Repair of UV-damaged DNA by competent and incompetent Bacillus subtilis cells.
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Bacillus subtilis↗
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Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. I. Construction of a RIP1 deletion strain and isolation of temperature-sensitive mutants.
A protocol has been devised to permit mutational analysis of the Rieske iron-sulfur protein of the mitochondrial cytochrome bc1 complex of Saccharomyces cerevisiae. The gene for this iron-sulfur protein (RIP1) has recently been cloned and sequenced (Beckmann, J. D., Ljungdahl, P. O., Lopez, J. L., and Trumpower, B. L. (1987) J. Biol. Chem. 262, 8901-8909). We have constructed a stable yeast deletion strain, JPJ1, in which the chromosomal copy of RIP1 was displaced by the yeast LEU2 gene by homologous recombination. A linear DNA fragment containing the LEU2 gene was inserted at the breakpoints of an 800-base pair deletion of the iron-sulfur protein gene and used to transform a leu- yeast strain. Leu+ transformants were obtained which were unable to grow on nonfermentable carbon sources. Southern analysis of the transformant, JPJ1, confirmed that the chromosomal copy of the RIP1 gene was deleted and replaced by the LEU2 gene. The genotype of JPJ1 was confirmed by genetic crosses. JPJ1 cannot grow on nonfermentable carbon sources but can be complemented to respiratory competence and transformed by yeast vectors containing the wild type RIP1 gene. The ability to complement strain JPJ1 with episomally encoded iron-sulfur protein provided the basis of a selection protocol by which mutagenized plasmids containing the RIP1 gene were assayed for mutations affecting respiratory growth. Five mutants of RIP1 were identified by their ability to complement JPJ1 to temperature-sensitive respiratory growth. DNA sequence analysis demonstrated that temperature-sensitive respiratory growth resulted from single point mutations within the protein coding region of RIP1. These mutations altered a single amino acid residue in each case. Mutations were dispersed throughout the terminal two-thirds of the protein. Each mutation was recessive and did not affect fermentative growth on dextrose. However, each mutation exerted unique temperature-sensitive growth characteristics on media containing the nonfermentable carbon source glycerol.
Amino Acid Sequence↗
Conditions for transformation of human fibroblast cells: an overview.
Low passage (low population doubling) human diploid fibroblasts respond to carcinogen and mutagen treatment, with higher passage level human cells remaining refractory to the insult. A cell cycle dependency for an optimize response to the carcinogen of competent responsive low passage cells is associated with early S phase. The process of fixation of the damage in dividing young cells could be more efficient due to intrinsic sensitivity of young cells towards carcinogens. However, specific DNA-carcinogen adduct analysis does not reveal any qualitative or quantitative difference. These low passage carcinogen initiated human cells progress towards the expression of a malignant phenotype. There is little evidence to suggest that these abnormal phenotypes exhibit an infinite lifespan using the selection pressures for isolation of the transformed phenotypes. However, the lifespan of these treated cells is extended beyond those of the untreated cells. In conclusion, criteria can be established to measure the expression of progression of these carcinogen initiated cells towards a malignant phenotype.
Antigens, Neoplasm↗
Synthesis of biologically active rat transforming growth factor I.
The hypothesis that some transformed cells produce endogenous transforming growth factors (TGFs) has been supported by isolation of peptide factors from transformed cells. One group of TGFs, TGF-I, produced only by transformed cells, displays sequence homology with the functionally related mouse submaxillary epidermal growth factor ( mEGF ). Both TGF-I and mEGF exhibit similar activities in competition for binding to the EGF receptor, stimulation of DNA synthesis and cell growth. Another group of TGFs, TGF-II (also known as TGF beta), present both in normal and transformed cells, is structurally and functionally unrelated to TGF-I or EGF, and does not compete for binding to the EGF receptor or induce cell growth. However, TGF-I or EGF in the presence of TGF-II produces a synergistic effect that is responsible for the observed phenotypic transformation of NRK fibroblasts. The complete amino acid sequence of rat TGF-I ( rTGF -I) from transformed Fischer rat embryo fibroblasts, has recently been determined. Using this proposed sequence, we have now prepared synthetic rTGF -I by the solid-phase synthesis method and find that it exhibits chemical and biological properties indistinguishable from those of natural rTGF -I. Since synthetic rTGF -I is free of any biological contaminants, our findings provide independent evidence that rTGF -I is an active principle in the transformation of cells.
