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PubMed1985

Amplification of a chromosomal region in Bacillus subtilis.

We report on the amplification in Bacillus subtilis of a defined DNA sequence after exposure of the bacteria to increasing levels of antibiotic. The experimental system consisted of transformation of competent cells with a plasmid (pRHA39) unable to replicate in the host and carrying the alpha-amylase gene derived from B. subtilis. Selection of transformants resistant to 5 micrograms of chloramphenicol per ml resulted in the isolation of strains with the plasmid integrated into the chromosome at the site of homology, by a Campbell type mechanism. Starting from such a nontandem duplication, amplification was achieved by growing the bacteria in increasing concentrations of chloramphenicol. By dilution, Southern blotting, and hybridization to a radioactive probe, we estimated a copy number of about 10 for the amplified sequence of samples grown in the presence of 50 micrograms of chloramphenicol per ml. No free plasmid could be detected in the amplified strains. The extent of the amplified region was the same for all transformants, and the endpoints appeared to be the same in all isolates. As a consequence of the amplification, there was a noticeable increase in amylase production, and the amount of enzyme produced correlated with gene dosage. The amplification did not occur in a recE genetic background.

Alleles↗
PubMed1989

Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation.

Two human cell lines (termed rho 0), which had been completely depleted of mitochondrial DNA (mtDNA) by long-term exposure to ethidium bromide, were found to be dependent on uridine and pyruvate for growth because of the absence of a functional respiratory chain. Loss of either of these two metabolic requirements was used as a selectable marker for the repopulation of rho 0 cells with exogenous mitochondria by complementation. Transformants obtained with various mitochondrial donors exhibited a respiratory phenotype that was in most cases distinct from that of the rho 0 parent or the donor, indicating that the genotypes of the mitochondrial and nuclear genomes as well as their specific interactions play a role in the respiratory competence of a cell.

Cell Fusion↗
PubMed1995

Genetic competence in Escherichia coli requires poly-beta-hydroxybutyrate/calcium polyphosphate membrane complexes and certain divalent cations.

In earlier studies of genetic competence in Escherichia coli induced with calcium-containing buffers, a strong correlation was found between transformation efficiency and the formation of poly-beta-hydroxybutyrate/calcium polyphosphate (PHB/Ca2+/PPi) complexes in the plasma membranes. In this study, we replaced Ca2+ with one of a number of other cations--monovalent, divalent, and trivalent--and found significant numbers of transformants (transformation efficiency, > 10(5)/micrograms of pBR322 DNA) only when the cells had high levels of PHB/Ca2+/PPi and the medium contained at least one of the divalent cations Ca2+, Mn2+, Sr2+, or Mg2+. Cells with high levels of the complexes were not competent when the medium did not contain these cations, but the cations were also ineffectual when the cells had few complexes. Surprisingly, Mn, Sr, and Mg were not incorporated into the complexes in place of Ca. These results indicate that PHB/Ca2+/PPi complexes and the above-mentioned divalent cations each have essential but disparate roles in genetic competence. Moreover, the strong selectivity of PHB/PPi for Ca2+ suggests the binding sites in the complexes are ionophoretic.

Calcium↗
PubMed1991

Analysis of p53 mutants for transcriptional activity.

The wild-type p53 protein functions to suppress transformation, but numerous mutant p53 proteins are transformation competent. To examine the role of p53 as a transcription factor, we made fusion proteins containing human or mouse p53 sequences fused to the DNA binding domain of a known transcription factor, GAL4. Human and mouse wild-type p53/GAL4 specifically transactivated expression of a chloramphenicol acetyltransferase reporter in HeLa, CHO, and NIH 3T3 cells. Several mutant p53 proteins, including a mouse p53 mutant which is temperature sensitive for suppression, were also analyzed. A p53/GAL4 fusion protein with this mutation was also transcriptionally active only at the permissive temperature. Another mutant p53/GAL4 fusion protein analyzed mimics the mutation inherited in Li-Fraumeni patients. This fusion protein was as active as wild-type p53/GAL4 in our assay. Two human p53 mutants that arose from alterations of the p53 gene in colorectal carcinomas were 30- to 40-fold less effective at activating transcription than wild-type p53/GAL4 fusion proteins. Thus, functional wild-type p53/GAL4 fusion proteins activate transcription, while several transformation competent mutants do so poorly or not at all. Only one mutant p53/GAL4 fusion protein remained transcriptionally active.

