X-Linked Female-Sterile Loci in DROSOPHILA MELANOGASTER

We have examined the number of X-linked loci specifically required only during oogenesis. Complementation analyses among female-sterile (fs) mutations obtained in two mutagenesis screens--GANS' and MOHLER's--indicate that any fs locus represented by two or more mutant alleles in GANS' collection are usually present in MOHLER's collection. However, when a locus is represented by a single allele in one collection, it is generally not present in the other collection. We propose that this discrepancy is due to the fact that most "fs loci" represented by less than two mutant alleles are, in fact, vital (zygotic lethal) genes, and that the fs alleles are hypomorphic mutations of such genes. In support of this hypothesis we have identified lethal alleles at 12 of these "fs loci." The present analysis has possibly identified all maternal-effect lethal loci detectable by mutations on the X chromosome and has allowed us to reevaluate the number of "ovary-specific fs" loci in the Drosophila genome. Finally, germline clone analysis of a large number of fs mutations was performed in order to estimate the relative contribution of germline and somatic cell derivatives to oogenesis and to embryonic development. All the maternal-effect lethal loci tested are germline-dependent.     

The role of X-linked lethal and viable male-sterile mutations in male gametogenesis of Drosophila melanogaster: genetic analysis.

Summary The possibility that viable male-sterile mutations occur in vital genes and the role played by lethal mutations and viable male-steriles in male gametogenesis were studied. Five sterile loci were identified among the 30 most proximal vital loci of the X-chromosome and two of them were shown to be allelic with lethal mutations. Fertility tests on gynanders for nonautonomous lethal mutations proved that vital genes operate autonomously in male gonads, independently of their effect on somatic tissues. Fertility tests of ts lethals, shifted to the nonpermissive temperature after the TSP, showed that 40% of vital genes function in male gonads. It is further shown that about the same proportion of vital genes is operating in female gonads and that the two groups overlap by about 70%. The role of viable and lethal male sterile mutations in the control and regulation of male gametogenesis is discussed in detail.This work was supported by the Israel Commission for Basic Sciences.     

The Question of the Total Gene Number in DROSOPHILA MELANOGASTER

A statistical analysis has been carried out on the distribution and allelism of nearly 500 sex-linked, X-ray-induced, cytologically normal and rearranged lethal mutations in Drosophila melanogaster that were obtained by G. Lefevre. The mutations were induced in four different regions of the X chromosome: (1) 1A1-3E8, (2) 6D1-8A5, (3) 9E1-11A7 and (4) 19A1-20F4, which together comprise more than one-third of the entire chromosome.--The analysis shows that the number of alleles found at different loci does not fit a Poisson distribution, even when the proper procedures are taken to accommodate the truncated nature of the data. However, the allele distribution fits a truncated negative binomial distribution quite well, with cytologically normal mutations fitting better than rearrangement mutations. This indicates that genes are not equimutable, as required for the data to fit a Poisson distribution.--Using the negative binomial parameters to estimate the number of genes that did not produce a detectable lethal mutation in our experiment (n0) gave a larger number than that derived from the use of the Poisson parameter. Unfortunately, we cannot estimate the total numbers of nonvital loci, loci with undetectable phenotypes and loci having extremely low mutabilities. In any event, our estimate of the total vital gene number was far short of the total number of bands in the analyzed regions; yet, in several short intervals, we have found more vital genes than bands; in other intervals, fewer. We conclude that the one-band, one-gene hypothesis, in its literal sense, is not true; furthermore, it is difficult to support, even approximately.--The question of the total gene number in Drosophila will, not doubt, eventually be solved by molecular analyses, not by statistical analysis of mutation data or saturation studies.     

