Nucleotide sequence of the virG locus of the Agrobacterium tumefaciens plasmid pTiC58

The nucleotide sequence of the virG locus of the nopaline type plasmid pTiC58 of Agrobacterium tumefaciens has been determined. It contains an open reading frame (ORF) of 759 nucleotides and has 77% homology to the virG sequences of octopine type plasmids. Differences between the sequences of the two types of Ti plasmids in the region of virG are located predominantly outside the ORF. The amino acid sequences inferred from the two virG genes show 80% homology to each other and each shows the same moderate homologies to amino acid sequences derived from genes in a family of two-component regulatory systems. Specific differences in nucleotide and amino acid sequences as well as a structure-function model for the gene product are discussed.     

Characterization of the virB operon of an Agrobacterium tumefaciens Ti plasmid: nucleotide sequence and protein analysis

The virulence regulon of the Agrobacterium tumefaciens TiC58 plasmid is composed of six operons, virA, virB, virG, virC, virD and virE, which direct the transfer of T-DNA into plant cells. The 9.5 kbp virB operon is the largest of these operons and its entire nucleotide sequence was determined and found to contain eleven open reading frames (ORFs). Gene fusions of each VirB ORF to T7 phi 10 were made and overexpressed in Escherichia coli to confirm that they encode proteins of predicted size. Hydrophobic analysis of these peptide sequences revealed nine proteins that contain hydrophobic spanning regions including signal-peptide-like sequences. These data suggest that the majority of VirB proteins may associate with bacterial cell membranes, while the two additional proteins possess a potential ATP-binding site. Strong homologies in amino acid sequences were observed between nopaline- and octopine-type plasmids. Specific differences in amino acid sequence encoded by VirB ORFs of nopaline and octopine Ti plasmid and a functional role of the gene products are discussed.     

Homologies between herpesvirus of turkey and Marek's disease virus type-1 DNAs within two co-linearly arranged open reading frames, one encoding glycoprotein A

The genomes of two avian herpesviruses, Marek's disease virus type 1 (MDV1) and herpesvirus of turkey (HVT), share close homology only within certain DNA regions. One such homologous region of HVT DNA was cloned and sequenced. Two open reading frames (ORFs) were found in the long unique region, ORF1 encoding the glycoprotein A (gA), and ORF2 encoding a still unidentified protein. These two HVT-ORFs are located at almost the same positions as the homologous MDV1-ORFs. The nucleotide sequence homologies between HVT and MDV1 were 73% and 68% for ORF1 and ORF2, respectively. Both the 5'- and 3'-noncoding regions, however, are less conserved. The third letter within every codon of ORF1 and ORF2 showed a mismatch of greater than 50% between the two viruses. The amino acid (aa) sequence homologies between the corresponding putative viral proteins are 83% and 80% for ORF1 (gA) and ORF2, respectively. More than 90% homology was observed in the C-terminal region of ORF1 (gA). Furthermore, the deduced aa sequences for both of the ORFs in these two viruses showed considerable homology to two adjoining genes in herpes simplex virus type 1, the glycoprotein C and UL45 genes.     

Comparative analysis of the plasmids from two isolates of "Candidatus Phytoplasma australiense"

Two plasmids from the plant-pathogenic mollicute "Candidatus Phytoplasma australiense" were completely sequenced from two isolates derived from different plant hosts. Plasmid pPAPh2 (3607bp) was obtained from Phormium showing Phormium yellow leaf symptoms and pPASb11 (3635bp) from strawberry showing strawberry lethal yellows symptoms. The plasmids varied in their copy number and nucleotide sequence yet contained the same four open reading frames (ORFs). The deduced amino acid sequence derived from ORF1 shares similarity with hypothetical proteins encoded on the plasmids from onion yellows and beet leafhopper-transmitted virescence agent phytoplasmas. The deduced amino acid sequences of both ORF2 and ORF3 share similarity with functionally unknown proteins on the chromosome of onion yellows phytoplasma. An ORF with a similar sequence to ORF2 is also present on the chromosome of "Ca. P. australiense." The deduced amino acid sequence derived from ORF4 is most similar to replication proteins encoded by other phytoplasma plasmids and by geminiviruses, the only protein on the plasmids for which a putative function can be assigned. The identities of the deduced amino acid sequences of ORF1, ORF2, ORF3, and ORF4 between pPAPh2 and pPASb11 were 89, 68, 91, and 68%, respectively; the differences being consistent with the subgroup status of the parental phytoplasmas.     