Animals↗
PubMed1993

A novel determinant (comA) essential for natural transformation competence in Neisseria gonorrhoeae and the effect of a comA defect on pilin variation.

A novel genetic determinant (comA) has been identified and found to be required for the transformation of piliated Neisseria gonorrhoeae. Mutants in comA of strain MS11 grow normally and are DNA-uptake proficient but blocked in the translocation of DNA into the cytoplasm. Here we show by site-specific mutagenesis and genetic complementation that only one of two open reading frames identified in comA is essential for competence: it encodes a protein (ComA) with a predicted size of 74 kDa. The comA gene maps upstream of the iga locus and is transcribed in the opposite orientation, probably under the control of a putative sigma 54-type promoter. While DNA probes specific for the N. gonorrhoeae iga locus reveal only a little cross-reactivity with commensal Neisseria species, the neighbouring comA gene appears to be present in most of them. ComA fusion proteins were obtained by in vitro translation. The synthesized gene products migrated atypically in SDS gels indicating its strong hydrophobicity. Several transmembrane alpha-helices were predicted from the amino acid sequence of ComA which, in the context of an observed sequence similarity with other inner membrane proteins, suggests a location for the protein in the inner membrane. Using piliated and non-piliated comA mutants the consequences of transformation deficiency on pilin phase variation were assessed. We show that the comA defect affects some but not all types of DNA rearrangements associated with pilE variation. The results are in agreement with previous observations supporting the notion that multiple recombination pathways contribute to the variability of pilE.

Amino Acid Sequence↗
PubMed2003

Lactic acid production by Rhizopus oryzae transformants with modified lactate dehydrogenase activity.

Rhizopus oryzae is capable of producing high levels of lactic acid by the fermentation of glucose. Yields typically vary over 60-80%, with the remaining glucose diverted primarily into ethanol fermentation. The goal of this work was to increase lactate dehydrogenase (LDH) activity, so lactic acid fermentation could more effectively compete for available pyruvate. Three different constructs, pLdhA71X, pLdhA48XI, and pLdhA89VII, containing various lengths of the ldhA gene fragment, were transformed into R. oryzae. This fungus rarely integrates DNA used for transformation, but instead relies on extra-chromosomal replication in a high-copy number. Plasmid pLdhA48XI was linearized prior to transformation in order to facilitate integration into the pyrG gene used for selection. Isolates transformed with ldhA containing plasmid were compared with both the wild-type parent strain and the auxotrophic recipient strain containing vector only. All isolates transformed with pLdhA71X or pLdhA48XI had multiple copies of the ldhA gene that resulted in ldhA transcript accumulation, LDH specific activity, and lactic acid production higher than the controls. Integration of plasmid pLdhA48XI increased the stability of the strain, but did not seem to offer any benefit for increasing lactic acid production. Since lactic acid fermentation competes with ethanol and fumaric acid production, it was not unexpected that increased lactic acid production was always concomitant with decreased ethanol and fumaric acid. Plasmid pLdhA71X, containing a large ldhA fragment (6.1 kb), routinely yielded higher levels of lactic acid than the smaller region (3.3 kb) used to construct plasmid pLdhA48XI. The greatest levels of ldhA transcript and enzyme production occurred with isolates transformed with plasmid pLdhA89VII. However, these transformants always produced less lactic acid and higher amounts of ethanol, fumaric, and glycerol compared with the control.

Cloning, Molecular↗
PubMed1976

Effect of pH on competence development and deoxyribonucleic acid uptake in Streptococcus sanguis (Wicky).

Streptococcus sanguis (Wicky) cells, strain WE4, developed little or no competence and failed to autolyze in permissive conditions when treated with competence factor (CF) below PH 7.0. This lack of activity was directly correlated with the inability of the cells to bind or take up CF at pH values of 5.5, 6.0, and 6.5. On the other hand, competent cells bound deoxyribonucleic acid molecules maximally below pH 7.0 and transformed maximally at pH 6.5. Deoxyribonucleic acid was optimally bound to cells in a deoxyribonuclease-resistant form at pH values between 7.0 and 8.5. Concomitant with this binding, undefined acid-soluble DNA fragments appeared in the culture menstrua. CF binding and uptake by cells was not only influenced by low pH but also by low temperature. At 0 C, WE4 cells bound only 4% of the input CF and took up less than 1% into a trypsin-insensitive state compared to cells treated at 37 C. Cells treated with CF at 0 C did not autolyze when transferred to permissive conditions. The results presented in this report extend earlier findings that showed that competence development and autolysis are related to the uptake of CF.

