of synthetic deleterious loci (SDL) has been controversial. Here, we derive the expected frequencies for SDL under a mutation-selection balance for the complete haploid model and selected cases of the diploid model. We have also obtained simple approximations that demonstrate good fit to exact solutions based on numerical iterations. In the haploid case, equilibrium frequencies of carrier haplotypes (individuals with only a single mutation) are comparable to analogous single-locus results, after allowing for the effects of linkage. Frequencies in the diploid case, however, are much higher and more comparable to the square root of the single-locus results. In particular, when selection operates only on the double-mutant homozygote and linkage is not too tight, the expected frequency of the carriers is approximately the quartic root of the ratio between the mutation rate and the selection coefficient of the synthetics. For a reasonably wide set of models, the frequencies of carriers can be on the order of a few percent. The equilibrium frequencies of these deleterious alleles can be relatively high because, with SDL, both dominance and epistasis act to shield carriers from exposure to selection. We also discuss the possible role of SDL in maintaining genetic variation and in hybrid breakdown.
Reassigning Cysteine in the Genetic Code of Escherichia coli
Authors: Döring Volker , Marlière Philippe
Published: April 2021

We investigated directed deviations from the universal genetic code. Mutant tRNAs that incorporate cysteine at positions corresponding to the isoleucine AUU, AUC, and AUA and methionine AUG codons were introduced in Escherichia coli K12. Missense mutations at the cysteine catalytic site of thymidylate synthase were systematically crossed with synthetic suppressor tRNACys genes coexpressed from compatible plasmids. Strains harboring complementary codon/anticodon associations could be stably propagated as thymidine prototrophs. A plasmid-encoded tRNACys reading the codon AUA persisted for more than 500 generations in a strain requiring its suppressor activity for thymidylate biosynthesis, but was eliminated from a strain not requiring it. Cysteine miscoding at the codon AUA was also enforced in the active site of amidase, an enzyme found in Helicobacter pylori and not present in wild-type E. coli. Propagating the amidase missense mutation in E. coli with an aliphatic amide as nitrogen source required the overproduction of Cys-tRNA synthetase together with the complementary suppressor tRNACys. The toxicity of cysteine miscoding was low in all our strains. The small size and amphiphilic character of this amino acid may render it acceptable as a replacement at most protein positions and thus apt to overcome the steric and polar constraints that limit evolution of the genetic code.
Modulation of MSL1 Abundance in Female Drosophila Contributes to the Sex Specificity of Dosage Compensation
Authors: Chang Kimberly A , Kuroda Mitzi I
Published: April 2021

Dosage compensation in Drosophila is the mechanism by which X-linked gene expression is made equal in males and females. Proper regulation of this process is critical to the survival of both sexes. Males must turn the male-specific lethal (msl)-mediated pathway of dosage compensation on and females must keep it off. The msl2 gene is the primary target of negative regulation in females. Preventing production of MSL2 protein is sufficient to prevent dosage compensation; however, ectopic expression of MSL2 protein in females is not sufficient to induce an insurmountable level of dosage compensation, suggesting that an additional component is limiting in females. A candidate for this limiting factor is MSL1, because the amount of MSL1 protein in females is reduced compared to males. We have identified two levels of negative regulation of msl1 in females. The predominant regulation is at the level of protein stability, while a second regulatory mechanism functions at the level of protein synthesis. Overcoming these control mechanisms by overexpressing both MSL1 and MSL2 in females results in 100% female-specific lethality.
LUSH Odorant-Binding Protein Mediates Chemosensory Responses to Alcohols in Drosophila melanogaster
Authors: Kim Min-Su , Repp Allen , Smith Dean P
Published: April 2021

The molecular mechanisms mediating chemosensory discrimination in insects are unknown. Using the enhancer trapping approach, we identified a new Drosophila mutant, lush, with odorant-specific defects in olfactory behavior. lush mutant flies are abnormally attracted to high concentrations of ethanol, propanol, and butanol but have normal chemosensory responses to other odorants. We show that wild-type flies have an active olfactory avoidance mechanism to prevent attraction to concentrated alcohol, and this response is defective in lush mutants. This suggests that the defective olfactory behavior associated with the lush mutation may result from a specific defect in chemoavoidance. lush mutants have a 3-kb deletion that produces a null allele of a new member of the invertebrate odorant-binding protein family, LUSH. LUSH is normally expressed exclusively in a subset of trichoid chemosensory sensilla located on the ventral-lateral surface of the third antennal segment. LUSH is secreted from nonneuronal support cells into the sensillum lymph that bathes the olfactory neurons within these sensilla. Reintroduction of a cloned wild-type copy of lush into the mutant background completely restores wild-type olfactory behavior, demonstrating that this odorant-binding protein is required in a subset of sensilla for normal chemosensory behavior to a subset of odorants. These findings provide direct evidence that odorant-binding proteins are required for normal chemosensory behavior in Drosophila and may partially determine the chemical specificity of olfactory neurons in vivo.
A Screen to Identify Drosophila Genes Required for Integrin-Mediated Adhesion
Authors: Walsh Edmund P , Brown Nicholas H
Published: April 2021