Amino Acid Sequence↗
Cloning and characterization of the Haemophilus influenzae Rd rec-1+ gene.
The Haemophilus influenzae Rd rec-1+ gene was cloned from a partial chromosomal digest into a plasmid vector as a 20-kilobase-pair (kbp) BstEII fragment and then subcloned. The smallest subclone with rec-1+ activity carried a 3.1-kbp EcoRI fragment. The identity of the rec-I gene in these clones was confirmed by transforming an Rd strain carrying a leaky rec-1 mutation (recA4) to resistance to methyl methanesulfonate (MMS) by using whole or digested plasmids. It was demonstrated that the Rec+ phenotype of the MMSr transformants was linked to the strA, novAB, and mmsA loci, as expected if the recA4 allele had been replaced by rec-1+. In growing cultures (rec-1 or rec+), all rec-1+-carrying plasmids induced near-maximal levels of transformability when their hosts reached stationary phase; these levels are 100 to 1,000 times higher than the values seen with strains not carrying a Rec plasmid. Transfer of the 3.1-kbp subclone was greatly reduced compared with transfer of similarly sized vector plasmids, and the resulting transformants grew slowly; this suggests an explanation of my failure to directly clone this fragment from chromosomal DNA digests. Transfer of a rec-1+ plasmid to a very poorly genetically transformable H. influenzae Rb strain resulted in greatly increased transformability. Transfer of such plasmids to a noncompetent H. influenzae Rc strain did not render this strain competent. It is suggested that transformability of Rd and Rb strains is limited by rec-1 expression but that the noncompetence of Rc has some other basis.
Aerobiosis↗
In vitro assay for HCV serine proteinase expressed in insect cells.
AIM: To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells. METHODS: The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition. After the recombinant bacmids had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells. The bacmids DNA was verified to replicate in insect cells and packaged into baculovirus particles via PCR and electronic microscopic analysis. The insect cells infected with recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was soluted in N-lauryl sarcosine sodium (NLS) and purified by metal-chelated-affinity chromatography, then the antigenicity of recombinant protease was determined by enzyme-linked immunoabsorbant assay and its enzymatic activity was detected. RESULTS: The HCV NS3 protease domain was expressed in insect cells at high level and it was partially solved in NLS. Totally 0.2 mg recombinant serine proteinase domain with high purity was obtained by metal-chelated-affinity chromatography from 5 x 10(7) cells, and both antigenicity and specificity of the protein were evaluated to be high when used as antigen to detect hepatitis C patients' sera in indirect ELISA format. In vitro cleavage assay corroborated its enzymatic activity. CONCLUSION: The recombinant HCV NS3 proteinase expressed by insect cells is a membrane-binding protein with good antigenicity and enzymatic activity.
Animals↗
Isolation and characterization of a competence operon associated with transformation and adhesion in Helicobacter pylori.
To identify adhesion-associated loci, we screened 1500 mutant strains of a Helicobacter pylori mutant library. A mutant that showed decreased adhesion to two gastric epithelial cell lines was identified. Inverse polymerase chain reaction (PCR) revealed that the interrupted locus of this mutant was an hp0015 homolog of H. pylori strain 26695. DNA sequencing and reverse-transcription PCR revealed that hp0015 and two downstream genes (hp0016 and hp0017) were a transcriptional unit. Deletion and complementation constructs revealed that hp0016 and hp0017 were involved in natural transformation, but not in adhesion. Hp0015 was associated with both adhesion and natural transformation. The reduction of adhesion to human gastric tissues in the hp0015 mutant was similar to that of the babA2 knockout mutants and greater than that of the sabA knockout mutants. Co-culture of a wild-type strain with AGS cells revealed that 19 genes in AGS cells were upregulated; however, five of the 19 genes were not induced by co-culture with hp0015 mutants. These results indicate that hp0015 is associated with adhesion, and hp0015, hp0016, and hp0017 are associated with natural transformation. Hp0017 has been named as virB4/comB4. Therefore, hp0015 and hp0016 are named as comB2 and comB3, respectively.
Adhesins, Bacterial↗
Incompatibility and transforming efficiency of ColE1 and related plasmids.
