Michael Theile

Mutagenesis by simian virus 40. I. Detection of mutations in Chinese hamster cell lines using different resistance markers

The mutagenic action of SV40 in permanent lines of Chinese hamster cells (CHO-K1 and V79) was investigated with the aid of different resistance markers. The markers studied had resistance to 8-azaguanine (25 and 30 mug/ml), aminopterin (3.3--5.5X10(-3) mug/ml), colchicine (6.5 and 7.0X10(-2) mug/ml) and 5-bromodeoxyuridine (50--120 mug/ml), respectively. After virus infection the mutation frequencies were increased by one (azaguanine, aminopterin) and two (colchicine) orders of magnitude as compared with spontaneous mutation frequencies. In contrast, it was not possible to enhance the frequency of mutation to BUdR resistance. On the other hand, the ability to proliferate in HAT medium was induced in three of five BUdR-resistant cell clones by infection with SV40. The resistance induced by SV40 was stable when isolated clones were cultured under non-selective conditions. Mechanisms are proposed that may be responsible for the mutagenic action of SV40.

Virus-induced gene mutations of eukaryotic cells

SummaryMost animal viruses studied so far induce chromosomal aberrations. In addition, adenoviruses, papovaviruses, and retroviruses are known to induce gene mutations like mutagenic bacteriophages. At least in one case studied retrovirus induced mutagenesis involves gene and/or scripton splitting analogous to the mutagenic mechanism of action of mutatorphage Mu and other movable DNA elements. On the contrary, several results obtained by independent means indicate that Simian virus 40, a papovavirus, does not act by splitting the affected gene but presumably by generation of base pair substitutions or of other minor DNA damages leading to amino acid substitutions. The mechanisms involved are still unknown. There a some hints, however, that these mechanisms might have some step(s) in common with processes leading to malignancy. In fact those viruses proved unequivocally so far to be capable of inducing gene mutations are oncogenic viruses.

Mutagenesis by Simian virus 40. II. Changes in substrate affinities in mutant hypoxanthine-guanine phosphoribosyl transferase enzymes at different pH values

A number of 8-azaguanine-resistant clones selected from Chinese hamster cells infected with SV 40, and supposed to originate by virus infection was investigated to demonstrate and analyze genetic alterations occurring in the cells after infection. All resistant clones tested showed reduced but detectable activity levels of the enzyme hypoxanthine-guanine phosphoribosyltransferase. The extent of reduction in the activity was not identical for different substrates. In all the clones tested, spontaneous mutants included, the pH optimum for the enzymic reaction with guanine was shifted to lower values. The reduced enzymic activities of resistant clones correlated with their colony-forming ability in corresponding selective media. The results support the suggestion that SV 40 is able to induce gene mutations.

Feasibility of simultaneous fluorescence immunophenotyping and fluorescence in situ hybridization study for the detection of estrogen receptor expression and deletions of the estrogen receptor gene in breast carcinoma cell lines

Abstract For the first time, combined immunophenotyping and fluorescence in situ hybridization (FISH) technique according to the ”fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasms” (FICTION) technique have been successfully applied in solid tumors. Thus, we were able to visualize the antigen expression of cells with chromosomal deletions of a tumor suppressor region directly. In six breast carcinoma cell lines, we investigated the correlation between estrogen receptor (ER) expression status and deletions of the estrogen receptor gene (ESR). To screen for deletions of the ESR gene, dual-color FISH was performed with a YAC (yeast artificial chromosome) probe containing the ESR gene and, as internal control, with a centromeric probe of chromosome 6. Deletions of the ESR gene were detected in four of six cell lines. For direct comparison of ER expression with the copy number of the ESR gene at the single cell level, immunophenotyping with mouse anti-human ER antibody was combined with FISH with the YAC probe containing the ESR gene according to the FICTION technique. There was no correlation between lack of or reduced ER expression and deletions of the ESR gene. One cell line with deletions of the ESR gene did express ER on the protein level, while another cell line without a deletion did not. Cells with deletions of the ESR gene were either ER expression positive or negative. The staining intensity of ER expression was not associated with the copy number of the ESR gene. Thus, this FICTION study unequivocally shows that deletions of the ESR gene are not the major cause of absent or reduced ER expression in breast carcinoma cell lines.

Detection of 6q deletions in breast carcinoma cell lines by fluorescence in situ hybridization

Abstract Dual-color fluorescence in situ hybridization was performed to detect the frequency and extent of 6q deletions in ten breast carcinoma cell lines. In five cell lines, the 6q deletions involved large regions extending from 6q12–q16 to 6q27, and in one the deletion extended from the region distal to YAC 751G10 at 6q25.1 to 6q27. In two cell lines, 6q deletions occurred only in cells with polysomy 6, indicating that such deletions might be secondary chromosomal aberrations and reflect late genetic changes in breast carcinomas. In addition, an overrepresentation of 6q21–q22.2 was detected in one cell line.

