bluuebunny/crossref_metadata_2025 huggingface dataset:
A genetic model for control of hypertriglyceridemia and apolipoprotein B levels in the Johns Hopkins colony of St. Thomas Hospital rabbits.
Authors: Beaty T H , Prenger V L , Virgil D G , Lewis B , Kwiterovich P O , Bachorik P S
Published: January 2021

The St. Thomas Hospital (STH) rabbit has been previously shown to have a Mendelian form of hypertriglyceridemia, accompanied by accelerated atherosclerosis, and these animals may serve as a useful model for human dyslipoproteinemia syndromes. Here we describe the establishment of a new colony of these STH animals, and present genetic analysis of triglyceride (TG) and apolipoprotein B (apoB) levels. Segregation analysis of TG in 39 STH animals and 24 controls gave evidence of Mendelian segregation for an allele leading to both elevated TG levels and increased variability in these levels. Predicted means from the most parsimonious model for the Johns Hopkins STH colony were quite similar to that seen in the original London colony, and this model accounted for 80% of the variation in TG seen in the sample. This hypertriglyceridemia locus indirectly influenced the mean apoB levels in these rabbits, and segregation analysis of mean apoB levels suggested a second locus controlling apoB levels. Analysis of residual apoB levels (adjusted for predicted effects of the hypertriglyceridemia locus) revealed clearer evidence for a second locus controlling mean apoB levels in this colony. Arguments for two distinct genetic mechanisms operating in these STH animals are presented.
Chi enhances heteroduplex DNA levels during recombination.
Authors: Holbeck S L , Smith G R
Published: January 2021

The major pathway of homologous recombination in Escherichia coli, the RecBCD pathway, is stimulated by Chi sites. To determine whether Chi enhances an early or late step in recombination, we measured formation of heteroduplex DNA (hDNA) in extracts of lambda-infected E. coli. Chi elevated hDNA levels in these extracts, supporting a role for Chi early (before hDNA formation) in recombination. RecA protein and RecBCD enzyme were both necessary for detection of hDNA, indicating that they, too, act early. Analysis of a panel of recBCD mutants indicated that Chi-nicking activity was needed for Chi's stimulation of hDNA formation. These results support a previously proposed model of recombination. Further results suggested that RecBCD enzyme has an additional role late in recombination.
Spontaneous amplification of the ADH4 gene in Saccharomyces cerevisiae.
Authors: Dorsey M , Peterson C , Bray K , Paquin C E
Published: January 2021

Five spontaneous amplifications of the ADH4 gene were identified among 1,894 antimycin A-resistant mutants isolated from a diploid strain after growth at 15 degrees. Four of these amplifications are approximately 40-kb linear extrachromosomal palindromes carrying telomere homologous sequences at each end similar to a previously isolated amplification. ADH4 is located at the extreme left end of chromosome VII, and the extrachromosomal fragments appear to be the fusion of two copies of the end of this chromosome. The fifth amplification is a chromosomal amplification carrying an extra copy of ADH4 on both homologs of chromosome VII. These results suggest that the ADH system can be used to study amplification in Saccharomyces cerevisiae.
Identification of functionally related genes that stimulate early meiotic gene expression in yeast.
Authors: Su S S , Mitchell A P
Published: January 2021

Meiosis and spore formation in the yeast Saccharomyces cerevisiae are associated with increased expression of sporulation-specific genes. One of these genes, IME2, encodes a putative protein kinase that is a positive regulator of other sporulation-specific genes. We have isolated mutations that cause reduced expression of an ime2-lacZ fusion gene. We found mutations in IME1, a known positive regultor of IME2, and MCK1, a known positive regulator of IME1. We also isolated recessive mutations in 12 other genes, which we designate RIM (Regulator of IME2) genes. Our analysis indicates that the defects in rim1, rim8, rim9 and rim13 mutants are a consequence of diminished IME1 expression and can be suppressed by expression of IME1 from the heterologous ACT1 promoter. These rim mutations also reduced expression of an ime1-HIS3 fusion, in which the HIS3 gene is expressed from the IME1 promoter, and caused reduced levels of IME1 RNA. Although the rim1, rim8, rim9 and rim13 mutant phenotypes are similar to those of mck1 mutants, we found that the defects in ime2-lacZ expression and sporulation of the mck1 rim double mutants were more severe than either single mutant. In contrast, the defects of the rim rim double mutants were similar to either single mutant. The rim1, rim8, rim9 and rim13 mutants also display slow growth at 17 degrees and share a smooth colony morphology that is not evident in mck1 mutants or isogenic wild-type strains. We suggest that RIM1, RIM8, RIM9 and RIM13 encode functionally related products that act in parallel to MCK1 to stimulate IME1 expression.
Statistical tests of neutrality of mutations.
Authors: Fu Y X , Li W H
Published: January 2021

