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PubMed1982

Identification and cloning of a yeast nuclear gene (CBP1) involved in expression of mitochondrial cytochrome b.

Nuclear pet mutants of Saccharomyces cerevisiae deficient in mitochondrial respiration have been studied genetically and biochemically. Seven noncomplementing mutations leading to a deficiency of mitochondrial cytochrome b have been assigned to a single complementation group (group 60). Examination of mitochondrial RNA by blot hybridization on diazobenzyloxymethyl-paper has revealed that group 60 mutants produce a large number of novel apocytochrome b transcripts not detected in wild-type yeast. The product of the gene affected in the mutants, therefore, appears to be required either for correct transcription or for processing of apocytochrome b premessenger RNA. The gene has been designated CBP1. A representative mutant from complementation group 60 (N5-26) has been transformed to respiratory competency with a recombinant plasmid pool consisting of random fragments of wild-type yeast nuclear DNA inserted into a vector capable of replicating in yeast and Escherichia coli. The complementation of the N5-26 mutation has been shown for a number of independent transformants to be due to the presence of plasmid DNA. The plasmid pG60/T10 was further characterized to have a nuclear DNA insert of 6.7 kilobase pairs. This plasmid complements the mutations of all group 60 mutants, thus confirming that it contains the CBP1 gene.

Cytochrome b Group↗
PubMed2002

Escherichia coli cells penetrated by chrysotile fibers are transformed to antibiotic resistance by incorporation of exogenous plasmid DNA.

A suspension of recipient Escherichia coli cells in stationary phase, chrysotile asbestos, and pUC18 donor DNA spread over the surface of a Luria-Bertani agar plate using a streak bar several times, resulted in intracellular uptake of the plasmid DNA by the E. coli cells. The transformation efficiency was highest with a duration of cell exposure to chrysotile of more than 60 s and an agar concentration of 2%. To improve chrysotile-mediated transformation efficiency, we systematically optimized various conditions and parameters. In comparison to chrysotile exposure without cations, exposure with cations produced up to 100-fold more transformants. Optimized conditions resulted in 10(6) transformants/ micro g pUC18 DNA. The drastic physical change due to 'quick drying on the surface of the agar plate' when cells were exposed to chrysotile, was essential for chrysotile-mediated transformation. We suggest that DNA uptake mediated by chrysotile asbestos is the result of a mechanical physical transformation of E. coli, since the E. coli cells are not chemically competent. Electron microscopy of cells exposed to chrysotile suggested penetration of the E. coli membrane by chrysotile fibers. It is suggested that E. coli transformation by the plasmid DNA was the result of penetration by chrysotile fibers to which plasmid DNA is bound or adsorbed.

Agar↗
PubMed2004

Harnessing natural transformation in Actinobacillus pleuropneumoniae: a simple method for allelic replacements.

We have investigated the use of a natural transformation protocol to introduce mutations into Actinobacillus pleuropneumoniae serotypes 1 and 5b. For both strains tested, we recovered 1 in 10(8) transformants during culture in rich medium, a result that was independent of the growth phase. This low frequency of transformation of A. pleuropneumoniae did not increase when bacteria were grown under conditions known to be optimal for induction of competence in Haemophilus influenzae. Using linearised plasmid DNA containing a kanamycin cassette inserted into the sodC gene of A. pleuropneumoniae serotype 1, we showed that natural transformation can be used as a simple method for introducing allele replacements into this bacterium, and can be used to transfer mutations from one serotype to another.

Actinobacillus pleuropneumoniae↗
PubMed1987

Transformation of Xanthomonas campestris pathovar campestris with plasmid DNA.

Procedures for the introduction of plasmid DNA into Gram-negative bacteria have been adapted and optimized to permit transformation of the plant pathogen Xanthomonas campestris pathovar campestris with the cloning vector pKT230 and other broad-host-range plasmids. The technique involves CaCl2-induced competence and heat shock and is similar to that routinely used for Escherichia coli. Wild-type X. c. campestris strains appear to restrict incoming unmodified DNA, so that plasmid DNA for transformation must be prepared from X. c. campestris (into which it has previously been introduced by conjugation). To overcome this disadvantage a restriction-deficient mutant has been isolated.

