[Competence in Escherichia coli cells. I. Recovery of transfectivity in incompetent Escherichia coli cells following treatment with the cultural fluid from competent cells].
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Coliphages↗
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Inhibition of transformation by sodium metaperiodate in Streptococcus sanguis (Wicky): demonstration that deoxyribonucleic acid binding sites contain amino acids.
Addition of sodium metaperiodate to competent Streptococcus sanguis (Wicky) cells at 0 or 37 C revealed that the deoxyribonucleic acid binding sites consist of amino acids.
Amino Acids↗
Characterization of genetic transformation in Streptococcus oralis NCTC 11427: expression of the pneumococcal amidase in S. oralis using a new shuttle vector.
We have worked out conditions for the study of competence development and genetic transformation in Streptococcus oralis NCTC 11427 (type strain), a species that contains choline in the cell wall. The peak of competence was found at the early exponential phase of growth and the optimal conditions for transformation were achieved with shuttle plasmids prepared from S. pneumoniae or from Escherichia coli serving as donor DNA. Transformation with dye-buoyant density gradient purified plasmid preparations followed first-order kinetics. The pneumococcal amidase can be expressed in S. oralis harbouring a plasmid carrying the lytA gene. This enzyme lysed the cell wall of the transformed cell in the presence of detergents.
Amidohydrolases↗
Construction of BAC and BIBAC libraries and their applications for generation of SSR markers for genome analysis of chickpea, Cicer arietinum L.
Large-insert bacterial artificial chromosome (BAC) libraries, plant-transformation-competent binary BAC (BIBAC) libraries, and simple sequence repeat (SSR) markers are essential for many aspects of genomics research. We constructed a BAC library and a BIBAC library from the nuclear DNA of chickpea, Cicer arietinum L., cv. Hadas, partially digested with HindIII and BamHI, respectively. The BAC library has 14,976 clones, with an average insert size of 121 kb, and the BIBAC library consists of 23,040 clones, with an average insert size of 145 kb. The combined libraries collectively cover ca. 7.0 x genomes of chickpea. We screened the BAC library with eight synthetic SSR oligos, (GA)10, (GAA)7, (AT)10, (TAA)7, (TGA)7, (CA)10, (CAA)7, and (CCA)7. Positive BACs were selected, subcloned, and sequenced for SSR marker development. Two hundred and thirty-three new chickpea SSR markers were developed and characterized by PCR, using chickpea DNA as template. These results have demonstrated that BACs are an excellent source for SSR marker development in chickpea. We also estimated the distribution of the SSR loci in the chickpea genome. The SSR motifs (TAA)n and (GA)n were much more abundant than the others, and the distribution of the SSR loci appeared non-random. The BAC and BIBAC libraries and new SSR markers will provide valuable resources for chickpea genomics research and breeding (the libraries and their filters are available to the public at http://hbz.tamu.edu).
Base Sequence↗
Effect of intrastrain variation in the amount of capsular polysaccharide on genetic transformation of Streptococcus pneumoniae: implications for virulence studies of encapsulated strains.
The degrees of competence of opaque and transparent colony variants of Streptococcus pneumoniae were compared. The transparent variants were transformed at 9- to 670-fold-higher rates than the opaque variants, independent of the DNA incorporated, due to decreased expression of capsular polysaccharide. Genetic transformation, therefore, tends to select for a less-encapsulated subpopulation.
Bacterial Capsules↗
Isolation and characterization of Tn917lac-generated competence mutants of Bacillus subtilis.
We isolated 28 mutants of Bacillus subtilis deficient in the development of competence by using the transposon Tn917lacZ as a mutagen. The mutant strains were poorly transformable with plasmid and chromosomal DNAs but were normally transducible and exhibited wild-type resistance to DNA-damaging agents. The mutations were genetically mapped, and the mutants were characterized with respect to their abilities to bind and take up radiolabeled DNA. All were defective in uptake, and some failed to bind significant amounts of DNA. The abilities of the mutant strains to resolve into two buoyant density classes on Renografin gradients were studied. Most resolved normally, but several banded in Renografin only at the buoyant density of noncompetent cells. The genetic mapping studies and the other analyses suggested that the mutations define a minimum of seven distinct com genes.
Bacillus subtilis↗
Neoplastic transformation of human lung fibroblast MRC-5 SV2 cells induced by benzo[a]pyrene and confluence culture.
