Suppression of the Escherichia coli dnaA46 mutation by amplification of the groES and groEL genes

Summary A hybrid phage (Sda1), containing an 8.1 kb EcoRI DNA fragment from the Escherichia coli chromosome, was selected on the basis of its ability to suppress bacterial thermosensitivity caused by the dnaA46 mutation. We have shown that this suppression is due to a recA ⁺-dependent amplification of the 8.1 kb fragment; consistent with this observation, cloning of the 8.1 kb fragment into a high copy number plasmid (pBR325) leads also to suppression of dnaA46. In the suppressed strains growing at high temperature, bidirectional replication starts in or near the oriC region and requires the presence of the DnaA polypeptide. These findings suggest that the overproduction of a gene product(s), encoded by the cloned 8.1 kb fragment, can restore dnaA-dependent initiation of replication at high temperature in the oriC region. Genetic mapping shows that the groES (mopB) and groEL (mopA) genes are located on the 8.1 kb suppressor fragment. Further analysis, including in vitro mutagenesis and subcloning, demonstrates that the amplification of the groES and groEL genes is both necessary and sufficient to suppress the temperature sensitive phenotype of the dnaA46 mutation.     

Cloning and expression in Escherichia coli K-12 of the biosynthetic dehydroquinase function of the arom cluster gene from the eucaryote, Aspergillus nidulans

Summary A 1.35 Md DNA HindIII fragment containing part of the arom gene cluster or cluster gene of Aspergillus nidulans encoding biosynthetic dehydroquinase (5-dehydroquinate hydrolyase) has been cloned in plasmid pBR322 on the basis of functional expression in Escherichia coli. The fungal fragment on pBR322, designated pHK29, complements a corresponding E. coli dehydroquinase structural gene (aroD) mutation. pHK29 contains one BamHI, HpaII, PstI, SmaI, XhoI and surprisingly, one HindIII site since pHK29 hybrid Aspergillus DNA is a HindIII fragment itself. The biosynthetic dehydroquinase activity extracted from E. coli strains, containing pHK29, had properties similar to those of the enzyme activity from Aspergillus. The protein specified by pHK29 appears to be 80 Kd. No increase of dehydroquinase activity was found in polynucleotide phosphorylase deficient strains (pnp) of E. coli.Standard Abbreviations Used SSC Standard saline citrate (3 M Sodium Chloride, 0.15 M Sodium citrate) - EDTA Ethylenediaminetetra acetic acid - DTT Dithiothreitol - PMSF Phenyl methyl sulphonyl fluoride - TEMED N N N N, Tetramethylethylenediamine - Md Megadaltons - Kd Kilodaltons     

Cloning of bacterial DNA replication genes in bacteriophage lambda

Summary Recombinant lambda phages containing the genes for dnaZ protein (the subunit of DNA polymerse III holoenzyme), primase (dnaG protein) and dnaC protein from Escherichi coli and Salmonella typhimurium were isolated. Each gene cloned from S. typhimurium has extensive DNA sequence homology to the corresponding E. coli gene. Clones selected by complementation of a dnaA temperature-sensitive mutant appear similar to other isolated suppressors of dnaA (Projan and Wechsler 1981). Derivatives of each cloned fragment suitable for overproduction of the protein were constructed. Of those tested, only the phage containing the E. coli dnaZ gene resulted in significant overproduction.Abbreviations DTT dithiothreitol - Ec Escherichia coli - EDTA ethylene diamine tetra acetic acid - kb kilobase 1,000 bases or base-pairs - moi multiplicity of infection - pol I E. coli DNA polymerase I - pol III holoenzyme E. coli DNA polymerase III holoenzyme - pri dnaG, primase-coding gene - SSB single-strand binding protein - St Salmonella typhimurium - sup gene coding for suppressor - ts temperature-sensitive     

Cloning and characterization of the alkaline phosphatase positive regulator gene (phoB) of Escherichia coli

