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During screening for antigenic proteins in Burkholderia pseudomallei, a novel insertion sequence, IS Bp1, was found by sequence similarity searches. IS Bp1 contains two overlapping ORFs of 261 bp ( orfA) and 852 bp ( orfB), encoding 87 and 284 amino acid residues, respectively, and an imperfect inverted repeat. The putative protein encoded by orfA (OrfA) is similar to the OrfA in insertion sequences of the IS 3 family in other bacteria, showing 49% and 76% amino acid identity and similarity, respectively, with the transposase encoded by IS D1 of Desulfovibrio vulgaris vulgaris. The putative protein encoded by orfB (OrfB) is similar to the OrfB in insertion sequences of the IS 3 family in other bacteria, showing 43% and 62% amino acid identity and similarity, respectively, with the transposase encoded by IS 1222 of Enterobacter agglomerans. Sequence analysis of OrfA showed the presence of an alpha-helix-turn-alpha-helix motif, as well as the putative leucine zipper at its 3' end, for possible DNA binding to the terminal inverted repeats. Sequence analysis of OrfB showed the presence of a DDE motif of aspartic acid, aspartic acid, and glutamic acid, a highly conserved motif present in OrfB of other members of the IS 3 family. Furthermore, several other conserved amino acid residues, including the arginine residue located seven amino acids downstream from the glutamic acid residue, were observed. PCR amplification of the IS Bp1 gene showed a specific band in 65% of the 26 B. pseudomallei strains tested. Southern blot hybridization after XhoI or SacI digestion showed nine different patterns of hybridization. The number of copies of IS Bp1 in those strains that possessed the insertion sequence ranged from three to 12. Using several insertion sequences and a combination of insertion-sequence-based and non-insertion-sequence-based methods such as ribotyping will probably increase the discriminatory power of molecular typing in B. pseudomallei. 相似文献
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大眼狮鲈鱼皮肤肿瘤(WDS)在秋季发生春季萎缩的季节性生长特点与大眼狮鲈鱼皮肤肿瘤病毒 (WDSV) 辅助基因的表达有关. 其中辅助基因orfA的表达产物OrfA蛋白可能是1个潜在的肿瘤相关因子, OrfA与细胞周期蛋白cyclin A和cyclin D同源,也被称为逆转录病毒周期蛋白(retroviral-cyclin).为了深入研究OrfA的作用和功能,我们构建了诱饵表达载体pGBKT7-orfA,采用酵母双杂交技术,从HeLa cDNA文库中筛选与其相互的蛋白因子. 通过2轮高强度压力的筛选、β-半乳糖苷酶活性验证、回转验证以及测序分析,最终获得了1个与OrfA相互作用的转录因子E2F5. OrfA C端78个氨基酸的多肽或者缺失C端78个氨基酸的OrfA突变体均不能在酵母双杂交系统中与E2F5相互作用. 这些发现为进一步研究OrfA在肿瘤萎缩中的功能奠定了基础. 相似文献
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伯克霍尔德氏菌(Burkholderiasp.)JT1500对2-萘酸(2-naphthoate)生物降解的关键步骤之一是通过2-萘酸加单氧酶羟化2-萘酸生成1-羟基-2-萘酸(1-hydroxy-2-naphthoate)。在已确定2-萘酸加单氧酶基因及其功能的基础上对含有该基因的一个4.8kb长度的基因簇进行了克隆测序。该序列上含有4个可能的阅读框orfB、orfC、orfD、orfA。序列比对发现,orfA序列与JaponicumUSDA110和RalstoniaeutrophaHF39中的加单氧酶基因同源性较高,orfB序列与BordetllapertussisTohamaI、RalstoniasolanacearumGMI1000和BordetellabronchisepticaRB50等菌中的黄素还原酶基因有一定的同源性。酶活分析发现只含基因orfA的重组大肠杆菌SA细胞提取液有很低的加氧活性,含基因orfB的重组子SB细胞提取液没有加氧活性,但在反应体系中同时加入SA和SB的细胞提取液后,其加氧活性显著增强,包含片段orfB orfA的重组子SB A在黄素(FMN、FAD)存在的情况下也表现出很强的加氧活性;在厌氧条件下,能检测出SB细胞提取液的黄素还原活性。基于以上信息,认为2-萘酸加单氧酶基因簇含有两个重要的组分黄素还原酶基因(nmoB)和加单氧酶基因(nmoA)。2-萘酸加单氧酶Nmo羟化2-萘酸的过程为先由黄素还原酶(NmoB)在NADH存在的条件下将黄素(FMN、FAD)还原为还原型黄素(FMNH2、FADH2),然后加单氧酶(NmoA)利用还原型黄素和O2羟化底物2-萘酸,生成1-羟基-2-萘酸。NmoB是NmoA的偶联蛋白。 相似文献
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Functional organization and insertion specificity of IS607, a chimeric element of Helicobacter pylori 总被引:4,自引:0,他引:4
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Kersulyte D Mukhopadhyay AK Shirai M Nakazawa T Berg DE 《Journal of bacteriology》2000,182(19):5300-5308
A search by subtractive hybridization for sequences present in only certain strains of Helicobacter pylori led to the discovery of a 2-kb transposable element to be called IS607, which further PCR and hybridization tests indicated was present in about one-fifth of H. pylori strains worldwide. IS607 contained two open reading frames (ORFs) of possibly different phylogenetic origin. One ORF (orfB) exhibited protein-level homology to one of two putative transposase genes found in several other chimeric elements including IS605 (also of H. pylori) and IS1535 (of Mycobacterium tuberculosis). The second IS607 gene (orfA) was unrelated to the second gene of IS605 and might possibly be chimeric itself: it exhibited protein-level homology to merR bacterial regulatory genes in the first approximately 50 codons and homology to the second gene of IS1535 (annotated as "resolvase," apparently due to a weak short recombinase motif) in the remaining three-fourths of its length. IS607 was found to transpose in Escherichia coli, and analyses of sequences of IS607-target DNA junctions in H. pylori and E. coli indicated that it inserted either next to or between adjacent GG nucleotides, and generated either a 2-bp or a 0-bp target sequence duplication, respectively. Mutational tests showed that its transposition in E. coli required orfA but not orfB, suggesting that OrfA protein may represent a new, previously unrecognized, family of bacterial transposases. 相似文献
