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At least 343 records · Page 19Linked to original sources
PubMed2006

Proposal of a method for the genetic transformation of Gordonia jacobaea.

AIMS: Gordonia jacobaea is a recently isolated bacterial species with potential industrial application on account of its ability to store large quantities of trans-canthaxanthin. Its genetic manipulation is, however, difficult and cumbersome owing to the presence of mycolic acids in the cell wall and, especially, because of current lack of knowledge about its basic genetics. The present work describes a method for the genetic transformation of G. jacobaea. METHODS AND RESULTS: Gordonia jacobaea was grown in media supplemented with different glycine, penicillin G and isoniazid concentrations. The temperature, carbon source, growth phase and ultrasounds were analyzed for improving the method efficiency. The cells were finally transformed by electroporation. Finally, the method was applied to Brevibacteriumlactofermentum and Gordonia bronchialis. CONCLUSIONS: The growth of G. jacobaea in the presence of glycine and isoniazid is essential for obtaining electrocompetents cells. The temperature, growth phase and ultrasounds appeared as the main factors for increasing the transformation efficiency. The use of shuttle plasmids became necessary. The method described can be used with other Corynebacteria species. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the importance of the CNM group (Corynebacteria, Nocardia and Mycobacteria genera) in different areas such as industry, bioremediation improve the knowledge of their molecular mechanisms are becoming essential. The method described here improves the genetic manipulation of this group of bacteria.

Anti-Bacterial Agents↗
PubMed1990

Genetic transformation in Streptococcus pneumoniae: nucleotide sequence and predicted amino acid sequence of recP.

We present the complete nucleotide sequence of recP, a locus required for high-efficiency recombination of chromosomal DNA during genetic transformation in Streptococcus pneumoniae. The sequence was determined by using plasmid DNA templates and synthetic oligonucleotide primers. The locus contained a single large open reading frame, ORF1, of 1,968 base pairs (bp). ORF1 is included within the recP locus previously mapped genetically and accounts for 94% of its extent. The predicted molecular weight of the largest polypeptide encoded within ORF1, 71,662, coincided with that measured previously (72,000) for the product of in vitro transcription-translation of the cloned recP locus. A Shine-Dalgarno sequence (AGAAAGGA; delta G = -17 kcal [ca. -71.1 kJ]) lay 6 bp upstream of ORF1. A sequence (TTGcat-17 bp-TATAAT) similar to the E. coli sigma-70 promoter consensus was located 52 bp upstream of ORF1. This putative promoter overlapped a structure consisting of two perfect inverted 10-bp repeats and a loop of 8 bp.

Amino Acid Sequence↗
PubMed1966

Evidence for the involvement of membranous bodies in the processes leading to genetic transformation in Bacillus subtilis.

Wolstenholme, David R. (Max-Planck-Institut für Biologie, Tübingen, Germany), Cornelius A. Vermeulen, and Gerhardus Venema. Evidence for the involvement of membranous bodies in the processes leading to genetic transformation in Bacillus subtilis. J. Bacteriol. 92:1111-1121. 1966.-Data obtained from electron microscopic autoradiographs of profiles of cells of a Bacillus subtilis population exposed to H(3)-thymidine-labeled donor deoxyribonucleic acid (DNA) during the phase of maximal competence indicated that molecules originating from absorbed DNA are closely associated with membranous bodies, particularly with those situated in the cytoplasm, but that most if not all of the radioactive molecules are outside the bodies. It is suggested that membranous bodies produce enzymes essential to the eventual incorporation of transforming DNA into the bacterial genome, or to the breakdown and utilization or expulsion of absorbed DNA not incorporated as transformant (or to both processes). During the phase of maximal competence, the total number of membranous bodies seen in profiles increased continuously to as much as 2.3 times the numbers found during earlier stages of culture. This increase was not accounted for by a decrease in bacterial cell volume, but resulted from an actual increase in total volume of membranous bodies. The number of membranous bodies visibly connecting plasma membrane and nuclear region increased during maximal competence to as much as 30 times the numbers found in earlier stages. As both increases were found in the absence of donor DNA and only began after maximal competence was attained, it seemed most probable that they were an expression of a physiological state influenced by the continuing deficiency of nutrients in the growth medium during this phase of culture.

Autoradiography↗
PubMed1997

Replication, expression and segregation of plasmid-borne DNA in genetically transformed malaria parasites.