Replicons derived from the ColE1 plasmid are incompatible with one another, but are compatible with their naturally occurring relatives ColK and CloDF13. The incompatibility results in loss, by segregation, of one or the other ColE1 plasmid. In most cases, the smaller derivatives tend to displace the larger ones, and the rate of displacement depends on the difference in size. One mini-plasmid retains only 19% of the sequences of ColE1, yet it exerts strong incompatibility: other ColE1 plasmids are rapidly lost when it is introduced into the host. The region essential for ColE1 incompatibility is deduced to lie within 700 base pairs of the origin of replication. The transforming efficiency of any ColE1 plasmid is markedly lowered when another incompatible replicon is resident in the competent cells, even when the transforming plasmid is much smaller than the resident. A model of incompatibility is proposed to account for these effects.
Colicins↗
Regulation of c-myc and c-fos mRNA levels by polyomavirus: distinct roles for the capsid protein VP1 and the viral early proteins.
The levels of c-myc, c-fos, and JE mRNAs accumulate in a biphasic pattern following infection of quiescent BALB/c 3T3 mouse cells with polyomavirus. Maximal levels of c-myc and c-fos mRNAs were seen within 1 hr and were nearly undetectable at 6 hr after infection. At 12 hr after infection mRNA levels were again maximal and remained elevated thereafter. Empty virions (capsids) and recombinant VP1 protein, purified from Escherichia coli, induced the early but not the late phase of mRNA accumulation. Virions, capsids, and recombinant VP1 protein stimulated [3H]thymidine nuclear labeling and c-myc mRNA accumulation in a dose-responsive manner paralleling their affinity for the cell receptor for polyoma. The second phase of mRNA accumulation is regulated by the viral early gene products, as shown by polyomavirus early gene mutants and by a transfected cell line (336a) expressing middle tumor antigen upon glucocorticoid addition. These results suggest that polyomavirus interacts with the cell membrane at the onset of infection to increase the levels of mRNA for cellular genes associated with cell competence for DNA replication, and subsequently these levels are maintained by the action of the early viral proteins.
Animals↗
Relationship between lysogeny, spontaneous induction, and transformation efficiencies in Bacillus subtilis.
The low transformation efficiency of Bacillus subtilis 168 lysogenic for phages ø105 or SPO2 is shown to result from the induction of lytic phage replication in competent cells. Lysogenic competent cells have a higher rate of spontaneous prophage induction than noncompetent cells. Mutants of ø105 and SPO2 which form lysogens resistant to spontaneous induction were isolated, and these lysogens exhibited higher transformation levels than those formed by wild-type phage. These results suggest that the physiological state of competence in B. subtilis promotes prophage derepression leading to cell death and the loss of potential transformants.
Bacillus subtilis↗
Transformation in Escherichia coli: cryogenic preservation of competent cells.
Escherichia coli cells prepared for transformation by treatment with cold 0.1 M CaCl2 remained viable and competent after storage at -82 degrees C in 15% glycerol; thawed-cell samples yielded up to 10(6) transformants per microgram of plasmid deoxyribonucleic acid.
DNA, Bacterial↗
Macromolecular synthesis in Bacillus subtilis during development of the competent state.
Rates of deoxyribonucleic acid, ribonucleic acid, and protein synthesis were examined in purified competent cells of Bacillus subtilis during the development of the transformable state. To become competent, a cell must depart from the normal course of vegetative growth and pass through a precompetent phase beginning as early as 90 to 180 min before the appearance of transformability. While in the precompetent state, the cell decreases its rate of deoxyribonucleic acid synthesis and lowers its ratio of ribonucleic acid synthesis to protein synthesis. This altered pattern of synthesis eventually leads to a decreased buoyant density of precompetent cells. Once a cell has become both precompetent and low in density, it can be converted to a competent (transformable) cell. The early alterations in macromolecular synthesis were found in two competence regimens, one utilizing a nutritional step-down and one free of such a shift. The data imply that the precompetent state is a generalized characteristic of the B. subtilis transformation system and is not specific to the procedure used to allow competence development. Since precompetence-specific events occur very early in a competence regimen, we conclude that the induction of precompetence is unrelated to sporulation or a nutritional shift.
Bacillus subtilis↗
Gangliosides in DNA virus-transformed and spontaneously transformed tumorigenic mouse cell lines.
