TherecA-dependent spontaneous degradation of proximal F merogenotes inEscherichia coli

Proximal F’ elements of KLF-1 type are relatively stable inEscherichia coli rec A recipients. In such merodiploids the transferability of F’-DNA and the plasmid determined fertility functions are expressed. When introduced into the wild typerecA ⁺ cells the F′-DNA is degraded and several classes of DNA molecules of molar mass about 66 Mg/mol and lower exist in the cell in 1–2 copies, per bacterial chromosome. As was detected by complementation analysis, the chromosomal genes determining the host specificity for DNA (hsd) originally located on the F’ element seem to be salvaged during the process of DNA degradation probably by recombination with the bacterial chromosome.     

Plasmid control of recombination in E. coli K 12

Summary The recombination proficiency of three recipient strains of Escherichia coli K 12 carrying different plasmids was investigated by conjugal mating with Hfr Cavalli. Some plasmids (e.g. R1drd 19, R6K) caused a marked reduction in the yield of recombinants formed in crosses with Hfr but did not reduce the ability of host strains to accept plasmid F104. The effect of plasmids on recombination was host-dependent. In Hfr crosses with AB1157 (R1-19) used as a recipient the linkage between selected and unselected proximal markers of the donor was sharply decreased. Plasmid R1-19 also decreased the yield of recombinants formed by recF, recL, and recB recC sbcA mutants, showed no effect on the recombination proficiency of recB recC sbcB mutant, and increased the recombination proficiency of recB, recB recC sbcB recF, and recB recC sbcB recL mutants. An ATP-dependent exonuclease activity was found in all tested recB recC mutants carrying plasmid R1-19, while this plasmid did not affect the activity of exonuclease I in strain AB1157 and its rec ^– derivatives. The same plasmid was also found to protect different rec ^– derivatives of the strain AB1157 against the lethal action of UV light. We suppose that a new ATP-dependent exonuclease determined by R1-19 plays a role in both repair and recombination of the host through the substitution of or competition with the exoV coded for by the genes recB and recC.     

Recombination between an Flac and a mini-FKm plasmid deleted for an origin of replication

The mini-F plasmid specifying resistance to kanamycin (Km), pML31, contains an origin of replication at kilobase coordinate 42.6 in the F DNA sequences. In previous research we found that this origin could be deleted by recombinant DNA techniques without the loss of plasmid maintenance functions. In this report we show that the deleted plasmid, designated pMF21, has normal incompatibility properties and a recA⁺-dependent ability to form cointegrates with an Flac plasmid. By comparison, pML31 does not form cointegrates with the Flac plasmid at a detectable frequency. The frequency for spontaneous loss of the Lac⁺ phenotype in strains containing pMF21:Flac cointegrates resembles that of the Flac plasmid; however, in some Lac⁻ variants the Km^r phenotype is retained. Examination of the plasmid DNA in four of these Lac⁻Km^r clones revealed two with normal pMF21 plasmids and two with plasmids intermediate in size between pMF21 and the Flac.     

Genetic and physical characterization of the ColIb plasmid using ColIb-R222 hybrids

Summary The presence of the ColIb plasmid in Escherichia coli cells inhibits the growth of bacteriophages BF23 and T5 (Ibf phenotype; inhibition of BF23 and T5 growth). To understand this abortive infection, we devised a method of isolating mutants that were defective in some ColIb phenotypes including Ibf. This method consisted of transduction of the tet (Tc^r; tetracycline resistance) or cml (Cm^r; chloramphenicol resistance) gene of plasmid R222 with phage P22 into ColIb, construction of Tc^rCm^rIbf⁺ Imm⁺ (immunity to colicin Ib) Cib^- (no production of colicin Ib) recombinants by crossing between the transductants, and isolation of deletion mutants from the recombinants by phage P1 transduction. By this procedure, pKM25-2 (Tc^rCm^sIbf^-Imm^-Cib^-) and pKM25-1 (Tc^rCm^sIbf⁺Imm⁺Cib^-) were isolated. Construction of the cleavage map of the ColIb plasmid by restriction endonucleases and comparative analyses of the DNA fragments produced from the mutant plasmids revealed that the genes determining Ibf and Imm mapped on a 4.60 Mdal HindIII fragment (H-3) and the gene determining Cib on a 1.71 Mdal EcoRI fragment (E-12).These results together with other observations (Wilkins et al. 1981; Hama personal communication) also show the approximate positions of the genes for Rep (replication), Inc (incompatibility), and Sog (suppression of dnaG) as well as Ibf, Imm, and Cib phenotypes on the cleavage map of the ColIb plasmid.Preliminary data were reported in the 1979 Annual Meeting of the Japan Molecular Biology Society (Uemura and Mizobuchi, Abst Ann Mol Biol Meet 1979, p 36)     

