bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Analysis of rolled, Which Encodes a Drosophila Mitogen-Activated Protein Kinase
Authors: Lim Young-Mi , Nishizawa Kimiko , Nishi Yoshimi , Tsuda Leo , Inoue Yoshihiro H , Nishida Yasuyoshi
Published: April 2021

Genetic and molecular characterization of the dominant suppressors of D-rafC110 on the second chromosome identified two gain-of-function alleles of rolled (rl), which encodes a mitogen-activated protein (MAP) kinase in Drosophila. One of the alleles, rlSu23, was found to bear the same molecular lesion as rlSem, which has been reported to be dominant female sterile. However, rlSu23 and the current stock of rlSem showed only a weak dominant female sterility. Detailed analyses of the rl mutations demonstrated moderate dominant activities of these alleles in the Torso (Tor) signaling pathway, which explains the weak dominant female sterility observed in this study. The dominant rl mutations failed to suppress the terminal class maternal-effect mutations, suggesting that activation of Rl is essential, but not sufficient, for Tor signaling. Involvement of rl in cell proliferation was also demonstrated by clonal analysis. Branching and integration of signals in the MAP kinase cascade is discussed.
Developmental Genetics of the Essential Drosophila Nucleoporin nup154: Allelic Differences Due to an Outward-Directed Promoter in the P-Element 3′ End
Authors: Kiger Amy A , Gigliotti Silvia , Fuller Margaret T
Published: April 2021

Drosophila nup154 encodes a predicted nucleoporin homologous to yeast Nup170p, Nup157p, and vertebrate Nup155, all of which are major components of the nuclear pore complex (NPC). Unlike its yeast homologs, nup154 is essential for viability. Animals with strong loss-of-function nup154 mutations caused by P-element insertion in the 5′-UTR of the gene died as larvae with small discs, brains, and testes. nup154 mRNA expression appeared developmentally regulated in tissues of wild-type embryos, larvae, and adults, suggesting that new nup154 synthesis is required when assembly of new NPCs is required, as in proliferating or growing tissues. Two additional nup154 alleles also associated with different P-element inserts in the 5′-UTR were viable but had strong loss-of-function sterile phenotypes, including failure to maintain spermatogenic stem cells and failure to progress into vitellogenic stages of oogenesis. Lethality vs. viability correlated with orientation of the P-element inserts in the different alleles. Transcript analysis by 5′-RACE suggested a mechanism for allelic differences: an outward-directed promoter internal to the P-element 3′ end able to drive sufficient expression of the nup154 transcript for viability but not for fertility.
The Molecular Genetics of Red and Green Color Vision in Mammals
Authors: Yokoyama Shozo , Radlwimmer F Bernhard
Published: April 2021

To elucidate the molecular mechanisms of red-green color vision in mammals, we have cloned and sequenced the red and green opsin cDNAs of cat (Felis catus), horse (Equus caballus), gray squirrel (Sciurus carolinensis), white-tailed deer (Odocoileus virginianus), and guinea pig (Cavia porcellus). These opsins were expressed in COS1 cells and reconstituted with 11-cis-retinal. The purified visual pigments of the cat, horse, squirrel, deer, and guinea pig have λmax values at 553, 545, 532, 531, and 516 nm, respectively, which are precise to within ±1 nm. We also regenerated the “true” red pigment of goldfish (Carassius auratus), which has a λmax value at 559 ± 4 nm. Multiple linear regression analyses show that S180A, H197Y, Y277F, T285A, and A308S shift the λmax values of the red and green pigments in mammals toward blue by 7, 28, 7, 15, and 16 nm, respectively, and the reverse amino acid changes toward red by the same extents. The additive effects of these amino acid changes fully explain the red-green color vision in a wide range of mammalian species, goldfish, American chameleon (Anolis carolinensis), and pigeon (Columba livia).
Genealogical Evidence for Positive Selection in the nef Gene of HIV-1
Authors: de A. Zanotto Paolo M , Kallas Esper G , de Souza Robson F , Holmes Edward C
Published: April 2021

The pattern and process of evolution in the nef gene of HIV-1 was analyzed within and among patients. Using a maximum likelihood method that allows for variable intensity of selection pressure among codons, strong positive selection was detected in a hemophiliac patient over 30 mo of infection. By reconstructing the process of allele substitution in this patient using parsimony, the synapomorphic amino acid changes separating each time point were found to have high probabilities of being under positive selection, with selective coefficients of at least 3.6%. Positive selection was also detected among 39 nef sequences from HIV-1 subtype B. In contrast, multiple pairwise comparisons of nonsynonymous and synonymous substitution rates provided no good evidence for positive selection and sliding window analyses failed to detect most positively selected sites. These findings demonstrate that positive selection is an important determinant of nef gene evolution and that genealogy-based methods outperform pairwise methods in the detection of adaptive evolution. Mapping the locations of positively selected sites may also be of use in identifying targets of the immune response and hence aid vaccine design.
Local Recombination and Mutation Effects on Molecular Evolution in Drosophila
Authors: Takano-Shimizu Toshiyuki
Published: April 2021