Summary The positive regulator gene (phoB) for alkaline phosphatase of Escherichia coli was cloned into the EcoRI site of pBR322 from the E. coli chromosome by a shotgun method. phoB was then constructed in vitro by replacing the C fragment of gtC by the phoB chromosomal fragment obtained from the hybrid plasmid. When the phoB mutant was lysogenized by phoB, the lysogen became PhoB⁺. The integration site of the phage was identified by P1 phage transduction to be around phoB site on the chromosome. From these results, we conclude that the cloned gene is phoB and not a gene which suppresses phenotypically phoB mutation when it is in a multi-copy state. The restriction map was constructed. Based on this information, several PhoB deletion plasmids and smaller PhoB⁺ plasmids were constructed in vitro. By examining PhoB phenotype when these plasmids were introduced into phoB mutant, we could define the phoB gene locus in 2 kb on the restriction map of the cloned chromosomal fragment. Cells carrying the multi-copy phoB gene produced alkaline phosphatase qualitatively under normal phosphate regulation. The phoB gene product was identified by the maxicell method as a protein with a molecular weight of approximately 31,000 daltons.     

Cloning and mapping of the RAD50 gene of Saccharomyces cerevisiae

Summary The RAD50 gene was cloned as a 4.8 kb fragment in the 2 derived plasmid pFL1. The gene resides in a 3.9 kb segment that was subcloned into the plasmid YRp7. The cloned gene complements the deficiency caused by the rad50-1 mutation with respect to -rays, MMS resistance and UV-induced mitotic recombination. Restoration of the Rad⁺ phenotype occurs when the cloned gene is on a freely replicating multiple-copy plasmid or in the integrated form.Mapping of the cloned gene following integration of the 2 plasmid, and of the subclone in plasmid YRp7, showed it to be located on the left arm of chromosome XIV. Tetrad analysis of various crosses involving tow different strains carrying rad50-1 showed the mutation to map next to pet2 on chromosome XIV, and not on the right arm of chromsome IV, as previously published.     

Cloning of a gene involved in cellulose biosynthesis in Acetobacter xylinum: Complementation of cellulose-negative mutants by the UDPG pyrophosphorylase structural gene

Summary Three cellulose-negative (Cel^-) mutants of Acetobacter xylinum strain ATCC 23768 were complemented by a cloned 2.8 kb DNA fragment from the wild type. Biochemical analysis of the mutants showed that they were deficient in the enzyme uridine 5-diphosphoglucose (UDPG) pyrophosphorylase. The analysis also showed that the mutants could synthesize (1-4)-glucan in vitro from UDPG, but not in vivo from glucose. This result was expected, since UDPG is known to be the precursor for cellulose synthesis in A. xylinum. In order to analyze the function of the cloned gene in more detail, its biological activity in Escherichia coli was studied. These experiments showed that the cloned fragment could be used to complement an E. coli mutant deficient in the structural gene for UDPG pyrophosphorylase. It is therefore clear that the cloned fragment must contain this gene from A. xylinum. This is to our knowledge the first example of the cloning of a gene with a known function in cellulose biosynthesis from any organism, and we suggest the gene be designated celA.     

Molecular cloning and nucleotide sequence of the mutT mutator of Escherichia coli that causes A:T to C:G transversion

Summary The Escherichia coli mutator gene mutT, which causes A:TC:G transversion, was cloned in pBR 322. mutT ⁺ plasmids carry a 0.9 kb PvuII DNA fragment derived from the E. coli chromosome. Specific labelling of plasmid-encoded proteins by the maxicell method revealed that mutT codes for a polypeptide of about 15,000 daltons. The protein was overproduced when the mutT gene was placed under the control of the lac regulatory region on a multicopy runaway plasmid. The nucleotide sequence of the mutT gene was determined by the dideoxy method.Abbreviations Ap ampicillin - IPTG isopropyl--d-thiogalactopyranoside - kb kilobase pair(s) - kDa kilodalton(s) - SDS sodium dodecyl sulphate - Tc tetracycline     