Mutations in the genealogy of the sequences in a random sample from a population can be classified as external and internal. External mutations are mutations that occurred in the external branches and internal mutations are mutations that occurred in the internal branches of the genealogy. Under the assumption of selective neutrality, the expected number of external mutations is equal to theta = 4Ne mu, where Ne is the effective population size and mu is the rate of mutation per gene per generation. Interestingly, this expectation is independent of the sample size. The number of external mutations is likely to deviate from its neutral expectation when there is selection while the number of internal mutations is less affected by the presence of selection. Statistical properties of the numbers of external mutations and of internal mutations are studied and their relationships to two commonly used estimates of theta are derived. From these properties, several new statistical tests based on a random sample of DNA sequences from the population are developed for testing the hypothesis that all mutations at a locus are neutral.
A mutational analysis of killer toxin resistance in Saccharomyces cerevisiae identifies new genes involved in cell wall (1-->6)-beta-glucan synthesis.
Authors: Brown J L , Kossaczka Z , Jiang B , Bussey H
Published: January 2021

Recessive mutations leading to killer resistance identify the KRE9, KRE10 and KRE11 genes. Mutations in both the KRE9 and KRE11 genes lead to reduced levels of (1-->6)-beta-glucan in the yeast cell wall. The KRE11 gene encodes a putative 63-kD cytoplasmic protein, and disruption of the KRE11 locus leads to a 50% reduced level of cell wall (1-->6)-glucan. Structural analysis of the (1-->6)-beta-glucan remaining in a kre11 mutant indicates a polymer smaller in size than wild type, but containing a similar proportion of (1-->6)- and (1-->3)-linkages. Genetic interactions among cells harboring mutations at the KRE11, KRE6 and KRE1 loci indicate lethality of kre11 kre6 double mutants and that kre11 is epistatic to kre1, with both gene products required to produce the mature glucan polymer at wild-type levels. Analysis of these KRE genes should extend knowledge of the beta-glucan biosynthetic pathway, and of cell wall synthesis in yeast.
Genetic studies of mei-1 gene activity during the transition from meiosis to mitosis in Caenorhabditis elegans.
Authors: Clandinin T R , Mains P E
Published: January 2021

Genetic evidence suggests that the mei-1 locus of Caenorhabditis elegans encodes a maternal product required for female meiosis. However, a dominant gain-of-function allele, mei-1(ct46), can support normal meiosis but causes defects in subsequent mitotic spindles. Previously identified intragenic suppressors of ct46 lack functional mei-1 activity; null alleles suppress only in cis but other alleles arise frequently and suppress both in cis and in trans. Using a different screen for suppressors of the dominant ct46 defect, the present study describes another type of intragenic mutation that also arises at high frequency. These latter alleles appear to have reduced meiotic activity and retain a weakened dominant effect. Characterization of these alleles in trans-heterozygous combinations with previously identified mei-1 alleles has enabled us to define more clearly the role of the mei-1 gene product during normal embryogenesis. We propose that a certain level of mei-1 activity is required for meiosis but must be eliminated prior to mitosis. The dominant mutation causes mei-1 activity to function at mitosis; intragenic trans-suppressors act in an antimorphic manner to inactivate multimeric mei-1 complexes. We propose that inactivation of meiosis-specific functions may be an essential precondition of mitosis; failure to eliminate such functions may allow ectopic meiotic activity during mitosis and cause embryonic lethality.
Nuclear mutations in Saccharomyces cerevisiae that affect the escape of DNA from mitochondria to the nucleus.
Authors: Thorsness P E , Fox T D
Published: January 2021

We have inserted a yeast nuclear DNA fragment bearing the TRP1 gene and its associated origin of DNA replication, ARS1, into the functional mitochondrial chromosome of a strain carrying a chromosomal trp1 deletion. TRP1 was not phenotypically expressed within the organelle. However, this Trp- strain readily gave rise to respiratory competent Trp+ clones that contained the TRP1/ARS1 fragment, associated with portions of mitochondrial DNA (mtDNA), replicating in their nuclei. Thus the Trp+ clones arose as a result of DNA escaping from mitochondria and migrating to the nucleus. We have isolated 21 nuclear mutants in which the rate of mtDNA escape is increased by screening for increased rates of papillation to Trp+. All 21 mutations were recessive and fell into six complementation groups, termed YME1-YME6. In addition to increasing the rate of mtDNA escape, yme1 mutations also caused a heat-sensitive respiratory deficient phenotype at 37 degrees and a cold-sensitive growth defect on complete glucose medium at 14 degrees. While the other yme mutations had no detectable growth phenotypes, synergistic interactions were observed in two double mutant combinations: a yme1, yme2 double mutant failed to respire at 30 degrees and a yme4, yme6 double mutant failed to respire at all temperatures tested. None of the respiratory defects were caused by loss of functional mtDNA. These findings suggest that yme1, yme2, yme4 and yme6 mutations alter mitochondrial functions and thereby lead to an increased rate of DNA escape from the organelle.
Estimation of mitotic stability in conidial fungi: a theoretical framework.
Authors: Basten C J , Asmussen M A
Published: January 2021

