Sequence analysis of the 17-kilodalton-antigen gene from Rickettsia rickettsii.

DNA obtained from the Sheila Smith strain of Rickettsia rickettsii was digested to completion with the restriction endonucleases BamHI and SalI and ligated with the plasmid vector pUC19. The ligation mixture was used to transform Escherichia coli. A total of 465 bacterial clones were screened for antigen production with hyperimmune rabbit serum. One of the reactive clones, containing a recombinant plasmid designated pSS124, was solubilized and subjected to immunoblot analysis and revealed expression of a 17-kilodalton protein reactive with anti-R. rickettsii serum that comigrated with an antigen from R. rickettsii. A 1.6-kilobase PstI-BamHI fragment from pSS124 was subcloned and continued to direct synthesis of the 17-kilodalton antigen. The nucleotide sequence was determined for this 1.6-kilobase subclone, which encompassed the gene encoding the polypeptide as well as flanking regions containing potential regulatory sequences. The open reading frame consisted of 477 nucleotides that specified a 159-amino-acid protein with a calculated molecular weight of 16,840. The deduced amino acid sequence contained a hydrophobic sequence near the amino terminus that resembled signal peptides described for E. coli. The carboxy terminus was hydrophilic in nature and probably contained the exposed epitopes.     

A gene at 59 minutes on the Escherichia coli chromosome encodes a lipoprotein with unusual amino acid repeat sequences.

We report a 1.432-kb DNA sequence at 59 min on the Escherichia coli chromosome that connects the published sequences of the pcm gene for the isoaspartyl protein methyltransferase and that of the katF or rpoS (katF/rpoS) gene for a sigma factor involved in stationary-phase gene expression. Analysis of the DNA sequence reveals an open reading frame potentially encoding a polypeptide of 379 amino acids. The polypeptide sequence includes a consensus bacterial lipidation sequence present at residues 23 to 26 (Leu-Ala-Gly-Cys), four octapeptide proline- and glutamine-rich repeats of consensus sequence QQPQIQPV, and four heptapeptide threonine- and serine-rich repeats of consensus sequence PTA(S,T)TTE. The deduced amino acid sequence, especially in the C-terminal region, is similar to that of the Haemophilus somnus LppB lipoprotein outer membrane antigen (40% overall sequence identity; 77% identity in last 95 residues). The LppB lipoprotein binds Congo red dye and has been proposed to be a virulence determinant in H. somnus. Utilizing a plasmid construct with the E. coli gene under the control of a phage T7 promoter, we demonstrate the lipidation of this gene product by the incorporation of [3H]palmitic acid into a 42-kDa polypeptide. We also show that treatment of E. coli cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 46-kDa precursor. We thus designate the protein NlpD (new lipoprotein D). E. coli cells overexpressing NlpD bind Congo red dye, suggesting a common function with the H. somnus LppB protein. Disruption of the chromosomal E. coli nlpD gene by insertional mutagenesis results in decreased stationary-phase survival after 7 days.     

Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli

The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.     

灰色链霉菌启动子活性片段在大肠杆菌中的亚克隆及其序列分析

通过对重组质粒No.8-1的亚克隆,灰色链霉菌插入到大肠杆菌载体质粒中的序列已缩小到410bp,仍具有启动子功能。序列分析表明,启动子活性片段的G C碱基组成为50.5%。内含链霉菌启动子区域常有的正向重复序列;有1个Alul位点,1个Clal位点,2个Mbol位点,3个Nla Ⅲ位点,1个Pvu Ⅱ位点;具有类似于E.coli启动子的保守序列-10区和-35区,两者间隔18bp;在相应位置上分别有一段序列与E.coli的SD序列和在苄铅青链霉菌的SEP(Streptomyces-E.coil-type promoter)序列中存在的保守序列具有一定的相似性。     

Sequence elements determining ampC promoter strength in E. coli

A number of spontaneous up-promoter mutations have been isolated in the ampC beta-lactamase gene of Escherichia coli. The mutants were analyzed by DNA sequencing, and the level of ampC gene expression was determined. Six mutants with a 21-fold increase in promoter strength compared with the wild-type were mutated in the -35 promoter region from TTGTCA to the consensus sequence TTGACA . The -10 region sequence TACAAT was mutated to the consensus sequence TATAAT in three mutants exhibiting an ampC promoter seven times stronger than the wild-type. We have previously described a 1-bp insertion mutant ( Jaurin et al., 1981) that changes the inter-region distance to the consensus 17 bp. Thus, all the up-mutations found in the ampC promoter represent corrections of the three major discrepancies between the ampC promoter and the consensus E. coli promoter. We conclude that the three consensus elements of E. coli promoters, the -35 and -10 regions and an optimal inter-region distance of 17 bp, are the main elements determining the promoter strength.     

Defining the consensus sequences of E.coli promoter elements by random selection.

The consensus sequence of E.coli promoter elements was determined by the method of random selection. A large collection of hybrid molecules was produced in which random-sequence oligonucleotides were cloned in place of a wild-type promoter element, and functional -10 and -35 E.coli promoter elements were obtained by a genetic selection involving the expression of a structural gene. The DNA sequences and relative levels of function for -10 and -35 elements were determined. The consensus sequences determined by this approach are very similar to those determined by comparing DNA sequences of naturally occurring E.coli promoters. However, no strong correlation is observed between similarity to the consensus and relative level of function. The results are considered in terms of E.coli promoter function and of the general applicability of the random selection method.     

