bluuebunny/crossref_metadata_2025 huggingface dataset:
The bereft Gene, a Potential Target of the Neural Selector Gene cut, Contributes to Bristle Morphogenesis
Authors: Hardiman Kirsten E , Brewster Rachel , Khan Shaema M , Deo Monika , Bodmer Rolf
Published: April 2021

The neural selector gene cut, a homeobox transcription factor, is required for the specification of the correct identity of external (bristle-type) sensory organs in Drosophila. Targets of cut function, however, have not been described. Here, we study bereft (bft) mutants, which exhibit loss or malformation of a majority of the interommatidial bristles of the eye and cause defects in other external sensory organs. These mutants were generated by excising a P element located at chromosomal location 33AB, the enhancer trap line E8-2-46, indicating that a gene near the insertion site is responsible for this phenotype. Similar to the transcripts of the gene nearest to the insertion, reporter gene expression of E8-2-46 coincides with Cut in the support cells of external sensory organs, which secrete the bristle shaft and socket. Although bft transcripts do not obviously code for a protein product, its expression is abolished in bft deletion mutants, and the integrity of the bft locus is required for (interommatidial) bristle morphogenesis. This suggests that disruption of the bft gene is the cause of the observed bristle phenotype. We also sought to determine what factors regulate the expression of bft and the enhancer trap line. The correct specification of individual external sensory organ cells involves not only cut, but also the lineage genes numb and tramtrack. We demonstrate that mutations of these three genes affect the expression levels at the bft locus. Furthermore, cut overexpression is sufficient to induce ectopic bft expression in the PNS and in nonneuronal epidermis. On the basis of these results, we propose that bft acts downstream of cut and tramtrack to implement correct bristle morphogenesis.
Larger Genetic Differences Within Africans Than Between Africans and Eurasians
Authors: Yu Ning , Chen Feng-Chi , Ota Satoshi , Jorde Lynn B , Pamilo Pekka , Patthy Laszlo , Ramsay Michele , Jenkins Trefor , Shyue Song-Kun , Li Wen-Hsiung
Published: April 2021

The worldwide pattern of single nucleotide polymorphism (SNP) variation is of great interest to human geneticists, population geneticists, and evolutionists, but remains incompletely understood. We studied the pattern in noncoding regions, because they are less affected by natural selection than are coding regions. Thus, it can reflect better the history of human evolution and can serve as a baseline for understanding the maintenance of SNPs in human populations. We sequenced 50 noncoding DNA segments each ∼500 bp long in 10 Africans, 10 Europeans, and 10 Asians. An analysis of the data suggests that the sampling scheme is adequate for our purpose. The average nucleotide diversity (π) for the 50 segments is only 0.061% ± 0.010% among Asians and 0.064% ± 0.011% among Europeans but almost twice as high (0.115% ± 0.016%) among Africans. The African diversity estimate is even higher than that between Africans and Eurasians (0.096% ± 0.012%). From available data for noncoding autosomal regions (total length = 47,038 bp) and X-linked regions (47,421 bp), we estimated the π-values for autosomal regions to be 0.105, 0.070, 0.069, and 0.097% for Africans, Asians, Europeans, and between Africans and Eurasians, and the corresponding values for X-linked regions to be 0.088, 0.042, 0.053, and 0.082%. Thus, Africans differ from one another slightly more than from Eurasians, and the genetic diversity in Eurasians is largely a subset of that in Africans, supporting the out of Africa model of human evolution. Clearly, one must specify the geographic origins of the individuals sampled when studying π or SNP density.
Sequence Variation and Haplotype Structure Surrounding the Flowering Time Locus FRI in Arabidopsis thaliana
Authors: Hagenblad Jenny , Nordborg Magnus
Published: April 2021

Linkage disequilibrium in highly selfing organisms is expected to extend well beyond the scale of individual genes. The pattern of polymorphism in such species must thus be studied over a larger scale. We sequenced 14 short (0.5-1 kb) fragments from a 400-kb region surrounding the flowering time locus FRI in a sample of 20 accessions of Arabidopsis thaliana. The distribution of allele frequencies, as quantified by Tajima’s D, varies considerably over the region and is incompatible with a standard neutral model. The region is characterized by extensive haplotype structure, with linkage disequilibrium decaying over 250 kb. In particular, recombination is evident within 35 kb of FRI in a haplotype associated with a functionally important allele. This suggests that A. thaliana may be highly suitable for linkage disequilibrium mapping.
A Molecular Cytogenetic Map of Sorghum Chromosome 1: Fluorescence in Situ Hybridization Analysis With Mapped Bacterial Artificial Chromosomes
Authors: Islam-Faridi M N , Childs K L , Klein P E , Hodnett G , Menz M A , Klein R R , Rooney W L , Mullet J E , Stelly D M , Price H J
Published: April 2021

