1. Supplementation of cultures of Erenwthecium ashbyii with ribitol leads to a twofold increase in riboflavin formation compared with unsupplemented cultures or those supplemented with ribose or ribulose phosphate. Addition of unlabelled ribitol decreases the incorporation of [1-14C]ribose into riboflavin, indicating that free ribitol is preferred to ribose for incorporation into riboflavin. 2. The enzymes ribitol kinase, D-ribose reductase, D-ribose 5'-phosphatase and GMP nucleosidase were demonstrated in the cell-free extracts. Ribitol induces the formation of ribitol kinase. The enzyme is activated in vitro by the flavinogenic purines, guanine and xanthine. D-Ribose reductase shows a specific requirement for NADPH and forms free ribitol from ribose. 3. The activities of ribitol kinase, ribose 5'-phosphatase and GMP nucleosidase reach their maximal values before riboflavin formation reaches a maximum. 4. [U-_4C]GMP is taken up intact by the culture ofE. ashbyii and is incorporated into riboflavin as well as into a blue fluorescent compound. The radioactivity from this compound is incorporated into riboflavin by the cell-free extract of E. ashbyii.Micro-organisms have been used extensively to study the biosynthesis of riboflavin, since some of them produce this vitamin in large excess. Although considerable information is available on the biosynthesis of riboflavin, the mode of insertion of the ribitol moiety into the vitamin molecule still remains obscure (Bacher & Lingens, 1970;Baugh & Krumdieck, 1969;Goodwin, 1963; Goodwin & Jones, 1956;Harvey & Plaut, 1966;Maley & Plaut, 1959;McNutt, 1954;Plaut, 1961). The present investigation was undertaken to study the effect of availability of ribitol and guanine on enzymic constitution and flavinogenesis in Eremothecium ashbyii. Materials and Methods OrganismA flavinogenic strain of Eremothecium ashbyii (no. 0944), obtained from the Type Culture Collection, Tokyo, Japan, was maintained on Sabourauds' agar (Difco Laboratories, Detroit, Mich., U.S.A.) slant cultures. The yeast was cultivated in the liquid fermentation medium as described by Goodwin & Pendlington (1954), supplemented with 0.5% vitamin-free Casamino acids (Difco Laboratories). The inoculated flasks were incubated at 28 ±20C in a dark incubator; unless otherwise specified, the cultures were incubated for 4 days. The surface cultures grown in the above liquid medium ('normal medium') are termed 'normal cultures'; when ribose (5 or lOmg/ 50ml) or ribitol (5 or lOmg/50ml) or guanine (3 mg/50ml) was included in this medium, the culVol. 130 tures are referred to as 'ribose-supplemented', 'ribitol-supplemented' or 'guanine-supplemented' respectively. Radioactivity experimentsTo study the fate of [1-14C]ribose during growth and flavinogenesis, the organisms were grown for 48h in the non-radioactive normal medium. Cells were collected by centrifugation and resuspended in the fermented broth to give a concentration ofapprox. 0.2mg dry wt./ml of suspension. Samples (5ml) from this suspension were distributed in lOOm...

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Recently, identification and molecular cloning of a host cellular gene designated GOR from chimpanzees experimentally infected with non-A, non-B hepatitis (NANBH) agent was reported. It was further demonstrated that there is a close association between the immune response to an antigenic peptide of GOR (GOR2) and NANBH. In order to define the specificity of the immune response, in the present study we have identified an additional epitope in the GOR gene sequence, upstream from GOR2, and studied its correlation with the immune response to hepatitis C virus (HCV) in NANBH patients. An enzyme-linked immunoassay (EIA) was developed which utilizes synthetic peptides designated spGOR346 and spGOR2 as the serological target for the detection of anti-GOR antibodies in patient serum samples from various hepatic and non-hepatic disease categories. GOR peptides identified 80-90% of the NANBH samples that were positive for HCV C100-3 and about 70% of the NANBH samples that were positive by Abbott prototype second-generation HCV antibody assay. Among a normal donor population(s), only 2-3% of the samples were positive for antibodies to GOR sequences, whereas from the patient categories unrelated to viral hepatitis as well as various nonhepatic diseases, the immune response to both GOR peptides was closely associated with the presence of antibodies to HCV. The data indicate that antibodies to GOR is a marker associated with NANBH.