Expression of a bacterial modification methylase gene in yeast

Methylation of specific cytosines in the DNA is generally believed to play some role in the regulation of gene expression in eukaryotes. However, some eukaryotes, such as Drosophila and yeast (S. Hattman, personal communication) seem not to contain 5-methylcytosine in their DNA. It would be interesting to test, how gene expression in such organisms would respond to the methylation of specific cytosines in the genome. As a first step towards this goal, we have introduced the gene encoding the Bacillus sphaericus R modification methylase, which methylates the internal cytosine within the recognition sequence 5'-GGCC, into yeast cells. Southern-type hybridization to DNAs isolated from the transformed yeast clones revealed that the yeast plasmid carrying the prokaryotic methylase gene, as well as the two chromosomal genes tested (his3 and leu2) were methylated, whereas the bulk of the yeast DNA remained largely unmethylated. This indicates that the Bacillus sphaericus modification methylase was expressed in yeast but it modified only certain parts of the yeast DNA.     

Escherichia coli dam—dcm—菌株的研究进展

E.coli dam-dcm是一种在分子生物学技术中被广泛应用的菌株之一。Dam和Dcm是两种甲基转移酶，Dam识别GATC位点而Dcm识别CCA（T）GG位点[1]，在E.coli细胞的生命活动中，dam基因产物在DNA的错配修复中具有重要作用，另外，dam的甲基化作用和DNA的复制和基因表达的调节等细胞生命活动过程，Dcm甲基化的生物学功能在很短的补丁修复有很重要的作用。Streptomyces(链霉菌），Bacillus(芽胞杆菌）和Paracoccus（副球菌）的转化通过用dam和dcm位点没有甲基化的DNA可以得到很大的改善[6]。因为5－甲基胞嘧啶对肼有抗生，所以 从dcm缺陷的菌株分离到的DNA用Mazam和Gilbert法进行测序，结果更好[10]。     

Retrovirus genomes methylated by mammalian but not bacterial methylase are non-infectious

The biological importance of DNA methylation for gene expression in eukaryotes is becoming increasingly evident, and a direct role of methylation in gene expression has been suggested by an analysis of the infectivity of integrated retroviral genomes in a transfection assay. These studies, however, did not address whether specific methylatable residues are involved in gene regulation. Methylation by sequence-specific bacterial DNA methylases has been shown to suppress the expression of some genes, but not others. To investigate the effect of methylation on gene expression without having to rely on sequence-specific methylases, a rat liver enzyme was used to methylate in vitro all C-G dinucleotides of a proviral genomic clone. This treatment reduced the biological activity of Moloney murine leukaemia virus (M-MuLV) proviral DNA by more than three orders of magnitude, whereas complete methylation of 35 HpaII sites in the same DNA had only a marginal effect. The rat methylase-induced inactivation was reversible, as treatment of recipient cells with 5-azacytidine rendered the non-infectious viral genomes biologically active. This suggests that methylation in other C-G dinucleotides than those detectable with restriction enzymes can be crucial for gene expression.     

The structure of trp pseudorepressor at 1.65A shows why indole propionate acts as a trp 'inducer'

The trp repressor is a small dimeric regulatory protein which controls the expression of three operons in Escherichia coli. The inactive aporepressor protein must bind two molecules of L-tryptophan to form the active repressor. If desamino analogues of L-tryptophan such as indole propionate (IPA) are substituted for L-tryptophan, an inactive pseudorepressor is formed. Because the desamino analogues thus cause derepression of operons under control of the trp repressor, they appear to be 'inducers'. We have determined the crystal structure of the pseudorepressor and refined it to 1.65 A. The molecular structure was compared to that of the nearly isomorphous orthorhombic form of the repressor. Surprisingly, the indole ring of IPA is in the same position as the indole ring of L-tryptophan in the repressor, but is 'flipped over'. As a result, the carboxyl group of IPA is oriented toward the DNA-binding surface of the protein and is in a position where it sterically and electrostatically repels the phosphate backbone of both operator and non-operator DNA. This explains why IPA acts as an apparent trp inducer.     

Single-site enzymatic cleavage of yeast genomic DNA mediated by triple helix formation.

Physical mapping of chromosomes would be facilitated by methods of breaking large DNA into manageable fragments, or cutting uniquely at genetic markers of interest. Key issues in the design of sequence-specific DNA cleaving reagents are the specificity of binding, the generalizability of the recognition motif, and the cleavage yield. Oligonucleotide-directed triple helix formation is a generalizable motif for specific binding to sequences longer than 12 base pairs within DNA of high complexity. Studies with plasmid DNA show that triple helix formation can limit the operational specificity of restriction enzymes to endonuclease recognition sequences that overlap oligonucleotide-binding sites. Triple helix formation, followed by methylase protection, triple helix-disruption, and restriction endonuclease digestion produces near quantitative cleavage at the single overlapping triple helix-endonuclease site. As a demonstration that this technique may be applicable to the orchestrated cleavage of large genomic DNA, we report the near quantitative single-site enzymatic cleavage of the Saccharomyces cerevisiae genome mediated by triple helix formation. The 340-kilobase yeast chromosome III was cut uniquely at an overlapping homopurine-EcoRI target site 27 base pairs long to produce two expected cleavage products of 110 and 230 kilobases. No cleavage of any other chromosome was detected. The potential generalizability of this technique, which is capable of near quantitative cleavage at a single site in at least 14 megabase pairs of DNA, could enable selected regions of chromosomal DNA to be isolated without extensive screening of genomic libraries.     