Drosophila integrins have essential adhesive roles during development, including adhesion between the two wing surfaces. Most position-specific integrin mutations cause lethality, and clones of homozygous mutant cells in the wing do not adhere to the apposing surface, causing blisters. We have used FLP-FRT induced mitotic recombination to generate clones of randomly induced mutations in the F1 generation and screened for mutations that cause wing blisters. This phenotype is highly selective, since only 14 lethal complementation groups were identified in screens of the five major chromosome arms. Of the loci identified, 3 are PS integrin genes, 2 are blistered and bloated, and the remaining 9 appear to be newly characterized loci. All 11 nonintegrin loci are required on both sides of the wing, in contrast to integrin α subunit genes. Mutations in 8 loci only disrupt adhesion in the wing, similar to integrin mutations, while mutations in the 3 other loci cause additional wing defects. Mutations in 4 loci, like the strongest integrin mutations, cause a “tail-up” embryonic lethal phenotype, and mutant alleles of 1 of these loci strongly enhance an integrin mutation. Thus several of these loci are good candidates for genes encoding cytoplasmic proteins required for integrin function.
Deletion Mapping of the Head Tilt (het) Gene in Mice: A Vestibular Mutation Causing Specific Absence of Otoliths
Authors: Bergstrom Rebecca A , You Yun , Erway Lawrence C , Lyon Mary F , Schimenti John C
Published: April 2021

Head tilt (het) is a recessive mutation in mice causing vestibular dysfunction. Homozygotes display abnormal responses to position change and linear acceleration and cannot swim. However, they are not deaf. het was mapped to the proximal region of mouse chromosome 17, near the T locus. Here we report anatomical characterization of het mutants and high resolution mapping using a set of chromosome deletions. The defect in het mutants is limited to the utricle and saccule of the inner ear, which completely lack otoliths. The unique specificity of the het mutation provides an opportunity to better understand the development of the vestibular system. Complementation analyses with a collection of embryonic stem (ES)- and germ cell-induced deletions localized het to an interval near the centromere of chromosome 17 that was indivisible by recombination mapping. This approach demonstrates the utility of chromosome deletions as reagents for mapping and characterizing mutations, particularly in situations where recombinational mapping is inadequate.
Organization and Expression of the Mitochondrial Genome in the Nicotiana sylvestris CMSII Mutant
Authors: Lelandais Christine , Albert Béatrice , Gutierres Sophie , De Paepe Rosine , Godelle Bernard , Vedel Fernand , Chétrit Philippe
Published: April 2021

Previous analyses suggested that the Nicotiana sylvestris CMSII mutant carried a large deletion in its mitochondrial genome. Here, we show by cosmid mapping that the deletion is 60 kb in length and contains several mitochondrial genes or ORFs, including the complex I nad7 gene. However, due to the presence of large duplications in the progenitor mitochondrial genome, the only unique gene that appears to be deleted is nad7. RNA gel blot data confirm the absence of nad7 expression, strongly suggesting that the molecular basis for the CMSII abnormal phenotype, poor growth and male sterility, is the altered complex I structure. The CMSII mitochondrial genome appears to consist essentially of one of two subgenomes resulting from recombination between direct short repeats. In the progenitor mitochondrial genome both recombination products are detected by PCR and, reciprocally, the parental fragments are detected at the substoichiometric level in the mutant. The CMSII mtDNA organization has been maintained through six sexual generations.
Identification of Trait-Improving Quantitative Trait Loci Alleles From a Wild Rice Relative, Oryza rufipogon
Authors: Xiao Jinhua , Li Jiming , Grandillo Silvana , Ahn Sang Nag , Yuan Longping , Tanksley Steven D , McCouch Susan R
Published: April 2021