Characterization of a small cryptic plasmid isolated from a methicillin-resistant strain of Staphylococcus aureus

The nucleotide sequence of a small (1613 bp) plasmid, pOX2000, isolated from a methicillin-resistant strain of Staphylococcus aureus has been determined. The sequence contains only one large ORF and the predicted amino acid sequence shows homology to the REP proteins of some other small staphylococcal plasmids. In addition there are two palindromic sequences, palA and palJ, that are similar to but not identical with the palindromes known from other staphylococcal plasmids to be involved in lagging strand initiation and possibly leading strand termination, respectively. Preliminary functional analysis of pOX2000 has been carried out by assessing the effect of interrupting the sequence at three unique restriction endonuclease sites. The plasmid pOX2000, and its relationship to other small staphylococcal plasmids, is discussed.     

Nucleotide sequence of diatom plasmids: identification of open reading frames with similarity to site-specific recombinases

We have determined the nucleotide sequence of two small circular DNA plasmids, pCf1 and pCf2 [22], from the marine diatom Cylindrotheca fusiformis. pCf1 is 4273 bp, and pCf2 is 4079 bp in size. In each plasmid, all of the major open reading frames (ORFs) are encoded on the same DNA strand. Two ORFs are similar, comparing the two plasmids. ORF218 (pCf1) and ORF217 (pCf2) share 80% amino acid identity and ORF482 (pCf1) and ORF484 (pCf2) share 54% amino acid identity. ORF218/217 shows significant similarity (28–31% amino acid identity) to the Tn3 class of resolvases. Resolvases are most commonly found in bacterial transposons. However, two other features found in the Tn3 class of transposon are missing in the plasmids; an ORF encoding a transposase and terminal inverted repeat sequences. This, and data mapping the portions of the plasmids that hybridize to genomic chloroplast DNA, suggest that the plasmids do not contain active transposons. By analogy with the R46 plasmid from Enterobacter [5, 6], another potential role for the resolvases encoded by pCf1 and pCf2 is the conversion of multimeric forms of the plasmid to monomers. The similarity of ORF218/217 to resolvases documents the first identification of a potential coding function in an algal plasmid.     

Complete nucleotide sequences and comparison of the structural genes of two 2-haloalkanoic acid dehalogenases from Pseudomonas sp. strain CBS3.

The nucleotide sequences of two DNA segments from Pseudomonas sp. strain CBS3 that code for two different haloalkanoic acid halidohydrolases were determined. Two open reading frames with coding capacities of 227 amino acids (corresponding to a molecular mass of 25,401 Da) and 229 amino acids (corresponding to a molecular mass of 25,683 Da) were identified as structural genes of 2-haloalkanoic acid dehalogenases I (dehCI) and II (dehCII) by comparison with the N-terminal amino acid sequences of these enzymes. Comparison of the two sequences revealed 45% homology on the DNA level and 37.5% homology on the amino acid level. No homology with other known protein or nucleotide sequences was found.     

Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene

The genes responsible for expression of type 1C fimbriae have been cloned from the uropathogenic Escherichia coli strain AD110 in the plasmid vector pACYC184. Analysis of deletion mutants from these plasmids showed that a 7-kb DNA fragment was required for biosynthesis of 1C fimbriae. Further analysis of this DNA fragment showed that four genes are present encoding proteins of 16, 18.5, 21 and 89 kDal. A DNA fragment encoding the 16-kDal fimbrial subunit has been cloned. The nucleotide sequence of the structural gene and of the C- and N-terminal flanking regions was determined. The structural gene codes for a polypeptide of 181 amino acids, including a 24-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the 1C subunit gene were compared with the sequences of the fimA gene, encoding the type 1 fimbrial subunit of E. coli K-12. The data show absolute homology at the N- and C-termini; there is less, but significant homology in the region between the N- and C-termini. Comparison of the amino acid compositions of the 1C and FimA subunit proteins with those of the F72 and PapA proteins (subunits for P-fimbriae) revealed that homology between these two sets of fimbrial subunits is also maximal at the N- and C-termini.     

