Characterization of the terminal sequences flanking the transposon that carries the Escherichia coli enterotoxin STII gene

The Escherichia coli enterotoxin STII gene is flanked by two repeat sequences, approx. 600 bp each and 8 kb apart. This 9-kb DNA fragment has been shown to transpose as a unit and is thus considered a transposon. It is presently designated as Tn4521. In this study, the two terminal sequences of Tn4521 cloned in pPS1 were localized, isolated, and characterized. The two terminal sequences were found to be composed of IS2 sequences and were in an inverted repeat orientation. However, neither repeat contained a complete IS2. The LTR contained bp 1-722, whereas the RTR contained bp 17-536 and 969-1327, all three of the IS2 sequence.     

Isolation of a novel transposon which carries the Escherichia coli enterotoxin STII gene.

The Escherichia coli heat-stable enterotoxin STII gene in P307 is flanked by inverted repeat sequences, suggesting that the STII gene is part of a transposon. To study the transposability, a DNA fragment containing the putative STII transposon has been cloned. Results of transposition assays indicated that the STII gene can transpose from one plasmid to another. The size of the transposon has been determined to be approximately 9 kilobases. The structure and the location of the STII gene in clinical isolates of Escherichia coli have been investigated by restriction enzyme analyses. The structural genes of STII from different clinical isolates appear to be uniform in size, but the flanking sequences are heterogeneous. This result suggests that the STII genes in different isolates are not on the same transposon as observed in P307.     

Transposition of DNA fragments flanked by two inverted Tn1 sequences: translocation of the plasmid RP4::Tn1 region harboring the Tcr marker

We have demonstrated the possibility of transposition of the plasmid RP4::Tn1 fragment (21.2 kb) carrying the tetracycline resistance (Tcr) gene and flanked by two Tn1 copies. The new transposon, designated Tn1756, bears lethal genes that kill host cells. Therefore, its transposition can only be revealed in the presence of lethality-compensating helper regions of the plasmid RP4. Thus, RP4::Tn1 consists of two transposons, Tn1755 (Tn1-Kmr-Tn1) and Tn1756 (Tn1-Tcr-Tn1), sharing the Tn1 sequences. Both of these transposons are capable of recA-independent translocation to other plasmids. Therefore, transposition of DNA fragments flanked by two inverted Tn1 sequences does not depend on Tn1 orientation.     

Detection of heat-stable and heat-labile enterotoxin genes of Escherichia coli in diarrhoeic faecal samples of mithun (Bos frontalis) calves by polymerase chain reaction

Aims:  To find out the prevalence of different serogroups of Escherichia coli ( E. coli ) and to detect heat-stable (ST) and heat-labile (LT) enterotoxin genes of enterotoxigenic E. coli (ETEC) from the faeces of mithun calves with diarrhoea.
Methods and Results:  Faecal samples obtained from 65 diarrhoeic mithun calves of under 2 months of age were examined for E. coli using polymerase chain reaction (PCR). Fifty-four E. coli isolates were obtained from those samples, which belonged to 38 different serogroups. Out of 54 isolates tested by PCR, two isolates (3·70%) belonging to serogroups O26 and O55 were found to possess gene that code for ST enterotoxin and one isolate (1·85%) belonging to serogroup O125 was found to carry LT enterotoxin gene.
Conclusions:  Escherichia coli isolates from diarrhoeic mithun calves were found to possess ST and LT enterotoxin genes, which are designated as ETEC, and these isolates can be detected through PCR using specific primers.
Significance and Impact of the Study:  This study reports the isolation of ETEC possessing ST and LT enterotoxin genes for the first time and ETEC could be a cause of diarrhoea in mithun calves leading to calf mortality.     

Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257

Trimethoprim resistance mediated by the Staphylococcus aureus multi-resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789-790 bp), the outside two of which are flanked by directly repeated 8-bp target sequences. IS257 has imperfect terminal inverted repeats of 27-28 bp and encodes for a putative transposase with two potential alpha-helix-turn-alpha-helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram-negative bacteria and with ISS1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim-resistant DHFRs from Gram-negative bacteria and with chromosomally encoded DHFRs from Gram-positive and Gram-negative bacteria. 5' to dfrA is a thymidylate synthetase gene, designated thyE.     

