The novel gene(s) ARD of plasmid pKM101: alleviation of EcoK restriction

Summary The host-controlled K restriction of unmodified phage was 10-100-fold alleviated in the wild-type strain E. coli K12, carrying plasmid pKM101 of incompability group N. pKM101-mediated release of K restriction was also observed in lexA ^-, recA ^-, and recB ^- strains of E. coli K12. By restriction mapping Tn5 insertions in pKM101, which reduced pKM101-mediated alleviation of restriction, were shown to be located within the BglIIB fragment approximately 11 kb anticlockwise from the RI site of pKM101. We have termed the gene(s) promoting the alleviation of K restriction of phage ard (alleviation of restriction of DNA). It was shown (1) that ard function affected only the EcoK restriction system and not the EcoB, EcoRI, EcoRIII, or EcoPI system, (2) ard gene(s) did not mediate EcoK type modification of DNA and did not increase the modification activity of the EcoK system in a way similar to that observed with gene ral of bacteriophage .     

Functional characterization of the H-2Dp gene product

A clone containing the D ^p gene was used to transform L cells. The D^p product expressed was identified by two-dimensional gel electrophoreis and flow cytometry. The D^p product expressed by the L cells was recognized by D^P-specific flow cytometry. The D^p product expressed by the L cells was recognized by D^p-specific but not K^p-specific killer T cells. This killing was inhibited by monoclonal antibodies specific for D^p but not K^p or K^k antigens. Similarly, lymphocytic choriomeningitis virus (LCMV) killer T cells from B 10.P mice were able to kill LCMV-infected L12a cells, but not LCMV-infected Ltk⁺. Again only D^p monoclonal antibodies could inhibit this killing.     

Molecular cloning of the T4 genome: organization and expression of the tail fiber gene cluster 34--38

Summary T4 derivatives that carry T4 tail fiber genes 34–38 have been isolated and characterized by genetic, structural and functional analysis. 32 T4 recombinants were identified by a marker rescue screen of 310 T4 clones generated by restriction of partial cytosine-containing T4 DNA with either HindIII or EcoRI and ligation into appropriately cleaved vectors. These tests defined 15 recombinant classes with respect to the contiguous stretches of genome recovered. Restriction enzyme structural analysis identified 7 HindIII fragments and 7 EcoRI fragments, established a restriction map covering about 11 kb, and indicated the orientation of the DNA inserts within the vectors. The cloned tail fiber genes are expressed efficiently from promoters and complement in vivo T4 phage carrying amber mutations in the tail fiber genes. Polypeptides corresponding to gp34-gp38 have been detected by SDS polyacrylamide gel electrophoresis of ³⁵S-labeled extracts of appropriate T4 recombinant infected UV-treated host cells. The genetic, structural and functional maps of the T4 tail fiber gene cluster have been correlated, and provide a rational approach to genetically directed DNA sequence analysis of genes 34–38 and their mutant variants that affect the assembly, structure and function of the tail fibers.     

Selective destruction by formaldehyde fixation of an H-2Kb serological determinant involving lysine 89 without loss of T-cell reactivity

In preparation for functional analyses, a study of the binding of H-2K^b-specific monoclonal antibodies (mAb) to formaldehyde (FOR)-fixed H-2^b spleen or tumor cells revealed that three of nine mAb tested had lost reactivity with the FOR-fixed cells, whereas the reactivity of the other mAb generally did not diminish. Comparison of the reactivity of these mAb on untreated H-2K ^bm mutant cells and on FOR-treated H-2K^b cells suggests that for three mAb the total loss of reactivity on the latter could be a consequence of the alteration by FOR of lysine 89, which is substituted by alanine in mutant bm3. H-2KP^b-specific alloreactive polyclonal or monoclonal CTL, all of which had retained reactivity with bm3 target cells, had also retained reactivity with FOR-fixed H-2^b cells as indicated by cold target inhibition studies. The H-2K^b-specific CTL were probably reactive with conformational determinants of H-2K^b, which are dependent on the integrity of both the 1 and the 2 domains of the H-2K^b molecule. Results are compatible with FOR treatment selectively affecting a serological determinant in the 1 domain without affecting conformational-type CTL determinants.Abbreviations used in this paper CTL cytotoxic T lymphocyte - FOR formaldehyde - PBS phosphatebuffered saline - FCS fetal calf serum - mAb monoclonal antibody - TNBS trinitrobenzene sulfonate     

Map location of the Escherichia coli origin of replication.

