Transformation of yeast by a replicating hybrid plasmid.

Chimaeric plasmids have been constructed containing a yeast plasmid and fragments of yeast nuclear DNA linked to pMB9, a derivative of the ColEl plasmid from E. coli. Two plasmids were isolated which complement leuB mutations in E. coli. These plasmids have been used to develop a method for transforming a leu2 strain of S. cerevisiae to Leu+ with high frequency. The yeast transformants contained multiple plasmid copies which were recovered by transformation in E. coli. The yeast plasmid sequence recombined intramolecularly during propagation in yeast.     

Giant linear plasmids in Streptomyces which code for antibiotic biosynthesis genes

A number of examples of circular plasmids with specific functions are known in both prokaryotes and eukaryotes. Several linear plasmids have also been identified, but these are all relatively small: large linear plasmids cannot be separated from chromosomal DNA by conventional techniques. There are several cases where the genetic evidence suggests that a character is encoded by a plasmid but no plasmid can be physically detected. This has been the case for antibiotic synthesis genes in Streptomyces; in particular a plasmid SCP1 in Streptomyces coelicolor has been shown to be involved in methylenomycin production by genetic evidence. We report here the application of orthogonal-field-alternation gel electrophoresis to the isolation of linear plasmids from Streptomyces. We have discovered a large linear plasmid of around 520 kilobases in Streptomyces lasaliensis and subsequently similar giant linear plasmids in other Streptomyces strains. We have confirmed that genes for methylenomycin biosynthesis are located on a series of giant linear plasmids in S. coelicolor. These observations may bear on the genetic variability and unstable genetic character of Streptomyces species.     

用STM证实质粒pUC18DNA分子新构象的存在

用清高妥液法从大肠杆菌ＢＨ１０１细胞制备出的多拷贝质粒ＰＵＣ１８ＤＮＡ分子的构象具有复杂的多样性，我们在对其进行ＳＴＭ研究计，证实了一种不同于已知构象的新构象的存在。     

三种新的穿梭质粒pCC10、pCCT7和pST16的构建和性质研究

利用大肠杆菌质粒pUC18、pTG206和金黄色葡萄球菌质粒pC194在体外构建3个嵌合质粒pCC10、pCCT7和pST16.3个新质粒均是大肠杆菌和枯草杆菌的穿梭质粒。它们在大肠杆菌中呈Ap~rCm~r型,在枯草杆菌中则为Cm~rAp~5型。其中pCCT7和pST16含有xylE基因,可作为启动子探测质粒。所有的新质粒都具有来自pUC18的多酶克隆位点,适合于在大肠杆菌和枯草杆菌中重组外源DNA以及比较它们的表达情况。     

Independent transfer of mitochondrial plasmids in Neurospora crassa

In the ascomycete fungus Neurospora, the distribution of homologous mitochondrial plasmid DNAs in different species and among mitochondrial types of N. crassa suggests that these molecules have moved between lineages of clonally propagated mtDNA. Here we report direct evidence for independent inheritance of mitochondrial plasmids by sexual reproduction which may help explain the distribution of these molecules among mitochondrial lineages.     

CpG DNA enhances the immune responses elicited by the DNA vaccine against foot-and-mouth disease virus in guinea pigs

CpG DNA is DNA sequence that has immune stimulatory effects. Several lines of investigation over the past few years indicate that CpG DNA plays an important role in the induction of immune responses to DNA vaccines. In this study, CpG DNA-containing synthetic oligodeoxynucleotide (CpG-ODN) was cloned into the eukaryotic expression plasmid encoding a fusion protein containing b- galactosidase from E. coli and immunogenic epitopes of foot- and-mouth disease virus (FMDV) type O, and the immune responses induced by the plasmid were assayed. The results showed that guinea pigs immunized with the recombinant plasmid containing CpG-ODN generated a higher level of FMDV-neutralizing antibody and a stronger T cell proliferative response and protection against viral challenge than those receiving the plasmid containing no CpG-ODN. Our study demonstrated that it is an effective route to enhance the efficacy of DNA vaccines by inserting exogenous CpG DNA into the plasmids, and the DNA vaccine developed here is a promising candidate to prevent FMDV infection.     