Conservation of a long open reading frame in two Neurospora mitochondrial plasmids

The nucleotide sequence of a specific region of the mitochondrial plasmid from the Neurospora intermedia Varkud-lc strain was determined. Analysis of the sequence revealed the presence of a long (up to 710 amino acids) ORF. This ORF is almost identical to a previously characterized ORF in the mitochondrial plasmid from the Neurospora crassa Mauriceville-lc strain. When the ORFs from the two plasmids are compared over their entire length of 2,133 bp, only 34 nucleotide substitutions are found (greater than 98% identity). These substitutions result in only nine amino acid replacements in the protein sequences predicted from the two ORFs. Though no function can be assigned to the putative products of these ORFs, their high conservation of nucleotide and deduced amino acid sequence suggest that they are under selective pressure, presumably to preserve the function of some protein.      

Characterization and replication properties of the Zymomonas mobilis ATCC 10988 plasmids pZMO1 and pZMO2

The complete nucleotide sequences of two small cryptic Zymomonas mobilis ATCC 10988 plasmids (pZMO1 and pZMO2) were determined. The plasmids showed 67% homology to each other at their nucleotide level. Plasmid pZMO1 was 1651 bp long with 38% G + C content and contained an open reading frame (ORFZMO1) of 1044 nucleotides. ORFZMO1 is predicted to encode a polypeptide of 348 amino acids and shows a high degree of homology with gram-negative replication proteins of rolling circle replicating plasmids, which belong to the pC194/pUB110 family. Plasmid pZMO2 was found to be 1669 bp long, with a 38.5% G + C content, and it contained an ORF of 552 nucleotides (ORFZMO2) encoding a putative polypeptide of 184 amino acids. This polypeptide also shows a high degree of homology with the replication proteins of RCR plasmids of gram-negative bacteria, but only at their N-termini. The region necessary for replication of both plasmids was determined by stability tests under nonselective conditions, following cloning in pBR325 and introduction in Z. mobilis ATCC 10988 by pRK2013 assisted conjugation. Double- and single-strand origin regions were predicted by sequence analysis. Detection of single-stranded DNA in the extract of exponentially growing cells confirmed experimentally the rolling circle replication mode of at least pZMO2.     

湖南沙子岭猪SLA-DR基因克隆及生物信息学分析

为了克隆湖南沙子岭猪SLA-DRA和SLA-DRB基因, 分析SLA基因特性和抗原多态性, 评价湖南沙子岭猪在异种移植中的应用前景奠定基础。应用RT-PCR分别扩增湖南沙子岭猪SLA-DRA和SLA-DRB基因, 鉴定后克隆到PUCm-T载体并进行测序, 用NCBI中的BLAST和ExPASY中相关软件进行生物信息学分析。得到的湖南沙子岭猪SLA-DRA 和SLA-DRB 基因特异性基因片段, 大小分别为1 177 bp和909 bp。生物信息学分析发现, 所扩增的SLA-DRA 和SLA-DRB 基因片段均包含完整的开放阅读框, 分别编码252和266个氨基酸残基。将湖南沙子岭猪SLA-DRA和SLA-DRB基因在GenBank 登录, 登录号分别为EF143987和EF143988。同源性分析发现, 湖南沙子岭猪SLA-DRA 和SLA-DRB 与人类相应的DRA、DRB相比, 核苷酸序列同源性分别为83% 和83 %, 编码氨基酸同源性分别为83% 和79%。与GenBank 登录的其他猪种相比, SLA-DRA基因同源性最高可达100 %, 而SLA-DRB基因具有多态性。     

Nucleotide sequence of Marchantia polymorpha chloroplast DNA: a region possibly encoding three tRNAs and three proteins including a homologue of E. coli ribosomal protein S14.

The nucleotide sequence of a region of Marchantia polymorpha chloroplast DNA was determined. On this DNA sequence (3.38kb), three open reading frames (ORFs) and three putative tRNA genes were detected in the following order: -ORF701-tRNASer(UGA)-ORF702-tRNAGly(GCC)-initiator tRNAMet(CAU)-ORF703-. The ORF703 is composed of 100 codons in which those for lysine (15%) and arginine (11%) are abundant, and could be accounted for as a counterpart of E. coli ribosomal protein S14 since they share 45% homology in the amino acid sequences. The ORF701 appears to code for a membrane protein, showing a periodic appearance of seven clusters of hydrophobic amino acids. Although the mechanisms remain unknown, the ORF701 causes a streptomycin-sensitive phenotype in resistant mutants of E. coli. The ORFs and tRNA genes are separated from each other by extremely AT-rich spacers containing sequences of dyad symmetry. The third letter positions of the codons in the ORFs are also rich in A and T residues.     