Acquisition and maintenance of enterotoxin plasmids in wild-type strains of Escherichia coli

Enterotoxigenic strains of Escherichia coli (ETEC) may produce a heat-labile enterotoxin (LT), a heat-stable enterotoxin (ST) or both enterotoxins. Certain serogroups are represented more frequently than others in ETEC isolated from humans. The transfer of three plasmids encoding enterotoxin production (Ent) to 22 non-toxigenic E. coli strains of many different O:H serotypes was studied. The Ent plasmids encoded ST (TP276), or LT (TP277), or ST + LT (TP214), and all carried antibiotic-resistance determinants. Twenty-one recipient strains acquired TP214, 18 acquired TP277 and 14 acquired TP276. Strains of those serotypes to which ETEC in diarrhoeal studies commonly belong neither acquired nor maintained Ent plasmids with a higher frequency than strains of those serotypes to which ETEC rarely belong. The recipient strains, with one exception, all expressed ST, or LT, or ST and LT, when they had acquired the appropriate plasmid; a non-motile strain belonging to O serogroup 88 expressed LT but failed to express ST when it acquired TP214 or TP277.     

Conserved structural features are found upstream from the three co-ordinately regulated discoidin I genes of Dictyostelium discoideum

The discoidin I genes of Dictyostelium form a small, co-ordinately regulated multigene family. We have sequenced and compared the upstream regions of the DiscI-alpha, -beta and -gamma genes. For the most part the upstream regions of the three genes are non-homologous. The upstream sequences of the beta and gamma genes are exceedingly A + T-rich, while those of the alpha gene are less so. All three genes have a relatively G + C-rich region 20 to 40 base-pairs in length, found approximately 200 base-pairs 5' to the messenger RNA start site. This G + C-rich region 5' to the beta and gamma genes is flanked by short inverted repeats. Within this region, there is an 11 base-pair exact homology between the alpha and gamma genes, and a less perfect homology between these genes and the beta gene. The homology is flanked at a short distance by interspersed G and T residues. The gamma gene is greater than 90% A + T for greater than 800 base-pairs upstream. Further upstream there is a G + C-rich region that is also found inverted approximately 3.5 X 10(3) base-pairs away. The gamma and beta genes are tandemly linked, and the entire approximately 500 base-pair intergene region between the 3' end of the gamma gene and the 5' end of the beta gene is A + T-rich (approximately 90%) with the exception of the homology region 5' to the gamma gene. We demonstrate also the presence of a discoidin I pseudogene fragment having only 139 base-pairs of discoidin homology with greater than 8% mismatch. It is flanked upstream by five 39 base-pair G + C-rich repeats, and downstream by sequences that are extremely A + T-rich. We discuss the possible significance of the conserved G + C-rich structures on discoidin I gene expression.     

表达大肠杆菌K88ac-ST1-LTB融合蛋白基因工程菌株的构建

利用PCR技术,从大肠杆菌C83902质粒中扩增出K88ac基因、ST1突变基因和LTB基因,通过分离、纯化、内切酶酶切、连接和转化,构建了含K88ac-ST1-LTB融合基因表达载体的重组菌株BL21（DE3）（pXKST3LT5)。经酶切鉴定和DNA序列分析证实,构建的重组质粒pXKST3LT5中含有K88ac-ST1-LTB融合基因,且基因序列和阅读框架均正确。经ELISA检测,重组菌株表达的K88ac-ST1-LTB融合蛋白能够被ST1单抗、LTB和K88ac抗体识别。经乳鼠灌胃试验证实,表达的融合蛋白已丧失天然ST1肠毒素的活性。免疫实验结果表明,K88ac-ST1-LTB融合蛋白能够诱发小白鼠产生抗体,该抗体具有中和天然ST1肠毒素的毒性作用,表明构建的重组菌株可以作为预防仔猪黄、白痢基因工程菌苗的候选菌株。     

Isolation and characterization of six different chicken actin genes.

Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene.     

Diarrhea in neonatal piglets caused by K99+ST+ enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli strains were isolated from the feces of 34.4% of 64 diarrheaic neonatal piglets on seven farms. Of a total of 518 isolates, 86 (16.6%) were enterotoxigenic and grouped into four phenotypes: K99+ST+ (K99 pilus antigen and heat-stable enterotoxin producing, 36 strains), ST+ (37 strains), K88+LT+ (K88 pilus antigen and heat-labile enterotoxin producing, 11 strains), and K88+ST+ (2 strains). K99+ST+ and ST+ isolates showed multiple drug resistance and most of those (58.3% and 56.8%, respectively) belonged to O serogroup 101. A K99+ST+ isolate proved to be capable of inducing diarrhea and death in hysterectomy-produced colostrum-deprived piglets when orally inoculated.     