Mitotic stability refers to the probability that genetic elements are transmitted to both daughters during mitosis. This is of practical importance in molecular genetics because autonomous cloning vectors should be transmitted at high frequency during mitosis. In filamentous coencytic fungi it is difficult to quantify mitotic stability because a fluctuation test is not feasible. We show how to get around this problem by formulating a general model of the transmission of nuclear genetic elements through the course of conidiogenesis. We derive formulas by two different methods for the expected proportion of conidiospores that retain the element as a function of its mitotic stability and the number of generations of spore production. An important by-product yields the exact probability distributions for the number of conidiospores retaining elements at each stage of conidiophore development. We outline, and illustrate through specific numerical examples, how to use these formulas to estimate mitotic stability. Although we use Aspergillus nidulans as our biological paradigm, the same general framework can be extended to other fungal species, and possibly to less closely related systems as well.
The influence of primary and secondary DNA structure in deletion and duplication between direct repeats in Escherichia coli.
Authors: Trinh T Q , Sinden R R
Published: January 2021

We describe a system to measure the frequency of both deletions and duplications between direct repeats. Short 17- and 18-bp palindromic and nonpalindromic DNA sequences were cloned into the EcoRI site within the chloramphenicol acetyltransferase gene of plasmids pBR325 and pJT7. This creates an insert between direct repeated EcoRI sites and results in a chloramphenicol-sensitive phenotype. Selection for chloramphenicol resistance was utilized to select chloramphenicol resistant revertants that included those with precise deletion of the insert from plasmid pBR325 and duplication of the insert in plasmid pJT7. The frequency of deletion or duplication varied more than 500-fold depending on the sequence of the short sequence inserted into the EcoRI site. For the nonpalindromic inserts, multiple internal direct repeats and the length of the direct repeats appear to influence the frequency of deletion. Certain palindromic DNA sequences with the potential to form DNA hairpin structures that might stabilize the misalignment of direct repeats had a high frequency of deletion. Other DNA sequences with the potential to form structures that might destabilize misalignment of direct repeats had a very low frequency of deletion. Duplication mutations occurred at the highest frequency when the DNA between the direct repeats contained no direct or inverted repeats. The presence of inverted repeats dramatically reduced the frequency of duplications. The results support the slippage-misalignment model, suggesting that misalignment occurring during DNA replication leads to deletion and duplication mutations. The results also support the idea that the formation of DNA secondary structures during DNA replication can facilitate and direct specific mutagenic events.
Reverse genetics of Drosophila RNA polymerase II: identification and characterization of RpII140, the genomic locus for the second-largest subunit.
Authors: Hamilton B J , Mortin M A , Greenleaf A L
Published: January 2021

We have used a reverse genetics approach to isolate genes encoding two subunits of Drosophila melanogaster RNA polymerase II. RpII18 encodes the 18-kDa subunit and maps cytogenetically to polytene band region 83A. RpII140 encodes the 140-kDa subunit and maps to polytene band region 88A10:B1,2. Focusing on RpII140, we used in situ hybridization to map this gene to a small subinterval defined by the endpoints of a series of deficiencies impinging on the 88A/B region and showed that it does not represent a previously known genetic locus. Two recently defined complementation groups, A5 and Z6, reside in the same subinterval and thus were candidates for the RpII140 locus. Phenotypes of A5 mutants suggested that they affect RNA polymerase II, in that the lethal phase and the interaction with developmental loci such as Ubx resemble those of mutants in the gene for the largest subunit, RpII215. Indeed, we have achieved complete genetic rescue of representative recessive lethal mutations of A5 with a P-element construct containing a 9.1-kb genomic DNA fragment carrying RpII140. Interestingly, the initial construct also rescued lethal alleles in the neighboring complementation group, Z6, revealing that the 9.1-kb insert carries two genes. Deleting coding region sequences of RpII140, however, yielded a transformation vector that failed to rescue A5 alleles but continued to rescue Z6 alleles. These results strongly support the conclusion that the A5 complementation group is equivalent to the genomic RpII140 locus.
Codon usage bias and base composition of nuclear genes in Drosophila.
Authors: Moriyama E N , Hartl D L
Published: January 2021

The nuclear genes of Drosophila evolve at various rates. This variation seems to correlate with codon-usage bias. In order to elucidate the determining factors of the various evolutionary rates and codon-usage bias in the Drosophila nuclear genome, we compared patterns of codon-usage bias with base compositions of exons and introns. Our results clearly show the existence of selective constraints at the translational level for synonymous (silent) sites and, on the other hand, the neutrality or near neutrality of long stretches of nucleotide sequence within noncoding regions. These features were found for comparisons among nuclear genes in a particular species (Drosophila melanogaster, Drosophila pseudoobscura and Drosophila virilis) as well as in a particular gene (alcohol dehydrogenase) among different species in the genus Drosophila. The patterns of evolution of synonymous sites in Drosophila are more similar to those in the prokaryotes than they are to those in mammals. If a difference in the level of expression of each gene is a main reason for the difference in the degree of selective constraint, the evolution of synonymous sites of Drosophila genes would be sensitive to the level of expression among genes and would change as the level of expression becomes altered in different species. Our analysis verifies these predictions and also identifies additional selective constraints at the translational level in Drosophila.
VNTR allele frequency distributions under the stepwise mutation model: a computer simulation approach.
Authors: Shriver M D , Jin L , Chakraborty R , Boerwinkle E
Published: January 2021