We used structural genomic resources for Sorghum bicolor (L.) Moench to target and develop multiple molecular cytogenetic probes that would provide extensive coverage for a specific chromosome of sorghum. Bacterial artificial chromosome (BAC) clones containing molecular markers mapped across sorghum linkage group A were labeled as probes for fluorescence in situ hybridization (FISH). Signals from single-, dual-, and multiprobe BAC-FISH to spreads of mitotic chromosomes and pachytene bivalents were associated with the largest sorghum chromosome, which bears the nucleolus organizing region (NOR). The order of individual BAC-FISH loci along the chromosome was fully concordant to that of marker loci along the linkage map. In addition, the order of several tightly linked molecular markers was clarified by FISH analysis. The FISH results indicate that markers from the linkage map positions 0.0-81.8 cM reside in the short arm of chromosome 1 whereas markers from 81.8-242.9 cM are located in the long arm of chromosome 1. The centromere and NOR were located in a large heterochromatic region that spans ∼60% of chromosome 1. In contrast, this region represents only 0.7% of the total genetic map distance of this chromosome. Variation in recombination frequency among euchromatic chromosomal regions also was apparent. The integrated data underscore the value of cytological data, because minor errors and uncertainties in linkage maps can involve huge physical regions. The successful development of multiprobe FISH cocktails suggests that it is feasible to develop chromosome-specific “paints” from genomic resources rather than flow sorting or microdissection and that when applied to pachytene chromatin, such cocktails provide an especially powerful framework for mapping. Such a molecular cytogenetic infrastructure would be inherently cross-linked with other genomic tools and thereby establish a cytogenomics system with extensive utility in development and application of genomic resources, cloning, transgene localization, development of plant “chromonomics,” germplasm introgression, and marker-assisted breeding. In combination with previously reported work, the results indicate that a sorghum cytogenomics system would be partially applicable to other gramineous genera.
The Correlation Between Relatives on the Supposition of Genomic Imprinting
Authors: Spencer Hamish G
Published: April 2021

Standard genetic analyses assume that reciprocal heterozygotes are, on average, phenotypically identical. If a locus is subject to genomic imprinting, however, this assumption does not hold. We incorporate imprinting into the standard quantitative-genetic model for two alleles at a single locus, deriving expressions for the additive and dominance components of genetic variance, as well as measures of resemblance among relatives. We show that, in contrast to the case with Mendelian expression, the additive and dominance deviations are correlated. In principle, this correlation allows imprinting to be detected solely on the basis of different measures of familial resemblances, but in practice, the standard error of the estimate is likely to be too large for a test to have much statistical power. The effects of genomic imprinting will need to be incorporated into quantitative-genetic models of many traits, for example, those concerned with mammalian birthweight.
Analysis of Multilocus Zygotic Associations
Authors: Yang Rong-Cai
Published: April 2021

While nonrandom associations between zygotes at different loci (zygotic associations) frequently occur in Hardy-Weinberg disequilibrium populations, statistical analysis of such associations has received little attention. In this article, we describe the joint distributions of zygotes at multiple loci, which are completely characterized by heterozygosities at individual loci and various multilocus zygotic associations. These zygotic associations are defined in the same fashion as the usual multilocus linkage (gametic) disequilibria on the basis of gametic and allelic frequencies. The estimation and test procedures are described with details being given for three loci. The sampling properties of the estimates are examined through Monte Carlo simulation. The estimates of three-locus associations are not free of bias due to the presence of two-locus associations and vice versa. The power of detecting the zygotic associations is small unless different loci are strongly associated and/or sample sizes are large (>100). The analysis of zygotic associations not only offers an effective means of packaging numerous genic disequilibria required for a complete characterization of multilocus structure, but also provides opportunities for making inference about evolutionary and demographic processes through a comparative assessment of zygotic association vs. gametic disequilibrium for the same set of loci in nonequilibrium populations.
Identification of Six Loci in Which Mutations Partially Restore Peroxisome Biogenesis and/or Alleviate the Metabolic Defect of pex2 Mutants in Podospora
Authors: Ruprich-Robert Gwenaël , Berteaux-Lecellier Véronique , Zickler Denise , Panvier-Adoutte Arlette , Picard Marguerite
Published: April 2021

Peroxins (PEX) are proteins required for peroxisome biogenesis. Mutations in PEX genes cause lethal diseases in humans, metabolic defects in yeasts, and developmental disfunctions in plants and filamentous fungi. Here we describe the first large-scale screening for suppressors of a pex mutation. In Podospora anserina, pex2 mutants exhibit a metabolic defect [inability to grow on medium containing oleic acid (OA medium) as sole carbon source] and a developmental defect (inability to differentiate asci in homozygous crosses). Sixty-three mutations able to restore growth of pex2 mutants on OA medium have been analyzed. They fall in six loci (suo1 to suo6) and act as dominant, allele-nonspecific suppressors. Most suo mutations have pleiotropic effects in a pex2+ background: formation of unripe ascospores (all loci except suo5 and suo6), impaired growth on OA medium (all loci except suo4 and suo6), or sexual defects (suo4). Using immunofluorescence and GFP staining, we show that peroxisome biogenesis is partially restored along with a low level of ascus differentiation in pex2 mutant strains carrying either the suo5 or the suo6 mutations. The data are discussed with respect to β-oxidation of fatty acids, peroxisome biogenesis, and cell differentiation.
Coevolution of the Telomeric Retrotransposons Across Drosophila Species
Authors: Casacuberta Elena , Pardue Mary-Lou
Published: April 2021