Benzo[a]pyrene (BaP) is potent rodent carcinogen and a reputed human carcinogen. Although much is known about its metabolic activation leading to DNA damage, the mechanisms of its actions are not as well understood at a cellular level. In addressing this, we have established an in vitro model that follows the progression toward neoplastic transformation induced by BaP. The model uses immortal nontumorigenic human lung fibroblast MRC-5 SV2 cells as effectors, cocultured with a metabolically competent human lymphoblastoid line h1A1v2 (activator cells). Treatment of the coculture with BaP for 48 h induced a dose-dependent decrease in cloning efficiency of the MRC-5 SV2 cells; nevertheless, cultures continued to progress to confluence. At prolonged confluence culture (day 11), an elevation in the proportion of G2-M phase cells was detected by flow cytometry. By day 15, the G2-M phase peak disappeared, accordant with the appearance of a population with DNA content greater than the cells in G2-M phase. These changes in DNA ploidy were coincident with changes in morphology, specifically the appearance of enlarged and irregular-shaped nuclei. Confluence culture of BaP-treated MRC-5 SV2 cells for more than 2 weeks resulted in cell death; however, a few colonies survived the crisis to reach confluence again after an additional 10-14 days. The number of death-resistant colonies was proportional to the dose of BaP, with the majority of the cells exhibiting abnormal morphology. The degree of morphological change progressively increased with successive rounds of confluence. Cells that survived three rounds of confluence adopted a vastly different morphology, becoming polygonal, spindle, or other irregular-shaped, and acquired the ability to form large dense clumps that grew in an anchorage-independent manner. In parallel experiments, treatment with the vehicle alone (DMSO) resulted in substantially less death resistance and lower numbers of high-density clumps. Our studies demonstrate that a single pulse treatment of human MRC-5 SV2 cells with metabolically activated BaP increased DNA ploidy, induced resistance to confluence-initiated cell death and morphological change, and was accompanied by substantial changes in growth pattern under prolonged confluence culture. The multiple-step nature of this process is characteristic of the development of neoplastic disease, and prolonged confluence seemed to play a pivotal role in selecting for carcinogen-induced transformants.
Benzo(a)pyrene↗
Transformation in Bacillus subtilis: properties of DNA-binding-deficient mutants.
Transformation-deficient mutants of Bacillus subtilis were selected after replica plating on agar plates containing transforming DNA. Out of 24 mutants tested, 3 showed highly reduced abilities to bind donor DNA; the residual levels of binding were similar to those of noncompetent cells. Transformation and transfection were reduced to nondetectable levels in the mutants. However, transduction with phage SPP1 occurred at normal frequencies. The nuclease activities involved in entry of donor DNA were present in the mutants. Comparison of protein patterns by two-dimensional gel electrophoresis revealed the absence of one major protein in the mutants as compared with the wild-type strain. This protein (molecular weight, approximately 18,000; isoelectric point, 5.0) appeared to be membrane associated. The protein was specific for competent cells, suggesting that it is involved in the binding of donor DNA.
Bacillus subtilis↗
NCA3, a nuclear gene involved in the mitochondrial expression of subunits 6 and 8 of the Fo-F1 ATP synthase of S. cerevisiae.
Respiratory-competent nuclear mutants have been isolated which presented a cryosensitive phenotype on a non-fermentative carbon source, due to a dysfunctioning of the mitochondrial F1-Fo ATP synthase which results from a relative defect in subunits 6 and 8 of the Fo sector. Both proteins are mtDNA-encoded, but the defect is due to the simultaneous presence of a mutation in two unlinked nuclear genes (NCA2 and NCA3, for Nuclear Control of ATPase) promoting a modification of the expression of the ATP8-ATP6 co-transcript (formerly denoted AAP1-OLI2). This co-transcript matures at a unique site to give two cotranscripts of 5.2 and 4.6 kb in length: in the mutant, the 5.2-kb co-transcript was greatly lowered. NCA3 was isolated from a wild-type yeast genomic library by genetic complementation. The level of the 5.2-kb transcript, like the synthesis of subunits 6 and 8, was partly restored in the transformed strain. A 1011-nucleotide ORF was identified that encodes an hydrophilic protein of 35417 Da. Disruption of chromosomal DNA within the reading frame promoted a dramatic decrease of the 5.2-kb mRNA but did not abolish the respiratory competence of a wild-type strain. NCA3 is located on chromosome IV and produces a single 1780-b transcript.
Amino Acid Sequence↗
CROC-1 encodes a protein which mediates transcriptional activation of the human FOS promoter.