Wild species are valued as a unique source of genetic variation, but they have rarely been used for the genetic improvement of quantitative traits. To identify trait-improving quantitative trait loci (QTL) alleles from exotic species, an accession of Oryza rufipogon, a relative of cultivated rice, was chosen on the basis of a genetic diversity study. An interspecific BC2 testcross population (V20A/O. rufipogon//V20B///V20B////Ce64) consisting of 300 families was evaluated for 12 agronomically important quantitative traits. The O. rufipogon accession was phenotypically inferior for all 12 traits. However, transgressive segregants that outperformed the original elite hybrid variety, V20A/Ce64, were observed for all traits examined. A set of 122 RFLP and microsatellite markers was used to identify QTL. A total of 68 significant QTL were identified, and of these, 35 (51%) had beneficial alleles derived from the phenotypically inferior O. rufipogon parent. Nineteen (54%) of these beneficial QTL alleles were free of deleterious effects on other characters. O. rufipogon alleles at two QTL on chromosomes 1 and 2 were associated with an 18 and 17% increase in grain yield per plant, respectively, without delaying maturity or increasing plant height. This discovery suggests that the innovative use of molecular maps and markers can alter the way geneticists utilize wild and exotic germplasm.
Should We Expect Substitution Rate to Depend on Population Size?
Authors: Cherry Joshua L
Published: April 2021

The rate of nucleotide substitution is generally believed to be a decreasingfunction of effective population size, at least for nonsynonymous substitutions. This view was originally based on consideration of slightly deleterious mutations with a fixed distribution of selection coefficients. A realistic model must include the occurrence and fixation of some advantageous mutations that compensate for the loss of fitness due to deleterious substitutions. Some such models, such as so-called “fixed” models, also predict a population size effect on substitution rate. An alternative model, presented here, predicts the near absence of a population size effect on substitution rate. This model is based on concave log-fitness functions and a fixed distribution of mutational effects on the selectively important trait. Simulations of an instance of the model confirm the approximate insensitivity of the substitution rate to population size. Although much experimental evidence has been claimed to support the existence of a population size effect, the body of evidence as a whole is equivocal, and much of the evidence that is supposed to demonstrate such an effect would also suggest that it is very small. Perhaps the proposed model applies well to some genes and not so well to others, and genes therefore vary with regard to the population size effect.
The Mitochondrial Genome of the Hemichordate Balanoglossus carnosus and the Evolution of Deuterostome Mitochondria
Authors: Castresana Jose , Feldmaier-Fuchs Gertraud , Yokobori Shin-ichi , Satoh Noriyuki , Pääbo Svante
Published: April 2021

The complete nucleotide sequence of the mitochondrial genome of the hemichordate Balanoglossus carnosus (acorn worm) was determined. The arrangement of the genes encoding 13 protein, 22 tRNA, and 2 rRNA genes is essentially the same as in vertebrates, indicating that the vertebrate and hemichordate mitochondrial gene arrangement is close to that of their common ancestor, and, thus, that it has been conserved for more than 600 million years, whereas that of echinoderms has been rearranged extensively. The genetic code of hemichordate mitochondria is similar to that of echinoderms in that ATA encodes isoleucine and AGA serine, whereas the codons AAA and AGG, whose amino acid assignments also differ between echinoderms and vertebrates, are absent from the B. carnosus mitochondrial genome. There are three noncoding regions of length 277, 41, and 32 bp: the larger one is likely to be equivalent to the control region of other deuterostomes, while the two others may contain transcriptional promoters for genes encoded on the minor coding strand. Phylogenetic trees estimated from the inferred protein sequences indicate that hemichordates are a sister group of echinoderms.
Mouse Brachyury the Second (T2) Is a Gene Next to Classical T and a Candidate Gene for tct
Authors: Rennebeck Gabriela , Lader Eric , Fujimoto Atsushi , Lei Elissa P , Artzt Karen
Published: April 2021

The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T). A mutation in T2 disrupts notochord development, pointing to the existence of a second T/t complex gene involved in axis development. T2 encodes a novel protein that is disrupted by an insertion in T2Bob mice. Sequence analysis of T2 from several t haplotypes shows that they all share the same changed stop codon, and, thus, T2 is a candidate gene for the t complex tail interaction factor. T1, T2, and the unlinked t-int are distinct and unrelated loci, and mutations in these genes do not complement one another genetically. Either their products interact in the same pathway during the genesis of the embryonic axis, or the T/t region itself is truly complex.
Melanocortin Receptor 1 (MC1R) Mutations and Coat Color in Pigs
Authors: Kijas J M H , Wales R , Törnsten A , Chardon P , Moller M , Andersson L
Published: April 2021

