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PubMed2003

Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast.

The repair of chromosomal double-strand breaks (DSBs) can be accomplished through homologous recombination in most organisms. We report here that exogenous oligonucleotides can efficiently target for repair a single DSB induced in a chromosome of yeast. The efficiency of recombinational targeting leading to a desired DNA change can be as high as 20% of cells. The DSB was generated either by a regulatable I-SceI endonuclease just before transformation or appeared spontaneously at the site of a long inverted repeat composed of human Alu sequences. The approach used features of our previously described delitto perfetto system for selecting transformants with integrative recombinant oligonucleotides. The DSB repair mediated by pairs of complementary integrative recombinant oligonucleotides was efficient for targeting to homologous sequences that were close to or distant from the DSB and in the presence of a competing homologous chromosome in diploid cells. We also demonstrate that a DSB can strongly stimulate recombination with single-stranded DNA, without strand bias. These findings expand current models of DSB repair. In addition, we establish a high-throughput system for rapid genome-wide modification with oligonucleotides.

Binding Sites↗
PubMed1984

Mitochondrial membrane biogenesis: characterization and use of pet mutants to clone the nuclear gene coding for subunit V of yeast cytochrome c oxidase.

A nuclear pet mutant of Saccharomyces cerevisiae that is defective in the structural gene for subunit V of cytochrome c oxidase has been identified and used to clone the subunit V gene (COX5) by complementation. This mutant, E4-238 [24], and its revertant, JM110, produce variant forms of subunit V. In comparison to the wild-type polypeptide (Mr = 12,500), the polypeptides from E4-238 and JM110 have apparent molecular weights of 9,500 and 13,500, respectively. These mutations directly alter the subunit V structural gene rather than a gene required for posttranslational processing or modification of subunit V because they are cis-acting in diploid cells; that is, both parental forms of subunit V are produced in heteroallelic diploids formed from crosses between the mutant, revertant, and wild type. Several plasmids containing the COX5 gene were isolated by transformation of JM28, a derivative of E4-238, with DNA from a yeast nuclear DNA library in the vector YEp13. One plasmid, YEp13-511, with a DNA insert of 4.8 kilobases, was characterized in detail. It restores respiratory competency and cytochrome oxidase activity in JM28, encodes a new form of subunit V that is functionally assembled into mitochondria, and is capable of selecting mRNA for subunit V. The availability of mutants altered in the structural gene for subunit V (COX5) and of the COX5 gene on a plasmid, together with the demonstration that plasmid-encoded subunit V is able to assemble into a functional holocytochrome c oxidase, enables molecular genetic studies of subunit V assembly into mitochondria and holocytochrome c oxidase.

Cloning, Molecular↗
PubMed1977

Selective enrichment for genetic markers in DNA released by competent cultures of Bacillus subtilis.

Deoxyribonucleic acid is released into the growth medium by Bacillus subtilis at the time of competence. This DNA is enriched for the genetic markers which have previously been demonstrated to be elevated in membrane-DNA preparations and more recently in cell wall-DNA complexes. Furthermore, the purA16/leu-8 relative marker enrichment varies with time, reaching its highest point at the time of maximal competence. Enrichment remains elevated for at least 60 min further in the competence regimen. Thr results suggest that certain genetic markers of the B. subtilis chromosome are preferentially more available to the external medium as the development of competence proceeds.

Bacillus subtilis↗
PubMed1992

The first 124 nucleotides of the E7 coding sequences of HPV16 can render the HPV11 genome transformation competent.

The human papillomavirus (HPV) type 16 is associated with the majority of cervical tumors and is capable of oncogenically transforming cells in culture. HPV type 11 is rarely associated with malignant lesions and does not transform cells in vitro. While the E7 of HPV16 is necessary and sufficient for transformation of rodent cells, the E7 gene of HPV11 is not transforming. In the present report we demonstrate that the HPV11 genome, with the first 124 nucleotides of the HPV16 E7 open reading frame fused to the last 198 nucleotides of the HPV11 E7 open reading frame, becomes transformation competent.