The cloning of signal transducing molecules capable of activating the human FOS proto-oncogene promoter was achieved by co-transfecting a modified human FOS promoter-driven polyomavirus large T antigen gene (P(f)LAG-8) with a human brain cDNA library, incorporated into a replication-competent mammalian retroviral expression vector whose replication occurs in the presence of T antigen. In murine cells, transcriptional activation of the P(f)LAG-8 promoter by a biologically active, cDNA-encoded signalling molecule resulted in plasmid replication. Replicated plasmids, following selective cleavage of unreplicated plasmids by Dpn1, were recovered by transformation into competent bacteria. Successive P(f)LAG-8/cDNA library co-transfections, using library plasmids resulting from prior transfections, ultimately resulted in the identification of individual plasmids capable of transcriptionally activating the FOS promoter. DNA sequencing revealed the first plasmid, denoted CROC-1, to contain a 1.8-kb cDNA encoding a 16.5-kDa nuclear protein possessing a bipartite structure comprised an amino-terminal acidic domain and a carboxy-terminal basic domain, and displaying partial homology to the HMG domain of the TAF(II)250 transcription cofactor. Co-transfection of CROC-1 with various FOS/CAT reporter genes revealed that the human FOS promoter region spanning -56 to - 105, encompassing two identical 8-bp DR enhancer sequences, was necessary for CROC-1-mediated transcriptional activation. Results suggest that CROC-1 participates in intracellular signalling pathways involved in induction of the human FOS promoter.
3T3 Cells↗
Competence pheromone, oligopeptide permease, and induction of competence in Streptococcus pneumoniae.
An unmodified heptadecapeptide pheromone capable of eliciting competence for genetic transformation in Streptococcus pneumoniae has recently been identified and characterized. In considering possible signal-transduction mechanisms for the peptide, the previously characterized Ami oligopeptide permease and the three highly homologous oligopeptide-binding lipo-proteins. AmiA, AliA, and AliB, appeared to be good candidates for receptors. We therefore compared the spontaneous transformability of Ami, AliA and AliB mutants to that of an isogenic wild-type strain and we investigated the response of the various mutants to treatment with synthetic competence-stimulating peptide (CSP). Our results clearly demonstrate that neither Ami nor any of the three highly homologous oligopeptide-binding lipoproteins identified so far in S. pneumoniae are required for competence induction following treatment with synthetic CSP. Although the existence of a fourth unidentified oligopeptide-binding lipoprotein and/or a second oligopeptide permease operon could not be completely ruled out, we favour the hypothesis that CSP signal transmission rather involves a two-component regulatory system. Although none of the single or double Ami and All mutants tested appeared severely affected for competence, an exceptional aliB plasmid-insertion mutation abolished competence completely. In addition, the triple AmiA-AliA-AliB mutant differed from wild type in showing no sharp peak of competence but exhibiting transformability throughout the exponential phase of growth. These and previous observations are discussed and a general hypothesis is proposed to account for the modulation of competence by peptide permease mutants in S. pneumoniae.
Bacterial Proteins↗
Identification and characterization of genes required for competence in Neisseria meningitidis.
We have identified genes required for competence of Neisseria meningitidis, a naturally transformable human pathogen. Although not comprehensive, our analysis identified competence-defective mutants with transposon insertions in genes not previously implicated in this process in Neisseria.
DNA Helicases↗
Differential effects by Mad and Max on transformation by cellular and viral oncoproteins.
c-Myc is an essential component of the regulatory mechanisms controlling cell growth. Max is the obligatory partner of c-Myc for all its biological functions analysed to date. Recently two Max interacting proteins, Mad and Mxi1, have been identified. It has been suggested that these two proteins modulate c-Myc function, in the simplest model by competing with c-Myc for the interaction with Max. We have analysed different aspects of Mad function in comparison to Max. Native Mad/Max heterodimers bound specifically to a c-Myc/Max consensus DNA binding site. Furthermore Mad inhibited efficiently c-Myc, mutant p53, adenovirus E1a, or human papilloma virus type 16 transformation of rat embryo cells in cooperation with activated Ha-Ras. Myc transformed clones showed an increased cell cycle time and a reduced immortalization frequency after cotransfection with either mad or max. In contrast to Mad, Max did not inhibit E1a/Ha-Ras cotransformation but repressed c-Myc/Ha-Ras transformation efficiently. Mad delta N, an N-terminal deletion mutant of Mad, was as efficient in repressing c-Myc/Ha-Ras cotransformation as full length Mad but showed little inhibitory activity when assayed on E1a/Ha-Ras. Unlike wt Mad, Mad delta N had little effect on cell growth. Our data suggest that Mad affects cell growth at least in part by a c-Myc independent mechanism.