Isolation of a yeast gene, SRH1, that encodes a homologue of the 54K subunit of mammalian signal recognition particle

A 1.7 kilobase HindIII fragment of Saccharomyces cerevisiae DNA was cloned by cross-hybridization with the Escherichia coli secY gene. The complete nucleotide sequence of the 2.6 kb fragment of the yeast genomic DNA containing the cross-hybridizing HindIII fragment was determined. The sequence showed no apparent similarity with that of the E. coli secY gene with the exception of a completely matched sequence of 21 bp, but it contained a 1,623 nucleotide open reading frame coding for a protein of 541 amino acids with a calculated Mr of 59,600. The N-terminal portion of 303 residues of the predicted sequence was homologous to the cytosolic domain of the alpha-subunit of the signal recognition particle receptor (SR alpha), including consensus sequence elements for a GTP binding site, whereas the C-terminal portion of 238 residues had an unusual methionine-rich domain containing several repetitive sequences. An mRNA of 2.0 kb was detected on Northern blotting analysis. The predicted sequence was 48% identical with the reported sequences of the 54K subunit of the mammalian signal recognition particle (SRP54) (Romisch K. et al. (1989) Nature 340, 478-483; Bernstein, H.D. et al. (1989) Nature 340, 482-486). We designated this gene as SRH1 (SRP54 homologue). Gene disruption experiments showed that the SRH1 gene product is essential for cell growth.     

Activation of Helicobacter pylori ureA promoter by a hybrid Escherichia coli-H. pylori rpoD gene in E. coli.

We constructed and analyzed hybrid Escherichia coli-Helicobacter pylori rpoD genes in an E. coli rpoD mutant. It turned out that a hybrid consisting of E. coli rpoD with subdomain 4.2 of H. pylori rpoD (for -35 recognition) was functional. On the other hand, hybrids consisting of E. coli rpoD with domain 2 and the adjacent sequence of H. pylori rpoD (for core enzyme binding and -10 recognition) were non-functional. Intriguingly, a hybrid rpoD containing H. pylori subdomain 4.2 conferred higher activity for the H. pylori PureA as determined by xylE expression of PureA-xylE fusions, although the activity of the hybrid rpoD for the tac promoter was comparable to that of E. coli rpoD. The tsp of ureA in E. coli with the hybrid rpoD and E. coli rpoD were 15 and 17bp upstream from that in H. pylori, respectively. The comparison of PureA sequences in both E. coli and H. pylori indicated the existence of a -10 consensus sequence but little conservation of -35 sequences. Instead, the PureA in both H. pylori and E. coli contained an identical heptamer, GTTAATA, in the extended -35 region.     

The construction of a synthetic Escherichia coli trp promoter and its use in the expression of a synthetic interferon gene.

An 82 base pair DNA fragment has been synthesised which contains the E. coli trp promoter and operator sequences and also encodes the first Shine Dalgarno sequence of the trp operon. This DNA fragment is flanked by EcoRI and ClaI/TaqI cohesive ends and is thus easy to clone, transfer between vector systems and couple to genes to drive their expression. It has been cloned into plasmid pAT153, producing a convenient trp promoter vector. We have also joined the fragment to a synthetic IFN-alpha 1 gene, using synthetic oligonucleotides to generate a completely natural, highly efficient bacterial translation initiation signal on the promoter proximal side of the IFN gene. Plasmids carrying this construction enable E. coli cells to express IFN-alpha 1 almost constitutively and with significantly higher efficiency than from a lacUV5 promoter based system.     

Expression of hepatitis B virus surface antigen P31 gene in Escherichia coli

A hepatitis B virus surface antigen (HBsAg) P31-coding DNA was constructed from a DNA fragment of the plasmid pHBr330 containing the entire hepatitis B virus (HBV) adr DNA and a chemically synthesized adaptor. The P31 gene was inserted into an expression vector, pTRP771, having an Escherichia coli tryptophan operon (trp) promoter to give a recombinant plasmid pTRP P31-R. The distance between the Shine-Dalgarno (SD) sequence and the initiation codon of P31 gene was adjusted to 9 bp. The expression level of HBsAg by E. coli 294[pTRP P31-R] was significantly elevated, in contrast to that of HBsAg by E. coli 294[pTRP SS-6]. Western blotting analysis has shown that E. coli[pTRP P31-R] synthesizes a specific polypeptide P31 of about 31 kDal, which reacts with anti-HBsAg antibody. The binding studies with polyalbumins from various species have also suggested that HBsAg P31 specifically binds to polymerized human serum albumin.     

Molecular cloning and expression of a novel family A endoglucanase gene from Fibrobacter succinogenes S85 in Escherichia coli

A Fibrobacter succinogenes S85 gene that encodes endoglucanase hydrolysing CMC and xylan was cloned and expressed in Escherichia coli DH5 by using pUC19 vector. Recombinant plasmid DNA from a positive clone hydrolysing CMC and xylan was designated as pCMX1, harboring 2,043 bp insert. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The nucleotide sequence accession number of the cloned gene sequence in Genbank is U94826. The endoglucanase gene cloned in this study does not have amino sequence homology to the other endoglucanase genes from F. succinogenes S85, but does show sequence homology to family 5 (family A) of glycosyl hydrolases from several species. The ORF encodes a polypeptide of 654 amino acids with a measured molecular weight of 81.3 kDa on SDS-PAGE. Putative signal sequences, Shine-Dalgarno-type ribosomal binding site and promoter sequences (-10) related to the consensus promoter sequences were deduced. The recombinant endoglucanase by E. coli harboring pCMX1 was partially purified and characterized. N-terminal sequences of endoglucanase were Ala-Gln-Pro-Ala-Ala, matched with deduced amino sequences. The temperature range and pH for optimal activity of the purified enzyme were 55 approximately 65 degrees C and 5.5, respectively. The enzyme was most stable at pH 6 but unstable under pH 4 with a K(m) value of 0.49% CMC and a V(max) value of 152 U/mg.