Cloning of the replication gene P of bacteriophage lambda: Effects of increased P-protein synthesis on cellular and phage DNA replication

Summary A restriction fragment of DNA carrying the P gene was cloned in the high copy number plasmid RSF2124. Cells harbouring this new plasmid RSF2124/E complement Pam80 phage. A lac promoter-operator region (lacP), produced by EcoRI digestion of plasmid pKB252, was inserted into RSF2124/glE such that induction of the lac promoter by IPTG or lactose leads to increased production of the P gene product. A high amount of P protein in E. coli cells results in a slow inhibition of bacterial DNA synthesis, suggesting that the initiation reaction is blocked by P protein. Synthesis of DNA proceeds normally under these conditions.Nonsuppressing groPA15 mutant bacteria which are unable to support the replication of wild-type (wt), acquire the ability to replicate Pam80 phage but not wt when they are transformed with a plasmid carrying the P gene. When harbouring a plasmid containing the mutant Pamber 80 gene, groPA15 mutants are able to support the replication of wt phage when infected at a high multiplicity. Pam80 phage does not multiply in these cells.     

Construction and some properties of packageable plasmid F

Summary A derivative of plasmid F which is packageable in phage coat was constructed using techniques of in vitro recombination. This plasmid is composed of three DNA fragments generated by restriction enzyme EcoRI: a miniF fragment (fragment f5 of F'lac) which is able to replicate autonomously, a DNA fragment from Staphylococcus Plasmid that carries the -lactamase gene, and a portion of guaA (B) transducing phage DNA carrying cohesive ends (cos site) along with almost all the late genes but devoid of all those genes and sites that are needed for replication, regulation, and recombination. The hybrid plasmid has a molecular weight of 2.7×10⁷ daltons, about 84% size of phage genome, and can be packaged in coat when helper phage replicates in the plasmid-carrier cell. The package plasmid and the helper phage particles are separated by CsCl density gradient centrifugation. The replication characteristics of the recombinant plasmid are all those of F including the copy number, incompatibility, and curing with acidine orange. The packaged plasmid is injected into an F^- cell and establishes a plasmid state with normal efficiency. In F⁺ or Hfr cells, the resident F factor hinders this process.     

Relationship of vector insert size to homologous integration during transformation of Neurospora crassa with the cloned am (GDH) gene

Summary We used lambda and plasmid vectors containing the am ⁺ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. A total of 199 transformants were examined with the potential to yield am ^– transformants by homologous recombination. When we used vectors that had 9.1 kb of homology with the chromosomal DNA, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a plasmid that had been linearized prior to transformation. When vectors with up to 5.1 kb of homology were used, very few transformants (1 of 89 tested) resulted from homologous recombination. Of a sample of 29 ectopic integration events obtained by transformation with the 9.1 kb fragment cloned in a vector, 18 included a major part (usually almost all) of both arms of lambda with the entire Neurospora 9.1 kb insert between them. Four included only long arm sequence together with an adjacent segment of the insert containing the am gene. The remaining seven were the result of multiple integrations. There was no evidence of circularization of the vector prior to integration. All transformants that had multiple copies of the am gene appeared to be subject to the RIP process, which causes multiple mutations in duplicated sequences during the sexual cycle.     

Structure and function of the repressor of bacteriophage lambda

Summary The high-affinity mutant cI gene of cIha (Nag et al. 1984) was cloned in the multicopy plasmid pBR322. In the resulting plasmid, pMD 102, a lacUV5 promoter was inserted giving the lacUV5-cIha fusion plasmid pMD 205. Bacteria carrying pMD 102 and pMD 205 contain 2.5 and 15 times, respectively, the level of repressor in a monolysogen of cIha. Results of the study of certain properties of the bacteria carrying these plasmids suggest that the ha repressor also has a higher affinity for the virulent mutant operators as well as the prm promoter of .     

Cloning bacterial genes with plasmid λdv

Summary Fragments ofEscherichia coli DNA carrying genes for -galactosidase, or for biosynthesis of guanine or biotin were recombined in vitro with dv DNA. The cloned recombinant molecules recovered from transformedE. coli cells consisted of a biologically functional bacterial DNA fragment and, except for dv-bio30-7, two dv monomer units: one of the dv units was used as the insertion site for the bacterial DNA, whereas the other was intact, and seemed to be responsible for the replication of the recombinant plasmid. The process which gives rise to these recombinant molecules at high frequency from mixtures of monomeric dv DNA's and bacterial DNA fragments is discussed.     

Isolation and characterization of lambda b221poriCasnA, a plaque-forming specialized transducing phage carrying the origin of replication of the Escherichia coli chromosome

Summary A specialized transducing phage, b221poriCasnA has been isolated carrying oriC the origin of chromosomal replication of Escherichia coli. All phage genes required for lytic growth are retained, thus the phage is capable of lytic growth. The presence of the oriC locus confers upon infecting phage DNA the ability to replicate as a plasmid using only host DNA replication functions. The presence of both oriC and asnA markers has allowed the development of a plaque assay for origin function which can be used to identify mutants at these loci. Comparison of restriction endonuclease cleavage sites present on b221poriCasnA DNA to those on tis parent, b221 rex::Tn10 suggests the steps involved in the formation of the transducing phage.     

Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was experessed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded -casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plamid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.Abbreviations cfu colony forming units - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] Dedicated to Prof. Dr. G. Drews on the occasion of his 65th birthday     

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.     

The target of the negative regulator of pMB1 replication overlaps with part of the repressor coding sequence

Summary Plasmid mutants (svir), insensitive to inhibition by the repressor of initiation of pMB1 replication, have been selected by exploiting their ability to support growth in the presence of repressor and inhibitor of plasmid replication. The alteration in the mechanism that controls plasmid replication causes a change in the plasmid copy number. svir mutants are dominant, as expected for mutants in the target of a repressor, but at the same time they are unable to synthesise a repressor active on the wild-type target. This lack of cross interaction between svir mutants and a co-resident wild-type plasmid results in their compatibility. These findings are explained by postulating that the target of the inhibitor of pMB1 replication coincides with part of the DNA segment that codes for the inhibitor itself. As a consequence single base pair changes in the target result in altered repressor molecules.     

Cloning and characterization of the Escherichia coli chromosomal region surrounding the dnaG gene, with a correlated physical and genetic map of dnaG generated via transposon Tn5 mutagenesis

Summary A 24 kilobase pair region of the E. coli chromosome surrounding the dnaG gene has been cloned and characterized. A phage library was first constructed by ligating a Sau3A (GATC) partial DNA digest of the entire E. coli chromosome into the BamHI (G GATCC) cloning vector charon 28. Partial digestion was performed to generate overlapping chromosomal fragments and to allow one to walk along the chromosome. This library was probed with a nick-translated plasmid (pRRB1) containing the rpoD gene, which maps adjacent to dnaG at 66 min. Four bacteriophages: 3, 4, 5, 6 that hybridized to the probe were isolated from the 2,500 plaques screened. One phage recombinant 4, was shown to contain the dnaG gene. Three recombinant plasmids containing dnaG: pGL444, pGL445, pBS105, were constructed via subcloning of 4 using different restriction fragments. Plasmids pGL444 and pBS105 were subjected to transposon Tn5 mutagenesis and 88 Tn5 inserts into the cloned region were isolated. The location of the Tn5 inserts were mapped by restriction enzyme analysis of the plasmids and the insertion mutations were checked for ability to complement a dnaGts chromosomal marker at nonpermissive 40° C. In this manner a correlated physical and genetic map of dnaG was determined. A large number of Tn5 inserts map to a specific 900 b.p. region which we propose may be involved in the regulation of dnaG gene expression.     

Molecular cloning of the uvrD gene of Escherichia coli that controls ultraviolet sensitivity and spontaneous mutation frequency

Summary The uvrD gene of Escherichia coli that control UV sensitivity and spontaneous mutation frequency has been cloned with phage as vector. The increased sensitivity to ultraviolet light (UV) of uvrD3, uvrE502, recL152, and pdeB41 mutants, high mutability of uvrD3 and pdeB41 mutants, and conditional lethality of strain TS41 that carried pdeB41, polA1, and sup126 mutations were all suppressed by lysogenization of the mutant cells with uvrD ⁺. These results were consistent with the idea that the uvrD, uvrE, recL, and pdeB mutations are alleles of the uvrD gene. In addition to the uvD gene, uvrD ⁺ carried the corA gene that controls transport of Mg⁺⁺, Mn⁺⁺, and Co⁺⁺ through the cell membrane. Hybrid plasmids carrying both uvrD and corA genes were also constructed by using pKY2289 as a cloning vehicle. Orientational isomers that carried the same 12.0 kb fragment in the opposite direction were equally efficient in complementing the UvrD^- as well as CorA^- defects of the transformed host cells, suggesting that the DNA insert contains all the genetic signals needed to express the two gene products. Insertion of the sequence into recombinant plasmids was performed to generate appropriate restriction endonuclease target sites in the cloned DNA fragments.     

Reduced expression of a distal gene of the prn gene cluster in deletion mutants of Aspergillus nidulans: genetic evidence for a dicistronic messenger in an eukaryote.

Summary Temperature sensitive dnaAts46 mutants, in which initiation of chromosome replication is blocked at 42° C, are unable to maintain a dv plasmid at the permissive temperature unless the plasmid carries a mutation in gene P of the type permitting phage to grow in groP (dnaB) bacteria. The growth rate of dnaAts46 mutants seems to be impaired by the presence of the dvP mutant plasmid.Cold sensitive dnaAcos mutants which overinitiate replication at low temperature and grow normally only at 40° and above, can maintain efficiently dvP ⁺ plasmids as well as dvP mutants. Cold sensitivity of dnaAcos mutants is suppressed by the presence of the plasmid dvP ⁺ and by certain dvP mutants, but not by others.The gene P product seems to act by reducing the initiation potential of both types of dnaA mutants, aggravating the initiation defect in dnaAts46 and correcting the overinitiation of dnaAcos.     

