A mitochondrial nuclease is modified in Drosophila mutants (mus308) that are hypersensitive to DNA crosslinking agents

Summary The mus308 mutants of Drosophila have previously been demonstrated to be defective in an enzyme that is designated Nuclease 3 (Boyd et al. 1990b). In this study that enzyme is shown to be present in mitochondria of both wild-type flies and embryos. Since the mus308 mutants are hypersensitive to DNA crosslinking agents, Nuclease 3 is potentially required for resistance of the mitochondrial genome to such agents. In support of this hypothesis, electron microscopic studies of mus308 mutant flies that had been exposed to nitrogen mustard revealed an increased frequency of mitochondrial abnormalities. Further investigation of the defect at the enzymological level revealed that the mutants possess a new nuclease activity that is apparently a modified form of the wild-type protein. In the earlier study, enzyme extracts from mus308 mutants were found to lack an enzyme with a pl of approximately 6.2. More precisely defined assay conditions in this study revealed the appearance of a new nuclease activity with a higher pI in extracts from mutants. This observation, together with the finding that only the normal enzyme form is present in heterozygous individuals, supports the hypothesis that the mus308 locus is not the structural gene for the enzyme. Rather, the mus308 gene product is necessary for Nuclease 3 to assume the lower pI. Nuclease 3 has been partially purified and characterized from wild-type embryos. Its activity is stimulated by Mg⁺⁺ and ATP. Optimum activity is found at a pH of 5.5 and a NaCl concentration of 50–100 mM. Nuclease 3 exhibits a temperature optimum of 42°C and is insensitive to N-ethylmaleimide. The enzyme is probably membrane-associated because it exhibits a strong tendency to aggregate and detergent is required for full solubilization.     

Genetic characterization of the mei-41 locus in Drosophila melanogaster

Summary The mutagen-sensitive mutant mus(1)104 ^D1 of Drosophila melanogaster maps to a position on the X chromosome very close to the meiotic mutant mei-41 ^D5 . Both mutants have been characterized as mutagen-sensitive and defective in post-replication repair. In the present report we show by complementation studies that mus(1)104 and mus(1)103 are allelic with mei-41. In addition, two reported alleles of mus(1)104 lie between the mei-41 alleles A10 and D5. The size of the mei-41 locus is estimated to be about 0.1 centimorgans (cM). Because several alleles of mei-41 have been shown to reduce recombination and increase meiotic chromosome loss and nondisjunction, mus(1)104 ^D1 females were examined for defects in meiosis. Although there was no evidence for reduced recombination on the second chromosome in homozygous mus(1)104 ^D1 females, heterozygous mus(1)104 ^D1 /mei-41 ^>D5 and mus(1)104 ^D1 /deficiency females showed reduced levels of recombination. However, there was no evidence of an increase in nondijunction in these females.We dedicate this article to the memory of Larry Sandler, who passed away suddenly on February 7, 1987     

Cytogenetical analysis of the 2B3-4-2B11 region of the X chromosome of Drosophila melanogaster

Summary Of 13 ecs mutations, which affect female fertility, as revealed by complementation analysis, 7 are chromosome rearrangements involving the br complementation group. The other six show no cytologically detectable rearrangements and behave as completely or partially noncomplementing ecs alleles. All viable combinations of these 13 mutations were characterized by partial or complete female sterility. Viable heterozygotes carrying any of these mutations and the rearrangements Df(1)sta, T(1,3)sta, Df(1)St490, previously localized distal to the ecs locus, were also sterile. Using deletions and an electrophoretic mobility variant from the Staket strain, a minor chorion gene S70 has been mapped. It had been thought this gene was located in the 2B3-5 region, and corresponded to the ecs locus. However, in the present study, this gene was shown to map in the region removed by Df(1)sta (1E1-2-2B3-4) but outside that removed by Df(1)At127 (1E1-2-2A1-2), i.e. within the 2A1-2-2B3-4 region which is distal to the ecs locus. Rearrangements and point mutations at the ecs locus that result in female sterility had no effect on synthesis of the chorion protein s70. It may therefore be suggested that the chorion protein gene is not functionally associated with the ecs locus and that sterility is caused not by disruptions of the chorion protein gene but by lesions in the ecs gene itself. Thus, an ecs product, which controlls cell sensitivity to ecdysterone is also necessary for female fertility. Data on the locations of lesions affecting female fertility indicate that at least two elements at the ecs locus are essential for this function: a cis-acting distal zone with no effect on viability and a sequence within the essential part of the ecs locus. A defect in either of these zones or their separation by chromosomal rearrangement leads to female sterility.     