纳豆激酶基因的克隆及其在大肠杆菌中的表达

纳豆激酶是一种良好的天然蛋白酶类溶栓物质。国内外许多学者对该酶进行了基因工程研究,但在克隆表达过程中出现了许多长短不同的基因片段。本研究通过原产日本的优质纳豆中分离鉴定出高产纳豆杆菌N07并提取该菌株的全基因组;通过PCR手段扩增出能编码纳豆激酶信号肽,前导肽和成熟肽的前纳豆激酶酶原基因NK1,以及能编码纳豆激酶成熟肽的纳豆激酶基因NK2,构建了纳豆激酶基因的表达载体pET30a-NK1和pET30a-NK2,转化E.coli BL21后在大肠杆菌中表达,并进行了活性分析。结果发现,纳豆激酶酶原基因片段NK1能成功表达出有活性的分泌型纳豆激酶;而纳豆激酶基因片段NK2的表达产物为无活性的包涵体。在对NK1和NK2的比较研究后可知,纳豆激酶酶原基因片段NK1能在大肠杆菌中很好的分泌表达,这将为纳豆激酶基因工程的深入研究奠定基础。     

Detection in vivo of protein-DNA interactions within the lac operon of Escherichia coli

Studies of the sequence-specific binding of proteins to DNA have so far relied on in vitro experiments using cloned restriction fragments containing the relevant DNA sequences. We have applied the genomic sequencing technique of Church and Gilbert to show that the interactions observed in vitro occur in vivo. We use this approach to study the binding of regulatory proteins to the lac operon in vivo and detect changes in the reactivity (inhibition or enhancement) of guanines to methylation by dimethyl sulphate caused by the proximity of proteins to the N-7 atom of these guanines. We can detect the simultaneous binding of the catobolite gene activator protein (CAP) and the Lac repressor to their specific recognition sequences, and following induction of the lac operon we observe effects that are related to RNA polymerase binding or RNA elongation. We have successfully used oligonucleotide probes as short as 17 bases to display genomic sequence.     

Expression and characterization of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is a common lethal genetic disease that manifests itself in airway and other epithelial cells as defective chloride ion absorption and secretion, resulting at least in part from a defect in a cyclic AMP-regulated, outwardly-rectifying Cl- channel in the apical surface. The gene responsible for CF has been identified and predicted to encode a membrane protein termed the CF transmembrane conductance regulator (CFTR). Identification of a cryptic bacterial promoter within the CFTR coding sequence led us to construct a complementary DNA in a low-copy-number plasmid, thereby avoiding the deleterious effects of CFTR expression on Escherischia coli. We have used this cDNA to express CFTR in vitro and in vivo. Here we demonstrate that CFTR is a membrane-associated glycoprotein that can be phosporylated in vitro by cAMP-dependent protein kinase. Polyclonal and monoclonal antibodies directed against distinct domains of the protein immunoprecipitated recombinant CFTR as well as the endogenous CFTR in nonrecombinant T84 cells. Partial proteolysis fingerprinting showed that the recombinant and non-recombinant proteins are indistinguishable. These data, which establish several characteristics of the protein responsible for CF, will now enable CFTR function to be studied and will provide a basis for diagnosis and therapy.     

Endogenous viral genes are non-essential in the chicken.

DNA sequences homologous to the genomes of type C retroviruses are widespread among vertebrates. Ten genetic loci containing endogenous viral DNA sequences have been documented in the white Leghorn chicken alone. Six of these genetic loci are associated with the production of virus or of viral proteins in embryonic fibroblasts (refs 2--4, and S.M.A., L, B. Crittenden and E.G.B., in preparation) and one of the loci may be expressed in the erythroblasts of 5-day-old embryos. The abiquitous presence of endogenous viral genes among vertebrate species and the association of their expression with development of the haematopoietic system in the mouse have led to the proposal that these genes are involved in ontogeny. In addition, the genes may be implicated in oncogenesis as in the case of the AKR mouse in which a high incidence of spontaneous leukaemia is associated with the expression of endogenous murine laukaemia virus genomes. We report here the production of a fertile rooster which lacks avian leukosis virus-related endogenous viral genes and which seems to be completely normal and healthy. Thus, endogenous viral genes are apparently not essential for the normal development of the chicken. An endogenous virus-free state has also been reported for three species of jungle fowl and for the B-type viral genes of the mouse.     