As in other eukaryotes, telomeres in Drosophila melanogaster are composed of long arrays of repeated DNA sequences. Remarkably, in D. melanogaster these repeats are produced, not by telomerase, but by successive transpositions of two telomere-specific retrotransposons, HeT-A and TART. These are the only transposable elements known to be completely dedicated to a role in chromosomes, a finding that provides an opportunity for investigating questions about the evolution of telomeres, telomerase, and the transposable elements themselves. Recent studies of D. yakuba revealed the presence of HeT-A elements with precisely the same unusual characteristics as HeT-Amel although they had only 55% nucleotide sequence identity. We now report that the second element, TART, is also a telomere component in D. yakuba; thus, these two elements have been evolving together since before the separation of the melanogaster and yakuba species complexes. Like HeT-Ayak, TART yak is undergoing concerted sequence evolution, yet they retain the unusual features TART mel shares with HeT-Amel. There are at least two subfamilies of TART yak with significantly different sequence and expression. Surprisingly, one subfamily of TART yak has >95% sequence identity with a subfamily of TART mel and shows similar transcription patterns. As in D. melanogaster, other retrotransposons are excluded from the D. yakuba terminal arrays studied to date.
Environment Dependence of Mutational Parameters for Viability in Drosophila melanogaster
Authors: Fry James D , Heinsohn Stefanie L
Published: April 2021

The genomic rate of mildly deleterious mutations (U) figures prominently in much evolutionary and ecological theory. In Drosophila melanogaster, estimates of U have varied widely, from <0.1 to nearly 1 per zygote. The source of this variation is unknown, but could include differences in the conditions used for assaying fitness traits. We examined how assay conditions affect estimates of the rates and effects of viability-depressing mutations in two sets of lines with accumulated spontaneous mutations on the second chromosome. In each set, the among-line variance in egg-to-adult viability was significantly greater when viability was assayed using a high parental density than when it was assayed using a low density. In contrast, the proportional decline in viability due to new mutations did not differ between densities. Two other manipulations, lowering the temperature and adding ethanol to the medium, had no significant effects on either the mean decline or among-line variance. Cross-environment genetic correlations in viability were generally close to one, implying that most mutations reduced viability in all environments. Using data from the low-density, lower-bound estimates of U approached the classic, high values of Mukai and Ohnishi; at the high density, U estimates were similar to recently reported low values. The difference in estimated mutation rates, taken at face value, would imply that many mutations affected fitness at low density but not at high density, but this is shown to be incompatible with the observed high cross-environment correlations. Possible reasons for this discrepancy are discussed. Regardless of the interpretation, the results show that assay conditions can have a large effect on estimates of mutational parameters for fitness traits.
The Drosophila melanogaster Translational Repressor Pumilio Regulates Neuronal Excitability
Authors: Schweers Brett A , Walters Karina J , Stern Michael
Published: April 2021

Maintenance of proper neuronal excitability is vital to nervous system function and normal behavior. A subset of Drosophila mutants that exhibit altered behavior also exhibit defective motor neuron excitability, which can be monitored with electrophysiological methods. One such mutant is the P-element insertion mutant bemused (bem). The bem mutant exhibits female sterility, sluggishness, and increased motor neuron excitability. The bem P element is located in the large intron of the previously characterized translational repressor gene pumilio (pum). Here, by several criteria, we show that bem is a new allele of pum. First, ovary-specific expression of pum partially rescues bem female sterility. Second, pum null mutations fail to complement bem female sterility, behavioral defects, and neuronal hyperexcitability. Third, heads from bem mutant flies exhibit greatly reduced levels of Pum protein and the absence of two pum transcripts. Fourth, two previously identified pum mutants exhibit neuronal hyperexcitability. Fifth, overexpression of pum in the nervous system reduces neuronal excitability, which is the opposite phenotype to pum loss of function. Collectively, these findings describe a new role of pum in the regulation of neuronal excitability and may afford the opportunity to study the role of translational regulation in the maintenance of proper neuronal excitability.
Recombination and Gene Conversion in a 170-kb Genomic Region of Arabidopsis thaliana
Authors: Haubold Bernhard , Kroymann Jürgen , Ratzka Andreas , Mitchell-Olds Thomas , Wiehe Thomas
Published: April 2021

Arabidopsis thaliana is a highly selfing plant that nevertheless appears to undergo substantial recombination. To reconcile its selfing habit with the observations of recombination, we have sampled the genetic diversity of A. thaliana at 14 loci of ~500 bp each, spread across 170 kb of genomic sequence centered on a QTL for resistance to herbivory. A total of 170 of the 6321 nucleotides surveyed were polymorphic, with 169 being biallelic. The mean silent genetic diversity (πs) varied between 0.001 and 0.03. Pairwise linkage disequilibria between the polymorphisms were negatively correlated with distance, although this effect vanished when only pairs of polymorphisms with four haplotypes were included in the analysis. The absence of a consistent negative correlation between distance and linkage disequilibrium indicated that gene conversion might have played an important role in distributing genetic diversity throughout the region. We tested this by coalescent simulations and estimate that up to 90% of recombination is due to gene conversion.
Dasheng: A Recently Amplified Nonautonomous Long Terminal Repeat Element That Is a Major Component of Pericentromeric Regions in Rice
Authors: Jiang Ning , Bao Zhirong , Temnykh Svetlana , Cheng Zhukuan , Jiang Jiming , Wing Rod A , McCouch Susan R , Wessler Susan R
Published: April 2021

