Nucleotide sequence of the genetic loci encoding subunits of Bradyrhizobium japonicum uptake hydrogenase.

An indispensable part of the hydrogen-recycling system in Bradyrhizobium japonicum is the uptake hydrogenase, which is composed of 34.5- and 65.9-kDa subunits. The gene encoding the large subunit is located on a 5.9-kilobase fragment of the H2-uptake-complementing cosmid pHU52 [Zuber, M., Harker, A.R., Sultana, M.A. & Evans, H.J. (1986) Proc. Natl. Acad. Sci. USA 83, 7668-7672]. We have now determined that the structural genes for both subunits are present on this fragment. Two open reading frames are present that correspond in size and deduced amino acid sequence to the hydrogenase subunits, except that the small-subunit coding region contains a leader peptide of 46 amino acids. The two genes are separated by a 32-nucleotide intergenic region and likely constitute an operon. Comparison of the deduced amino acid sequences of the B. japonicum genes with those from Desulfovibrio gigas, Desulfovibrio baculatus, and Rhodobacter capsulatus indicates significant sequence identity.     

Cloning and mapping of a gene for translational initiation factor IF2 in Escherichia coli.

A novel method, not relying on genetic complementation of a mutation, was used to clone a gene for translational initiation factor IF2. Two clones from a cosmid library of total Escherichia coli DNA were isolated for their ability to overproduce IF2 in vivo as determined by quantitative immunoblotting. "Maxicell" analysis of cosmid-encoded proteins and specific immune precipitation of the labeled proteins showed that the structural gene for IF2 (inf B) had been cloned. Subcloning fragments from the original cosmids located the inf B gene to a 4.8-kilobase pair HindIII/BamHI fragment. This fragment has been inserted into an integration-deficient recombinant lambda phage that lysogenizes by homology. By mapping the point of lysogenization on the E. coli chromosome, inf B has been located at 68 min, very close to argG, nusA, rpsO, and pnp. Because the gene for initiation factor IF3 is located at 38 min on the chromosome, the genes for translational initiation factors are not grouped together.     

Plasmid-directed synthesis of enzymes required for D-mannitol transport and utilization in Escherichia coli.

A transformant Escherichia coli colony bank [Clarke, L. & Carbon, J. (1976) Cell 9, 91-99] has been screened for hybrid ColE1 plasmids carrying the genes for D-mannitol utilization. Two of the plasmids, pLC11-7 and pLC15-48, were shown to contain the mannitol operon, which includes the structural genes for the mannitol-specific enzyme II of the phosphotransferase system and mannitol-1-phosphate dehydrogenase. One E. coli strain harboring plasmid pLC15-48 overproduced mannitol-1-phosphate dehydrogenase activity 4- to 5-fold. However, there was no corresponding increase in mannitol enzyme II activity. Plasmid pLC15-48 was shown to direct the synthesis of two polypeptides in E. coli minicells in the presence of cyclic AMP and mannitol. The larger (Mr = 60,000) was membrane bound and was specifically precipitated by antibody directed against purified mannitol-specific enzyme II. The smaller (Mr = 40,000) was soluble and had an electrophoretic mobility indistinguishable from that of the major component in a partially purified mannitol-1-phosphate dehydrogenase preparation. These data are consistent with previous genetic studies of the mannitol locus and confirm an independent conclusion [Jacobson, G. R., Lee, C. A. & Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 249-252] that mannitol enzyme II consists of a single type of polypeptide chain that has a Mr of 60,000. The plasmid pLC15-48 DNA was characterized by mapping of restriction endonuclease cleavage sites.     

Extensive sequence homology in the DNA coding for elongation factor Tu from Escherichia coli and the Chlamydomonas reinhardtii chloroplast.