Variable numbers of tandem repeats (VNTRs) are a class of highly informative and widely dispersed genetic markers. Despite their wide application in biological science, little is known about their mutational mechanisms or population dynamics. The objective of this work was to investigate four summary measures of VNTR allele frequency distributions: number of alleles, number of modes, range in allele size and heterozygosity, using computer simulations of the one-step stepwise mutation model (SMM). We estimated these measures and their probability distributions for a wide range of mutation rates and compared the simulation results with predictions from analytical formulations of the one-step SMM. The average heterozygosity from the simulations agreed with the analytical expectation under the SMM. The average number of alleles, however, was larger in the simulations than the analytical expectation of the SMM. We then compared our simulation expectations with actual data reported in the literature. We used the sample size and observed heterozygosity to determine the expected value, 5th and 95th percentiles for the other three summary measures, allelic size range, number of modes and number of alleles. The loci analyzed were classified into three groups based on the size of the repeat unit: microsatellites (1-2 base pair (bp) repeat unit), short tandem repeats [(STR) 3-5 bp repeat unit], and minisatellites (15-70 bp repeat unit). In general, STR loci were most similar to the simulation results under the SMM for the three summary measures (number of alleles, number of modes and range in allele size), followed by the microsatellite loci and then by the minisatellite loci, which showed deviations in the direction of the infinite allele model (IAM). Based on these differences, we hypothesize that these three classes of loci are subject to different mutational forces.
A genetic linkage map of Saccharum spontaneum L. 'SES 208'.
Authors: al-Janabi S M , Honeycutt R J , McClelland M , Sobral B W
Published: January 2021

The arbitrarily primed polymerase chain reaction was used to detect single-dose polymorphisms that, in turn, were used to generate a linkage map of a polyploid relative of cultivated sugarcane, Saccharum spontaneum 'SES 208' (2n = 64). The mapping population was composed of 88 progeny from a cross between SES 208 and a diploidized haploid derived from SES 208 by anther culture, ADP 85-0068. This cross allowed direct analysis of meiosis in SES 208 and gametic segregation ratios to be observed. One hundred twenty-seven 10-mer oligonucleotide primers of arbitrary sequence were selected from a pool of 420 primers used to screen the mapping parents. Three hundred thirty-six of the 420 primers amplified 4,540 loci or 13.5 loci per primer. The selected 127 primers revealed 2,160 loci of which 279 were present in SES 208 and absent in ADP 85-0068 and easily scored. Two hundred and eight (74.6%) of these 279 polymorphisms were single-dose polymorphisms (i.e., they displayed 1:1 segregation, chi 2 at 98% confidence level). Linkage analysis (theta = 0.25, LOD = 9.0 for two-point analysis, then theta = 0.25, LOD = 6.0 for multipoint analysis) of single-dose polymorphisms placed them into 42 linkage groups containing at least 2 markers. These single-dose markers span 1,500 contiguous centimorgans (cM) with 32 markers remaining unlinked (15.4%). From this 208-marker map we estimated the genome size of SES 208 to be 2,500 cM. The map has a predicted coverage of 85.1% at 30 cM, meaning that any new marker placed has an 85.1% chance of being within 30 cM of an existing marker. Furthermore, we show that SES 208 behaves like an autopolyploid because (i) the ratio of single-dose markers to higher dose markers fit the assumption of auto-octaploidy and (ii) the absence of repulsion phase linkages. This is the first genetic map constructed directly on a polyploid species for which no diploid relatives are known.
A single amino acid difference is sufficient to elicit vegetative incompatibility in the fungus Podospora anserina.
Authors: Deleu C , Clavé C , Bégueret J
Published: January 2021

Vegetative incompatibility is known to limit heterokaryosis in filamentous fungi. It results from genetic differences between incompatible strains at specific loci. The proteins encoded by the two incompatible alleles het-s and het-S of the fungus Podospora anserina differ from each other by 14 amino acids. Two approaches have been used to identify how many and which of these differences are necessary to elicit incompatibility. Twelve alleles of the het-s locus of wild-type isolates of P. anserina and of the related species Podospora comata have been sequenced to determine the extent of the variability of genes controlling s and S specificities. Expression of hybrid het-s/het-S genes and site-specific mutagenesis revealed that the specificities of het-s and het-S are under the control of a limited number of amino acid differences. The results show that vegetative incompatibility between s and S strains can be attributed to a single amino acid difference in the proteins encoded by the het-s locus.
Genetic evidence for functional interactions between actin noncomplementing (Anc) gene products and actin cytoskeletal proteins in Saccharomyces cerevisiae.
Authors: Vinh D B , Welch M D , Corsi A K , Wertman K F , Drubin D G
Published: January 2021

