Cloning and identification of the product of the dnaE gene of Escherichia coli

We successively subcloned the dnaE gene of Escherichia coli into pBR322, resulting in a plasmid that contains 4.6 kilobases of E. coli DNA. This plasmid can complement a dnaE temperature-sensitive mutation. A restriction map of the dnaE gene and the surrounding 10.7-kilobase region of the E. coli chromosome was determined. A unique HindIII restriction endonuclease site within the cloned segment of DNA was identified as a site required for expression of the dnaE gene. By using the maxicell plasmid-directed protein synthesizing system, we demonstrated that dnaE codes for the alpha subunit of DNA polymerase III.     

Identification, characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures.

We identified and cloned an Escherichia coli gene called htrA (high temperature requirement). The htrA gene was originally discovered because mini-Tn10 transposon insertions in it allowed E. coli growth at 30 degrees C but prevented growth at elevated temperatures (above 42 degrees C). The htrA insertion mutants underwent a block in macromolecular synthesis and eventually lysed at the nonpermissive temperature. The htrA gene was located at approximately 3.7 min (between the fhuA and dapD loci) on the genetic map of E. coli and between 180 and 187.5 kilobases on the physical map. It coded for an unstable, 51-kilodalton protein which was processed by removal of an amino-terminal fragment, resulting in a stable, 48-kilodalton protein.     

Isolation of recombinant plasmids and phage carrying the lexA gene of Escherichia coli K-12

The lexA gene of Escherichia coli K-12 was cloned from the plasmid pLC44-14 into pBR322. Plasmids carrying lexA+ were selected by their ability to complement a recessive tsl mutation, which is believed to be a mutation in lexA. The smallest lexA+ recombinant plasmid, pJL21, contained an EcoRI-PstI fragment 2.9 kilobases (kb) in length; two larger plasmids also contained this fragment, and genetic material to one or both sides of the EcoRI-PstI fragment. Plasmids homologous to pJL21, but carrying a dominant mutation, lexA3, or one of three recessive amber mutations in lexA, termed spr, were also isolated. To clone the EcoRI-PstI fragment onto a lambda vector, the PstI end was first converted to an EcoRI end by attachment of a 100-base pair PstI-EcoRI fragment isolated from the plasmid ColE1; the resultant EcoRI fragment was then cloned into the lambda vector lambda gt4. A restriction map of pLC44-14 was obtained for nine restriction enzymes. The orientation of this map was determined relative to the E. coli genetic map by complementation of the gene ubiA+ and by comparison with restriction enzyme digests of another plasmid, pLC11-9, which carries dnaB, a gene closely linked to lexA, but does not carry lexA.     

Localization of the Escherichia coli rnt gene encoding RNase T by using a combination of physical and genetic mapping.

The rnt gene encoding RNase T was cloned on a 13-kilobase BamHI fragment. Restriction analysis of the fragment and comparison of it with the Escherichia coli restriction map localized rnt to kilobase coordinates 1733 to 1746, corresponding to about 36 min on the genetic map. The map location was confirmed by cotransduction with the nearby zdg-229::Tn10 and ksgB1 markers.     

Cloning and mapping of the manganese superoxide dismutase gene (sodA) of Escherichia coli K-12.

An Escherichia coli gene bank composed of large DNA fragments (about 40 kilobases) was constructed by using the small cosmid pHC79. From it, a clone was isolated for its ability to overproduce superoxide dismutase. The enzyme overproduced was manganese superoxide dismutase, as determined by electrophoresis and antibody precipitation. Maxicell analysis and two-dimensional O'Farrell polyacrylamide gel electrophoresis demonstrated that the structural gene, sodA, of manganese superoxide dismutase was cloned. Subcloning fragments from the original cosmid located the sodA gene within a 4.8-kilobase EcoRI-BamHI fragment. This fragment was inserted into a lambda phage which was deleted for the att region and consequently could only lysogenize by recombination between the cloned bacterial DNA insertion and the bacterial chromosome. Genetic mapping of the prophage in such lysogens indicated that the chromosomal sodA locus lies near 87 min on the E. coli map.     

