Two Haemophilus influenzae Rd genes that complement the recA-like mutation rec-1.

Two Haemophilus influenzae Rd genes each complemented the pleiotropic defects of the recA-like mutation rec-1. One gene, fec, was isolated on a 3.6-kilobase-pair EcoRI restriction fragment by complementation of the Fec- phenotype of bacteriophage lambda. The other gene, rec, was identified on a 3.1-kilobase-pair EcoRI fragment by Southern hybridization by using recA-like gene probes from Erwinia carotovora and Pseudomonas aeruginosa PAO. In a rec-1 strain of H. influenzae, the cloned genes restored resistance to UV irradiation, transformation by chromosomal DNA, and spontaneous release of HP1 prophage to wild-type levels. The fec and rec genes were located on the cloned segments by insertion and deletion mutagenesis and subcloning. The restriction endonuclease cleavage maps of the two DNAs were similar but not identical. Southern hybridization demonstrated that the two EcoRI restriction fragments contained homologous DNA sequences, but a fec gene-specific probe was prepared. Each gene encoded a 38,000-dalton polypeptide.     

Cloning of plasmid genes encoding resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus CH34.

A 238-kilobase-pair plasmid, pMOL30, confers resistance to cadmium, zinc, and cobalt salts in Alcaligenes eutrophus CH34. After Tn5 mutagenesis, restriction nuclease analysis, and Southern DNA-DNA hybridization, a 9.1-kilobase-pair EcoRI fragment was found to harbor all of these resistance properties and was cloned into the broad-host-range hybrid plasmid pRK290. When transferred to a plasmid-free derivative of CH34, the hybrid plasmid conferred the same degree of resistance as the parent plasmid pMOL30. In two other Alcaligenes strains, the hybrid plasmid was expressed, but to a lower degree than in CH34 derivatives.     

Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin resistance determinant

The replication region of a 28-kilobase-pair (kbp) cryptic plasmid from Lactococcus lactis subsp. lactis biovar diacetylactis SSD207 was cloned in L. lactis subsp. lactis MG1614 by using the chloramphenicol resistance gene from the streptococcal plasmid pGB301 as a selectable marker. The resulting 8.1-kbp plasmid, designated pVS34, was characterized further with respect to host range, potential cloning sites, and location of replication gene(s). In addition to lactococci, pVS34 transformed Lactobacillus plantarum and, at a very low frequency, Staphylococcus aureus but not Escherichia coli or Bacillus subtilis. The 4.1-kbp ClaI fragment representing lactococcal DNA in pVS34 contained unique restriction sites for HindIII, EcoRI, XhoII, and HpaII, of which the last three could be used for molecular cloning. A region necessary for replication was located within a 2.5-kbp fragment flanked by the EcoRI and ClaI restriction sites. A 3.8-kbp EcoRI fragment derived from a nisin resistance plasmid, pSF01, was cloned into the EcoRI site of pVS34 to obtain a nisin-chloramphenicol double-resistance plasmid, pVS39. From this plasmid, the streptococcal chloramphenicol resistance region was subsequently eliminated. The resulting plasmid, pVS40, contains only lactococcal DNA. Potential uses for this type of a nisin resistance plasmid are discussed.     

Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin resistance determinant.

The replication region of a 28-kilobase-pair (kbp) cryptic plasmid from Lactococcus lactis subsp. lactis biovar diacetylactis SSD207 was cloned in L. lactis subsp. lactis MG1614 by using the chloramphenicol resistance gene from the streptococcal plasmid pGB301 as a selectable marker. The resulting 8.1-kbp plasmid, designated pVS34, was characterized further with respect to host range, potential cloning sites, and location of replication gene(s). In addition to lactococci, pVS34 transformed Lactobacillus plantarum and, at a very low frequency, Staphylococcus aureus but not Escherichia coli or Bacillus subtilis. The 4.1-kbp ClaI fragment representing lactococcal DNA in pVS34 contained unique restriction sites for HindIII, EcoRI, XhoII, and HpaII, of which the last three could be used for molecular cloning. A region necessary for replication was located within a 2.5-kbp fragment flanked by the EcoRI and ClaI restriction sites. A 3.8-kbp EcoRI fragment derived from a nisin resistance plasmid, pSF01, was cloned into the EcoRI site of pVS34 to obtain a nisin-chloramphenicol double-resistance plasmid, pVS39. From this plasmid, the streptococcal chloramphenicol resistance region was subsequently eliminated. The resulting plasmid, pVS40, contains only lactococcal DNA. Potential uses for this type of a nisin resistance plasmid are discussed.     

Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli.

A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the beta-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichia coli. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the beta-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium beta-galactosidase gene in E. coli was not subject to glucose repression. By using transposon Tn5 mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for beta-galactosidase expression in E. coli. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and Klebsiella pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus II, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but Tn5 insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of beta-galactosidase in E. coli.     

Expression and nucleotide sequence of the Lactobacillus bulgaricus beta-galactosidase gene cloned in Escherichia coli.

The Lactobacillus bulgaricus beta-galactosidase gene was cloned on a ca. 7-kilobase-pair HindIII fragment in the vector pKK223-3 and expressed in Escherichia coli by using its own promoter. The nucleotide sequence of the gene and approximately 400 bases of 3'- and 5'-flanking sequences was determined. The amino acid sequence of the beta-galactosidase, deduced from the nucleotide sequence of the gene, yielded a monomeric molecular mass of ca. 114 kilodaltons, slightly smaller than the E. coli lacZ and Klebsiella pneumoniae lacZ enzymes but larger than the E. coli evolved (ebgA) beta-galactosidase. The cloned beta-galactosidase was found to be indistinguishable from the native enzyme by several criteria. From amino acid sequence alignments, the L. bulgaricus beta-galactosidase has a 30 to 34% similarity to the E. coli lacZ, E. coli ebgA, and K. pneumoniae lacZ enzymes. There are seven regions of high similarity common to all four of these beta-galactosidases. Also, the putative active-site residues (Glu-461 and Tyr-503 in the E. coli lacZ beta-galactosidase) are conserved in the L. bulgaricus enzyme as well as in the other two beta-galactosidases mentioned above. The conservation of active-site amino acids and the large regions of similarity suggest that all four of these beta-galactosidases evolved from a common ancestral gene. However, these enzymes are quite different from the thermophilic beta-galactosidase encoded by the Bacillus stearothermophilus bgaB gene.     

Cloning of a carbofuran hydrolase gene from Achromobacter sp. strain WM111 and its expression in gram-negative bacteria.

A 14-kilobase-pair (kbp) EcoRI DNA fragment that encodes an enzyme capable of rapid hydrolysis of N-methylcarbamate insecticides (carbofuran hydrolase) was cloned from carbofuran-degrading Achromobacter sp. strain WM111. When used to probe Southern blots containing plasmid and total DNAs from WM111, this 14-kbp fragment hybridized strongly to a 14-kbp EcoRI fragment from the greater than 100-kbp plasmid harbored by this strain but weakly to EcoRI-digested total DNA from Achromobacter sp. strain WM111, indicating that the gene for N-methylcarbamate degradation (mcd) is plasmid encoded. Further subcloning localized the mcd gene on a 3-kbp ScaI-ClaI fragment. There was little or no expression of this gene in the alternative gram-negative hosts Pseudomonas putida, Alcaligenes eutrophus, Acinetobacter calcoaceticus, and Achromobacter pestifer. Western blotting (immunoblotting) of the protein products produced by low-level expression in P. putida confirmed that this 3-kbp fragment encodes the two 70+-kilodalton protein products seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified carbofuran hydrolase.     

Molecular genetic analysis of the major histocompatibility complex in an ELA typed horse family

Restriction fragment length polymorphism was studied in an ELA typed horse family which included a stallion, a mare with two full-sibs, another mare with three full-sibs and, in addition, three paternal half-sibs. DNA samples from all individuals were investigated by Southern blot analysis using three restriction enzymes (EcoRI, HindIII or TaqI) and human cDNA class I, class II (DR beta) and class III (C4) probes. In addition, a genomic class II DQ alpha probe was used. Fragments hybridized with the various probes revealed the existence of DNA sequences homologous to HLA class I, DR beta, DQ alpha and C4 genes in the horse. Polymorphic fragments were found when DNA was hybridized with class I and class II probes irrespective of the enzyme used; but hybridization with the C4 probe did not reveal variability. All polymorphic fragments segregated according to the ELA serological specificities, thus indicating a close linkage between the different revealed subregions. Banding patterns suggest that the horse possesses about 20-30 class I genes, probably more than one DR beta and DQ alpha genes and possibly only one C4 gene. The high degree of polymorphism observed suggests that molecular DNA typing may represent a potentially powerful aid to decision in parentage control determination.     