两种策略实现1,3-丙二醇关键酶基因的共表达

甘油脱水酶(GDHt)和1,3-丙二醇氧化还原酶(PDOR)是甘油歧化为1,3-丙二醇(1,3-PD)的两个关键酶。采用多顺反子重组和质粒共存两种策略,对来自克雷伯肺炎杆菌(Klebsiela pneumoniae)的两个关键酶进行共表达。构建表达载体pET-28a-dhaB1B2B3-dhaT将两酶基因dhaB1B2B3和dhaT用SD序列相隔,在E.coli BL21(DE3)高水平共表达了GDHt 3个亚基和PDOR,表达蛋白分别约占菌体总蛋白的18%、9%、7%和9%。质粒pET-28a-dhaB1B2B3和pET-22b-dhaT共转化E.coli BL21(DE3)得到稳定的双质粒系统,48h后84%的细胞能同时含有两种质粒,GDHt 3亚基和PDOR分别约占菌体总蛋白的16%、8%、6%和14%。两种酶在两种表达方法下均显示高于原始菌株的酶活力。     

The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA

Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.     

青鳉鱼早期胚胎中外源DNA的复制行为的研究

利用显微注射技术将外源ＤＮＡ与标有生物素的ｄＡＴＰ一起导入处于１细胞期的青鱼（Ｍｅｄａｋａ）受精卵细胞质中，鱼卵在２５℃孵化一天至胚孔封闭期，从胚胎细胞质中提取ＤＮＡ，经分子杂交发现外源ＤＮＡ在胚胎中可进行复制。ＤＮＡ样品再经核酸电镜分析，发现了几种类型的复制分子，并发现多复制叉及成熟前复制现象。由此认为外源ＤＮＡ可以多种方式在青鱼早期胚胎细胞质中进行复制。     

High levels of unintegrated HIV-1 DNA in brain tissue of AIDS dementia patients

In the host cell, retroviral DNAs exist in three main forms: unintegrated linear, unintegrated circular, and integrated (the provirus). High levels of unintegrated forms of retroviral DNA often correlate with superinfection and accompanying cytopathic effects, as, for example, in the case of feline acquired immunodeficiency. In culture, HIV-1 infection also results in high levels of unintegrated viral DNA although direct correlations with cytopathicity have not been made. The low frequency of HIV-1-infected cells in patients has made it difficult to determine the structure of the viral DNA in fresh tissue samples from AIDS patients by standard methods such as Southern hybridization. The PCR technique however, which allows the detection of viral DNA at levels far below that possible by other hybridization methods is, in its conventional form, of limited use for quantitative analysis. To study the amount and form of HIV-1 DNA in primary tissue of AIDS patients we have therefore modified the PCR method. Our results indicate that each of the three species of viral DNA are detectable in blood and brain of AIDS patients, and that in autopsy samples from patients with HIV encephalitis there is a considerably higher proportion of unintegrated viral DNA.     

Structures of ParB bound to DNA reveal mechanism of partition complex formation

The faithful inheritance of genetic information, which is essential for all organisms, requires accurate DNA partition (segregation) at cell division. In prokaryotes, partition is mediated by par systems, for which the P1 plasmid system of Escherichia coli is a prototype comprising a partition site and two proteins, ParA and ParB. To form the partition complex necessary for segregation, P1 ParB must recognize a complicated arrangement of A-box and B-box DNA motifs located on opposite ends of a sharply bent parS partition site of approximately 74 bp (refs 3-7). Here we describe structures of ParB bound to partition sites. ParB forms an asymmetric dimer with extended amino-terminal HTH (helix-turn-helix) domains that contact A-boxes. The two HTH domains emanate from a dimerized DNA-binding module composed of a six-stranded beta-sheet coiled-coil that binds B-boxes. Strikingly, these individual DNA-binding modules rotate freely about a flexible linker, enabling them to contact several arrangements of A- and B-boxes. Most notably, each DNA-binding element binds to and thus bridges adjacent DNA duplexes. These unique structural features of ParB explain how this protein can bind complex arrays of A- and B-box elements on adjacent DNA arms of the looped partition site.     