I studied the cause of the significant difference in the synonymous-substitution pattern found in the achaete-scute complex genes in two Drosophila lineages, higher codon bias in Drosophila yakuba, and lower bias in D. melanogaster. Besides these genes, the functionally unrelated yellow gene showed the same substitution pattern, suggesting a region-dependent phenomenon in the X-chromosome telomere. Because the numbers of A/T → G/C substitutions were not significantly different from those of G/C → A/T in the yellow noncoding regions of these species, a AT/GC mutational bias could not completely account for the synonymous-substitution biases. In contrast, we did find an ~14-fold difference in recombination rates in the X-chromosome telomere regions between the two species, suggesting that the reduction of recombination rates in this region resulted in the reduction of the efficacy of selection in D. melanogaster. In addition, the D. orena yellow showed a 5% increase in the G + C content at silent sites in the coding and noncoding regions since the divergence from D. erecta. This pattern was significantly different from those at the orena Adh and Amy loci. These results suggest that local changes in recombination rates and mutational pressures are contributing to the irregular synonymous-substitution patterns in Drosophila.
Unusual Haplotype Structure at the Proximal Breakpoint of In(2L)t in a Natural Population of Drosophila melanogaster
Authors: Andolfatto Peter , Wall Jeffrey D , Kreitman Martin
Published: April 2021

The existence of temporally stable frequency clines for In(2L)t in natural populations of Drosophila melanogaster suggests a role for selection in the maintenance of this polymorphism. We have collected nucleotide polymorphism data from the proximal breakpoint junction regions of In(2L)t to infer its evolutionary history. The finding of a novel LINE-like element near the In(2L)t breakpoint junction in sampled inverted chromosomes supports a transposable element-mediated origin for this inversion. An analysis of nucleotide variation in a Costa Rican population sample of standard and inverted chromosomes indicates a unique and relatively recent origin for In(2L)t. Additional In(2L)t alleles from three geographically diverse populations reveal no detectable geographic differentiation. Low levels of In(2L)t nucleotide polymorphism suggest a recent increase in the inversion's frequency in tropical populations. An unusual feature of our sample of standard alleles is a marked heterogeneity in levels of linkage disequilibrium among polymorphic sites across the breakpoint region. We introduce a test of neutral equilibrium haplotype structure that corrects both for multiple tests and for an arbitrarily chosen window size. It reveals that an ~1.4-kb region immediately spanning the breakpoint has fewer haplotypes than expected under the neutral model, given the expected level of recombination in this genomic region. Certain features of our data suggest that the unusual pattern in standard chromosomes is the product of selection rather than demography.
A Fluorescent Quantitative PCR Approach to Map Gene Deletions in the Drosophila Genome
Authors: Chiang Pei-Wen , Wei Wan-Li , Gibson Kristi , Bodmer Rolf , Kurnit David M
Published: April 2021

We report the application of TaqMan quantitative PCR (QPCR) to map Drosophila chromosome deficiencies by discrimination of twofold copy number differences. For a model system, we used this technology to confirm the X chromosomal mapping of Dspt6 given the autosomal mapping of Dspt4. We then used this technique on both preexisting deletion mutant flies and flies that we generated with deletions to demonstrate the presence or absence of Dspt6, Dspt4, and swa in various deletion mutant flies. In contrast with in situ hybridization studies, QPCR both vitiates the need to do these more intricate studies, and it is more accurate as the site of deletion can be known down to the 102-bp level. We then successfully applied the technique to the analysis of transcription, demonstrating that the amount of Dspt6 or Dspt4 transcriptional product depended directly on the dosage of the Dspt6 or Dspt4 gene, respectively. The rapidity and precision of this method demonstrates its applicability in Drosophila genetics, the rapid and accurate mapping of Drosophila deletion mutants.
The Genetic Architecture of Selection Response: Inferences From Fine-Scale Mapping of Bristle Number Quantitative Trait Loci in Drosophila melanogaster
Authors: Nuzhdin Sergey V , Dilda Christy L , Mackay Trudy F C
Published: April 2021

Quantitative trait loci (QTL) affecting responses and correlated responses to selection for abdominal and sternopleural bristle number have been mapped with high resolution to the X and third chromosomes. Advanced intercross recombinant isogenic chromosomes were constructed from high and low selection lines in an unselected inbred background, and QTL were detected using composite interval mapping and high density transposable element marker maps. We mapped a total of 26 bristle number QTL with large effects, which were in or immediately adjacent to intervals previously inferred to contain bristle number QTL on these chromosomes. The QTL contributing to response to selection for high bristle number were not the same as those contributing to response to selection for low bristle number, suggesting that distributions of allelic effects per locus may be asymmetrical. Correlated responses were more often attributable to loose linkage than pleiotropy or close linkage. Bristle number QTL mapping to the same locations have been inferred in studies with different parental strains. Of the 26 QTL, 20 mapped to locations consistent with candidate genes affecting peripheral nervous system development and/or bristle number. This facilitates determining the molecular basis of quantitative variation and allele frequencies by associating molecular variation at the candidate genes with phenotypic variation in bristle number in samples of alleles from nature.
The Function of the Broad-Complex During Drosophila melanogaster Oogenesis
Authors: Tzolovsky George , Deng Wu-Min , Schlitt Thomas , Bownes Mary
Published: April 2021