We describe here genetic interactions between mutant alleles of Actin-NonComplementing (ANC) genes and actin (ACT1) or actin-binding protein (SAC6, ABP1, TPM1) genes. The anc mutations were found to exhibit allele-specific noncomplementing interactions with different act1 mutations. In addition, mutant alleles of four ANC genes (ANC1, ANC2, ANC3 and ANC4) were tested for interactions with null alleles of actin-binding protein genes. An anc1 mutant allele failed to complement null alleles of the SAC6 and TPM1 genes that encode yeast fimbrin and tropomyosin, respectively. Also, synthetic lethality between anc3 and sac6 mutations, and between anc4 and tpm1 mutations was observed. Taken together, the above results strongly suggest that the ANC gene products contribute to diverse aspects of actin function. Finally, we report the results of tests of two models previously proposed to explain extragenic noncomplementation.
Responsiveness to exogenous cAMP of a Saccharomyces cerevisiae strain conferred by naturally occurring alleles of PDE1 and PDE2.
Authors: Mitsuzawa H
Published: January 2021

The Saccharomyces cerevisiae strain P-28-24C, from which cAMP requiring mutants derived, responded to exogenously added cAMP. Upon the addition of cAMP, this strain showed phenotypes shared by mutants with elevated activity of the cAMP pathway. Genetic analysis involving serial crosses of this strain to a strain with another genetic background revealed that the responsiveness to cAMP results from naturally occurring loss-of-function alleles of PDE1 and PDE2, which encode low and high affinity cAMP phosphodiesterases, respectively. In addition, P-28-24C was found to carry a mutation conferring slow growth that lies in CYR1, which encodes adenylate cyclase, and the slow growth phenotype caused by the cyr1 mutation was suppressed by the pde2 mutation. Therefore P-28-24C is fortuitously a pde1 pde2 cyr1 triple mutant. Responsiveness to cAMP conferred by pde mutations suggests that S. cerevisiae cells are permeable to cAMP to some extent and that the apparent absence of effect of exogenously added cAMP on wild-type cells is due to immediate degradation by cAMP phosphodiesterases.
Cloning of flagellar genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis.
Authors: Tam L W , Lefebvre P A
Published: January 2021

Chlamydomonas is a popular genetic model system for studying many cellular processes. In this report, we describe a new approach to isolate Chlamydomonas genes using the cloned nitrate reductase gene (NIT1) as an insertional mutagen. A linearized plasmid containing the NIT1 gene was introduced into nit1 mutant cells by glass-bead transformation. Of 3000 Nit+ transformants examined, 74 showed motility defects of a wide range of phenotypes, suggesting that DNA transformation is an effective method for mutagenizing cells. For 13 of 15 such motility mutants backcrossed to nit- mutant strains, the motility phenotype cosegregated with the Nit+ phenotype, indicating that the motility defects of these 13 mutants may be caused by integration of the plasmid. Further genetic analysis indicated that three of these mutants contained alleles of previously identified loci: mbo2 (move backward only), pf13 (paralyzed flagella) and vfl1 (variable flagellar number). Three other abnormal-flagellar-number mutants did not map to any previously described loci at which mutations produce similar phenotypes. Genomic sequences flanking the integrated plasmid in the mbo2 and vfl1 mutants were isolated and used as probes to obtain wild-type genomic clones, which complemented the motility defects upon transformation into cells. Our results demonstrate the potential of this new approach for cloning genes identified by mutation in Chlamydomonas.
A note on the estimation of the effective number of additive and dominant loci contributing to quantitative variation.
Authors: Ollivier L , Janss L L
Published: January 2021

A method of estimating the number of loci contributing to quantitative variation has been proposed by S. Wright in 1921. The method makes use of the means of inbred lines and the variances of their F1, F2 and backcrosses. The method has been extended to crosses between outbreeding populations by R. Lande in 1981. Additive gene action is one of the major assumptions required for obtaining valid estimates. It is shown here that this assumption may be relaxed. One can estimate both a total number of effective loci and a number of dominant loci (the latter only when the parents are inbred) by comparing the variances of the F1, F2 and backcrosses. Numerical illustrations are given, based on crossbreeding data.
Effects of multiple retrovirus insertions on quantitative traits of mice.
Authors: Keightley P D , Evans M J , Hill W G
Published: January 2021

To assess the potential to generate quantitative genetic variation by insertional mutagenesis in a vertebrate, lines of mice in which many provirus vector inserts segregated at a low initial frequency on an inbred background (insert lines) were subjected to divergent artificial selection on body weight at 6 weeks and responses and heritability estimates compared to control lines lacking inserts. Heritability estimates were more than 1.5 times greater in the insert lines than in the controls, but because the phenotypic variance was substantially higher in the insert lines the genetic variance was about 3 times greater. Realized heritability estimates tended to be lower than heritabilities estimated by an animal model which utilizes information in covariances between all relatives in the data set. A surprisingly large response to selection occurred in the inbred control line. Insert lines were about 20% less fertile than controls. Division of the selection lines into inbred sublines in the later generations of the experiment revealed substantially greater variation among sublines of the insert lines than among the controls. Heritabilities were similar to typical estimates for the trait in outbred populations. In conclusion, there was clear evidence of extra variation deriving from inserts, which has yet to be attributed to individual genes.
Deletion mapping of four loci defined by N-ethyl-N-nitrosourea-induced postimplantation-lethal mutations within the pid-Hbb region of mouse chromosome 7.
Authors: Rinchik E M , Carpenter D A , Long C L
Published: January 2021