The melanocortin receptor 1 (MC1R) plays a central role in regulation of eumelanin (black/brown) and phaeomelanin (red/yellow) synthesis within the mammalian melanocyte and is encoded by the classical Extension (E) coat color locus. Sequence analysis of MC1R from seven porcine breeds revealed a total of four allelic variants corresponding to five different E alleles. The European wild boar possessed a unique MC1R allele that we believe is required for the expression of a wild-type coat color. Two different MC1R alleles were associated with the dominant black color in pigs. MC1R*2 was found in European Large Black and Chinese Meishan pigs and exhibited two missense mutations compared with the wild-type sequence. Comparative data strongly suggest that one of these, L99P, may form a constitutively active receptor. MC1R*3 was associated with the black color in the Hampshire breed and involved a single missense mutation D121N. This same MC1R variant was also associated with EP, which results in black spots on a white or red background. Two different missense mutations were identified in recessive red (e/e) animals. One of these, A240T, occurs at a highly conserved position, making it a strong candidate for disruption of receptor function.
Extreme Structural Heterogeneity Among the Members of a Maize Retrotransposon Family
Authors: Marillonnet Sylvestre , Wessler Susan R
Published: April 2021

A few families of retrotransposons characterized by the presence of long terminal repeats (LTRs) have amplified relatively recently in maize and account for >50% of the genome. Surprisingly, none of these elements have been shown to cause a single mutation. In contrast, most of the retrotransposon-induced mutations isolated in maize are caused by the insertion of elements that are present in the genome at 2-50 copies. To begin to understand what limits the amplification of this mutagenic class of LTR-retrotransposons, we are focusing on five elements previously identified among 17 mutations of the maize waxy gene. One of these elements, Stonor, has sustained a deletion of the entire gag region and part of the protease domain. Missing sequences were recovered from larger members of the Stonor family and indicate that the deletion probably occurred during retrotransposition. These large elements have an exceptionally long leader of 2 kb that includes a highly variable region of ∼1 kb that has not been seen in previously characterized retrotransposons. This region serves to distinguish each member of the Stonor family and indicates that no single element has yet evolved that can attain the very high copy numbers characteristic of other element families in maize.
The Anatomy of a Hypoxic Operator in Saccharomyces cerevisiae
Authors: Deckert Jutta , Torres Ana Maria Rodriguez , Hwang Soo Myung , Kastaniotis Alexander J , Zitomer Richard S
Published: April 2021

Aerobic repression of the hypoxic genes of Saccharomyces cerevisiae is mediated by the DNA-binding protein Rox1 and the Tup1/Ssn6 general repression complex. To determine the DNA sequence requirements for repression, we carried out a mutational analysis of the consensus Rox1-binding site and an analysis of the arrangement of the Rox1 sites into operators in the hypoxic ANB1 gene. We found that single base pair substitutions in the consensus sequence resulted in lower affinities for Rox1, and the decreased affinity of Rox1 for mutant sites correlated with the ability of these sites to repress expression of the hypoxic ANB1 gene. In addition, there was a general but not complete correlation between the strength of repression of a given hypoxic gene and the compliance of the Rox1 sites in that gene to the consensus sequence. An analysis of the ANB1 operators revealed that the two Rox1 sites within an operator acted synergistically in vivo, but that Rox1 did not bind cooperatively in vitro, suggesting the presence of a higher order repression complex in the cell. In addition, the spacing or helical phasing of the Rox1 sites was not important in repression. The differential repression by the two operators of the ANB1 gene was found to be due partly to the location of the operators and partly to the sequences between the two Rox1-binding sites in each. Finally, while Rox1 repression requires the Tup1/Ssn6 general repression complex and this complex has been proposed to require the aminoterminal regions of histones H3 and H4 for full repression of a number of genes, we found that these regions were dispensable for ANB1 repression and the repression of two other hypoxic genes.
Genetic Characterization of the Drosophila melanogaster Suppressor of deltex Gene: A Regulator of Notch Signaling
Authors: Fostier Maggy , Evans Dana A P , Artavanis-Tsakonas Spyros , Baron Martin
Published: April 2021

