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PubMed1977

Factors affecting genetic transformation of Neisseria gonorrhoeae.

Piliated gonococci were competent in genetic transformation in all stages of growth in minimal and enriched media, but nonpiliated cells were almost totally incompetent. Uptake of deoxyribonucleic acid into a deoxyribonuclease-insensitive state was observed only in competent piliated cells. Competence was not affected by washing of competent cells or treatment of competent cells with proteolytic enzymes. Expression of competence required presence of any of several different monovalent or divalent cations, as well as a utilizable source of energy. Efforts to produce genotypically or phenotypically competent derivatives of nonpiliated cells were unsuccessful. These experiments are consistent with the idea that pili may play a role in the irreversible uptake of transforming deoxyribonucleic acid by the gonococcus, but fail to provide evidence for other types of competence factors.

Cations, Divalent↗
PubMed1979

Single-stranded regions in Streptococcus pneumoniae chromosomal deoxyribonucleic acid and their relation to transformation.

Deoxyribonucleic acid (DNA) in lysates of both completent and noncompetent streptococcus pneumoniae cells was characterized by chromatography on benzoylated, naphthoylated diethylaminoethyl-cellulose columns, by sensitivity to Aspergillus oryzae S1 endonuclease, and by sucrose gradient analysis. The DNAs from both competent and noncompetent cells were found to contain similar extents of single-stranded regions. These single-stranded regions appeared to be intact, unpaired regions in double-stranded DNA rather than gaps, nicks, or unpaired ends in the DNA. Inhibition of cells with rifampin prior to lysis increased the amount of such single strandedness in the DNA. Lysates made at various times after [14C]thymidine-labeled cells had bound [3H]thymidine-labeled transforming DNA were also characterized by benzoylated, naphthoylated diethylaminoethyl-cellulose chromatography. Changes in the elution profiles of DNA from cells exposed to homospecific (S. pneumoniae) donor DNA were indicative of the formation of complexes between donor DNA and the single-stranded regions of recipient DNA. In contrast, profiles of DNA from cells exposed to heterospecific (S. sanguis) DNA did not show significant changes, indicating that few such donor-recipient complexes were formed during heterospecific transformation.

Aminoglycosides↗
PubMed2005

Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA.

AIM: To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity. METHODS: Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coli DH5alpha, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using Lipofectamine2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot. RESULTS: The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot. CONCLUSION: The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.

Adhesins, Bacterial↗
PubMed1988

Oligonucleotide site-directed mutagenesis in Xenopus egg extracts.

Addition of M13mp18 single-stranded DNA annealed with an oligonucleotide to a Xenopus egg extract results in a rapid and efficient incorporation of the oligonucleotide in a complete double-stranded supercoiled molecule. Both the efficiency of DNA synthesis and the recovery of complete double-stranded molecules are increased relative to the reaction carried out by the classical technique using the E. coli Klenow DNA polymerase, DNA ligase, dNTPs, ATP and ions. Site specific mutagenesis was assayed by reverting a point mutation in the lacz region of M13mp18. The color assay described by Messing and sequencing of the DNA extracted from isolated plaques was used to check for the reversion. A 2 hr incubation of the heteroduplex carrying the mutagenic oligonucleotide in the Klenow-ligase-dNTP mixture allows a recovery of 6% mutant phage after transformation of competent cells with the reaction products. Using the Xenopus egg extract, 83% mutant phage were recovered after the same incubation time, in reactions entirely performed in parallel. The Xenopus extract is stable and contains all components required for the assay, including all ionic and protein factors; thus the only addition is the annealed DNA. Such an eukaryotic system is therefore an attractive alternative to the reconstituted prokaryotic DNA polymerase-DNA ligase system for site specific mutagenesis.

Animals↗
PubMed1970

Cellular metabolism in genetic transformation of pneumococci: requirement for protein synthesis during induction of competence.

