The fatty acid synthase (FAS) gene was cloned from liver of grass carp (Ctenopharyngodon idella) by degenerate oligonucleotide primed PCR. The obtained cDNA fragment was 683 bp, which encoded 227 amino acids. Then, grass carps with initial body weight of (134.89 ± 12.12) g were fed diet supplemented with 0, 20, 40 g kg )1 fat to investigate the impacts of dietary fat levels on growth, liver FAS enzyme activity and mRNA expression. After 8 weeks feeding, final body weights of the three groups were 344.11, 347.23 and 373.02 g. Compared with control group (0 g kg )1 ), growth rate of 40 g kg )1 fat group was increased by 14.03%, and feed conversion rate decreased by 11.32% (P < 0.05), liver FAS enzyme activity of 20, 40 g kg )1 fat groups were reduced by 33%, 64% (P < 0.05), and FAS mRNA expression level reduced by 18%, 74% (P < 0.05), respectively. Results above showed that 40 g kg )1 fat addition can significantly improve growth performance of grass carp. Liver FAS activity and mRNA expression tended to be inhibited by the increasing dietary fat level. Fat containing high levels of polyunsaturated fatty acids had strongly inhibitory effects on liver FAS activity and gene expression.

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We have developed a viral RNA (vRNA) dot blot assay for identified a subset of candidate high-titer producer cell rapid identification of high-titer retrovirus vector production clones. In three independent screens the supernatant with by packaging cell clones. The procedure employs Trizol LS the highest biological titer was produced by one of the prereagent to purify vRNA from packaging cell supernatants, viously defined candidate high-titer producer clones. Our a sensitive dot blot assay, and PhosphorImager technology procedure greatly facilitates virus titration by: (1) rapidly elito quantify packaged viral genomes in 2 days. Experiments minating the vast majority of low-titer producer cell clones; performed on viral supernatants of known biological titer (2) accurately identifying the subset of candidate high-titer demonstrated that the vRNA dot blot assay was extremely producer clones for further biological titration and assesssensitive and that dot intensity correlated directly with viral ment of the proviral genomic structure; and (3) reducing titer. It is often necessary to analyze approximately 100 laborious tissue culture manipulations to a minimum. Furvirus producing cell clones, making this method useful as thermore, the reliance of this method on molecular deteca rapid screen to identify the highest virus producing tion makes it ideally suited for the isolation of high-titer clones. The vRNA dot blot assay consistently clones lacking a drug selection marker.

The AcNPV orf69 gene encodes a protein that contains an S-adenosylmethionine (AdoMet)-dependent methyltransferase signature motif. More significantly, ORF69 shows high conservation at residues diagnostic for (nucleoside 2-O)-methyltransferase activity. To analyze the function of this protein, which was renamed MTase1, it was overexpressed in Escherichia coli and purified to homogeneity. Photo cross-linking experiments showed that MTase1 bound AdoMet, and functional assays demonstrated cap 0-dependent methyltransferase activity. In vivo expression assays in insect cells showed that MTase1 was synthesized during the late phase of infection and that its expression was dependent on viral DNA replication. Primer extension analysis identified a late promoter motif, ATAAG, at the transcription start site. A mutant virus was constructed by inserting the lacZ gene into the coding region of mtase1. Immunoblot analysis confirmed that MTase1 was not synthesized in these cells, and single-step growth curves revealed that the rate of virus replication in tissue culture was not affected by the absence of MTase1.