The Broad-Complex (BR-C) is an early ecdysone response gene that functions during metamorphosis and encodes a family of zinc-finger transcription factors. It is expressed in a dynamic pattern during oogenesis. Its late expression in the lateral-dorsal-anterior follicle cells is related to the morphogenesis of the chorionic appendages. All four zinc-finger isoforms are expressed in oogenesis, which is consistent with the abnormal appendage phenotypes resulting from their ectopic expression. We investigated the mechanism by which the BR-C affects chorion deposition by using BrdU to follow the effects of BR-C misexpression on DNA replication and in situ hybridization to ovarian mRNA to evaluate chorion gene expression. Ectopic BR-C expression leads to prolonged endoreplication and to additional amplification of genes, besides the chorion genes, at other sites in the genome. The pattern of chorion gene expression is not affected along the anterior-posterior axis, but the follicle cells at the anterior of the oocyte fail to migrate correctly in an anterior direction when BR-C is misexpressed. We conclude that the target genes of the BR-C in oogenesis include a protein essential for endoreplication and chorion gene amplification. This may provide a link between steroid hormones and the control of DNA replication during oogenesis.
Time Course Study of the Chromosome-Type Breakage-Fusion-Bridge Cycle in Maize
Authors: Zheng Yin-Zhou , Roseman Robin R , Carlson Wayne R
Published: April 2021

The B chromosome of maize has been used in a study of dicentric chromosomes. TB-9Sb is a translocation between the B and chromosome 9. The B-9 of TB-9Sb carries 60% of the short arm of 9. For construction of dicentrics, a modified B-9 chromosome was used, B-9-Dp9. It consists of the B-9 chromosome plus a duplicated 9S region attached to the distal end. In meiosis, fold-back pairing and crossing over in the duplicated region gives a chromatid-type dicentric B-9 that subsequently initiates a chromatid-type breakage-fusion-bridge cycle. In the male, it forms a single bridge in anaphase II of meiosis and at the first pollen mitosis. However, the cycle is interrupted by nondisjunction of the B centromere at the second pollen mitosis, which sends the B-9 dicentric to one pole and converts it from a chromatid dicentric to a chromosome dicentric. As expected, the new dicentric undergoes the chromosome-type breakage-fusion-bridge cycle and produces double bridges. A large number of plants with chromosome dicentrics were produced in this way. The presence of double bridges in the root cells of plants with a chromosome dicentric was studied during the first 10 wk of development. It was found that the number of plants and cells showing double bridges declined steadily over the 10-wk period. Several lines of evidence indicate that there was no specific developmental time for dicentric loss. “Healing” of broken chromosomes produced by dicentric breakage accounted for much of the dicentric loss. Healing produced a wide range of derived B-9 chromosomes, some large and some small. A group of minichromosomes found in these experiments probably represents the small end of the scale for B-9 derivatives.
The Identification of Wos2, a p23 Homologue That Interacts With Wee1 and Cdc2 in the Mitotic Control of Fission Yeasts
Authors: Muñoz Manuel J , Bejarano Eduardo R , Daga Rafael R , Jimenez Juan
Published: April 2021

The Wee1 kinase inhibits entry into mitosis by phosphorylation of the Cdc2 kinase. Searching for multicopy suppressors that abolish this inhibition in the fission yeast, we have identified a novel gene, here named wos2, encoding a protein with significant homology to human p23, an Hsp90-associated cochaperone. The deletion mutant has a modest phenotype, being heat-shock sensitive. Using antibodies raised against bacterially produced protein, we determined that Wos2 is very abundant, ubiquitously distributed in the yeast cell, and its expression dropped drastically as cells entered into early stationary phase, indicating that its function is associated with cell proliferation. In proliferating cells, the amount of Wos2 protein was not subjected to cell cycle regulation. However, in vitro assays demonstrated that this Hsp90 cochaperone is potentially regulated by phosphorylation. In addition to suppressing Wee1 activity, overproduction of Wos2 displayed synthetic lethality with Cdc2 mutant proteins, indicating that this Hsp90 cochaperone functionally interacts with Cdc2. The level of Cdc2 protein and its associated H1 kinase activity under synthetic lethal conditions suggested a regulatory role for this Wos2-Cdc2 interaction. Hsp90 complexes are required for CDK regulation; the synergy found between the excess of Wos2 and a deficiency in Hsp90 activity suggests that Wos2 could specifically interfere with the Hsp90-dependent regulation of Cdc2. In vitro analysis indicated that the above genetic interactions could take place by physical association of Wos2 with the single CDK complex of the fission yeast. Expression of the budding yeast p23 protein (encoded by the SBA1 gene) in the fission yeast indicated that Wos2 and Sba1 are functionally exchangeable and therefore that properties described here for Wos2 could be of wide significance in understanding the biological function of cochaperone p23 in eukaryotic cells.
Analysis of the Seven-Member AAD Gene Set Demonstrates That Genetic Redundancy in Yeast May Be More Apparent Than Real
Authors: Delneri Daniela , Gardner David C J , Oliver Stephen G
Published: April 2021