Metabolic inhibitors have differential effects on various phases of genetic transformation in pneumococci. Evidence is presented suggesting that, in addition to the competence factor, another specific protein or class of proteins is essential for the development of cellular "competence." The precise role of this protein(s) in genetic transformation is not known, but it seems essential for some function subsequent to the interaction of competence factor and cells.

Amino Acids↗
PubMed1982

Action of restriction endonucleases on transforming DNA of Haemophilus influenzae.

Cleavage of DNA from Haemophilus influenzae with restriction endonucleases caused inactivation of transforming ability to an extent that depended on the genetic marker and the enzyme. The rate of inactivation, but not the final level of survival, depended on the concentration of enzyme in the restriction digest. In general, the greatest extent of inactivation of transforming activity was obtained with endonucleases that are known to produce the shortest fragments. We electrophoresed restriction digests of H. influenzae DNA in agarose gels and assayed transforming activity of DNA extracted from gel slices. In this way, we determined the lengths of restriction fragments that contain genetic markers of H. influenzae. For the marker that we studied most thoroughly (nov), the shortest restriction fragment that possessed detectable transforming activity was a 0.9-kilobase pair fragment produced by endonuclease R . PstI. The shortest marker-bearing restriction fragment that retained substantial transforming activity (50% of value for undigested DNA) was a 2.1-kilobase pair EcoRI fragment bearing the kan marker. Among marker-bearing restriction fragments 1 to 4 kilobase pairs in length, survival of transforming activity varied 10,000-fold. We relate these observations to the recent findings by Sisco and Smith (Proc. Natl. Acad. Sci. U.S.A. 76:972-976, 1979) that efficient entry of DNA into competent H. influenzae cells appears to require the presence of a recognition sequence that is scattered throughout the Haemophilus genome in many more copies than in unrelated genomes.

Base Sequence↗
PubMed1978

Genetic transformation of pilation and virulence into Neisseria gonorrhoeae T4.

Genetic transformation of nonpilated strains of Neisserai gonorrhoeae to pilated forms is described. The transformants displayed phenotypic T1 and T2 colonial morphology on agar and possessed pili visualized by electron microscopy. When T1 or T2 transformant cells were injected into 11-day-old chicken embryos, they exhibited virulence characteristics only slightly less than the parental donor strains, though the parental recipient strains were avirulent. Competence was maximal in the late log phase of growth, and the frequency of transformation of clonal T4s to pilation and virulence approached 2%. DNA extracted from transformants could be used to transform other T4 cells. In the course of this work, a shift to a novel colonial type, designated T2-T3 wrinkled, was observed as a consequence of growth of T4 in presence of enzymatic digests of either DNA or RNA, nucleases or individual deoxy- or ribonucleosides. In sharp distinction to the parental T4, these novel organisms were very pilated; however, they were only minimally virulent. Various nucleic acid analogs could neither induce nor inhibit this population shift. Additionally, DNA extracted from this T2-T3 wrinkled variant could be used to transform genetically both T1 and T4 gonococci to the new morphology.

Conjugation, Genetic↗
PubMed2006

Intracytoplasmic maturation of the human immunodeficiency virus type 1 reverse transcription complexes determines their capacity to integrate into chromatin.

