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At least 127 records · Page 7Linked to original sources
PubMed1981

Isolation of Bacillus subtilis genes from a charon 4A library.

A library of Bacillus subtilis chromosomal deoxyribonucleic acid (DNA) was constructed, using lambda charon 4A as a cloning vector. Partially cleaved Bacillus subtilis DNA was prepared by partial methylation with EcoRI methylase, followed by complete EcoRI endonuclease digestion. More than 95% of the phage particles carried B. subtilis DNA inserts. When this library was screened for transforming activity, using competent cells, 70% of the genetic markers tested were found in a sample of 1,710 plaques. Cloned genetic loci were found to be about 100-fold more efficient in transforming activity than chromosomal DNA. Intact phage particles containing the pheA locus were found to be able to transform competent recipients with approximately the same efficiency as phage DNA. Transformation by intact particles was insensitive to deoxyribonuclease.

Bacillus subtilis↗
PubMed1990

Integrated defective replication units of hepatitis B virus.

Stable transformants of the human hepatoma cell line HepG2 were established that constitutively transcribe a DNA unit consisting of a stretch of hepatitis B virus DNA and of nonviral DNA conferring resistance to neomycin. Previously it had been shown that upon cotransfection of such transformants with replication-competent HBV DNA, transcripts of such units become reverse transcribed, demonstrating that DNA constructs can function as defective replication units. Transformed cell lines stably transcribing the defective replication units could be shown to use the transcriptional starts for the viral pregenome and for the large core antigen at a ratio of 9:1. Upon the induction of replicative processes in the transformed cells by transfection with replication-competent wild type (wt) DNA, defective pregenomes transcribed from the integrated state became included in the pool of replicating nucleic acids.

Blotting, Southern↗
PubMed1982

DNA-mediated gene transfer in Chinese hamster ovary cells: clonal variation in transfer efficiency.

Thymidine kinase-deficient Chinese hamster ovary (CHO) cells were genetically transformed with the BamHI restriction fragment encoding the thymidine kinase gene of herpes simplex virus (HSV-tk). We have observed considerable clonal variation among independent CHO sublines with respect to transformation competence for the DNA-mediated gene transfer of HSV-tk. Transformation frequencies greater than or equal to 3 X 10(-4) were observed consistently in one subline, with a transformation efficiency of approximately 1 transformant per ng viral gene. The frequency and efficiency of transformation we observed in this system are at least 10-fold greater than those previously reported for DNA-mediated transformation of CHO cells by HSV-tk. All of the CHO HSV-tk+ transformants examined were stable for the transferred genotype in the absence of selection, and all showed evidence of co-transformation by unselected plasmid pBR322 sequences.

Animals↗
PubMed1989

[Molecular cloning of butirosin biosynthesis genes. I. Construction and characterization of recombinant plasmid DNA No. 2 and No. 11].

B. circulans NRRL-B 3312 is a butirosin-producing strain, butirosin being one of aminoglycoside antibiotics. We used shotgun strategy for molecular cloning of butirosin biosynthesis genes. EcoRI digests of B. circulans NRRL-B 3312 chromosome DNA were ligated to EcoRI digests of plasmid pUB110 with T4-DNA ligase. Recombinant DNA was transformed into competent cells of B. subtilis 168. Kanr transformants were selected on medium containing kanamycin (5 micrograms/ml). Results showed that kanamycin and neomycin resistance levels were higher than that of pUB110 for both No. 2 and No. 11 recombinant plasmid DNA. Restriction enzyme maps were constructed for two recombinant plasmid DNA No. 2 and No. 11 respectively. Southern hybridization showed that there were some homogenous DNA sequences between No. 2 and No. 11 recombinant plasmid DNA and B. circulans NRRL-B 3312 DNA.

Anti-Bacterial Agents↗
PubMed1985

Processing of donor DNA during Haemophilus influenzae transformation: analysis using a model plasmid system.