Saccharomyces cerevisiae has seven genes encoding proteins with a high degree (>85%) of amino-acid sequence identity to the aryl-alcohol dehydrogenase of the lignin-degrading, filamentous fungus, Phanerochaete chrysosporium. All but one member of this gene set are telomere associated. Moreover, all contain a sequence similar to the DNA-binding site of the Yap1p transcriptional activator either upstream of or within their coding sequences. The expression of the AAD genes was found to be induced by chemicals, such as diamide and diethyl maleic acid ester (DEME), that cause an oxidative shock by inactivating the glutathione (GSH) reservoir of the cells. In contrast, the oxidizing agent hydrogen peroxide has no effect on the expression of these genes. We found that the response to anti-GSH agents was Yap1p dependent. The very high level of nucleotide sequence similarity between the AAD genes makes it difficult to determine if they are all involved in the oxidative-stress response. The use of single and multiple aad deletants demonstrated that only AAD4 (YDL243c) and AAD6 (YFL056/57c) respond to the oxidative stress. Of these two genes, only AAD4 is likely to be functional since the YFL056/57c open reading frame is interrupted by a stop codon. Thus, in terms of the function in response to oxidative stress, the sevenfold redundancy of the AAD gene set is more apparent than real.
The STE12α Homolog Is Required for Haploid Filamentation But Largely Dispensable for Mating and Virulence in Cryptococcus neoformans
Authors: Yue Changli , Cavallo Lora M , Alspaugh J Andrew , Wang Ping , Cox Gary M , Perfect John R , Heitman Joseph
Published: April 2021

Cryptococcus neoformans is a fungal pathogen that causes meningitis in immunocompromised hosts. The organism has a known sexual cycle, and strains of the MATα mating type are more virulent than isogenic MATa strains in mice, and they are more common in the environment and infected hosts. A C. neoformans homolog of the STE12 transcription factor that regulates mating, filamentation, and virulence in Saccharomyces cerevisiae and Candida albicans was identified previously, found to be encoded by a novel region of the MATα mating type locus, and shown to enhance filamentous growth when overexpressed. We have disrupted the C. neoformans STE12 gene in a pathogenic serotype A isolate. ste12 mutant strains exhibit a severe defect in filamentation and sporulation (haploid fruiting) in response to nitrogen starvation. In contrast, ste12 mutant strains have only modest mating defects and are fully virulent in two animal models compared to the STE12 wild-type strain. In genetic epistasis experiments, STE12 functions in a MAP kinase cascade to regulate fruiting, but not mating. Thus, the C. neoformans STE12α transcription factor homolog plays a specialized function in haploid fruiting, but it is dispensable or redundant for mating and virulence. The association of the MATα locus with virulence may involve additional genes, and other transcription factors that regulate mating and virulence remain to be identified.
Lack of Nucleotide Polymorphism in the Y-Linked Sperm Flagellar Dynein Gene Dhc-Yh3 of Drosophila melanogaster and D. simulans
Authors: Zurovcova Martina , Eanes Walter F
Published: April 2021

We studied levels of intra- and interspecific nucleotide variation associated with a Y-linked gene in five members of the Drosophila melanogaster subgroup. Using published sequence for 348 bp of the Dhc-Yh3 gene, and degenerate PCR primers designed from comparisons of the sea urchin and Chlamydomonas flagellar dynein genes, we recovered a 1738-bp region in D. melanogaster. Analyses of sequence variation in a worldwide collection of 11 lines of D. melanogaster and 10 lines of D. simulans found only a single silent polymorphism in the latter species. The synonymous site divergence per site for Dhc-Yh3 is comparable to values for X and autosomal genes. Assuming a Wright-Fisher population model, the lack of variation is statistically less than expected using appropriately reduced estimates of θ from the X and autosomes. Because the Y chromosome encodes only six known genes, genetic hitchhiking associated with background selection is unlikely to explain this low variation. Conversely, adaptive hitchhiking, as associated with sex-ratio chromosomes, or a large variance in male fertility may reduce the polymorphism on the Y chromosome. Codon bias is very low, as seen for other genes in regions of low recombination.
A Selective Screen Reveals Discrete Functional Domains in Drosophila Nanos
Authors: Arrizabalaga Gustavo , Lehmann Ruth
Published: April 2021

The Drosophila protein Nanos encodes an evolutionarily conserved protein with two zinc finger motifs. In the embryo, Nanos protein function is required for establishment of the anterior-posterior body pattern and for the migration of primordial germ cells. During oogenesis, Nanos protein is involved in the establishment and maintenance of germ-line stem cells and the differentiation of oocyte precursor cells. To establish proper embryonic patterning, Nanos acts as a translational regulator of hunchback RNA. Nanos' targets for germ cell migration and development are not known. Here, we describe a selective genetic screen aimed at isolating new nanos alleles. The molecular and genetic analysis of 68 new alleles has allowed us to identify amino acids critical for nanos function. This analysis shows that the CCHC motifs, which coordinate two metal ions, are essential for all known functions of Nanos protein. Furthermore, a region C-terminal to the zinc fingers seems to constitute a novel functional domain within the Nanos protein. This “tail region” of Nanos is required for abdomen formation and germ cell migration, but not for oogenesis.
Inbreeding Load, Average Dominance and the Mutation Rate for Mildly Deleterious Alleles in Mimulus guttatus
Authors: Willis John H
Published: April 2021

