Dependence of efficiency of DNA transfer and recombinat formation on cell cycle of Hfr in Escherichia coli K-12.

The efficiency of DNA transfer during the Hfr cell cycle, studied with the use of 3H-thymidine, is the highest in the first half of the cycle. The efficiency of recombinant formation in the Hfr cell cycle demonstrates a similar periodicity only when the ratio of 1 Hfr cell to 8 or more F- cells in mating mixture is maintained. The absence of such changes in the number of recombinants during the cell cycle of a donor with relative excess of Hfr cells seems to be caused by limitation of the number of recombinants by the competent recipent cell fraction.     

Studies on bacterial conjugational crosses via model populations. III. Reproduction of results of Hfr x F- crosses in Escherichia coli

Hfr x F- recombinational genome interactions are simulated by a computer program. Genotype spectrums of selected samples from Hfr x F- crosses of Escherichia coli, found in the published literature, are reproduced. Relevance of the findings for interpretation of conjugational results in Escherichia coli is discussed.     

F deoxyribonucleic acid superinfected into phenocopies of donor strains.

When F+ donor cells of Escherichia coli are conjugated with F-, F+, or Hfr recipients under the conditions of phenocopy mating, the male recipients are found capable of accepting the F episome as effectively as the F- recipients. The F deoxyribonucleic acid (DNA) superinfected into the male recipients is converted to the covalently closed, circular duplex form, as in the F- recipients. It is also found that the synthesis of the strand complementary to the transferred single strand and its subsequent conversion to the covalently closed, circular duplex occur effectively in male recipients as well as in female recipients. Under these mating conditions, F-ilv+ episome superinfected to F+ and Hfr cells is diluted out during growth, whereas F-ilv+ transferred into F-cells is replicated and established in almost all progeny cells. These results suggest that the incompatibility of the F episome is not due to the reduction in the rate of the conversion of transferred single-straned F DNA to covalently closed, circular duplex, but, rather, to an inhibition of further replication of the covalently closed, circular F DNA.     

Physiology of L-asparaginase synthesis in recombinants of Escherichia coli A-1.

A mating between Escherichia coli 4318 (thi leu Las- Hfr) and E. coli A-1 (Met- Las+ F-) resulted in the formation of prototrophic recombinants having L-asparaginase activities at three distinct levels. The physiology of L-asparaginase synthesis in these recombinants is decribed. One class of recombinants produced significantly more L-asparaginase than E. coli A-1. L-Asparaginase synthesis in the recombinants was inhibited by the presence of dissolved oxygen in the medium and was transiently repressed by the presence of glucose in the same manner as that observed in the parental strains. L-Asparaginase activity was increased by the addition of oxalacetate as well as other members of the tricarboxylic acid cycle.     

Hyper-Recombining Recipient Strains in Bacterial Conjugation

Using a direct enrichment and screening procedure, mutants of Escherichia coli have been isolated in which recombination frequencies for several intragenic Hfr X F- crosses are significantly higher (twofold to sixfold) than in the parental strains. These hyper-recombination mutations comprised five new mutS- and one new mutL- allele. Together with other known mut- alleles, they were analyzed for effects on intragenic recombination using several types of crosses. Hyper-recombination was found for mutS-, mutL-, mutH (= mutR)- and mutU (= uvrD)-, with the largest effects seen for certain alleles of uvrD; these resulted in over 20-fold excesses in recombinant production for Hfr X F- crosses and F'-chromosome homogenotization. Spontaneous mutator ability was not always correlated with degree of hyper-recombination.     

In vivo studies on the interaction of RecBCD enzyme and lambda Gam protein.