As part of a long-term effort to refine the physical and functional maps of the Fes-Hbb region of mouse chromosome 7, four loci [l(7)1Rn, l(7)2Rn, l(7)3Rn, l(7)4Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutations were mapped by means of trans complementation crosses to mice carrying lethal deletions of the mouse chromosome-7 albino (c) locus. Each locus was assigned to a defined subregion of the deletion map at the distal end of the Fes-Hbb interval. Of particular use for this mapping were preimplantation-lethal deletions having distal breakpoints localized between pid and Omp. Hemizygosity or homozygosity for each of the ENU-induced lethals was found to arrest development after uterine implantation; the specific time of postimplantation death varied, and depended on both the mutation itself and on whether it was hemizygous or homozygous. Based on their map positions outside of and distal to deletions that cause death at preimplantation stages, these ENU-induced mutations identify loci, necessary for postimplantation development, that could not have been discovered by phenotypic analyses of mice homozygous for any albino deletion. The mapping of these loci to specific genetic intervals defined by deletion breakpoints suggests a number of positional-cloning strategies for the molecular isolation of these genes. Phenotypic and genetic analyses of these mutations should provide useful information on the functional composition of the corresponding segment of the human genome (perhaps human 11q13.5).
Evolution of multilocus genetic structure in Avena hirtula and Avena barbata.
Authors: Allard R W , García P , Sáenz-de-Miera L E , Pérez de la Vega M
Published: January 2021

Avena barbata, an autotetraploid grass, is much more widely adapted than Avena hirtula, its diploid ancestor. We have determined the 14-locus genotype of 754 diploid and 4751 tetraploid plants from 10 and 50 Spanish sites, respectively. Allelic diversity is much greater in the tetraploid (52 alleles) than in the diploid (38 alleles): the extra alleles of the tetraploid were present in nonsegregating heteroallelic quadriplexes. Seven loci were monomorphic for the same allele (genotypically 11) in all populations of the diploid: five of these loci were also monomorphic for the same allele (genotypically 1111) in all populations of the tetraploid whereas two loci each formed a heteroallelic quadriplex (1122) that was monomorphic or predominant in the tetraploid. Seven of the 14 loci formed one or more highly successful homoallelic and/or heteroallelic quadriplexes in the tetraploid. We attribute much of the greater heterosis and wider adaptedness of the tetraploid to favorable within-locus interactions and interlocus (epistatic) interactions among alleles of the loci that form heteroallelic quadriplexes. It is difficult to account for the observed patterns in which genotypes are distributed ecogeographically except in terms of natural selection favoring particular alleles and genotypes in specific habitats. We conclude that natural selection was the predominant integrating force in shaping the specific genetic structure of different local populations as well as the adaptive landscape of both the diploid and tetraploid.
A method for calibrating molecular clocks and its application to animal mitochondrial DNA.
Authors: Lynch M , Jarrell P E
Published: January 2021

A generalized least-squares procedure is introduced for the calibration of molecular clocks and applied to the complete mitochondrial DNA sequences of 13 animal species. The proposed technique accounts for both nonindependence and heteroscedasticity of molecular-distance data, problems that have not been taken into to account in such analyses in the past. When sequence-identity data are transformed to account for multiple substitutions/site, the molecular divergence scales linearly with time, but with substantially more variation in the substitution rate than expected under a Poisson model. Significant levels of divergence are predicted at zero divergence time for most loci, suggesting high levels of site-specific heterozygosity among mtDNA molecules establishing in sister taxa. For nearly all loci, the baseline heterozygosity is lower and the substitution rate is higher in mammals relative to other animals. There is considerable variation in the evolutionary rate among loci but no compelling evidence that the average rate of mtDNA evolution is elevated with respect to that of nuclear DNA. Using the observed patterns of interspecific divergence, empirical estimates are derived for the mean coalescence times of organelles colonizing sister taxa.
The Drosophila maternal effect locus deadhead encodes a thioredoxin homolog required for female meiosis and early embryonic development.
Authors: Salz H K , Flickinger T W , Mittendorf E , Pellicena-Palle A , Petschek J P , Albrecht E B
Published: January 2021

This study describes the identification, function and molecular characterization of deadhead, a Drosophila thioredoxin homolog. Although in vitro studies have shown that thioredoxin can post-translationally regulate the activity of many different proteins, we find that this homolog is not essential for viability. The phenotypic analysis of two different mutations which eliminate function suggests that dhd is essential for female meiosis. The majority of eggs laid by females homozygous for null mutations are fertilized but fail to complete meiosis. A small number of escaper embryos initiate development and display a range of phenotypes suggesting functions in both preblastoderm mitosis and head development. Our analysis of deadhead's RNA expression pattern is consistent with its maternal effect function: the RNA is predominately expressed in the nurse cells of the ovary, is maternally deposited into the egg, but does not appear to be zygotically expressed during embryogenesis. Thus both our genetic and molecular data are consistent with a function during meiosis and preblastoderm mitosis. Whether the head defect indicates an additional function or is an indirect consequence of earlier defects remains to be determined.
Recovery of heritable, transposon-induced, mutant alleles of the rf 2 nuclear restorer of T-cytoplasm maize.
Authors: Schnable P S , Wise R P
Published: January 2021