The goal of this study is to provide information on the genetics of inbreeding depression in a primarily outcrossing population of Mimulus guttatus. Previous studies of this population indicate that there is tremendous inbreeding depression for nearly every fitness component and that almost all of this inbreeding depression is due to mildly deleterious alleles rather than recessive lethals or steriles. In this article I assayed the homozygous and heterozygous fitnesses of 184 highly inbred lines extracted from a natural population. Natural selection during the five generations of selfing involved in line formation essentially eliminated major deleterious alleles but was ineffective in purging alleles with minor fitness effects and did not appreciably diminish overall levels of inbreeding depression. Estimates of the average degree of dominance of these mildly deleterious alleles, obtained from the regression of heterozygous fitness on the sum of parental homozygous fitness, indicate that the detrimental alleles are partially recessive for most fitness traits, with h~∼0.15 for cumulative measures of fitness. The inbreeding load, B, for total fitness is ~1.0 in this experiment. These results are consistent with the hypothesis that spontaneous mildly deleterious mutations occur at a rate >0.1 mutation per genome per generation.
Isolation and Characterization of Nrf1p, a Novel Negative Regulator of the Cdc42p GTPase in Schizosaccharomyces pombe
Authors: Murray Janet M , Johnson Douglas I
Published: April 2021

The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24ts mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1+, encoded an ~15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe Δnrf1 mutant was viable but overexpression of nrf1+ in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1+ also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.
Cytologically Integrated Physical Restriction Fragment Length Polymorphism Maps for the Barley Genome Based on Translocation Breakpoints
Authors: Künzel Gottfried , Korzun Larissa , Meister Armin
Published: April 2021

We have developed a new technique for the physical mapping of barley chromosomes using microdissected translocation chromosomes for PCR with sequence-tagged site primers derived from >300 genetically mapped RFLP probes. The positions of 240 translocation breakpoints were integrated as physical landmarks into linkage maps of the seven barley chromosomes. This strategy proved to be highly efficient in relating physical to genetic distances. A very heterogeneous distribution of recombination rates was found along individual chromosomes. Recombination is mainly confined to a few relatively small areas spaced by large segments in which recombination is severely suppressed. The regions of highest recombination frequency (≤1 Mb/cM) correspond to only 4.9% of the total barley genome and harbor 47.3% of the 429 markers of the studied RFLP map. The results for barley correspond well with those obtained by deletion mapping in wheat. This indicates that chromosomal regions characterized by similar recombination frequencies and marker densities are highly conserved between the genomes of barley and wheat. The findings for barley support the conclusions drawn from deletion mapping in wheat that for all plant genomes, notwithstanding their size, the marker-rich regions are all of similar gene density and recombination activity and, therefore, should be equally accessible to map-based cloning.
Genetic Analysis of Salt-Tolerant Mutants in Arabidopsis thaliana
Authors: Quesada Víctor , Ponce María Rosa , Micol José Luis
Published: April 2021

Stress caused by the increased salinity of irrigated fields impairs plant growth and is one of the major constraints that limits crop productivity in many important agricultural areas. As a contribution to solving such agronomic problems, we have carried out a large-scale screening for Arabidopsis thaliana mutants induced on different genetic backgrounds by EMS treatment, fast neutron bombardment, or T-DNA insertions. From the 675,500 seeds we screened, 17 mutant lines were isolated, all but one of which yielded 25–70% germination levels on 250 mm NaCl medium, a condition in which their ancestor ecotypes are unable to germinate. Monogenic recessive inheritance of NaCl-tolerant germination was displayed with incomplete penetrance by all the selected mutants, which fell into five complementation groups. These were named SALOBREÑO (SAÑ) and mapped relative to polymorphic microsatellites, the map positions of three of them suggesting that they are novel genes. Strains carrying mutations in the SAÑ1-SAÑ4 genes display similar responses to both ionic effects and osmotic pressure, their germination being NaCl and mannitol tolerant but KCl and Na2SO4 sensitive. In addition, NaCl-, KCl-, and mannitol-tolerant as well as abscisic-acid-insensitive germination was displayed by sañ5, whose genetic and molecular characterization indicates that it carries an extremely hypomorphic or null allele of the ABI4 gene, its deduced protein product lacking the APETALA2 DNA binding domain.
Mutator-Suppressible Alleles of rough sheath1 and liguleless3 in Maize Reveal Multiple Mechanisms for Suppression
Authors: Girard Lisa , Freeling Michael
Published: April 2021

