Characterization of a novel DNA damage-inducible gene of Saccharomyces cerevisiae , DIN7 , which is a structural homolog of the RAD2 and RAD27 DNA repair genes

A number of DNA damage-inducible genes (DIN) have been identified in Saccharomyces cerevisiae. In the present study we describe isolation of a novel gene, Din7, the expression of which is induced by exposure of cells to UV light, MMS (methyl methanesulfonate) or HU (hydoxyurea). The DNA sequence of DIN7 was determined. By comparison of the predicted Din7 amino acid sequence with those in databases we found that it belongs to a family of proteins which includes S. cerevisiae Rad2 and its Schizosaccharomyces pombe and human homologs Rad13 and XPGC; S. cerevisiae Rad27 and its S. pombe homolog Rad2, and S. pombe Exo I. All these proteins are endowed with DNA nuclease activity and are known to play an important function in DNA repair. The strongest homology to Din7 was found with the Dhs1 protein of S.␣cerevisiae, the function of which is essentially unknown. The expression of the DIN7 gene was studied in detail using a DIN7-lacZ fusion integrated into a chromosome. We show that the expression level of DIN7 rises during meiosis at a time nearly coincident with commitment to recombination. No inducibility of DIN7 was found after treatment with DNA-damaging agents of cells bearing the rad53-21 mutation. Surprisingly, a high basal level of DIN7 expression was found in strains in which the DUN1 gene was inactivated by transposon insertion. We suggest that a form of Dun1 may be a negative regulator of the DIN7 gene expression. Received: 30 May 1996 / Accepted: 26 September 1996     

RAD51 homologues in Xenopus laevis: two distinct genes are highly expressed in ovary and testis

The RAD51 gene is a eukaryotic counterpart of the Escherichia coli recA gene which is involved in genetic recombination. Two distinct Xenopus laevis RAD51 cDNA clones (XRAD51.1 and XRAD51.2) were isolated from an oocyte cDNA library using the human RAD51 cDNA (HsRAD51) as a probe. Sequence analysis revealed that 98.2% of the amino-acid residues were identical between XRAD51.1 and XRAD51.2, and that both were 95% identical to HsRAD51. Both of the XRAD51 genes were expressed at a higher level in ovary and testis than in other somatic tissues, suggesting their involvement in meiotic recombination. The expression of XRAD51.1 was about eightfold in excess of that of XRAD51.2 in all of the tissues examined. Analysis of the rates of synonymous substitution in the coding sequences of the two XRAD51 suggests that these two genes diverged about 50 million years ago. The structural similarities of the XRAD51 proteins to RecA in E. coli and Rad51 in yeasts or vertebrates are discussed.     

Identification of the DNA damage-responsive elements of therhp51+ gene, arecA andRAD51 homolog from the fission yeastSchizosaccharomyces pombe

TheSchizosaccharomyces pombe rhp51 ⁺ gene encodes a recombinational repair protein that shares significant sequence identities with the bacterial RecA and theSaccharomyces cerevisiae RAD51 protein. Levels ofrhp51 ⁺ mRNA increase following several types of DNA damage or inhibition of DNA synthesis. Anrhp51::ura4 fusion gene was used to identify the cis-acting promoter elements involved in regulatingrhp51 ⁺ expression in response to DNA damage. Two elements, designated DRE1 and DRE2 (fordamage-responsiveelement), match a decamer consensus URS (upstream repressing sequence) found in the promoters of many other DNA repair and metabolism genes fromS. cerevisiae. However, our results show that DRE1 and DRE2 each function as a UAS (upstream activating sequence) rather than a URS and are also required for DNA-damage inducibility of the gene. A 20-bp fragment located downstream of both DRE1 and DRE2 is responsible for URS function. The DRE1 and DRE2 elements cross-competed for binding to two proteins of 45 and 59 kDa. DNase I footprint analysis suggests that DRE1 and DRE2 bind to the same DNA-binding proteins. These results suggest that the DRE-binding proteins may play an important role in the DNA-damage inducibility ofrhp51 ⁺ expression.     

Plasmid multimerization is dependent on RAD52 activity in Saccharomyces cerevisiae

Summary A mutant plasmid, pX, derived from the 1453 base pair small plasmid, YARp1 (or TRP1 RI circle), consists of 849 base pairs of DNA bearing the TRP1 gene and the ARS1 sequence of Saccharomyces cerevisiae and, unlike YARp1 and other commonly used yeast plasmids, highly multimerizes in a S. cerevisiae host. The multimerization of pX was dependent on RAD52, which is known to be necessary for homologous recombination in S. cerevisiae. Based upon this observation, a regulated system of multimerization of pX with GAL1 promoter-driven RAD52 has been developed. We conclude that the regulated multimerization of pX could provide a useful model system to study genetic recombination in the eukaryotic cell, in particular to investigate recombination intermediates and the effects of various trans-acting mutations on the multimerization and recombination of plasmids.     