The incorporation of homologous and heterologous hypoxanthine-guanine phosphoribosyltransferase into mutant cells

Experiments are described leading to partial compensation of a deficiency in the enzyme hypoxanthine-guanine phosphoribosyltransferase in mutant cells by supplying the cells with exogenous purified enzymes. DEAE-dextran is an effective helper agent, whereas poly (L-lysine), lysolecithin and amphotericin B seem to inhibit the entry of the enzymes of their activity. Enzyme preparation from Chinese hamster was found to have different effects in different mutant cell lines. In mutant Chinese hamster cells, the electrophoretic activity pattern remains unchanged for the Chinese hamster enzyme, but changes progressively to faster-moving activity peaks for the human enzyme after several hours. The metabolic effect of the incorporated enzyme is in the range between 3 and 4% of the normal cellular enzyme activity which corresponds to a 10--20 fold increase of hypoxanthine-guanine phosphoribosyltransferase activity in the mutant cells.

Simian Virus 40-induced Mutagenesis: Action of the Early Viral Region

Earlier results have demonstrated a mutagenic activity of simian virus 40 (SV40) in mammalian cells. To analyse this ability further, the effect of SV40 DNA fragments, introduced into Chinese hamster cells, on the frequency of mutations at the hypoxanthine phosphoribosyltransferase locus and other loci was studied. It was found that the mutagenic effect was substantially maintained when the viral genome had been replaced by a fragment comprising the T antigen-coding region and the early promoter-enhancer region; was strongly reduced or abolished when the promoter region including upstream sequences in this fragment had been replaced by the chicken lysozyme gene promoter or both enhancer elements were deleted, and was abolished in an SV40 replication origin-defective mutant in which the structure of the T antigen-binding site II was affected. It may be concluded that SV40-induced mutagenesis depends on the expression of the early region of the genome and on a function involved in specific binding of large T antigen to viral DNA. Since origin-defective mutants of SV40 were reported as being able to transform cells, the functions of transformation and mutation do not seem to correlate.

Influence of superinfection on the photoreversible phase of UV induced lysogenic bacteria

Summary1.Lysogenic induction by UV light can be reversed by photoreactivation. UV-treated E. coli K12 (λ)+uvr+ and uvr cells are sensitive to photoreactivation for a given time after irradiation. This sensitivity suddenly disappears at the end of this time.2.The photoreversible period of UV induction is more than twice as long in uvr cells as it is in uvr+ cells.3.The photoreversible period can be reduced by superinfection with λc mutants after irradiation. This effect is positively correlated with the multiplicity of superinfection. Such a reduction does not occur when superinfection is carried out with wild-type phages or with heteroimmune derivatives.4.We concluded that during the photoreversible period of UV induction oligonucleotides are excised or synthesized and gaps are formed during excision repair and post replication repair of UV damage; these might react with E. coli recA protein thereby activating it to induce its won synthesis, to cleave phage repressors and to exert its other SOS functions.

Die Hybridisierung von tierischen somatischen Zellen

Jahrzehntelang, bevor die Mikrobengenetik und insbesondere die Bakteriengenetik unser genetisches Wissen revolutionierten, war die Analyse genetischer Gesetzmasigkeiten eine sorgfaltige Analyse der Ergebnisse sexueller Reproduktionen. Erst die Entdeckung parasexueller Prozesse1) in Bakterien und vor allem in fila-mentosen Pilzen veranlaste vor nunmehr 20 Jahren Genetiker wie Guido Pontecorvo, Curt Stern und Joshua Lederberg zu der optimistischen Prognose, das solche parasexuellen Vorgange eventuell auch bei in vitro kultivierten tierischen Zellen nachweisbar sind und eine echte Alternative zu der sexuellen Reproduktion darstellen konnten. Mit der erfolgreichen Hybridisierung tierischer somatischer Zellen in vitro haben diese Spekulationen eine glanzende Bestatigung erfahren. Von einer biologischen Kuriositat hat sich die Hybridisierung tierischer somatischer Zellen zu einer Methode entwickelt, die detaillierte Einblicke in molekularbiologische Pro-, zesse einer Vielzahl biologischer Phanomene erlaubt.

Gentransfer bei tierischen somatischen Zellen

In der belebten Welt ist die zellulare Aufnahme von informativen Makromolekulen, inklusive DNA, extrem selten. Vor allem die Zellen, die aus der Teilung somatischer Zellen hervorgehen, behalten ihr genetisches Programm in strikter Reservation fur sich selbst und ihre Nachkommen. Der Zell-zu-Zell-Transfer biologischer Informationen vollzieht sich bei hoheren Organismen gleichsam an den Polenden des biologischen Spektrums: das eine Ende umschliest im wesentlichen alle sexuellen Prozesse (Keimzellen), wahrend das andere Ende durch die Invasion viraler Nukleinsaure in die Zelle charakterisiert wird. Abgesehen von diesen Ereignissen ist die lebende Zelle gewohnlich eine feste Barriere, sowohl fur den Austritt, als auch fur das Eindringen von Molekulen in der Grose von Nukleinsauren oder deren informativen Fragmenten. Wahrend bei hoheren Organismen die sexuellen Prozesse haufig den einzigen Weg fur deren Reproduktion darstellen scheint ein Zell-zu-Zell-Transfer von Nukleinsauren bei Bakterien ein zwar seltener, nichtsdestoweniger jedoch gangiger Prozes zu sein.