人类基因组miRNA转录调控区保守性DNA序列生物信息学分析

MicroRNA(miPNA)的表达调控方式一直是一个有争议的问题,为了研究miRNA潜在的转录调控特点,本文通过Sanger网站miRNA数据库获得人类miRNA的信息,并建立miRNA相关信息数据库,用MEME和Wordspy两个软件对其上游2 000 bp序列进行保守性分析,得到保守性的DNA序YU(motif),用TESS软件分析保守性DNA序列,预测其转录因子结合情况.通过比较位于基因间、反义链和内含子中的三类不同miRNA转录调控区的保守性和自主转录能力的差异,结果发现位于基因间、反义链上的miRNA上游调控区的保守性比位于内含子的miRNA高,在miRNA的转录调控区存在RNA聚合酶Ⅱ类型的转录因子结合位点,miRNA还表现出自身独特的转录调控方式.通过分析,我们还得到了miRNA表达调控中一些重要的转录因子以及独特的调控序列.本研究结果为miRNA转录调控机制的进一步研究提供了理论依据.     

Evaluation of the Extent of Homologous Chloroplast DNA Sequences in the Mitochondrial Genome of Cowpea (Vigna unguiculata L.)

Southern blot hybridization techniques were used to estimate the extent of chloroplast DNA sequences present in the mitochondrial genome of cowpea (Vigna unguiculata L.) The entire mitochondrial chromosome was homogeneously labeled and used to probe blotted DNA fragments obtained by extensive restriction of the tobacco chloroplast genome. The strongest cross-homologies were obtained with fragments derived from the inverted repeat and the atpBE cluster regions, although most of the clones tested (spanning 85% of the tobacco plastid genome) hybridized to mitochondrial DNA. Homologous chloroplast DNA restriction fragments represent a total of 30 to 68 kilobase pairs, depending upon the presence or absence of tRNA-encoding fragments. Plastid genes showing homology with mitochondrial DNA include those encoding ribosomal proteins, RNA polymerase, subunits of photosynthetic complexes, and the two major rRNAs.     

Replication of the Baculovirus Genome

The review describes the current state of studying the baculovirus DNA replication. The structural organization of replication initiation sites and replication intermediates are considered. Attention is focused on virus replication factors, including DNA polymerase, helicase, IE-1, LEF-1, LEF-2, and LEF-3.     

Interspersed Homologous DNA of Autographa californica Nuclear Polyhedrosis Virus Enhances Delayed-Early Gene Expression

The five regions of homologous DNA which are interspersed in the genome of the baculovirus Autographa californica nuclear polyhedrosis virus increased the expression of a delayed-early gene of this virus. Although this activity was first observed as a 10-fold trans effect, the homologous region 5 (hr5) enhanced the expression of linked genes 1,000-fold. The hr5 enhancer also exhibited the other characteristics associated with viral enhancer elements, including orientation independence and the abilities to function at a distance from the linked promoter, to regulate heterologous promoters, and to increase the number of RNA polymerase molecules transcribing the linked genes. The expression of the immediate-early regulatory gene was not enhanced by cis-linked hr5, although the enhancer function may require the immediate-early regulatory gene product. The hr5 enhancer was relatively insensitive to competition by an excess of enhancer molecules. The nucleotide sequence of hr5 revealed two different conserved repeats separated by nonhomologous DNA. Deletion analysis of the hr5 enhancer indicated that a 30-base-pair inverted repeat was essential for enhancer function.     

原癌基因ras在玉米中同源序列的检出及其荧光原位杂交定位

原癌基因ｒａｓ是动物中抑制细胞凋亡的重要基因之一。以人的ｒａｓ基因为探针,通过Ｓｏｕｔｈｅｒｎ杂交技术,确证玉米和水稻中均含有ｒａｓ基因的同源序列;同时,运用荧光原位杂交技术,首次对ｒａｓ基因在玉米中的同源序列进行了定位。结果表明：ｒａｓ探针在第２号和第７号染色体上均检出了杂交信号,信号检出率分别为１０．８５％和１４．１５％,杂交信号与着丝粒的百分距离分别为 ５４．９２± １．９０和 ９４．６２± ２．７７。上述研究结果为植物细胞凋亡的进一步研究提供了线索和帮助。     

Acquisition of Sequences Homologous to Host DNA by Closed Circular Simian Virus 40 DNA III. Host Sequences