DNA, Bacterial↗
PubMed1985

DNA-mediated gene transfer efficiency is enhanced by ionizing and ultraviolet irradiation of rodent cells in vitro. I. Kinetics of enhancement.

The enhancement effects of ionizing and ultraviolet radiation on the efficiency of DNA-mediated gene transfer were studied. The established cell line, Rat-2, consists of cells that are density-dependent contact-inhibited and produce flat monolayers in vitro. When these cells are infected with SV40 virus, a small fraction of cells becomes morphologically "transformed" due to the stable expression of the viral A-gene. Rat-2 cells are competent for DNA-mediated gene transfer, deficient in thymidine kinase activity (TK-), and will die in HAT selective media. Confluent Rat-2 cells were transfected with purified SV40 viral DNA (via calcium phosphate precipitation), irradiated with either X rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were nonlinear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X rays or 330 MeV/amu argon particles at the Berkeley BEVALAC showed a higher frequency of HAT+ colonies/survivor than unirradiated transfected cells. In both cases the enhancement contained a linear and a higher order component in dose, but the argon ions were at least twice more efficient than X rays in producing enhancement per unit dose. Rat-2 cells transfected with pOT-TK5, X-irradiated, and assayed for either TK transformation or A-gene transformation showed the same dose dependence for radiation enhancement. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK transformation by both X rays and ultraviolet radiation. SV40 A-gene products are not necessary for the radiation enhancement of the efficiency of gene transfer. This in vitro system will be used to study the lesions produced by ionizing radiation on mammalian cell DNA that may act as substrates for integration of exogenously introduced plasmid DNA.

Animals↗
PubMed2001

A ComGA-dependent checkpoint limits growth during the escape from competence.

In Bacillus subtilis, competence for transformation develops in 5-10% of the cells in a stationary phase culture. These cells exhibit a prolonged lag in the resumption of growth and cell division during the escape from competence. To better understand the basis of this lag, we have characterized competent cultures microscopically. To distinguish the minority of competent cells, a translational fusion between ComK, the competence transcription factor, and the green fluorescent protein (GFP) was used as a marker. Only 5-10% of the cells in a competent culture were fluorescent, indicating that ComK synthesis is an all or nothing event. To validate the identification of competent cells, we demonstrated the coincident expression of comEA, a late competence gene, and comK-gfp. Competent cells resemble stationary phase cells; the majority are single (not in chains), contain single nucleoids, and rarely contain FtsZ rings. Upon dilution into fresh medium, competent cells maintain this appearance for about 2 h. In contrast, the majority of non-competent cells rapidly resume growth, exhibiting chaining, nuclear division and FtsZ-ring formation. The late competence protein ComGA is required for the competence-related block in chromosome replication and cell division. In the competent cells of a comGA mutant culture, chromosomal replication and FtsZ-ring formation were no longer blocked, although competent comGA mutant cells were abnormal in appearance. It is likely that one role for ComGA is to prevent growth, chromosome replication and cell division until ComK can be eliminated by degradation. A mutation in the ATP-binding site of comGA inactivated the protein for transformation but did not prevent it from inhibiting DNA replication and cell division. The buoyant density difference between competent and non-competent cells depends on the competence-specific growth arrest.

Bacillus subtilis↗
PubMedDate not supplied

p53 and DNA polymerase alpha compete for binding to SV40 T antigen.

The large T antigen (T) of simian virus 40 is a multifunctional protein required for both viral DNA replication and cellular transformation. T antigen forms specific protein complexes with the host protein p53 in both virus-infected and transformed cells. p53 has recently been shown to be an oncogene, but its normal function is not clear. We previously established a radioimmunoassay to study the newly described complex between T antigen and DNA polymerase alpha, and have noted a similarity between the antigenic changes induced in T by the binding of both p53 and polymerase. We now extend this analysis to a larger collection of anti-T antibodies and formally establish that p53 and DNA polymerase alpha can compete for binding to the SV40 T antigen. At a critical concentration of the three components it is possible to detect a trimeric complex of T, p53 and DNA polymerase alpha. Our observations have important implications for the control by these nuclear oncogenes of viral and cellular DNA synthesis and viral host range in both normal and transformed cells. We present a model for the action of p53 in growth control.