Location of a gene (ssrA) for a small, stable RNA (10Sa RNA) in the Escherichia coli chromosome.

The gene for 10Sa RNA, which is a major small, stable RNA in Escherichia coli, is a unique gene in the E. coli chromosome. The 10Sa RNA gene (ssrA) has been located between 2,760 and 2,761 kilobases on the E. coli genome.     

Structure of the gene for human von Willebrand factor

von Willebrand factor is a large multimeric plasma protein composed of identical subunits which contain four types of repeated domains. von Willebrand factor is essential for normal hemostasis, and deficiency of von Willebrand factor is the most common inherited bleeding disorder of man. Four human genomic DNA cosmid libraries and one bacteriophage lambda library were screened with von Willebrand factor cDNA probes. Twenty positive overlapping clones were characterized that span the entire von Willebrand factor gene. A high-resolution restriction map was constructed for approximately 75% of the locus and a total of approximately 33.8 kilobases was sequenced on both strands including all intron-exon boundaries. The gene is approximately 178 kilobases in length and contains 52 exons. The exons vary from 40 to 1379 base pairs in length, and the introns vary from 97 base pairs to approximately 19.9 kilobases in length. The signal peptide and propeptide (von Willebrand antigen II) of von Willebrand factor are encoded by 17 exons in approximately 80 kilobases of DNA while the mature subunit of von Willebrand factor and 3' noncoding region are encoded by 35 exons in the remaining approximately 100 kilobases of the gene. A number of repetitive sequences were identified including 14 Alu repeats and a approximately 670-base pair TCTA simple repeat in intron 40 that is polymorphic. Regions of the gene that encode homologous domains have similar structures, supporting a model for their origin by gene segment duplication.     

Genetic organization of plasmid R1162 DNA involved in conjugative mobilization.

DNA involved in the mobilization of broad-host-range plasmid R1162 was localized to a region of 2.7 kilobases within coordinates 3.4 to 6.1 kilobases on the R1162 map. By examining the transfer properties of plasmids containing cloned fragments of DNA from within this region, we showed that at least four trans-active products and a cis-active site (oriT) were involved in mobilization. A cloned DNA fragment of 155 base pairs was capable of providing full oriT activity. This fragment was located within 600 base pairs of DNA containing the origin of replication of R1162, and its nucleotide sequence and that of neighboring DNA were determined. Activation of oriT required R1162-encoded, trans-acting products. Deletions which resulted in the loss of one or more of these had a variable effect on transfer efficiency and indicated the presence of both essential and nonessential Mob products. Regions encoding these products flanked oriT and in one case appeared to overlap a gene essential for plasmid replication. The implications of these findings with respect to the broad host range of R1162 are discussed.     

Sub-cloning of the wild-type proAB region of the Escherichia coli genome

The genes proA and proB encoding the first two enzymes of the proline biosynthetic sequence in Escherichia coli were subcloned from a ColE1 hybrid plasmid containing 23.3 kilobases of genomic DNA. proA and proB are contiguous and constitute a single operon transcribed in the direction proB-proA. The pro operon is contiguous with the gene phoE. Hybridization experiments showed no homology between proAB of E. coli and the other regions of the E. coli genome or with the DNA of several other bacterial species.     

Cloning of the pif region of the F sex factor and identification of a pif protein product

This paper reports a detailed investigation of the pif region of the F factor responsible for inhibition of development of T7 and related "female-specific" phages. We have mapped a series of pif::Tn5 insertions to a region between 39.6 and 42.8 kilobases on the physical map of F. All pif::Tn5 insertions plated T7 at full efficiency; most were clustered in a 1.8-kilobase interval on both sides of the EcoRI site located at F coordinate 40.3 kilobases. A 5.2-kilobase Pst-I fragment with F coordinates 38.9 to 44.1 has been cloned into a pSC101 vector to create the Pif+ plasmid pGS103. A series of Pif- deletion mutants and nonsense mutants were isolated from pGS103. Using minicells carrying pGS103 or its derivatives, we have identified a 70,000-dalton pif protein.     

