The Role of DNA Repair Genes in Recombination between Repeated Sequences in Yeast

The presence of repeated sequences in the genome represents a potential source of karyotypic instability. Genetic control of recombination is thus important to preserve the integrity of the genome. To investigate the genetic control of recombination between repeated sequences, we have created a series of isogenic strains in which we could assess the role of genes involved in DNA repair in two types of recombination: direct repeat recombination and ectopic gene conversion. Naturally occurring (Ty elements) and artificially constructed repeats could be compared in the same cell population. We have found that direct repeat recombination and gene conversion have different genetic requirements. The role of the RAD51, RAD52, RAD54, RAD55, and RAD57 genes, which are involved in recombinational repair, was investigated. Based on the phenotypes of single and double mutants, these genes can be divided into three functional subgroups: one composed of RAD52, a second one composed of RAD51 and RAD54, and a third one that includes the RAD55 and RAD57 genes. Among seven genes involved in excision repair tested, only RAD1 and RAD10 played a role in the types of recombination studied. We did not detect a differential effect of any rad mutation on Ty elements as compared to artificially constructed repeats.     

染色体上聚集的microRNAs具有更多共同的靶基因

miRNAs是一类-22nt、在转录后调节mRNAs表达的内源性非编码RNA。人类miRNA具有成簇聚集于染色体上的特点。文章系统分析了人基因组簇内miRNA各成员的预测靶基因之间的关系,发现簇内miRNA各成员具有更多共同的靶基因,表明簇内miRNA各成员功能相似。仔细分析簇内miRNA和mRNA的结合位点,发现有两种类型：一种是簇内miRNAs可能竞争性结合同一个靶基因的同一个结合位点：另一种是簇内miRNAs可能协同结合于同一个靶基因的不同结合位点。     

Extragenic Suppressors of Saccharomyces Cerevisiae Prp4 Mutations Identify a Negative Regulator of Prp Genes

The PRP4 gene encodes a protein that is a component of the U4/U6 small nuclear ribonucleoprotein particle and is necessary for both spliceosome assembly and pre-mRNA splicing. To identify genes whose products interact with the PRP4 gene or gene product, we isolated second-site suppressors of temperature-sensitive prp4 mutations. We limited ourselves to suppressors with a distinct phenotype, cold sensitivity, to facilitate analysis of mutants. Ten independent recessive suppressors were obtained that identified four complementation groups, spp41, spp42, spp43 and spp44 (suppressor of prp4, numbers 1-4). spp41-spp44 suppress the pre-mRNA splicing defect as well as the temperature-sensitive phenotype of prp4 strains. Each of these spp mutations also suppresses prp3; spp41 and spp42 suppress prp11 as well. Neither spp41 nor spp42 suppresses null alleles of prp3 or prp4, indicating that the suppression does not occur via a bypass mechanism. The spp41 and spp42 mutations are neither allele- nor gene-specific in their pattern of suppression and do not result in a defect in pre-mRNA splicing. Thus the SPP41 and SPP42 gene products are unlikely to participate directly in mRNA splicing or interact directly with Prp3p or Prp4p. Expression of PRP3-lacZ and PRP4-lacZ gene fusions is increased in spp41 strains, suggesting that wild-type Spp41p represses expression of PRP3 and PRP4. SPP41 was cloned and sequenced and found to be essential. spp43 is allelic to the previously identified suppressor srn1, which encodes a negative regulator of gene expression.     

MouseSprr2Genes: A Clustered Family of Genes Showing Differential Expression in Epithelial Tissues

Small proline-rich (SPR) proteins are structural components of the cornified cell envelope of stratified squamous epithelia. They are subdivided into three families, i.e., SPR1, SPR2, and SPR3, of which the SPR2 family is the most complex. To understand the significance of this complexity, we have isolated 11 mouseSprr2genes, constructed a provisional physical map of theSprr2locus on mouse Chromosome 3, and examined the expression patterns of theSprr2genes in mouse epithelial tissues. The 11Sprr2sequences are highly conserved with a central domain containing a variable number of repeats.In situhybridization showed theSprr2expression to be confined to epithelia. RT-PCR using primers specific for each of the 11Sprr2members demonstrated varying degrees of expression among the individualSprr2members in different tissues. The correlation between the physical location of the genes in theSprr2locus and their expression patterns suggests multiple levels of controlled expression.     

沙眼衣原体质粒调控基因的上游序列有广泛的ChxR结合位点

目的:利用凝胶阻滞实验分析沙眼衣原体质粒调控基因的上游DNA序列与ChxR 的相互作用;方法:利用表达载体p ET-21b在大肠杆菌BL21(DE3)中重组表达ChxR ,重组蛋白的N端有T7标签、C端有6×His标签,用TALON金属亲和介质纯化重组蛋白;PCR扩增沙眼衣原体质粒调控基因的上游区约150 bp,连接p CR2.1载体,重组载体经单酶切,制备3'端生物素标记探针;用引物延伸实验确定glgA的转录起始位点,并合成5条glgA上游区的30 bp重叠探针;用凝胶阻滞实验分析ChxR 与以上探针的相互作用。结果:4个质粒调控基因的上游均有多个ChxR 结合位点,特别是glgA上游区至少有6个结合位点;glgA上游的ChxR 结合位点多位于启动子和启动子下游区。结论:ChxR 可能参与质粒调控基因的转录;在glgA转录中,ChxR 可能起转录抑制子的作用。     

An Ergot Alkaloid Biosynthesis Gene and Clustered Hypothetical Genes from Aspergillus fumigatus

