Hybrid plasmid pBS251 containing genes for n-alkane degradation

The strain of Pseudomonas aeruginosa BS316 utilizing H-alkanes of the C6-C12 series (Alk+) harbours the 96 Md plasmid pBS250. The use of plasmid RP4 to mobilize Alk+ markers in conjugal transfer to Pseudomonas aeruginosa and Pseudomonas putida has resulted in isolation of transconjugants resistant to antibiotics (due to genes coded by plasmid RP4) and capable of growth on H-alkanes. A transconjugant from this series harbours plasmid pBS251, a derivative of plasmid RP4 containing the genes for octane and octanol catabolism. A fragment of DNA inserted into RP4 has a mol mass 3.8 Md, possesses two restriction sites for EcoRI, one site for PstI, is not restricted by SmaI and BamHI restriction endonucleases, and is localized in the region 4.5-5.7 Md on the physical map of plasmid RP4.     

F factor inhibition of conjugal transfer of broad-host-range plasmid RP4: requirement for the protein product of pif operon regulatory gene pifC.

By the use of deletions, point mutations, and gene fusions, we show that the protein product of the F factor pifC gene is responsible for F factor inhibition of plasmid RP4 conjugal transfer. Deletion analysis of pif sequences carried by pSC101-F chimeric plasmids demonstrated that removal of all or part of the pifC coding sequence greatly decreased or abolished the ability of these plasmids to inhibit RP4 transfer. Amber mutations in the pifC gene eliminated inhibition in an Su- host strain but not in and Su+ (supF) host. Plasmids carrying nonpolar pifC mutations did not decrease the efficiency of RP4 transfer when present in trans. Whereas pifC+ plasmids inhibited RP4 transfer, the presence of RP4 in the same cell as F' lac increased F'lac Pif activity approximately 1,000-fold. This effect most likely resulted from the binding of the pifC product to RP4 DNA and concomitant derepression of the F factor pif operon. PifC inhibited trans mobilization of pMS204, a nonconjugative plasmid carrying the RP4 oriT locus, by the RP1 derivative pUB307. pMS204 had no trans effect on pif operon expression, whereas pUB307 increased F'lac Pif expression, as did RP4. Our results suggest that the pifC product inhibits expression of one or more RP4 genes, the products of which are required for conjugal transfer of RP4 and are required in trans for mobilization of nonconjugal RP4 oriT containing plasmids.     

trans activation of the latent Epstein-Barr virus (EBV) genome after transfection of the EBV DNA fragment.

Transfection of Epstein-Barr virus (EBV)-nonproducer Raji cells with the BamHI Z fragment of EBV DNA induced antigens that were detected with human antiserum against EBV-specific early antigens. Northern blot analysis of transfected cells revealed that one intense RNA band hybridized with the BamHI H and F fragments but not with the BamHI Z fragment. Cooperation between the BamHI H, F, and BamHI Z regions was also confirmed in baby hamster kidney cells that were cotransfected with both fragments. These results indicate that the transfected BamHI Z fragment of EBV DNA induces a trans-acting factor which activates the gene expression of the BamHI H and F region and that the BamHI Z region possibly plays an important role in the latency of EBV.     

Beta-lactamase genes of Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae: cloning and expression in Streptomyces lividans

Genes encoding extracellular beta-lactamases (EC 3.5.2.6) of Gram-positive Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae have been cloned into Streptomyces lividans. The beta-lactamase gene of S. badius was initially isolated on a 7 kb BamHI fragment and further located on a 1300 bp DNA segment. An 11 kb BamHI fragment was isolated encompassing the S. cacaoi beta-lactamase gene, which was subcloned to a 1250 bp DNA fragment. The beta-lactamase gene of S. fradiae was cloned on an 8 kb BamHI fragment and mapped to a 4 kb DNA segment. Each of the three BamHI fragments encompassing the beta-lactamase genes hybridized to a BamHI fragment of the corresponding size in chromosomal DNA from the respective strain used for cloning. The activities of the three beta-lactamases were predominantly found to be extracellular in the S. lividans recombinants. The S. badius and S. cacaoi beta-lactamases exhibited a 10-100-times lower activity in S. lividans, whereas the S. fradiae beta-lactamase showed an approximately 10-fold higher activity in the cloned state, compared with the activities found in the original strains.     