The Notch receptor signaling pathway regulates cell differentiation during the development of multicellular organisms. A number of genes are known to be components of the pathway or regulators of the Notch signal. One candidate for a modifier of Notch function is the Drosophila Suppressor of deltex gene [Su(dx)]. We have isolated four new alleles of Su(dx) and mapped the gene between 22B4 and 22C2. Loss-of-function Su(dx) mutations were found to suppress phenotypes resulting from loss-of-function of Notch signaling and to enhance gain-of-function Notch mutations. Hairless, a mutation in a known negative regulator of the Notch pathway, was also enhanced by Su(dx). Phenotypes were identified for Su(dx) in wing vein development, and a role was demonstrated for the gene between 20 and 30 hr after puparium formation. This corresponds to the period when the Notch protein is involved in refining the vein competent territories. Taken together, our data indicate a role for Su(dx) as a negative regulator of Notch function.
Retrotransposon-Related DNA Sequences in the Centromeres of Grass Chromosomes
Authors: Miller Joseph T , Dong Fenggao , Jackson Scott A , Song Junqi , Jiang Jiming
Published: April 2021

Several distinct DNA fragments were subcloned from a sorghum (Sorghum bicolor) bacterial artificial chromosome clone 13I16 that was derived from a centromere. Three fragments showed significant sequence identity to either Ty3/gypsy- or Ty1/copia-like retrotransposons. Fluorescence in situ hybridization (FISH) analysis revealed that the Ty1/copia-related DNA sequences are not specific to the centromeric regions. However, the Ty3/gypsy-related sequences were present exclusively in the centromeres of all sorghum chromosomes. FISH and gel-blot hybridization showed that these sequences are also conserved in the centromeric regions of all species within Gramineae. Thus, we report a new retrotransposon that is conserved in specific chromosomal regions of distantly related eukaryotic species. We propose that the Ty3/gypsy-like retrotransposons in the grass centromeres may be ancient insertions and are likely to have been amplified during centromere evolution. The possible role of centromeric retrotransposons in plant centromere function is discussed.
Molecular Population Genetics of the rp49 Gene Region in Different Chromosomal Inversions of Drosophila subobscura
Authors: Rozas Julio , Segarra Carmen , Ribó Griselda , Aguadé Montserrat
Published: April 2021

Nucleotide variation at the ribosomal protein 49 (rp49) gene region has been studied in 75 lines of Drosophila subobscura belonging to four chromosomal arrangements (Ost, O3+4, O3+4+8, and O3+4+23). The location of the rp49 gene region within the inversion loop differs among heterokaryotypes: it is very close to one of the breakpoints in heterozygotes involving Ost chromosomes, while it is in a more central position in all other heterokaryotypes. The distribution of nucleotide polymorphism in the different arrangements is consistent with a monophyletic origin of the inversions. The data also provide evidence that gene conversion and possibly double crossover are involved in shuffling nucleotide variation among gene arrangements. The analyses reveal that the level of genetic exchange is higher when the region is located in a more central position of the inverted fragment than when it is close to the breakpoints. The pairwise difference distributions as well as the negative values of Tajima's and Fu and Li's statistics further support the hypothesis that nucleotide variation within chromosomal arrangements still reflects expansion after the origin of the inversions. Under the expansion model, we have estimated the time of origin of the studied inversions.
Organization of the Large Mitochondrial Genome in the Isopod Armadillidium vulgare
Authors: Raimond Roland , Marcadé Isabelle , Bouchon Didier , Rigaud Thierry , Bossy Jean-Pierre , Souty-Grosset Catherine
Published: April 2021

The mitochondrial DNA (mtDNA) in animals is generally a circular molecule of ∼15 kb, but there are many exceptions such as linear molecules and larger ones. RFLP studies indicated that the mtDNA in the terrestrial isopod Armadillidium vulgare varied from 20 to 42 kb. This variation depended on the restriction enzyme used, and on the restriction profile generated by a given enzyme. The DNA fragments had characteristic electrophoretic behaviors. Digestions with two endonucleases always generated fewer fragments than expected; denaturation of restriction profiles reduced the size of two bands by half; densitometry indicated that a number of small fragments were present in stoichiometry, which has approximately twice the expected concentration. Finally, hybridization to a 550-bp 16S rDNA probe often revealed two copies of this gene. These results cannot be due to the genetic rearrangements generally invoked to explain large mtDNA. We propose that the large A. vulgare mtDNA is produced by the tripling of a 14-kb monomer with a singular rearrangement: one monomer is linear and the other two form a circular dimer. Densitometry suggested that these two molecular structures were present in different proportions within a single individual. The absence of mutations within the dimers also suggests that replication occurs during the monomer phase.
Suppressors of the Arabidopsis lsd5 Cell Death Mutation Identify Genes Involved in Regulating Disease Resistance Responses
Authors: Morel Jean-Benoit , Dangl Jeffery L
Published: April 2021