The histidine tRNA genes of yeast

Yeast has at least seven nuclear histidine tRNA genes although there is a single tRNAHis. We have sequenced three of the histidine tRNA genes. The genes have identical coding sequences and the DNA anti-codon sequence GTG corresponds to the GUG anti-codon in tRNAHis. None of the three yeast histidine tRNA genes has an intervening sequence. Two of the three genes contain repeated DNA elements in the region adjacent to the 5' end of the histidine tRNA gene. One of the elements, sigma, is 18 base pairs (bp) from the 5' end of each of these genes, sigma elements are highly conserved and flanked by 5-bp repeats. The other element, delta, is at variable distances from the tRNA gene; one is 439 bp from a histidine tRNA gene and the other is 52 bp from a histidine tRNA gene. These solo delta elements are quite divergent when compared with delta s associated with transposon yeast elements and are not flanked by 5-bp repeats.     

Bacteriophage conversion of heat-labile enterotoxin in Escherichia coli.

A temperate phage designated obeta1 (omicron beta) was mitomycin C induced and isolated from heat-labile enterotoxin (LT)-producing Escherichia coli E2631-C2. Phage obeta1 infected the nonlysogenic, nontoxigenic, mitomycin C-sensitive strain of E. coli K-12 (CSH38) and converted it to lysogeny and enterotoxigenicity. After the establishment of lysogeny, E. coli CSH38(obeta1) produced produced LT and phage particles at maximal levels following mitomycin C induction. The LT Tox+ character is carried by the temperate phage obeta1.     

产肠毒素大肠杆菌的热敏肠毒素B亚基(LT-B)和耐热肠毒素(ST)基因融合及其表达

采用基因重组技术构建了表达产肠毒素大肠杆菌(ETEC)的耐热肠毒素(ST)基因和热敏肠毒素B亚基(LT-B)基因融合抗原的疫苗候选株。将ST基因的5’端与LT-B基因的3’端连接,并置于同一阅读框。编码ST的基因是通过PCR从pSLM004质粒中扩增得到的,含有ST的pro序列(其编码ST前体的pro区域),并应用寡核苷酸定点突变技术将编码ST的第14位氨基酸残基发生突变,使ST的第14位氨基酸残基Ala突变为Leu。在所构建的结构中,于LT-B和proST之间分别插入了不同长度的氨基酸Linkers。表达的融合多肽同时具有ST和LT-B的抗原性,并保留结合GM-1神经节苷脂的能力,且无LT和ST的生物毒性。表达的融合蛋白免疫动物,能诱导产生相应的特异性抗体。     

Restriction endonuclease map of corynebacteriophage omega ctox+ isolated from the Park-Williams no. 8 strain of Corynebacterium diphtheriae.

The toxigenic corynebacteriophage omega tox+ was isolated from the hypertoxigenic Park-Williams no. 8 (PW8) strain of Corynebacterium diphtheriae and compared with the toxigenic corynebacteriophage beta tox+. The physical size and host range of both phages were found to be identical. An endonuclease restriction map of omega tox+ was constructed, and the locations of the cohesive ends (cos), phage attachment site (attP), and the diphtheria tox operon were identified. The genome of omega tox+ was found to differ from that of beta tox+ in three regions. In addition, omega tox+ was shown to be integrated into two nontandem corynebacterial phage attachment sites (attB1, attB2) in the PW8 chromosome. The differences in the restriction endonuclease digestion maps of omega tox+ and beta tox+ and the contribution of double lysogeny are discussed in relation to the hypertoxigenicity of the PW8 strain.     

Identification and cloning of the conjugative transfer region of Staphylococcus aureus plasmid pGO1.