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PCR analysis of the pandemic strain of Vibrio parahaemolyticus, KX-V237 (total genome sequenced) showed a subculture where the size of the amplicons had increased. The purpose of this study was to analyze the mechanism of this change. We found a 1,243-bp DNA sequence inserted in one of the pandemic marker genes in this strain. The inserted DNA sequence possessed the genetic structures shared by insertion sequences (ISs) of the IS3 family. This IS had 26-bp imperfect terminal inverted repeats (IRs) and two partially overlapping reading frames, orfA and orfB. OrfA codes for a helix-turn-helix, OrfA and OrfAB produced by translational frameshifting code for leucine zipper motifs, and OrfB codes for a DDE motif. orfA and orfB were homologous to those in the IS3 family. This IS was named ISVpa2. Southern blot analysis showed the copy number of ISVpa2 in our stock culture and its subculture of KX-V237 was three and four, respectively; whereas it was only one in the reported genome sequence. Analysis of the flanking sequences for seven ISVpa2 copies showed ISVpa2 is capable of inserting at multiple sites and ISVpa2 causes genetic rearrangements including insertional inactivation of the target gene and adjacent deletion. ISVpa2 created 3-base duplications upon insertion. PCR, hybridization, and nucleotide sequence analyses showed ISVpa2 homologs were detected in all of the 62 other strains of V. parahaemolyticus examined; and in some strains of Vibrio vulnificus (98% identity), Vibrio penaeicida (86% identity), and Vibrio splendidus (87% identity); but was not in 25 other species in the genus Vibrio. The data demonstrate that ISVpa2 is a transpositionally active IS discovered for the first time in V. parahaemolyticus and suggest that ISVpa2 may be transferred among the species of the genus Vibrio. 相似文献
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An in vitro system has been developed which supports efficient integration of transposon circles derived from the bacterial insertion sequence IS911. Using relatively pure preparations of IS911-encoded proteins it has been demonstrated that integration into a suitable target required both the transposase, OrfAB, a fusion protein produced by translational frameshifting between two consecutive open reading frames, orfA and orfB, and OrfA, a protein synthesized independently from the upstream orfA. Intermolecular reaction products were identified in which one or both transposon ends were used. The reaction also generated various intramolecular transposition products including adjacent deletions and inversions. The circle junction, composed of abutted left and right IS ends, retained efficient integration activity when carried on a linear donor molecule, demonstrating that supercoiling in the donor molecule is not necessary for the reaction. Both two-ended integration and a lower level of single-ended insertions were observed under these conditions. The frequency of these events depended on the spacing between the transposon ends. Two-ended insertion was most efficient with a natural spacing of 3 bp. These results demonstrate that transposon circles can act as intermediates in IS911 transposition and provide evidence for collaboration between the two major IS911-encoded proteins, OrfA and OrfAB. 相似文献
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Transposase-induced excision and circularization of the bacterial insertion sequence IS911. 总被引:4,自引:0,他引:4
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We have investigated the role of three IS911-specified proteins in transposition in vivo: the products of the upstream (OrfA) and downstream (OrfB) open reading frames, and a transframe protein (OrfAB) produced by -1 translational frameshifting between orfA and orfB. The production of OrfAB alone is shown to lead both to excision and to circularization of the element and to be sufficient for intermolecular transposition into a plasmid target. Simultaneous and independent production of OrfA is shown to stimulate OrfAB-mediated intermolecular transposition while greatly reducing the appearance of transposon circles. We have not been able to detect a role for OrfB. Although under certain conditions, the vector plasmid undergoes precise resealing after IS911 excision, the data suggest that this is not normally the case and that the donor plasmid is not generally conserved. The use of IS911 derivatives carrying mutations in the terminal 2 bp suggested that circle formation represents a site-specific intramolecular transposition event. We present a model which explains both intra- and intermolecular transposition events in terms of a single reaction mechanism of the 'cut and paste' type. 相似文献
