Nucleotide sequence of the bacteriophage T4 gene 57 and a deduced amino acid sequence.

A 693 basepair cloned fragment of bacteriophage T4 DNA, which supports specifically growth of T4 amber mutants in gene 57, has been sequenced. A polypeptide can be deduced from this sequence, that is either 54 or 60 amino acids long depending which of two AUG codons, 18 nucleotides apart, are used for initiation. The size of this deduced polypeptide is compatible with the size of a single polypeptide (based on polyacrylamide gel electrophoresis) synthesized in vivo in E. coli under the direction of the cloned T4 DNA fragment.     

Identification of the Escherichia coli K-12 cpxA locus as a single gene: construction and analysis of biologically-active cpxA gene fusions

In the accompanying communication we showed that a 2 kb EcoRI-BamHI restriction fragment from the pfkA-rha interval of the Escherichia coli K-12 chromosome fully complemented a chromosomal cpxA mutation when the fragment was cloned in pBR325. The same fragment cloned in pBR322 lacked any complementing activity. We show here that minicells containing the pBR325 derivative (pRA310) synthesized a 33 kDa polypeptide, designated phi 33, that was not synthesized in minicells containing the pBR322 derivative (pRA311) or either of the parent plasmids. Synthesis of the phi 33 polypeptide did not occur in minicells containing Tn5 insertion alleles of pRA310 that inactivated its cpxA complementing activity. These insertions mapped within the vector cat (chloramphenicol acetyltransferase gene) sequence immediately adjacent to the EcoRI site of pRA310 and within the 700-800 bp of the cloned EcoRI-BamHI fragment immediately adjacent to the EcoRI site. Tn5 insertions located within the fragment but closer to the BamHI terminus affected neither the cpxA complementing activity of pRA310 nor synthesis of the phi 33 polypeptide in minicells. Plasmid pRA311 could be converted to a plasmid with cpxA complementing activity by cloning into its EcoRI site a restriction fragment containing a hybrid trp-lacUV5 promoter, the lacZ ribosome binding site, and the first eight lacZ codons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and sequencing of a gene encoding a glutamate and aspartate carrier of Escherichia coli K-12.

A gene encoding a carrier protein for glutamate and aspartate was cloned into Escherichia coli K-12 strain BK9MDG by using the high-copy-number plasmid pBR322. The gene (designated gltP) is probably identical to a gene recently cloned from E. coli B (Y. Deguchi, I. Yamato, and Y. Anraku, J. Bacteriol. 171:1314-1319). A 1.6-kilobase DNA fragment containing gltP was subcloned into the expression plasmids pT7-5 and pT7-6, and its product was identified by a phage T7 RNA polymerase-T7 promoter coupled system (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078) as a polypeptide with an apparent mass of 38 kilodaltons. A portion of the gltP polypeptide was associated with the cytoplasmic membrane. The nucleotide sequence of the 1.6-kilobase fragment was determined. It contained an open reading frame capable of encoding a highly hydrophobic polypeptide of 395 amino acids, containing four possible transmembrane segments. Uptake of glutamate and aspartate was increased 5.5- and 4.5-fold, respectively, in strains containing gltP plasmids. Glutamate uptake was insensitive to the concentration of Na+ and was inhibited by L-cysteate and beta-hydroxyaspartate. These results suggest that gltP is a structural gene for a carrier protein of the Na(+)-independent, binding-protein-independent glutamate-aspartate transport system.     

The DNA [adenine-N6]methyltransferase (Dam) of bacteriophage T4

A functional bacteriophage T4 dam+ gene, which specifies a DNA [adenine-N6]methyltransferase (Dam), was cloned on a 1.8-kb HindIII fragment [Schlagman and Hattman, Gene 22 (1983) 139-156]. Sequence analysis [Macdonald and Mosig, EMBO J. 3 (1984) 2863-2871] revealed two overlapping in-phase open reading frames (ORFs). The 5' proximal ORF initiates translation at an AUG and encodes a 30-kDa polypeptide, whereas the downstream ORF initiates translation at a GUG and encodes a 26-kDa polypeptide. Analysis of BAL 31 deletions in our original dam+ clone has verified that at least one of these overlapping ORFs, in fact, encodes T4 Dam. To investigate where T4 Dam translation is initiated, we have constructed plasmids in which a tac or lambda PL promoter is placed 5' to either the longer ORF or just the shorter ORF. Only clones which contain a promoter in front of the longer ORF produce active T4 Dam. This indicates that the 26-kDa polypeptide alone cannot be T4 Dam. Additional experiments suggest that only the 30-kDa polypeptide is required for enzyme activity and that the shorter ORF is not translated in plasmid-carrying cells. We also present evidence that T4 Dam is capable of methylating 5'-GATC-3', GATm5C, and GAThmC sequences; non-canonical sites (e.g., GACC) are also methylated, but much less efficiently.     