A new and unusual family of LTR elements, Dasheng, has been discovered in the genome of Oryza sativa following database searches of ~100 Mb of rice genomic sequence and 78 Mb of BAC-end sequence information. With all of the cis-elements but none of the coding domains normally associated with retrotransposons (e.g., gag, pol), Dasheng is a novel nonautonomous LTR element with high copy number. Over half of the ~1000 Dasheng elements in the rice genome are full length (5.6–8.6 kb), and 60% are estimated to have amplified in the past 500,000 years. Using a modified AFLP technique called transposon display, 215 elements were mapped to all 12 rice chromosomes. Interestingly, more than half of the mapped elements are clustered in the heterochromatic regions around centromeres. The distribution pattern was further confirmed by FISH analysis. Despite clustering in heterochromatin, Dasheng elements are not nested, suggesting their potential value as molecular markers for these marker-poor regions. Taken together, Dasheng is one of the highest-copy-number LTR elements and one of the most recent elements to amplify in the rice genome.
Functional Dissection of the Global Repressor Tup1 in Yeast: Dominant Role of the C-Terminal Repression Domain
Authors: Zhang Zhizhou , Varanasi Ushasri , Trumbly Robert J
Published: April 2021

In the yeast Saccharomyces cerevisiae, Tup1, in association with Cyc8 (Ssn6), functions as a general repressor of transcription. Tup1 and Cyc8 are required for repression of diverse families of genes coordinately controlled by glucose repression, mating type, and other mechanisms. This repression is mediated by recruitment of the Cyc8-Tup1 complex to target promoters by sequence-specific DNA-binding proteins. We created a library of XhoI linker insertions and internal in-frame deletion mutations within the TUP1 coding region. Insertion mutations outside of the WD domains were wild type, while insertions within the WD domains induced mutant phenotypes with differential effects on the target genes SUC2, MFA2, RNR2, and HEM13. Deletion mutations confirmed previous findings of two separate repression domains in the N and C termini. The cumulative data suggest that the C-terminal repression domain, located near the first WD repeat, plays the dominant role in repression. Although the N-terminal repression domain is sufficient for partial repression, deletion of this region does not compromise repression. Surprisingly, deletion of the majority of the histone-binding domain of Tup1 also does not significantly reduce repression. The N-terminal region containing potential α-helical coiled coils is required for Tup1 oligomerization and association with Cyc8. Association with Cyc8 is required for repression of SUC2, HEM13, and RNR2 but not MFA2 and STE2.
Genetic Interaction Between Calcineurin and Type 2 Myosin and Their Involvement in the Regulation of Cytokinesis and Chloride Ion Homeostasis in Fission Yeast
Authors: Fujita Masaaki , Sugiura Reiko , Lu Yabin , Xu Linxiao , Xia Yujie , Shuntoh Hisato , Kuno Takayoshi
Published: April 2021

Calcineurin plays a critical role in Ca2+ signaling in various cell types. In fission yeast, calcineurin is required for cytokinesis and chloride ion homeostasis. However, most of its physiological functions remain obscure. A genetic screen was performed to identify genes that share an essential function with calcineurin. We screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and to a high concentration of chloride ion and isolated a mutant, cis2-1/myp2-c2, which contains a novel allele of the myp2+/myo3+ gene that encodes a type 2 myosin heavy chain. The myp2-c2 mutant showed morphological defects similar to those associated with a calcineurin deletion mutant, such as multiseptated and branched cells. Consistently, myp2-null cells were hypersensitive to chloride ion and showed the multiseptated phenotype in the presence of immunosuppressants or at high chloride concentrations. Overexpression of constitutively active calcineurin suppressed the chloride ion-sensitive growth defect and cytokinesis abnormality of the myp2-c2 mutant and myp2-null cells. Interestingly, the essential myosin light chain mutant cdc4-8 failed to grow and could not form a normal contractile ring in the presence of immunosuppressants. Furthermore, calcineurin-null cells exhibited aberrant contractile rings, suggesting impaired contraction of the rings. These results indicate that calcineurin is involved in the regulation of cytokinesis and that chloride ion homeostasis is mediated by type 2 myosin.
Meiotic Silencing by Unpaired DNA: Properties, Regulation and Suppression
Authors: Shiu Patrick K T , Metzenberg Robert L
Published: April 2021

In Neurospora, a gene not paired with a homolog in prophase I of meiosis generates a signal that transiently silences all sequences homologous to it by a process called meiotic silencing by unpaired DNA (MSUD). Thus a deletion mutation in a heterozygous cross is formally “ascus-dominant” because its unpaired wild-type partner silences itself. We describe in detail the isolation of a mutation, Sad-1UV, that suppresses the dominance of various ascus-dominant mutations. Additional dominant, semidominant, and recessive Sad-1 alleles have been generated by RIP; the DNA of the dominant RIP alleles becomes methylated, but dim-2-dependent methylation is not necessary for silencing. The barrenness of homozygous Sad-1 crosses is not due to the failure to silence unpaired mating-type mat A-2 mat A-3 genes. Transcripts of sad-1+ can be detected during the sexual phase in a homozygous wild-type cross, indicating that the gene is expressed even if all DNA can pair normally. Meiotic silencing is confined to the ascus in which DNA is unpaired, and silencing does not spread to neighboring asci in a fruiting body of mixed genetic constitution.
Chromosomal Map of the Model Legume Lotus japonicus
Authors: Pedrosa Andrea , Sandal Niels , Stougaard Jens , Schweizer Dieter , Bachmair Andreas
Published: April 2021