RETRACTED ARTICLE: The effect of the <Emphasis Type="Italic">mus309</Emphasis> mutation,defective in DNA double-strand break repair,on crossing over in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> suggests a mechanism for the centromere effect of crossing over

The mus309 gene in Drosophila melanogaster encodes a RecQ helicase which is involved in DNA double-strand break (DSB) repair. In a brood pattern analysis, it was observed that in mus309 mutant females, the frequency of single crossovers in the central cv–v interval of the X chromosome was reduced in young females but returned to the level of the wild type control as the females aged. In the proximal v–f interval, the frequency of single crossovers was increased during the entire experimental period. In particular, it was observed that the frequency of double crossovers, as well as the coefficient of coincidence first increased but then gradually decreased, finally reaching the level of the control flies, as the females aged. Map distances increased due to the mus309 mutation in both gene interval studies, but they did not change as the females aged, a result suggesting that the mus309 gene controls the distribution of DSBs to be repaired as crossovers instead of non-crossovers. The results suggest a mechanism for the centromere effect of crossing over in Drosophila, viz the fact the frequency of meiotic crossing over reduces with the age of the female, and that the reduction is more pronounced the closer the interval is to the proximal heterochromatin of the chromosome arm. According to the model suggested, the centromere effect is simply a matter of the balance between different pathways of the repair of the DSBs of DNA.     

hsc70-4基因促进家蚕核型多角体病毒复制增殖

【目的】热休克应答(heatshockresponse,HSR)是机体细胞应对环境压力的一种重要防御策略,鉴定热休克蛋白在杆状病毒侵染宿主过程中的功能,并揭示其作用的分子机制,为探明宿主与病毒相互作用的分子基础提供理论依据。【方法】通过分子克隆技术对Bmhsc70-4基因进行克隆,并利用BioEdit及GeneDoc对其进行多序列比对分析;分别通过真核表达和基于CRISPR/Cas9的基因编辑系统对Bmhsc70-4基因进行过表达和敲除;利用荧光定量PCR技术检测相应基因的表达量;通过对Caspase-9和Caspase-3/7活性的检测确定Bmhsc70-4基因对细胞凋亡的影响;通过免疫荧光验证BmHSC70-4和BmIAP的共定位情况,并进一步通过免疫共沉淀验证它们的相互作用。【结果】Bmhsc70-4基因开放阅读框为1950bp,编码649个氨基酸,在昆虫间具有较高的保守性;BmNPV能够诱导Bmhsc70-4基因上调表达,过表达Bmhsc70-4基因能够促进BmNPV的增殖,敲除Bmhsc70-4基因能够抑制BmNPV的增殖,表明Bmhsc70-4基因的表达利于BmNPV的增殖;Bmhsc70-4基因具有抑制家蚕细胞凋亡的功能;荧光共定位显示BmHSC70-4和BmIAP共定位于细胞质中,免疫共沉淀结果表明两者可以相互作用;BmNPV侵染过程中Bmhsc70-4基因能够促进Bmiap基因的表达。【结论】Bmhsc70-4基因具有抑制家蚕细胞凋亡的功能,在BmNPV侵染家蚕细胞过程中,能够与BmIAP相互作用,并促进BmNPV复制增殖。     

The glucose repression and RAS-cAMP signal transduction pathways of Saccharomyces cerevisiae each affect RNA processing and the synthesis of a reporter protein

Previously we reported that mutations in the Saccharomyces cerevisiae REG1 gene encoding a negative regulator of glucose-repressible genes, suppress the RNA processing defects and temperature-sensitive growth of rna1-1 and prp cells. This result and the fact that growth on non-glucose carbon sources also suppresses rna1-1 led us to propose that RNA processing and export of RNA from the nucleus are responsive to carbon source regulation. To understand how carbon source affects these processes, we used p70, an antigen regulated by REGI and by glucose availability, as a reporter. We found that the response of p70 to glucose availability is mediated by both the SNFI-SSN6-dependent glucose repression and the RAS-cAMP pathways. These results led us to test whether the RAS-cAMP pathway interacts with RNA1. We found that suppression of rnal-1 appears to be mediated, at least in part, by the RAS-cAMP pathway.     

Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae

In the koji molds Aspergillus sojae and Aspergillus oryzae, exogenous DNA is integrated in the genome, in most cases irrespective of the sequence homology, suggesting that DNA integration occurs predominantly through a nonhomologous end joining pathway where two ku genes, namely, ku70 and ku80, play a key role. To determine the effect of ku gene disruption on the gene targeting frequency, we constructed ku70-, ku80-, and ku70–ku80-disrupted strains of A. sojae and A. oryzae. The gene targeting frequency of the tannase gene in ku70 and ku80 strains of both Aspergillus species was markedly enhanced as compared with that of the parental strains. The gene targeting frequency of the aflR and ku80 genes was also enhanced in an A. sojae ku70 background. Therefore, the koji mold strains with ku-disrupted genes will be excellent tools as hosts for efficient gene targeting.     

Ectopic expression of the<Emphasis Type="Italic"> Suppressor of Underreplication</Emphasis> gene inhibits endocycles but not the mitotic cell cycle in<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis>

The Suppressor of Underreplication ( SuUR) gene contributes to the regulation of DNA replication in regions of intercalary heterochromatin in salivary gland polytene chromosomes. In the SuUR mutant these regions complete replication earlier than in wild type and, as a consequence, undergo full polytenization. Here we describe the effects of ectopic expression of SuUR using the GAL4-UAS system. We demonstrate that ectopically expressed SuUR exerts qualitatively distinct influences on polyploid and diploid tissues. Ectopic expression of SuUR inhibits DNA replication in polytene salivary gland nuclei, and reduces the degree of amplification of chorion protein genes that occurs in the follicle cell lineage. Effects caused by ectopic SuUR in diploid tissues vary considerably; there is no obvious effect on eye formation, but apoptosis is observed in the wing disc, and wing shape is distorted. The effect of ectopic SuUR expression is enhanced by mutations in the genes E2F and mus209 ( PCNA). Differential responses of polyploid and diploid cells to ectopic SuUR may reflect differences in the mechanisms underlying mitotic cell cycles and endocycles.Communicated by G. P. Georgiev     

Complete sequence of p53 gene in 20 patients with lung cancer: comparison with chemosensitivity and immunohistochemistry

In this study the entirep53 complementary DNA has been sequenced in 20 non-small cell lung carcinomas (NSCLC) and the results correlated with chemosensitivity, immunohistochemistry and clinical data. Ten patients had mutations inp53, 8 missense mutations and 2 nonsense mutations. The method discovered two mutations never described previously and two other mutations that have never been described before in connection with NSCLC tumours. Chemosensitivity data, according to a short-term assay (FMCA), indicated that tumours with p53 mutation were more resistant to cisplatin and cyclophosphamide. Immunohistochemical studied demonstrated a 70% concordance between over-expression of p53 protein and mutation inp53. No conclusions or trends could be drawn from the immunohistochemical studies ofBcl-2 andBax.     

Involvement of DnaK protein in mini-F plasmid replication: temperature-sensitive seg mutations are located in the dnaK gene

Summary The seg mutants (seg-1 and seg-2) of Escherichia coli cannot support the replication of the F factor and mini-F plasmids at 42°C. We cloned the wild-type E. coli chromosomal DNA fragment complementing the seg-1 and seg-2 mutations and found that both mutations were complemented by the wild-type dnaK gene coding for a heat shock protein. Transduction with phage P1 indicated that the seg-2 mutation is located at about 0.3 min in the region containing the dnaK gene in the order trpR-thrA-seg-2-leuB, consistent with the locus of the dnaK gene. Cloning and sequencing of the dnaK gene of the seg mutants showed that there was one base substitution within the dnaK gene in each mutant causing an amino acid substitution. These results indicate that the seg gene in which the seg-1 and seg-2 mutations occurred is identical to the dnaK gene. The mini-F plasmid pXX325 did not transform a dnaK null mutant to ampicillin resistance at 30°C in contrast to plasmids pBR322, pACYC184 and pSC101, which did. The active dnaK (seg) gene product is therefore essential for replication of the mini-F plasmid at both 30° and 42°C.     