Insertions of Mutator transposons into maize genes can generate suppressible alleles. Mu suppression is when, in the absence of Mu activity, the phenotype of a mutant allele reverts to that of its progenitor. Here we present the characterization of five dominant Mu-suppressible alleles of the knox (knotted1-like homeobox) genes liguleless3 and rough sheath1, which exhibit neomorphic phenotypes in the leaves. RNA blot analysis suggests that Mu suppression affects only the neomorphic aspect of the allele, not the wild-type aspect. Additionally, Mu suppression appears to be exerting its effects at the level of transcription or transcript accumulation. We show that truncated transcripts are produced by three alleles, implying a mechanism for Mu suppression of 5′ untranslated region insertion alleles distinct from that which has been described previously. Additionally, it is found that Mu suppression can be caused by at least three different types of Mutator elements. Evidence presented here suggests that whether an allele is suppressible or not may depend upon the site of insertion. We cite previous work on the knox gene kn1, and discuss our results in the context of interactions between Mu-encoded products and the inherently negative regulation of neomorphic liguleless3 and rough sheath1 transcription.
Effective Population Sizes for Cytoplasmic and Nuclear Genes in a Gynodioecious Species: The Role of the Sex Determination System
Authors: Laporte Valérie , Cuguen Joël , Couvet Denis
Published: April 2021

Equations are derived for the effective sizes of gynodioecious populations with respect to both nuclear and cytoplasmic genes (Nec and Nen, respectively). Compared to hermaphroditism, gynodioecy generally reduces effective population sizes for both kinds of loci to an extent depending on the frequency of females, the sex determination system, and the selfing rate of hermaphrodites. This reduction is due to fitness differences between the sexes and is highly influenced by the mode of inheritance of this fitness. In absence of selfing, nuclear gynodioecy results in a reduction of Nec that depends strongly on the dominance of male sterility alleles, while Nen remains equal to the census number (N). With cytonuclear gynodioecy, both cytoplasmic and nuclear effective sizes are reduced, and at the extreme, dioecy results in the minimum Nec values and either minimum or maximum Nen values (for low or high frequency of females, respectively). When selfing occurs, gynodioecy either increases or decreases Nen as compared to hermaphroditism with the same selfing rate of hermaphrodites. Unexpectedly, Nec also varies with the selfing rate. Thus the genetic sex-determination system appears as a major factor for the nuclear and cytoplasmic genetic diversities of gynodioecious species.
Sip5 Interacts With Both the Reg1/Glc7 Protein Phosphatase and the Snf1 Protein Kinase of Saccharomyces cerevisiae
Authors: Sanz Pascual , Ludin Katja , Carlson Marian
Published: April 2021

The Snf1 protein kinase is an essential component of the glucose starvation signalling pathway in Saccharomyces cerevisiae. We have used the two-hybrid system to identify a new protein, Sip5, that interacts with the Snf1 kinase complex in response to glucose limitation. Coimmunoprecipitation studies confirmed the association of Sip5 and Snf1 in cell extracts. We found that Sip5 also interacts strongly with Reg1, the regulatory subunit of the Reg1/Glc7 protein phosphatase 1 complex, in both two-hybrid and coimmunoprecipitation assays. Previous work showed that Reg1/Glc7 interacts with the Snf1 kinase under glucose-limiting conditions and negatively regulates its activity. Sip5 is the first protein that has been shown to interact with both Snf1 and Reg1/Glc7. Genetic analysis showed that the two-hybrid interaction between Reg1 and Snf1 is reduced threefold in a sip5Δ mutant. These findings suggest that Sip5 facilitates the interaction between the Reg1/Glc7 phosphatase and the Snf1 kinase.
Antagonism of Ultraviolet-Light Mutagenesis by the Methyl-Directed Mismatch-Repair System of Escherichia coli
Authors: Liu Hongbo , Hewitt Stephen R , Hays John B
Published: April 2021

Previous studies have demonstrated that the Escherichia coli MutHLS mismatch-repair system can process UV-irradiated DNA in vivo and that the human MSH2·MSH6 mismatch-repair protein binds more strongly in vitro to photoproduct/base mismatches than to “matched” photoproducts in DNA. We tested the hypothesis that mismatch repair directed against incorrect bases opposite photoproducts might reduce UV mutagenesis, using two alleles at E. coli lacZ codon 461, which revert, respectively, via CCC → CTC and CTT → CTC transitions. F′ lacZ targets were mated from mut+ donors into mutH, mutL, or mutS recipients, once cells were at substantial densities, to minimize spontaneous mutation prior to irradiation. In umu+ mut+ recipients, a range of UV fluences induced lac+ revertant frequencies of 4–25 × 10−8; these frequencies were consistently 2-fold higher in mutH, mutL, or mutS recipients. Since this effect on mutation frequency was unaltered by an Mfd− defect, it appears not to involve transcription-coupled excision repair. In mut+ umuC122::Tn5 bacteria, UV mutagenesis (at 60 J/m2) was very low, but mutH or mutL or mutS mutations increased reversion of both lacZ alleles roughly 25-fold, to 5–10 × 10−8. Thus, at UV doses too low to induce SOS functions, such as Umu2′D, most incorrect bases opposite occasional photoproducts may be removed by mismatch repair, whereas in heavily irradiated (SOS-induced) cells, mismatch repair may only correct some photoproduct/base mismatches, so UV mutagenesis remains substantial.
A Homologue of the Recombination-Dependent Growth Gene, rdgC, Is Involved in Gonococcal Pilin Antigenic Variation
Authors: Mehr Ian J , Long Cynthia D , Serkin Carla D , Seifert H Steven
Published: April 2021