BACKGROUND: The early events of the HIV-1 life cycle include entry of the viral core into target cell, assembly of the reverse transcription complex (RTCs) performing reverse transcription, its transformation into integration-competent complexes called pre-integration complexes (PICs), trafficking of complexes into the nucleus, and finally integration of the viral DNA into chromatin. Molecular details and temporal organization of these processes remain among the least investigated and most controversial problems in the biology of HIV. RESULTS: To quantitatively evaluate maturation and nuclear translocation of the HIV-1 RTCs, nucleoprotein complexes isolated from the nucleus (nRTC) and cytoplasm (cRTC) of HeLa cells infected with MLV Env-pseudotyped HIV-1 were analyzed by real-time PCR. While most complexes completed reverse transcription in the cytoplasm, some got into the nucleus before completing DNA synthesis. The HIV-specific RNA complexes could get into the nucleus when reverse transcription was blocked by reverse transcriptase inhibitor, although nuclear import of RNA complexes was less efficient than of DNA-containing RTCs. Analysis of the RTC nuclear import in synchronized cells infected in the G2/M phase of the cell cycle showed enrichment in the nuclei of RTCs containing incomplete HIV-1 DNA compared to non-synchronized cells, where RTCs with complete reverse transcripts prevailed. Immunoprecipitation assays identified viral proteins IN, Vpr, MA, and cellular Ini1 and PML associated with both cRTCs and nRTCs, whereas CA was detected only in cRTCs and RT was diminished in nRTCs. Cytoplasmic maturation of the complexes was associated with increased immunoreactivity with anti-Vpr and anti-IN antibodies, and decreased reactivity with antibodies to RT. Both cRTCs and nRTCs carried out endogenous reverse transcription reaction in vitro. In contrast to cRTCs, in vitro completion of reverse transcription in nRTCs did not increase their integration into chromatin. CONCLUSION: These results suggest that RTC maturation occurs predominantly in the cytoplasm. Immature RTCs containing RT and incomplete DNA can translocate into the nucleus during mitosis and complete reverse transcription, but are defective for integration.

Cell Division↗
PubMed2004

[Construction of phage display antibody library to MCF-7 cells and screening of single-chain antibodies against breast cancer cells].

The aim of this study is to construct a phage display single-chain variable fragment (scFv) library against breast cancer cells and screen the specific antibodies against MCF-7 cells from the library. The BALB/C mice were immunized with MCF-7 cells. Total RNA of spleens was isolated. The heavy-chain (VH) and light-chain variable region genes (VL) of the antibodies were amplified by RT-PCR and joined into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The assembled scFv fragments were cloned into the phagemids(pCANTAB5E) and the recombinant phagemids were used to transform competent E. coli TG1. The transformed TG1 cells were infected by helper phage M13KO7 and the recombinant phagemids were rescued. The scFv fusion proteins were displayed on the surfaces of the recombinant phages. A phage display antibody library of repertoire of 1.2 x 10(6) clones was constructed. The specific antibodies against MCF-7 cells were enriched by 75 times after five rounds of affinity selection. Ten recombinant phages clones that exhibited specific binding to MCF-7 cells were identified. The specificity of those phage clones was analyzed by reactivity against HepG2 cells and Hela cells by ELISA. One of the selected phage clones against MCF-7cells was used to infect E. coli TOP10 to produce the soluble scFv antibodies after induction with IPTG. The strategy of construction and screening of antibody library directed against the whole tumor cells described in this report should be generally applicable to generate tumor cell-specific antibodies.

Animals↗
PubMed1996

Detection of potential cytokine gene(s) in an Alu-PCR library from the human chromosome 4.

Jumping and linking libraries have been constructed in order to have more information of chromosome 4. Two types of jumping libraries (Not I and Xma III jumping) and one linking library were prepared. The jumping clones represent DNA regions between two rare-cutting enzymes sites (such is Not I or Xma III) whereas linking clones represent only the area which surrounds the rare-cutting site. These clones were used as target DNA in Alu-PCR. The Alu-PCR fragments were cloned into pGEM-T vector and transformed into Escherichia coli JM 109 competent cells. The colonies were screened for inserts by restriction analysis. More than 50% of the colonies were shown to contain inserts. Inserts from each library were screened by PCR. One of the jumping libraries appeared to have inserts containing CpG islands and AT-rich sequences.

Chromosomes, Human, Pair 4↗
PubMed1985

Evidence for multiple steps in neoplastic transformation of normal and preneoplastic Syrian hamster embryo cells following transfection with Harvey murine sarcoma virus oncogene (v-Ha-ras).