Further data on the polarity of the paba1 locus of Aspergillus nidulans

Newly mapped paba1 alleles are better distributed on the fine-structure map than those mapped earlier. They provide a proximal, a middle and a distal cluster, of four, one and four sites respectively (Fig. 1). The recombination fractions indicate map expansion (Fig. 2). The large samples of paba ⁺ recombinants classified show that classes I and I III are positively correlated with increased recombination fraction, and classes I II and I II III are negatively correlated (Fig. 3). No polarity reversal was found. The results are explained in terms of a hybrid DNA model for crossing over.     

Intramolecular homologous recombination of linearized plasmids in Escherichia coli K12

Summary The efficacy of linear DNA as a substrate for general homologous recombination was demonstrated using BamHI-linearized pKLC8.5, a plasmid that carries internal direct repeats flanking the unique BamHI site. An analogous plasmid, pKLC2.31, was used in a parallel and comparative study of intramolecular homologous recombination in circular DNA substrates. When the rec ⁺ wild-type strain, AB1157, and its isogenic rec ^– derivatives were transformed with linear pKLC8.5 DNA, intramolecular homologous recombination was independent of recA, recB, recN, recO and exonuclease III (xth-1) functions. Although the recBCsbcA and recBCsbcBC cells were both very recombination proficient, only linear but not circular DNA was used as substrate for intramolecular homologous recombination in the recBCsbcA cells. In both the recBCsbcA and recBCsbcBC genetic backgrounds, the recombination frequencies for linearized pKLC8.5 DNA were 100%. A notable difference between the two strains was that none of the recBCsbcA transformants obtained with circular pKLC8.5 DNA were Tc^s recombinants, whereas 11% of the corresponding recBCsbcBC transformants were Tc^s recombinants. The sbcB mutation was responsible for the recombination proficiency of the recBCsbcBC cells. Unlike the case in recBCsbcA cells, intramolecular homologous recombination of linear DNA in the recBCsbcBC cells was dependent on recA and recF as well as recN and recO gene functions, but was independent of recJ and reeL gene functions.     

Trans-dominance of dnaA mutants in Escherichia coli.

Summary Temperature sensitivity of growth and DNA synthesis was tested in merogenotes heterozygous for thednaA allele. All combinations tested (FdnaA⁺/dnaA5, FdnaA⁺/dnaA46, FdnaA⁺/dnaA204, FdnaA5/dnaA⁺, FdnaA204/dnaA⁺) were temperature sensitive. The mutantdnaA allele is thus trans-dominant to the wild type allele.     

Properties of a temperature sensitive plasmid in Citrobacter freundii

Summary The properties of a strain of Citrobacter freundii with a deletion of the gal, chl D, bio, uvr B, chl A region of the chromosome, harbouring a temperature sensitive plasmid F _{ts 114} ¹ aro G⁺ gal ⁺ chl D⁺ bio ⁺ uvr B⁺, originating from Escherichia coli, are described.The isolation of strains with an integrated plasmid was tried by incubation of the partial diploid strain at the restrictive temperature and selection for retained plasmid properties. However C. freundii Hfr strains were not obtained, since only fragments of the plasmid were integrated. Integration occurred at seven different sites of the chromosome and resulted in an inactivation of the gene in which the fragment was integrated. Mutants with deficiencies for arginine, isoleucine and valine, tryptophan, guanine and either tryptophan or tyrosine were obtained. In another type the deficiency, resulting from integration, could not be identified, whereas in the seventh type integration had occurred in one or more non-essential genes, because no deficiency was present. Release of the integrated fragment occurred in such a way that gene activity was restored. The released fragment was lost or was integrated again at one of the other six integration sites, resulting in another mutant type.     