Fine-structure mapping of herpes simplex virus type 1 temperature-sensitive mutations within the short repeat region of the genome.

Cloned herpes simplex virus type 1 (HSV-1) DNA fragments were used to fine-structure map the temperature-sensitive (ts) lesions from four mutants, ts T, D, c75, and K, by marker rescue. These mutants all overproduced immediate-early viral polypeptides at the nonpermissive temperature. Although one of these viruses, ts K, gave a more restricted infected-cell polypeptide profile under these conditions than the other three, no complementation was detected between pairwise crosses of these mutants in the yield test. Recombination, however, was obtained between all mutant pairs except ts T and D. In physical mapping experiments, ts+ virus was recovered from cells coinfected with DNA of ts T, D, or c75 and BamHI fragment k from wild-type strain 17 HSV-1 DNA cloned in pAT153, whereas ts K was rescued by cloned HSV-1 BamHI-y. Both of these cloned DNA fragments contained sequences from the short repeat region of the HSV-1 genome. The ts mutations were more precisely mapped by marker rescue, using restriction enzyme fragments within BamHI-k and -y from cloned DNA. The smallest fragment able to rescue a mutant was 320 base pairs long. The order of the four mutations derived from these studies was consistent with the assignment by genetic recombination. All four lesions mapped within the coding sequences of the immediate-early polypeptide Vmw IE 175 (ICP4) which lie outside the "a" sequence. The results showed that mutations in different regions of the gene encoding Vmw IE 175 could produce similar phenotype effects at the nonpermissive temperature.     

Cloning and DNA sequence of a plasmid-determined citrate utilization system in Escherichia coli.

The citrate utilization determinant from a large 200-kilobase (kb) naturally occurring plasmid was previously cloned into the PstI site of plasmid vector pBR325 creating the Cit+ tetracycline resistance plasmid pWR61 (15 kb). Tn5 insertion mutagenesis analysis of plasmid pWR61 limited the segment responsible for citrate utilization to a 4.8-kb region bordered by EcoRI and PstI restriction nuclease sites. The 4.8-kb fragment was cloned into phage M13, and the DNA sequence was determined by the dideoxyribonucleotide method. Within this sequence was a 1,296-base-pair open reading frame with a preceding ribosomal binding site. The 431-amino-acid polypeptide that could be translated from this open reading frame would be highly hydrophobic. A second long open reading frame with the potential of encoding a 379-amino-acid polypeptide preceded the larger open reading frame. Portions of the 4.8-kb fragment were further subcloned with restriction endonucleases BglII and BamHI, reducing the minimum size needed for a citrate-positive phenotype to a 1.9-kb BamHI-BglII fragment (which includes the coding region for the 431-amino-acid polypeptide, but only the distal 2/3 of the reading frame for the 379-amino-acid polypeptide). Citrate utilization results from a citrate transport activity encoded by the plasmid. With the 4.8-kb fragment (as with larger fragments) the citrate transport activity was inducible by growth on citrate. On transfer from glucose, succinate, malate, or glycerol medium to citrate medium, the Cit+ Escherichia coli strains showed a delay of 36 to 48 h before growth.     