The distribution of randomly recovered X-ray-induced sex-linked genetic effects in Drosophila melanogaster

Cytogenetic analysis of more than 1500 randomly recovered lethal X chromosomes derived from 2000 and 3000 r X-ray exposures of post-meiotic male germ cells has made possible a plot of the distribution in different regions of the X chromosome of: (1) gene mutations associated with cytologically normal chromosomes, (2) mutations associated with chromosomal rearrangement breakpoints, (3) deficiencies, and (4) rearrangement breakpoints whether or not they are associated with mutations. The distribution of point mutations, vital loci and rearrangement breakpoints in different regions of the X chromosome is not proportional to either the number of bands or the relative DNA content. Further, the density of vital loci (those capable of mutating to a lethal allele) is quite different in some regions as compared to others. For example, vital loci in the 3AB region, which has been thoroughly studied by Judd and others, are at least as numerous as bands; whereas, the 3CD region, equally long, has only two vital loci. Other regions densely populated with vital loci include 1B, 1F-2A, 10A, 11A, and 19EF; sparsely populated regions include 6EF and 10B-10E. It seems reasonable to conclude that the recovered X-ray-induced mutants available for analysis do not represent a random sample of those initially induced in the exposed male germ cells.     

人工心脏瓣膜置换术后近期死亡因素的Logistic回归模型与分析

主要研究人工心脏瓣膜置换手术中导致病人术后近期死亡的重要影响因素．目的是通过统计学方法分析病人身体状况与治疗手段的数据,探讨导致病人死亡的因素,为临床上改善治疗方法,减少或消除危险因素,降低手术的死亡率提供参考．通过Logistic回归模型,对所有变量作单因素分析和相关因素分析,在此基础上,结合医学背景知识,对入选因素进行多因素的逐步筛选;对所得模型作残差分析,用刀切法加以检验,保证了模型的正确性。     

Genetic and Cytogenetic Analysis of the ADH Region in DROSOPHILA MELANOGASTER

Eighteen Adh-negative mutations were selected with 1-pentyn-3-ol after feeding of formaldehyde. Twelve of the 18 were shown by cytological and genetic analysis to be deletions. Cytological examination of the deletions allowed us to localize the Adh gene to a region including bands 35B3-5 on the left arm of chromosome 2. The deletions were also used to order known visible loci located near Adh.--The vital loci near Adh were also investigated. A total of 109 lethal mutations were generated with EMS and 33 of these, localized within a region defined by the overlap of two of the deletions, were found to belong to 13 complementation groups. If one includes three other loci known to belong there (el, Adh and Sco) a total of 16 complemetation groups have been identified in the region close to Adh.     

Multiple Allele Approach to the Study of Genes in DROSOPHILA MELANOGASTER That Are Involved in Imaginal Disc Development

The phenotypes of five different lethal mutants of Drosophila melanogaster that have small imaginal discs were analyzed in detail. From these results, we inferred whether or not the observed imaginal disc phenotype resulted exclusively from a primary imaginal disc defect in each mutant. To examine the validity of these inferences, we employed a multiple-allele method. Lethal alleles of the five third-chromosome mutations were identified by screening EMS-treated chromosomes for those which fail to complement with a chromosome containing all five reference mutations. Twenty-four mutants were isolated from 13,197 treated chromosomes. Each of the 24 was then tested for complementation with each of the five reference mutants. There was no significant difference in the mutation frequencies at these five loci. The stage of lethality and the imaginal disc morphology of each mutant allele were compared to those of its reference allele in order to examine the range of defects to be found among lethal alleles of each locus. In addition, hybrids of the alleles were examined for intracistronic complementation. For two of the five loci, we detected no significant phenotypic variation among lethal alleles. We infer that each of the mutant alleles at these two loci cause expression of the null activity phenotype. However, for the three other loci, we did detect significant phenotypic variation among lethal alleles. In fact, one of the mutant alleles at each of these three loci causes no detectable imaginal disc defect. This demonstrates that attempting to assess the developmental role of a gene by studying a single mutant allele may lead to erroneous conclusions. As a byproduct of the mutagenesis procedure, we have isolated two dominant, cold-sensitive mutants.     