A preparation of serially passaged simian virus 40 (SV40) DNA, in which at least 66% of the molecules contain covalently linked cellular DNA sequences, was digested to completion with the Hemophilus influenzae restriction endonuclease. Polyacrylamide gel electrophoresis of the digest showed that the majority of the cleavage products migrated as nine classes of fragments, each class defined by a particular molecular weight. These classes of fragments differ in molecular weight from the fragments produced by the action of the same enzyme on plaque-purified virus DNA. Three classes of fragments were present in less than equimolar amounts relative to the original DNA. The remaining six classes of fragments each contain more than one fragment per original DNA molecule. DNA-DNA hybridization analysis (using the filter method) of the isolated cleavage products demonstrated the presence of highly reiterated cell DNA sequences in two of the nine classes of fragments. A third class of fragments hybridized with high efficiency only to serially passaged SV40 DNA; the level of hybridization to plaque-purified virus DNA was low and there was essentially no hybridization with cell DNA immobilized on filters. It is suggested that this class of fragments contains unique host sequences. It was estimated that at least 27% of the sequences in the substituted SV40 DNA molecules studied are host sequences. The majority of these are probably of the nonreiterated type.     

Centromere-Like Regions in the Budding Yeast Genome

Accurate chromosome segregation requires centromeres (CENs), the DNA sequences where kinetochores form, to attach chromosomes to microtubules. In contrast to most eukaryotes, which have broad centromeres, Saccharomyces cerevisiae possesses sequence-defined point CENs. Chromatin immunoprecipitation followed by sequencing (ChIP–Seq) reveals colocalization of four kinetochore proteins at novel, discrete, non-centromeric regions, especially when levels of the centromeric histone H3 variant, Cse4 (a.k.a. CENP-A or CenH3), are elevated. These regions of overlapping protein binding enhance the segregation of plasmids and chromosomes and have thus been termed Centromere-Like Regions (CLRs). CLRs form in close proximity to S. cerevisiae CENs and share characteristics typical of both point and regional CENs. CLR sequences are conserved among related budding yeasts. Many genomic features characteristic of CLRs are also associated with these conserved homologous sequences from closely related budding yeasts. These studies provide general and important insights into the origin and evolution of centromeres.     

Possible Molecular Detent in the DNA Structure at Regulatory Sequences

Abstract

A common feature that appears in a number of DNA sites where proteins interact is the sequence GTG/CAC. In the lac operator this sequence leads to a region with a higher imino proton exchange rate well below the optical melting temperature. It is suggested that this reflects a structural feature recognized by proteins that bind specific sites on the DNA molecule.     

Tandem and Interspersed Repeats Contribute to the Mosaic Structure of Segmental Duplications in the Human Genome

Intrachromosomal and interchromosomal segmental duplications account for more than 5% of the human genome. To analyze the processes resulting in the complex mosaic structure of duplicons, a draft human genome sequence was searched for duplicated segments of a genomic fragment of the pericentric region of the chromosome 21 short arm. The duplicons found consist of modules having paralogs in various genome regions. Module ends are flanked with various tandem or interspersed repeats, which are more unstable as compared with unique sequences. In most cases, the boundaries of duplicated segments exactly coincide with or are in close proximity to hot spots of various rearrangements within repeats or boundaries between repeats and unique sequences or between two different repeats. Homologous recombination between repetitive elements was assumed to be the major mechanism contributing to the mosaic structure of duplicons.     

Complete Sequence and Enhancer Function of the Homologous DNA Regions of Autographa californica Nuclear Polyhedrosis Virus

The nucleotide sequence of the five regions of homologous DNA in the genome of Autographa californica nuclear polyhedrosis virus DNA was determined. The homology of repeated sequences within a region was 65 to 87%, and the consensus sequences for each region were 88% homologous to each other. Sequences proximal to the EcoRI sites were most conserved, while the distal sequences were least conserved. The EcoRI sites formed the core of a 28-base-pair imperfect inverted repeat. All homologous regions functioned as enhancers in a transient expression assay. A single EcoRI minifragment located between EcoRI-Q and -L enhanced the expression of 39CAT as efficiently as the regions containing numerous EcoRI repeats did.     

