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PubMed1982

Diploid state of phenotypically recombinant progeny arising after protoplast fusion in Bacillus subtilis.

After fusion of Bacillus subtilis protoplasts the phenotypically recombinant clones isolated, whether immediately or as segregants of complementing diploid clones, have in common the following properties. They appear independently of the recN+ gene, most often as the result of apparently non-reciprocal recombination occurring in genetic intervals encompassing the origin and the terminus of replication. First indicated by reciprocal fusion crosses between ø105-lysogenic and ø105-sensitive strains, the diploidy of the recombinants was confirmed by studying the transforming activities of their DNA. These experiments establish heterozygosity at eight loci scattered on the chromosome map. By revealing the presence of the trpF+ allele in trpF7 recombinants, the results also strongly suggest that stable phenotypic recombinants may arise by genetic inactivation. Two possible genetic structures for these recombinants are discussed, one implying total inactivation of one recombinant chromosome, the other a segmentary inactivation of one unrecombined chromosome. Whatever the structure, genetic stability is not a reliable sign of haploidy in bacterial clones produced after protoplast fusion.

Bacillus subtilis↗

PubMed1982

Isolation and characterization of the nucleoid of non-complementing diploids from protoplast fusion in Bacillus subtilis.

Nucleoids of non-complementing diploids (Ncd) from protoplast fusion of B. subtilis were isolated. Their purified DNA banded in neutral CsCl gradient as a single unimodal peak of buoyant density 1.711 g/cm3, a value which is similar to that of the DNA purified from the original parental strains, suggesting that methylation of bases is not a significant factor in chromosome inactivation. Nucleoids released from a Ncd clone give two peaks in a sucrose gradient with a characteristic S value for each nucleoid. That is in contrast to nucleoids from the haploid parents whose sedimental patterns show only one peak. Both nucleoid preparations from Ncd strains assayed for transformation activity show the fast sedimenting nucleoid devoid of transformation activity while the slow nucleoid was active in transformation for the alleles carried by the genome which is expressed in vivo. Both nucleoids of the Ncd strains are transcribed in vivo. The RNA associated with the inactive chromosome is synthesized by the RNA polymerase of the active one. This study provides evidence that inactivation of one parental genome in the Ncd strain may be related with the tertiary organization of its DNA.

Bacillus subtilis↗

PubMed1991

Continuous production of tissue plasminogen activator (t-PA) by human embryonic lung diploid fibroblast, IMR-90 cells, using a ceramic bed reactor.

Ceramic pieces composed of 99.5% Al2O3, 3 to 6 mm long, were found to be a good matrix for growth of the human embryonic lung diploid fibroblast, IMR-90 cells. The tissue plasminogen activator (t-PA) was secreted in DME medium containing proteose peptone as a t-PA inducer. In addition, production of t-PA was enhanced by increasing extracellular CaCl2, from 3.6 to 5.4 mM. In order to eliminate negative feed-back control caused by t-PA produced and thus raise productivity, perfusion cultivation was performed using a ceramic-packed bed column, with a recirculating vessel. The recirculating vessel was used to mix fresh medium with spent medium, and to control dissolved oxygen concentrations in the extracellular environment by stirring. In continuous production using the packed bed column with 2 kg of ceramics (phi = H = 150 mm), increasing dilution rate to 0.5 day-1 could reduce product inhibition at 3-4 x 10(5) cells/ml. Cellular productivity of 560 IU/10(6) cells/day was obtained over 40 days and corresponded to the volumetric productivity of 183 IU/ml/day.

Biotechnology↗

PubMed1987

Diploid and tetraploid precursors of megakaryocytes in normal human bone marrow detected by immunofluorescence.

A sequential preparation method is described which allows immunological identification, morphological characterization, cytophotometric determination of relative DNA content of the megakaryocyte lineage as well as quantitation of megakaryocyte precursors in human bone marrow aspirates. We compared several monoclonal (anti-GP IIIa and HD 19) and polyclonal (A225, RAHPS) antiplatelet antibodies for immunofluorescent staining. Among the identified cells, a small number of cells showing a diploid and tetraploid DNA content were found which must be regarded as promegakaryoblasts, representing 2.5-4.7% of all megakaryocytes. The heterogenous morphology of these precursors in panoptically stained smears is described.

Bone Marrow Cells↗

PubMed1989

Benzo[a]pyrene-diol-epoxide-induced anchorage-independence in diploid human fibroblasts. Analysis of cellular protooncogenes.