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Programmed translational frameshifting and initiation at an AUU codon in gene expression of bacterial insertion sequence IS911. 总被引:9,自引:0,他引:9
The proteins expressed by insertion sequence IS911, a member of the widespread IS3 family of elements, have been analyzed. The results indicate that three major species are produced from two consecutive reading frames. A protein of Mr 11,500, ORFA, is synthesized from an upstream reading frame. A larger protein, ORFAB, uses the same initiation codon and is produced by a -1 programmed translational frameshift between orfA and a downstream frame, orfB, whose amino acid sequence shows significant homology with retroviral integrase proteins. The orfB frame is also expressed independently in two alternative forms: the first uses a rare AUU initiation codon in the orfB phase whereas the second appears to initiate in the orfA phase and is produced by a -1 frameshift mechanism similar to that used in ORFAB expression. A specific IS911 integration reaction using a minimal active junction composed of 51 base-pairs of the right inverted repeat and a flanking phase lambda sequence resembling a second end in inverted orientation has been developed to analyze the functions of these proteins by transcomplementation in vivo. The orfA and orfB frames are shown to be essential and production of ORFAB is shown to stimulate integration in this system, suggesting that this fusion protein is the IS911 transposase. 相似文献
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IS629 is 1,310 bp in length with a pair of 25-bp imperfect inverted repeats at its termini. Two partially overlapping open reading frames, orfA and orfB, are present in IS629, and two putative translational frameshift signals, TTTTG (T4G) and AAAAT (A4T), are located near the 3'-end of orfA. With the lacZ gene as the reporter, both T4G and A4T motifs are determined to be a -1 frameshift signal. Two peptides representing the two transframe products designated OrfAB' and OrfAB, are identified by a liquid chromatography-tandem mass spectrometric approach. Results of transposition assays show that OrfAB' is the transposase and that OrfAB aids in the transposition of IS629. Pulse-chase experiments and Escherichia coli two-hybrid assays demonstrate that OrfAB binds to and stabilizes OrfAB', thus increasing the transposition activity of IS629. This is the first transposable element in the IS3 family shown to have two functional frameshifted products involved in transposition and to use a transframe product to regulate transposition. 相似文献
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Kalia A Mukhopadhyay AK Dailide G Ito Y Azuma T Wong BC Berg DE 《Journal of bacteriology》2004,186(22):7508-7520
Prokaryotic insertion sequence (IS) elements behave like parasites in terms of their ability to invade and proliferate in microbial gene pools and like symbionts when they coevolve with their bacterial hosts. Here we investigated the evolutionary history of IS605 and IS607 of Helicobacter pylori, a genetically diverse gastric pathogen. These elements contain unrelated transposase genes (orfA) and also a homolog of the Salmonella virulence gene gipA (orfB). A total of 488 East Asian, Indian, Peruvian, and Spanish isolates were screened, and 18 and 14% of them harbored IS605 and IS607, respectively. IS605 nucleotide sequence analysis (n = 42) revealed geographic subdivisions similar to those of H. pylori; the geographic subdivision was blurred, however, due in part to homologous recombination, as indicated by split decomposition and homoplasy tests (homoplasy ratio, 0.56). In contrast, the IS607 populations (n = 44) showed strong geographic subdivisions with less homologous recombination (homoplasy ratio, 0.2). Diversifying selection (ratio of nonsynonymous change to synonymous change, >1) was evident in approximately 15% of the IS605 orfA codons analyzed but not in the IS607 orfA codons. Diversifying selection was also evident in approximately 2% of the IS605 orfB and approximately 10% of the IS607 orfB codons analyzed. We suggest that the evolution of these elements reflects selection for optimal transposition activity in the case of IS605 orfA and for interactions between the OrfB proteins and other cellular constituents that potentially contribute to bacterial fitness. Taken together, similarities in IS elements and H. pylori population genetic structures and evidence of adaptive evolution in IS elements suggest that there is coevolution between these elements and their bacterial hosts. 相似文献