Lotus japonicus is a model plant for the legume family. To facilitate map-based cloning approaches and genome analysis, we performed an extensive characterization of the chromosome complement of the species. A detailed karyotype of L. japonicus Gifu was built and plasmid and BAC clones, corresponding to genetically mapped markers (see the accompanying article by Sandal  et al. 2002, this issue), were used for FISH to correlate genetic and chromosomal maps. Hybridization of DNA clones from 32 different genomic regions enabled the assignment of linkage groups to chromosomes, the comparison between genetic and physical distances throughout the genome, and the partial characterization of different repetitive sequences, including telomeric and centromeric repeats. Additional analysis of L. filicaulis and its F1 hybrid with L. japonicus demonstrated the occurrence of inversions between these closely related species, suggesting that these chromosome rearrangements are early events in speciation of this group.
Antioxidant Gene Expression in Active and Sedentary House Mice (Mus domesticus) Selected for High Voluntary Wheel-Running Behavior
Authors: Bronikowski Anne M , Morgan Theodore J , Garland Theodore , Carter Patrick A
Published: April 2021

We present liver mRNA levels of the two antioxidant enzymes catalase (CAT) and Mn-superoxide dismutase (SOD2) in four treatment groups of house mice assayed by RNase protection at 20 months of age. These groups were mice from four replicate selection and four replicate control lines from the sixteenth generation of selective breeding for high voluntary wheel running, housed with or without running wheels from age 3 weeks through 20 months. Exercising control females had induced CAT expression; SOD2 exhibited a similar pattern in females from two of the four control lines. Exercising male mice had induced CAT expression, but not SOD2 expression, irrespective of genetic background. We discuss these results with respect to both evolutionary (genetic) and training (exercise-induced) adaptations and explore predictions of these results in relation to the oxidative-damage theory of senescence.
Using the P{wHy} Hybrid Transposable Element to Disrupt Genes in Region 54D-55B in Drosophila melanogaster
Authors: Mohr Stephanie E , Gelbart William M
Published: April 2021

Understanding the function of each gene in the genome of a model organism such as Drosophila melanogaster is an important goal. The development of improved methods for uncovering the mutant phenotypes of specific genes can accelerate achievement of this goal. The P{wHy} hybrid transposable element can be used to generate nested sets of precisely mapped deletions in a given region of the Drosophila genome. Here we use the P{wHy} method to generate overlapping, molecularly defined deletions from a set of three P{wHy} insertions in the 54E-F region of chromosome 2. Deletions that span a total of 0.5 Mb were identified and molecularly mapped precisely. Using overlapping deletions, the mutant phenotypes of nine previously uncharacterized genes in a 101-kb region were determined, including identification of new loci required for viability and female fertility. In addition, the deletions were used to molecularly map previously isolated lethal mutations. Thus, the P{wHy} method provides an efficient method for systematically determining the phenotypes of genes in a given region of the fly genome.
A Green Fluorescent Protein Reporter Genetic Screen That Identifies Modifiers of Hox Gene Function in the Drosophila Embryo
Authors: Merabet Samir , Catala Francoise , Pradel Jacques , Graba Yacine
Published: April 2021

Hox genes encode evolutionarily conserved transcription factors that play fundamental roles in the organization of the animal body plan. Molecular studies emphasize that unidentified genes contribute to the control of Hox activity. In this study, we describe a genetic screen designed to identify functions required for the control of the wingless (wg) and empty spiracles (ems) target genes by the Hox Abdominal-A and Abdominal-B proteins. A collection of chromosomal deficiencies were screened for their ability to modify GFP fluorescence patterns driven by Hox response elements (HREs) from wg and ems. We found 15 deficiencies that modify the activity of the ems HRE and 18 that modify the activity of the wg HRE. Many deficiencies cause ectopic activity of the HREs, suggesting that spatial restriction of transcriptional activity is an important level in the control of Hox gene function. Further analysis identified eight loci involved in the homeotic regulation of wg or ems. A majority of these modifier genes correspond to previously characterized genes, although not for their roles in the regulation of Hox targets. Five of them encode products acting in or in connection with signal transduction pathways, which suggests an extensive use of signaling in the control of Hox gene function.
An Overexpression Screen in Drosophila for Genes That Restrict Growth or Cell-Cycle Progression in the Developing Eye
Authors: Tseng Ai-Sun Kelly , Hariharan Iswar K
Published: April 2021