Summary Working with restriction fragments obtained directly from the Escherichia coli K12 chromosome, the EcoRI-HindIII restriction map of the section of the chromosome containing the replication origin has been extended by 14 kilobase pairs (kb) to cover 56kb. Within this newly mapped portion, the liv and rrnC cistrons have been identified by (1) hybridization of individual restriction fragmenents to the ilv-transducing phage dilv5 and (2) a comparison of the restriction map of this region with the EcoRI map of dilv5 and the HindIII map of the plasmid pJC110, a ColE1-ilv hybrid. The replication origin is located approximately 30 kb from the ilvE gene and 20 kb from the rrnC 16S rRNA cistron. This places the origin near 82.7 min on the genetic map, close to uncA.     

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.     

Host specificity of DNA produced by Escherichia coli. XII. The two restriction and modification systems of strain 15T-

Summary E. coli 15T^- carries two distinct sets of DNA restriction and modification activities. The genetic information for system A is contained in the bacterial chromosome and linked to the thr region. This fact suggests host specificity A to be related to those of strains K and B. The genes controlling system 15 are on a plasmid which is related to phage Pl: it competes with Pl for stable inheritance in the carried state and it genetically recombines with Pl. This recombination may produce plasmid genomes with newly assorted characters (see Table 3). One of them is an active, Pl-like prophage with the 15-specific instead of the parental Pl-specific restriction and modification characters. Superinfection of 15T^- with Pl may also result in curing of the bacteria from the restriction plasmid.Bot A- and 15-specific restrictions and modifications act on bacterial DNA, on the DNA of various sex factors and on the DNA of certain bacteriophages, e.g. of phage . Phage 82 DNA is sensitive only to 15-specific restriction, but not to A-specific restriction.Independently of the A- and 15-specific restrictions, the growth of phage in E. coli 15T^- encounters another limitation of yet unknown nature. No such limitation is observed either with phage 82 or with mutants of occurring at a frequency of about 10^-5.     

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.     

Molecular cloning of Bacillus subtilis genes involved in DNA metabolism

Summary Different clones carrying a chromosomal DNA fragment able to transform Bacillus subtilis mutants dnaA13, dnaB19, dnaG5, recG40 and polA42 to a wild-type phenotype were isolated from a library constructed in plasmid pJH101. A recombinant clone carrying a chromosomal fragment able to transform dnaC mutants was obtained from a Charon 4A library. A restriction map of the cloned DNA fragments was constructed. The 11.3 kb cloned DNA fragment of plasmid pMP60-13 containing the wild-type sequence of dnaG5 was shown to transform a recF33 mutant as well.     

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.     

UV-induced allevation of lambda restriction in Escherichia coli K-12: kinetics of induction and specificity of this SOS function

Summary In UV-irradiated cells of Escherichia coli K-12 a partial release of the restriction of non-modified phage is observed when the cells are recA ⁺ lexA ⁺. We show here that the induction of this restriction allevation (RA) also depends on the recBC enzyme and that the expression of RA requires protein synthesis. Maximum expression was reached within 60 to 90 min after irradiation. Experiments are presented which show that upon UV-irradiation a signal is created which triggers the development of RA when protein synthesis is allowed. This signal decayed with a half-life of only a few minutes in cells treated with chloramphenicol. The decay kinetics were similar in uvr ⁺ and uvrA mutants. RA appeared to be specific for EcoK insofar as no allevation of restriction by EcoRI, EcoRII and EcoP1 occurred. During maximum expression of RA no gross reduction of the activities of the recBC enzyme (exonuclease V) and the restriction endonuclease EcoK was observed and no new DNA modifying activity appeared in the cells. Since, in fully expressed cells, up to 75% of the infecting DNA was converted to acid-soluble material within 20 min after infection we suggest that only a small specific fraction of infections may undergo RA.     