To fully exploit the transfection technology developed for Plasmodium we investigated the features of replication, expression and segregation of an episomally maintained DNA construct during a sexual blood stage development in genetically transformed parasites of P. berghei. Using DNA in situ hybridisation techniques we were able to show that the introduced DNA construct is located in the nucleus of the parasite and is not segregating uniformly during schizogony. Replication of the construct mainly takes place between 16 and 24 h after invasion of the merozoites, coinciding with chromosomal replication. Furthermore the plasmid-borne DHFR/TS gene is constitutively transcribed throughout the asexual blood stage development. Hence the DHFR/TS promoter would appear to be a useful tool in the study of (over)expression of introduced genes and performing complementation studies in transfected parasites during the complete a sexual blood stage development of P. berghei.

Animals↗
PubMed1994

Genetic transformation of the Lyme disease agent Borrelia burgdorferi with coumarin-resistant gyrB.

No useful method to genetically manipulate Borrelia burgdorferi, the causative agent of Lyme disease, has been developed previously. We have used resistance to the coumarin antibiotic coumermycin A1, an inhibitor of DNA gyrase, as a genetic marker to monitor the transformation of B. burgdorferi by electroporation. Introduction of site-directed mutations into the gyrB gene demonstrated that transformation was successful, provided evidence that homologous recombination occurs on the chromosome, and established that mutations at Arg-133 of DNA gyrase B confer coumermycin A1 resistance in B. burgdorferi. The coumermycin A1-resistant gyrB marker and genetic transformation can now be applied toward dissecting the physiology and pathogenesis of the Lyme disease agent on a molecular genetic level.

Aminocoumarins↗
PubMed1991

Genetic transformation in Streptococcus pneumoniae: nucleotide sequence analysis shows comA, a gene required for competence induction, to be a member of the bacterial ATP-dependent transport protein family.

The complete nucleotide sequence of comA, a gene required for induction of competence for genetic transformation in Streptococcus pneumoniae, was determined by using plasmid DNA templates and synthetic oligonucleotide primers. The sequence contained a single large open reading frame, ORF1, of 2,151 bp. ORF1 was included within the comAB locus previously mapped genetically and accounted for 50% of its extent. The predicted molecular weight of the largest polypeptide encoded within ORF1, 80,290, coincided with that measured previously (77,000) for the product of in vitro transcription-translation of the cloned comA locus. A Shine-Dalgarno sequence (AAAGGAG, delta G = -14 kcal) lay immediately upstream of ORF1. A sequence (TTtAat-17 bp-TAaAAT) similar to the Escherichia coli sigma 70 promoter consensus was located 410 bp upstream of ORF1. The deduced protein sequence of ComA showed a very strong similarity to the E. coli hemolysin secretion protein, HlyB, and strong similarities to other members of the family of ATP-dependent transport proteins, including the mammalian multidrug resistance P-glycoprotein. These similarities suggest that ComA functions in the transport of some molecule, possibly pneumococcal competence factor itself.

Adenosine Triphosphate↗
PubMed1994

Identification of Moraxella bovis and related species from calves with IBK and goats by qualitative genetic transformation assay.

Eight Moraxella bovis strains isolated from bovine pink eye, 16 Moraxella bovis related strains isolated from healthy goats nasal flora, one nonhemolytic Moraxella sp. isolated from bovine conjunctivitis and different collection strains of Moraxella and Branhamella genus were studied through the combined use of qualitative genetic transformation assay and the ability to grow on a moraxella bovis defined medium (medium MB). Crude DNA extracted from the strains studied were used to transform two mutant auxotroph competent strains of Moraxella bovis CIP 103741 and CIP 103743. Non-specific positive transformation was obtained with mutant assay strain CIP 103743 when treated with DNA from caprine or bovine Moraxella strains, collection strains of Moraxella bovis, Moraxella lacunata, Moraxella nonliquefaciens and strains of Branhamella genus, whereas specific transformation was observed with mutant assay CIP 103741 when treated only with DNA from all the Moraxella bovis (collection and clinical isolates) and 14 of the 16 caprine Moraxella bovis related strains. The specificity and the simplicity of the test make it suitable for use in clinical laboratories.

Animals↗
PubMed1980

Genetic transformation of Streptococcus sanguis (Challis) with cryptic plasmids from Streptococcus ferus.

