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At least 127 records · Page 7Linked to original sources
PubMed1998

Genetic transformation of germinated conidia of the thermophilic fungus Humicola grisea var. thermoidea to hygromycin B resistance.

Germinated conidia of the thermophilic fungus Humicola grisea var. thermoidea were transformed to hygromycin B resistance using the plasmid pAN7.1. Transformation was achieved using lithium acetate treatment or electroporation. The efficiency of transformation was up to 32 and 25 transformants per microgram of plasmid DNA with the two methods, respectively. Transformants obtained by the lithium acetate method were more stable and showed a high copy number of the hph gene integrated into their genome. The other transformants, from the electroporation procedure, were stable, but unable to grow in the presence of high levels of hygromycin, and detection of the hph gene was only possible by polymerase chain reaction analysis.

Dose-Response Relationship, Drug↗
PubMed1999

Construction and analysis of a library for random insertional mutagenesis in Streptococcus pneumoniae: use for recovery of mutants defective in genetic transformation and for identification of essential genes.

To explore the use of insertion-duplication mutagenesis (IDM) as a random gene disruption mutagenesis tool for genomic analysis of Streptococcus pneumoniae, a large mutagenic library of chimeric plasmids with 300-bp inserts was constructed. The library was large enough to produce 60,000 independent plasmid clones in Escherichia coli. Sequencing of a random sample of 84 of these clones showed that 85% of the plasmids had inserts which were scattered widely over the genome; 80% of these plasmids had 240- to 360-bp inserts, and 60% of the inserts targeted internal regions of apparent open reading frames. Thus, the library was both complex and highly mutagenic. To evaluate the randomness of mutagenesis during recombination and to test the usefulness of the library for obtaining specific classes of nonessential genes, this library was used to seek competence-related genes by constructing a large pneumococcal transformant library derived from 20,000 mutagenic plasmids. After we screened the mutants exhaustively for transformation defects, 114 competence-related insertion mutations were identified. These competence mutations hit most previously known genes required for transformation as well as a new gene with high similarity to the Bacillus subtilis competence gene comFA. Mapping of the mutation sites at these competence loci showed that the mutagenesis was highly random, with no apparent hot spots. The recovery of a high proportion of competence genes and the absence of hot spots for mutational hits together show that such a transformant library is useful for finding various types of nonessential genes throughout the genome. Since a promoterless lacZ reporter vector was used for the construction of the mutagenic plasmid library, it also serves as a random transcriptional fusion library. Finally, use of a valuable feature of IDM, directed gene targeting, also showed that essential genes, which can be targets for new drug designs, could be identified by simple sequencing and transformation reactions. We estimate that the IDM library used in this study could readily achieve about 90% genome coverage.

Bacillus subtilis↗
PubMed1981

Genetic transformation of Streptococcus mutans.

Three strains of Streptococcus mutans belonging to serotypes a, c, and f were transformed to streptomycin resistance by deoxyribonucleic acids derived from homologous and heterologous streptomycin-resistant strains of S. mutans and Streptococcus sanguis strain Challis. Homologous transformation of S. mutans was less efficient than heterologous transformation by deoxyribonucleic acids from other strains of S. mutans.

DNA, Bacterial↗
PubMed1970

Mutants of Diplococcus pneumoniae that lack deoxyribonucleases and other activities possibly pertinent to genetic transformation.

Mutants of Diplococcus pneumoniae that lacked the two major deoxyribonucleases of the cell-one an endonuclease, the other an exonuclease preferentially active on native deoxyribonucleic acid (DNA)-were obtained. The development of a method for detecting mutant colonies, based on the binding of methyl green to DNA, facilitated isolation of the mutants. Neither enzyme was essential for growth of the cells, for repair of ultraviolet damage, or for any phase of DNA-mediated transformation. Residual deoxyribonuclease activity in the double mutant corresponded to an exonuclease, approximately one-fifth as active as the major exonuclease, that attacked native and denatured DNA equally well. This activity appeared to be associated with the DNA-polymerase enzyme. A mutant that apparently lacked a cell wall lytic enzyme was also fully transformable. A mutant strain that was four times more sensitive to ultraviolet light than the wild type also transformed normally. Recipient cells of this strain were deficient in the repair of ultraviolet-irradiated transforming DNA. Mutants were found which, unlike the wild type, integrated donor markers only with high efficiency, thereby indicating that a particular cellular component that is susceptible to loss by mutation, such as an enzyme, is responsible for low integration efficiency.

Cell Wall↗
PubMed1973

Competence for deoxyribonucleic acid uptake and deoxyribonuclease action external to cells in the genetic transformation of Diplococcus pneumoniae.

A mutant of Diplococcus pneumoniae that apparently does not require activator can become competent for uptake of deoxyribonucleic acid (DNA) when grown in dilute cultures or in the presence of trypsin. Development of competence in both mutant and wild strains is temperature dependent, being 10-fold greater at 30 C than at 37 C. Induction of competence on a shift from 37 to 30 C requires protein synthesis and the presence of Mg(2+) and Ca(2+); uptake of DNA does not require protein synthesis. Competence decays exponentially at higher temperatures. As well as taking up DNA, competent cells release oligonucleotide fragments of donor DNA in the medium external to the cells. Normal strains release fragments comparable in amount to the DNA taken up; but, in a mutant selected for inability to degrade DNA in agar, the amount of fragments formed external to the cells is only 40% of DNA uptake. Requirements for external deoxyribonuclease action are identical to those for DNA uptake: prior development of competence and the presence during treatment with DNA of Mg(2+) ions and a source of energy.