Physical characterisation of the "Rac prophage" in E. coli K12.

Summary We confirm the hypothesis of Low (1973) that many E. coli K 12 strains contain a prophage (the Rac prophage) located a few minutes clockwise of the trp operon on the genetic map. We have used restriction endonucleases and ³²P-labelled probes to construct a physical map of this prophage. Some E. coli K 12 strains, including AB1157, have lost the entire prophage, apparently by a specific deletion. This is consistent with prophage excision by site-specific recombination. reverse (rev) phages (Zissler et al., 1971) are recombination proficient derivatives of phage in which the phage recombination functions have been replaced by analogous functions (RecE) derived from the host chromosome (Gottesman et al., 1974; Gillen et al., 1977). Our data support the origin of rev phages by recombination between and the Rac prophage following excision of the Rac prophage from the E. coli chromosome.Important experimental data are included in the Figure legends.     

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.     

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.     

Mutations of coliphage lambda affecting the expression of replicative functions O and P

Summary A selection technique, using the thermoinducible prophage CI857Nsus7 Nsus53, has lead to the characterization of a new class of prophage mutations (called r), which prevent host killing upon thermal induction.N-defective r mutants efficiently complement i⁴³⁴ or O and P mutants, but not the corresponding mutants of i²¹. Complementation data suggest that the i²¹ hybrid fails to provide the positive regulatory mechanism dependent on the N-gene product, since it cannot activate the Q gene of a N-defective mutant. Thus, it seems possible that r mutants cannot express genes O and P unless the N-gene product is present in the cell. This interpretation is supported by the fact that r mutants are not defective and form plaques when their N-gene is functional. r mutation confer a clear phenotype, and map in the y-CII region. Results of a density gradient analysis suggest that they result from small insertions of DNA. Induced N-defective r prophages appear to be only poorly transcribed on strand H.Complementation tests performed in a strain lysogenic for indicate that the C17 mutation can suppress a r mutation in a cis position, even in the absence of the N-gene product.These results suggest that the expression of genes O and P, in addition to gene Q, is under the positive regulation of the N-gene product.     

Construction and physical mapping of plasmids containing the MetA gene of Escherichia coli K-12

Summary Plasmids containing the metA gene of E. coli K-12 were constructed in vitro using pBR322 as the cloning vehicle and metA transducing phage as the source of metA DNA. EcoRI digests of pBR322 and metA20 were joined by ligase and plasmids carrying the metA gene were selected after transformation in a metA deletion strain. Recombinant DNA molecules contained one pBR322 fragment and one metA20 fragment of 12.2 kb which was present in either of two possible orientations. Plasmids constructed by BamH1 digestion of metA2 contained a single bacterial DNA fragment of 5.8 kb inserted in the tet gene. Insertion of the metA fragment led to loss of resistance to tetracycline in one orientation and partial resistance in the opposite orientation.     

The Use of Competitive PCR Mimic to Evaluate a Limulus Lambda Phage Genomic DNA Library

1. A phage genomic DNA library for Limulus (L.) polyphemus brain was constructed using the GEM-12 vector and the host strain KW251.2. The primary library contained approximately 1.275 × 10⁶ independent clones, increasing upon amplfication to 6.66 × 10⁹ pfu/ml in a total volume of 58 ml.3. A total of 28 clones was randomly chosen for a determination of the average size of inserts in the library. All clones contained inserts and the average size was 14.9 kb, ranging from 11.7 to 28.0 kb. The library provides a 10-fold equivalent of the L. polyphemus genome.4. A new approach for evaluating a genomic DNA library was developed, in which competitive PCR MIMIC was employed to determine the target gene copy number in both constructed library and brain genomic DNA. The putative protein kinase C (PKC) was selected as the target gene because its partial sequence of cDNA was recently cloned from L. polyphemus brain in our laboratory (Cao et al., 1998). A 419-bp fragment of nonhomologous sequence derived from putative PKC and a 306-bp fragment from plasmid pUC 18 were generated for use as target and competitor in PCR MIMIC, respectively.5. Within the genomic library DNA, a 0.8 value was obtained for the copy number of the putative PKC gene that was detected in 0.1 amol of one equivalent L. polyphemus genome in terms of the average recombinant molecular weight. In the genomic DNA, a single copy of putative PKC was found in 0.1 amol of one coverage for the L. polyphemus genome. Thus, it was implied that nearly 80% genetic resource was incorporated into the library. This percentage was termed the incorporation rate.6. Based on these findings, we suggest that the incorporation rate is an essential factor for evaluating genomic libraries, particularly, when using partial digestion with restriction enzymes for library construction.     