Regulation and expression of the bacteriophage mu mom gene: mapping of the transactivation (dad) function to the C region

Expression of the bacteriophage Mu mom gene is under tight regulatory control. One of the factors required for mom gene expression is the trans-acting function (designated Dad) provided by another Mu gene. To facilitate studies on the signals mediating mom regulation, we have constructed a mom-lacZ fusion plasmid which synthesizes beta-galactosidase only when the Mu Dad transactivating function is provided. lambda pMu phages carrying different segments of the Mu genome have been assayed for their ability to transactivate beta-galactosidase expression by the fusion plasmid. The results of these analyses indicated that the Dad transactivation function is encoded between the leftmost EcoRI site and the lys gene of Mu; this region includes the C gene, which is required for expression of all Mu late genes. Cloning of an approx. 800-bp fragment containing the C gene produced a plasmid which could complement MuC- phages for growth and could transactivate the mom-lacZ fusion plasmid to produce beta-galactosidase. These results suggest that the C gene product mediates the Dad transactivation function.     

Characterization and regulation of Schizosaccharomyces pombe gene encoding thioredoxin

A cDNA coding thioredoxin (TRX) was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization. The 438 bp EcoRI fragment, which was detected by Southern hybridization, reveals an open reading frame which encodes a protein of 103 amino acids. The genomic DNA encoding TRX was also isolated from S. pombe chromosomal DNA using PCR. The cloned sequence contains 1795 bp and encodes a protein of 103 amino acids. However, the C-terminal region obtained from the cDNA clone is -Val-Arg-Leu-Asn-Arg-Ser-Leu, whereas the C-terminal region deduced from the genomic DNA appears to contain -Ala-Ser-Ile-Lys-Ala-Asn-Leu. This indicates that S. pombe cells contain two kinds of TRX genes which have dissimilar amino acid sequences only at the C-terminal regions. The heterologous TRX 1C produced from the cDNA clone could be used as a subunit of T7 DNA polymerase, while the TRX 1G from the genomic DNA could not. The upstream sequence and the region encoding the N-terminal 18 amino acids of the genomic DNA were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357 to generate the fusion plasmid pYKT24. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by hydrogen peroxide, menadione and aluminum chloride. It indicates that the expression of the cloned TRX gene is induced by oxidative stress.     

Restriction mapping of a chlorobenzoate degradative plasmid and molecular cloning of the degradative genes

The genes for the degradation of 3-chlorobenzoic acid ( 3Cba ) are present in a 110-kb plasmid pAC27 . A circular map is established using the restriction endonucleases EcoRI, HindIII and Bg/II. The map is derived from the results obtained by partial restriction digestion, complete single and double restriction digestion and finally confirmed with hybridization of the digested fragments using different purified fragments as probes. The 3Cba degradative genes are found to be clustered in one region of the map (EcoRI fragment A) as judged by molecular cloning with a broad host range vector pLAFRI . A portion of the 3Cba degradative gene cluster appears to undergo ready recombination with the chromosome, even in a recA host, suggesting the probable transposable nature of such gene cluster.     

Euglena gracilis chloroplast transfer RNA transcription units. Nucleotide sequence analysis of a tRNAThr-tRNAGly-tRNAMet-tRNASer-tRNAGln gene cluster

The arrangement and the nucleotide sequence of the tRNA genes in the 2.0-kilobase-pair EcoRI restriction fragment EcoQ of Euglena gracilis Klebs, strain Z Pringsheim chloroplast DNA have been determined. This fragment, cloned in pBR325 to form the plasmid pEZC300, contains five tRNA genes. The DNA insert of this plasmid, a known tRNA gene locus (Orozco, E.M., Jr., and Hallick, R.B. (1982) J. Biol. Chem. 257, 3258-3264) has been mapped by Southern gel analysis using a 32P-labeled oligodeoxynucleotide tRNA gene probe. The DNA sequence of 870 base pairs (bp) from EcoQ containing the entire tRNA gene locus was determined. The organization of this tRNA gene cluster on the E. gracilis chloroplast chromosome is tRNAUUGGln-14-BP spacer-RNAGCUSer-175-bp spacer-tRNACAUMet-12-bp spacer-tRNAGCCGly-5-bp spacer-tRNAUGUThr. The tRNAUUGGln and tRNAGCUSer gene sequences are of the opposite polarity as the other three gene sequences, but of the same polarity as the rRNA genes. The tRNAMet gene is a putative initiator tRNA. The five tRNA genes are separated and flanked by A-T-rich spacer sequences. This gene arrangement is consistent with the model that E. gracilis chloroplast tRNA genes are transcribed into multicistronic tRNA precursors. The DNA sequences have been used to deduce the primary and secondary structures of the tRNAs.     