Bacterial Proteins↗
PubMed2005

[Construction of a virulence-related gene library of Streptococcus mutans by suppression subtractive hybridization].

OBJECTIVE: To construct a suppression subtractive library of virulence-related genes from c serotype Streptococcus mutans (S. mutans), and lay foundations for screening the virulent genes. METHODS: After being isolated from virulent and avirulent strain of S. mutans respectively, the intact and high-pure genomic DNA was digested with three appropriate four-base-cutting restriction endonueleases to produce fragments of optimal length. The digested DNA of the virulent strain ligated with adaptor was used as tester DNA, and that of the avirulent strain as driver DNA. Then the suppression subtractive hybridization was carried out, and the efficiency of ligation and subtraction detected respectively. The subtracted fragments were inserted into vector pCR2. 1 using T/A cloning kit, and transformed into E. coli TOP10F' competent cells. Those white colonies were selected to construct the suppression subtractive library. RESULTS: Alu I chosen from three restriction endonucleases was verified to be suitable for preparing restriction fragments from S. mutans genomic DNA. Through electrophoresis of Alu I -digested DNA, a smear ranged from 0.1 to 2.0 kb was observed. The ligation efficiency of tester DNA with adaptor was at least higher than 25 percent. The subtraction efficiency of suppression subtractive hybridization confirmed the success in enrichment of differential genes between virulent and avirulent strain of S. mutans. In the subtracted group, the appearance time of the 23S rRNA gene both in tester and driver DNA was later than that in the unsubtracted group by six cycles. It suggested that suppression subtractive hybridization happened indeed. After the subtracted fragments were cloned, 96 colonies were picked up for constructing the suppression subtractive library of virulence-related genes of S. mutans. CONCLUSION: Suppression subtractive hybridization allows rapid and easy construction of virulence-related gene library of S. mutans.

Escherichia coli↗
PubMed1963

ALTERATION IN TRANSFORMABILITY OF DIPLOCOCCUS PNEUMONIAE AFTER THE ACQUISITION OF GENETIC DETERMINANTS INDUCING RESISTANCE TO ERYTHROMYCIN.

Sirotnak, Francis M. (Sloan-Kettering Institute for Cancer Research, New York, N.Y.), Ramona B. Lunt, and Dorris J. Hutchison. Alteration in transformability of Diplococcus pneumoniae after the acquisition of genetic determinants inducing resistance to erythromycin. J. Bacteriol. 86:735-739. 1963.-The genetic alteration of a highly transformable (competent) recipient strain of Diplococcus pneumoniae by the transformation of at least two of three identified erythromycin-resistance determinants (Ery(a), Ery(b), Ery(c)) results in a marked decrease (Ery(a-b) recombinant) or complete loss (Ery(a-c) recombinant) in the ability to be transformed. The occurrence of transformable cells in cultures of the R6 Ery(a-b) recombinant strains, although greatly diminished, still varies during growth in a manner characteristic of the fully competent parent strain. Both Ery(b) and Ery(c) determinants appear to be linked to Ery(a). In experiments using P(32)-labeled deoxyribonucleic acid (DNA), data correlating DNA uptake with transformation show a decrease or loss in uptake capacity of the erythromycin-resistant strains.

DNA↗
PubMed1995

The rec locus, a competence-induced operon in Streptococcus pneumoniae.