Molecular cloning and DNA sequence analysis of Escherichia coli topB, the gene encoding topoisomerase III

Escherichia coli contains two type 1 topoisomerases, topoisomerase I and III. Although topoisomerase III can be purified as a potent decatenase, its role in DNA metabolism is unclear. In order to address this issue, the gene encoding topoisomerase III from E. coli has been molecularly cloned and its DNA sequence determined. The cloned fragment of DNA contains an open reading frame that can encode a polypeptide of 73.2 kDa. The first 20 amino acids of this open reading frame are identical to those of topoisomerase III as determined by amino-terminal gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame, using a bacteriophage T7 transient expression system, results in the accumulation of a 74-kDa polypeptide. Soluble extracts prepared from cells overexpressing this gene product show a dramatic increase in topoisomerase activity when compared with control extracts. We propose that this gene be designated topB.     

Genetic, functional and sequence analysis of the xylR and xylS regulatory genes of the TOL plasmid pWW0

Mutant derivatives of a plasmid, pCF20, which carries the XhoI-D fragment of the TOL plasmid pWW0 have been isolated using Tn5 transposon mutagenesis. Insertion mutations of the xylR and xylS regulatory genes of the catabolic pathway have been isolated and characterized and their ability to induce catechol 2,3-oxygenase activity determined. Analysis of the insertion mutants and also segments of the XhoI-D fragment cloned into plasmid pUC8 in maxicells has identified a 68 kDa polypeptide product encoded by the xylR gene. No clear candidate for the xylS polypeptide was observed. The nucleotide sequence of the xylS region, the intergenic region and part of the xylR region has been determined and open reading frames (ORFs) assigned for both genes. The ORF designated xylS appears capable of encoding a polypeptide of approximately 37 kDa.     

Cloning and expression in minicells of the determinant of resistance to fosfomycin from the transposon Tn2921

The fosfomycin resistance determinant of the transposon Tn2921 has been localized and cloned into the plasmid pBR322. A DNA fragment of 1 kb contains all the information required for full expression of the resistance. The level of resistance was unaffected by the plasmid copy number and by the orientation in which the fragment was cloned. Studies in minicells showed that this 1-kb fragment directed the synthesis of a single polypeptide with a molecular mass of 16 kDa.     

Cloning of the gene for delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni

We have cloned an approx. 5-kb fragment of Pseudomonas testosteroni DNA containing the structural gene of delta 5-3-ketosteroid isomerase into the EcoRI site of the lambda gt11 genome. Escherichia coli infected with these recombinant phages produce a polypeptide which is recognized by antiserum raised against the purified isomerase. Four of the recombinant lambda gt11 clones contain significant levels of isomerase activity and produce an immunopositive polypeptide of the same apparent Mr as the native isomerase obtained from P. testosteroni. The approx. 5-kb fragment hybridizes to synthetic 21-mer and 17-mer oligodeoxynucleotide mixtures corresponding to the 5' and 3' regions, respectively, of the expected nucleotide sequence of the gene.     

Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping.

Tn5-tagged invasion plasmid DNA (pWR110) from Shigella flexneri serotype 5 (strain M90T) was cloned into the expression vector lambda gt11. Recombinant phage (lambda gt11Sfl) expressing pWR110-encoded polypeptide antigens were identified by using rabbit antisera directed against S. flexneri M90T invasion plasmid antigens. Antigens encoded by lambda gt11Sfl recombinant phage were characterized by reacting affinity-purified antibodies, eluted from nitrocellulose-bound plaques of lambda gt11Sfl recombinants, with virulent, wild-type S. flexneri M90T polypeptides in Western blot analyses. lambda gt11Sfl clones directing the synthesis of complete, truncated, and beta-galactosidase fusion versions of three previously identified outer membrane polypeptides (57-, 43-, and 39-kilodalton [kDa] antigens) were isolated. A fourth polypeptide, similar in size to the 57-kDa antigen (ca. 58 kDa) but unrelated as determined by DNA homology and serological measurements, was also identified. Southern blot analysis of S. flexneri M90T invasion plasmid DNA hybridized with lambda gt11Sfl insert DNA probes was used to construct a map of invasion plasmid antigen genes (ipa) corresponding to the 57-kDa (ipaB), 43-kDa (ipaC), and 39-kDa (ipaD) polypeptides. Genes ipaB, ipaC and ipaD mapped to contiguous 4.6-kilobase (kb) and 1.0-kb HindIII fragments contained within a larger (23-kb) BamHI fragment. The ipaH gene, which encodes the synthesis of the 58-kDa polypeptide, did not map in or near the ipaBCD gene cluster, suggesting a distinct location of ipaH on the invasion plasmid.     

Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

The gene for the catalytic subunit of cellulose synthase from Acetobacter xylinum has been cloned by using an oligonucleotide probe designed from the N-terminal amino acid sequence of the catalytic subunit (an 83 kDa polypeptide) of the cellulose synthase purified from trypsin-treated membranes of A. xylinum. The gene was located on a 9.5 kb HindIII fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the HindIII fragment led to the identification of an open reading frame of 2169 base pairs coding for a polypeptide of 80 kDa. Fifteen amino acids in the N-terminal region (positions 6 to 20) of the amino acid sequence, deduced from the DNA sequence, match with the N-terminal amino acid sequence obtained for the 83 kDa polypeptide, confirming that the DNA sequence cloned codes for the catalytic subunit of cellulose synthase which transfers glucose from UDP-glucose to the growing glucan chain. Trypsin treatment of membranes during purification of the 83 kDa polypeptide cleaved the first 5 amino acids at the N-terminal end of this polypeptide as observed from the deduced amino acid sequence, and also from sequencing of the 83 kDa polypeptide purified from membranes that were not treated with trypsin. Sequence analysis suggests that the cellulose synthase catalytic subunit is an integral membrane protein with 6 transmembrane segments. There is no signal sequence and it is postulated that the protein is anchored in the membrane at the N-terminal end by a single hydrophobic helix. Two potential N-glycosylation sites are predicted from the sequence analysis, and this is in agreement with the earlier observations that the 83 kDa polypeptide is a glycoprotein [13]. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.     

Molecular cloning and sequence analysis of the crtB gene of Thermus thermophilus HB27, an extreme thermophile producing carotenoid pigments.

We have cloned and sequenced a 1.5-kb chromosomal fragment of Thermus thermophilus which promoted the overproduction of carotenoids in T. thermophilus. An open reading frame (ORF-A) coding for a polypeptide with 289 amino acids was responsible for carotenoid overproduction. The putative ORF-A protein showed significant homology with the amino acid sequences of crtB gene products (phytoene syntheses) of other microorganisms. The clone containing the ORF-A on a multicopy plasmid produced about three times as much carotenoid as that produced by the host strain, suggesting that the crtB gene product is a rate-limiting enzyme for carotenoid biosynthesis in T. thermophilus.     

Expression of the 4-chlorobenzoate dehalogenase genes from Pseudomonas sp.-CBS3 in Escherichia coli and identification of the gene translation products.

The genes encoding the 4-chlorobenzoate dehalogenase of Pseudomonas sp. strain CBS3 were, in an earlier study, cloned in Escherichia coli DH1 with the cosmid vector pPSA843 and then mobilized to the 4-chlorobenzoate dehalogenase minus strain Pseudomonas putida KT2440. In this paper we report on the expression of 4-chlorobenzoate dehalogenase in these clones and on the polypeptide composition of the active enzyme. The dehalogenase activity in whole cells suspended in 3.2 mM 4-chlorobenzoate (30 degrees C) was determined to be approximately 27 units (micromoles 4-hydroxybenzoate produced per minute) per 100 g of E. coli-pPSA843 cells and approximately 28 units per 100 g of P. putida-pPSA843 cells. Dehalogenase activity in fresh cellular extracts (pH 7.4, 30 degrees C) prepared from the E. coli and P. putida clones was unstable and at least 20-fold lower than that observed with the whole cells. The polypeptide components of the dehalogenase were identified by selective expression of the cloned dehalogenase genes and analysis of the gene translation products. Analysis of dehalogenase activity in omega insertion mutants and deletion mutants circumscribed the dehalogenase genes to a 4.8-kilobase (4.8 kb) stretch of the 9.5-kb DNA fragment. Selective expression of the dehalogenase genes from a cloned 4.8-kb DNA fragment in a maxicell system revealed a 30-kDa polypeptide as one of the components of the dehalogenase system. Selective expression of the dehalogenase genes using the T7 polymerase promoter system revealed the 30-kDa polypeptide and 57- and 16-kDa polypeptide products. Determination of which of the three polypeptides were translated in deletion mutants provided the relative positions of the encoding genes on a single DNA strand and the direction in which they are transcribed.     