We screened for genes that, when overexpressed in the proliferating cells of the eye imaginal disc, result in a reduction in the size of the adult eye. After crossing the collection of 2296 EP lines to the ey-GAL4 driver, we identified 46 lines, corresponding to insertions in 32 different loci, that elicited a small eye phenotype. These lines were classified further by testing for an effect in postmitotic cells using the sev-GAL4 driver, by testing for an effect in the wing using en-GAL4, and by testing for the ability of overexpression of cycE to rescue the small eye phenotype. EP lines identified in the screen encompass known regulators of eye development including hh and dpp, known genes that have not been studied previously with respect to eye development, as well as 19 novel ORFs. Lines with insertions near INCENP, elB, and CG11518 were characterized in more detail with respect to changes in growth, cell-cycle phasing, and doubling times that were elicited by overexpression. RNAi-induced phenotypes were also analyzed in SL2 cells. Thus overexpression screens can be combined with RNAi experiments to identify and characterize new regulators of growth and cell proliferation.
mei-P22 Encodes a Chromosome-Associated Protein Required for the Initiation of Meiotic Recombination in Drosophila melanogaster
Authors: Liu Hao , Jang Janet K , Kato Naohiro , McKim Kim S
Published: April 2021

Double-strand breaks (DSB) initiate meiotic recombination in a variety of organisms. Here we present genetic evidence that the mei-P22 gene is required for the induction of DSBs during meiotic prophase in Drosophila females. Strong mei-P22 mutations eliminate meiotic crossing over and suppress the sterility of DSB repair-defective mutants. Interestingly, crossing over in mei-P22 mutants can be restored to almost 50% of wild-type by X irradiation. In addition, an antibody-based assay was used to demonstrate that DSBs are not formed in mei-P22 mutants. This array of phenotypes is identical to that of mei-W68 mutants; mei-W68 encodes the Drosophila Spo11 homolog that is proposed to be an enzyme required for DSB formation. Consistent with a direct role in DSB formation, mei-P22 encodes a basic 35.7-kD protein, which, when examined by immunofluorescence, localizes to foci on meiotic chromosomes. MEI-P22 foci appear transiently in early meiotic prophase, which is when meiotic recombination is believed to initiate. By using an antibody to C(3)G as a marker for synaptonemal complex (SC) formation, we observed that SC is present before MEI-P22 associates with the chromosomes, thus providing direct evidence that the development of SC precedes the initiation of meiotic recombination. Similarly, we found that MEI-P22 foci did not appear in a c(3)G mutant in which SC does not form, suggesting that DSB formation is dependent on SC formation in Drosophila. We propose that MEI-P22 interacts with meiosis-specific chromosome proteins to facilitate DSB creation by MEI-W68.
Role of the Unfolded Protein Response Pathway in Regulation of INO1 and in the sec14 Bypass Mechanism in Saccharomyces cerevisiae
Authors: Chang Hak J , Jones Elizabeth W , Henry Susan A
Published: April 2021

INO1, encoding inositol 1-phosphate synthase, is the most highly regulated of a class of genes containing the repeated element, UASINO, in their promoters. Transcription of UASINO-containing genes is modulated by the availability of exogenous inositol and by signals generated by alteration of phospholipid metabolism. The unfolded protein response (UPR) pathway also is involved in INO1 expression and the ire1Δ and hac1Δ mutants are inositol auxotrophs. We examined the role of the UPR in transmitting a signal generated in response to inositol deprivation and to alteration of phospholipid biosynthesis created in the sec14ts cki1Δ genetic background. We report that the UPR is required for sustained high-level INO1 expression in wild-type strains, but not for transient derepression in response to inositol deprivation. Moreover, the UPR is not required for expression or regulation of INO1 in response to the change in lipid metabolism that occurs in the sec14ts cki1Δ genetic background. Thus, the UPR signal transduction pathway is not involved directly in transcriptional regulation of INO1 and other UASINO-containing genes. However, we discovered that inactivation of Sec14p leads to activation of the UPR, and that sec14 cki1 strains exhibit defective vacuolar morphology, suggesting that the mechanism by which the cki1Δ mutation suppresses the growth and secretory defect of sec14 does not fully restore wild-type morphology. Finally, synthetic lethality involving sec14 and UPR mutations suggests that the UPR plays an essential role in survival of sec14 cki1 strains.
An Allelic Series of Mutations in the Kit ligand Gene of Mice. I. Identification of Point Mutations in Seven Ethylnitrosourea-Induced KitlSteel Alleles
Authors: Rajaraman S , Davis W S , Mahakali-Zama A , Evans H K , Russell L B , Bedell M A
Published: April 2021

An allelic series of mutations is an extremely valuable genetic resource for understanding gene function. Here we describe eight mutant alleles at the Steel (Sl) locus of mice that were induced with N-ethyl-N-nitrosourea (ENU). The product of the Sl locus is Kit ligand (or Kitl; also known as mast cell growth factor, stem cell factor, and Steel factor), which is a member of the helical cytokine superfamily and is the ligand for the Kit receptor tyrosine kinase. Seven of the eight ENU-induced KitlSl alleles, of which five cause missense mutations, one causes a nonsense mutation and exon skipping, and one affects a splice site, were found to contain point mutations in Kitl. Interestingly, each of the five missense mutations affects residues that are within, or very near, conserved α-helical domains of Kitl. These ENU-induced mutants should provide important information on structural requirements for function of Kitl and other helical cytokines.
The Control of Natural Variation in Cytosine Methylation in Arabidopsis
Authors: Riddle Nicole C , Richards Eric J
Published: April 2021