By using the basic methodology initially published by Kretschmer et al. (J. Bacteriol. 124:225-231, 1975), we have been able to introduce phenotypically cryptic plasmids from Streptococcus ferus (formerly Streptococcus mutans subsp. ferus) into Streptococcus sanguis by genetic transformation. In this system, the entry of the cryptic plasmids is selected indirectly. This is effected with transforming deoxyribonucleic acid mixtures in which the cryptic plasmid deoxyribonucleic acid is present in an approximate 10-fold molar excess with respect to a plasmid (pVA1) known to confer erythromycin resistance. Under such conditions, 5 to 10% of the pVA1-containing erythromycin-resistant transformants were cotransformed with cryptic plasmid deoxyribonucleic acid. pVA1 may be selectively eliminated by growth of its S. sanguis host strain at 42 degrees C, enabling the construction of isogenic strains with and without S. ferus cryptic plasmids. Comparative physiological studies of such strains have failed to reveal any plasmid-conferred phenotypes in S. sanguis. With this procedure, we have been able to physically separate two small cryptic plasmids (2.4 x 10(6) and 2.8 x 10(6) daltons) of S. ferus. Although these plasmids were found naturally to exist in a single S. ferus host, they were able to replicate independently of one another in S. sanguis. Restriction enzyme fingerprinting indicated that these plasmids did not share a common ancestry.

Bacteriocins↗
PubMed2000

Regulation of competence for genetic transformation in Streptococcus pneumoniae: expression of dpnA, a late competence gene encoding a DNA methyltransferase of the DpnII restriction system.

The chromosomal DpnII gene cassette of Streptococcus pneumoniae encodes two methyltransferases and an endonuclease. One methyltransferase acts on double-stranded and the other on single-stranded DNA. Two mRNAs are transcribed from the cassette. One, a SigA promoter transcript, includes all three genes; the other includes a truncated form of the second methyltransferase gene (dpnA) and the endonuclease gene. The truncated dpnA, which is translated from the second start codon in the full gene, was shown to produce active enzyme. A promoter reporter plasmid for S. pneumoniae was devised to characterize the promoter for the second mRNA. This transcript was found to depend on a promoter that responded to the induction of competence for genetic transformation. The promoter contains the combox sequence recognized by a SigH-containing RNA polymerase. As part of the competence regulon, the dpnA gene makes a product able to methylate incoming plasmid strands to protect them from the endonuclease and allow plasmid establishment. Its function differs from most genes in the regulon, which are involved in DNA uptake. Comparison of R6 and Rx strains of S. pneumoniae showed the temperature dependence of transformation in R6 to result from temperature sensitivity of the uptake apparatus and not the development of competence.

Base Sequence↗
PubMed1998

[Genetic transformation of plants, recombination processes, and gene expression regulation in transgenic plants].

Recent studies in molecular biology and gene engineering were conducive to accumulation of numerous facts which help better understand the processes of plant genome functioning and follow up the effects of recombination processes in purposefully modified genome of a transformed plant on its expression. This review analyzes the major approaches to studies and results attained in plant gene engineering and plant genome imprinting.

DNA Methylation↗
PubMed2003

Stable genetic transformation of Vigna mungo L. Hepper via Agrobacterium tumefaciens.

Vigna mungo is one of the large-seeded grain legumes that has not yet been transformed. We report here for the first time the production of morphologically normal and fertile transgenic plants from cotyledonary-node explants inoculated with Agrobacterium tumefaciens carrying binary vector pCAMBIA2301, the latter of which contains a neomycin phosphotransferase ( nptII) gene and a beta-glucuronidase (GUS) gene ( uidA) interrupted with an intron. The transformed green shoots, selected and rooted on medium containing kanamycin, tested positive for nptII and uidA genes by polymerase chain reaction (PCR) analysis. These shoots were established in soil and grown to maturity to collect the seeds. Mechanical wounding of the explants prior to inoculation with Agrobacterium, time lag in regeneration due to removal of the cotyledons from explants and a second round of selection at the rooting stage were found to be critical for transformation. Analysis of T(0) plants showed the expression and integration of uidA into the plant genome. GUS activity in leaves, roots, flowers, anthers and pollen grains was detected by histochemical assay. PCR analysis of T(1) progeny revealed a Mendelian transgene inheritance pattern. The transformation frequency was 1%, and 6-8 weeks were required for the generation of transgenics.

Agrobacterium tumefaciens↗