Erythromycin A, a clinically important polyketide antibiotic, is produced by the Gram-positive bacterium Saccharopolyspora erythraea. In an arrangement that seems to be generally true of antibiotic biosynthetic genes in Streptomyces and related bacteria like S. erythraea, the ery genes encoding the biosynthetic pathway to erythromycin are clustered around the gene (ermE) that confers self-resistance on S. erythraea. The aglycone core of erythromycin A is derived from one propionyl-CoA and six methylmalonyl-CoA units, which are incorporated head-to-tail into the growing polyketide chain, in a process similar to that of fatty-acid biosynthesis, to generate a macrolide intermediate, 6-deoxyerythronolide B. 6-Deoxyerythronolide B is converted into erythromycin A through the action of specific hydroxylases, glycosyltransferases and a methyltransferase. We report here the analysis of about 10 kilobases of DNA from S. erythraea, cloned by chromosome 'walking' outwards from the erythromycin-resistance determinant ermE, and previously shown to be essential for erythromycin biosynthesis. Partial sequencing of this region indicates that it encodes the synthase. Our results confirm this, and reveal a novel organization of the erythromycin-producing polyketide synthase, which provides further insight into the mechanism of chain assembly.

Sequencing of the eryA region of the erythromycin biosynthetic gene cluster from Saccharopolyspora erythraea has revealed another structural gene (ORF B), in addition to the previously characterised ORF A , which appears to encode a component of 6-deoxyerythronolide-B synthase, the enzyme that catalyses the first stage in the biosynthesis of the polyketide antibiotic erythromycin A. The nucleotide sequence of ORF B, whch lies immediately adjacent to ORF A , has been determined. The predicted gene product of ORF B is a polypeptide of 374417 Da (3568 amino acids), which is highly similar to the product of ORF A and which likewise contains a number of separate domains, each with substantial amino acid sequence similarity to components of known fatty-acid synthases and polyketide synthases. The order of the predicted active sites along the chain from the N-terminus is 3-oxoacyl-synthase -acyltransferase -acyl-carrier-protein -3-oxoacyl-synthase -acyltransferase -dehydratase -enoylreductase -oxoreductase -acyl-carrier-protein. The position of the dehydratase active site has been pinpointed for the first time for any polyketide synthase or vertebrate fatty-acid synthase. The predicted domain structure of 6-deoxyerythronolide-B synthase is strikingly similar to that previously established for vertebrate fatty-acid synthases. This analysis of the sequence supports the view that the erythromycin-producing polyketide synthase contains three multienzyme polypeptides, each of which accomplishes two successive cycles of polyketide chain extension. In this scheme, the role of the ORF B gene product is to accomplish extension cycles 3 and 4.

The erA' A resion of the ¢rythromycin biosynthetic gene cluster of Saccharopolyspora erythraea has previo-sl~t been shown to contain thr~e large open reading frames (ORFs) that encode the components of 6-deoxyerythronolide B synthase (DEBS). Pol;,clonal antibodies were raised against recombinant proteins obtained by overexpression of 3' regions of the ORF2 and ORF3 genes. In Western blotting experiments, each antiserum reacted strongly with a different hish molecular weight protein in extracts of erythromycin-producins S. erythraea cells. These putative DEB$ 2 and DEBS 3 proteins were purified and subjected to N-terminal sequence analysis, The protein sequences were entirely consistent with the translation start sites predicted from the DNA sequences ofORFs 2 and 3. A third high molecular weight protein co.purified with DEBS 2 and DEBS 3 and had an N-terminal sequence that matched a protein sequence translated from the DNA sequen~ some 155 base pairs upstream from the previously proposed start codon of ORFI.