To study competence and the process of transformation (TFN) in pneumococci, we developed a method for isolating TFN- mutants using insertional inactivation coupled with fusions to the gene for alkaline phosphatase (phoA). One TFN- mutant transformed 2 log units less efficiently than the parent strain. Reconstitution of the mutated region revealed a locus, rec, that contains two polycistronic genes, exp10 and the previously identified recA (B. Martin, J. M. Ruellan, J. F. Angulo, R. Devoret, and J. P. Claverys, Nucleic Acids Res. 20:6412, 1992). Exp10 is likely to be a membrane-associated protein, as it has a prokaryotic signal sequence and an Exp10-PhoA fusion localized with cell membranes. On the basis of sequence similarity, pneumococcal RecA is a member of bacterial RecA proteins responsible for homologous recombination of DNA. DNA-RNA hybridization analysis showed that this locus is transcribed as a polycistronic message, with increased transcription occurring during competence. With an Exp10-PhoA chimera used as a reporter, there was a 10-fold increase in the expression of the rec locus during competence while there was only minimal expression under growth conditions that repressed competence. The TFN- mutant containing the exp10-phoA fusion produced activator, a small extracellular polypeptide that induces competence, and the expression of rec was induced in response to activator. Therefore, the rec locus is directly required for genetic transformation and is regulated by the cell signaling mechanism that induces competence.

Alkaline Phosphatase↗
PubMed1992

DH11S: an Escherichia coli strain for preparation of single-stranded DNA from phagemid vectors.

A new E. coli strain DH11S [mcrA delta(mrr-hsdRMS-mcrBC) delta(lac-proAB) delta(rec1398) deoR rpsL srl- thi-/F'proAB+ lacIqZ delta M 15] has been constructed. Transformation of DH11S competent cells with any of several different phagemid vectors [pSPORT1, pBluescript II SK(+), pGEM11Zf(+)] results in the production of highly purified single-stranded DNAs upon the addition of M13KO7 helper phage. Contamination by double-stranded DNAs was observed with all the other studied strains (XL1-Blue, JM109, DH5 alpha F'IQ). The optimal yield of single-stranded DNA production was obtained when glycerol stocks made from stationary phase cells or single colonies from overnight ampicillin plates of DH11S containing the phagemid vector were infected with M13KO7 helper phage using a wide range (1 to 100) of multiplicities of infection. Five different pSPORT1 clones containing cDNA inserts of various lengths (0.3 kb to 2.0 kb) were compared using these four different bacterial strains. The use of strain DH11S results in the best yields and quality of single-stranded DNA. Therefore, DH11S appears to be the best all-around host for various applications that require single-stranded DNA such as DNA sequencing, in vitro mutagenesis and construction of subtractive cDNA libraries.

Coliphages↗
PubMed1995

Competence for genetic transformation in Streptococcus pneumoniae: organization of a regulatory locus with homology to two lactococcin A secretion genes.

Regulation of competence for genetic transformation in Streptococcus pneumoniae involves the comAB locus and a small extracellular protein, the competence factor (CF). The comA or comB mutations block both spontaneous competence induction and elaboration of CF, yet permit induction of competence by added CF and subsequent transformation at normal levels. Sequence and genetic studies showed that the com locus comprised the comA and comB genes, encoding 77- and 50-kDa proteins, respectively, and demonstrated that they were closely flanked by genes and DNA not required for competence regulation. In-frame deletion of comA demonstrated that the translation product of this gene is required for normal competence regulation; deletion-replacement mutations showed that no com gene lay in the 0.2-kb gap between comB and purC or within 2.5 kb upstream from comA. Strong sequence similarities (51-59% identities) showed that ComA and the proteins, PdcD and LcnC, which act in the secretion of pediocin A-1 and lactococcin A, respectively, form a subfamily within the large ABC-transporter protein family. ComB was found to be homologous to a single known protein, LcnD, required for secretion of the peptide antibiotic lactococcin A. Thus, the comAB locus displays homology to two lactococcin A secretion genes, but is devoid of additional linked com genes. The results suggest that the mechanism for CF production is similar to that for the small peptide bacteriocins, lactococcin A and pediocin A-1, but that its genetic organization is unusual in being split into at least two separate operons.

ATP-Binding Cassette Transporters↗
PubMed1985

The in vivo formation of nonbilayer lipid phase in E. coli membranes during the development of Ca2-dependent competence.

31P-NMR studies on E. coli cells reveal the in vivo formation of nonbilayer lipid structures, presumably of H11 phase, during Ca2-dependent competence induction. The data suggest the involvement of these structures in the exogenous DNA transfer into the cells during genetic transformation and transfection. The suggestion is supported by in vitro experiments in which the liposomes composed of different phospholipid species bind 14C-DNA in DNase and wash resistant form in conditions promoting the hexagonal phase formation.

Calcium↗
PubMed2000

[Production of phage-displayed single chain variable fragments of monoclonal antibody MGb1].