Amino Acid Sequence↗
PubMed1995

Induction of apoptosis in HeLa cells by trans-activation-deficient p53.

The p53 tumor suppressor protein is a transcriptional activator, which can mediate apoptotic cell death in a variety of cell types. To determine whether sequence-specific trans-activation is a prerequisite for the induction of apoptosis by p53, the apoptotic effects of various p53 deletion mutants were monitored in an assay based on the transient transfection of HeLa cells. A truncated protein (p53dl214), containing only the first 214 amino-terminal residues of murine p53, induced extensive apoptosis, albeit at a slower rate than trans-activation-competent wild-type p53. p53dl214 also suppressed the transformation of rat fibroblasts by several oncogene combinations and particularly by myc plus ras and HPV E7 plus ras. p53dl214 lacks a major portion of the DNA-binding domain and cannot activate p53-responsive promoters. Moreover, a human p53 protein carrying mutations in residues 22 and 23 also triggered HeLa cell apoptosis, despite failing to induce significant activation of relevant p53 target promoters. These data suggest the existence of two p53-dependent apoptotic pathways--one requiring activation of specific target genes, and the other independent of sequence-specific trans-activation. The latter pathway may actually be totally uncoupled from the binding of p53 to its consensus DNA sites. The relative contribution of trans-activation-independent apoptosis to tumor suppression by p53 may be dictated by the specific genetic lesions present in the particular tumor.

Animals↗
PubMed2004

Evidence for the active role of a novel nuclease from Helicobacter pylori in the horizontal transfer of genetic information.

Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach, causes gastritis, and is associated with ulcers and gastric cancer. H. pylori is naturally competent for transformation. Natural genetic transformation is believed to be essential for the genetic plasticity observed in this species. While the relevance of horizontal gene transfer in H. pylori adaptiveness and antibiotic resistance is well documented, the DNA transformation machinery components are barely known. No enzymatic activity associated with the transformation process has been determined experimentally and described. We isolated, microsequenced, and cloned a major DNA nuclease from H. pylori. This protein, encoded by the open reading frame hp0323, was expressed in Escherichia coli. The purified protein, NucT, has a cation-independent thermostable nuclease activity that preferentially cleaves single-stranded DNA. NucT is associated with the membrane. NucT-deficient H. pylori strains are one or more orders of magnitude less efficient than the parental strain for transformation with either chromosomal or self-replicating plasmid DNA. To the best of our knowledge, NucT is the first nuclease identified in a gram-negative natural transformation system, and its existence suggests that there is a mechanism of DNA processing and uptake similar to the mechanisms in well-studied gram-positive systems.

Amino Acid Sequence↗
PubMed1968

Competence and deoxyribonucleic acid uptake in Bacillus subtilis.

The distribution of uninucleate and multinucleate cells of Bacillus subtilis, fractionated by zonal centrifugation, shows that the uninucleate cells are most likely to be competent. Only the competent fraction of the population incorporates deoxyribonucleic acid.

Bacillus subtilis↗
PubMed2002

Helicobacter pylori interstrain restriction-modification diversity prevents genome subversion by chromosomal DNA from competing strains.

Helicobacter pylori, bacteria that colonize the human gastric mucosa, possess a large number of genes for restriction-modification (R-M) systems, and essentially, every strain possesses a unique complement of functional and partial R-M systems. Nearly half of the H.pylori strains studied possess an active type IIs R-M system, HpyII, with the recognition sequence GAAGA. Recombination between direct repeats that flank the R-M cassette allows for its deletion whereas strains lacking hpyIIRM can acquire this cassette through natural transformation. We asked whether strains lacking HpyII R-M activity can acquire an active hpyIIRM cassette [containing a 1.4 kb kanamycin resistance (aphA) marker], whether such acquisition is DNase sensitive or resistant and whether restriction barriers limit acquisition of chromosomal DNA. Our results indicate that natural transformation and conjugation-like mechanisms may contribute to the transfer of large (4.8 kb) insertions of chromosomal DNA between H.pylori strains, that inactive or partial R-M systems can be reactivated upon recombination with a functional allele, consistent with their being contingency genes, and that H.pylori R-M diversity limits acquisition of chromosomal DNA fragments of >/=1 kb.