In mouse cell lines transformed by SV40 virus, a decrease was observed in the higher ganglioside homologues disialo-ceramidetetrahexoside and monosialo-ceramidetetrahexoside. Such change was observed only in cells which carry the virus genome, and it correlated with increased saturation density in tissue culture and with rejection in immunologically competent syngeneic host. This indicates that the change is one of the virus-regulated functions, and it is postulated that it relates to the rejection of the virus-transformed cells.
Animals↗
Electron microscopy of single-stranded structures in the DNA of competent Haemophilus influenzae cells.
Chromosomal DNAs from exponential-phase and competent cells of Haemophilus influenzae were examined by electron microscopy to determine whether the chromosome undergoes structural changes during competence development. Single-stranded gaps and single-stranded tails formed in chromosomal DNA during competence development. The generation of gaps was dependent on the rec-2 function. Since the rec-2 mutant is defective in the translocation of donor DNA, it was inferred that the gaps were involved in the translocation step of transformation. The generation of single-stranded tails was independent of the rec-1 and rec-2 genes. Therefore, these structures were assumed to play no direct role in the interaction of donor and recipient DNAs during transformation. Gaps were preferentially associated with a readily denaturable, possibly A + T-rich fraction of the genome. This finding raised the possibility that hot spots for transformation might be associated with A + T-rich DNA.
Chromosomes, Bacterial↗
Nonhomologous DNA end joining in Schizosaccharomyces pombe efficiently eliminates DNA double-strand-breaks from haploid sequences.
Cells of higher eucaryotes are known to possess mechanisms of illegitimate recombination which promote the joining between nonhomologous ends of broken DNA and thus may serve as basic tools of double-strand-break (DSB) repair. Here we show that cells of the fission yeast Schizosaccharomyces pombe also contain activities of nonhomologous DNA end joining resembling the ones found in higher eucaryotes. Nonhomologous end joining activities were detected by transformation of linearized self-replicating plasmids in yeast cells employing a selection procedure which only propagates transformants carrying recircularized plasmid molecules. Linear plasmid substrates were generated by duplicate restriction cuts carrying either blunt ends or 3' or 5' protruding single strands (PSS) of 4 nt which were efficiently joined in any tested combination. Sequence analysis of joined products revealed that junctional sequences were shortened by 1 to 14 nt. Two mechanisms may account for junction formation (i) loss of terminal nucleotides from PSS tails to produce blunt ends which can be joined to abutting ends and (ii) interactions of DNA termini at patches of sequence homologies (1-4 bp) by formation of overlap intermediates which are subsequently processed. A general feature of the yeast joining system is that end joining can only be detected in the absence of sequence homology between the linear substrate and host genome. In the presence of homology, nonhomologous DNA end joining is efficiently competed by activities of homologous recombination.
Base Sequence↗
Competence-stimulating activity in sterile filtrates from Hemophilus influenzae.
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DNA, Bacterial↗
Relationship between competence and doubling time in Bacillus subtilis cultures.
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Bacillus subtilis↗
Transforming growth factor type beta can act as a potent competence factor for AKR-2B cells.
Transforming growth factor type beta (TGF beta) is a pleiotropic regulator of cell growth with specific high-affinity cell-surface receptors on a large number of cells; its mechanism of action, however, is poorly defined. In this report, we utilized the mouse fibroblast line AKR-2B to explore the question of the temporal requirements during the cell cycle in regard to both the growth inhibitory and the growth stimulatory action of TGF beta. The results indicate that AKR-2B cells are most sensitive to the inhibitory action of TGF beta during early to mid-G1. In addition, TGF beta need be present only briefly (as little as 1 min) in order to exert its inhibitory effect on EGF-induced DNA synthesis. Likewise, the stimulatory effect of TGF beta in the absence of EGF requires only an equally brief exposure to TGF beta. Use of homogeneous 125I-labeled TGF beta in a cell-binding assay demonstrates that TGF beta bound to cell-surface receptors can readily exchange into the culture medium T1/2 = 120 min), helping to rule out the possibility that persistent receptor-bound TGF beta is the source of a continuous stimulus. The data indicate that TGF beta exposure induces a stable state in the cell (T1/2 = 20 h) similar to but distinct from the state of "competence" induced by platelet-derived growth factor (PDGF).
Animals↗