Measuring the rate of conjugal plasmid transfer in a bacterial population using quantitative PCR

Horizontal transfer of genes between species is an important mechanism for bacterial genome evolution. In Escherichia coli, conjugation is the transfer from a donor (F⁺) to a recipient (F⁻) cell through cell-to-cell contact. We demonstrate what we believe to be a novel qPCR method for quantifying the transfer kinetics of the F plasmid in a population by enumerating the relative abundance of genetic loci unique to the plasmid and the chromosome. This approach allows us to query the plasmid transfer rate without the need for selective culturing with unprecedented single locus resolution. We fit the results to a mass action model where the rate of plasmid growth includes the lag time of newly formed F⁺ transconjugants and the recovery time between successive conjugation events of the F⁺ donors. By assaying defined mixtures of genotypically identical donor and recipient cells at constant inoculation densities, we extract an F plasmid transfer rate of 5 × 10⁻¹⁰ (cells/mL · min)⁻¹. We confirm a plasmid/chromosome ratio of 1:1 in homogenous F⁺ populations throughout batch growth. Surprisingly, in some mixture experiments we observe an excess of F plasmid in the early saturation phase that equilibrates to a final ratio of one plasmid per chromosome.     

cis-Dominant, glutamine synthetase constitutive mutations of Escherichia coli independent of activation by the glnG and glnF products

Summary Mutants resistant to 80 M L-methionine-DL-sulfoximine (MS) were isolated on glucose-minimal 15 mM NH ₊ ⁴ medium plates from Escherichia coli cells which were hypersensitive to this concentration of the analogue by virtue of their harboring glnG mutations. MS-resistant mutants derived from strain MX902 carried, in addition to its glnG74::Tn5 allele, mutations tightly linked to glnA, as shown by P1-mediated transduction experiments. One particular allele, gln-76, which suppressed the MS-sensitivity conferred by glnG74::Tn5 but not its Ntr^– phenotype (inability to transport and utilize compounds such as arginine or proline as the only nitrogen sources), was shown to allow constitutive expression of glutamine synthetase in the absence not only of a functional glnG product but also of a functional glnF product. This behavior was found to be cis-dominant in complementation experiments with F'14 merogenotes. In an otherwise wild-type genetic background as in MX929 (gln-76 glnA ⁺ glnL⁺ glnG⁺ glnF⁺), however, normal activation, mediated by the glnG and glnF products was preferred over that mediated by gln-76.     

Spermatogonial exchange involving the X but not the y chromosome inDrosophila Melanogaster

Summary The distal uninverted portion of In(1)sc⁸, which carriesy ⁺ andac ⁺, is occasionally lost during spermatogonial divisions. This is accomplished by exchange between the protion of the proximal heterochromatin that has been removed distally by the inversion and some other heterochromatin in the complement (see alsoLindsley 1955b).. The majority of the recombiants recovered from males carrying In(1)sc⁸ arise through exchange with the Y chromosome (12/15). The majority of the recombinants recovered from males carring In(1)sc^8L, EN^R, which is characterized by a heterochromatic second arm, do not arise through exchange with the Y chromosome (18/22). The absolute frequencies of Y involvement with In(1)sc⁸ (7/105067) and In(1)sc^8L, EN^R,(2/38588), however, are comparable. The heterochromatic constitution of the recombinants examined is consistent with the hypothesis that an observed excess of recombinants recoverred from In(1)sc^8L, EN^R as compared with In(1)sc⁸ is accounted for by Y independent recombinants and is the consequence of exchange between the second heterochromatin arm of In(1)EN^R and the distal heterochromatin of In(1)sc^8L. A maximum of six different regions of exchange between these two regions may be inferred from the constitution of the recombinants. This inference is considered to support the hypothesis that pairing and exchange between heterochromatic regions are not strictly homologous.With 6 Figures in the TextOperated by Union Carbide Nuclear Company for the U.S. Atomic Energy Commission.Part of the material was presented to the Graduate School of the California Institute of Technology in partial fulfillment of requirements for the degree of Doctor of Philosophy supported by an Atomic Energy Commission predoctoral fellowship. Further experimentation has been pursued under a National Research Council postdoctoral fellowship at the University and under a National Science Foundation postdoctoral fellowship at the University of Missouri. Experimentation was completed at Oak Ridge.     