A plasmid system was used to investigate the processing of donor DNA during transformation of competent Haemophilus influenzae. Using biochemical and genetic methods, we have determined that portion of a donor plasmid molecule that, on average, can become integrated into a homologous recipient plasmid during transformation. Our results show that (i) transformation efficiency decreases linearly with donor DNA length over the range of 11 to 3.5 kilobase pairs, (ii) transformation efficiency decreases exponentially with size for donor molecules less than 3.5 kilobase pairs in length, and (iii) 5'-end label, but not 3'-end label, can be specifically incorporated into the resident homologous region. We present a model for donor processing during entry that encompasses and explains these observations.

DNA Restriction Enzymes↗
PubMed2001

[Mechanism of transformation in Pichia methanolica yeast: transforming and nontransforming genes].

Two types of genes were found in the study of transformation in yeast Pichia methanolica: transforming (Trg) and nontransforming (Ntg) genes. Transforming genes (P-ADE7,4 and S-LEU2), as linear DNA molecules, can transform competent cells with high efficiency inversely proportional to the molecule size. Nontransforming genes (P-ADE5 and H-LEU2) transform P. methanolica cells at an extremely low rate even when they are combined with transforming genes. The analysis showed that linear DNA molecules with Trg and Ntg can be either rearranged and integrated in random sites of the recipient genome or form circular plasmids, which are capable of autonomous replication irrespective of the presence of specific replicative elements.

Gene Deletion↗
PubMed1982

Iron- and molybdenum-repressible outer membrane proteins in competent Azotobacter vinelandii.

Azotobacter vinelandii produced three major proteins of 93,000, 85,000, and 81,000 daltons and a minor 77,000-dalton protein in the outer membrane of Fe-limited cells, and these cells were competent for transformation by DNA. The synthesis of these proteins was repressed in Fe-sufficient medium. Mo limitation of nitrogen-fixing cells resulted in the hyperproduction of a 44,000-dalton protein and the production of a minor 77,000-dalton protein in the outer membrane. Mo limitation enhanced competence in Fe-limited medium and induced competence in Fe-sufficient medium. The 44,000-dalton protein was replaced by a 45,000-dalton protein when Fe-sufficient medium also contained NH4+, but the cells were noncompetent. The synthesis of these proteins was repressed in Mo-sufficient medium and by NH4+ in Fe-limited medium. All of the culture supernatants contained a blue-white fluorescent material (absorbance maximum, 214 nm) which appeared to coordinate Fe3+, Fe2+, MoO4(2-), WO3(2-), and VO3(-).

Azotobacter↗
PubMed1980

Properties and transforming activities of two plasmids in Streptococcus pneumoniae.

Two plasmids from group B streptococcus were introduced into pneumococcus (Streptococcus pneumoniae) and examined for copy number, stability, and some features of the process by which they transform pneumococcal recipients. The 3.6 Mdal pMV158 (tet) was present at a minimum of 12 to 16 copies per chromosome and was never observed to be cured. The 20 Mdal pIP501 (cat erm) had a minimum copy number of 3 to 4 per chromosome and was lost spontaneously at a frequency near 0.03 per division. The presence of novobiocin increased this frequency 2 to 3-fold. Competence for chromosomal transformation and the membrane endonuclease needed for normal DNA entry were required for plasmid transformation. Plasmid transformants segregated transformed cells one generation ahead of chromosomal transformants. Both single and multiple hit components of the transformation reaction kinetics were observed, but the latter could not be seen in the presence of competing chromosomal DNA. The major of the transforming activity behaved as covalently closed circular DNA in dye-buoyancy gradients. Although most of the activity for both plasmids sedimented in sucrose gradients more rapidly than did monomeric closed circular DNA, a significant fraction was found at a position suggesting that it may have been due to monomeric plasmids.

Centrifugation, Density Gradient↗
PubMed1982

Sequence-specific DNA uptake in transformation of Neisseria gonorrhoeae.