An endo-exonuclease activity of yeast that requires a functional RAD52 gene

Summary Extracts of Rad⁺ and radiation-sensitive (rad) mutants of the yeast Saccharomyces cerevisiae were examined for total Mg²⁺-dependent alkaline deoxyribonuclease activity and the presence of a nuclease that crossreacts immunologically with an antiserum raised against an endoexonuclease from Neurospora crassa, an enzyme exhibiting both deoxyribo- and ribonuclease activities. No significant differences were observed in total deoxyribonuclease activity between Rad⁺ and rad mutants. The antibody precipitable activity, however, was found to be 30%–40% of the total alkaline deoxyribonuclease activity in logarithmically growing Rad⁺ cells. Extracts of stationary phase cells were lacking in antibody precipitable activity. Using immunoblot methods, a 72 kDa crossreacting protein was identified from logarithmically growing cells that was absent from stationary phase cells. In all radiation-sensitive mutants examined, except rad52, at least 20% of total activity was precipitable. Extracts from logarithmically growing rad52 mutants, including a rad52::LEU2 insertion mutant, exhibited less than 10% of the Rad⁺ precipitable activity; however, some crossreacting material was detected. Although, the level of endo-exonuclease activity is influenced by the RAD52 gene, it is not the product of this gene. The total deoxyribonuclease and the antibody precipitable endo-exonuclease activities were also followed during meiosis. Unlike the Rad⁺ strain which had previously been shown to have increased levels of total and immunoprecipitable endo-exonuclease as cells underwent meiosis, the rad52 mutant exhibited no increases in either category of nuclease activity. Given the importance of the RAD52 gene in repair, recombination and mutagenesis, the endo-exonuclease may be a significant component of these processes.     

Recombination and mutagenesis in rad6 mutants of Saccharomyces cerevisiae: Evidence for multiple functions of the RAD6 gene

Summary The rad6-1 and rad6-3 mutants are highly UV sensitive and show an increase in spontaneous and UV induced mitotic heteroallelic recombination in diploids. Both rad6 mutants are proficient in spontaneous and UV induced unequal sister chromatid recombination in the reiterated ribosomal DNA sequence and are deficient in UV induced mutagenesis. In contrast to the above effects where both mutants appear similar, rad6-1 mutants are deficient in sporulation and meiotic recombination whereas rad6-3 mutants are proficient. The differential effects of these mutations indicate that the RAD6 gene is multifunctional. The possible role of the RAD6 gene in error prone excision repair of UV damage during the G1 phase of the cell cycle in addition to its role in postreplication repair is discussed.     

The RAD52 gene is not required for the function of the DEL1 mutator gene in Saccharomyces cerevisiae

Summary The DEL1 mutator in Saccharomyces cerevisiae leads to the formation of deletions adjacent to itself (Liebman et al. 1979). Here we show that the frequency of these DEL1-promoted deletions is not altered by the presence of the recombination-deficient mutation, rad52-1. This indicates that generalized recombination is not required for the formation of deletions in DEL1 yeast strains.     

Polymorphism of fecundity genes (BMPR1B, BMP15 and GDF9) in the Indian prolific Black Bengal goat

The Black Bengal is a prolific goat breed in India. Natural mutations in prolific sheep breeds have shown that the transforming growth factor beta (TGF-β) super family ligands such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and their type I receptor (bone morphogenetic protein receptor, BMPR1B) are crucial for ovulation and as well as for increasing litter size. Mutations in any of these genes increased prolificacy in sheep. Based on the known mutation information in sheep PCR primers were designed to screen known polymorphism in 88 random Black Bengal goats. Only the BMPR1B gene was polymorphic. Three genotypes of animals were detected in tested animals with mutant (FecB^B) and wild type (FecB⁺) alleles were 0.57 and 0.43, respectively. Non-carrier, heterozygous carrier and homozygous carrier Black Bengal does had 2.7, 3.04 and 3.11 kids, respectively. All known point mutations of BMP15 and GDF9 genes were monomorphic in the animals tested. These results preliminarily showed that the BMPR1B gene might be a major gene that influences prolificacy of Black Bengal goats.     