The interaction between the RecBCD enzyme of Escherichia coli and the lambda Gam protein was investigated. Two types of experiments were done. In one type, Gam protein was produced by transient induction of the cells lysogenic for lambda cI857gam+. The presence of Gam protein, which inhibits RecBCD nuclease, enabled these cells to support the growth of a gene 2 mutant of bacteriophage T4 (T4 2). The lysogens overproducing the RecB subunit of RecBCD enzyme could titrate Gam protein and thus prevent the growth of T4 2. In contrast, the lysogens overproducing either RecC or RecD retained their capacity for growth of T4 2. It is therefore concluded that the RecB subunit is capable of binding Gam protein. In the second type of experiments, Gam protein was provided by derepressing the gamS gene on the plasmid pSF117 (S. A. Friedman and J. B. Hays, Gene 43:255-263, 1986). The presence of this protein did not interfere with the growth of wild-type cells (which were F-). Gam protein had a certain effect on recF mutants, whose doubling time became significantly longer. This suggests that the recF gene product plays an important role in maintenance of viability of the Gam-expressing cells. Gam protein exerted the most striking effect on growth of Hfr bacteria. In its presence, Hfr bacteria grew extremely slowly, but their ability to transfer DNA to recipient cells was not affected. We showed that the effect on growth of Hfr resulted from the interaction between the RecBCD-Gam complex and the integrated F plasmid.     

Mechanism of conjugation and recombination in bacteria. XVIII. Polarity of donor DNA strands transferred to the recipient as determined by DNA-RNA hybridization.

Polarity of donor DNA strand transferred into recipient during conjugation in Escherichia coli K-12 was determined by DNA-3H-RNA hybridization. Lambda prophage was used as a marker. The defective lysogen Hfr H (lambdat11) as a donor and thermosensitive F- CR34 dnaB strain as recipient were used. Two sets of hybridization experiments, with 1-strand specific lambda mRNA and lambda mRNA specific for both phage strands but with large excess of r-strand specific mRNA, were carried out. Strand 1 of lambda DNA was detected preferentially in recipient cells mated at restrictive temperature, when Hfr transferred its genophore in the order gal-lambda-bio. Thus the genophore is transferred with 5'OH at its origin.     

Overexpression of E2F-1 inhibits progression of gastric cancer in vitro

E2F-1 plays a critical role in cell cycle regulation and other biological processes in cells. E2F-1 mediates apoptosis and suppresses tumorigenesis in many tissue types, but there are few data available on E2F-1 expression and its relationship to tumor kinetics in gastric cancer. To gain better insight into the involvement of E2F-1 in the biological characteristics of gastric tumors, we investigated the effect of E2F-1 overexpression on the progression of gastric carcinoma cells. A gastric cancer cell line stably overexpressing E2F-1 (MGC-803/E2F-1) was established. The influence of E2F-1 overexpression on in vitro cell growth was assessed by measuring cell survival, colony formation, and cell cycle progression. The results clearly show that overexpression of E2F-1 significantly inhibits cell growth and proliferation, blocking entry into the S-phase of the cell cycle. MGC-803/E2F-1 cells also had a higher apoptotic rate than control cells. In addition, E2F-1 reduced the motility and invasion of gastric cancer cells.     

Deregulated expression of cloned transcription factor E2F-1 in Chinese hamster ovary cells shifts protein patterns and activates growth in protein-free medium

Engineering of the cell cycle can be an effective means for bypassing growth factor requirements of animal cells. Cloned human E2F-1 from Nalm 6 cells was subcloned into pRc/CMV and transfected into Chinese hamster ovary (CHO) cells. Ten stable transfectant clones isolated from cells cultured under neomycin-resistance selection pressure all expressed significantly higher amounts of E2F-1 than control cells as determined by Western analysis. Confocal immunofluorescent microscopy and Southern analysis of several clones also provided evidence for the expression of cloned E2F-1 in these cells. CHO K1:E2F-1 cells are able to proliferate on well-defined serum- and protein-free basal medium and exhibit an S-phase extended by 65% compared to CHO K1 cells mitogenically stimulated by basic fibroblast growth factor (bFGF). Two-dimensional electrophoresis of the intracellular proteins of E2F-1 clones shows an increase in 236 gene products compared to CHO K1 control cells, further verifying a functional regulatory role of cloned E2F-1 in CHO cells. Among these upregulated species is the cell cycle regulatory protein, cyclin A, which has already been shown to be regulated by E2F-1 in human fibroblasts. Overexpression of cloned E2F-1 in CHO cells is a potentially useful new strategy for bypassing serum requirements in mammalian cell culture. Furthermore, such cell cycle control stimulus-protein pattern response data can contribute to a clearer understanding of complex multigene networks involved in mammalian cell cycle regulation. (c) 1996 John Wiley & Sons, Inc.     

cis-Dominant, transfer-deficient mutants of the Escherichia coli K-12 F sex factor.

