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PubMed2005

Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation.

Human artificial chromosomes (HACs) provide a unique opportunity to study kinetochore formation and to develop a new generation of vectors with potential in gene therapy. An investigation into the structural and the functional relationship in centromeric tandem repeats in HACs requires the ability to manipulate repeat substructure efficiently. We describe here a new method to rapidly amplify human alphoid tandem repeats of a few hundred base pairs into long DNA arrays up to 120 kb. The method includes rolling-circle amplification (RCA) of repeats in vitro and assembly of the RCA products by in vivo recombination in yeast. The synthetic arrays are competent in HAC formation when transformed into human cells. As short multimers can be easily modified before amplification, this new technique can identify repeat monomer regions critical for kinetochore seeding. The method may have more general application in elucidating the role of other tandem repeats in chromosome organization and dynamics.

Cell Line, Tumor↗
PubMed1974

Electron microscope and autoradiographic study of ultrastructural aspects of competence and deoxyribonucleic acid absorption in Bacillus subtilis: ultrastructure of competent and noncompetent cells and cellular changes during development of competence.

By means of electron microscope autoradiography of component cultures of Bacillus subtilis exposed to [(3)H]thymidine-labeled transforming deoxyribonucleic acid competent and noncompetent cells can be distinguished. Competence is not limited to a specific phase of the cell division cycle. With serial section electron microscopy of competent and noncompetent cells, two types of mesosomal structures are observed: mesosomes connected to the plasma membrane only (plasma membrane mesosomes) and mesosomes which are additionally connected to the nuclear bodies (nuclear mesosomes). The two types show different cellular distributions. Especially the number of nuclear mesosomes is higher in competent than in noncompetent cells. This, and the observation that the increase and decrease of competence is correlated with both the number of cells carrying nuclear mesosomes and the number of nuclear mesosomes per cell, suggests that mesosomes are involved in the acquisition of competence.

Absorption↗
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↗
PubMed1990

Transforming growth factor-beta activities in 'in vivo' lines of hormone-dependent and independent mammary adenocarcinomas induced by medroxyprogesterone acetate in BALB/c mice.

We have determined the presence of transforming growth factor-beta (TGF-beta)-like polypeptides in mammary adenocarcinomas induced by medroxyprogesterone acetate (MPA) in BALB/c mice. In hormone-dependent tumors (HD) from nontreated and MPA-treated mice a high molecular weight (43 kDa) transforming activity was purified by Bio-Gel P-60 chromatography. This TGF was able to confer the neoplastic phenotype on NRK-49F cells without the addition of epidermal growth factor (EGF), though its activity was potentiated by EGF. It did not compete for binding to the EGF receptor, had no mitogenic activity on monolayer cultures of NRK fibroblasts, and was a potent inhibitor of DNA synthesis induced in these cells by EGF and insulin. In HD and hormone-independent tumors (HI) another TGF with a Mr of 13 kDa was isolated. This transforming activity showed the same biological properties as 43 kDa TGF, with the exception that in the absence of EGF it did not stimulate soft agar growth of NRK-49F cells. The synthesis of both factors in 'in vivo' HD tumors seems to be under MPA control, since it is much lower in HD tumors from MPA-treated mice. Further purification of the 13 and 43 kDa TGFs by hydrophobic interaction HPLC demonstrated that each one eluted in a different position, and that their elution profile differed from the TGF-beta from human platelets. The biological activity of the 13 and 43 kDa TGFs was not neutralized by a specific anti-TGF-beta antibody.

Adenocarcinoma↗
PubMed1982

Non-immortalizing P3J-HR-1 Epstein-Barr virus: a deletion mutant of its transforming parent, Jijoye.