TFS1: A suppressor of cdc25 mutations in Saccharomyces cerevisiae

Summary The TFS1 gene of Saccharomyces cerevisiae is a dosage-dependent suppressor of cdc25 mutations. Overexpression of TFS1 does not alleviate defects of temperature-sensitive adenylyl cyclase (cdc35) or ras2 disruption mutations. The ability of TFS1 to suppress cdc25 is allele specific: the temperature-sensitive cdc25-1 mutation is suppressed efficiently but the cdc25-5 mutation and two disruption mutations are only partially suppressed. TFS1 maps to a previously undefined locus on chromosome XII between RDN1 and CDC42. The DNA sequence of TFS1 contains a single long open reading frame encoding a 219 amino acid polypeptide that is similar in sequence to two mammalian brain proteins. Insertion and deletion mutations in TFS1 are haploviable, indicating that TFS1 is not essential for growth.     

Genetic and fine structure analysis of unc-26(IV) and adjacent regions in Caenorhabditis elegans

Summary The genetic organization of unc-26(IV) and adjacent regions was studied in Caenorhabditis elegans. We constructed a fine structure genetic map of unc-26(IV), a gene that affects locomotion and pharyngeal muscle movement but not muscle structure. Eleven alleles were positioned relative to each other recombinationally and were classified according to phenotypic severity. The unc-26 gene spans at least 0.026 map units, which is exceptionally large for a C. elegans gene. All but one allele, e205, are amorphic alleles. Interestingly, e205 is hypomorphic but also suppressible by the amber suppressor sup-7. Nineteen lethal mutations in the unc-26 region were isolated and characterized. The unc-26 region is subdivided into four zones by five deficiency breakpoints. These mutations fall into 15 complementation groups. The stages of development affected by these mutations were determined.     

Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii.

Summary We have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes. Wild-type C. reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence. Antibiotic-resistant transformants were selected under conditions permissive for growth of nonphotosynthetic mutants. Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence. A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated. Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene. Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques. We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation. The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.     

Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli

Summary DNA fragments of 3.4 kb containing the gyrB gene were cloned from Escherichia coli KL-16 and from spontaneous nalidixic acid-resistant mutants. The mutations (nal-24 and nal-31) had been determined to be in the gyrB gene by transduction analysis. Nucleotide sequence analysis of the cloned DNA fragments revealed that nal-24 was a G to A transition at the first base of the 426th codon of the gyrB gene, resulting in an amino acid change from aspartic acid to asparagine, and nal-31 was an A to G transition at the first base of the 447th codon, resulting in an amino acid change from lysine to glutamic acid. This indicates that mutations in the gyrB gene are responsible for nalidixic acid resistance.     

Regulation of homologous integration in yeast by the DNA repair proteins Ku70 and RecQ

The product of the BLM gene, which is mutated in Bloom syndrome in humans, and the Saccharomyces cerevisiae protein Sgs1 are both homologous to the Escherichia coli DNA helicase RecQ, and have been shown to be involved in the regulation of homologous recombination. Mutations in these genes result in genome instability because they increase the incidence of deletions and translocations. We present evidence for a genetic interaction between SGS1 and YKU70, which encodes the S. cerevisiae homologue of the human DNA helicase Ku70. In a yku70 mutant background, sgs1 mutations increased sensitivity to DNA breakage induced either by treatment with camptothecin or by the expression of the restriction enzyme EcoRI. The yku70 mutation caused a fourfold increase in the rate of double-strand break (DSB)-induced target integration as that seen in the sgs1 mutant. The combination of yku70 and sgs1 mutations additively increased the rate of the targeted integration, and this effect was completely suppressed by deletion of RAD51. Interestingly, an extra copy of YKU70 partially suppressed the increase in targeted integration seen in the sgs1 single mutant. These results suggest that Yku70 modulates the repair of DSBs associated with homologous recombination in a different way from Sgs1, and that the inactivation of RecQ and Ku70 homologues may enhance the frequency of gene targeting in higher eukaryotes.     