Independent transfer of mitochondrial chromosomes and plasmids during unstable vegetative fusion in Neurospora

The sporadic distribution of similar introns in organelle, nuclear ribosomal RNA and bacteriophage genes suggests that at least some of these introns are mobile genetic elements. Some plasmids in fungal mitochondria contain intron-like sequences and, like introns, they have scattered distributions within and among species. The occurrence and evolutionary importance of such horizontal transfer of DNA, not only between fungi, but among a wide range of organisms have been matters of much discussion. Here, we report experimental evidence for transfer of Neurospora mitochondrial plasmids from one mitochondrial genotype to another at high frequency during unstable vegetative cell fusion. Exchange of mitochondrial and nuclear genomes can also occur. These observations suggest that vegetative fusion may have a more important role in the mitochondrial genetic structure of natural populations than is generally thought, and may provide an explanation for the distribution of certain plasmids and possibly other mobile genetic elements.     

Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells

Epstein-Barr virus (EBV) infects human B lymphocytes, transforming the infected cells into dividing blasts that can proliferate indefinitely. The viral genome of 172 kilobase pairs (kbp) is a plasmid in most transformed cells. We have identified a region of EBV DNA, termed oriP (nucleotides 7,333-9,109 of strain B95-8), which acts in cis to permit linked DNAs to replicate as plasmids in cells containing EBV DNA. We have postulated the existence of a trans-acting gene allowing oriP function. Here we report that this gene lies in a 2.6-kbp region of the viral genome (nucleotides 107, 567-110, 176) which encodes the EBNA-1 antigen. We show that circular DNAs containing oriP, the EBNA-1 gene and a selectable marker replicate autonomously in cells derived from at least four developmental lineages and from at least three species. We also find that the one-third of the EBNA-1 gene repetitive in sequence is not essential for the trans-acting function that EBNA-1 gives oriP.     

耐盐细菌质粒的研究

本实验以1株耐盐细菌My23（耐18％NaCl）作为研究对象,采用碱裂解法对其进行质粒提取,结果表明可以提取得大小2个不同质粒;改变常规提取方法中的部分环节,如加入STE清洗菌体的培养基成分,溶菌酶的使用,以及加入碱裂解液Ⅰ后静置20min,对该耐盐菌质粒的提取有较好的作用。经SDS法将小质粒消除后的菌株与野生型菌株的耐盐功能比较,发现提取出的小质粒与耐盐无关。将大肠杆菌DH5α制备成感受态细胞,将耐盐菌的质粒进行转化,结果进一步表明耐盐性状与质粒没有关系。     

萘亚胺类化合物对DNA光敏剪切作用的初步研究

研究了Ｎ－（Ｎ,Ｎ－二甲基）－乙胺－１,８－萘硫酰亚胺和Ｎ－（Ｎ,Ｎ－二甲基）－乙胺－１,８－萘酰亚胺对小牛胸腺ＤＮＡ的嵌入结合反应,并求取了嵌入常数,它们可以把质粒ｐＵＣ１９的超螺旋ＤＮＡ剪切为开环ＤＮＡ,在紫外光作用下,这种能力会得到不同程度的提高。     

基因筛选克隆表达原花青素降解酶及其酶解条件优化的初步研究

针对化学法制备低聚原花青素存在环保压力和副产物多等问题，研究了通过生物酶法降解高聚原花青素来制备低聚原花青素的方法。首先通过基因筛选方法筛选出SananA和EcnanA基因用于酶法降解高聚原花青素，将SananA和EcnanA基因连接在不同质粒载体表达蛋白的结果表明，SananA基因连接在pETDuet-1载体上时的蛋白表达效果较优。进而，利用纯化的SananA蛋白优化高聚原花青素降解条件，结果显示较优的酶解条件为温度50℃、pH=10、反应时间8 h，在此条件下原花青素单体积累量为35.67 mg/g，二聚体积累量为14.49 mg/g。     

Asymmetric gene conversion at inserted segments on yeast mitochondrial DNA.