The P3J-HR-1 strain of Epstein-Barr virus (EBV) fails to immortalize human lymphocytes. We wished to understand the nature of the genomic alterations which correlated with the loss of this ability. As a first step, the heterogeneity of DNA molecules in the P3J-HR-1 line was eliminated by cell cloning. Then a physical map was prepared of virion DNA from one cell clone, designated FF452-3. By comparison with the genomes of two EBVs, B95-8 and FF41, which are competent to immortalize lymphocytes, we identified a total of eight modifications of BamHI and EcoRI restriction endonuclease fragments of EBV (FF452-3) DNA consisting of insertions, deletions, or loss of a restriction endonuclease recognition site. To determine which of these alterations might be responsible for the loss of transforming phenotype, we examined homologous DNA fragments of the Jijoye strain of EBV, the progenitor of the HR-1 strain which still retains the ability to immortalize lymphocytes. We also studied viral DNA in lymphocytes transformed in vitro by Jijoye virus. Six of the eight alterations were found both in Jijoye and in clonal HR-1 DNA and were presumably genomic traits characteristic of this lineage of EBV. A small deletion in the BamHI-K fragment of HR-1 DNA was not found in Jijoye virion DNA, but this deletion was present in intracellular Jijoye DNA. Thus only one major genomic lesion in HR-1 DNA, a deletion of at least 2.4 x 10(6) molecular weight of DNA from a fused BamHI-H-Y fragment, consistently distinguished Jijoye DNA from its non-immortalizing P3J-HR-1 derivative. This deletion is likely to affect EBV genes which are directly or indirectly involved in immortalizing lymphocytes.

Base Sequence↗
PubMed1973

Chromatographically fractionated complementary strands of Bacillus subtilis deoxyribonucleic acid: transformation of hybrids.

The annealing properties as measured by the restoration of transforming activity and hypochromicity of methylated albumin-kieselguhr (MAK)-fractionated complementary strands of Bacillus subtilis deoxyribonucleic acid (DNA) are presented. Temperature-absorbance measurements performed on annealed mixtures of various L and H strand fractions indicated the existence of a complementarity gradient between the two MAK peaks. The markers purA16, leu-8, metB(5), thr-5, and the linked marker hisB(2)-try-2 exhibited different bimodal distributions on MAK columns. The transforming efficiency of heteroduplex mixtures, prepared by cross-annealing resolved complementary strands of wild-type and recipient DNA, was compared. The transforming efficiency of the wild-type L and H strands was equal in one preparation and unequal in a second preparation. It was found that in the second strand preparation the heteroduplex DNA containing the H strand from wild type was more efficient for all of the markers tested. The variations in transforming efficiencies of the complementary strands in heteroduplex molecules reported here and by others are due in part to strands of unequal length and probably to the self-annealing property of the H strands. At present, no conclusion could be made regarding the existence of strand selection bias during integration of donor DNA in competent B. subtilis cells.

Bacillus subtilis↗
PubMed1994

Immortalization of BALB/c mouse embryo fibroblasts alters SV40 large T-antigen interactions with the tumor suppressor p53 and results in a reduced SV40 transformation-efficiency.

In order to analyse the immortalizing and transforming potential of simian virus 40 (SV40), we compared the transformation efficiencies of SV40 in primary and in established BALB/c mouse fibroblasts. Five independently isolated clones of freshly immortalized normal fibroblasts (FTE cells) were established from precrisis BALB/c mouse embryo fibroblasts (pMEF cells) according to the protocol for establishing 3T3 cells (Todaro & Green, 1963). These cells expressed a wild-type p53 and were indistinguishable in all parameters analysed from original 3T3 cells kept in our laboratory. Using abortive infection to control gene dosage, followed by selection of transformed cells by cloning in soft agar, SV40 was able to transform primary cells with a much higher efficiency than 3T3 or FTE cells. Analysis of this unexpected result revealed that the different transformation efficiencies of SV40 in primary and established cells correlated with an altered cellular response to SV40 infection regarding metabolic stabilization of p53 complexed to large T during abortive infection. Whereas p53 in pMEF cells became stabilized upon abortive infection with SV40, p53 in 3T3 and FTE cells remained unstable. Our results strongly favour the hypothesis that metabolic stabilization and the ensuing higher levels of p53 in abortively infected cells enhance the transforming competence of large T.

Animals↗
PubMed1991

Immunofluorescent detection of bovine papillomavirus E4 antigen in the cytoplasm of cells permissive in vitro for viral DNA amplification.

The E4 gene of several human papillomavirus types is expressed in association with vegetative viral DNA synthesis in differentiated epidermal cells. To develop reagents to study expression of the bovine papillomavirus type 1 (BPV-1) E4 gene in warts and in virus-transformed cell lines, rabbit polyclonal antiserum was raised to the BPV-1 E4 antigen produced as a fusion polypeptide in Escherichia coli. By immunoblotting analysis of productively infected bovine fibropapilloma tissue, E4-related proteins of 16K, 21K, 30K and 42K were detected. In some but not all C127 cell lines transformed by BPV-1 or by a replication-competent BPV-1 deletion mutant, cytoplasmic E4 antigen with a predominantly perinuclear localization was detected by immunofluorescence analysis in a subpopulation of cells in stationary-phase cultures. The E4-expressing cells were identified by their grossly enlarged size to represent the same cell subpopulation shown earlier to support BPV-1 DNA amplification. The observation of synthesis of the E4 protein in association with viral DNA amplification in this system provides further evidence that there is a switch in viral early region gene expression in a subpopulation of division-arrested cells, which may accurately reflect events occurring during the vegetative phase of BPV-1 replication in terminally differentiated cells in vivo.