Considerable DNA sequence homology can be detected between the Escherichia coli genes coding for translational components and Chlamydomonas reinhardtii chloroplast DNA. Labeled chloroplast DNA was found to hybridize to restriction fragments of the transducing phage lambda fus3 that code for elongation factor Tu. The chloroplast probe also reacts with fragments coding for ribosomal proteins carried by this phage. The region homologous to the elongation factor genes was located on the physical map of the chloroplast genome by probing restriction fragments of chloroplast DNA with cloned fragments, labeled in vitro, carrying the E. coli elongation factor Tu genes.     

lambda altSF: a phage variant that acquired the ability to substitute specific sets of genes at high frequency

We report the isolation of lambda altSF, a variant of Escherichia coli phage lambda that substitutes sets of genes at high frequency. Two forms of the variant phage have been studied: lambda altSF lambda, which exhibits the immunity (repressor recognition) of phage lambda, and lambda altSF22, which exhibits the immunity of Salmonella phage P22. Lysates made from single plaques of lambda altSF lambda contain 10-30% phage of the P22 form. Similarly, lysates from single plaques of lambda altSF22 contain as much as 1% phage of the lambda form. Heteroduplex analyses reveal the following features of the lambda altSF chromosomes: (i) each form has the immunity genes appropriate to its immune phenotype, (ii) the substituted segments include genes involved in regulation and replication, and (iii) the alt phages have unusual additions and substitutions of DNA not normally found associated with either immunity region. In the case of lambda altSF lambda, there is a small insertion in the region of the cI gene. Because revertants that lose this inserted DNA concomitantly lose the ability to substitute, we conclude that the insertion plays a role in the substitution process. In the case of change from lambda altSF lambda to lambda altSF22, the substituting P22 genes are derived from the E. coli host. We have identified a set of Salmonella phage P22 genes in a standard nonlysogenic strain of E. coli K-12 that is apparently carried in a silent form. The reason for this lack of expression is not obvious, because this P22 material includes structural genes and associated promoters and is potentially active. When this set of genes substitutes for the analogous set of genetic material on the genome of lambda altSF lambda, the P22 genes are expressed in a normal manner.     

Cloned genomic DNA sequences from Mycoplasma hyorhinis encoding antigens expressed in Escherichia coli.

A library of cloned Mycoplasma hyorhinis genomic sequences was constructed by incorporation of EcoRI digestion fragments of mycoplasma DNA into the lambda Charon 4A bacteriophage vector. Immunological screening of recombinant phage plaques identified clones containing genes encoding mycoplasma antigenic structures expressed in an Escherichia coli host. Two such recombinant phage isolates, lambda Ch4A-MhrG1 and lambda Ch4A-MhrG28, were defined and found to contain distinct genomic sequences by analysis of restriction endonuclease fragments. Inoculation of mice with recombinant gene products from lambda Ch4A-MhrG1 yielded antiserum selectively recognizing a Mr 29,500 trypsin-sensitive mycoplasma constituent. This established a means for producing selected immunogenic mycoplasma component in a bacterial host. The cloned genomic sequences of M. hyorhinis encoding expressed mycoplasma antigens represent molecular probes that can be characterized both by specific DNA sequences and by the antigenic structure of corresponding gene products. These genomic fragments define initial physical markers of the M. hyorhinis genome and may be useful in assessing antigenic and molecular genetic relationships within the genus Mycoplasma and among other members of the class Mollicutes.     

Viable Molecular Hybrids of Bacteriophage Lambda and Eukaryotic DNA

A bacteriophage lambda strain has been constructed and a method developed by which DNA from potentially any source can be covalently inserted through EcoRI cohesive ends into the middle of the lambda DNA. These hybrid DNAs can infect nonrestricting Escherichia coli cells and can then propagate as plaque-forming phage. A unique feature of this lambda strain is that extra DNA in the middle of its genome is required for plaque formation. A large number of such phages have been produced with E. coli DNA and Drosophila melanogaster DNA.     

In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase.

DNA from lambdagt-lambdaB bacteriophage was cleaved with EcoRI endonuclease and fragments from EcoRI-digested E. coli DNA were inserted. This DNA was used to infect E. coli, and phages containing the gene for DNA ligase were isolated by genetic selection. Two different hybrids were found with the same E. coli segment inserted in opposite orientations. Both hybrids produced similar levels of ligase as measured in crude extracts of infected cells.     