T (Texas) cytoplasm is associated with a mitochondrial disruption that is phenotypically expressed during microsporogenesis resulting in male sterility. Restoration of pollen fertility in T-cytoplasm maize is controlled by dominant alleles at two unlinked, complementary, nuclear-encoded genes, rf1 and rf2. As a first step in the molecular isolation of the rf2 gene, 178,300 gametes derived from plants that carried the Mutator, Cy or Spm transposon families were screened for rf2 mutant alleles (rf2-m) via their inability to restore pollen fertility to T-cytoplasm male-sterile maize. Seven heritable rf2-m alleles were recovered from these transposon populations. Pedigrees and restriction fragment length polymorphism (RFLP)-based analyses indicated that all seven rf 2-m alleles were derived independently. The ability to obtain rf 2-m derivatives from Rf2 suggests that Rf2 alleles produce a functional product necessary to restore pollen fertility to cmsT. Molecular markets flanking the rf1 and rf2 loci were used to decipher segregation patterns in progenies segregating for the rf2-m alleles. These analyses provided preliminary evidence of a weak, third restorer gene of cmsT that can substitute for Rf1.
Detection of genetic interference: simulation studies and mouse data.
Authors: Weeks D E , Ott J , Lathrop G M
Published: January 2021

Genetic chiasma interference occurs when the occurrence of one crossover (or chiasma) influences the probability of another crossover occurring nearby. We investigated, by simulation studies, the power of three statistical methods to detect interference. Neither the traditional three-locus method nor a multiplicative model approach are very powerful, while a multilocus-feasible map function approach is more powerful, particularly as the number of loci increases. We show that the power to detect interference is quite sensitive to the underlying type of interference. When we tested for interference in two mouse data sets (from chromosomes 1 and 12), we found significant evidence of positive interference.
Enhancement of telomere-plasmid segregation by the X-telomere associated sequence in Saccharomyces cerevisiae involves SIR2, SIR3, SIR4 and ABF1.
Authors: Enomoto S , Longtine M S , Berman J
Published: January 2021

We have previously shown that circular replicating plasmids that carry yeast telomere repeat sequence (TG1-3) tracts segregate efficiently relative to analogous plasmids lacking the TG1-3 tract and this efficient segregation is dependent upon RAP1. While a long TG1-3 tract is sufficient to improve plasmid segregation, the segregation efficiency of telomere plasmids (TEL-plasmids) is enhanced when the X-Telomere Associated Sequence (X-TAS) is also included on the plasmids. We now demonstrate that the enhancement of TEL-plasmid segregation by the X-TAS depends on SIR2, SIR3, SIR4 and ABF1 in trans and requires the Abf1p-binding site within the X-TAS. Mutation of the Abf1p-binding site within the X-TAS results in TEL-plasmids that are no longer affected by mutations in SIR2, SIR3 or SIR4, despite the fact that other Abf1p-binding sites are present on the plasmid. Mutation of the ARS consensus sequence within the X-TAS converts the X-TAS from an enhancer element to a negative element that interferes with TEL-plasmid segregation in a SIR-dependent manner. Thus, telomere associated sequences interact with TG1-3 tracts on the plasmid, suggesting that the TASs have an active role in modulating telomere function.
Transient, meiosis-induced expression of the rec6 and rec12 genes of Schizosaccharomyces pombe.
Authors: Lin Y , Smith G R
Published: January 2021

Two meiotic recombination genes, rec6 and rec12, from Schizosaccharomyces pombe have been cloned by genetic complementation and their DNA sequences determined. Gene replacements demonstrated that the cloned fragments contained the rec6 and rec12 genes. Further analysis showed that the functional rec6 gene was within a 1.3-kb fragment and rec12 within a 1.7-kb fragment. The nucleotide sequences of these fragments revealed open reading frames (ORFs) predicting 143 amino acids for the rec6 gene product and 139 amino acids for the rec12 gene product. After pat1-114 temperature-induced meiosis, the transcripts of rec6 and rec12 were induced to maximal levels at 2-3 hr, at about the time of premeiotic DNA synthesis, but were present at much lower levels before and after this time. The transient induction of the transcripts and the phenotypes of the mutants suggest that the rec6 and rec12 gene products are involved primarily in the early steps of meiotic recombination in S. pombe. Near the rec6 gene are two additional ORFs potentially encoding proteins with homology to ribosomal protein S7 of Saccharomyces cerevisiae (ORF137) and to the homeodomain of developmental regulatory proteins (ORF201). The roles of these S. pombe ORFs remain to be determined.
Evidence that TUP1/SSN6 has a positive effect on the activity of the yeast activator HAP1.
Authors: Zhang L , Guarente L
Published: January 2021

The activity of the yeast transcriptional activator HAP1 is controlled by heme and the heme effect is mediated through the heme domain of HAP1. In this report, we show that HAP1 activity is significantly reduced in strains deleted of TUP1 or SSN6, and addition of a heme analog does not allow HAP1 to regain its full activity. Deletion of the heme domain alleviates the requirement for TUP1/SSN6. The results suggest that TUP1/SSN6 have a positive effect on the activity of HAP1 and this effect is mediated through the heme domain. Although TUP1/SSN6 generally repress transcription of many genes, our data indicate they may have positive effect on the expression of certain genes.
Effects of the maleless mutation on X and autosomal gene expression in Drosophila melanogaster.
Authors: Hiebert J C , Birchler J A
Published: January 2021