Cloning and characterization of the hemA gene for synthesis of δ-aminolevulinic acid in Xanthomonas campestris pv. phaseoli

The gene from Xanthomonas campestris pv. phaseoli that is involved in the C5 pathway of -amino-levulinic acid (ALA) of Escherichia coli. Subcloning of deletion fragments from the initial 2.5-kilobase (kb) chromosomal fragment allowed the isolation of a 1.6-kb fragment that could complement the hemM mutation. Nucleotide sequence analysis of the 1.6-kb DNA fragment revealed an open reading frame that encodes a polypeptide of 426 amino acid residues, and the deduced molecular mass of this polypeptide is 46768 Da. The amino acid sequence shows a high degree of homology of the HemA protein, which is glutamyl-tRNA reductase, to other organisms. Thus, we examined the complementation test of the cloned gene from Xanthomonas with a hemA mutation of E. coli and found that the gene complemented the hemA mutation. These results suggest that the cloned gene is hemA and the gene from Xanthomonas also complements both hemA and hemM mutations, as in the case of the E. coli hemA. Correspondence to: Y. Murooka     

Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase

Summary The Bacillus subtilis cdd gene encoding cytidine/2-deoxycytidine deaminase has been located by transduction at approximately 225 degrees on the chromosome, and the gene order rpC-lys-cdd-aroD was established. The gene was isolated from a library of B. subtilis DNA cloned in D69 by complementation of an Escherichia coli cdd mutation. Minicell experiments revealed a molecular mass of 14000 dalton for the cytidine deaminase subunit encoded by the cloned DNA fragment. The molecular weight of the native enzyme was determined to be 58000, suggesting that it consists of four identical subunits. The nucleotide sequence of 1170 bp, including the cdd gene, was determined. An open reading frame encoding a polypeptide with a calculated molecular mass of 14800 dalton was deduced to be the coding region for cdd. The deduced amino acid composition of the 136-amino acid-long subunit shows that it contains six cysteine residues. A computer search in the GenBank DNA sequence library revealed that the 476 bp HindIII fragment containing the putative promoter region and the first ten codons of cdd is identical to the P43 promoter-containing fragment previously isolated by Wang and Doi (1984). They showed that the fragment contained overlapping promoters transcribed by B. subtilis 43 and 37 RNA polymerase holoenzymes during growth and stationary phase.Abbreviations SDS sodium dodecyl sulphate - Ap ampicillin resistance - Tet^r tetracycline resistance - Km^r kanamycin resistance     

Molecular cloning of the phosphate (inorganic) transport (pit) gene of Escherichia coli K12. Identification of the pit+ gene product and physical mapping of the pit-gor region of the chromosome

Summary The pit ⁺ gene, encoding the phosphate (inorganic) transport system of Escherichia coli, was isolated from a library of E. coli genes inserted in the cosmid vector pHC79. A 25.5-kb chromosomal DNA fragment was shown also to carry the gor locus encoding glutathione oxidoreductase. Physical mapping placed the two genes about 10 kb apart, confirming bacteriophage P1 mapping of the 77-min region. Subcloning and deletion analysis indicated that the entire pit ⁺ gene was located within a 2.2-kb Sal1-Ava1 fragment. The pit ⁺ gene product was identified by SDS-polyacrylamide gel electrophoresis as a 39-kdal inner membrane protein by two methods: (i) ³⁵S-methionine-labelling of minicells carrying pit ⁺ plasmids or plasmids from which all or part of the pit ⁺ gene was deleted. (ii) Overproduction of the Pit protein using a thermoinducible runaway replication plasmid.Complementation of the pit-1 mutant allele using a unit-copy-number pit ⁺ plasmid indicated that the pit-1 mutation is recessive.Strains carrying a multicopy pit ⁺ plasmid show a 10-fold increase in the initial rate of phosphate uptake; however there is no change in the steady-state level of ³²Pi accumulation.Abbreviations kb kilobase-pairs - kdal kilodalton - Pi inorganic phosphate - G3P sn-glycerol-3-phosphate - LB Luria broth - Tc tetracycline - Cm chloramphenicol - Ap ampicillin - UV ultraviolet light - TE 10 mM Tris.HCl, pH 8.0, 1 mM EDTA - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Cloning and characterization of the alkA gene of Escherichia coli that encodes 3-methyladenine DNA glycosylase II