Characterization of two Azospirillum brasilense Sp7 plasmid genes homologous to Rhizobium meliloti nodPQ

Bacteria belonging to the Azospirillum genus are nitrogen fixers that colonize the roots of grasses, but do not cause the formation of differentiated structures. Sequences from total DNA of several Azospirillum strains are homologous to restriction fragments containing Rhizobium meliloti nodulation genes. A 10-kilobase (kb) EcoRI fragment from A. brasilense Sp7, sharing homology with a 6.8-kb EcoRI fragment carrying nodGEFH and part of nodP of R. meliloti 41, was cloned in pUC18 to yield pAB502. The nucleotide sequence of a 3.5-kb EcoRI-SmaI fragment of the pAB502 insert revealed 60% homology with R. meliloti nodP and nodQ genes. The nodP gene product shares no homology to any known protein sequence. The Azospirillum nodQ gene product shares homology with a family of initiation and elongation factors as does the R. meliloti nodQ gene product. Since the nodQ gene overlaps the nodP gene, the two genes might be cotranscribed. Azospirillum contains large plasmids, and the nodPQ genes were found on the 90-MDa plasmid (p90). A translational nodP-lacZ fusion was constructed in the broad host range plasmid pGD926. No beta-galactosidase activity was detected in Escherichia coli, but the fusion was functional in Azospirillum and constitutively expressed. Deletions and mutations of nodPQ did not modify growth, nitrogen fixation, or interaction with wheat seedlings.     

Characteristics of hybrid plasmids carrying the genes of colicinogenic plasmid Collb-P9 responsible for colicin Ib synthesis and inhibition of phage T5 development

The EcoRI and HindII restriction endonucleases and pBR325 vector plasmid were used to obtain a set of hybrid plasmids containing ColIb-P9 fragments carrying the characters for colicin Ib synthesis and immunity and the ability to inhibit T5 phage growth. The genes responsible for colicin synthesis and immunity are closely linked and localized in the EcoRI fragment with a molecular weight of 1.85 MD (pIV41) or in the HindII fragment of 2.4 MD (pIV1). The clones containing these plasmids show an increased level of both spontaneous and mitomycin C-induced colicin synthesis and an increased level of immunity due to a larger dosage of the genes. The genes controlling T5 growth inhibition are localized in other restriction fragments of ColIb DNA: the EcoRI fragment of 1.45 MD (pIV7) and the HindII fragment of 4.3 MD (pIV5). We have demonstrated by means of hybrid plasmids that T5 growth inhibition is not connected with the colicin Ib synthesized in infected cells and is controlled by other specific product(s) of the ColIb plasmid genes. T5 phage growth was as efficient in clones containing plasmids with cloned colicin Ib genes as in a strain without plasmids. An investigation of the expression of the genes inhibiting T5 phage growth in an in vitro protein synthesis system has revealed a protein with a molecular weight of 36 000 which seems to take part in the process.     

环状芽孢杆菌α—羟基—γ—氨丁酰酰化酶基因的克隆和表达

本文报道了以质粒pUB110为载体,以枯草芽孢杆菌(Bacillus subtilis 168)作为受体菌,对丁酰苷菌素产生菌(Bacillus circulans NRRL-B3312)总DNA进行了鸟枪克隆,在所获得的转化子中,No.733转化子经薄层层析,生物显迹和质谱分析表明,它具有将卡那霉素A生物转化成为丁胺卡那霉素的能力,说明该转化子所含重组质粒pUBC733的插入片段中含有a-羟基-r-氨丁酰(HABA)酰化酶基因,HABA酰化酶基因已经在枯草芽孢杆菌中获得了克隆和表达。该重组质粒分子量为7.3kb,插入片段为2.8kb,经Southern分子杂交确证此片段确来源于环状芽孢杆菌,已构建了该质粒限制性内切酶图谱。