Yeast Nucleosome DNA Pattern: Deconvolution from Genome Sequences of S. cerevisiae

Abstract

Positional correlation analysis for the complete genome of Saccharomyces cerevisiae is performed with the aim to reveal possible chromatin-related sequence features. A strong periodicity with the period 10.4 bases is detected in the distance histograms for the dinucleotides AA and TT, with the characteristic decay distance of approximately 50 base pairs. The oscillations are observed as well in the distributions of other dinucleotides. However, the respective amplitudes are small, consistent with secondary effects, due to dominant periodicity of AA and TT. The observations are in accord with earlier data on the chromatin sequence periodicities and nucleosome DNA sequence patterns. The autocorrelations of AA and TT dinucleotides in yeast include also a counter-phase component. A tentative DNA sequence pattern for the yeast nucleosomes is suggested and verified by comparison of its autocorrelation plots with the respective natural autocorrelations. The nucleosome mapping guided by the pattern is in accord with experimental data on the linker length distribution in yeast.     

Homologous Recombinational Repair Factors Are Recruited and Loaded onto the Viral DNA Genome in Epstein-Barr Virus Replication Compartments

Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.Replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA)-binding protein, is a heterotrimeric complex composed of three tightly associated subunits of 70, 32, and 14 kDa (referred as to RPA70, RPA32, and RPA14, respectively) that is essential for DNA replication, recombination, and all major types of DNA repair (4). RPA participates in such diverse pathways through its ability to interact with DNA and numerous proteins involved in its processing. During DNA replication, RPA associates with ssDNA at forks and facilitates nascent-strand DNA synthesis by replicative DNA polymerases localized at replication foci during S phase. Under DNA-damaging conditions, RPA binds to ssDNA at damaged sites and interacts with repair and recombination components to process double-strand DNA breaks (DSBs) and other lesions (6, 14, 21, 32, 38, 41).RPA undergoes both DNA damage-independent and -dependent phosphorylation on the N-terminal 33 residues of RPA32. Unstressed cell cycle-dependent phosphorylation occurs during the G₁/S-phase transition and in M phase, primarily at the conserved cyclin-CDK phosphorylation sites of Ser-23 and Ser-29 in the N terminus of the RPA32 subunit (13, 15). In contrast, stress-induced hyperphosphorylation of RPA is much more extensive. Nine potential phosphorylation sites within the N-terminal domain of RPA32, Ser-4, Ser-8, Ser-11/Ser-12/Ser-13, Thr-21, Ser-23, Ser-29, and Ser-33, in response to DNA-damaging agents, have been suggested (33, 54). Although this region of RPA32 is not required for the ssDNA-binding activity of RPA (5, 22), a phosphorylation-induced subtle conformation change in RPA, resulting from altered intersubunit interactions, regulates the interaction of RPA with both interacting proteins and DNA (30). The hyperphosphorylated form of RPA32 is unable to localize to replication centers in normal cells, while binding to DNA damage foci is unaffected (46). Therefore, RPA phosphorylation following damage is thought to both prevent RPA from catalyzing DNA replication and potentially serve as a marker to recruit repair factors to sites of DNA damage. RPA localizes to nuclear foci where DNA repair is occurring after DNA damage and is essential for multiple DNA repair pathways, participating in damage recognition, excision, and resynthesis reactions (4, 56).Mammalian cells can repair DSBs by homologous recombination (HR) or by nonhomologous end joining. HR is an accurate repair process, the first step of which is the resection of the 5′ ends of the DSB to generate 3′ ssDNA overhangs. This reaction is carried out by the Mre11/Rad50/Nbs1 (MRN) complex, which not only functions as a damage sensor upstream of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) activation but also plays a role in DSB repair (4). RPA and members of the RAD52 epistasis group of gene products, such as Rad51, Rad52, and Rad54, bind to the resulting 3′ ssDNA strands and form a helical, nucleoprotein filament that facilitates the invasion of a damaged DNA strand into the homologous double-stranded DNA partner. The human Rad51 protein is a structural and functional homolog of the Escherichia coli RecA protein, which promotes homologous pairing and strand transfer reactions in vitro. Both Rad51 and Rad52 bind specifically to the terminal regions of tailed duplex DNA, the substrate thought to initiate recombination in vivo. Furthermore, nucleoprotein filaments of Rad51, formed on tailed DNA, catalyze strand invasion of homologous duplex DNA in a reaction that is stimulated by Rad52 and RPA (3).Epstein-Barr virus (EBV) is a human herpesvirus that infects B lymphocytes, inducing their continuous proliferation. In B-lymphoblastoid cell lines, there is no production of virus particles, which is termed latent infection (52). Reactivation from latency is characterized by the expression of lytic genes, and one of the first detectable changes is the expression of the BZLF1 immediate-early gene product, which trans-activates viral promoters (16), leading to an ordered cascade of viral early and late gene expression. This lytic EBV DNA replication occurs in discrete sites in nuclei, called replication compartments, in which seven viral replication proteins are assembled (44). The viral genome is amplified several hundredfold by the viral replication machinery and is thought to generate highly branched replication intermediates through HR coupled with viral DNA replication (48). With the progression of lytic replication, the replication compartments become larger and appeared to fuse to form large globular structures that eventually filled the nucleus at late stages of infection (8, 45).We previously isolated latently EBV-infected Tet-BZLF1/B95-8 cells in which the exogenous BZLF1 protein is conditionally expressed under the control of a tetracycline-regulated promoter, leading to a highly efficient induction of lytic replication (28). Using this system, we have demonstrated that the induction of the EBV lytic program results in the inhibition of replication of cellular DNA in spite of the replication of viral DNA (28) and elicits a cellular DNA damage response, with the activation of the ATM-Chk2-p53 DNA damage transduction pathway (29). The DNA damage sensor MRN complex and phosphorylated ATM are recruited and retained in viral replication compartments (29).Here we report that RPA32 is extensively phosphorylated after EBV lytic replication is induced, with the phosphorylation sites in accordance with those for ATM. Phosphorylated RPA, Rad51, and Rad52, which are involved in HR repair (HRR), are recruited and retained in viral replication compartments as well as the MRN complex. Furthermore, DSBs could be demonstrated to occur during viral genome synthesis in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference (RNAi) knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that HR and/or repair of viral DNA genome might occur, coupled with DNA replication, to facilitate viral genome synthesis.     