Rare conjugational progeny formed by crossing each of five Hfr strains with a recA-F- strain have been characterized. Selection was made for a proximal Hfr marker, taking strict precautions to prevent transfer of recA+ to the zygotes. Most of the progeny were found to be F' strains containing deletion mutant plasmids. With two exceptions, these mutant plasmids have lost all of the tra genes, which are required to confer conjugational donor ability upon a host. In addition, all but the exceptional mutant plasmids were found to be very poorly transmissible from transient heterozygotes which also contain a wild-type F' plasmid. The poor transmissibility is a cis-dominant transfer-defective phenotype which may result from deletion of all or part of the origin of transfer replication (ori), or of a gene determining a cis-acting protein. The two exceptional mutant plasmids may carry short deletions of some of the tra genes or polar tra mutations. The remaining progeny were nonmutant F' strains and F- strains. The frequency with which the F- strains were recovered permits us to estimate that the maximum amount of recombination possible in a recA56 zygote is 10(-6) that of a recA+ zygote.     

Chromosome transfer and Hfr formation by F in rec+ and recA strains of Escherichia coli K12.

We attempted to assess the role of Hfr clones in chromosome transfer by F⁺ populations. We thought that any Hfr-independent component of fertility might be affected to a different extent by the recA mutation than was the Hfr component. However, the rate of Hfr formation and the efficiency of chromosome transfer were reduced to an equal extent (× 100-fold) by the recA mutation. Such experiments therefore provide no evidence for an Hfr-independent component. It appeared that Type II strains, which were thought to suffer a defect in Hfr formation, actually produced fertile clones but had a secondary defect which affected the persistence of these clones. Thus, evidence from Type II strains is also not useful for examining the quantitative contribution of Hfr cells to F⁺ transfer.     

Tryptophan-requiring parental strains yield Salmonella typhimurium x Escherichia coli hybrid recombinants with functional tryptophan operons.

Crosses between an Escherichia coli Hfr trp strain and three Salmonella typhimurium F- trp strains produced some trp+ hybrids in which the tryptophan operon is composed of genes from both parental species.     

Localized mutagenesis with bacteriophage Mu: method for increasing the frequency of specific bacterial mutants.

A method is described for markedly enriching a bacterial population for cells containing any given Mu insertion mutation. The method involves the transfer of a small piece of deoxyribonucleic acid from a Mu-infected Hfr donor donor strain to a suitable F- strain and a subsequent selection of those recombinant organisms that have received a Mu prophage from the donor. The method is particularly usefule for isolating mutants whose selection requires "brute-force" assay, since only a few hundred colonies have to be screened.     

Active nuclear import and export pathways regulate E2F-5 subcellular localization

Epidermal keratinocyte differentiation is accompanied by differential regulation of E2F genes, including up-regulation of E2F-5 and its concomitant association with the retinoblastoma family protein p130. This complex appears to play a role in irreversible withdrawal from the cell cycle in differentiating keratinocytes. We now report that keratinocyte differentiation is also accompanied by changes in E2F-5 subcellular localization, from the cytoplasm to the nucleus. To define the molecular determinants of E2F-5 nuclear import, we tested its ability to enter the nucleus in import assays in vitro using digitonin-permeabilized cells. We found that E2F-5 enters the nucleus through mediated transport processes that involve formation of nuclear pore complexes. It has been proposed that E2F-4 and E2F-5, which lack defined nuclear localization signal (NLS) consensus sequences, enter the nucleus in association with NLS-containing DP-2 or pRB family proteins. However, we show that nuclear import of E2F-5 only requires the first N-terminal 56 amino acid residues and is not dependent on interaction with DP or pRB family proteins. Because E2F-5 is predominantly cytoplasmic in undifferentiated keratinocytes and in other intact cells, we also examined whether this protein is subjected to active nuclear export. Indeed, E2F-5 is exported from the nucleus through leptomycin B-sensitive, CRM1-mediated transport, through a region corresponding to amino acid residues 130-154. This region excludes the DNA- and the p130-binding domains. Thus, the subcellular distribution of E2F-5 is tightly regulated in intact cells, through multiple functional domains that direct nucleocytoplasmic shuttling of this protein.     

