Molecular cloning and identification of the laspartomycin biosynthetic gene cluster from Streptomyces viridochromogenes

The biosynthetic gene cluster for laspartomycins, a family of 11 amino acid peptide antibiotics, has been cloned and sequenced from Streptomyces viridochromogenes ATCC 29814. Annotation of a segment of 88912bp of S. viridochromogenes genomic sequence revealed the putative lpm cluster and its flanking regions which harbor 43 open reading frames. The lpm cluster, which spans approximately 60 kb, consists of 21 open reading frames. Those include four NRPS genes (lpmA/orf18, lpmB/orf25, lpmC/orf26 and lpmD/orf27), four genes (orfs 21, 22, 24 and 29) involved in the lipid tail biosynthesis and attachment, four regulatory genes (orfs 13, 19, 32 and 33) and three putative exporters or self-resistance genes (orfs 14, 20 and 30). In addition, the gene involved in the biosynthesis of the nonproteinogenic amino acid Pip was also identified in the lpm cluster while the genes necessary for the biosynthesis of the rare residue diaminopropionic acid (Dap) were found to reside elsewhere on the chromosome. Interestingly, the dabA, dabB and dabC genes predicted to code for the biosynthesis of the unusual amino acid diaminobutyric acid (Dab) are organized into the lpm cluster even though the Dab residue was not found in the laspartomycins. Disruption of the NRPS lpmC gene completely abolished laspartomycin production in the corresponding mutant strain. These findings will allow molecular engineering and combinatorial biosynthesis approaches to expand the structural diversity of the amphomycin-group peptide antibiotics including the laspartomycins and friulimicins.     

柞蚕核型多角体病毒PstI-B、C片段克隆及序列分析

克隆并测序分析了柞蚕核型多角体病毒(Antheraea pernyinucleopolyhedrovirus,ApNPV)PstI-B和C片段。结果表明:ApNPVPstI-B片段长7406bp,编码7个开放阅读框(open readingframes,orf),包括p87、he65、pnk/pnl、odv-ec43基因及黄杉毒蛾核型多角体病毒(Orgyiapseudotsugatamulticapsid nucleopolyhedrovirus,OpMNPV)orf107、orf108同源区。ApNPVPstⅠ-C长6663bp,编码11个orf,包括pk-1、orf1629、polh、lef-2、ptp-2、ctl-1、ptp-1基因及OpMNPVorf5、orf7、orf8和orf11同源区。在鉴定的18个杆状病毒基因中,he65和orf1629基因分化较大;polh和lef-2基因较保守。ApNPV是已知的第3个编码pnk/pnl基因、第4个同时编码ptp-1和ptp-2两个基因的杆状病毒。     

Marchantia polymorpha mitochondrial orf identifies transcribed sequence in angiosperm mitochondrial genome

Heterologous hybridizations performed using nine Marchantia polymorpha mitochondrial orfs and the sdh4 gene against angiosperm mtDNA suggested that three of them and the sdh4 gene have been conserved in the mitochondrial genome of different angiosperm species. Solanum tuberosum mtDNA fragments, which hybridized to M. polymorpha orf207 and sdh4 gene, were cloned, sequenced, and their expressions evaluated by Northern and RT-PCR. Hybridizing fragments to sdh4 gene and orf207 from potato mtDNA were shown to be transcribed, but only in the case of sdh4 gene was there homology between the protein encoded by the DNA sequence from M. polymorpha and the potato mitochondrial genome. M. polymorpha orf207 showed little similarity to an open reading frame from potato mtDNA, named here orf78. The putative proteins encoded by both orf207 and orf78 were not related, indicating that these orfs do not constitute homologous sequences.     