We explore the extent and sources of epigenetic variation in cytosine methylation in natural accessions of the flowering plant, Arabidopsis thaliana, by focusing on the methylation of the major rRNA gene repeats at the two nucleolus organizer regions (NOR). Our findings indicate that natural variation in NOR methylation results from a combination of genetic and epigenetic mechanisms. Genetic variation in rRNA gene copy number and trans-acting modifier loci account for some of the natural variation in NOR methylation. Our results also suggest that divergence and inheritance of epigenetic information, independent of changes in underlying nucleotide sequence, may play an important role in maintaining natural variation in cytosine methylation.
The Effect of Population History on the Lengths of Ancestral Chromosome Segments
Authors: Chapman Nicola H , Thompson Elizabeth A
Published: April 2021

An isolated population is a group of individuals who are descended from a founding population who lived some time ago. If the founding individuals are assumed to be noninbred and unrelated, a chromosome sampled from the population can be represented as a mosaic of segments of the original ancestral types. A population in which chromosomes are made up of a few long segments will exhibit linkage disequilibrium due to founder effect over longer distances than a population in which the chromosomes are made up of many short segments. We study the length of intact ancestral segments by obtaining the expected number of junctions (points where DNA of two distinct ancestral types meet) in a chromosome. Assuming random mating, we study analytically the effects of population age, growth patterns, and internal structure on the expected number of junctions in a chromosome. We demonstrate that the type of growth a population has experienced can influence the expected number of junctions, as can population subdivision. These effects are substantial only when population sizes are very small. We also develop an approximation to the variance of the number of junctions and show that the variance is large.
Enzyme Kinetics, Substitutable Resources and Competition: From Biochemistry to Frequency-Dependent Selection in lac
Authors: Lunzer Mark , Natarajan Arvind , Dykhuizen Daniel E , Dean Antony M
Published: April 2021

Trade-offs in catalytic efficiency at the lac permease of Escherichia coli produce alleles with different substrate specializations that are selectively favored on different galactosides. We show that differential resource utilization during competition for mixtures of galactosides produces frequency-dependent selection at lac. However, the polymorphism is protected only in a narrow range of galactoside ratios despite intense selection on the pure galactosides. Hence, stabilizing frequency-dependent selection protecting natural allozyme polymorphisms through differential resource utilization will be sporadic and ephemeral in randomly changing environments. A comparison of predictions, based on first principles, with experimental outcomes reveals an additional, unanticipated source of weak selection.
Escherichia coli Strains (ndk) Lacking Nucleoside Diphosphate Kinase Are Powerful Mutators for Base Substitutions and Frameshifts in Mismatch-Repair-Deficient Strains
Authors: Miller Jeffrey H , Funchain Pauline , Clendenin Wendy , Huang Tiffany , Nguyen Anh , Wolff Erika , Yeung Annie , Chiang Ju-Huei , Garibyan Lilit , Slupska Malgorzata M , Yang Hanjing
Published: April 2021

Nucleoside diphosphate (NDP) kinase is one of the enzymes that maintains triphosphate pools. Escherichia coli strains (ndk) lacking this enzyme have been shown to be modest base substitution mutators, and two members of the human family of NDP kinases act as tumor suppressors. We show here that in E. coli strains lacking NDP kinase high levels of mispairs are generated, but most of these are corrected by the mismatch-repair system. Double mutants that are ndk mutS, lacking both the NDP kinase and mismatch repair, have levels of base substitutions 15-fold higher and levels of certain frameshifts up to 10-fold higher than those of the respective mutations in mutS strains that are NDP kinase proficient. A sequence analysis of the specificity of base substitution mutations generated in ndk and ndk mutS backgrounds as well as other experiments suggests that NDP kinase deficiency stimulates polymerase errors that lead to A:T → G:C transitions and that the editing capacity of cells may be affected, leading to additional uncorrected mispairs and to A:T → T:A transversions.
Transcription and Double-Strand Breaks Induce Similar Mitotic Recombination Events in Saccharomyces cerevisiae
Authors: González-Barrera Sergio , García-Rubio María , Aguilera Andrés
Published: April 2021

We have made a comparative analysis of double-strand-break (DSB)-induced recombination and spontaneous recombination under low- and high-transcription conditions in yeast. We constructed two different recombination substrates, one for the analysis of intermolecular gene conversions and the other for intramolecular gene conversions and inversions. Such substrates were based on the same leu2-HOr allele fused to the tet promoter and containing a 21-bp HO site. Gene conversions and inversions were differently affected by rad1, rad51, rad52, and rad59 single and double mutations, consistent with the actual view that such events occur by different recombination mechanisms. However, the effect of each mutation on each type of recombination event was the same, whether associated with transcription or induced by the HO-mediated DSB. Both the highly transcribed DNA and the HO-cut sequence acted as recipients of the gene conversion events. These results are consistent with the hypothesis that transcription promotes initiation of recombination along the DNA sequence being transcribed. The similarity between transcription-associated and DSB-induced recombination suggests that transcription promotes DNA breaks.
The Ubiquitin-Dependent Targeting Pathway in Saccharomyces cerevisiae Plays a Critical Role in Multiple Chromatin Assembly Regulatory Steps
Authors: Harkness Troy A A , Davies Gerald F , Ramaswamy Vijay , Arnason Terra G
Published: April 2021