Studies on bacterial conjugational crosses via model populations. I. The model populations

A procedure is presented in which model populations, simulating progenies obtained from Escherichia coli Hfr x F- crosses, are generated. The procedure seems to be appropriate for visualizing hidden features of the genetic analysis, which are not detected by the conjugational crosses.     

Convenient transduction of recA with bacteriophage T4GT7

The generalized transducing phage T4GT7 grew well on recA strains of Escherichia coli and transduced recA into F- and Hfr strains of E. coli at high frequency.     

Specific Action of Sodium Dodecyl Sulfate on the Sex Factor of Escherichia coli K-12 Hfr Strains

A specific action of sodium dodecyl sulfate (SDS) on the sex (F) factor in the integrated state of Escherichia coli K-12 Hfr H strain is reported. Growth of Hfr cells in Penassay Broth containing SDS results in the elimination of part or all of the F factor, yielding low and nonfertile variants of defective Hfr type and F⁺ cells and also F⁻ derivatives. Appearance of such variants was generally observed after the culture reached stationary phase. The frequencies of F⁻ cells then increased. F⁻ cells were usually isolated as the major population among survivors. Some defective variants of Hfr cells with an intermediate fertility between standard Hfr and F⁺ cells had lost sensitivity toward the male-specific ribonucleic acid phage M12. Other defective Hfr variants with as much or less fertility than standard F⁺ cells had also all lost sensitivity to phage M12. On single-colony isolation, they segregated nonfertile female H cells which, when infected with F, could restore high fertility with oriented transfer of the chromosome the same as that of the original Hfr H. Also, sensitivity to phage M12 was regained. Female H cells were characterized as those lacking fertility but still retaining a small segment of F or sfa locus at the original part of the chromosome, where newly infected F could attach. Similar results were obtained with two other Hfr strains. A possible mechanism of the specific action of SDS is discussed.     

Stability of the carrier state in bacteriophage M13-infected cells.

Bacteriophage M13-infected carrier cells were shown to be unstable to prolonged growth under all conditions. Carrier Hfr cells were transferred in dilute culture (10(3) to 10(4)/ml), where reinfection was impossible and the physiology of the cell was minimally altered. After an initial period of about 10 generations, during which all cells in the culture remained infected, there was exponential decay in the proportion of infected cells in the culture. Uninfected cells that appeared were M13 sensitive. Hfr and F' males were also transferred serially at high cell densities (10(7) to 10(9)/ml), where high levels of phage should permit reinfection. The proportion of phage-producing cells in the cultures remained constant for 7 to 15 generations and then dropped exponentially on further growth. Non-phage-producing cells appearing in the culture were refractory to infection by M13; in some cases cells scored as non-phage producers for 20 generations were observed to produce phage on further growth in liquid culture. F'trp+ males infected with M13 lost trp+ function almost immediately; this was not regained in these experiments. Infected cells grown in dilute culture or on plates remained infected longer, produced more PFU per cell for a longer period, and retained trp+ function in F'trp+ males for over 90 generations. Non-phage-producing cells that appeared were sometimes phage resistant, sometimes phage sensitive. The existence of a phage-related material accumulating at high cell densities and affecting expression of free episomes, episomal expression in Hfr males, and phage synthesis itself is suggested.