Piliated, competent gonococci are known to preferentially take up homologous transforming DNA into the cell. We examined the mechanism for DNA uptake with pFA10, a hybrid 11.5-kilobase (kb) penicillin-resistant (Pcr) plasmid composed of heterologous DNA from a 7.2-kb Pcr plasmid and homologous DNA from a 4.2-kb gonococcal cryptic plasmid. The presence of the gonococcal cryptic plasmid DNA in the hybrid resulted in markedly increased transformation efficiencies in isogenic crosses as compared with the parent 7.2-kb Pcr plasmid. Uptake of 32P-end-labeled MspI or TaqI restriction fragments of the hybrid was limited to fragments entirely derived from the 4.2-kb gonococcal cryptic plasmid, indicating that DNA uptake was probably dependent on the presence of a specific DNA sequence. Since Haemophilus DNA did not inhibit transformation by the hybrid Pcr plasmid, the gonococcal DNA uptake sequence is different from the known sequence involved in homologous DNA uptake by Haemophilus spp.

Base Sequence↗
PubMed1998

Cell surface localization and processing of the ComG proteins, required for DNA binding during transformation of Bacillus subtilis.

The comG operon of Bacillus subtilis encodes seven proteins essential for the binding of transforming DNA to the competent cell surface. We have explored the processing of the ComG proteins and the cellular localization of six of them. All of the proteins were found to be membrane associated. The four proteins with N-terminal sequence motifs typical of type 4 pre-pilins (ComGC, GD, GE and GG) are processed by a pathway that requires the product of comC, also an essential competence gene. The unprocessed forms of ComGC and GD behave like integral membrane proteins. Pre-ComGG differs from pre-ComGC and pre-ComGD, in that it is accessible to proteolysis only from the cytoplasmic face of the membrane and at least a portion of it behaves like a peripheral membrane protein. The mature forms of these proteins are translocated to the outer face of the membrane and are liberated when peptidoglycan is hydrolysed by lysozyme or mutanolysin. ComGG exists in part as a disulphide-cross-linked homodimer in vivo. ComGC was found to possess an intramolecular disulphide bond. The previously identified homodimer form of this protein is not stabilized by disulphide bond formation. ComGF behaves as an integral membrane protein, while ComGA, a putative ATPase, is located on the inner face of the membrane as a peripheral membrane protein. Possible roles of the ComG proteins in DNA binding to the competent cell surface are discussed in the light of these and other results.

Amino Acid Sequence↗
PubMed1983

Protease-sensitive transfection of Streptococcus pneumoniae with bacteriophage Cp-1 DNA.

The transfecting activity of pneumococcal phage Cp-1 DNA was destroyed by treatment with proteolytic enzymes, although these enzymes did not affect transfection with bacteriophage Dp-4 DNA. This transfection was stimulated by calcium ions. Protease-treated Cp-1 DNA competes for binding and uptake with transforming pneumococcal DNA as well as with transfecting Dp-4 DNA to approximately the same extent as does untreated Cp-1 DNA. In addition, [3H]thymidine-labeled Cp-1 DNA, treated with proteases or untreated, was absorbed with the same efficiency. These data suggest that uptake of Cp-1 DNA is not affected by protease treatment. [3H]thymidine-labeled Cp-1 DNA showed remarkable resistance against surface nuclease activity of competent wild-type cells. The monomeric form of the Cp-1 DNA-protein complex showed a linear dose response in transfection.

Bacteriophages↗
PubMed1989

Heat shock at an elevated temperature improves transformation efficiency of protoplasts from Podospora anserina.

We have developed an improved transformation procedure for the filamentous fungus Podospora anserina. This procedure is based on the observation that a heat shock at an elevated temperature (48 degrees C) improves the competence of P. anserina protoplasts for transformation 5- to 10-fold. This is observable only if the heat shock is applied before the addition of transforming DNA. An increase in competence is observed immediately after the heat shock, and heat-shocked cells are still competent after 20-30 min. The mechanism by which heat shock improves competence remains unclear. The modified transformation procedure gives as many as 200-500 stable transformants per microgram of plasmid DNA containing the P. anserina ura5 gene. This should allow direct cloning of P. anserina genes from a cosmid library.