Vital Genes That Flank Sex-Lethal, an X-Linked Sex-Determining Gene of DROSOPHILA MELANOGASTER

The X-chromosome:autosome balance in D. melanogaster appears to control both sex determination and dosage compensation through effects on a maternally influenced sex-linked gene called Sex-lethal (Sxl; 1-19.2). To facilitate molecular and genetic analysis of Sxl, we attempted to determine the locations of all ethyl methanesulfonate (EMS)-mutable genes vital to both sexes in the region between 6E1 and 7B1. This area includes approximately 1 cM of the genetic map on each side of Sxl and was reported by C. B. Bridges to contain 26 salivary gland polytene chromosome bands. The region appears rather sparsely populated with genes vital to both sexes, since the 122 recessive lethal mutations we recovered fell into only nine complementation groups. From one to 38 alleles of each gene were recovered. There was a preponderance of embryonic lethals in this area, although the lethal periods of loss-of-function mutations included larval, pupal and adult stages as well. Since the screen required that mutations be recessive and lethal to males, our failure to recover new Sxl alleles was the result expected for a gene with a female-specific function. An attempt was made to identify recessive male-specific lethals in this region, but none were found. Precise map positions were determined for eight of the nine vital genes. An interesting feature of the map is the location of Sxl in the middle of a 0.6- to 0.7-cM interval that appears to be devoid of genes vital to both sexes. The genetic location was determined of breakpoints near Sxl for all available chromosome rearrangements. Sxl is most likely located just to the left of band 7A1. We determined the relationship of our EMS-induced mutations in these nine genes to alleles induced by others. From this we conclude that the various genes appear to differ significantly from each other in their relative sensitivity to mutation by EMS vs. X rays.     

Pest control by the introduction of a conditional lethal trait on multiple loci: potential, limitations, and optimal strategies

Advances in genetics have made it feasible to genetically engineer insect strains carrying a conditional lethal trait on multiple loci. We model the release into a target pest population of insects carrying a dominant and fully penetrant conditional lethal trait on 1-20 loci. Delaying the lethality for several generations after release allows the trait to become widely spread in the target population before being activated. To determine effectiveness and optimal strategies for such releases, we vary release size, number of generations until the conditional lethality, nonconditional fitness cost resulting from gene insertions, and fitness reduction associated with laboratory rearing. We show that conditional lethal releases are potentially orders of magnitude more effective than sterile male releases of equal size, and that far smaller release sizes may be required for this approach than necessary with sterile males. For example, a release of male insects carrying a conditional lethal allele that is activated in the F4 generation on 10 loci reduces the target populatioin to 10(-4) of no-release size if there are initially two released males for every wild male. We show how the effectiveness of conditional lethal releases decreases as the nonconditional fitness reduction (i.e., fitness reduction before the trait becomes lethal) associated with the conditional lethal genes increases. For example, if there is a 5% nonconditional fitness cost per conditional lethal allele, then a 2:1 (released male:wild male) release with conditional lethal alleles that are activated in the F4 generation reduces the population to 2-5% (depending on the degree of density dependence) of the no-release size. If there is a per-allele reduction in fitness, then as the number of loci is increased there is a trade-off between the fraction of offspring carrying at least one conditional lethal allele and the fitness of the released insects. We calculate the optimal number of loci on which to insert the conditional lethal gene given various conditions. In addition, we show how laboratory-rearing fitness costs, density-dependence, and all-male versus male-female releases affect the efficiency of conditional lethal releases.     

Fine gentic structure of the 2D3-2F5 region of the X-chromosome of Drosophila melanogaster

Summary 97 lethal and semilethal mutations were induced by ethyl methanesulfonate, nitrosomethyl urea and -irradiation in the 2D3-F5 region of the X-chromosome of D. melanogaster. Approximately 1 per cent of the tested X-chromosomes carried a lethal in the 2D3-2F5 region. The mutation frequencies per band or DNA content in this region and the whole X-chromosome are equal.Complementation analysis revealed at least 10 functionally independent essential loci in this region including about 10 bands. The data presented in this study support the one bandone gene hypothesis.The Pgd locus coding for 6-phosphogluconate dehydrogenase (6PGD) is mapped in the 2D3 (or 2D4) band. Isolation of 11 lethal or semilethal point mutations with null or reduced 6PGD activity shows that the Pgd locus is a vital one.     

The fbpABC locus of Neisseria gonorrhoeae functions in the periplasm-to-cytosol transport of iron.