The rnh gene is essential for growth of Escherichia coli.

We have determined that a functional gene coding for ribonuclease H seems to be essential for cell growth in Escherichia coli. A strain was made with two copies of the rnh gene by lysogenizing an E. coli strain with a lambda phage bearing a copy of the rnh gene. Inactivation of one of the two copies of the rnh gene was accomplished by transformation with a linear DNA molecule that had the gene for chloramphenicol acetyltransferase inserted near the middle of the rnh gene. In recombinants that had an inactive gene replacing the normal chromosomal rnh gene, the lambda rnh prophage supplies an intact functional copy of the rnh gene. Curing the cells of the lambda rnh prophage left the cell with an inactive rnh gene and resulted in cell death. An intact functional rnh gene provided on a plasmid permits normal curing, and cured survivors were readily obtained. The technique described is probably generally applicable for assessing the requirement for other E. coli genes.     

Evidence that a high molecular weight replicative DNA polymerase is conserved during evolution.

Using a technique developed recently to detect DNA polymerase activity in situ after NaDodSO4 gel electrophoresis (Spanos, A., Sedgwick, S. G., Yarranton, g. T., Hübscher, U. & Banks, G. R. (1981) Nucleic Acids Res. 9, 1825-1839), we present evidence that a high Mr (greater than or equal to 125,000) polypeptide is responsible for chromosomal DNA replication in prokaryotes, lower eukaryotes and high eukaryotes. Not only extracts from Escherichia coli, Ustilago maydis, Drosophila melanogaster, rat neurones, calf thymus, human fibroblast, and HeLa cells possess such high Mr activities, but also highly purified E. coli DNA polymerase III core enzyme, U. maydis DNA polymerase, and D. melanogaster embryo and calf thymus DNA alpha polymerases. The evidence that these activities are responsible for chromosomal DNA replication is genetical (E. coli, U. maydis, and D. melanogaster); also, the high Mr activity disappears from rat neurones during differentiation from an actively dividing precursor cell to a postmitotically mature neurone. Furthermore, when limited proteolysis is allowed to occur, a defined and remarkably similar pattern of intermediate Mr activities is generated in lower eukaryotic and high eukaryotic extracts and, to some extent, in prokaryotic extracts. In higher eukaryotic extracts, a low Mr activity of approximately 35,000 is also generated. Protease inhibitors can retard formation of these catalytically active proteolytic fragments. We propose that the replicative DNA polymerase complex of both prokaryotes and eukaryotes contains a high Mr polypeptide responsible for chain elongation which might be conserved during evolution and which is extremely sensitive to proteolytic cleavage.     

Immunogenic (tum-) variants of mouse tumor P815: cloning of the gene of tum- antigen P91A and identification of the tum- mutation.

Mutagen treatment of mouse P815 tumor cells produces tum- variants that are rejected by syngeneic mice because these variants express new surface antigens. These "tum- antigens" are recognized by cytolytic T lymphocytes but induce no detectable antibody response. Transfection of P815 cell line P1.HTR with DNA of tum- variant P91 yielded transfectants expressing tum- antigen P91A. They were detected by their ability to stimulate proliferation of cytolytic T lymphocytes [W?lfel, T., Van Pel, A., De Plaen, E., Lurquin, C., Maryanski, J. L. & Boon, T. (1987) Immunogenetics 26, 178-187]. A cosmid library of a cell line expressing antigen P91A was transfected into P1.HTR. Transfectants expressing the antigen were obtained. By packaging directly the DNA of a transfectant with lambda phage extracts, we obtained a small cosmid population containing as major component a cosmid that transferred the expression of P91A. The assay of various restriction fragments of this cosmid led to the isolation of an 800-base-pair fragment containing the P91A sequence required for transfection. Comparison with a homologous cDNA showed that this fragment contained only one of the several exons of the P91A gene. The normal and the tum- forms of the gene differ by one nucleotide located in this 137-base-pair exon. The essential role of this mutation, which produces an amino acid change, was confirmed by site-directed mutagenesis. No significant sequence similarity was found between the 800-base-pair fragment and any recorded gene.     