Cloning and regulation of Erwinia herbicola pigment genes.

The genes coding for yellow pigment production in Erwinia herbicola Eho10 (ATCC 39368) were cloned and localized to a 12.4-kilobase (kb) chromosomal fragment. A 2.3-kb AvaI deletion in the cloned fragment resulted in the production of a pink-yellow pigment, a possible precursor of the yellow pigment. Production of yellow pigment in both E. herbicola Eho10 and pigmented Escherichia coli clones was inhibited by glucose. When the pigment genes were transformed into a cya (adenylate cyclase) E. coli mutant, no expression was observed unless exogenous cyclic AMP was provided, which suggests that cyclic AMP is involved in the regulation of pigment gene expression. In E. coli minicells, the 12.4-kb fragment specified the synthesis of at least seven polypeptides. The 2.3-kb AvaI deletion resulted in the loss of a 37K polypeptide and the appearance of a polypeptide of 40 kilodaltons (40K polypeptide). The synthesis of the 37K polypeptide, which appears to be required for yellow pigment production, was not repressed by the presence of glucose in the culture medium, as was the synthesis of other polypeptides specified by the 12.4-kb fragment, suggesting that there are at least two types of gene regulation involved in yellow pigment synthesis. DNA hybridization studies indicated that different yellow pigment genes exist among different E. herbicola strains. None of six pigmented plant pathogenic bacteria examined, Agrobacterium tumefaciens C58, Cornyebacterium flaccumfaciens 1D2, Erwinia rubrifaciens 6D364, Pseudomonas syringae ATCC 19310, Xanthomonas campestris 25D11, and "Xanthomonas oryzae" 17D54, exhibited homology with the cloned pigment genes.     

盘状结构域受体2胞外区的可溶性表达、纯化和功能鉴定

盘状结构域受体2（discoidin domain receptor 2,DDR2）是一种与肿瘤细胞转移相关的蛋白酷氨酸激酶,其配体为纤维性胶原,胶原对DDR2的活化上调细胞中基质金属蛋白酶1（MMP－1）的表达。为研究DDR2在类风温性关节炎(rteumatoid arthritis,RA）软骨破坏和肿瘤转移中的作用,尝试了在大肠杆菌中表达一段DDR2胞外区（命名DB）,并进行了可溶性部分的纯化和功能鉴定,以备将来用作DDR2的特异性阻断剂。获得了一株表达GST－DB融合蛋白的大肠杆菌克隆;其表达的蛋白质中可溶性部分约占全部融合蛋白的13％;经GST融合蛋白特异性亲和珠纯化后,获得了纯度约86.1%的可溶性GST－DB融合蛋白;竞争结合抑制实验显示,GST－DB具有阻断Ⅱ型胶原和细胞表面天然DDR2受体结合的功能;细胞实验表明,GST－DB有抑制Ⅱ型胶原刺激下的类风湿性关节炎滑膜细胞和NIH3T3细胞分泌MMP－1的作用。以上结果提示,表达的融保蛋白GST－DB具有抑制天然DDR2功能的作用;DDR2在滑膜细胞和NIH3T3细胞中介导Ⅱ型胶原刺激下的MMP－1的分泌。     

Cloning, expression and characterisation of a recombinant triosephosphate isomerase from Taenia solium

We isolated and characterised the cDNA that encodes the glycolytic enzyme, triosephosphate isomerase from Taenia solium. A 450 bp DNA fragment was obtained by the polymerase chain reaction using a cDNA from larval stage as template and degenerate oligonucleotides designed from conserved polypeptide sequences from TPIs of several organisms. The fragment was used to screen a T. solium larval stage cDNA library. The isolated cDNA, encoding a protein of 250 amino acids shares 44.8-59.6% positional identity with other known TPIs, in which the catalytic enzyme residues were conserved. The complete coding sequence of the T. solium TPI cDNA was cloned into the expression vector pRSET and expressed as a fusion protein with an N-terminal tail of six histidine residues. The catalytic activity of the purified protein was similar to other TPI enzymes. Northern and Southern blot analysis suggest that in T. solium, single gene exists for triosephosphate isomerase and that the gene is expressed in all stages of the parasite.