Neoplastic development of Syrian hamster embryo (SHE) cells in culture is a multistep process in which intermediate or preneoplastic cells can be identified and isolated. In an attempt to further characterize normal and preneoplastic cells, we have compared their susceptibilities to neoplastic transformation following transfection with cloned DNA of the oncogenic virus, Harvey murine sarcoma virus (HaMSV). Normal SHE cells, which are stably nontumorigenic when injected in nude mice, are competent to take up and express exogenous DNA as demonstrated by transfection experiments with pSV2-neo DNA and certain viral DNAs. SHE cells treated with 5 micrograms of HaMSV DNA per dish remained nontumorigenic. Colonies of SHE cells, isolated after cotransfection with HaMSV and pSV2-neo DNA and selection for G418 antibiotic resistance, expressed Harvey murine sarcoma virus oncogene (v-Ha-ras) RNA and were initially morphologically altered; however, all colonies senesced when subcultured. In contrast, transfection of the cells with polyoma virus DNA alone or HaMSV DNA plus MC29 viral DNA (pSVv-myc) and then injection of the cells into nude mice resulted in progressively growing tumors of hamster origin within 3 to 5 weeks. A preneoplastic cell line, DES-4, isolated after treatment of SHE cells with the human carcinogen diethylstilbestrol, was chosen for comparative analyses. These immortalized cells are nontumorigenic and excellent recipients for exogenous DNA. In contrast to SHE cells, DES-4 cells were highly susceptible to neoplastic transformation following transfection with HaMSV DNA. To further investigate the role of HaMSV DNA in the neoplastic transformation of DES-4 cells and to determine whether this occurred as a single step, clones of DES-4 cells cotransfected with pSV2-neo and HaMSV DNAs were selected by antibiotic resistance and characterized. There was a good correlation between tumorigenicity and expression of v-Ha-ras DNA; however, the clones were highly variable in terms of their latency periods in vivo and anchorage-independent growth. Neither of these two parameters correlated with the level of expression of v-Ha-ras RNA. All of the cell lines derived from tumors and reinoculated into nude mice had short latency periods in vivo, were highly anchorage independent, and had high levels of v-Ha-ras expression. These results suggest that, in these experiments, v-Ha-ras expression was necessary, but not sufficient, for the tumorigenicity of DES-4 cells and that additional changes in the cells were acquired.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Isolation and characterization of a bacteriophage factor that confers competence for genetic transformation to an exfoliative toxin-producing strain of Staphylococcus aureus.

Competence for genetic transformation in an exfoliative toxin-producing strain of Staphylococcus aureus was shown to be dependent on a virion factor that was isolated from a crude bacteriophage 80 alpha lysate. This competence-conferring factor was completely separated from infectious virus particles after either centrifugation through a neutral sucrose velocity gradient or fractionation on a Sepharose 2B gel. Since the competence-conferring factor tends to aggregate, optimal separation was obtained after treatment of the phage factor with the detergent Nonidet P-40. The competence-conferring factor had a molecular weight between 3 X 10(6) and 20 X 10(6) and an approximate sedimentation coefficient of 252. The factor was neutralized after interaction with antiserum prepared against isolated infectious 80 alpha virions. Electron microscopy of transforming cells that were exposed to isolated competence-conferring factor revealed a significant number of abnormally long and aggregated phage tail-like structures attached to the surface of recipient cells. This phenomenon was only observed in the presence of donor DNA, indicating that a phage tail-DNA-surface receptor complex might be one of the early steps in DNA-mediated transformation of S. aureus.

Bacterial Proteins↗
PubMed1997

[Amplification, clone and identification of the specific fragments of tumor metastasis-suppressor gene nm23-H1 and nm23-H2].