Escherichia coli extract-catalyzed recombination in switch regions of mouse immunoglobulin genes.

We have shown that Escherichia coli extracts catalyze recombination between mouse immunoglobulin mu and alpha genes inserted separately in lambda phage vectors carrying different genetic markers. Most of the recombination sites in the inserts are located in the switch regions of the heavy chain genes, as previously found in the expressed genes of myeloma cells. The recombination took place at relatively high frequency (10(-4)). The recombinational system in E. coli or lambda phage seems to prefer short nucleotide sequences similar to those used in the class switch recombination.     

Initiation of bacteriophage lambda DNA replication in vitro with purified lambda replication proteins.

We have developed a soluble enzyme system that replicates exogenously added plasmid DNA (lambda dv) bearing the replication origin of the bacteriophage lambda chromosome. The system contains pure phage lambda O and P replication proteins and a partially purified mixture of Escherichia coli replication proteins [the enzyme system of Fuller, R.S., Kaguni, J.M. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 7370-7374). The features of lambda dv replication in this system closely resemble the known characteristics of phage lambda DNA replication in vivo. The system (i) depends completely on exogenously supplied DNA, (ii) specifically replicates supercoiled plasmid DNA that contains a lambda replication origin, (iii) depends on both the lambda O protein and the lambda P protein, (iv) depends on RNA polymerase, (v) depends on host replication proteins (e.g., primase, dnaB protein, and several others that function in the priming of DNA synthesis in E. coli) as judged by antibody inhibitions, and (vi) replicates as much as 32% of added lambda dv plasmid DNA through a single complete round to generate catenated daughter molecules. Furthermore, replication of lambda dv DNA in vitro requires DNA gyrase and an ATP-regenerating system. It is notable that addition of lambda O and P proteins to the mixture of E. coli replication proteins inhibits replication of plasmids bearing the origin of the E. coli chromosome. Exploitation of this enzyme system should allow a detailed investigation of the biochemical mechanisms involved in bacteriophage lambda DNA replication and its regulation.     

Genetic and biochemical analysis of gonococcal IgA1 protease: cloning in Escherichia coli and construction of mutants of gonococci that fail to produce the activity.

The biological significance of bacterial extracellular proteases that specifically cleave human IgA1 is unknown. We have prepared a gene bank of gonococcal chromosomal DNA in Escherichia coli K-12 using a cosmid cloning system. Among these clones, we have identified and characterized an E. coli strain that elaborates an extracellular endopeptidase that is indistinguishable from gonococcal IgA1 protease in its substrate specificity and action on human IgA1. Analysis of recombinant plasmids and examination of plasmid-specific peptides in minicells have shown that the IgA1 protease activity in E. coli is associated with expression of a Mr 140,000 peptide. We have isolated IgA1 protease-deficient mutants of Neisseria gonorrhoeae by reintroduction of physically defined deletions of the cloned gene into the gonococcal chromosome by transformation.     

Specialized transduction of colicin E1 DNA in Escherichia coli K-12.

Genetic studies were made on E. coli K-12 TM96, which carries recombinant molecules constructed by in vitro combination of colicin E1 DNA and a DNA fragment of E. coli for guanine synthesis derived from transducing phage. The recombinant molecules existed as stable plasmids within the cell and contained genes for colicin E1 immunity and the guaA enzyme (xanthosine 5'-monophosphate aminase) together with a part of the lambda genome, R through J: (R-A-F-J)+. A block of the lambda genome, int through Q, was not detected in the recombinant molecule. Thus, this recombinant molecule was named ColEl-coslambda-guaA, and the specialized tranduction of the ColEl-coslambda-guA DNA into various E. coli K-12 cells by lambda phage was described. Lysates prepared by lytic infection of lambda phage onto TM96 or by induction of TM96(lambda) lysogens contained transducing particles which could transduce gua-deleted E. coli to stable guaA+ cells. These transductants were proved to have similar genetic properties as those of TM96. The frequency of transduction was not affected by the presence of an attachement site for lambda, prophage lambda, colicin E1 plasmids, or the recA property within gua-deleted recipient cells. Transducing particles were resistant to EDTA treatment and most of them had an average density of about 1.472. This value corresponds to that of lambda phage particles, which contain about 72% of the lenght of lambda DNA.     