Treatment of diploid human fibroblasts with stereoisomeric benzo[alpha]pyrene anti and syn diol epoxides has been shown to induce anchorage-independent clones of cells with a dose dependence and frequency [(0.5-12) X 10(-4)] not significantly different from mutations at the hypoxanthine-guanine phosphoribosyltransferase locus [(1-8) X 10(-4)] in these cells. The majority of the anchorage-independent clones that were picked retained their mutagen-induced, anchorage-independent phenotype through at least 20 generations of expansion in monolayer culture. No variant cells showing extended life-span were detected among survivors in any of the mutagen treatment groups (less than 1.6 X 10(-7) frequency). Extensive analysis of a pool of 15 cellular protooncogenes (Ha-ras, Ki-ras, N-ras, mos, fos, fes, myc, abl, sis, myb, erbA, erbB, src, raf, N-myc), using Southern and northern blot analysis, was done to determine whether mutagen-induced rearrangement, amplification or overexpression of any of these genes was responsible for the mutagen-induced, anchorage-independent phenotype. We found no evidence that the genomic arrangement or expression level of any of these genes had been altered, thus indicating that an alternative form of mutation, or an alternative gene not included in this screening was responsible for the mutagen-induced, anchorage-independent phenotype.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1984

Complementation and genetic inactivation: two alternative mechanisms leading to prototrophy in diploid bacterial clones.

Evidence for diploidy at loci located all around the Bacillus subtilis chromosome previously led us to refer to the prototrophic bacterial clones produced by fusion of polyauxotrophic protoplasts as complementing diploid clones (Lévi-Meyrueis et al. 1980; Sanchez-Rivas 1982). In this paper, evidence is presented that gene inactivation may occur in such clones, as judged from the unequal expression of three unselected markers and their low transforming activity in cell lysates, an established property of inactivated genes (Bohin et al. 1982). The insensitivity to protease treatment of the lysates and also the low transforming activity observed with purified DNA may indicate that chromosome inactivation does not necessarily result from the mere attachment of proteins to DNA. Cotransfer by transformation of similarly expressed genes, initially located on separate chromosomes, suggests that genetic recombination has taken place, resulting in the reassortment of active and inactive genes on separate chromosomes. Several genetic structures compatible with the observations are presented which illustrate that prototrophy may result from such reassortment as well as from functional complementation.

Bacillus subtilis↗

PubMed1984

Catalytic properties of alcohol dehydrogenase isozymes specified by duplicate genes in the diploid plant Stephanomeria exigua.

The alcohol dehydrogenase (ADH) isozymes induced in flooded roots of the diploid plant Stephanomeria exigua are specified by tightly linked genes comprising a complex locus, Adh1. Individuals homozygous for a complex with two active genes which specify electrophoretically different subunits have three ADH-1 isozymes, two intragenic homodimers and an intergenic heterodimer. Individual isozymes were partially purified from plants homozygous for several different Adh1 complexes and apparent Km values for acetaldehyde, ethanol, NAD, and NADH and responses to temperature, pH, and two different alcohols were determined. The two homodimeric enzymes specified by a particular Adh1 complex generally differed in one or more of the properties studied, and in three of four cases, intergenic heterodimers differed significantly from intermediacy, often having lower Km values than either homodimer. None of the isozymes studied could be considered greatly divergent or defective. Constraints on evolution of duplicate genes which form intergenic heterodimers are considered.

Alcohol Dehydrogenase↗

PubMed1981

Escape efficiency of diploid and polyploid frogs: a comparison of Odontophrynus cultripes (2n = 22) and O. americanus (4n = 44).

The escape efficiency of two closely related species of frogs, Odontophrynus cultripes (2n = 22) and the tetraploid O. americanus (4n = 44), were compared in a shuttle box and under simulated naturalistic conditions. O. americanus was generally superior to O. cultripes, and females tended to outperform males within both species. The relative inefficiency of O. Cultripes escape behavior was examined in light of the animals' having an elaborate, passive defense mechanism in the form of well marked venom glands. Escape efficiency was highly variable in both species. Possessing twice the amount of DNA, the tetraploid behavioral variation was paradoxically less than that of the diploid, but compatible with what has been found for morphological characters in other organisms.

Animals↗

PubMed1984

Increased glycolysis in ageing cultured human diploid fibroblasts.

With increasing population doubling in vitro, human diploid fibroblasts exhibited a highly significant increase in glucose uptake from the growth medium and a corresponding increase in lactate production. The switch to glycolysis occurred prior to the onset of changes in intracellular glucose and lactate concentrations or in the specific activity of the glycolytic regulatory enzyme, pyruvate kinase. It also preceded the morphological alterations held to be characteristic of cellular senescence.

