Walleye dermal sarcoma virus: expression of a full-length clone or the rv-cyclin (orf a) gene is cytopathic to the host and human tumor cells.

Walleye dermal sarcoma virus (WDSV) is etiologically associated with a skin tumor, walleye dermal sarcoma (WDS), which develops in the fall and regresses in the spring. WDSV genome contains, in addition to gag, pol and env, three open reading frames (orfs) designated orf a (rv-cyclin), orf b and orf c. Unintegrated linear WDSV provirus DNA isolated from infected tumor cells was used to construct a full-length WDSV provirus clone pWDSV, while orf a was cloned into pSVK3 to construct the expression vector porfA. Stable co-transfection of a walleye cell line (W12) with pWDSV and pcDNA3 generated fewer and smaller G418-resistant colonies compared to the control. By Northern blot analysis, several small transcripts (2.8, 1.8, 1.2, and 0.8 kb) were detected using a WDSV LTR-specific probe. By RT-PCR and Southern blot analysis, three cDNAs (2.4, 1.6 and 0.8 kb) were identified, including both orf a and orf b messenger. Furthermore stable co-transfection of both a human lung adenocarcinoma cell line (SPC-A-1) and a cervical cancer cell line (HeLa) with pcDNA3 and ether porfA or pWDSV also generated fewer and smaller G418-resistant colonies. We conclude that expression of the full-length WDSV clone or the orf a gene inhibits the host fish and human tumor cell growth, and Orf A protein maybe a potential factor which contributes to the seasonal tumor development and regression. This is the first fish provirus clone that has been expressed in cell culture system, which will provide a new in vitro model for tumor research and oncotherapy study.

Effects of ghrelin gene genotypes on the growth traits in Chinese cattle.

Ghrelin is an important peptide that stimulates food intake and regulates energy balance of animals. Single nucleotide polymorphisms of ghrelin gene in three Chinese cattle populations were investigated through PCR-SSCP and DNA sequencing. Five over-lapped DNA fragments were analyzed and a total of three ones exhibited different genotypes. Three genotypes and four SNPs (-415 A > G, -414 T > C, -321 C > A, and -172 A > G) were found on the -544 to +35 bp region (G-1) of ghrelin gene. On the locus of -1037 to -509 bp (G-2), two genotypes and one SNP (-726 A > T) were discovered. And in the exon1, exon2, and intron1 (G-4 locus, (+4 to +427)), two genotypes and one SNP were detected (+205 C > T, located in intron1). Positions of the five SNPs in the 5' regulatory region might be the transcription factor binding sites. The SNPs at -415 and -414 in the core binding sequence were found to cause the change of the site. Though the SNP at -172 did not change the binding site, it generated one new site at the same time. The frequencies of the genotypes varied differently in the three breeds. Results of ANOVA showed that G-1 was correlative to the ischium width (IW) of Nanyang cattle aged 18 months (p = 0.043). The least square analysis between genotypes at G-1 locus and growth traits in Nanyang cattle showed that the individuals (aged 18 months) with C genotype had greater IW than that of the other two genotypes. The C genotype might serve as one potential candidate genetic marker for cattle growth and development.

An efficient and rapid method for gene cloning from eukaryotic genomic DNA using overlap-PCR: With an example of cattle Ghrelin gene.

In this study, overlap-PCR, an efficient and rapid method, was used to clone cattle Ghrelin gene CDS (coding sequence) from genomic DNA. The procedure included seven primers and three-step PCRs. Cattle Ghrelin gene consists of four exons and the CDS contains 351 bps. In the first step three PCRs were performed to generate extended exon1, exon2, and exon3 that contained overlapped nucleotides and were used as the templates for second ligation PCR. Secondly, exon1 and exon2 were spliced together. And it was same with exon3 and exon4. Lastly, the four exons were linked together with outermost primers and the templates from the second step. Comparison analysis on the obtained CDS of Ghrelin gene and cDNA by RT-PCR showed that the two sequences were same. As an efficient and rapid method, overlap-PCR is feasible and acceptable for gene cloning from genomic DNA.

[Polymorphisms of ZAG gene with growth traits in Jiaxian red cattle].

The main function of ZAG gene is to enable the decomposition of fat, and reduced fat content. In this study, polymorphisms of four loci (Z1, Z2, Z3, Z4) at the coding region of the bovine ZAG gene were detected in 145 Jiaxian red cattle, and polymorphisms were found on Z1, Z3, Z4 loci. The fragments showing different SSCP patterns were sequenced, nd a total of six SNPs (C115T, A3257G, A4013G, T4027C, C4032T, and T4120C) were found. The Z3 locus of ZAG gene was at Hardy-Weinberg equilibrium, while Z1 and Z4 loci of ZAG gene were not at Hardy-Weinberg disequilibrium. The association analysis of different genotypes with some of the growth and development traits showed that the individuals with genotype AC had a larger body length, heart girth, circumference of cannon bone, body weight than the individuals with genotypes AA and AB (P<0.05 or P<0.01, respectively) in Z4 locus. It is suggest that Z4 locus may be one of the marker-assisted selection used as growth traits in Jiaxian red cattle.

Effect of genetic variations of the POU1F1 gene on growth traits of Nanyang cattle.

PCR-RFLP was applied to analyze the effect of the genetic variations of the POU1F1 gene on growth traits of 100 Nanyang cattle. The results showed that the 451 bp PCR product digested with Hinf I demonstrated polymorphism in the population, which was at Hardy-Weinberg equilibrium. Moreover, the frequencies of alleles A/B in the Nanyang population were 0.465/0.535. The association of the variations of the POU1F1 gene with the growth traits in the population was analyzed. The following parameters were greater in individuals with a genotype BB than in those with an genotype AB: birth weight, average weight increase before ablactation, body height at 12 months, body weight, body length, and chest girth at 6 months and 12 months (P<0.05). The body weight at 12 months was higher in the BB individuals than in the AA individuals (P <0.05). The body weight and body sizes also showed a trend of allele B> allele A in the other age groups. Therefore, the genotype BB maybe a dominant genotype and the allele B may be a dominant allele. These results imply that the allele B of the POU1F1 gene is likely to positively affect the growth traits.

[Morphological differentiations of the gills of two Gymnocypris przewa-lskii subspecies in different habitats and their functional adaptations].

Gill morphologies of two subspecies of Gymnocypris przewalskii (Gymnocypris przewalskii przewalskii and Gymnocypris przewalskii ganzihonensis) in different habitats were analyzed under scanning electron microscope. Results indicated that G. p. przewalskii had numerous long and dense-lined gill rakers while G. p. ganzihonensis had few short and scatter-lined gill rakers. There were no significant differences in distance between gill filaments (DBF) and distance gill lamella (DBL) between the two subspecies, but gill filaments of G. p. przewalskii were longer than in G. p. ganzihonensis. The electron microscopic study indicated that the pavement epithelium cells of G. p. przewalskii were well defined as irregular ovals, but were hexagonal in G. p. ganzihonensis. Moreover, G. p. przewalskii had more chloride cells than G. p. ganzihonensis, and mucous cells were only found on the surface of gill filaments of G. p. przewalskii. The morphological differences between the two subspecies of G. przewalskii are adaptations to their corresponding diets and habitats.