Cloning, nucleotide sequence, and overexpression of the bacteriophage T4 regA gene.

The bacteriophage T4 regA gene codes for a regulatory protein that controls the expression of a number of T4 early genes, apparently at the level of translation. Restriction fragments containing the regA structural gene have been cloned into phage M13, and the nucleotide sequence has been determined. Translation of the DNA sequence predicted that regA protein contains 122 amino acids, with a Mr of 14,620. A DNA fragment carrying 85% of the coding sequence of regA has been cloned into the phage lambda leftward promoter PL expression vector pAS1, and a high level of truncated regA protein was produced by nalidixic acid induction. Protein chemical studies of the truncated regA protein gave results consistent with the nucleotide sequence of the regA gene. Subsequently, an intact regA gene was cloned into plasmid pAS1 and overexpressed. The regA protein produced in this way regulates the level of T4 45 and 44 proteins when their corresponding genes are carried on the same plasmid as the regA gene.     

Uptake of homologous single-stranded fragments by superhelical DNA: a possible mechanism for initiation of genetic recombination.

Superhelical [3-H]DNA (replicative form I, RFI) of bacteriophage phiX174 slowly but spontaneously took up 32-P-labeled homologous single-stranded fragments at 4 degrees. Uptake was accelerated by heating to 75 degrees. RFI did not take up single-stranded fragments derived from DNA of Escherichia coli or from separated strands of phage lambda. Uptake was inhibited by low concentrations of ethidium bromide. Relaxed circular phiX174 DNA did not take up homologous fragments. Per molecule of RFI, the complexes contained as much as 90 nucleotide residues of homologous fragment. The 32-P-lebeled fragments were largely resistant to digestion by exonuclease I, and were not displaced by heating complexes at 60 degrees for 1 min in 16 mM or 100 mM NaCl. Under comparable conditions of temperature and salt all of the fragments were displaced from complexes in which at least one phosphodiester bond was cleaved by pancreatic DNase, but a significant fraction of the fragments was retained in complexes that were relaxed by digestion with S1 nuclease. These observations are interpreted to mean that S1 nuclease digested the plus (viral) strand of the recipient RF at the site of uptake in some instances. Transfection of E. coli by heterozygous complexes produced recombinant progeny, thereby showing that genetic information can be transferred from the fragment of plus strand to progeny plus strands. We propose that both uptake of a third strand by superhelical DNA and the action of nucleases on the resulting complex may simulate early steps in genetic recombination.     

Genetic engineering production of H37Rv M. tuberculosis proteins]

A complete library of M. tuberculosis H37Rv genes was produced by incorporating the DNA fragments of M. tuberculosis H37Rv into the lambda pSI phasmid. For this, DNA isolated from the mycobacteria was treated by EcoRI restrictases and the fragments of 8-17 thousand nucleotide pairs were crosslinked with the phasmid DNA. Hybrid DNA molecules were packed into the caspids from the proteins of E. coli BHB2688 and BHB2690 strains. By estimates, this library contained 98% of M. tuberculosis H37Rv genome so that any required gene can be found at 0.99 probability. The needed genes were sought by monoclonal antibodies against a protein with a molecular mass of 17-19 kDa (IT-12, IT-51, IT-54) obtained from the WHO. The protein gene was also produced by the method for raising the end sequences using synthesis of two oligonucleotides SP30 complementary to segments that limit this gene in the presence of Taq-DNA-polymerase and DNA of M. tuberculosis H37Rv. Copies of a gene (MT-1) were produced by denaturation, firing and raising. This gene was incorporated in the puC119 plasmid and expressed in E. coli cells, the direction of reading being checked up. A protein with a molecular mass of 19 kDa was detected in E. coli extracts with the expressed pMT-2 plasmid using monoclonal antibodies IT-12 and IT-54 in enzyme-linked immunoassay and immunoblotting.