A series of DNA primers specific for the specific fragments of nm23-H1 and nm23-H2 were designed and synthesized. The specific fragments of nm23-H1(185 bp) and nm23-H2(145 bp) were amplified from human blood DNA by using polymerase chain reaction (PCR). The recovered PCR products were treated with Klenow fragment, inserted into pGEM-3zf(+) vector with blunt-end ligation, and then transformed into competent cell JM109. The positive colonies were directly identified by colour screening on indicator plates. The recombinant plasmids were digested by Alu I and identified by PCR. The results showed that the authors had obtained the specific fragments of nm23-H1 and nm23-H2 respectively, and these specific fragments could be used for study the expression of nm23-H1 and nm23-H2 seperately.

Cloning, Molecular↗
PubMed1974

Role of the phi 11 phage genome in competence of Staphylococcus aureus.

Both phage ø11 and 83A, when present as prophage or when used as helper phage, induce competence for transfection and transformation to the same level in Staphylococcus aureus, strain 8325-4. Cells lysogenized with certain temperature-sensitive (ts) mutants of phage ø11 show competence at the nonpermissive temperature (41 C) without production of infectious phages. Phage ø11ts allele 31 can neither as a prophage nor as a helper phage develop competence under nonpermissive conditions. This mutant appears, therefore, to be mutated in the region of the phage genome controlling competence. The competence level for both transfection and transformation is increased by superinfecting strain 8325-4 (ø11) or 8325-4 (83A) at high multiplicities with phage ø11 with some of its mutants or with phage 83A. This superinfection enhancement appears to require protein synthesis but not deoxyribonucleic acid synthesis as judged from studies with inhibitors of macromolecular synthesis. Besides the phage particle, no extracellular or cell-bound factors so far detected can induce competence. The phage-induced product conferring competence is rapidly synthesized by strain 8325-4 (tsø11(31)) after shift to permissive conditions, but requires deoxyribonucleic acid and protein synthesis to be expressed. Recombination between the sus mutants of phage ø11 of Kretschmer and Egan and tsø11(31) indicate that competence is controlled by an early gene in the lytic cycle which may be expressed also in lysogenic cells. The phage product inducing competence appears to have a half-life of 10 to 15 min in the conditional lethal mutant at shift to nonpermissive temperature. Ultraviolet inactivation of phage ø11 infectivity occurs more rapidly than inactivation of competence induction. In fact, the number of transformants is increased at low doses of irradiation. Competence induction is, however, decreased at high does of ultraviolet irradiation.

Azo Compounds↗
PubMed1999

Smad3 inhibits transforming growth factor-beta and activin signaling by competing with Smad4 for FAST-2 binding.

Transcriptional regulation by transforming growth factor-beta and activin is mediated by interaction of Smad2 and Smad3 with specific transcription factors and/or DNA elements. However, Smad3 behaves differently from Smad2 in regulating transcription by a winged-helix transcription factor, FAST-2, on an activin-responsive element (ARE) in the Xenopus Mix.2 promoter. Smad3 alone was able to stimulate the ARE through FAST-2, but inhibited the ARE transactivation mediated by Smad2/Smad4 following receptor activation. We characterized the functional domains that are involved in these two activities of Smad3. Deletion of the MH1 domain as well as mutations of four lysine residues in the MH1 domain abrogated the inhibitory activity of Smad3, but did not compromise the self-stimulatory function. In contrast, deletion of the MH2 domain or a point mutation of glycine 379 within this domain obliterated the self-stimulatory activity of Smad3, but not the inhibitory activity. In an electrophoretic mobility shift assay, we found that Smad3 was able to associate with the FAST-2.ARE complex and that this association was dependent on FAST-2. In addition, Smad3 was not able to directly bind the ARE in a DNase I protection assay, in which FAST-2 binds the ARE around a motif (TGTGTATT) previously characterized to associate with the human FAST-1 protein. Interestingly, Smad4 was also able to directly associate with the FAST-2.ARE complex through binding with FAST-2. In a gel shift assay, the association of FAST-2 with Smad4 was mutually exclusive from the association with Smad3. Taken together, these data indicate that Smad3 exerts the inhibitory activity by competitive association with FAST-2.