By in vitro recombination we have constructed hybrid plasmids which can suppress the increased methylmethane sulfonate sensitivity caused by the alkA1 mutation in Escherichia coli. Since the cloned DNA fragment was mapped at 44 to 45 min of the E. coli K12 genetic map, an area where the alkA gene is located, we conclude that the cloned DNA fragment contains the alkA gene itself but not other gene(s) that suppresses the alkA mutation. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the alkA codes for a polypeptide whose molecular weight is about 30,000. When cells harboring the alkA+ plasmids were grown in the presence of low doses of a simple alkylating agent (adapted condition), the activity of 3-methyladenine DNA glycosylase II was increased. The enzyme activity was copurified with the Mr 30,000 polypeptide. These results indicate that the alkA gene codes for 3-methyladenine DNA glycosylase II. Taking advantage of overproduction of the alkA protein in adapted cells that harbor multicopy plasmids carrying the alkA+ gene, 3-methyladenine DNA glycosylase II has been purified to apparent physical homogeneity.     

Isolation of a recombinant lambda phage carrying nusA and surrounding region of the Escherichia coli K-12 chromosome

Summary A recombinant bacteriophage lambda, argG-6, has been isolated which carries the argG gene and neighbouring loci on an EcoRI-generated 15.5 Kb DNA fragment from the Escherichia coli chromosome. The locations of the argG, nusA and pnp genes on the 15.5 Kb DNA fragment have been determined. In the case of nusA, a Tn5 insertion and sub-cloning of restriction fragments were used to locate the gene. The gene products of nusA and pnp have been identified on one- and two-dimensional polyacrylamide gels. The clockwise gene order was found to be argG-nusA-pnp.     

Cloning of Bacillus subtilis leucina A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli.

Summary The leucine genes of Bacillus subtilis have been cloned directly from the chromosomal DNA into Escherichia coli leuB cells by selection for the Leu⁺ phenotype using RSF2124 as a vector plasmid. The hybrid plasmid designated RSF2124-B·leu contained a 4.2 megadalton fragment derived from B. subtilis DNA, including the leu genes. The fragment had one site susceptible to EcoRI^* and another site susceptible to BamNI endonuclease. Among the three fragments produced by EcoRI^* and BamNI endonucleases, the 1.2 megadalton fragment had the ability to transform B. subtilis leuA, leuB and leuC auxotrophs to leu ⁺. However, B. subtilis ilvB and ilvC auxotrophs were not rescued even by the whole 4.2 megadalton fragment present in the hybrid plasmid. -Isopropylmalate dehydrogenase (leuB gene product) activity found in E. coli cells containing the hybrid plasmid was about 60% of that in E. coli wild type cells, despite the high copy number (7.8) of the plasmid per chromosome observed.     

A DNA sequence from Dictyostelium discoideum complements ura3 and ura5 mutations of Saccharomyces cerevisiae

Summary A 3.7 kilobase fragment of Dictyostelium discoideum genomic DNA has been cloned by its ability to complement a yeast ura5 mutation affecting the activity of orotidine-5-phosphate carboxy-lyase (EC 4.1.1.23). This fragment also complements a yeast ura5 mutation that leads to a defect in orotate phosphoribosyl transferase (EC 2.4.2.10). The orotidine-5-phosphate carboxy-lyase and the orotate phosphoribosyl transferase activities that result from Dictyostelium gene expression in yeast have been detected. The size of the DNA required for both complementations has been localised to a segment of less than 2 kb. A unique Dictyostelium RNA species of 1,600 base pairs hybridises to this fragment. In vitro deletions in this fragment lead to the simultaneous loss of the two activities. The two enzymatic activities coelute as a protein of 120.000 daltons during gel filtration of a Dictyostelium extract. These results favour the existence, on the cloned Dictyostelium DNA fragment, of a unique structural gene which codes for a bifunctional enzyme carrying the two activities, orotidine-5-phosphate carboxy-lyase and orotate phosphoribosyl transferase.Abbreviations bp basepair - kb kilobasepair - MOPS Morpholino propane sulfonic acid     