Different molecular weight forms of the protein product of the yeast mitochondrial gene var 1 are shown at arise by a process of asymmetric gene conversion. These different forms can be accounted by two DNA segments, 36 and 57 base pairs long, present in one allelic form of the var 1 structural gene, which can be inserted independently and at different frequencies into other var 1 alleles.     

The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria

Bacillus anthracis is an endospore-forming bacterium that causes inhalational anthrax. Key virulence genes are found on plasmids (extra-chromosomal, circular, double-stranded DNA molecules) pXO1 (ref. 2) and pXO2 (ref. 3). To identify additional genes that might contribute to virulence, we analysed the complete sequence of the chromosome of B. anthracis Ames (about 5.23 megabases). We found several chromosomally encoded proteins that may contribute to pathogenicity--including haemolysins, phospholipases and iron acquisition functions--and identified numerous surface proteins that might be important targets for vaccines and drugs. Almost all these putative chromosomal virulence and surface proteins have homologues in Bacillus cereus, highlighting the similarity of B. anthracis to near-neighbours that are not associated with anthrax. By performing a comparative genome hybridization of 19 B. cereus and Bacillus thuringiensis strains against a B. anthracis DNA microarray, we confirmed the general similarity of chromosomal genes among this group of close relatives. However, we found that the gene sequences of pXO1 and pXO2 were more variable between strains, suggesting plasmid mobility in the group. The complete sequence of B. anthracis is a step towards a better understanding of anthrax pathogenesis.     

Evolution of a bacteria/plasmid association

Associations between bacteria and their accessory elements (viruses, plasmids and transposons) range from antagonistic to mutualistic. A number of previous studies have demonstrated that plasmid carriage reduces bacterial fitness in the absence of selection for specific functions such as antibiotic resistance. Many studies have demonstrated increased fitness of evolving microbial populations in laboratory environments, but we are aware of only one study in which fitness gains were partitioned between a plasmid and its host. Here, we examine the evolution of an association between a plasmid and its bacterial host. Carriage of the non-conjugative plasmid pACYC184 initially reduced the fitness of Escherichia coli B in the absence of antibiotic. We then cultured plasmid-bearing bacteria for 500 generations in the presence of antibiotic. The fitness of each combination of host and plasmid, with and without the culture history, was determined by competing it against a baseline strain. The results indicate adaptation by the host genome, but no plasmid adaptation. We also competed the evolved host, transformed with the baseline plasmid, against its isogenic plasmid-free counterpart. The plasmid now increased the fitness of its host.     

Efficient transformation and expression of gfp gene in the edible mushroom Pleurotus nebrodensis

An efficient transformation method mediated by PEG-protoplasts was developed for the newly commercial edible mushroom Pleu-rotus nebrodensis. Two plasmids were used to co-transform protoplasts of P. nebrodensis. One plasmid is pAN7-1 containing a positive selectable marker gene hph conferring hygromycin B resistance. Another plasmid is pBIue-GFP containing a reporter gene gfp conferring green fluorescent protein. PCR and Southern blot analysis showed that hph gene or/and gfp gene were integrated into the genome of P.nebrodensis transformants. The transformation efficiency of the positive selectable marker gene hph was 3 transformants per microgram of plasmid pAN7-1 DNA, which was about 30 times higher than that previously reported in thoroughly studied Pleurotus species such as Pleurotus ostreatus. The transformation efficiency of the reporter gene gfp was 9 transformants per microgram of plasmid pBlu-GFP DNA. The co-transformation efficiency was 23.68%. This is the first report that a "reporter" gene, green fluorescent protein gene can be successfully stably expressed in this Pleurotus species.