Host specificity of DNA produced by Escherichia coli. XVI. Phage lambda DNA carries a single site of affinity for A-specific restriction and modification

Summary Bacteria with A-specific restriction plate unmodified phage with an efficiency of 10^-2. One mutational event can produce restriction insensitive (sA^o) mutants of . These differ from the original sA form of by no other property than their response to A-host specificity. Two-parental phage crosses involving sA and sA^o, respectively, as non-selective marker allowed to map sA between genes cII and O. These data indicate that sA is the only site on DNA with affinity for A-specific restriction. DNA is thus an interesting substrate in in vitro A-specific restriction and modification. Using an assay based on the infectivity of DNA on helper-infected bacteria, A-specific modification activity was found in partially purified sonicates of bacteria with A-host specificity. In parallel to modification, ³H-methyl label from s-adenosylmethionine, the only cofactor required for modification, was transferred to unmodified DNA. No association of radioactivity was observed in control experiments with DNA from either modified ·A or from asA^o mutant. These data suggest that A-specific modification is brought about by DNA methylation and that the sA^o mutation not only abolished the affinity for A-specific restriction, but also for A-specific modification.     

Molecular cloning of the fnr gene of Escherichia coli K12

Summary Mutations in the fnr gene of Escherichia coli have pleiotropic effects leading to deficiencies in the reduction of fumarate and nitrate, hydrogen production and the ability to grow anaerobically with fumarate or nitrate as terminal electron acceptors. Transducing phages (fnr) carrying the wild-type fnr gene were isolated from populations of artificially-constructed recombinant lambda phages by their ability to complement the lesions of fnr mutants. The fnr phages restored anaerobic growth with fumarate and nitrate as electron acceptors and, as prophages, they promoted normal synthesis of fumarate reductase, nitrate reductase and hydrogenase in fnr mutants. Five independently-isolated fnr phages each contained a R.HindIII fragment (11.5 kilobases) that possessed three internal R.EcoRI targets and had inserted with the same orientation relative to the phage. A physical map of the fnr region was constructed by restriction analysis and flanking fragments were identified by DNA:DNA hybridization.     

Cloning of the dnaB gene of Escherichia coli: the dnaB gene of groPB534 and groPB612 and the replication of phage lambda

Summary Fragments of the E. coli chromosome that carry the dnaB groPB534 or groPB612 alleles have been cloned into a cosmid vector. The resulting recombinant plasmids contained the genes uvrA, groP (B534 or B612), and lexA. Further subcloning into high copy number plasmids, during which the uvrA and lexA genes were removed successively, yielded a groPB534 and groPB612 DNA fragment of about 2.4 kb each. Both fragments contained an overlapping 1.8 kb segment of DNA in which the sites of all restriction enzymes tested were identical. The size of these dnaB gene fragments were further delimited by deletion analysis.In E. coli groPB534 in which wild-type and A mutants do not replicate (Georgopoulos and Herskowitz 1971) phage replication is rescued if the strain contains the groPB534 gene on high copy number plasmids. On the contrary, in E. coli groPB612, which is temperature-sensitive for its groP character, replication of and A is abolished at 30° C if the strain contains the groPB612 recombinant plasmid. On the other hand, replication of B remains unaffected whether or not the groP strains harbor the isogenic dnaB gene-containing plasmid. The results suggest that within the cell not only the quality but also the relative amounts of dnaB and P protein are crucial for phage replication.     

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.     

Tandem duplication induced by an unusual ampA1-, ampC-transducing lambda phage: A probe to initiate gene amplification

Summary Secondary attachment site -lysogens were isolated in an Escherichia coli strain carrying multiple tandem 9.8 kb repeats. The repeat carried the structural gene for chromosomal -lactamase, ampC. One lysogen produced lysates with amp-transducing activity. Three types of phages with different densities were obtained from this lysogen. The one with the lowest density was found to be a helper cI857S7 phage. The other two phages showed identical restriction endonuclease fragmentation patterns. The difference in density was due to the presence or absence of phage tail. In damp the right cohesive end segment was deleted in a random fashion with the majority ending between 81.0% and 82.4% of . The chromosomal segment of damp was most likely located at the attachment site. The damp DNA was compared to that of a ColE1 hybrid carrying the chromosomal amp segment and a ColE1 hybrid carrying the same 9.8 kb amp repeat as the lysogen from which damp was isolated. It was found that the chromosomal part of damp constituted 9.8 kb, i.e. the size of one repeat. Moreover, the novel joint between adjacent repeats was present. In a attB-deleted E. coli K-12 strain, lysogenic for damp, highly ampicillin-resistant mutants occurred at an exceedingly high frequency. They were found to contain in the chromosome an amplified 9.8 kb repeat. This suggested that integration of the novel joint from damp into the amp region gives rise to an amplifiable duplication. In E. coli lysogenized for damp at attB highly ampicillin-resistant clones were also found at a high frequency. These clones carried multiple tandem repeats of damp DNA, each with an intact right end segment.