Activin Receptors↗
PubMed1986

Generation and characterization of a recombinant Moloney murine leukemia virus containing the v-myc oncogene of avian MC29 virus: in vitro transformation and in vivo pathogenesis.

A new retrovirus consisting of the v-myc oncogene sequences of avian MC29 virus inserted into the genome of Moloney murine leukemia virus (M-MuLV) was generated. This was accomplished by constructing a recombinant DNA clone containing the desired organization, introducing the recombinant DNA into mouse NIH 3T3 cells, and superinfecting the cells with replication-competent M-MuLV. The construction was designed so that an M-MuLV gag-myc fusion protein would be produced. The resulting virus, M-MuLV(myc), morphologically transformed uninfected NIH 3T3 cells. Stocks of M-MuLV(myc)-M-MuLV were infected into secondary mouse embryo cultures. M-MuLV(myc) induced striking growth and proliferation of hematopoietic cells. These cells were of the myeloid lineage by morphology, phagocytic properties, and surface staining with Mac-1 and Mac-2 monoclonal antibodies. They resembled mature macrophages, although they displayed minor properties of immaturity. The myeloid cells were transformed in comparison with uninfected myeloid cells since they were less adherent and had unlimited proliferative capacity and reduced growth factor requirements. The transformed myeloid cells with proliferative potential were actually myeloid progenitors which apparently underwent terminal differentiation to macrophages. It was possible to derive a permanent line of factor-independent macrophages from M-MuLV(myc)-transformed myeloid cells. M-MuLV(myc) also immortalized and morphologically transformed mouse embryo fibroblasts. These in vitro properties closely resembled the biological activity of MC29 virus in avian cells and suggested that the nature of the v-myc oncogene was an important determinant in transformation specificity. Neonatal NIH Swiss mice inoculated intraperitoneally with M-MuLV(myc)-M-MuLV only developed lymphoblastic lymphoma characteristic of the M-MuLV helper alone, and no acute fibrosarcomas or myeloid tumors resulted. In light of the strong myeloid transformation observed in vitro, the absence of acute in vivo myeloid disease was noteworthy. Interestingly, when a derivative of M-MuLV(myc) carried by a nonpathogenic amphotropic MuLV helper was inoculated, T lymphomas developed with long latency. Molecular hybridization confirmed that these tumors contained M-MuLV(myc).

Animals↗
PubMed1986

Reciprocal and non-reciprocal homologous recombination between Escherichia coli chromosomal DNA and ultraviolet light-irradiated plasmid DNA.

Plasmid DNA substrates were used to study ultraviolet (UV)-induced recombination events in Escherichia coli host cells. Plasmids derived from pBR322, containing all or part of the lac operon of E. coli, were irradiated with ultraviolet light before transformation into E. coli strains of different recA and lacY genotypes. Recombinational exchanges were identified by phenotypic changes in lactose utilization and were confirmed by restriction analysis of isolated plasmids. Ultraviolet-induced reciprocal plasmid-chromosome recombination occurred at a slightly higher frequency then non-reciprocal chromosome-to-plasmid recombination, and at a much higher frequency than non-reciprocal plasmid-to-chromosome recombination. These frequencies did not depend on segregative mechanisms. The asymmetry of non-reciprocal exchange was not due to the particular arrangement of wild-type and lacY1 alleles because the same results were observed when these were interchanged. The host recA gene was required for plasmid-chromosome recombination, and slightly enhanced plasmid survival. Evidence for plasmid replication prior to recombination was found in reciprocal recombinants, but rarely in the non-reciprocal recombinants analyzed. Irradiation of competent bacterial host cells prior to transformation did not effectively induce plasmid-chromosome recombination.

DNA, Bacterial↗