In a screen designed to isolate Saccharomyces cerevisiae strains defective for in vitro chromatin assembly, two temperature-sensitive (ts) mutants were obtained: rmc1 and rmc3 (remodeling of chromatin). Cloning of RMC1 and RMC3 revealed a broad role for the ubiquitin-dependent targeting cascade as the ubiquitin-protein ligases (E3s), the anaphase promoting complex (APC; RMC1 encodes APC5) and Rsp5p, respectively, were identified. Genetic studies linked the rmc1/apc5 chromatin assembly defect to APC function: rmc1/apc5 genetically interacted with apc9Δ, apc10Δ, and cdc26Δ mutants. Furthermore, phenotypes associated with the rmc1/apc5 allele were consistent with defects in chromatin metabolism and in APC function: (i) UV sensitivity, (ii) plasmid loss, (iii) accumulation of G2/M cells, and (iv) suppression of the ts defect by growth on glucose-free media and by expression of ubiquitin. On the other hand, the multifunctional E3, Rsp5p, was shown to be required for both in vitro and in vivo chromatin assembly, as well as for the proper transcriptional and translational control of at least histone H3. The finding that the distinctly different E3 enzymes, APC and Rsp5p, both play roles in regulating chromatin assembly highlight the depth of the regulatory networks at play. The significance of these findings will be discussed.
The Est1 Subunit of Saccharomyces cerevisiae Telomerase Makes Multiple Contributions to Telomere Length Maintenance
Authors: Evans Sara K , Lundblad Victoria
Published: April 2021

The telomerase-associated Est1 protein of Saccharomyces cerevisiae mediates enzyme access by bridging the interaction between the catalytic core of telomerase and the telomere-binding protein Cdc13. In addition to recruiting telomerase, Est1 may act as a positive regulator of telomerase once the enzyme has been brought to the telomere, as previously suggested by the inability of a Cdc13-Est2 fusion protein to promote extensive telomere elongation in an est1-Δ strain. We report here three classes of mutant Est1 proteins that retain association with the telomerase enzyme but confer different in vivo consequences. Class 1 mutants display a telomere replication defect but are capable of promoting extensive telomere elongation in the presence of a Cdc13-Est2 fusion protein, consistent with a defect in telomerase recruitment. Class 2 mutants fail to elongate telomeres even in the presence of the Cdc13-Est2 fusion, which is the phenotype predicted for a defect in the proposed second regulatory function of EST1. A third class of mutants impairs an activity of Est1 that is potentially required for the Ku-mediated pathway of telomere length maintenance. The isolation of mutations that perturb separate functions of Est1 demonstrates that a telomerase holoenzyme subunit can contribute multiple regulatory roles to telomere length maintenance.
The Role of the Carboxyterminal Domain of RNA Polymerase II in Regulating Origins of DNA Replication in Saccharomyces cerevisiae
Authors: Gauthier Laura , Dziak Renata , Kramer David J H , Leishman David , Song Xiaomin , Ho Jason , Radovic Maja , Bentley David , Yankulov Krassimir
Published: April 2021

MCM (minichromosome maintenance) proteins function as a replication licensing factor (RLF-M), which contributes to limiting initiation of DNA replication to once per cell cycle. In the present study we show that a truncation of the pol II CTD in a S. cerevisiae strain harboring a mutation in mcm5 partially reverses its ts phenotype and improves maintenance of CEN/ARS minichromosomes. We correlate this phenotype to effects on DNA replication rather than to effects on transcription or specific gene expression. We also demonstrate that a similar truncation of the CTD reduces minichromosome stability and impairs stimulation of DNA replication by trans-activators and that tethering of recombinant pol II CTD to an origin of replication has a significant stimulatory effect on minichromosome stability. Furthermore, we show that pol II is recruited to ARS1. We propose that in S. cerevisiae a mechanism of coordinating pol II transcription and DNA replication is mediated by the CTD of pol II.
The DrosophilaslamdanceGene: A Mutation in an Aminopeptidase Can Cause Seizure, Paralysis and Neuronal Failure
Authors: Zhang HaiGuang , Tan Jeff , Reynolds Elaine , Kuebler Daniel , Faulhaber Sally , Tanouye Mark
Published: April 2021

We report here the characterization of slamdance (sda), a Drosophila melanogaster “bang-sensitive” (BS) paralytic mutant. This mutant exhibits hyperactive behavior and paralysis following a mechanical “bang” or electrical shock. Electrophysiological analyses have shown that this mutant is much more prone to seizure episodes than normal flies because it has a drastically lowered seizure threshold. Through genetic mapping, molecular cloning, and RNA interference, we have demonstrated that the sda phenotype can be attributed to a mutation in the Drosophila homolog of the human aminopeptidase N (APN) gene. Furthermore, using mRNA in situ hybridization and LacZ staining, we have found that the sda gene is expressed specifically in the central nervous system at particular developmental stages. Together, these results suggest that the bang sensitivity in sda mutants is caused by a defective APN gene that somehow increases seizure susceptibility. Finally, by using the sda mutation as a sensitized background, we have been able to identify a rich variety of sda enhancers and other independent BS mutations.