Calcium↗
PubMed1975

Constitution of the cell envelope of Haemophilus influenzae in relation to competence for genetic transformation.

Cell envelopes of Haemophilus influenzae have been prepared by breakage in a French pressure cell followed by differential centrifugation. The envelope fraction may be resolved into an inner-membrane (light) and an outer-membrane (heavy) fraction on density gradients. Envelopes from competent cells possess elevated levels of lipopolysaccharide with a composition different from that of log-phase cell envelopes. Three apparently new polypeptides have been observed in envelopes from competent cells by gel electrophoresis in sodium dodecyl sulfate; additional quantitative alterations in the profiles of membrane polypeptides also company the development of the capacity to transport deoxyribonucleic acid. Most of the polypeptide changes are confined to the outer membrane; one new polypeptide is associated with the inner cytoplasmic membrane of competent cells. Protein synthesis during competence developement is rquired for the change in lipopolysaccharides and in the envelope polypeptides to occur.

Bacterial Proteins↗
PubMed1976

Synthesis of envelope polypeptides by Haemophilus influenzae during development of competence for genetic transformation.

Six polypeptides with apparent molecular weights of 95,000, 90,000, 80,000, 67,000, 64,000, and 43,000 were found to be characteristic of the cell envelopes of competent Haemophilus influenzae, and were synthesized entirely during the period of competence development. Two polypeptides with apparent molecular weights of 58,500 and 40,500 were synthesized during growth as well as during competence development, but were only associated with the envelope fraction of cells that had developed competence. The kinetics of synthesis of the competence-related envelope polypeptides showed a lag period of approximately 20 min. The observation of this lag period raises the question as to whether some of these competence-related polypeptides might be involved in the process of deoxyribonucleic acid uptake, since the development of this property also exhibits a sigmoid time course during competence development.

Bacterial Proteins↗
PubMed1984

Optimal conditions for genetic transformation of the cyanobacterium Anacystis nidulans R2.

Under optimal conditions, the cyanobacterium Anacystis nidulans R2 was transformed to ampicillin resistance at frequencies of greater than 10(7) transformants per microgram of plasmid (pCH1) donor DNA. No stringent period of competency was detected, and high frequencies of transformation were achieved with cultures at various growth stages. Transformation increased with time after addition of donor DNA up to 15 to 18 h. The peak of transformation efficiency (transformants/donor molecule) occurred at plasmid concentrations of 125 to 325 ng/ml with an ampicillin resistance donor plasmid (pCH1) and 300 to 625 ng/ml for chloramphenicol resistance conferred by plasmid pSG111. The efficiency of transformation was enhanced by excluding light during the incubation or by blocking photosynthesis with the electron transport inhibitor 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) or the uncoupler carbonyl cyanide-m-chlorophenyl hydrazone. Preincubation of cells in darkness for 15 to 18 h before addition of donor DNA significantly decreased transformation efficiency. Growth of cells in iron-deficient medium before transformation enhanced efficiency fourfold. These results were obtained with selection for ampicillin (pCH1 donor plasmid)- or chloramphenicol (pSG111 donor plasmid)-resistant transformants. Approximately 1,000 transformants per microgram were obtained when chromosomal DNA from an herbicide (DCMU)-resistant mutant was used as donor DNA. DCMU resistance was also transferred to recipient cells by using restriction fragments of chromosomal DNA from DCMU-resistant mutants. This procedure allowed size classes of fragments to be assayed for the presence of the DCMU resistance gene.

Ampicillin↗
PubMed1987

Genetic transformation in the methanogen Methanococcus voltae PS.

Mutations causing requirements for histidine, purine, and vitamin B12 were obtained in strain PS of Methanococcus voltae (archaebacteria) upon irradiation with UV or gamma rays. The first two mutations were shown to revert at low frequencies and were used to demonstrate the occurrence of transformation with homologous, wild-type DNA. The transformation rates obtained for these presumably chromosomal markers were in the range of 2 to 100 transformants per microgram of DNA. Mutants resistant to 2-bromoethanesulfonate and to 5-methyl-DL-tryptophan were also isolated.

Alkanesulfonates↗
PubMed1995

ComEA, a Bacillus subtilis integral membrane protein required for genetic transformation, is needed for both DNA binding and transport.

The competence-related phenotypes of mutations in each of the four open reading frames associated with the comE locus of Bacillus subtilis are described. comEA and comEC are required for transformability, whereas the products of comEB and of the overlapping comER, which is transcribed in the reverse direction, are dispensable. Loss of the comEA product decreases the binding of DNA to the competent cell surface and the internalization of DNA, in addition to exhibiting a profound effect on transformability. The comEC product is required for internalization but is dispensable for DNA binding. ComEA is shown to be an integral membrane protein, as predicted from hydropathy analysis, with its C-terminal domain outside the cytoplasmic membrane. This C-terminal domain possesses a sequence with similarity to those of several proteins known to be involved in nucleic acid transactions including UvrC and a human protein that binds to the replication origin of the Epstein-Barr virus.

Amino Acid Sequence↗
PubMed1977

Identification of Neisseria gonorrhoeae by genetic transformation: a clinical laboratory evaluation.

Transformation of a Neisseria gonorrhoeae auxotroph (uracil and arginine deficient) to prototrophy was attempted with wild-type deoxyribonucleic acid from 71 random clinical N. gonorrhoeae cultures. Of these 71 cultures, 97.1% transformed the nutritionally deficient mutant to prototrophy. The procedure was reliable and economical and offered several distinct advantages over other methods used for the confirmation of N. gonorrhoeae.

Neisseria gonorrhoeae↗