We have determined that the DNA sequence downstream of the well-characterized gonococcal fbp gene contains two open reading frames: one designated fbpB, which encodes a protein proposed to function as a cytoplasmic permease, and one designated fbpC, which encodes a protein proposed to function as a nucleotide-binding protein. The fpbABC operon composes an iron transport system that is homologous to the sfu and hit operons previously reported for Serratia marcescens and Haemophilus influenzae, respectively, and displays elements characteristic of ATP binding cassette transporters. The fpbABC operon differs from these loci in that it is lethal when overexpressed in Escherichia coli.     

Accumulation of U14 small nuclear RNA in Saccharomyces cerevisiae requires box C, box D, and a 5'', 3'' terminal stem.

U14 is one of several nucleolar small nuclear RNAs required for normal processing of rRNA. Functional mapping of U14 from Saccharomyces cerevisiae has yielded a number of mutants defective in U14 accumulation or function. In this study, we have further defined three structural elements required for U14 accumulation. The essential elements include the U14-conserved box C and box D sequences and a 5', 3' terminal stem. The box elements are coconserved among several nucleolar small nuclear RNAs and have been implicated in binding of the protein fibrillarin. New mutational results show that the first GA bases of the box C sequence UGAUGA are essential, and two vital bases in box D have also been identified. An intragenic suppressor of a lethal box C mutant has been isolated and shown to contain a new box C-like PyGAUG sequence two bases upstream of normal box C. The importance of the terminal stem was confirmed from new compensatory base changes and the finding that accumulation defects in the box elements can be complemented by extending the terminal stem. The results suggest that the observed defects in accumulation reflect U14 instability and that protein binding to one or more of these elements is required for metabolic stability.     

Genetics of 51d-52a, a Region Containing Several Maternal-Effect Genes and Two Maternal-Specific Transcripts in Drosophila

Two genomic clones exhibiting a maternal-specific pattern of expression map to cytological region 52A. To elucidate the function of these clones we have undertaken a mutagenesis of the cytological region 51D-52A. This paper presents the results of this screen and the preliminary analysis of female-sterile and lethal mutations isolated. A total of twelve complementation groups have been identified, four of which are defined exclusively by female-sterile alleles. Only one visible mutation was isolated, a recessive temperature-sensitive allele of Thickened-arista (Tarts). Several of the seven lethal loci display an embryonic lethal phase. Three of the four female-sterile loci affect chorion structure with one resulting in underamplification of the chorion genes, and two (possibly three) of the four female-steriles affect nuclear division/DNA replication. Thus it appears that this is a "developmentally important" region, possibly representing a clustering of genes involved in either DNA replication or nuclear division.     

Computational identification of RNA functional determinants by three-dimensional quantitative structure–activity relationships

Anti-infection drugs target vital functions of infectious agents, including their ribosome and other essential non-coding RNAs. One of the reasons infectious agents become resistant to drugs is due to mutations that eliminate drug-binding affinity while maintaining vital elements. Identifying these elements is based on the determination of viable and lethal mutants and associated structures. However, determining the structure of enough mutants at high resolution is not always possible. Here, we introduce a new computational method, MC-3DQSAR, to determine the vital elements of target RNA structure from mutagenesis and available high-resolution data. We applied the method to further characterize the structural determinants of the bacterial 23S ribosomal RNA sarcin–ricin loop (SRL), as well as those of the lead-activated and hammerhead ribozymes. The method was accurate in confirming experimentally determined essential structural elements and predicting the viability of new SRL variants, which were either observed in bacteria or validated in bacterial growth assays. Our results indicate that MC-3DQSAR could be used systematically to evaluate the drug-target potentials of any RNA sites using current high-resolution structural data.     

Development of nine new microsatellite loci for the American beaver, Castor canadensis (Rodentia: Castoridae), and cross-species amplification in the European beaver, Castor fiber

We developed nine new nuclear dinucleotide microsatellite loci for Castor canadensis. All loci were polymorphic, except for one. The number of alleles ranged from two to four and from five to 12 in populations from Arizona and Wisconsin, respectively. Average heterozygosity ranged from 0.13 to 0.86 per locus. Since cross-species amplification in Castor fiber was successful only in four loci, we tested also nine recently published C. canadensis loci in the Eurasian species. Eight of the published loci amplified; however, three were monomorphic. The number of alleles was lower in C. fiber than in C. canadensis at all loci tested.     