Cell Line↗

PubMed1991

Survival of diploid yeast cells to bleomycin in combination with UV-light or hyperthermia.

In the present work we have investigated the possible interaction between Bleomycin (B) and UV light or hyperthermia (HT) in the induction of lethal events in diploid yeast populations in the stationary phase of growth. UV and B acting as single agents determine sigmoid survival curves. The combination of UV + B produces different degrees of sensitization depending on dose ranges. For [B] = 7.5 micrograms/ml combined with different UV fluences an exponential course is observed, suggesting overlapping lesion specificity of the involved repair pathways (excision and recombination). The hyperthermia plus Bleomycin treatment produces different degrees of inactivation depending on the sequences. Maximal inactivation effect was obtained for the sequence B + HT. In the case of HT + B ([B] greater than 7.5 micrograms/ml) the obtained sensitization is lower.

Bleomycin↗

PubMed1991

Kinds and locations of mutations arising spontaneously in the coding region of the HPRT gene of finite-life-span diploid human fibroblasts.

Spontaneous thioguanine-resistant mutants were derived from populations of finite-life-span, diploid human fibroblasts by means of a fluctuation analysis. cDNA was prepared from mutant HPRT mRNA and amplified by the polymerase chain reaction, and the sequence of the product was analyzed. Exon deletions, which very likely arose from mutations in the intron splice site consensus sequences, were found in 10 of the 37 mutants examined (27% of the total). Among the 28 mutations in the coding sequence, base pair substitutions predominated (89%). With the exception of one base pair involved in a tandem mutation, all base pair substitutions resulted in alterations in the predicted amino acid sequence of the protein. In addition there were three frameshift mutations, consisting of the deletion of one or two base pairs. Although mutations occurred throughout the coding sequence, 50% (14/28) were found in the 5' portion of exon 3.

Base Sequence↗

PubMed1991

Adaptation and serial passage of porcine group C rotavirus in ST-cells, an established diploid swine testicular cell line.

A porcine group C rotavirus (strain Cowden-AmC-1) was adapted and serially passaged in an established diploid swine testicular cell line (ST cells). Growth of group C rotavirus in ST cells, which in maintenance medium required trypsin, but not pancreatin resulted in cytopathic effect characterized by cellular stranding and subsequent cell lysis. Active replication and assembly were confirmed by RNA profile analysis, immune electron microscopy and immunofluorescence. Adaptation of non-group A rotavirus to a continuous cell line has not previously been reported and should facilitate progress in diagnostic procedures and vaccine development.

Adaptation, Physiological↗

PubMed1979

Interferon response to human diploid cell strain rabies vaccines in man.

Circulating interferon was found in volunteers immunized with 3.0 ml and 1.0 ml doses of human diploid cell strain (HDCS) rabies vaccines. Peak interferon titres were observed 17--25 hours after primary vaccination and the response diminished upon revaccination. The response was dose-related and did not correlate with the antigenic value of the vaccines or with the titre of infections virus in the vaccines before inactivation.

Cell Line↗

PubMed1990

A recombinant vaccinia virus expressing hepatitis B virus middle surface protein. Restricted expression of HBV antigens in human diploid cells.

Several vaccinia virus recombinants inducing the synthesis of the middle surface (M) protein of hepatitis B virus (HBV) were constructed. One of them, denoted v137, was examined in some detail. The virus replicated nearly to the same extent in various cell lines, viz. human embryo diploid fibroblast LEP and MRC-5 cells, rabbit embryo fibroblast REF cells, TK- rat RAT-2 cells, and green monkey CV-1 cells. However, the production of M protein was found considerably lower in the human LEP and MRC-5 than in the other cells examined. In addition, the kinetics of M formation were different in these two cell systems, LEP cells lagging significantly behind CV-1 cells. The low-level production of M protein in LEP cells was not increased by repeated v137 passages in LEP cells, nor by a passage in a laboratory worker accidentally infected with the v137 virus, nor by shortening the leader sequence preceding the translation initiation codon. The greater part of the M antigen was found to be cell associated, more so in the cells of human than monkey origin. From the major HBV S antigen (HBsAg) isolated from the plasma of chronically infected subjects, the antigen released by cell destruction differed by binding to polymerized human albumin. This property was utilized in ELISA to detect anti-preS2 antibody. Rabbits inoculated intradermally with the v137 virus developed antibodies reactive in this assay as well as with a synthetic peptide corresponding in the amino acids 14-34 of the NH2 terminus of the HBsAg preS2 region.