The ergot alkaloids are a family of indole-derived mycotoxins with a variety of significant biological activities. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, and several fungi in the relatively distant taxon Clavicipitaceae (clavicipitaceous fungi) produce different sets of ergot alkaloids. The ergot alkaloids of these divergent fungi share a four-member ergoline ring but differ in the number, type, and position of the side chains. Several genes required for ergot alkaloid production are known in the clavicipitaceous fungi, and these genes are clustered in the genome of the ergot fungus Claviceps purpurea. We investigated whether the ergot alkaloids of A. fumigatus have a common biosynthetic and genetic origin with those of the clavicipitaceous fungi. A homolog of dmaW, the gene controlling the determinant step in the ergot alkaloid pathway of clavicipitaceous fungi, was identified in the A. fumigatus genome. Knockout of dmaW eliminated all known ergot alkaloids from A. fumigatus, and complementation of the mutation restored ergot alkaloid production. Clustered with dmaW in the A. fumigatus genome are sequences corresponding to five genes previously proposed to encode steps in the ergot alkaloid pathway of C. purpurea, as well as additional sequences whose deduced protein products are consistent with their involvement in the ergot alkaloid pathway. The corresponding genes have similarities in their nucleotide sequences, but the orientations and positions within the cluster of several of these genes differ. The data indicate that the ergot alkaloid biosynthetic capabilities in A. fumigatus and the clavicipitaceous fungi had a common origin.     

Clustered Genes for Galactose Metabolism from Streptococcus mutans Cloned in Escherichia coli

DNA cloned into Escherichia coli from a serotype c strain of Streptococcus mutans allowed a galKTE mutant to utilize galactose for growth. However, the DNA does not appear to encode enzymes of the Leloir pathway used by E. coli, but rather appears to encode enzymes of the tagatose phosphate pathway.     

DNA Sequences at a Glance

Data summarization and triage is one of the current top challenges in visual analytics. The goal is to let users visually inspect large data sets and examine or request data with particular characteristics. The need for summarization and visual analytics is also felt when dealing with digital representations of DNA sequences. Genomic data sets are growing rapidly, making their analysis increasingly more difficult, and raising the need for new, scalable tools. For example, being able to look at very large DNA sequences while immediately identifying potentially interesting regions would provide the biologist with a flexible exploratory and analytical tool. In this paper we present a new concept, the “information profile”, which provides a quantitative measure of the local complexity of a DNA sequence, independently of the direction of processing. The computation of the information profiles is computationally tractable: we show that it can be done in time proportional to the length of the sequence. We also describe a tool to compute the information profiles of a given DNA sequence, and use the genome of the fission yeast Schizosaccharomyces pombe strain 972 h⁻ and five human chromosomes 22 for illustration. We show that information profiles are useful for detecting large-scale genomic regularities by visual inspection. Several discovery strategies are possible, including the standalone analysis of single sequences, the comparative analysis of sequences from individuals from the same species, and the comparative analysis of sequences from different organisms. The comparison scale can be varied, allowing the users to zoom-in on specific details, or obtain a broad overview of a long segment. Software applications have been made available for non-commercial use at http://bioinformatics.ua.pt/software/dna-at-glance.     

Extragenic Suppressors of Mutations in the Cytoplasmic C Terminus of Sec63 Define Five Genes in Saccharomyces Cerevisae

Mutations in the SEC63 gene of Saccharomyces cerevisiae affect both nuclear protein localization and translocation of proteins into the endoplasmic reticulum. We now report the isolation of suppressors of sec63-101 (formerly npl1-1), a temperature-sensitive allele of SEC63. Five complementation groups of extragenic mutations, son1-son5 (suppressor of npl1-1), were identified among the recessive suppressors. The son mutations are specific to SEC63, are not bypass suppressors, and are not new alleles of previously identified secretory (SEC61, SEC62, KAR2) or nuclear protein localization genes (NPL3, NPL4, NPL6). son1 mutations show regional specificity of suppression of sec63 alleles. At low temperatures, son1 mutants grow slowly and show partial mislocalization of nuclear antigens. The SON1 gene maps to chromosome IV and encodes a nuclear protein of 531 amino acids that contains two acidic stretches and a putative nuclear localization sequence. We show that son1 mutations suppress sec63-101 by elimination of Son1p function.     

Clustered DNA damage induced by heavy ion particles.

Clustered DNA damage (locally multiply damaged site) is thought to be a critical lesion caused by ionizing radiation, and high LET radiation such as heavy ion particles is believed to produce high yields of such damage. Since heavy ion particles are major components of ionizing radiation in a space environment, it is important to clarify the chemical nature and biological consequences of clustered DNA damage and its relationship to the health effects of exposure to high LET particles in humans. The concept of clustered DNA damage emerged around 1980, but only recently has become the subject of experimental studies. In this article, we review methods used to detect clustered DNA damage, and the current status of our understanding of the chemical nature and repair of clustered DNA damage.     

Mapping of a Bradyrhizobium japonicum DNA Region Carrying Genes for Symbiosis and an Asymmetric Accumulation of Reiterated Sequences

Spontaneous kanamycin-sensitive derivatives were obtained from Bradyrhizobium japonicum (strain 110) carrying Tn5 insertions in symbiotic gene cluster I; the derivatives were shown to have deletions of cluster I plus flanking DNA which was indicated by the absence of different copies of the repeated sequences RSα and RSβ. The deletion endpoints were mapped using cloned wild-type DNA fragments containing RSα copies which also served as origins for overlapped cosmid cloning. The majority of the deletions resulted from recombinational fusion of two remote RSα copies. Novel types of repeated sequences (RSγ, RSδ, and RSε) occurring in 12, 10, and 4 copies per genome were detected. Seven, nine, and three copies of RSγ, RSδ, and RSε, respectively, were located near cluster I. It is concluded that the B. japonicum genome has an unusual DNA segment of >230 kilobase pairs characterized by the presence of repeated sequences and genes for symbiotic N₂ fixation.