IncP plasmids are most effective in mediating conjugation between Escherichia coli and streptomycetes

Mobilizable shuttle plasmids containing the origin of transfer (oriT) region of plasmid F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPalpha) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Streptomyces. The conjugative system of the IncPalpha plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 +/- 0.2) x 10(-2) S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPalpha plasmids is crucial in conjugative transfer, an assay with an IncQ-based mobilizable plasmid (RSF1010) specifying its own DNA-processing system was developed. Only the IncPalpha plasmid mobilized the construct to S. lividans indicating that the mating-pair formation system is primarly responsible for the promiscuous transfer of the plasmids between E. coli and Streptomyces. Dynamic of conjugative transfer from E. coli to S. lividans was investigated and exconjugants starting from the first hour of mating were obtained.     

Construction of a Tn5 derivative determining resistance to gentamicin and spectinomycin using a fragment cloned from R1033

A physical and genetic map of the IncP plasmid R1033 was constructed: restriction fragments were subcloned and antibiotic resistance genes were located. The map is consistent with previous reports that R1033 is a derivative of RP4 carrying a 16-kb transposon Tn1696 which contains the antibiotic-resistance determinants present on R1033 but not on RP4. A BamHI fragment from R1033, determining resistance to gentamicin, spectinomycin and streptomycin, was cloned into Tn5, replacing the central Bg/II fragment that determined kanamycin resistance, producing a recombinant transposon Tn5-GmSpSm. This was shown to transpose in Rhizobium leguminosarum at a frequency similar to that of the parental Tn5.     

Analysis of chromosome mobilization using hybrids between plasmid RP4 and a fragment of bacteriophage lambda carrying IS1

In vitro recombination was used to generate RP4 plasmids with an inserted restriction fragment of bateriophage λ. In some cases the λ DNA also carried the insertion sequence IS1. Comparisons were made between the abilities of these plasmids to mobilize the Escherichia coli K-12 chromosome in different genetic backgrounds. RP4-borne IS1 acting alone promoted chromosome transfer but with an efficiency 1% of that resulting from more extensive plasmid-chromosome homology. A recA mutation in the donor depressed the mobilization frequency below the level of detection. Correlation of the direction of chromosome transfer and the orientation of the cloned λ DNA allowed the direction of RP4 transfer to be determined. Studies on recombinants showed that in general they also acquired an intact, autonomous plasmid, suggesting the process of mobilization by RP4 may differ in certain features from chromosome transfer by F.     

Analysis of chromosome mobilization using hybrids between plasmid RP4 and a fragment of bacteriophage λ carrying IS1

In vitro recombination was used to generate RP4 plasmids with an inserted restriction fragment of bateriophage λ. In some cases the λ DNA also carried the insertion sequence IS1. Comparisons were made between the abilities of these plasmids to mobilize the Escherichia coli K-12 chromosome in different genetic backgrounds. RP4-borne IS1 acting alone promoted chromosome transfer but with an efficiency 1% of that resulting from more extensive plasmid-chromosome homology. A recA mutation in the donor depressed the mobilization frequency below the level of detection. Correlation of the direction of chromosome transfer and the orientation of the cloned λ DNA allowed the direction of RP4 transfer to be determined. Studies on recombinants showed that in general they also acquired an intact, autonomous plasmid, suggesting the process of mobilization by RP4 may differ in certain features from chromosome transfer by F.     

Genetic basis of non-transposition of Tn5 in Pseudomonas aeruginosa following mobilisation of RP4 Mob::Tn5 from Escherichia coli

Abstract A Tn 5 transposon mutagenesis system based on mobilization of the narrow-host-range plasmid pACYC184 from Escherichia coli by a chromosomally integrated promiscuous plasmid RP4 was found to be non-applicable to Pseudomonas aeruginosa recipients. Transposition following mobilization was based on cloning an RP4 DNA fragment (/ RP4 Mob) into pACYC184 and Tn 5 transposition into the fragment (/ RP4 Mob::Tn5). It was shown by DNA sub-cloning of RP4 Mob::Tn 5 on to a wide-host-range plasmid vector that mobilization was unaffected but that reduced survival of the vector or host following mobilization was responsible. However, mutagenesis was achieved by the provision of cloned RP4 Mob DNA in the P. aeruginosa recipients.     