Animals↗

PubMed1976

Liquid holding recovery in stationary and log phase cultures of diploid yeast exposed to gamma and alpha radiations.

The kinetics of liquid-holding recovery (LHR) in diploid yeast after gamma and alpha irradiation is studied. In case of stationary phase culture the rate and extent of LHR is found to be greater for gamma-ray-induced damage than for alpha-ray-induced damage. At 10% survival level, the half-time for recovery is 5.2 h for gamma-ray damage and 12 h for alpha-ray damage. Further, while the recovery factor for alpha damage reaches saturation at 5% survival level, that for gamma damage continues to increase as survival level decreases. Oxygen is required for the recovery process during LH after gamma irradiation. The cells can recover to the same extent from both oxygen-dependent and oxygen-independent components of damage. Log phase cells containing a high per cent of budding cells, however, exhibit negative liquid holding effect after gamma irradiation.

Alpha Particles↗

PubMed1994

Hepatitis B virus proteins expressed by recombinant vaccinia viruses: influence of preS2 sequence on expression surface and nucleocapsid proteins in human diploid cells.

Fifteen vaccinia virus (VV) recombinants derived from VV strains Praha, LIVP and DD (i.e. Dryvax Wyeth vaccine-derived) and expressing genes for S, preS2-S or c antigens of hepatitis B virus (HBV) were tested in monkey CV-1 cells and human diploid LEP cells. The production of infectious virus was found to be alike in all the recombinants and parental viruses as well. However, several recombinants produced markedly lesser amounts of S and preS2 antigens in LEP cells than in CV-1 cells. This reduction was independent of the parental virus used. There was, however, a relationship between the production of preS2 in CV-1 cells and the production of S and preS2 antigens in LEP cells; in general, recombinants efficiently inducing preS2 antigen formation in CV-1 cells produced markedly reduced amounts of S and preS2 antigens in LEP cells. Reduction of HBV antigen production in LEP cells was not apparent in recombinants expressing only S or c antigens of HBV, and the production of c antigen by double recombinants was not influenced by simultaneous expression of preS2 and S. The various recombinants also differed in the ratio of S:preS2 antigen formation. This difference seemed to be associated with the length of the untranslated leader sequence preceding preS2 but not with the parental virus or cell type used. The titers of antibodies against S and preS2 antigens induced in mice immunized with different recombinants differed markedly. The differences in the ratio of S:preS2 antigen production in vitro were not reflected in vivo by S:preS2 antibody ratio.

Animals↗

PubMed1984

Genetic analysis of control of proliferation in fibroblastic cells in culture. I. Isolation and characterization of mutants temperature-sensitive for proliferation or survival of untransformed diploid rat cell line 3Y1.

Mutants temperature sensitive for proliferation or survival were isolated from an untransformed diploid clone of fibroblastic rat cells (3Y1), according to an isolation protocol that selected for mutants defective at 38.5 degrees C (selection temperature) in undergoing the transition from quiescent to proliferating state while maintaining viability at 38.5 degrees C. Of the 108 temperature-sensitive clones isolated, 32 were examined for survival in sparse cultures at 39.8 degrees C (nonpermissive temperature) and classified into four classes. Results of temperature shift-up experiments suggest that functions defective in 11 of the 32 mutants are necessary not only for changing from the quiescent to proliferating state but also for maintenance of the proliferating state. Of the 32 mutants, 17 were assigned to eight complementation groups. Results of the physiological characterization of the representative mutants of each of the eight complementation groups are presented.

Animals↗

PubMed1985

Assignment of human gene encoding thymidylate synthase to chromosome 18 using interspecific cell hybrids between thymidylate synthase-negative mouse mutant cells and human diploid fibroblasts.

We have constructed interspecific somatic cell hybrids between a thymidine-auxotrophic mutant cell line of mouse FM3A cells that lacks thymidylate synthase and human diploid fibroblasts derived from a male patient with fragile X-linked mental retardation. Twenty primary hybrid clones were isolated independently, all of which exhibited the thymidine-prototrophic phenotype. Segregation of the hybrid cells in nonselective culture conditions gave rise to thymidine-auxotrophic hybrid clones. Both electrophoretic assay of thymidylate synthase activity and karyotype analysis of the segregants revealed a strong correlation between the expression of the human form of the enzyme and the presence of human chromosome 18. Thus, it is concluded that the functional gene for human thymidylate synthase, designated TS, is located on this chromosome.

Animals↗