An evolutionary relationship between the ColE5-099 and the ColE9-J plasmids revealed by nucleotide sequencing

The nucleotide sequence of a 1124 bp fragment of the ColE5-099 plasmid which encodes colicin E5 immunity, a lys gene involved in colicin release from the host cell, and the 3' end of the colicin E5 structural gene has been determined. Open reading frames corresponding to the three genes have been located by analogy with similar sequences from other E colicin plasmids. The location of these open reading frames corresponds with the position of the genes as determined by subcloning and transposon mutagenesis. The amino acid sequence of the carboxy-terminal 107 amino acid residues of the colicin E5 gene shows no homology with any other E colicin, suggesting a different mode of action in killing sensitive cells. A comparison of the nucleotide sequence of this region of the ColE5-099 plasmid with that of the equivalent region of the ColE9-J plasmid suggests a close evolutionary relationship between these two plasmids.     

Cloning and characterization of the MboII restriction-modification system

The two genes encoding the class IIS restriction-modification system MboII from Moraxella bovis were cloned separately in two compatible plasmids and expressed in E. coli RR1 delta M15. The nucleotide sequences of the MboII endonuclease (R.MboII) and methylase (M.MboII) genes were determined and the putative start codon of R.MboII was confirmed by amino acid sequence analysis. The mboIIR gene specifies a protein of 416 amino acids (MW: 48,617) while the mboIIM gene codes for a putative 260-residue polypeptide (MW: 30,077). Both genes are aligned in the same orientation. The coding region of the methylase gene ends 11 bp upstream of the start codon of the restrictase gene. Comparing the amino acid sequence of M.MboII with sequences of other N6-adenine methyltransferases reveals a significant homology to M.RsrI, M.HinfI and M.DpnA. Furthermore, M.MboII shows homology to the N4-cytosine methyltransferase BamHI.     

Yeast plasmids resembling 2 micron DNA: regional similarities and diversities at the molecular level.

The nucleotide sequence of two Zygosaccharomyces plasmids, pSB2 (5,415 base pairs), isolated from Zygosaccharomyces bailii, and pSM1 (5,416 base pairs), isolated from Zygosaccharomyces fermentati Naganishi, was determined. The predicted amino acid sequences of open reading frames among six yeast plasmids that resemble 2 microns DNA indicated regional sequence similarities among FLP proteins. Greater similarities were seen among Zygosaccharomyces plasmids (pSB2, pSB3, pSR1, and pSM1) than other combinations. A putative recognition site for the FLP enzyme of a Zygosaccharomyces plasmid also showed some conservation, especially in the 4 nucleotides flanking the central spacer region. From comparative studies of the sequences of putative genes of each plasmid, we propose an apparent phylogenetic relationship among yeast plasmids resembling 2 micron DNA. Among the Zygosaccharomyces plasmids, pSB2 and pSR1 are most closely related, since not only were the FLP enzymes of these two plasmids most closely related, but also the amino acid sequence of the putative P gene of pSR1 showed clear homology with that of open reading frame B of pSB2.     

Cloning and nucleotide sequence analysis of the chloramphenicol resistance gene on conjugative R plasmids from the fish pathogen Photobacterium damselae subsp. piscicida

Transferable resistance to various drugs was investigated in Photobacterium damselae subsp. piscicida from Japan. Drug resistances were transferred via plasmids of 100, 50, and 40 kb. Resistance to chloramphenicol (Cmr) was transferred on plasmids of all 3 sizes. The Cmr gene (cat) was cloned from the 50 kb plasmids pPDP8511 and pPDP9106 transferred from P. damselae subsp. piscicida strains isolated in different years and places in Japan. Subcloning localized the cat to within 1.5 kb HindIII-HincII (or PstI) fragments. Nucleotide sequences of the coding and flanking region of the cat were determined as 1607 bp (HindIII-HincII fragment) in pPDP8511 and 1568 bp (HindIII-PstI fragment) in pPDP9106, which corresponded with the sequence from nucleotides 40 to 1607 in pPDP8511. The nucleotide sequences identified an open reading frame (ORF) encoding 213 amino acid residues with a calculated molecular mass of about 24.8 kDa, a size consistent with the molecular mass of known cat gene products, and the ORF had maximum homology (99.5%) with a Type II CAT variant from Haemophilus influenzae.