Ascomycota↗
PubMed1993

Anti-SOS effects induced in Bacillus subtilis by a phi 105 mutant prophage.

The presence of the mutant prophage phi 105cts23 in Bacillus subtilis strains strongly affected several biological parameters including the viability of protoplasts and the establishment of plasmid pC194. A defective inducibility of the prophage after treatments that de-repress the SOS-like response were also observed. Although these alterations suggested a Rec-deficient phenotype, homologous recombination was not impaired in these lysogenic derivatives. In fact, chromosomal DNA transformation in these competent cells was more efficient than in cells carrying the wild type prophage: cell death due to prophage induction upon competence development was lower than expected. Alterations in the response to SOS-inducing agents and to osmotic stress correlated with the presence of this particular mutant prophage or the cloned thermosensitive repressor at the permissive temperature. The induction of an anti-SOS effect is discussed.

Bacillus Phages↗
PubMed2002

pilG Gene cluster and split pilL genes involved in pilus biogenesis, motility and genetic transformation in the cyanobacterium Synechocystis sp. PCC 6803.

The unicellular motile cyanobacterium Synechocystis sp. PCC 6803 exhibits phototactic motility that depends on the type IV-like thick pilus structure. By gene disruption analysis, we showed that a gene cluster of slr1041, slr1042, slr1043 and slr1044, whose predicted products are homologous to PatA, CheY, CheW and MCP, respectively, was more or less required for pilus assembly, motility and natural transformation competency with extraneous DNA. By sequence homology, the missing cheA-like gene in this cluster was identified as novel split genes, slr0073 and slr0322, at separate loci on the genome. This was confirmed by non-motile phenotype of their disruptants. Unique hyperpiliation was observed in the slr1042 and slr0073 disruptants, suggestive of their specific interaction with pilT1. The genes, thus identified as pil genes in this study, were designated pilG (slr1041), pilH (slr1042), pilI (slr1043), pilJ (slr1044), pilL-N (slr0073) and pilL-C (slr0322).

Amino Acid Sequence↗
PubMed1975

Physiological studies on the pneumococcal Forssman antigen: a choline-containing lipoteichoic acid.

The cell concentration and possible biological activities of the pneumococcal Forssman (F) antigen (membrane lipoteichoic acid) were examined in a number of physiological situations. In test tube cultures of pneumococci the concentration of the Forssman antigen per bacterium showed no significant fluctuations within a typical culture cycle. Purified F antigen had no effect on the activation of pneumococci to competence for genetic transformation, DNA mediated genetic transformation or adsorption of the pneumococcal phage Dp-1 to bacteria. Pneumococci grown in the presence of different amino alcohols (ethanolamine, N-monomethylethanolamine, or choline) exhibit differences with regard to both their ability to stimulate heterophile (haemolytic) antibody production in rabbits and in their ability to bind such antibodies. Choline-grown bacteria seem to cross-react with sheep red blood cells better than do the analogue-grown bacteria.

Adsorption↗
PubMed1991

Conversion of Bacillus subtilis 168 to a subtilin producer by competence transformation.

Subtilin is a ribosomally synthesized peptide antibiotic produced by Bacillus subtilis ATCC 6633. B. subtilis 168 was converted to a subtilin producer by competence transformation with chromosomal DNA from B. subtilis ATCC 6633. A chloramphenicol acetyltransferase gene was inserted next to the subtilin structural gene as a selectable marker. The genes that conferred subtilin production were derived from a 40-kb region of the B. subtilis ATCC 6633 chromosome that had flanking homologies to the B. subtilis 168 chromosome. The subtilin produced by the mutant was identical to natural subtilin in its biological activity, chromatographic behavior, amino acid composition, and N-terminal amino acid sequence.

Amino Acid Sequence↗