Molecular cloning of the T4 genome; organization and expression of the frd--DNA ligase region

Summary derivatives including the thymidylate synthetase (td) gene of T4 were selected by their ability to substitute for the thyA gene of E. coli. Two HindIII fragments of T4 DNA, but only one EcoRI fragment, are required for a functional td gene; one of the HindIII fragments includes a functional frd gene. The organisation of the EcoRI and HindIII fragments in the td region and their orientation with respect to the T4 genome have been deduced from genetic, physical, and functional evidence. The T4 genes can be transcribed from phage promoters and the T4td derivatives include genes specifying five T4 polypeptides. Three of these are identified as the products of the frd, td, and nrdA genes; two, neither of which appears to be the nrdB gene product, remain to be identified. Some td phages yield lysogens of thyA bacteria which are thymine-independent and some frd phages yield trimethoprim-resistant lysogens, indicating that the td and frd genes can be transcribed from included T4 DNA sequences. EcoRI fragments of DNA from the td and lig regions, used as probes, identified a single large HindIII fragment that joins the HindIII fragment carrying the DNA ligase gene to that carrying the td gene. Since this fragment, which must include genes coding for RNA ligase and polynucleotide kinase, could not be recovered in either phage or plasmid vectors, a derivative of it was used to identify the EcoRI fragments located between the td and DNA ligase genes. The order of these fragments within the T4 genome was deduced and all but one of them cloned in a vector. As none of these recombinants rescued T4 phage having mutations within the RNA ligase gene, the missing fragment may include this gene. Three adjacent EcoRI fragments, each of which has been cloned, are missing in a mutant of T4 deleted for the polynucleotide kinase gene.     

Cloning and characterization of genes coding for ribosomal RNA inCampylobacter jejuni

The DNA fragments coding for ribosomal RNA inCampylobacter jejuni have been cloned from a genomic library ofC. jejuni constructed inEscherichia coli. Clones carrying DNA Sequences for rRNA were identified by hybridization of 5-end-labeled rRNA fromC. jejuni to colony blots of transformants from this gene library. Cloned DNA sequences homologous to each of 5S, 16S, and 23S rRNA were idenfified by hybridization of labeled plasmid DNA to Northern blots of rRNA. The gene coding for 23S rRNA was found to be located on a 5.5kb HindIII fragment, while the 5S and 16S rRNA genes were on HindIII fragments of 1.65 and 1.7 kb, respecitively. The DNA fragment containing the 16S rRNA gene was characterized by restriction endonuclease mapping, and the location of the 16S rRNA gene on this fragment was determined by hybridization of 5-end-labeled rRNA to restriction fragments and also by DNA sequence determination. It appears that the major portion of the coding region for 16S rRNA is located on the 1.7-kb HindIII fragment, while a small portion is carried on an adjacent HindIII fragment of 7.5 kb. Cloned rRNA genes fromC. jejuni were used to study the organization of the rDNA inC. jejuni and other members of the genùsCampylobacter.     