Evidence for Base Substitutions and Repair of DNA Mismatch Damage Induced by Low Energy N^＋ Ion Beam Implantation in E. coli

Ever since the low energy N^ ion beam has been accepted, the mutations of ionizing radia-tion are attributable mainly to avoidance of DNA damages repair. Evidences based on in vivo proof results are limited. Using the E. coli wild type and mutator strains, the mutant frequencies suggest that base substitutions in rpoB gene are induced by the N^ implantation. A highly con-served region is selected to get the direct evidence for base substitutions by sequence of the high fi-delity PCR amplification products in mutants. Most of the mutants (90.9%, 40/44) have at least one base substitution in the amplification region. The evidences for CG to TA (55 %, 22/40), AT to GC (20%, 8/40) and TA to CG (5%, 2/40) transitions are identified. The transversions are AT to TA ( 15 %, 6/40) and GC to CG (5 %, 2/40). It is suggested that DNA cytosine methylase might play an important role in mismatch repair of DNA damage induced by N^ implantation by analysis of the mutant frequencies of mutator strains.     

铜绿假单胞菌的LexA蛋白在大肠杆菌中的高效表达与纯化

目的:对铜绿假单胞菌(Pseudomonas aeruginosa,PA)的阻遏蛋白LexA进行基因克隆、重组表达及蛋白纯化.方法:采用PCR法从PAO1株基因组中扩增出1 086 bp的LexA基因,将此基因片段插入到表达载体pET32a( )上,并在大肠杆菌BL21(DE3)中表达.包涵体洗涤后用8 mol/L尿素溶解,以镍离子亲合柱层析作为第1步纯化,Superdex75凝胶过滤层析作为第2步精细纯化,反相高相液相色谱法(HPLC)测定蛋白的浓度.结果:LexA以包涵体形式表达,表达量为45%,经镍离子亲合柱层析纯化后LexA蛋白纯度达到85%以上,回收率大于80%,镍离子亲合柱层析和凝胶过滤层析两步纯化目的蛋白,经HPLC测定,最终目的蛋白的纯度为98.97%.结论:在pET32a( )表达系统中实现了PA的LexA蛋白的高效表达,两步纯化法获得高纯度的目的蛋白,为进一步鉴定LexA靶位点奠定了基础.     

Epigenetic inheritance in plants

The function of plant genomes depends on chromatin marks such as the methylation of DNA and the post-translational modification of histones. Techniques for studying model plants such as Arabidopsis thaliana have enabled researchers to begin to uncover the pathways that establish and maintain chromatin modifications, and genomic studies are allowing the mapping of modifications such as DNA methylation on a genome-wide scale. Small RNAs seem to be important in determining the distribution of chromatin modifications, and RNA might also underlie the complex epigenetic interactions that occur between homologous sequences. Plants use these epigenetic silencing mechanisms extensively to control development and parent-of-origin imprinted gene expression.     

红斑丹毒丝菌C43065株spaA基因的克隆和表达

通过PCR从红斑丹毒丝菌C43065株基因组DNA中扩增出编码信号肽除外的成熟SpaA蛋白基因spaA,将其克隆到表达载体pET32a的BamHⅠ和Hind Ⅲ位点上,构建重组表达质粒pET-spaA,转化大肠杆菌BL21,在IPTG诱导下表达N端带有Trx标签的融合蛋白rSpaA,SDS-PAGE检测表达蛋白.DNA测序结果表明,spaA基因大小为1794 bp,编码由597个氨基酸残基组成的成熟SpaA蛋白,SDS-PAGE结果显示在大肠杆菌BL21中成功表达了分子量约为86 kDa的重组rSpaA,为进一步开展SpaA保护区域的研究奠定基础.     

DNA protection by stress-induced biocrystallization.

The crystalline state is considered to be incompatible with life. However, in living systems exposed to severe environmental assaults, the sequestration of vital macromolecules in intracellular crystalline assemblies may provide an efficient means for protection. Here we report a generic defence strategy found in Escherichia coli, involving co-crystallization of its DNA with the stress-induced protein Dps. We show that when purified Dps and DNA interact, extremely stable crystals form almost instantaneously, within which DNA is sequestered and effectively protected against varied assaults. Crystalline structures with similar lattice spacings are formed in E. coli in which Dps is slightly over expressed, as well as in starved wild-type bacteria. Hence, DNA-Dps co-crystallization is proposed to represent a binding mode that provides wide-range protection of DNA by sequestration. The rapid induction and large-scale production of Dps in response to stress, as well as the presence of Dps homologues in many distantly related bacteria, indicate that DNA protection by biocrystallization may be crucial and widespread in prokaryotes.     

Expression of human erythropoietin directed by mWAP promoter in mammary gland of transgenic mice

The present work has generated transgenic mice with a hybrid gene construct consisting of genomic sequences encodinghuman erythropoietin (hEPO) and governed by regulatory sequences of mousewhey acidic protein (mWAP). The construct proved effective by transient expression in lactating animal. After introducing hybrid gene construct into single-cell embryo via pronuclear microinjection, surviving embryo are reimplanted into pseudopregnant foster mother mouse. 58 mice of 86 generation zero mice obtained were identified to be positive by PCR-Southern blot and genomic DNA Southern blot methods. The integration rate is 67%.hEPO was expressed in the milk of 16 mice of 39 mice measured byhEPO ELISA kit The expression level gets over 15 μg/mL.