水稻OsWRKY89启动子紫外线-B反应元件区的鉴定

植物需要利用太阳光能进行光合作用,因而不可避免地受到紫外线-B(UV-B) 辐射的影响．为了鉴定水稻WRKY转 录因子OsWRKY89基因启动子中的UV-B反应相关元件,分析了转启动子不同缺失片段与gus融合基因的水稻幼苗,发现在该启动子中存在UV-B反应元件,位于基因翻译起始位点上游-1 213～-1 188之间的25 bp区域,碱基序列为AAGATCTACCATTGCTCTATAGCTT．结合OsWRKY89和UV-B诱导上调表达基因启动子序列分析发现,该元件区在水稻UV-B反应基因启动子上具有高度的保守性,而且与已知保守的光反应元件位置邻近,表明该区域在水稻UV-B反应的转录调控中可能具有重要功能．     

Identification of a Novel Mitogen-Inducible Gene (mig-6): Regulation during G1 Progression and Differentiation

We have identified a novel human gene (mig-6) that is rapidly induced upon mitogenic stimulation of quiescent fibroblasts. Serum induction is partially inhibited by protein synthesis inhibitors, indicating that mig-6 shares characteristics of both primary and secondary response genes. In contrast to most other mitogen-responsive genes, mig-6 mRNA expression is also regulated during normal cell cycle progression, showing a clear peak around mid-G₁. Consistent with the regulation of mig-6 expression during the cell cycle, terminal differentiation of HL-60 cells to either granulocytic or macrophage-like cells also leads to clear changes in the levels of mig-6 mRNA. These observations suggest that the mig-6 gene represents a useful tool for the analysis of cell cycle progression and perhaps terminal differentiation. As a first step toward a functional characterization we show that the Mig-6 polypeptide is located in the cytoplasm.     

人类新细胞因子CKLFSF2羧基端蛋白在大肠杆菌中的表达纯化、抗体制备与鉴定

哺乳动物细胞表达的人类新细胞因子——趋化素样因子超家族成员-2(CKLFSF2)存在分泌形式,位于CKLFSF2分子的羧基端,具有细胞趋化作用.为进一步研究CKLFSF2羧基端蛋白的结构和生物学功能及抗体制备,构建了GST-CKLFSF2C51原核表达质粒,经原核表达、亲和层析、凝胶过滤,获得GST-CKLFSF2C51融合蛋白和CKLFSF2羧基端蛋白(CKLFSF2C51),纯度可达到95%以上.GST-CKLFSF2C51融合蛋白用于制备多克隆抗体,ELISA方法检测抗体效价阳性,蛋白质印迹检测CKLFSF2哺乳动物细胞超表达细胞裂解液,获得特异性条带与预期大小一致.CKLFSF2C51经N端测序,质谱鉴定与预期结果一致,该蛋白质具有对PC-3细胞趋化的活性,并且该活性可被制备的多克隆抗体中和.上述结果表明,原核CKLFSF2羧基端蛋白具有与CKLFSF2真核表达蛋白类似的细胞趋化活性,原核CKLFSF2羧基端蛋白制备的多克隆抗体可用于免疫组织化学、蛋白质印迹检测,并能中和CKLFSF2蛋白的趋化活性作用.     

乳酸乳球菌fabZ1和fabZ2基因功能的鉴定

大肠杆菌(Escherichia coli)是Ⅱ型脂肪酸合成系统的模式生物,3-羟基脂酰ACP脱水异构酶(FabA)是不饱和脂肪酸合成中的关键酶.生物信息学分析表明,乳酸乳球菌(Lactococcus lactis)的基因组中没有标注为3-羟基脂酰ACP脱水异构酶的基因,但有两个标注为3-羟基脂酰ACP脱水酶基因LlfabZ1和LlfabZ2,其编码的蛋白质与EcFabZ的相似性分别为41%和45.1%,且都具有3-羟基脂酰ACP脱水酶两个保守的α螺旋结构.用携带LlfabZ1和LlfabZ2的质粒载体遗传互补大肠杆菌fabA温度敏感突变株CY57,在42℃下不能恢复生长,但无细胞抽提物的结果显示LlFabZ1能够使反-2-癸烯酰ACP异构成顺-3-癸烯酰ACP,而LlFabZ2则不能.互补大肠杆菌fabZ突变株HW7显示,在诱导的条件下,含有LlfabZ2的转化子能够恢复生长,而LlfabZ1则不能.体外重建脂肪酸合成反应及蛋白质活性测定表明,LlFabZ1具有3-羟基脂酰ACP脱水异构酶功能,而LlFabZ2只具有3-羟基脂酰ACP脱水酶功能.另外,未得到LlfabZ1和LlfabZ2的突变株,表明LlFabZ1和LlFabZ2可能是乳酸乳球菌脂肪酸合成酶系中的必不可少的关键蛋白.上述结果证实了乳酸乳球菌fabZ1和fabZ2两个基因在脂肪酸合成中的功能.