美洲商陆核基因组抗病毒蛋白基因的克隆及序列分析

通过ＰＣＲ扩增,从美洲商陆（Ｐｈｙｔｏｌａｃａａｍｅｒｉｃａｎａ）核基因组中克隆了商陆抗病毒蛋白基因,序列分析表明,该基因含８８５个核苷酸,与已报道的序列比较,核苷酸同源性为９９．３％。     

Restriction and electron microscopic analyses of deletion derivatives thermosensitive with respect to maintaining plasmid pEG1

Temperature-independent deletion mutants of temperature-sensitive in self-maintenance plasmid pEG-1, derived from R-factor RP4, are mapped using the restriction endonucleases PstI, SmaI, EcoRI, BamHI and the heteroduplex analysis. The pEG1 derivatives under study are found to have deletions in the area of ampicillin transposon Tn1 and nearby genes. The left and the right ends of these deletions boundaries are localized between 37.5 MD and 7.6 MD in the RP4 map. Thus far, the area of plasmid RP4 (37.5-7.6 MD) with Tn1 and, presumably, inc gene(s) in it does not have any genes needed for stable maintenance of R-factor. A conclusion is made that the gene RP4, which carries the mutation determining thermosensitive character of pEG1 maintenance and of inhibition of the cell growth, is localized between the EcoRI site and transposon Tn1 at a distance less than 1.0 MD from the latter.     

Polymorphic proteins encoded within BZLF1 of defective and standard Epstein-Barr viruses disrupt latency.

These experiments identify an Epstein-Barr virus-encoded gene product, called ZEBRA (BamHI fragment Z Epstein-Barr replication activator) protein, which activates a switch between the latent and replicative life cycle of the virus. Our previous work had shown that the 2.7-kilobase-pair WZhet piece of rearranged Epstein-Barr virus DNA from a defective virus activated replication when introduced into cells with a latent genome, but it was not clear whether a protein product was required for the phenomenon. We now use deletional, site-directed, and chimeric mutagenesis, together with gene transfer, to show that a 43-kilodalton protein, encoded in the BZLF1 open reading frame of het DNA, is responsible for this process. The rearrangement in defective DNA does not contribute to the structural gene for the protein. Similar proteins with variable electrophoretic mobility (37 to 39 kilodaltons) were encoded by BamHI Z fragments from standard, nondefective Epstein-Barr virus genomes. Plasmids expressing the ZEBRA proteins from B95-8 and HR-1 viruses were less efficient at activating replication in D98/HR-1 cells than those which contained the ZEBRA gene from the defective virus. It is not yet known whether these functional differences are due to variations in expression of the plasmids or to intrinsic differences in the activity of these polymorphic polypeptides.     

新城疫病毒F蛋白在昆虫细胞中的表达及其融细胞作用

用RT-PCR方法扩增出新城疫病毒标准强毒株F48E8的F基因,并将其克隆到pGEM-T载体,命名为pGEM-NDF。鉴定正确后,以BamHI和XbaI双酶切将F基因从pGEM-NDF中释放出来,并插入到pFastBacI载体中,得到重组转移载体pFast-NDF。然后将该重组质粒转入含有穿梭质粒的感受态DH10Bac中,通过转座作用获得重组穿梭质粒reBacmid-NDF。再用reBacmid-NDF转染Sf9昆虫细胞,获得含有新城疫病毒F48E8株F基因的重组杆状病毒。间接免疫荧光和Western-blot分析结果表明F蛋白在昆虫细胞中获得表达,而且主要表达于细胞膜上,并使感染重组杆状病毒的昆虫细胞在96h发生融合作用。动物试验表明,表达的F蛋白能够产生中和抗体。本文的研究结果为F蛋白的进一步开发奠定了基础。