Regional replication of the bacterial chromosome induced by derepression of prophage lambda

Summary Derepression of prophage in E. coli strain K12 results in constitutive synthesis of the enzymes directed by the nearby bacterial operon, gal (escape synthesis). Phage 82 fails to cause escape synthesis despite that it lysogenizes the strain K12 at the site identical to that of on the host chromosome. The reason for the observed difference between 82 and is studied in the light of the recent finding that escape synthesis in -lysogen is closely associated to phage-promoted replication of bacterial chromosome contiguous to the prophage including gal operon (escape replication). Excision-defective mutants from 82, 82int or 82xis, do initiate escape synthesis, suggesting that the prophage 82 is normally excised too quickly after induction to allow sufficient escape replication. In support of this, much more DNA hybridizable to bacterial DNA contained in gal accumulates after induction of 82int than after induction of 82. Studies with various hybrid phages between 82 and have suggested: 1. The occurrence of gal escape synthesis depends on the nature of the region between b2 and N in the map. 2. Regions of the 82 genome on both sides of the attachment site contribute independently to prevent gal escape synthesis. Implications of these results are discussed with regard to the factors involved in the prophage excision.The IIIrd article of this series is in Molec. Gen. Genet. 159, 185–190 (1978)     

Induction of lambdoid prophages by amino acid deprivation: Differential inducibility; Role of recA

Summary Lambda prophage in auxotrophic lysogens can be induced by omission of one or combinations of the required amino acids from the culture medium. Such amino acid deprivation can result in nearly as effective induction of lambda as thymine deprivation. Prophage 424 is also induced equally effectively under both conditions although to a lesser extent than lambda. By contrast prophage 21 and i²¹ are differentially induced effectively by thymine deprivation and virtually not at all during amino acid deprivation. The same differential induction of 21 and equivalent induction of and 424 occur when all three prophages are present in the same lysogen. Increasing the levels of repressor with a cI carrying-plasmid prevented amino acidless induction of as did the ind ^– mutation. A recA, but not a recB, mutation in the host prevented induction by amino acid deprivation. A recC mutant host showed increased spontaneous induction of and 21 prophages. The findings reported are used as an argument that the recA protease probably is not itself acting as the inducing protease and that a likely source of the observed specificity is an effector molecule. Different effector molecules may be produced in response to different exigent situations, to which the phage repressors may have evolved sensitivity. i⁸⁰ was inducible both by amino acid and thymine deprivation.     

The genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli

Summary The genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli (Ca⁺⁺, Mg⁺⁺ dependent ATPase, EC 3.6.1.3) were mapped through genetic, physical and functional analysis of specialized transducing phages asn (von Meyenburg et al. 1978). The ATP synthase genes, designated atp ¹, are located at 83.2 min in a segment of the chromosome between 3.5 and 11.3 kb left (counterclockwise) of the origin of replication oriC. The counterclockwise order of the genes for the eight subunits, the expression of which starts from a control region at 3.5 kb-L, was found to be: a, (c, b, ), , , (, ) which in the notation of Downie et al. (1981) reads atpB (E F H) A G (C D). The analysis was in part based on the isolation of new types of atp (unc, Suc^-) mutations. We made use of the fact that specialized transducing phages asn carrying oriC can establish themselves as minichromosomes rendering asnA cells Asn⁺, and that the resulting Asn⁺ cells grow slowly if the asn carries part or all of the atp operon. Selecting for fast growing strains mutations were isolated on the asn which either eliminated atp genes or affected their expression (promoter mutations). The relationship between these atp mutations and the cop mutations of Ogura et al. (1980), which also appear to map in front of or within the atp genes, is discussed.