The mutational effect of the maleless (mle) gene in Drosophila has been reexamined. Earlier work had suggested that mle along with other male-lethal genes was responsible for hypertranscription of the X chromosome in males to bring about dosage compensation. Prompted by studies on dosage sensitive regulatory genes, we tested for effects of mlets on the phenotypes of 16 X or autosomal mutations in adult escapers of lethality. In third instar larvae, prior to the major lethal phase of mle, we examined activities of 6 X or autosomally encoded enzymes, steady state mRNA levels of 15 X-linked or autosomal genes and transcripts from two large genomic segments derived from either the X or from chromosome 2 and present in yeast artificial chromosomes. In contrast to the previously hypothesized role, we detected pronounced effects of mle on the expression of both X-linked and autosomal loci such that a large proportion of the tested genes were increased in expression, while only two X-linked loci were reduced. The most prevalent consequence was an increase of autosomal gene expression, which can explain previously observed reduced X:autosome transcription ratios. These observations suggest that if mle plays a role in the discrimination of the X and the autosomes, it may do so by modification of the effects of dosage sensitive regulatory genes.
Molecular population genetics of sex determination genes: the transformer gene of Drosophila melanogaster.
Authors: Walthour C S , Schaeffer S W
Published: January 2021

The transformer locus (tra) produces an RNA processing protein that alternatively splices the doublesex pre-mRNA in the sex determination hierarchy of Drosophila melanogaster. Comparisons of the tra coding region among Drosophila species have revealed an unusually high degree of divergence in synonymous and nonsynonymous sites. In this study, we tested the hypothesis that the tra gene will be polymorphic in synonymous and nonsynonymous sites within species by investigating nucleotide sequence variation in eleven tra alleles within D. melanogaster. Of the 1063 nucleotides examined, two synonymous sites were polymorphic and no amino acid variation was detected. Three statistical tests were used to detect departures from an equilibrium neutral model. Two tests failed to reject a neutral model of molecular evolution because of low statistical power associated with low levels of genetic variation (Tajima/Fu and Li). The Hudson, Kreitman, and Aguade test rejected a neutral model when the tra region was compared to the 5'-flanking region of alcohol dehydrogenase (Adh). The lack of variability in the tra gene is consistent with a recent selective sweep of a beneficial allele in or near the tra locus.
Efficient recovery of ENU-induced mutations from the zebrafish germline.
Authors: Solnica-Krezel L , Schier A F , Driever W
Published: January 2021

We studied the efficiency with which two chemical mutagens, ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can induce mutations at different stages of spermatogenesis in zebrafish (Brachydanio rerio). Both EMS and ENU induced mutations at high rates in post-meiotic germ cells, as indicated by the incidence of F1 progeny mosaic for the albino mutation. For pre-meiotic germ cells, however, only ENU was found to be an effective mutagen, as indicated by the frequencies of non-mosaic mutant progeny at four different pigmentation loci. Several mutagenic regimens that varied in either the number of treatments or the concentration of ENU were studied to achieve an optimal ratio between the mutagenicity and toxicity. For the two most mutagenic regimens: 4 x 1 hr in 3 mM ENU and 6 x 1 hr in 3 mM ENU, the minimum estimate of frequencies of independent mutations per locus per gamete was 0.9-1.3 X 10(-3). We demonstrate that embryonic lethal mutations induced with ENU were transmitted to offspring and that they could be recovered in an F2 screen. An average frequency of specific-locus mutations of 1.1 X 10(-3) corresponded to approximately 1.7 embryonic lethal mutations per single mutagenized genome. The high rates of mutations achievable with ENU allow for rapid identification of large numbers of genes involved in a variety of aspects of zebrafish development.
Fitness, flux and phantoms in temporally variable environments.
Authors: Dean A M
Published: January 2021

The evolutionary problem of selection in temporally variable environments is addressed by investigating a metabolic model describing the approach to steady state of a flux emanating from a simple linear pathway of unsaturated enzymes catalyzing reversible monomolecular reactions. Analysis confirms previous claims that steps having no influence on the steady state flux may influence transient behavior, and that enzymes immune to natural selection at steady state may become subject to selection when fitness is a function of individual transient metabolic events. Indeed, calculations show that the beta-galactosidase of Escherichia coli, which exerts a negligible effect on the steady state lactose flux, controls the approach to steady state. However, after 6 sec the lactose flux is within 0.1% of steady state, and so an ever changing environment must be invoked to continually expose beta-galactosidase to selection. Analysis of the metabolic model undergoing multiple transient events reveals that fitness differences remain unaffected if enzyme activities remain constant and become minimized if enzyme activities differ among environments. Until suitable data become available, claims that metabolic behavior away from steady state necessarily exposes a far greater proportion of allozymes to natural selection should be treated with great skepticism.