ABSTRACT
Mutants of Actinomyces viscosus T14V lacking type 1 or type 2 fimbriae or both were selected by their failure to react with rabbit antibodies against either or both fimbrial antigens. Immunospecific double labeling with iron dextran and ferritin-conjugated antibodies showed two types of fimbriae on individual cells of the parent organism, a single type on mutant strains with type 1+2- and type 1-2+ fimbriae and no labeled or unlabeled fimbriae on a type 1-2- fimbria-deficient strain. The mutational loss of one fimbrial antigen did not appear to affect expression of the other, since bacteria with one or two types of fimbriae bound similar amounts of a monoclonal antibody directed against the fimbrial antigen present on both bacterial phenotypes. The strong adsorption of strains with type 1+2+ or 1+2- fimbriae to saliva-treated hydroxyapatite and weak adsorption of those with type 1-2+ or no fimbriae was consistent with the known involvement of type 1 fimbriae in this attachment process. Similarly, the A. viscosus lectin was clearly associated with the expression of type 2 fimbriae, since only the strains with type 1+2+ or 1-2+ fimbriae participated in lactose-sensitive coaggregations with Streptococcus sanguis 34. Further studies using the fimbria-deficient mutant strains showed that aggregation of A. viscosus T14V in the presence of sialidase-treated human saliva involved both types of fimbriae, whereas neither type was required for the lactose-resistant coaggregation of the organism with certain streptococcal strains.
Subject(s)
Actinomyces/genetics , Fimbriae, Bacterial/physiology , Antibodies, Bacterial/immunology , Antibodies, Monoclonal/immunology , Antigens, Bacterial/genetics , Microscopy, Electron , Mutation , Saliva/immunology , Saliva/microbiologyABSTRACT
A sensitive assay for O6-alkylguanine-DNA alkyltransferase activity in cell or tumor extracts has been devised. The theoretical basis of the new assay lies in the observation that certain restriction enzymes will not cleave DNA containing methylated bases. Thus, if a synthetic oligodeoxynucleotide with a restriction sequence containing O6-methylguanine were incubated with the restriction enzyme, this synthetic oligodeoxynucleotide should remain intact. However, if the guanine-O6 methyl group were first removed by O6-alkylguanine-DNA alkyltransferase present in certain cell or tissue extracts the synthetic oligodeoxynucleotide would be cleaved by the restriction enzyme. The parental oligodeoxynucleotide and its restriction products are separated from each other and analyzed on denaturing polyacrylamide gels. The extent of cleavage by the restriction enzyme is a direct assay of the content of O6-alkylguanine-DNA alkyltransferase in the cell/tumor extracts. The assay has been tested against cell culture and xenograft tumor systems and has performed in a predictive manner, correctly predicting five Mer- and three Mer+ phenotypes. Furthermore, the assay is quantitative and the number of molecules of the O6-alkylguanine-DNA alkyltransferase per cell estimated using this assay agrees with those that have been published.
Subject(s)
DNA Restriction Enzymes/metabolism , DNA/analysis , Methyltransferases/analysis , Neoplasms/enzymology , Base Sequence , Cells, Cultured , Humans , Methods , Neoplasm Transplantation , Neoplasms/genetics , O(6)-Methylguanine-DNA Methyltransferase , Oligodeoxyribonucleotides/analysis , Phenotype , Transplantation, HeterologousABSTRACT
Human fibroblasts and tumor cells with constitutive levels of the DNA repair protein O6-methylguanine-DNA methyltransferase were incubated with mM concentrations of the free base O6-methylguanine for up to 24 h. This treatment depleted the cells of their transferase activity, and sensitized the cells to killing by the antineoplastic drug 1-[2-chloroethyl]-1-nitrosourea. Cells constitutively lacking the methyltransferase were not sensitized to cell killing. Cell free extracts incubated with O6-methylguanine also lost methyltransferase activity. Other alkylpurines, such as O6-methylguanosine, S6-methylthioguanine, O6-ethylguanine, and 3-methyladenine, did not have this effect on extracts of human tumor cells, while O6-methylguanosine and O6-methylguanine inactivated purified methyltransferase from Escherichia coli. The data suggest that the free base O6-methylguanine is probably a substrate for the methyltransferase. Calculation of the second order rate constants for free base versus O6-methylguanine in DNA, and experiments in which the free base was mixed with DNA containing O6-methylguanine before reaction with methyltransferase, indicated that the base in DNA is about 4 X 10(7) better as a substrate than is the free base. These results demonstrate that DNA repair capacity of tumor cells can be diminished without DNA damage, and suggest a method for increasing the efficiency of chemotherapy.