The purpose of this study was to analyse the influence of the humoral immune response on the generation and clearance of hepatitis C virus (HCV) RNA containing particles in the blood of chronically infected patients. Blood samples were fractionated by sequential flotation ultracentrifugation and HCV RNA was recovered in three fractions: low density of < 1.063 g/ml, intermediate density of 1.063-1.21 g/ml, and high density of > 1.21 g/ml. Serum low-density lipoproteins co-fractionated with the low-density particles, and high-density lipoproteins co-fractionated with the intermediate-density particles. Immunoglobulins were found exclusively in the high-density fractions. In patients with congenital immunodeficiencies, with no or low serum antibodies to the virus, mean HCV RNA titres were equal in each fraction, at approximately 10(5) IU/ml. In antibody-positive, immunocompetent patients, however, virus titres in the low-density fraction and those in the high-density fraction were reduced or absent in most patients, suggesting that virus particles in these fractions are subject to antibody-mediated clearance. Particles of intermediate density were approximately equal in titre in both patient groups, suggesting that these particles are neither generated by, nor cleared, as a result of the humoral immune response. Immunoprecipitation experiments indicated that particles of intermediate density were not complexed with either high-density lipoprotein or immunoglobulins. Elucidation of the mechanisms by which these particles are generated and maintained in the blood may provide valuable insight into the mechanism of virus persistence.

In the absence of satisfactory cell culture systems for hepatitis C virus (HCV), virtually all that is known about the proteins of the virus has been learned by the study of recombinant proteins. Characterization of virus proteins from patients with HCV has been retarded by the low virus titre in blood and limited availability of infected tissue. Here, the authors have identified a primary infection in a liver transplanted into an immunodeficient patient with chronic HCV. The patient required re-transplant and the infected liver, removed 6 weeks after the initial transplant, had a very high titre of HCV, 5610 9 International Units (IU) per gram of liver. The density distribution of HCV in iodixanol gradients showed a peak at 1?04 g ml "1 with 73 % of virus below 1?08 g ml "1 . Full-length HCV RNA was detected by Northern blotting and the ratio between positive-and negative-strand HCV RNA was determined as 60. HCV was partially purified by precipitation with heparin/Mn 2+ and a single species of each of the three structural proteins, core, E1 and E2, was detected by Western blotting. The molecular mass of core was 20 kDa, which corresponds to the mature form from recombinant sources. The molecular mass of glycoprotein E1 was 31 kDa before and 21 kDa after deglycosylation with PNGase F or endoglycosidase H. Glycoprotein E2 was 62 kDa before and 36 kDa after deglycosylation, but E2-P7 was not detected. This was in contrast to recombinant sources of E2 which contain E2-P7. INTRODUCTIONInfection with hepatitis C virus (HCV) occurs worldwide and afflicts approximately 170 million people (Anonymous, 1997). Chronic infection, which occurs in 80 % of cases, can result in chronic active hepatitis, cirrhosis and hepatocellular carcinoma and, less commonly, extrahepatic autoimmune or immune complex diseases (reviewed by Major et al., 2001). Gene fragments from the virus were initially identified by screening a lambda phage expression library of cDNA from the nucleic acid extracted from infected chimpanzee serum against serum from a non-A non-B hepatitis patient (Choo et al., 1989). Subsequently, the whole virus genome of approximately 9500 nt was rescued, sequenced and shown to exhibit a similar genome organization to the flaviviruses and pestiviruses (Choo et al., 1991). This insight, the in vitro transcription of the genome and extensive studies of viral protein expression in eukaryotic expression systems indicate that the genome consists of a single open reading frame encoding a 3000 aa polyprotein flanked by highly conserved 59 and 39 untranslated regions. The putative structural proteins of the virus, core protein and two membrane glycoproteins, E1 and E2, lie at the amino-terminal end of the polyprotein and are released co-translationally by host cell signal peptidase. The nonstructural proteins of the virus, NS2 to NS5B, are located at the C-terminal end of the polyprotein and are released by virus proteases (reviewed by Major et al., 2001;Bartenschlager & Lohmann, 2000). In between the structural and non-structural...