Polymorphism in human T cell receptor alpha chain variable region genes.

Polymorphism of human T cell antigen receptor (TCR)alpha genes was detected by restriction fragment length polymorphism (RFLP). Individual TCR alpha gene segments showed limited polymorphism in that few restriction enzymes revealed polymorphism in genomic DNA samples and when RFLP were detected only two or three allelic forms were observed. A rabbit TCR alpha cDNA clone (VJR5) detected polymorphism in human DNA samples digested with PvuII or MspI. In order to characterize the human V alpha genes detected by the rabbit probe, genomic clones hybridizing with the VJR5 probe were isolated and characterized. A probe derived from a human genomic clone (HUTAVR5) hybridized with some but not all fragments detected by the rabbit VJR5 probe. The data suggest that the rabbit probe hybridized with two distinct human TCR alpha V genes and that polymorphism of each gene was detectable by only one restriction enzyme. In contrast to the limited polymorphism of TCR alpha genes detected by individual markers, extensive heterogeneity of TCR alpha genes was observed in the combination of markers present in haplotypes. Six RFLP were used as TCR alpha markers to define haplotypes in 20 parents and 53 offspring from 10 families and 17 different combinations of markers were observed. The observation that TCR alpha haplotypes include numerous combinations of markers that individually show limited polymorphism suggests that recombination may occur frequently within the TCR alpha gene complex.

The oligosaccharide chains of cobra venom factor are required for complement activation.

To examine the function of the carbohydrate chains of cobra venom factor (CVF), the molecule was enzymatically deglycosylated under non-denaturing conditions with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase). The deglycosylation of CVF chains seems to proceed independently of each other, leading to partially deglycosylated intermediates. Complete deglycosylation of CVF was found to abolish the activity of CVF. The deglycosylated molecule is unable to activate the alternative pathway of complement. Deglycosylated CVF no longer consumes the serum complement activity, it does not induce C3 activation in serum, nor does it induce complement-mediated hemolysis. These results indicate that the carbohydrate moieties of CVF are essential for its role in complement activation.

Cobra venom factor and human C3 share carbohydrate antigenic determinants.

As tools to study structural relationships of cobra venom factor (CVF) and human complement component C3, murine monoclonal antibodies to CVF were produced. In this paper we describe two of these monoclonal anti-CVF antibodies designated GV1.8 and GV1.10, both of which bind to carbohydrate epitopes. On immunoblotting, antibody GV1.8 binds to both the alpha- and beta-chains of CVF, whereas antibody GV1.10 binds only to the alpha-chain of CVF. After enzymatic deglycosylation of CVF with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase), both antibodies lose their ability to bind to the deglycosylated protein. Additionally, the free oligosaccharide chains of CVF are able to inhibit the binding of antibodies GV1.8 and GV1.10 to CVF on enzyme-linked immunosorbent assay, further demonstrating their carbohydrate specificity. Both monoclonal antibodies to CVF cross-react with human C3. Antibody GV1.8 binds to both chains of human C3 indicating that the shared antigenic epitope present on the two glycosylated chains of CVF is also present on the two chains of human C3. Antibody GV1.10 cross-reacts only with the beta-chain of human C3 which is the homologous chain to the alpha-chain of CVF. After enzymatic deglycosylation of human C3 by N-glycanase, both antibodies lose their ability to bind to the deglycosylated protein consistent with the carbohydrate nature of the recognized epitopes. These results indicate that CVF and human C3 share carbohydrate epitopes on their homologous and nonhomologous chains.

Susceptibility to fluorescent light-induced chromatid breaks associated with DNA repair deficiency and malignant transformation in culture.

The increased susceptibility of mouse cells to fluorescent light-induced chromatid damage following their spontaneous malignant transformation in culture could result from loss or inactivation of catalase that decomposes the photoproduct H2O2 or from impaired capacities to repair DNA damage. No consistent change in catalase activity with respect to neoplastic state could be established. To interpret the cytogenetic damage in terms of DNA strand breaks, we determined the incidence of chromatid breaks induced by light exposure during the G1 and late S-G2 phases of the cell cycle in normal and malignant derivatives of a C3H mouse cell line. Chromatid breaks at metaphase following light exposure during G1 would result from DNA strand breaks, cross-links, or base damage, whereas breaks following exposure during late S-G2 would result from single-or double-strand breaks. Both G1 and late S-G2 were susceptible in malignant cells but only G1 in normal. Since caffeine inhibits DNA repair, we compared its effects on light-induced chromatid damage in the normal and malignant cells to assess their DNA repair capacities. Treatment of normal cells with caffeine (50 microgram/ml) directly following five hr of light exposure in G1 increased the chromatid damage to that in malignant cells exposed with or without caffeine. Similarly, treatment of normal cells with caffeine during late S-G2 exposure increased chromatid damage to a level not significantly different from that in malignant cells exposed without caffeine. Caffeine had little influence on chromatid damage in malignant cells. The increased susceptibility of malignant mouse cells to fluorescent light-induced chromatid breaks thus appears to result from impaired capacities to repair DNA damage.