Animals↗
PubMed1991

Toxicity, mutagenicity, and mutational spectra of N-ethyl-N-nitrosourea in human cell lines with different DNA repair phenotypes.

We examined the toxicity, mutagenicity, and mutational spectra of N-ethyl-N-nitrosourea (ENU) in three Epstein-Barr virus-transformed human lymphoblastoid cell lines, each with a different DNA repair phenotype. One cell line lacks O6-alkylguanine-DNA alkyltransferase (AGT) activity; another, derived from a patient with xeroderma pigmentosum, complementation group A, lacks nucleotide exicision repair (NER) capability, and the third is competent in both repair functions. ENU-induced toxicity and mutagenicity at the hypoxanthine-guanine phosphoribosyltransferase locus were increased to a similar degree relative to the repair-competent cells in both AGT-deficient and NER-deficient cells. We determined the mutational spectra for ENU by identifying DNA sequence changes at the hypoxanthine-guanine phosphoribosyltransferase locus in at least 26 clones resistant to 6-thioguanine from each cell line. Of the characterized mutations, 89% were single-base pair substitutions. Transitions and transversions were found at AT and GC base pairs in all three cell lines. The biggest difference within the spectra was in the rate of transitions at GC base pairs. Compared to the repair-competent cell line, this mutation was elevated about 8-fold in the AGT-deficient cells and about 3-fold in the NER-deficient cells. We conclude that both AGT and NER play an important role in protecting human cells from the toxic and mutagenic effects of ENU. Furthermore, the mutational spectra suggest that both of these repair systems participate in the repair of O6-ethylguanine adducts.

Adolescent↗
PubMed1997

Interaction of human papillomavirus type 16 and adeno-associated virus type 2 co-infecting human cervical epithelium.

Recently, we hypothesized that the tumour-suppressive, human helper-virus-dependent, adeno-associated parvoviruses (AAV) may interfere with transforming functions of human papillomaviruses (HPV) in the development of cervical carcinoma. Here, we demonstrate that in cervical epithelium containing papillomavirus DNA, AAV DNA can be detected in a replication-competent form and that AAV proteins are expressed. In cultured cells containing integrated AAV-2 DNA, transfection of HPV-16 DNA induced rescue of infectious AAV-2, revealing helper functions of HPV-16. Similarly, cotransfection of HPV-16 and AAV-2 DNAs into human epithelial cell lines led to replication of AAV-2, and, in keratinocytes, to a cytopathic effect. These data suggest an interaction of the two viruses, possibly influencing the development of HPV-related lesions.

Animals↗
PubMed1993

Bacillus subtilis mutants defective in bacteriophage phi 29 head assembly.

Virus assembly mutants of asporogenous Bacillus subtilis defective in bacteriophage phi 29 head assembly were detected by the use of antibodies that reacted strongly with the free dodecameric phi 29 portal vertex composed of gene product 10 (gp10) but weakly with the portal vertex assembled into proheads or phage. Phage adsorption and the synthesis of phage proteins, DNA-gene product 3, and prohead RNA were normal in these mutants, but prohead and phage production was greatly reduced. The assembly defect was transferred to competent B. subtilis by transformation and transduction. PBS1 transduction showed that the vam locus was linked to Tn917 located at 317 degrees on the B. subtilis chromosome.

Bacillus Phages↗
PubMed1987

Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis.

Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding.

Animals↗
PubMed2004

[Expression of N domain of chromogranin A in Bacillus subtilis and its antifungal activity].