DNA Methylation↗
PubMed2000

The molecular chaperone activity of simian virus 40 large T antigen is required to disrupt Rb-E2F family complexes by an ATP-dependent mechanism.

The simian virus 40 large T antigen (T antigen) inactivates tumor suppressor proteins and therefore has been used in numerous studies to probe the mechanisms that control cellular growth and to generate immortalized cell lines. Binding of T antigen to the Rb family of growth-regulatory proteins is necessary but not sufficient to cause transformation. The molecular mechanism underlying T-antigen inactivation of Rb function is poorly understood. In this study we show that T antigen associates with pRb and p130-E2F complexes in a stable manner. T antigen dissociates from a p130-E2F-4-DP-1 complex, coincident with the release of p130 from E2F-4-DP-1. The dissociation of this complex requires Hsc70, ATP, and a functional T-antigen J domain. We also report that the "released" E2F-DP-1 complex is competent to bind DNA containing an E2F consensus binding site. We propose that T antigen disrupts Rb-E2F family complexes through the action of its J domain and Hsc70. These findings indicate how Hsc70 supports T-antigen action and help to explain the cis requirement for a J domain and Rb binding motif in T-antigen-induced transformation. Furthermore, this is the first demonstration linking Hsc70 ATP hydrolysis to the release of E2F bound by Rb family members.

Adenosine Triphosphate↗
PubMed2006

Nonhomologous chromosomal integration of foreign DNA is completely dependent on MUS-53 (human Lig4 homolog) in Neurospora.

Homologous integration of a foreign DNA segment into a chromosomal target sequence enables precise disruption or replacement of genes of interest and provides an effective means to analyze gene function. However, integration after transformation is predominantly nonhomologous in most species other than yeast. Here, we show that homologous integration in the filamentous fungus Neurospora requires the homologous-recombination proteins MEI-3 (yeast Rad51 homolog) and MUS-25 (yeast Rad54 homolog), whereas nonhomologous integration requires nonhomologous end-joining protein MUS-52 (yeast Ku80 homolog). Two additional minor integration pathways are present, one MEI-3-independent and homologous, the other MUS-52-independent and nonhomologous. Homologous and nonhomologous mechanisms compete when external DNA is integrated. In Neurospora, both nonhomologous integration pathways, MUS-52-dependent and MUS-52-independent, require MUS-53 (a homolog of human Lig4), which functions in the final step of nonhomologous end-joining. Because nonhomologous integration is eliminated in a LIG4-disrupted strain, integration occurs only at the targeted site in mus-53 mutants, making them an extremely efficient and safe host for gene targeting.

Chromosomes, Fungal↗
PubMed1976

Calcium-requiring step in the uptake of deoxyribonucleic acid molecules through the surface of competent pneumococci.

The conversion of surface-adsorbed deoxyribonucleic acid (DNA) molecules to a state in which they are inaccessible to exogenous deoxyribonuclease requires specifically calcium ions; magnesium ions cannot replace calcium ions. Virtually maximal levels of nuclease-resistant DNA binding and genetic transformation can be obtained in media free from magnesium and containing only calcium ions. It is suggested that the calcium-requiring process is the transport of DNA molecules across the plasma membrane. Magnesium ions stimulate both the loss of surface-adsorbed DNA to the medium and the extracellular degradation of DNA.

Calcium↗
PubMed1991

Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii.

We have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes. Wild-type C. reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence. Antibiotic-resistant transformants were selected under conditions permissive for growth of non-photosynthetic mutants. Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence. A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated. Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene. Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques. We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation. The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.

Animals↗