Conjugal Transfer of Genetic Information in Group N Streptococci

Streptococcus lactis strains ML3 and C₂O and S. lactis subsp. diacetylactis strains DRC3, 11007, and WM₄ were found to transfer lactose-fermenting ability to LM0230, an S. lactis C2 lactose-negative (Lac⁻) derivative which is devoid of plasmid deoxyribonucleic acid (DNA). Lactose-positive streptomycin-resistant (Lac⁺ Str^r) recombinants were found when the Lac⁺ Str^s donor was mixed with Lac⁻ Str^r LM0230 in solid-surface matings. Transduction and transformation were ruled out as the mechanism of genetic exchange in strains ML3, DRC3, 11007, and WM₄, nor was reversion responsible for the high number of Lac⁺ Str^r recombinants. Furthermore, chloroform treatment of the donor prevented the appearance of recombinants, indicating that transfer of lactose-fermenting ability required viable cell-to-cell contact. Strain C₂O demonstrated transduction as well as conjugation. Transfer of plasmid DNA during conjugation for all strains was confirmed by demonstrating the presence of plasmid DNA in the transconjugants by using agarose gel electrophoresis. In some instances, a cryptic plasmid was transferred in conjunction with the lactose plasmid by using strains DRC3, 11007, and WM₄. In S. lactis C2 × LM0230 matings, the Str^r marker was transferred from LM0230 to C2, suggesting conjugal transfer of chromosomal DNA. The results confirm conjugation as another mechanism of genetic exchange occurring in dairy starter cultures.     

Genetic rearrangements in plasmids specifying total degradation of chlorinated benzoic acids

Summary Growth in a chemostat of the 3-chlorobenzoatepositive Pseudomonas putida cells harboring the plasmid pAC25, in presence of cells harboring the TOL plasmid, allows emergence of cells that can also utilize 4-chlorobenzoate (4Cba). Isolation of plasmid DNA from such cells demonstrate the deletion of a 11kb (Kilobase pair) EcoR1 fragment from the pAC25 plasmid; a portion of the TOL plasmid (41.5 kb TOL^*) is also found to be transposed onto the chromosome of such cells. Further enrichment of the 4-chlorobenzoate-positive cells with 3,5-dichlorobenzoate (3,5-Dcb) as a sole carbon source has produced cells that can also slowly utilize 3,5-dichlorobenzoate. Isolation of plasmid DNA from such cells demonstrates the appearance of a second plasmid (pAC29). Restriction hybridization of pAC29 EcoRI fragments with pAC25 and TOL demonstrates that pAC29 is derived primarily by duplication of a segment of the pAC27 plasmid and a fragment from TOL, with further mutational divergence. Southern hybridization of the EcoRI-digested chromosomal DNA with ³²P-labeled TOL, pTS11 and pTS71 plasmid DNAs demonstrates the presence of the TOL^* transposon containing xylD, G, E and F genes in both 4Cba⁺ (pAC27⁺) and 3,5-DCb⁺ (pAC27⁺, pAC29⁺) cells. Isolation of plasmid DNA from 3,5-Dcb⁺ faster growing variants, obtained from slow-growing pAC27⁺ pAC29⁺ cells, demonstrates the presence of a single type of plasmid, with identical size and EcoRI digestion profile as pAC27. The implications of gene duplications and subsequent homologous recombination with regard to the biochemical pathway of 3,5-dichlorobenzoate degradation have been discussed.     