Identification of the pifC gene and its role in negative control of F factor pif gene expression

The pif region of the F factor includes two genes, pifA and pifB, that lead to abortive T7 infection. We have identified a new gene in this region, pifC, by constructing an in vitro fusion of pif DNA at 41.6 kilobases on the F factor physical map to the lacZ gene. A PifC-LacZ fusion protein of 149,000 daltons has been identified by immunoprecipitation and polyacrylamide gel electrophoresis. This allows us to assign the N terminus of pifC to 42.5 kilobases on the F map. Using fusions of pifC, pifA, and pifB to lacZ, we have studied the regulation of pif gene expression and have shown that the product of pifC negatively controls its own expression and that of pifA and pifB.     

Physical analysis of deletion mutations in the ilvGEDA operon of Escherichia coli K-12.

DNA-DNA hybridization of cloned segments of the Escherichia coli K-12 ilvGEDA operon to genomic blots was used to determine the physical dimensions of a series of deletion mutations of the ilvGEDA operon. The smallest mutation resulted from the deletion of approximately 200 base pairs from within ilvD, whereas the largest mutation resulted from the deletion of 17 kilobases including the rep gene. The structure of three of these mutants indicates that formation of the deletions was mediated by Tn5 (or Tn5-131) that is retained in the chromosome. This is the first observation of this type of Tn5-mediated event. Our analysis of the total acetohydroxy acid synthase activity of strains containing deletions of ilvG indicates that the truncated ilvG polypeptide of wild-type E. coli K-12 lacks enzyme activity. The small 200-base-pair deletion of ilvD confirms the presence of a strong polar site 5' to ilvA. The detailed structure of these deletions should prove useful for the investigation of other genes in this region. This genomic analysis demonstrates that the ilv restriction site map that was established previously by the analysis of recombinant bacteriophage and plasmids is identical to that on the genome.     

Characterization of degP, a gene required for proteolysis in the cell envelope and essential for growth of Escherichia coli at high temperature.

The degP gene, required for proteolysis in the cell envelope of Escherichia coli, maps at approximately 3.5 min on the chromosome. Null mutations in degP result in temperature-sensitive growth. In certain genetic backgrounds, expression of abnormal periplasmic or inner membrane proteins (protein fusions or proteins with internal deletions) enhances the temperature-sensitive phenotype. Such growth defects were used as a selection for cloning the degP gene into Mud4042 and pACYC184 plasmid vectors, and a restriction map was determined. Analysis of deletion and insertion mutations on one of these plasmids showed that the degP gene is approximately 1.5 kilobases in size. The plasmid-encoded DegP protein had an apparent molecular weight of 50,000, as determined by maxicell analysis. Protein fusions between DegP and alkaline phosphatase had high alkaline phosphatase enzymatic activity, indicating that DegP is a periplasmic or membrane protein.     

Molecular cloning of four tricarboxylic acid cyclic genes of Escherichia coli.

A fragment of DNA (3.1 kilobases [kb]) from a ColE1 Escherichia coli DNA hybrid plasmid containing the bacterial citrate synthase gene (gltA) was subcloned in both orientations into phage lambda vectors by in vitro recombination. The resulting phages were able to transduce gltA and, as prophages, complemented the lesion of a gltA mutant, showing that a functional gltA gene is contained in the 3.1-kb fragment. The segment of E. coli DNA cloned in these lambda gltA phages was extended in vivo by prophage integration and aberrant excision in the gltA region. Plaque-forming derivatives, carrying up to three additional tricarboxylic acid cycle genes, succinate dehydrogenase (sdh), 2-oxoglutarate dehydrogenase (sucA), and dihydrolipoamide succinyltransferase (sucB), were isolated and characterized by their transducing and complementing activities with corresponding mutants, and the order of the genes was confirmed as gltA-sdh-sucA-sucB. Physical maps of a variety of the transducing phages showed that the four tricarboxylic acid cycle genes are contained in a 12.8-kb segment of bacterial DNA. The four gene products, plus a possible succinate dehydrogenase small subunit, were identified in postinfection labeling studies, and the polarities of gene expression were defined as counterclockwise for gltA and clockwise for sdh, sucA, and sucB, relative to the E. coli linkage map.