Cell death is associated with the development of the plant disease resistance hypersensitive reaction (HR). Arabidopsis lsd mutants that spontaneously exhibit cell death reminiscent of the HR were identified previously. To study further the regulatory context in which cell death acts during disease resistance, one of these mutants, lsd5, was used to isolate new mutations that suppress its cell death phenotype. Using a simple lethal screen, nine lsd5 cell death suppressors, designated phx (for the mythological bird Phoenix that rises from its ashes), were isolated. These mutants were characterized with respect to their response to a bacterial pathogen and oomycete parasite. The strongest suppressors—phx2, 3, 6, and 11-1—showed complex, differential patterns of disease resistance modifications. These suppressors attenuated disease resistance to avirulent isolates of the biotrophic Peronospora parasitica pathogen, but only phx2 and phx3 altered disease resistance to avirulent strains of Pseudomonas syringae pv tomato. Therefore, some of these phx mutants define common regulators of cell death and disease resistance. In addition, phx2 and phx3 exhibited enhanced disease susceptibility to different virulent pathogens, confirming probable links between the disease resistance and susceptibility pathways.
Unpredictable Fitness Transitions Between Haploid and Diploid Strains of the Genetically Loaded Yeast Saccharomyces cerevisiae
Authors: Korona Ryszard
Published: April 2021

Mutator strains of yeast were used to accumulate random point mutations. Most of the observed changes in fitness were negative and relatively small, although major decreases and increases were also present. The average fitness of haploid strains was lowered by ∼25% due to the accumulated genetic load. The impact of the load remained basically unchanged when a homozygous diploid was compared with the haploid from which it was derived. In other experiments a heterozygous diploid was compared with the two different loaded haploids from which it was obtained. The fitness of such a loaded diploid was much less reduced and did not correlate with the average fitness of the two haploids. There was a fitness correlation, however, when genetically related heterozygous diploids were compared, indicating that the fitness effects of the new alleles were not entirely lost in the heterozygotes. It is argued here that to explain the observed pattern of fitness transitions it is necessary to invoke nonadditive genetic interactions that go beyond the uniform masking effect of wild-type alleles. Thus, the results gathered with haploids and homozygotes should be extrapolated to heterozygotes with caution when multiple loci contribute to the genetic load.
Propagation of a Novel Cytoplasmic, Infectious and Deleterious Determinant Is Controlled by Translational Accuracy in Podospora anserina
Authors: Silar Philippe , Haedens Vicki , Rossignol Michèle , Lalucque Hervé
Published: April 2021

Some mutant strains of the filamentous fungus Podospora anserina spontaneously present a growth impairment, which has been called Crippled Growth (CG). CG is caused by a cytoplasmic and infectious factor, C. C is efficiently transmitted during mitosis but is not transmitted to the progeny after sexual crosses. C is induced by stationary phase and cured by various means, most of which stress the cells. Translational accuracy is shown to tightly regulate the propagation of C during the active growth period, because its propagation in dividing hyphae is restricted to cells that display an increased translational accuracy. However, induction of C in stationary phase proceeds independently from the translational accuracy status of the strain. CG does not seem to be accompanied by mitochondrial DNA modifications, although C activates the action of the Determinant of Senescence, another cytoplasmic and infectious element, which causes a disorganization of the mitochondrial genome. In addition, presence of C drastically modifies the spectrum of the mitochondrial DNA rearrangements in AS6-5 mat–cultures during Senescence. C seems to belong to the growing list of unconventional genetic elements. The biological significance of such elements is discussed.
The Effect of Overdominance on Characterizing Deleterious Mutations in Large Natural Populations
Authors: Li Jin-Long , Li Jian , Deng Hong-Wen
Published: April 2021