Molecular cloning and expression ofBacillus subtilis bglS gene inSaccharomyces cerevisiae

A 2.7-kb EcoRI DNA fragment carrying aBacillus subtilis endo--1,3-1,4-glucanase gene (bglS) from theE. coli plasmid pFG1 was cloned into anEscherichia coli/yeast shuttle vector to construct a hybrid plasmid YCSH. The hybrid plasmid was used to transformSaccharomyces cerevisiae, and thebglS gene was expressed. Variation between levels ofbglS gene expression inS. cerevisiae was about 2.3-fold, depending on the orientation of the 2.7-kb DNA fragment. Assay of substrate specificity and optimal pH of the enzyme demonstrated that the enzyme encoded by YCSH (bglS) was identical with that found inB. subtilis, but the expression level ofbglS gene inS. cerevisiae (YCSH) was much lower than that inE. coli (YCSH).     

Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp. cellulosa

Summary A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in bacteriophage 47.1 and recombinants expressing carboxymethylcellulase (CMCase) activity isolated. A 7.3 kb partial EcoRI fragment, a 9.4 kb EcoRI fragment and a 5.8 kb HindIII fragment were subcloned from three different phages into pUC18 to yield recombinant plasmids pJHH1, pJHH3 and pGJH2 respectively. Cells of Escherichia coli harbouring these plasmids expressed CMCase activity. The positions of the CMCase genes in the three plasmids were determined by subcloning and transposon mutagenesis. pJHH1 contained two distinct DNA regions encoding CMCases, which were controlled by the same promoter. All four cloned enzymes cleaved p-nitrophenyl--D-glucopyranoside, although at a very low rate, but none exhibited exoglucanase activity. In common with other extracellular enzymes cloned in E. coli, all the CMCases were exported to the periplasmic space in the enteric bacterium. The carboxymethylcellulase genes encoded by pJHH1 and pJHH3, were subject to glucose repression in E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - Sm^r resistance to streptomycin - Km^r resistance to kanamycin - Ap^r resistance to ampicillin - Tc^r resistance to tetracycline - Cm^r resistance to chloramphenicol - CMCase carboxymethylcellulase     

Sequence divergence of an archaebacterial gene cloned from a mesophilic and a thermophilic methanogen

Summary A 1.6-kb fragment of DNA from the thermophilic, methane-producing, anaerobic archaebacteriumMethanobacterium thermoautotrophicum H has been cloned and sequenced. This DNA complements mutations in both the purE₁ and purE₂ loci ofEscherichia coli. The sequence of theM. thermoautotrophicum DNA predicts that complementation inE. coli results from the synthesis of a polypeptide with a molecular weight of 36,249. A polypeptide apparently of this molecular weight is synthesized inE. coli minicells containing recombinant plasmids that carry the cloned fragment of methanogen DNA. We have previously cloned and sequenced a purE-complementing gene from the mesophilic methanogenMethanobrevibacter smithii. The two methanogen-derived purE-complementing genes are 53% homologous and encode polypeptides that are 45% homologous in their amino acid sequences but would be 74% homologous if conservative amino acid substitutions were considered as maintaining sequence homology. The genome ofM. thermoautotrophicum has a molar G+C content of 49.7%, whereas the genome ofM. smithii is 30.6% G+C. Conservation of encoded amino acids while accommodating the very different G+C contents is accomplished by use of different codons that encode the same amino acid. The majority of base changes occur at the third codon position. The intergenic regions of the clonedM. thermoautotrophicum DNA contain sequences previously identified as ribosome binding sites and as putative methanogen promoters. Although the two purE-complementing genes are apparently derived from a common ancestor, only the gene fromM. smithii maintains a codon usage that conforms to the RNY rule.     

Nucleotide sequence of the region encompassing the int gene of a cryptic prophage and the dna Y gene flanked by a curved DNA sequence of Escherichia coli K12

Summary The nucleotide sequence of a 2.5 kb region encompassing a curved DNA segment (BENT-9) randomly cloned from the total Escherichia coli chromosome was determined. This region was found to contain the dnaY gene encoding a transfer RNA. The curved DNA structure was demonstrated to be located just upstream of the dnaY promoter. The results of sequencing further revealed that the int gene of a cryptic prophage, qsr, which has been shown to be present in the E. coli genome, is located next to the dnaY gene.