Insertion of Vector Sequences in the Genome of Transgenic Plants

Insertion of vector sequences of different lengths in the genome of transgenic tobacco and carrot plants occurring at a frequency of 35.2% was shown. No significant differences in insertion frequencies between the two species were observed. Integrated fragments of the vector DNA were stably inherited in the following generation resulted from self-pollination of original transformants.     

水稻基因组中MITEs的偏爱位置

已知微型倒转重复转座因子（ＭＩＴＥｓ）广泛分布于真核生物基因附近的非编码区。为探讨ＭＩＴＥｓ在基因组一级结构中的分布规律,通过对８２ｋｂ的水稻ＢＡＣ克隆ｌ１３３２全序列分析,精确鉴定了１０个蛋白质编码基因的位置和转录方向以及７个ＭＩＴＥｓ的们置,毫无例外的观察到,ＭＩＴＥｓ只要存在,总是位于最近的基因的上游 ,不管该基因与之相距多远、转录方向如何。另外还发现,１个ＭＴＩＥ被２个基因共享,由于ＭＩＴ     

Localization in the Tomato Genome of DNA Restriction Fragments Containing Sequences Homologous to the rRNA (45s), the Major Chlorophyll a/b Binding Polypeptide and the Ribulose Bisphosphate Carboxylase Genes

DNA restriction fragments containing sequences homologous to the ribosomal RNA (45s), the major chlorophyll a/b binding polypeptide (CAB) and the small subunit of ribulose bisphosphate carboxylase (RBCS) genes have been localized and mapped in the tomato nuclear genome by linkage analysis. Ribosomal RNA genes map to a single locus, R45s, which resides in a terminal position on the short arm of chromosome 2 and corresponds to the Nucleolar Organizer Region. The size of the 45s repeating unit is estimated to be approximately 9 kb in Lycopersicon esculentum and 11 kb in Lycopersicon pennellii. Five loci were found to contain CAB sequences. Two of the loci, Cab-1 (chromosome 2) and Cab-3 (chromosome 8), together accounted for more than 80% of the hybridization signal. These loci contain more than one CAB structural gene. The other three loci, Cab-2 (chromosome 8), Cab-4 (chromosome 7) and Cab-5 (chromosome 12), each account for <10% of the total signal and may contain only a single copy of the CAB structural sequence. Three loci were found to contain RBCS sequences. Rbcs-2 (chromosome 3) and Rbcs-3 (chromosome 2) were responsible for >80% of the signal, with the remainder being associated with Rbcs-1 (chromosome 2). Rbcs-2 and Rbcs-3 may contain more than one copy of the gene.