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S Q Cheng R E Musso R Liu D M Niemeyer J J Shaw F K McCleskey V G DelVecchio 《Plasmid》1999,42(1):42-44
We have cloned and identified an insertion sequence, IS1485, that was present in several members of the genus Enterococcus. IS1485 exists in varying copy numbers with at least 12 copies in E. durans (ATCC 11576), 3 copies in E. faecium (ATCC 19434), and one copy each in E. faecalis (ATCC 19433) and E. avium (ATCC 14024). It was also detected in clinical strains of E. gallinarum, E. casseliflavus, and E. saccharolyticus. IS1485 is 1366 bp in length, it has imperfect terminal inverted repeats with 25 of the terminal 39 residues matched, and it contains three open reading frames exceeding 50 codons, designated orfA, orfB, and orfC. The largest, orfB, was located 36 bp downstream and in the -1 reading frame relative to orfA; orfC is oriented in the opposite direction and overlaps orfA. The genetic organization of IS1485 resembles that of members of the IS3 family of transposable elements. Sequence homology exists with several members of the IS3 family especially with IS199 from Streptococcus mutans. 相似文献
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A full-length cDNA clone encoding osteocalcin from the bullfrog, Rana catesbeiana (bone Gla-protein, BGP) has been isolated, and the complete coding sequence for the 100-amino-acid pre-pro-osteocalcin protein was determined. The amino acid sequence of Rana catesbeiana osteocalcin, especially the mature 49-amino acid sequence, is closer to the mammalian than to the fish, Sparus osteocalcin. Rana mature osteocalcin has a similarity of 67% with human or 59% with rat osteocalcin, and only 42% with fish mature osteocalcin. The 51-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Arg-Arg sequence preceding the NH2-terminal Ser of the mature 49-amino-acid Rana osteocalcin. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, which targets vitamin K-dependent gamma-carboxylation of three specific Glu residues at positions 17, 21, and 24 in the mature protein. At the native protein expression levels, extraction from Rana cortical bone in the presence of protease inhibitor cocktail resulted in the isolation of two distinct forms of osteocalcin, P-1 and P-2, with a 3:2 distribution. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and amino acid sequence analysis of the N-terminal domain, we confirmed that P-1 is the intact 49-residue osteocalcin with N-terminal SNLRNAVFG., and that P-2 lacks four amino acids from the N-terminus, (NAVFG.). These results demonstrate the existence of a form of osteocalcin lacking four N-terminal amino acids in Rana bone, and that mature Rana osteocalcins remained highly conserved in their molecular evolution, especially with respect to the conservation of the C-terminal domain (residues 14-49). 相似文献
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Naka T Nakata N Maeda S Yamamoto R Doe M Mizuno S Niki M Kobayashi K Ogura H Makino M Fujiwara N 《Journal of bacteriology》2011,193(20):5766-5774
The Mycobacterium avium-M. intracellulare complex (MAIC) is divided into 28 serotypes by a species-specific glycopeptidolipid (GPL). Previously, we clarified the structures of serotype 7 GPL and two methyltransferase genes (orfA and orfB) in serotype 12 GPL. This study elucidated the chemical structure, biosynthesis gene, and host innate immune response of serotype 13 GPL. The oligosaccharide (OSE) structure of serotype 13 GPL was determined to be 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-β-hexose-(1 → 3)-4-O-methyl-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 2)-α-L-6-deoxy-talose by using chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) analyses. The structure of the serotype 13 GPL was different from those of serotype 7 and 12 GPLs only in O-methylations. We found a relationship between the structure and biosynthesis gene cluster. M. intracellulare serotypes 12 and 13 have a 1.95-kb orfA-orfB gene responsible for 3-O-methylation at the terminal hexose, orfB, and 4-O-methylation at the rhamnose next to the terminal hexose, orfA. The serotype 13 orfB had a nonfunctional one-base missense mutation that modifies serotype 12 GPL to serotype 13 GPL. Moreover, the native serotype 13 GPL was multiacetylated and recognized via Toll-like receptor 2. The findings presented here imply that serotypes 7, 12, and 13 are phylogenetically related and confirm that acetylation of the GPL is necessary for host recognition. This study will promote better understanding of the structure-function relationships of GPLs and may open a new avenue for the prevention of MAIC infections. 相似文献