The Long Intron 1 of Growth Hormone Gene from Reeves' Turtle (Chinemys reevesii) Correlates with Negatively Regulated GH Expression in Four Cell Lines.

Turtles grow slowly and have a long lifespan. Ultrastructural studies of the pituitary gland in Reeves' turtle (Chinemys reevesii) have revealed that the species possesses a higher nucleoplasmic ratio and fewer secretory granules in growth hormone (GH) cells than other animal species in summer and winter. C. reevesii GH gene was cloned and species-specific similarities and differences were investigated. The full GH gene sequence in C. reevesii contains 8517 base pairs (bp), comprising five exons and four introns. Intron 1 was found to be much longer in C. reevesii than in other species. The coding sequence (CDS) of the turtle's GH gene, with and without the inclusion of intron 1, was transfected into four cell lines, including DF-1 chicken embryo fibroblasts, Chinese hamster ovary (CHO) cells, human embryonic kidney 293FT cells, and GH4C1 rat pituitary cells; the turtle growth hormone (tGH) gene mRNA and protein expression levels decreased significantly in the intron-containing CDS in these cell lines, compared with that of the corresponding intronless CDS. Thus, the long intron 1 of GH gene in Reeves' turtle might correlate with downregulated gene expression.

Differential expression of microRNA in endothelial cells incubated with serum of hypertension patients with blood-stasis syndrome.

OBJECTIVE: To screen out blood-stasis syndrome (BSS)-associated microRNA and therefore determine the possible target for treating hypertension. METHODS: A high-energy sequencing method and digital gene expression sequencing theory were adopted to sequence microRNA (miRNA) and messenger RNA (mRNA), and to determine differential expression in human umbilical vein endothelial cells incubated with serum samples from hypertension patients with or without BSS, and healthy controls. The results were confirmed using gene prediction software. RESULTS: A total of 13 miRNAs and 11 mRNAs showed statistical difference both in the BSS/normal groups and BSS/non-BSS groups, respectively. Four pairs of target mRNA/miRNA were identified: FRMD4A/hsa-miR-34a, MAP3K14/hsa-miR-34a, PER1/hsa-miR-34a, and FGF2/hsa-miR-132. CONCLUSION: Four mRNA/miRNA pairs mentioned above seem to be involved in pathogenesis and maintenance of hypertension with BSS.

Constructing protein-protein interaction network of hypertension with blood stasis syndrome via digital gene expression sequencing and database mining.

OBJECTIVE: To construct a protein-protein interaction (PPI) network in hypertension patients with blood-stasis syndrome (BSS) by using digital gene expression (DGE) sequencing and database mining techniques. METHODS: DGE analysis based on the Solexa Genome Analyzer platform was performed on vascular endothelial cells incubated with serum of hypertension patients with BSS. The differentially expressed genes were filtered by comparing the expression levels between the different experimental groups. Then functional categories and enriched pathways of the unique genes for BSS were analyzed using Database for Annotation, Visualization and Integrated Discovery (DAVID) to select those in the enrichment pathways. Interologous Interaction Database (I2D) was used to construct PPI networks with the selected genes for hypertension patients with BSS. The potential candidate genes related to BSS were identified by comparing the number of relationships among genes. Confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), gene ontology (GO) analysis was used to infer the functional annotations of the potential candidate genes for BSS. RESULTS: With gene enrichment analysis using DAVID, a list of 58 genes was chosen from the unique genes. The selected 58 genes were analyzed using I2D, and a PPI network was constructed. Based on the network analysis results, candidate genes for BSS were identified: DDIT3, JUN, HSPA8, NFIL3, HSPA5, HIST2H2BE, H3F3B, CEBPB, SAT1 and GADD45A. Verified through qRT-PCR and analyzed by GO, the functional annotations of the potential candidate genes were explored. CONCLUSION: Compared with previous methodologies reported in the literature, the present DGE analysis and data mining method have shown a great improvement in analyzing BSS.

[Analysis on molecular evolution of MC1R gene in dog].

Melanocortin 1 receptor (MC1R) gene regulates pigment synthesis in mammals, and therefore is regarded as an important candidate gene for dog coat color. Based on MC1R amino acids and cDNA sequences of 10 vertebrate animals released by NCBI, molecular evolution of dog MC1R gene was analyzed with bioinformatic software and internet resource. Results showed that 10 vertebrate animals were divided into two major groups, a compact group A (7 mammals) and an incompact group B (chicken, zebrafish and fugu). This phylogenetic tree was consistent with putative evolutionary relationship within these 10 species. Positive selection was detected during the evolutionary process of dog (also cat and pig) from cattle by PAML branch model (omega = 90.8177), and five amino acids of 2V, 25E, 184N, 197V and 314L of dog MC1R were predicted under positive selection by site model. Comparative linkage analysis of chromosome showed that "ZFP276-MC1R-GAS8" linkage group was conservative in human, chimpanzee, chicken and dog.