Neisseria gonorrhoeae pilin undergoes high-frequency changes in primary amino acid sequence that aid in the avoidance of the host immune response and alter pilus expression. The pilin amino acid changes reflect nucleotide changes in the expressed gene, pilE, which result from nonreciprocal recombination reactions with numerous silent loci, pilS. A series of mini-transposon insertions affecting pilin antigenic variation were localized to three genes in one region of the Gc chromosome. Mutational analysis with complementation showed that a Gc gene with sequence similarity to the Escherichia coli rdgC gene is involved in pilus-dependent colony phase variation and in pilin antigenic variation. Furthermore, we show that the Gc rdgC homologue is transcriptionally linked in an operon with a gene encoding a predicted GTPase. The inability to disrupt expression of this gene suggests it is an essential gene (engA, essential neisserial GTPase). While some of the transposon mutations in rdgC and insertions in the 5′-untranslated portion of engA showed a growth defect, all transposon insertions investigated conferred an aberrant cellular morphology. Complementation analysis showed that the growth deficiencies are due to the interruption of RdgC expression and not that of EngA. The requirement of RdgC for efficient pilin variation suggests a role for this protein in specialized DNA recombination reactions.
The Saccharomyces cerevisiae DNA Recombination and Repair Functions of the RAD52 Epistasis Group Inhibit Ty1 Transposition
Authors: Rattray Alison J , Shafer Brenda K , Garfinkel David J
Published: April 2021

RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or δ) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA.
Roles for the Saccharomyces cerevisiae SDS3, CBK1 and HYM1 Genes in Transcriptional Repression by SIN3
Authors: Dorland Scott , Deegenaars Michelle L , Stillman David J
Published: April 2021

The Saccharomyces cerevisiae Sin3 transcriptional repressor is part of a large multiprotein complex that includes the Rpd3 histone deacetylase. A LexA-Sin3 fusion protein represses transcription of promoters with LexA binding sites. To identify genes involved in repression by Sin3, we conducted a screen for mutations that reduce repression by LexA-Sin3. One of the mutations identified that reduces LexA-Sin3 repression is in the RPD3 gene, consistent with the known roles of Rpd3 in transcriptional repression. Mutations in CBK1 and HYM1 reduce repression by LexA-Sin3 and also cause defects in cell separation and altered colony morphology. cbk1 and hym1 mutations affect some but not all genes regulated by SIN3 and RPD3, but the effect on transcription is much weaker. Genetic analysis suggests that CBK1 and HYM1 function in the same pathway, but this genetic pathway is separable from that of SIN3 and RPD3. The remaining gene from this screen described in this report is SDS3, previously identified in a screen for mutations that increase silencing at HML, HMR, and telomere-linked genes, a phenotype also seen in sin3 and rpd3 mutants. Genetic analysis demonstrates that SDS3 functions in the same genetic pathway as SIN3 and RPD3, and coimmunoprecipitation experiments show that Sds3 is physically present in the Sin3 complex.
Self-Inflicted Wounds, Template-Directed Gap Repair and a Recombination Hotspot: Effects of the mariner Transposase
Authors: Lohe Allan R , Timmons Courtney , Beerman Isabel , Lozovskaya Elena R , Hartl Daniel L
Published: April 2021

Aberrant repair products of mariner transposition occur at a frequency of ~1/500 per target element per generation. Among 100 such mutations in the nonautonomous element peach, most had aberrations in the 5′ end of peach (40 alleles), in the 3′ end of peach (11 alleles), or a deletion of peach with or without deletion of flanking genomic DNA (29 alleles). Most mariner mutations can be explained by exonuclease “nibble” and host-mediated repair of the double-stranded gap created by the transposase, in contrast to analogous mutations in the P element. In mariner, mutations in the 5′ inverted repeat are smaller and more frequent than those in the 3′ inverted repeat, but secondary mutations in target elements with a 5′ lesion usually had 3′ lesions resembling those normally found at the 5′ end. We suggest that the mariner transposase distinguishes between the 5′ and 3′ ends of the element, and that the 5′ end is relatively more protected after strand scission. We also find: (1) that homolog-dependent gap repair is a frequent accompaniment to mariner excision, estimated as 30% of all excision events; and (2) that mariner is a hotspot of recombination in Drosophila females, but only in the presence of functional transposase.
The Genetic Structure of the Raleigh Natural Population of Drosophila melanogaster Revisited
Authors: Kusakabe Shinichi , Yamaguchi Yumi , Baba Hiroshi , Mukai Terumi
Published: April 2021