The conjugative transfer (tra) genes of a 52-kilobase (kb) staphylococcal plasmid, pGO1, were localized by deletion analysis and transposon insertional inactivation. All transfer-defective (Tra-) deletions and Tn551 or Tn917 transposon insertions occurred within a 14.5-kb BglII fragment. Deletions and insertions outside this fragment all left the plasmid transfer proficient (Tra+). The tra region was found to be flanked by directly repeated DNA sequences, approximately 900 base pairs in length, at either end. Clones containing the 14.5-kb BglII fragment (pGO200) and subclones from this fragment were constructed in Escherichia coli on shuttle plasmids and introduced into Staphylococcus aureus protoplasts. Protoplasts could not be transformed with pGO200E (pGO200 on the staphylococcal replicon, pE194) or subclones containing DNA at one end of the tra fragment unless pGO1 or specific cloned tra DNA fragments were present in the recipient cell. However, once stabilized by sequences present on a second replicon, each tra fragment could be successfully introduced alone into other plasmid-free S. aureus recipients by conjugative mobilization or transduction. In this manner, two clones containing overlapping fragments comprising the entire 14.5-kb BglII fragment were shown to complement each other. The low-frequency transfer resulted in transconjugants containing one clone intact, deletions of that clone, and recombinants of the two clones. The resulting recombinant plasmid (pGO220), which regenerated the tra region intact on a single replicon, transferred at frequencies comparable to those of pGO1. Thus, all the genes necessary and sufficient for conjugative transfer of pGO1 are contained within a 14.5-kb region of DNA.     

Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis.

A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined. Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published. In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector. An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions. The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues. In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region. PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp. israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin. Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.     

Structure of two human beta-actin-related processed genes one of which is located next to a simple repetitive sequence.

From a human gene library we have isolated and sequenced a beta-actin-like pseudogene, H beta Ac-psi 2, which lacks intervening sequences and contains several mutations resulting in frame-shifts, stop codons and in a departure from the known beta-actin protein sequence. We have also extended our sequence work on the intronless human beta-actin-related pseudogene H beta Ac-psi 1 described previously and we find that both genes are processed genes ending in a poly(dA) tract and flanked by direct repeats. The gene H beta Ac-psi 2 is preceded by a 230-bp region in which the simple sequence 5'-GAAA-3' is repeated greater than 40 times. This satellite-like sequence is highly repetitive in the human genome.     

Novel one-step cloning vector with a transposable element: application to the Myxococcus xanthus genome.

A new strategy was developed for rapid cloning of genes with a transposon mutation library. We constructed a transposon designated TnV that was derived from Tn5 and consists of the gene coding for neomycin phosphotransferase II as well as the replication origin of an Escherichia coli plasmid, pSC101, flanked by Tn5 inverted repeats (IS50L and IS50R). TnV can transpose to many different sites of DNA in E. coli and Myxococcus xanthus and confers kanamycin resistance (Kmr) to the cells. From the Kmr cells, one-step cloning of a gene which is mutated as a result of TnV insertion can be achieved as follows. Chromosomal DNA isolated from TnV-mutagenized cells is digested with an appropriate restriction enzyme, ligated, and transformed into E. coli cells with selection for Kmr. The plasmids isolated contain TnV in the target gene. The plasmid DNA can then be used as a probe for characterization of the gene and screening of clones from a genomic library. We used this vector to clone DNA fragments containing genes involved in the development of M. xanthus.     

Release of heat-labile enterotoxin subunits by Escherichia coli.

Most of the heat-labile enterotoxin (LT) synthesized by Escherichia coli is cell associated; however, a small portion of LT (approximately 10%) is released by bacterial cells into the culture supernatant. The LT subunit B (LT-B) produced by a cloned LT-B gene (tox B) was released in amounts equal to the parent LT release. In contrast, no release of LT subunit A (LT-A) or its smaller derivatives was observed in strains containing cloned toxA genes. The data suggest that LT-B is necessary for the release of LT-A across the bacterial membrane.     

One of the copia genes is adjacent to satellite DNA in Drosophila melanogaster.

A method for purifying sequences adjacent to satellite DNA in the heterochromatin of D. melanogaster is described. A cloned DNA segment containing part of a copia gene adjacent to 1.688 g/cm3 satellite DNA has been isolated. The copia genes compose a repeated gene family which codes for abundant cytoplasmic poly(a)-containing RNA (Young and Hogness, 1977; Finnegan et al., 1978). We have identified two major poly (A)-containing RNA species [5.2 and 2.1 kilobases (kb)] produced by the copia gene family. The cloned segment contains copia sequences homologous to the 5' end of RNA within 0.65 kb of the 1.688 satellite DNA sequences. Seven different cloned copia genes from elsewhere in the genome have also been isolated, and a 5.2 kb region present in five of the clones was identified as copia by heteroduplex analysis. In addition, three ususual copies of copia were found: a "partial" copy of the gene (3.7 kb) which has one endpoint in common with the 5.2 kb unit; a copia gene flanked on one side by a 1.6 kb sequence and on the other by the same 1.6 kb sequence in the inverted orientation; and a copia gene flanked only on one side by the same sequence.