Animals↗
Cloned ural locus of Schizosaccharomyces pombe propagates autonomously in this yeast assuming a polymeric form.
DNA segments cloned from Schizosaccharomyces pombe by the ability to complement Escherichia coli pyrB mutations are shown to complement a ural mutation in S. pombe, thereby demonstrating that ural is the structural gene for aspartate transcarbamylase of S. pombe. Further, such segments combined with parts or all of pBR322 are shown to be capable of autonomous propagation in S. pombe. This suggests the existence of an autonomously replicating sequence (ars) in the vicinity of ural. Unlike the TRP1 segment cloned from Saccharomyces cerevisiae [Struhl, K., Stinchcomb, D. T., Scherer, S. & Davis, R. W. (1979) Proc. Natl. Acad. Sci. USA 76, 1035-1039], plasmids carrying the ural locus do not multiply as monomers but assume a polymeric form as large as a decamer to an icosamer in the yeast. Monomers are tandemly arranged in the polymer. Inversion of an inserted fragment or insertion of another segment into a competent plasmid greatly decreases the efficiency of such transformation, implying a role of the tertiary structure of the plasmids in the establishment of transformation of this kind.
Ascomycota↗
Plant genome complexity may be a factor limiting in situ the transfer of transgenic plant genes to the phytopathogen Ralstonia solanacearum.
The development of natural competence by bacteria in situ is considered one of the main factors limiting transformation-mediated gene exchanges in the environment. Ralstonia solanacearum is a plant pathogen that is also a naturally transformable bacterium that can develop the competence state during infection of its host. We have attempted to determine whether this bacterium could become the recipient of plant genes. We initially demonstrated that plant DNA was released close to the infecting bacteria. We constructed and tested various combinations of transgenic plants and recipient bacteria to show that the effectiveness of such transfers was directly related to the ratio of the complexity of the plant genome to the number of copies of the transgene.
Betaproteobacteria↗
Relationship of macromolecular synthesis to competence induction in a group H streptococcus.
Group H streptococcus strain Wicky, which was induced to competence for genetic transformation with competence factor (CF) derived from a related strain, displayed reduced rates of ribonucleic acid (RNA) and peptidoglycan synthesis. Pulse-labeling studies revealed that the inhibition of both RNA and peptidoglycan synthesis was maximal at the peak of competence and decreased as competence declined. These studies indicated that competence induction had only a slight effect on the rate of protein synthesis. Trypsin inactivation of CF prevented the reductions in synthesis normally elicited by CF preparations. If the addition of trypsin was delayed until 5 min after the addition of CF, competence induction and decreased synthesis of RNA and peptidoglycan were again apparent. Thus, the alterations in the synthesis of these macromolecules appeared to be related to the induction of competence. Further studies indicated that the apparent reductions in biosynthesis were not caused by decreased uptake of the labeled precursors by intact Wicky cells. In addition, these effects were probably not the result of turnover of macromolecules induced by CF. The lack of turnover of labeled peptidoglycan suggested that competence induction may not involve an autolysin.
Amino Acids↗
Agrobacterium-mediated root transformation is inhibited by mutation of an Arabidopsis cellulose synthase-like gene.
Agrobacterium-mediated plant genetic transformation involves a complex interaction between the bacterium and the host plant. Relatively little is known about the role plant genes and proteins play in this process. We previously identified an Arabidopsis mutant, rat4, that is resistant to Agrobacterium transformation. We show here that rat4 contains a T-DNA insertion into the 3'-untranslated region of the cellulose synthase-like gene CSLA9. CSLA9 transcripts are greatly reduced in the rat4 mutant. Genetic complementation of rat4 with wild-type genomic copies of the CSLA9 gene restores both transformation competence and the wild-type level of CSLA9 transcripts. The CSLA9 promoter shows a distinct pattern of expression in Arabidopsis plants. In particular, the promoter is active in the elongation zone of roots, the root tissue that we previously showed is most susceptible to Agrobacterium-mediated transformation. Disruption of the CSLA9 gene in the rat4 mutant results in reduced numbers and rate of growth of lateral roots and reduced ability of the roots to bind A. tumefaciens cells under certain conditions. No major differences in the linkage structure of the non-cellulosic polysaccharides could be traced to the defective CSLA9 gene.
Agrobacterium tumefaciens↗
De novo protein synthesis during the development of competence in Bacillus subtilis.
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Bacillus subtilis↗