The Raleigh natural population of Drosophila melanogaster was reanalyzed with special attention to possible dysgenic effects during the extraction of chromosomes. About 600 second chromosomes were extracted from the Raleigh natural population, half in the cytoplasm of wild-caught females (native genetic background) and half in the cytoplasm of the laboratory line, C160(In(2LR)SM1, Cy/In(2LR)bwV1) (foreign genetic background). We could not find significant differences between the two extraction schemes in the frequency of lethal second chromosomes (Q = 0.252 for the lines with the negative genetic background vs. 0.231 for the lines with the foreign genetic background) or in the homozygous detrimental (D) and lethal (L) loads (D = 0.210 vs. 0.251; L = 0.287 vs. 0.264). The effective size of the population was estimated to be ~19,000, based on the allelism rate of lethal-bearing chromosomes. The homozygous load markedly decreased in the 15 years since a previous study of the same population.
Functional Domains of the Drosophila Bicaudal-D Protein
Authors: Oh Junyoung , Baksa Katalin , Steward Ruth
Published: April 2021

The localization of oocyte-specific determinants in the form of mRNAs to the pro-oocyte is essential for the establishment of oocyte identity. Localization of the Bicaudal-D (Bic-D) protein to the presumptive oocyte is required for the accumulation of Bic-D and other mRNAs to the pro-oocyte. The Bic-D protein contains four well-defined heptad repeat domains characteristic of intermediate filament proteins, and several of the mutations in Bic-D map to these conserved domains. We have undertaken a structure-function analysis of Bic-D by testing the function of mutant Bic-D transgenes (Bic-DH) deleted for each of the heptad repeat domains in a Bic-D null background. Our transgenic studies indicate that only the C-terminal heptad repeat deletion results in a protein that has lost zygotic and ovarian functions. The three other deletions result in proteins with full zygotic function, but with affected ovarian function. The functional importance of each domain is well correlated with its conservation in evolution. The analysis of females heterozygous for Bic-DH and the existing alleles Bic-DPA66 or Bic-DR26 reveals that Bic-DR26 as well as some of Bic-DH transgenes have antimorphic effects. The yeast two-hybrid interaction assay shows that Bic-D forms homodimers. Furthermore, we found that Bic-D exists as a multimeric protein complex consisting of Egl and at least two Bic-D monomers.
Proteasome Mutants, pre4-2 and ump1-2, Suppress the Essential Function but Not the Mitochondrial RNase P Function of the Saccharomyces cerevisiae Gene RPM2
Authors: Lutz Mallory S , Ellis Steven R , Martin Nancy C
Published: April 2021

The Saccharomyces cerevisiae nuclear gene RPM2 encodes a component of the mitochondrial tRNA-processing enzyme RNase P. Cells grown on fermentable carbon sources do not require mitochondrial tRNA processing activity, but still require RPM2, indicating an additional function for the Rpm2 protein. RPM2-null cells arrest after 25 generations on fermentable media. Spontaneous mutations that suppress arrest occur with a frequency of ~9 × 10−6. The resultant mutants do not grow on nonfermentable carbon sources. We identified two loci responsible for this suppression, which encode proteins that influence proteasome function or assembly. PRE4 is an essential gene encoding the β-7 subunit of the 20S proteasome core. A Val-to-Phe substitution within a highly conserved region of Pre4p that disrupts proteasome function suppresses the growth arrest of RPM2-null cells on fermentable media. The other locus, UMP1, encodes a chaperone involved in 20S proteasome assembly. A nonsense mutation in UMP1 also disrupts proteasome function and suppresses Δrpm2 growth arrest. In an RPM2 wild-type background, pre4-2 and ump1-2 strains fail to grow at restrictive temperatures on nonfermentable carbon sources. These data link proteasome activity with Rpm2p and mitochondrial function.
DNA Damage-Inducible and RAD52-Independent Repair of DNA Double-Strand Breaks in Saccharomyces cerevisiae
Authors: Moore Carol Wood , McKoy Judith , Dardalhon Michelle , Davermann Darline , Martinez Marcia , Averbeck Dietrich
Published: April 2021

Chromosomal repair was studied in stationary-phase Saccharomyces cerevisiae, including rad52/rad52 mutant strains deficient in repairing double-strand breaks (DSBs) by homologous recombination. Mutant strains suffered more chromosomal fragmentation than RAD52/RAD52 strains after treatments with cobalt-60 γ irradiation or radiomimetic bleomycin, except after high bleomycin doses when chromosomes from rad52/rad52 strains contained fewer DSBs than chromosomes from RAD52/RAD52 strains. DNAs from both genotypes exhibited quick rejoining following γ irradiation and sedimentation in isokinetic alkaline sucrose gradients, but only chromosomes from RAD52/RAD52 strains exhibited slower rejoining (10 min to 4 hr in growth medium). Chromosomal DSBs introduced by γ irradiation and bleomycin were analyzed after pulsed-field gel electrophoresis. After equitoxic damage by both DNA-damaging agents, chromosomes in rad52/rad52 cells were reconstructed under nongrowth conditions [liquid holding (LH)]. Up to 100% of DSBs were eliminated and survival increased in RAD52/RAD52 and rad52/rad52 strains. After low doses, chromosomes were sometimes degraded and reconstructed during LH. Chromosomal reconstruction in rad52/rad52 strains was dose dependent after γ irradiation, but greater after high, rather than low, bleomycin doses with or without LH. These results suggest that a threshold of DSBs is the requisite signal for DNA-damage-inducible repair, and that nonhomologous end-joining repair or another repair function is a dominant mechanism in S. cerevisiae when homologous recombination is impaired.