Animals↗
PubMed1969

Mechanism of transfection with deoxyribonucleic acid from the temperate Bacillus bacteriophage phi-105.

Bacteriophage phi105 is a temperate phage for the transformable Bacillus subtilis 168. The infectivity of deoxyribonucleic acid (DNA) extracted from mature phi105 phage particles, from bacteria lysogenic for phi105 (prophage DNA), and from induced lysogenic bacteria (vegetative DNA) was examined in the B. subtilis transformation system. About one infectious center was formed per 10(8) mature DNA molecules added to competent cells, but single markers could be rescued from mature DNA by a superinfecting phage at a 10(3)- to 10(4)-fold higher frequency. Single markers in mature DNA were inactivated at an exponential rate after uptake by a competent cell. Prophage and vegetative DNA gave about one infectious center per 10(3) molecules added to competent cells. Infectious prophage DNA entered competent cells as a single molecule; it gave a majority of lytic responses. Single markers in sheared prophage DNA were inactivated at the same rate as markers in mature DNA. Prophage DNA was dependent on the bacterial rec-1 function for its infectivity, whereas vegetative DNA was not. The mechanism of transfection of B. subtilis with viral DNA is discussed, and a model for transfection with phi105 DNA is proposed.

Bacillus subtilis↗
PubMed2004

Method for introducing specific and unmarked mutations into the chromosome of Streptococcus pneumoniae.

This work describes a procedure for the generation of site-specific mutations into the chromosome of Streptococcus pneumoniae that does not involve the use of an antibiotic resistance marker. A linear fragment of transforming deoxyribonucleic acid (DNA) is constructed by polymerase chain reaction (PCR) (gene splicing by overlap extension) and used to transform competent cells of S. pneumoniae. Selection of transformants is performed by PCR, and typically, 1% of the transformed cells show the expected mutation. By this protocol it is possible to change a single base pair into the pneumococcal genome, as well as obtaining in-frame deletions and insertions.

Cell Division↗
PubMed2000

Trefoil factor expression in normal and diseased human salivary glands.

Trefoil factors are wound-healing peptides important in protection and healing of the human gastrointestinal tract. Their potential for therapy of gastrointestinal ulcers has been established. This study investigated the hypothesis that trefoil factors are also present in human salivary gland. Tissues from surgical biopsy specimens were collected fresh into ice and stored in liquid nitrogen. Breast, stomach, and colon constituted positive controls. Trefoil factor mRNAs were detected by reverse transcription polymerase chain reaction (RT-PCR) or by in situ hybridization (ISH) with formalin-fixed, paraffin-embedded sections. Amplified DNA fragments were ligated into pGEM-T Easy vector and used to transform competent Escherichia coli JM109, allowing sequencing to confirm identity of cloned fragments. Generation of amplifiable cDNA was confirmed using primers specific to the ubiquitously expressed abl gene. By RT-PCR, TFF1 (pS2) mRNA was detected in 14 of 15 glands, TFF3 (hITF) mRNA in 13, and TFF2 (hSP) in only 1 gland. ISH of 15 glands (7 of which had been studied by RT-PCR) showed the same pattern of expression and indicated that TFF1 mRNA was usually expressed at low levels by a few mucous cells, whereas TFF3 was produced abundantly by most mucous cells. There was no difference in patterns of expression comparing parotid, submandibular, and minor mucous glands. Nor was there an obvious relationship between trefoil factor expression and pathology, but those glands not expressing TFF1 or TFF3 had evidence of chronic inflammation or atrophy. Trefoil factors are likely to be important in healing, predisposition to, and therapy of, oral diseases.

Adult↗
PubMed1990

High efficiency introduction of plasmid DNA into glycine treated Enterococcus faecalis by electroporation.

A highly efficient electroporation system for Enterococcus faecalis was developed by systematically optimizing different parameters. One parameter found to be particularly critical for electroporation was cultivation of E. faecalis in medium containing a high glycine concentration, prior to electroporation. Osmotic stabilization of cells with 0.5 M sucrose was also found to be critical during glycine treatment. 10(6) transformants per microgram of plasmid DNA were consistently obtained within 48 h. Electrocompetent preparations of E.-faecalis could be stored at - 70 degrees C without loss of competence.

DNA, Bacterial↗
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↗