Chromogranin A (CGA) is a soluble protein existed in most secreted cells and neurons. It was recently found that the bovine CGA N terminal region has vasoinhibitory, antibacterial and antifungal activities. Since the need for effective antifungal agents increases in parallel with the expanding number of immunocompromised patients at risk for fungal infections, it becomes imperative to find antifungal compounds with low toxicity toward mammalian cells. To study the antifungal activity of CGA N terminal region, the DNA fragment encoding for the N terminal 1-76 amino acid sequence (CGA1-76) of human CGA was amplified by PCR technique. After DNA sequence analysis, the amplified DNA fragment was cloned into the Bacillus subtilis inducible and expression vector pSBPTQ constructed in this study and the resultant plasmid pSVTQ was then transformed into triple-protease deficient Bacillus subtilis strain DB403 competent cells. The transformants was screened on LB plates containing 10 microg/mL kanamycin. The positive transformant DB403 (pSVTQ) was grown on kanmycin containing 2 x MSR medium and sucrose was added to 2% final concentration for induction after 2h cultivation. The culture supernatant was used to run SDS-PAGE. The result of SDS-PAGE showed that the CGA1-76 was expressed by sucrose induction and the expressed product secreted into the medium with a yield of 5 mg/L. The expressed product reacts specifically with mouse anti CGA47-68 monoclonal antibody. The antifungal activity of the expressed product was examined by adding the culture supernatant to the fungal spore or Candida albican suspensions at appropriate proportion and found that the recombinant human CGA1-76 produced in Bacillus subtilis inhibits the growth of Fusarium sp. Alternaria sp. and Candida albican at the concerntration of 4 micromol/L. These results demonstrate that human CGA1-76 has expressed in Bacillus subtilis and the expressed product is immunogenic and has the antifungal activity.

Amino Acid Sequence↗
PubMed1990

Differential repair and replication of damaged DNA in ribosomal RNA genes in different CHO cell lines.

We studied the repair of psoralen adducts in the pol I-transcribed ribosomal RNA (rRNA) genes of excision repair competent Chinese hamster ovary (CHO) cell lines, their UV sensitive mutant derivatives, and their UV resistant transformants, which express a human excision repair gene. In the parental cell line CHO-AA8, both monoadducts and interstrand crosslinks are removed efficiently from the rRNA genes, whereas neither adduct is removed in the UV sensitive derivative UV5; removal of both adducts is restored in the UV resistant transformant CHO-5T4 carrying the human excision repair gene ERCC-2. In contrast, removal of psoralen adducts from the rRNA genes is not detected in another parental CHO cell line CHO-9, neither in its UV sensitive derivative 43-3B, nor in its UV resistant transformant 83-G5 carrying the human excision repair gene ERCC-1. In contrast to such intergenomic heterogeneity of repair, persistence of psoralen monoadducts during replication of the rRNA genes occurs equally well in all CHO cell lines tested. From these data, we conclude that: 1) the repair efficiency of DNA damage in the rRNA genes varies between established parental CHO cell lines; 2) the repair pathways of intrastrand adducts and interstrand crosslinks in mammalian cells share, at least, one gene product, i.e., the excision repair gene ERCC-2; 3) replicational bypass of psoralen monoadducts at the CHO rRNA locus occurs similarly on both DNA strands.

Animals↗
PubMed2005

Two distinct functions of ComW in stabilization and activation of the alternative sigma factor ComX in Streptococcus pneumoniae.

Natural genetic transformation in Streptococcus pneumoniae is controlled by a quorum-sensing system, which acts through the competence-stimulating peptide (CSP) for transient activation of genes required for competence. More than 100 genes have been identified as CSP regulated by use of DNA microarray analysis. One of the CSP-induced genes required for genetic competence is comW. As the expression of this gene depended on the regulator ComE, but not on the competence sigma factor ComX (sigma(X)), and as expression of several genes required for DNA processing was affected in a comW mutant, comW appears to be a new regulatory gene. Immunoblotting analysis showed that the amount of the sigma(X) protein is dependent on ComW, suggesting that ComW may be directly or indirectly involved in the accumulation of sigma(X). As sigma(X) is stabilized in clpP mutants, a comW mutation was introduced into the clpP background to ask whether the synthesis of sigma(X) depends on ComW. The clpP comW double mutant accumulated an amount of sigma(X) higher (threefold) than that seen in the wild type but was not transformable, suggesting that while comW is not needed for sigma(X) synthesis, it acts both in stabilization of sigma(X) and in its activation. Modification of ComW with a histidine tag at its C or N terminus revealed that both amino and carboxyl termini are important for increasing the stability of sigma(X), but only the N terminus is important for stimulating its activity.

Artificial Gene Fusion↗