Genetic and Molecular Analysis of the Proximal Region of the Mouse T-Complex Using New Molecular Probes and Partial T-Haplotypes

The t-complex is located on the proximal third of chromosome 17 in the house mouse. Naturally occurring variant forms of the t-complex, known as complete t-haplotypes, are found in wild mouse populations. The t-haplotypes contain at least four nonoverlapping inversions that suppress recombination with the wild-type chromosome, and lock into strong linkage disequilibrium loci affecting normal transmission of the chromosome, male gametogenesis and embryonic development. Partial t-haplotypes derived through rare recombination between t-haplotypes and wild-type homologs have been critical in the analysis of these properties. Utilizing two new DNA probes. Au3 and Au9, and several previously described probes, we have analyzed the genetic structure of several partial t-haplotypes that have arisen in our laboratory, as well as several wild-type chromosomes deleted for loci in this region. With this approach we have been able to further our understanding of the structural and dynamic characteristics of the proximal region of the t-complex. Specifically, we have localized the D17Tul locus as most proximal known in t-haplotypes, achieved a better structural analysis of the partial t-haplotype t6, and defined the structure and lethal gene content of partial t-haplotypes derived from the lethal tw73 haplotype.     

Gene Flow in Natural Populations of DROSOPHILA MELANOGASTER with Special Reference to Lethal Allelism Rates and Protein Variation

A simultaneous survey of 14 protein loci, together with frequencies and within- and between-population allelism rates of lethal chromosomes, was carried out in five (four Japanese and one Korean) natural populations and one cage population of Drosophila melanogaster. It was found that lethal allelism rates decrease rapidly as geographic distance between two populations increases, while variation at protein loci shows a remarkable similarity over all populations examined. These findings suggest that there are very high levels of gene flow in these natural populations and that selection at protein loci which can maintain substantial geographic variation, if present, is overshadowed by gene flow. There is no indication that invasion of D. melanogaster to the Far East occurred so recently that the frequencies of lethal chromosomes are still in nonequilibrium.     

Genetic Analysis of the Heterochromatin of Chromosome 3 in Drosophila Melanogaster. II. Vital Loci Identified through Ems Mutagenesis

Chromosome 3 of Drosophila melanogaster contains the last major blocks of heterochromatin in this species to be genetically analyzed. Deficiencies of heterochromatin generated through the detachment of compound-3 chromosomes revealed the presence of vital loci in the heterochromatin of chromosome 3, but an extensive complementation analysis with various combinations of lethal and nonlethal detachment products gave no evidence of tandemly repeated vital genes in this region. These findings indicate that the heterochromatin of chromosome 3 is genetically similar to that of chromosome 2. A more thorough genetic analysis of the heterochromatic regions has been carried out using the chemical mutagen ethyl methanesulfonate (EMS). Seventy-five EMS-induced lethals allelic to loci uncovered by detachment-product deficiencies were recovered and tested for complementation. In total, 12 complementation groups were identified, ten in the heterochromatin to the left of the centromere and two to the right. All but two complementation groups in the left heterochromatic block could be identified as separate loci through deficiency mapping. The interallelic complementation observed between some EMS-induced lethals, as well as the recovery of a temperature-sensitive allele for each of the two loci, provided further evidence that single-copy, transcribed vital genes reside in the heterochromatin of chromosome 3. Cytological analysis of three detachment-product deficiencies provided evidence that at least some of the genes uncovered in this study are located in the most distal segments of the heterochromatin in both arms. This study provides a detailed genetic analysis of chromosome 3 heterochromatin and offers further information on the genetic nature and heterogeneity of Drosophila heterochromatin.     

Delivery of Cas9 Protein into Mouse Zygotes through a Series of Electroporation Dramatically Increases the Efficiency of Model Creation

Previously we established Zygote Electroporation of Nucleases(ZEN) technology as an efficient and high-throughput way to generate genetically modified mouse models.However,there were significant variations of the targeting efficiency among different genomic loci using our previously published protocol.In this study,we improved the ZEN technology by delivering Cas9 protein into mouse zygotes through a series of electroporation.Using this approach,we were able to introduce precise nucleotide substitutions,large segment deletion and short segment insertion into targeted loci with high efficiency.