Phylogenetic and functional conservation of the NKR-P1F and NKR-P1G receptors in rat and mouse

Two clusters of rat Nkrp1 genes can be distinguished based on phylogenetic relationships and functional characteristics. The proximal (centromeric) cluster encodes the well-studied NKR-P1A and NKR-P1B receptors and the distal cluster, the largely uncharacterized, NKR-P1F and NKR-P1G receptors. The inhibitory NKR-P1G receptor is expressed only by the Ly49s3⁺ NK cell subset as detected by RT-PCR, while the activating NKR-P1F receptor is detected in both Ly49s3⁺ and NKR-P1B⁺ NK cells. The mouse NKR-P1G ortholog is expressed by both NKR-P1D⁻ and NKR-P1D⁺ NK cells in C57BL/6 mice. The rat and mouse NKR-P1F and NKR-P1G receptors demonstrate a striking, cross-species conservation of specificity for Clr ligands. NKR-P1F and NKR-P1G reporter cells reacted with overlapping panels of tumour cell lines and with cells transiently transfected with rat Clr2, Clr3, Clr4, Clr6 and Clr7 and mouse Clrc, Clrf, Clrg and Clrd/x, but not with Clr11 or Clrb, which serve as ligands for NKR-P1 from the proximal cluster. These data suggest that the conserved NKR-P1F and NKR-P1G receptors function as promiscuous receptors for a rapidly evolving family of Clr ligands in rodent NK cells.     

Variety of Hybrid Characters among Recombinants Obtained by Interspecific Protoplast Fusion in Streptomycetes

To discover whether the protoplast fusion method is useful or not for interspecific breeding, some methods were devised, and the appearance of various hybrids with different characters and the change of antibiotic activities in the recombinants obtained by the protoplast fusion were investigated. The purification of protoplasts, the choice of parental natural characters as selection markers, and the adoption of a replica method for selecting all types of recombinants were devised and used for these experiments. Protoplast fusion was done between S. griseus KCC S-0644 and each strain of 5 species that were clearly different species from S. griseus, in addition to being streptomycin sensitive (SM^s) and capable of L-arabinose utilization for growth (Ara⁺). Recombinants (SM^r, Ara⁺) obtained by protoplast fusion displayed a great variety of hybrids in their taxonomic characters, e.g., 21 recombinant strains obtained by the cross between S. griseus and S. griseoruber consisted of 14 types of hybrids. Antibiotic productivity was examined in all recombinants obtained. Although both parental species produced their respective antibiotics, 60% of the recombinants did not produce any antibiotic and 24% produced different antibiotics from those of their parents. Among those recombinants, it was also found that the distribution of the productivity of each antibiotic among the recombinants was entirely different from that of the allelo-character in each taxonomic feature.     

Influence of edeine on intergenic and interallelic recombination in Neurospora crassa

Summary The effect of edeine and the mutation ed ^r-2 to edeine resistance on genetic recombination in Neurospora crassa was investigated. For this purpose crosses between pairs of edeine sensitive and edeine resistant strains respectively were set up without or in the presence of the drug (0–750 g/ml). The genetic markers ylo-1, ad-1, pan-2 (B ₃ and B ₅) and tryp-2, all on linkage group VI, were used for scoring recombinants. These were ad ⁺, tryp ⁺ (intergenic recombination) and pan ⁺ (interallelic recombination).Frequencies of about 6–7% for intergenic and of about 0.4% for interallelic recombination were found in crosses between ed^s strains and ed^r strains respectively, if edeine was absent. However, crosses in the presence of edeine gave higher frequencies of both intergenic and interallelic recombination (about 12% intergenic and 1% interallelic with 180 to 200 g ed/ml).The pan⁺ prototrophs (interallelic recombinants) obtained in the different crosses were tested for distribution of outside markers. The data thus obtained revealed, that under the effect of both the mutation to edeine resistance and edeine itself the relative number of non-crossover (gene conversion) recombinants decrease in favour of crossover recombinants, and the relative number of double crossover recombinants (events outside the pan locus) decreases in favour of single crossover recombinants.It is concluded that a) edeine and the mutation ed ^r-2 to edeine resistance affect recombination via related pathways, and b) noncrossover and crossover recombinants are caused by different molecular mechanisms, in agreement with the work of other authors.     

Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3

Summary R124 and R124/3 are R plasmids that carry the genes for two different restriction and modification systems. The phenotype of strains carrying either of these plasmids along with the F'lac ⁺ plasmid, is restriction-deficient (Res^-). The Res^- phenotype is not due to selection of preexisting mutants but rather to a complex mutational event caused by the F plasmid. Restriction-deficient mutants carry extensive deletions and other DNA rearrangements. Tn7 insertion is used to locate the restriction gene. Many of the Res^- mutants are genetically unstable and revert at exceptionally high frequencies. Reversion is accompanied by DNA rearrangements which result in a net gain of 9 kb of DNA. F derivates of F⁺ which do not cause restriction-deficiency but do cause deletion were used to distinguish between the DNA rearrangements associated with restriction-deficiency and those associated with deletion. From Res⁺ revertants of strains carrying F'lac ⁺ and R124 or R124/3 we have isolated F plasmids that now carry the genes for the R124 or R124/3 restriction and modification systems. It is suggested that interaction between part of the F plasmid and that segment of the R plasmid which controls the switch in Res-Mod specificity which has been observed (Glover et al. 1983) is responsible for the production of restriction-deficiency.     

Expression of an excision repair gene in transformation of Bacillus subtilis.

Summary DNA of Bacillus subtilis proficient in excision repair (hcr ⁺) was introduced into Angiografinpurified competent cells of an excision repair-deficient strain UVS-1 (hcr-1). The hcr ⁺ gene was found to affect the UV-survival curve of the cells, giving rise to a UV-resistant component. However, a considerable number of colonies of the UV-resistant component consisted of cells that were not transformed to hcr ⁺ as judged by their sensitivity to mitomycin C (MC), UV, and by their ability to reactivate UV-irradiated M2 phages. This suggests that the hcr gene may be expressed without integration. The recA function of B. subtilis was necessary for expression of UV resistance to occur. When DNA-treated cells were selected for met ⁺ recombinants, the UV-resistant component was again found on the UV-survival curve and about half of the colonies of the UV-resistant component consisted of Hcr^- cells. This result was explained by an integration-segregation model for hcr ⁺ and met ⁺ genes. The effect of the hcr ⁺ gene was seen even when DNA was added after cells were irradiated with UV, although this effect was gradually diminished by delaying the time of DNA addition. A complementation effect was found between two excision repair mutations residing in two distant loci, using hcr-114 DNA as a donor and hcr-1 cells as a recipient.     

F plasmid incompatibility and copy number genes: Their map locations and interactions

The genes coding for vegetative F plasmid replication, replication control, and incompatibility are known to map between the kilobase coordinates 40.3 and 49.3 (abbreviated 40.3–49.3F). We have subdivided this region of the F genome by a combination of in vivo and in vitro genetic techniques and have constructed F:pSC101 hybrid plasmids which contain the F DNA sequences having the approximate coordinates 41–43, 43–46, and 46–49F. We find that hybrids with regions 43–46 and 46–49F are incompatible with an F′lac⁺ plasmid while the hybrid with the region 41–43F is compatible. We have also constructed similar F:pSC101 hybrid plasmids with the regions 43–46 and 46–49F derived from mini-F plasmid copy number mutants. We find that hybrids made from three independent F copy number mutants show a loss of the incompatibility function associated with the 43–46F region and retention of the incompatibility function associated with 46–49F region. Moreover, spontaneous revertants, selected for regain of the 43–46F incompatibility function, have also regained normal control over their copy numbers. We also find that copy number mutations map in the 43–46F region. From our results we conclude (i) that F contains at least two inc⁺ loci, designated incA⁺ (46–49F) and incB⁺ (43–46F), and (ii) that gene(s) regulating F copy number may be related to the incB⁺ gene(s).