Alternatives to the mutation-accumulation approach have been developed to characterize deleterious genomic mutations. However, they all depend on the assumption that the standing genetic variation in natural populations is solely due to mutation-selection (M-S) balance and therefore that overdominance does not contribute to heterosis. Despite tremendous efforts, the extent to which this assumption is valid is unknown. With different degrees of violation of the M-S balance assumption in large equilibrium populations, we investigated the statistical properties and the robustness of these alternative methods in the presence of overdominance. We found that for dominant mutations, estimates for U (genomic mutation rate) will be biased upward and those for h̄ (mean dominance coefficient) and s̄ (mean selection coefficient), biased downward when additional overdominant mutations are present. However, the degree of bias is generally moderate and depends largely on the magnitude of the contribution of overdominant mutations to heterosis or genetic variation. This renders the estimates of U and s̄ not always biased under variable mutation effects that, when working alone, cause U and s̄ to be underestimated. The contributions to heterosis and genetic variation from overdominant mutations are monotonic but not linearly proportional to each other. Our results not only provide a basis for the correct inference of deleterious mutation parameters from natural populations, but also alleviate the biggest concern in applying the new approaches, thus paving the way for reliably estimating properties of deleterious mutations.
Mechanisms of Double-Strand-Break Repair During Gene Targeting in Mammalian Cells
Authors: Ng Philip , Baker Mark D
Published: April 2021

In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin μ-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal μ-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal μ-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process: The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an “end” bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence.Formation of hDNA was frequently associated with gene targeting and, in most cases, began ∼645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.
Analysis of Mutations in the Yeast mRNA Decapping Enzyme
Authors: Tharun Sundaresan , Parker Roy
Published: April 2021

A major mechanism of mRNA decay in yeast is initiated by deadenylation, followed by mRNA decapping, which exposes the transcript to 5′ to 3′ exonucleolytic degradation. The decapping enzyme that removes the 5′ cap structure is encoded by the DCP1 gene. To understand the function of the decapping enzyme, we used alanine scanning mutagenesis to create 31 mutant versions of the enzyme, and we examined the effects of the mutations both in vivo and in vitro. Two types of mutations that affected mRNA decapping in vivo were identified, including a temperature-sensitive allele. First, two mutants produced decapping enzymes that were defective for decapping in vitro, suggesting that these mutated residues are required for enzymatic activity. In contrast, several mutants that moderately affected mRNA decapping in vivo yielded decapping enzymes that had at least the same specific activity as the wild-type enzyme in vitro. Combination of alleles within this group yielded decapping enzymes that showed a strong loss of function in vivo, but that still produced fully active enzymes in vitro. This suggested that interactions of the decapping enzyme with other factors may be required for efficient decapping in vivo, and that these particular mutations may be disrupting such interactions. Interestingly, partial loss of decapping activity in vivo led to a defect in normal deadenylation-dependent decapping, but it did not affect the rapid deadenylation-independent decapping triggered by early nonsense codons. This observation suggested that these two types of mRNA decapping differ in their requirements for the decapping enzyme.
Bayesian Mapping of Multiple Quantitative Trait Loci From Incomplete Outbred Offspring Data
Authors: Sillanpää Mikko J , Arjas Elja
Published: April 2021

A general fine-scale Bayesian quantitative trait locus (QTL) mapping method for outcrossing species is presented. It is suitable for an analysis of complete and incomplete data from experimental designs of F2 families or backcrosses. The amount of genotyping of parents and grandparents is optional, as well as the assumption that the QTL alleles in the crossed lines are fixed. Grandparental origin indicators are used, but without forgetting the original genotype or allelic origin information. The method treats the number of QTL in the analyzed chromosome as a random variable and allows some QTL effects from other chromosomes to be taken into account in a composite interval mapping manner. A block-update of ordered genotypes (haplotypes) of the whole family is sampled once in each marker locus during every round of the Markov Chain Monte Carlo algorithm used in the numerical estimation. As a byproduct, the method gives the posterior distributions for linkage phases in the family and therefore it can also be used as a haplotyping algorithm. The Bayesian method is tested and compared with two frequentist methods using simulated data sets, considering two different parental crosses and three different levels of available parental information. The method is implemented as a software package and is freely available under the name Multimapper/outbred at URL http://www.rni.helsinki.fi/~mjs/.
Maximal Stimulation of Meiotic Recombination by a Yeast Transcription Factor Requires the Transcription Activation Domain and a DNA-Binding Domain
Authors: Kirkpatrick David T , Fan Qingqing , Petes Thomas D
Published: April 2021

The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic recombination hotspot. Although the activity of this hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of recombination. We find that the recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein. We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4. In addition, we find recombination hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors. We suggest that yeast cells have two types of recombination hotspots, α (transcription factor dependent) and β (transcription factor independent).
Transgenic Inhibitors Identify Two Roles for Protein Kinase A in Drosophila Development
Authors: Kiger John A , Eklund Jennifer L , Younger Susan H , O’Kane Cahir J
Published: April 2021