OBJECTIVE: To lay a foundation for obtaining a tumor-targeting vehicle for in vivo study on diagnosis and treatment of gastric carcinoma by generating single chain variable fragments (ScFv) of monoclonal antibody MGb1 directed against the cancer. METHODS: mRNA was isolated from MGb1-producing mouse hybridoma cell line, and the variable regions of heavy and light chain cDNAs were amplified separately and assembled into ScFv DNAs with a specially constructed linker DNA by PCR. The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage, which display ScFv fragments as a fusion with gene 3 protein on the tips of the phage M13. After two rounds of panning with gastric carcinoma cell line KATO III highly expressing MGb1-binding antigen, the phage clones displayed ScFv fragments of the antibody were selected by enzyme-linked immunosorbent assay (ELISA) from the enriched phages. The affinity of the positive phage clones was detected by competition ELISA. RESULTS: The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. 17 phage clones displayed ScFv of MGb1 were selected from 40 enriched phage clones. 4 out of the 17 phage clones could strongly compete with the original hybridoma antibody MGb1 for binding to the antigen expressed on KATOIII cells. CONCLUSION: The phage-displayed ScFv fragments of monoclonal antibody MGb1 are successfully produced by phage antibody technology, which may be useful to widen the range of application of the antibody.

Animals↗
PubMed2003

[Soluble expression of peptide containing MUC1/Y-specific epitope in Escherichia coli and preparation of the antibody].

MUC1 mucin is a high molecular weight, type I transmembrane glycoprotein. High and aberrant expression of MUC1 is observed in various types of tumors, which make it an ideal target for tumor biotherapy as well as a biomarker for tumor diagnosis and prognosis. MUC1/Y is an isoform of MUC1 generated by alternative splicing. Specific expression of MUC1/Y in breast cancer as well as its involvement in tumor cell signal transduction have been reported. In order to purify peptides containing MUC1/Y-specific epitope in E. coli and prepare MUC1/Y-specific antibody, DNA fragment encoding the MUC1/Y-specific peptide was amplified by PCR using MUC1/Y full length cDNA as the template and cloned into fusion expression vector pGEX-2T, resulting pGEX-Y30. DNA sequencing was performed to confirm the correct amplification and orientation of the target sequence. Competent E. coli DH5alpha was transformed with pGEX-Y30 and the expression was induced for 4-5 hours in 0.2 mmol/L IPTG at 30 degrees C and 37 degrees C. Expressed proteins were released from the cells by ultrasonication or B-PER II reagent treatments. The fusion protein GST-Y30 were purified by affinity and anion exchange columns and identified by SDS-PAGE and Western-blotting. Polyclonal antibody was prepared by immunizing rabbits with the GST-Y30 protein for 4 times with intervals of 3 weeks and purified by GST column. Western blotting, ELISA and immunohistochemistry analysis were carried out using the purified antibody to confirm its MUC1/Y-binding capacity and specificity. The expressed fusion protein GST-Y30 is about 31 kD in size and represented about 20% of total cellular proteins. The majority of the GST-Y30 protein existed as soluble form when the induction was carried out at both 30 degrees C and 37 degrees C. After the two-step purification, the purity of GST-Y30 was about 94%. The titer of polyserum generated by GST-Y30 immunization was 1:320,000 by ELISA. The antiserum showed MUC1/Y specificity and can recognize MUC1/Y on MCF7 cell. The MUC1/Y-specific polyclonal antibody can be used for studying the role of MUC1/Y in carcinogenesis.

Animals↗
PubMed1981

[Comparative study of chromosome and plasmid transformation in Bacillus subtilis: the effect of lysozyme and polyethylene glycol].

The influence of lysozyme and polyethyleneglycol (PEG) on chromosomal and plasmid transformation was comparatively investigated. Treatment of competent cell culture with low concentrations of lysozyme which do not affect cell viability stimulates both types of transformation. When transforming DNA was treated with definite concentrations of PEG, inhibition of transformation using chromosomal DNA occurred. The similar treatment, however, stimulated transformation by oligomeric forms of plasmid DNA. Possibly, PEG alters the DNA conformation rendering it less sensitive to nuclease degradation. This promotes plasmid transformation but reduces the frequency of transformation by chromosomal DNA, because the changes resulting from nuclease action further DNA integration into the genome of a recipient.

Bacillus subtilis↗