柞蚕核型多角体病毒PstⅠ-B、C片断克隆及序列分析

本研究克隆并测序分析了柞蚕核型多角体病毒(Antheraea pernyi nucleopolyhedrovirus,ApNPV) PstⅠ-B和C片断。结果表明：ApNPV PstⅠ-B片断长7406 bp,编码7个开放阅读框(open reading frames, orf),包括p87、he65、pnk/pnl、odv-ec43基因及黄杉毒蛾核型多角体病毒病毒(Orgyia pseudotsugata multicapsid nucleopolyhedrovirus,OpMNPV) orf107、orf108同源区。ApNPV PstⅠ-C长6663 bp,编码11个orf,包括pk-1、orf1629、polh、lef-2、ptp-2、ctl-1、ptp-1及OpMNPV orf5、orf7、orf8和orf11同源区。ApNPV是已知的第三个编码pnk/pnl、第四个同时编码ptp-1和 ptp-2基因的杆状病毒。     

A novel gene, DSCR5, from the distal Down syndrome critical region on chromosome 21q22.2.

Based on a detailed sequence of the distal Down syndrome critical region (DSCR), we predicted and molecularly cloned a novel gene, designated DSCR5. We determined the sequences of expressed sequence tags (ESTs) that almost matched the predicted cDNA sequence of DSCR5. Northern blot analysis showed that DSCR5 is expressed in several tissues including the liver, skeletal muscle, heart, pancreas and testis. To determine the 5'-end of DSCR5, the oligo-capping method was employed. Combining the EST sequence data and that from the oligo-capping experiments, we obtained the full-length cDNA sequence of DSCR5. DSCR5 had at least four types of alternatively spliced variants. According to the number of exons, they could be classified into two subtypes: DSCR5alpha and DSCR5beta. DSCR5alpha includes three splice variant subtypes, DSCR5alpha1, alpha2 and alpha3, which each has different first non-coding exon. In addition, the most abundantly isolated form, DSCR5alpha1, shows microheterogeneity of the mRNA start site. Comparison of the sequences between the predicted cDNA and the molecularly cloned cDNA revealed that the computer programs had limited validity to correctly predict the terminal exons. Thus, molecular cloning should always be required to complement the inadequacy of the computer predictions.     

A tandemly-oriented late gene cluster within the vaccinia virus genome.

The nucleotide sequence of a 5.1 kilobase-pair fragment from the central portion of the vaccinia virus genome has been determined. Within this region, five complete and two incomplete open reading frames (orfs) are tightly-clustered, tandemly-oriented, and read in the leftward direction. Late mRNA start sites for the five complete orfs and one incomplete orf were determined by S1 nuclease mapping. The two leftmost complete orfs correlated with late polypeptides of 65,000 and 32,000 molecular weight previously mapped to this region. When compared with each other and with sequences present in protein data banks, the five complete orfs showed no significant homology matches amongst themselves or any previously reported sequence. The six putative promoters were aligned with three previously sequenced late gene promoters. While all of the nine are A-T rich, the only apparent consensus sequence is TAA immediately preceeding the initiator ATG. Identification of this tandemly-oriented late gene cluster suggests local organization of the viral genome.     

Characterization of a phage resistance plasmid, pLKS, of silage-making Lactobacillus plantarum NGRI0101

Phage contamination has resulted in abnormal fermentation in silage. We isolated a phage-resistant strain, Lactobacillus plantarum NGRI0101 from silage. The strain carried two plasmids, pLKL (6.8 kb) and pLKS (2.0 kb). By curing and retransformation of the plasmids, we clarified that pLKS has phage resistant activity, characterized as no adsorption inhibition. pLKS has 2,025 bp and three orfs, orfl23, orf132, and orf918. The predicted amino acid sequence of the orf918 product showed high similarity to those of Rep proteins of Pediococcus halophilus plasmid pUCL287 and Lactobacillus acidophilus plasmid pLA103. The replication origin (ori) was upstream from orf918. There was no gene similar to typical phage resistant genes encoded by known plasmids. The phage resistance of L. plantarum NGRI0101 may possibly be due to a plasmid-encoded abortive infection.