Molecular cloning and sequence analysis of mutant alleles of the fission yeast cdc2 protein kinase gene: Implications for cdc2+ protein structure and function

Summary The cdc2 ⁺ gene function plays a central role in the control of the mitotic cell cycle of the fission yeast Schizosaccharomyces pombe. Recessive temperature-sensitive mutations in the cdc2 gene cause cell cycle arrest when shifted to the restrictive temperature, while a second class of mutations within the cdc2 gene causes a premature advancement into mitosis. Previously the cdc2 ⁺ gene has been cloned and has been shown to encode a 34 kDa phosphoprotein with in vitro protein kinase activity. Here we describe the cloning of 11 mutant alleles of the cdc2 gene using two simple methods, one of which is presented here for the first time. We have sequenced these alleles and find a variety of single amino acid substitutions mapping throughtout the cdc2 protein. Analysis of these mutations has identified a number of regions within the cdc2 protein that are important for cdc2 ⁺ activity and regulation. These include regions which may be involved in the interaction of the cdc2 ⁺ gene product with the proteins encoded by the wee1 ⁺, cdc13 ⁺ and suc1 ⁺ genes.     

Involvement of the PS03 gene of Saccharomyces cerevisiae in intrachromosomal mitotic recombination and gene amplification

Using a genetic system of haploid strains of Saccharomyces cerevisiae carrying a duplication of the his4 region on chromosome III, the pso3-1 mutation was shown to decrease the rate of spontaneous mitotic intrachromosomal recombination 2- to 13-fold. As previously found for the rad52-1 mutant, the pso3-1 mutant is specifically affected in mitotic gene conversion. Moreover, both mutations reduce the frequency of spontaneous recombination. However, the two mutations differ in the extent to which they affect recombination between either proximally or distally located markers on the two his4 heteroalleles. In addition, amplifications of the his4 region were detected in the pso3-1 mutant. We suggest that the appearance of these amplifications is a consequence of the inability of the pso3-1 mutant to perform mitotic gene conversion.     

aPCR-SSCP and DNA sequencing detecting two silent SNPs at KAP8.1 gene in the cashmere goat

Keratin-associated proteins 8.1 gene (KAP8.1) is a structural gene responsible for the cashmere. KAP8.1 protein contains high glycine and tyrosine, which concerns regulation and function of the matrix structure fiber. In this study, the polymorphism of KAP8.1 gene was detected by methods of aPCR-SSCP (asymmetric polymerase chain reaction single-strand conformation polymorphism) and DNA sequencing in 791 individuals from two breeds. The results showed that there were two mutations in this gene. The mutations were described as c.63 T>G and c.66 C>G, which would result in two synonymous mutations in KAP8.1 protein. The findings go against previous research, in which there was not polymorphism at KAP8.1 gene. The reasons might be that different cashmere breeds were detected in two studies. Further analysis of results leads us to believe that the polymorphism of KAP8.1 gene might be relevant to fiber diameter.     

Analysis of the CYP21A1P pseudogene: indication of mutational diversity and CYP21A2-like and duplicated CYP21A2 genes

The CYP21A1P gene downstream of the XA gene, carrying 15 deteriorated mutations, is a nonfunctional pseudogene that shares 98% nucleotide sequence homology with CYP21A2 located on chromosome 6p21.3. However, these mutations in the CYP21A1P gene are not totally involved in each individual. From our analysis of 100 healthy ethnic Chinese (i.e., Taiwanese) (n = 200 chromosomes) using the polymerase chain reaction (PCR) products combined with an amplification-created restriction site (ACRS) method and DNA sequencing, we found that approximately 10% of CYP21A1P alleles (n = 195 chromosomes) presented the CYP21A2 sequence; frequencies of P30, V281, Q318, and R356 in that locus were approximately 24%, 21%, 11%, and 34%, respectively, and approximately 90% of the CYP21A1P alleles had 15 mutated loci. In addition, approximately 2.5% (n = 5 chromosomes) showed four haplotypes of the 3.7-kb TaqI-produced fragment of the CYP21A2-like gene and one duplicated CYP21A2 gene. We conclude that the pseudogene of the CYP21A1P mutation presents diverse variants. Moreover, the existence of the CYP21A2-like gene is more abundant than that of the duplicated CYP21A2 gene downstream of the XA gene and could not be distinguished from the CYP21A2–TNXB gene; thus, it may be misdiagnosed by previously established methods for congenital adrenal hyperplasia caused by a 21-hydroxylase deficiency.     

A new Escherichia coli gene, fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.