We have initiated an analysis of protein kinase A (PKA) in Drosophila using transgenic techniques to modulate PKA activity in specific tissues during development. We have constructed GAL4/UAS-regulated transgenes in active and mutant forms that encode PKAc, the catalytic subunit of PKA, and PKI(1-31), a competitive inhibitor of PKAc. We present evidence that the wild-type transgenes are active and summarize the phenotypes produced by a number of GAL4 enhancer-detector strains. We compare the effects of transgenes encoding PKI(1-31) with those encoding PKAr*, a mutant regulatory subunit that constitutively inhibits PKAc because of its inability to bind cyclic AMP. Both inhibitors block larval growth, but only PKAr* alters pattern formation by activating the Hedgehog signaling pathway. Therefore, transgenic PKI(1-31) should provide a tool to investigate the role of PKAc in larval growth regulation without concomitant changes in pattern formation. The different effects of PKI(1-31) and PKAr* suggest two distinct roles, cytoplasmic and nuclear, for PKAc in Hedgehog signal transduction. Alternatively, PKAr* may target proteins other than PKAc, suggesting a role for free PKAr in signal transduction, a role inhibited by PKAc in reversal of the classical relationship of these subunits.
Identification and Characterization of Schizosaccharomyces pombe asp1+, a Gene That Interacts with Mutations in the Arp2/3 Complex and Actin
Authors: Feoktistova Anna , McCollum Dannel , Ohi Ryoma , Gould Kathleen L
Published: April 2021

The Arp2/3 complex is an essential component of the actin cytoskeleton in yeast and is required for the movement of actin patches. In an attempt to identify proteins that interact with this complex in the fission yeast Schizosaccharomyces pombe, we sought high-copy suppressors of the S. pombe arp3-c1 mutant, and have identified one, which we have termed asp1+. The asp1+ open reading frame (ORF) predicts a highly conserved protein of 921 amino acids with a molecular mass of 106 kD that does not contain motifs of known function. Neither asp1+ nor its apparent Saccharomyces cerevisiae ortholog, VIP1, are essential genes. However, disruption of asp1+ leads to altered morphology and growth properties at elevated temperatures and defects in polarized growth. The asp1 disruption strain also is hypersensitive to Ca+ ions and to low pH conditions. Although Asp1p is not stably associated with the Arp2/3 complex nor localized in any discrete structure within the cytoplasm, the asp1 disruption mutant was synthetically lethal with mutations in components of the Arp2/3 complex, arp3-c1 and sop2-1, as well as with a mutation in actin, act1-48. Moreover, the vip1 disruption strain showed a negative genetic interaction with a las17Δ strain. We conclude that Asp1p/Vip1p is important for the function of the cortical actin cytoskeleton.
Population Structure and Dynamics of Magnaporthe grisea in the Indian Himalayas
Authors: Kumar J , Nelson R J , Zeigler R S
Published: April 2021

The population genetics of Magnaporthe grisea, the rice blast pathogen, were analyzed in a center of rice diversity (the Uttar Pradesh hills of the Indian Himalayas) using multilocus and single-, or low-copy, DNA markers. Based on DNA fingerprinting with the multilocus probe MGR586 and single-locus probes, 157 haplotypes clustered into 56 lineages (at ≥70% MGR586 band similarity, each with unique single-locus profiles) and high diversity indices were detected among 458 isolates collected from 29 sites during 1992–1995. Most valleys sampled had distinct populations (73% of the lineages were site specific) with some containing one or a few lineages, confirming the importance of clonal propagation, and others were very diverse. Widely distributed lineages suggested that migration occurs across the region and into the Indo-Gangetic plains. Repeated sampling at one site, Matli, (170 isolates, 1992–1995) yielded 19 lineages and diversity significantly greater than that reported from similar samples from Colombia and the Philippines. Analysis of allelic associations using pairwise comparisons and multilocus variance analysis failed to reject the hypothesis of gametic phase equilibrium. The Matli population shifted from highly diverse in 1992 to almost complete dominance by one lineage in 1995. Such population dynamics are consistent with recombination followed by differential survival of clonal descendants of recombinant progeny. At another site, Ranichauri, population (n = 84) composition changed from 2 to 11 lineages over 2 yr and yielded additional evidence for equilibrium. Sexually fertile and hermaphrodite isolates of both mating types were recovered from rice in both Matli and Ranichauri. We demonstrate that Himalayan M. grisea populations are diverse and dynamic and conclude that the structure of some populations may be affected to some extent by sexual recombination.