1.Construction of a cDNA library from Agkistrodon acutus venom gland and identification of Agkihagin, a novel transcript for metalloproteinase
Qinghua LIU ; Songnian HU ; Wei YIN ; Xingwen SU ; Xiaowei ZHANG ; Chenji LI ; Pengxin QIU ; Guangmei YAN
Chinese Journal of Pharmacology and Toxicology 2006;20(2):81-90
AIM To construct a non-normalized cDNA library from Agkistrodon acutus venom gland as an imtial step to develop new and more effective venom by genetic engineering technique for screening and expressing target genes. METHODS The total RNA was extracted from fresh venom gland using Trizol. mRNA was reversely transcripted to cDNA using superscriptⅡ reverse transcriptase. Second-strand synthesis was performed using DNA polymeraseⅠ. After adding EcoRⅠ adaptor, phosphorylating the end and digesting with XhoⅠ, the cDNA was collected in five fractions (<0.25 kb, 0.25-0.5 kb, 0.5-1 kb, 1-2 kb and >2 kb) using the QIAquick Gel Extraction kit and ligated to pBluescriptⅡ vectors. The five libraries obtained were plated by infecting E.coli DH10B, constructing a cDNA library of Agkistrodon acutus venom gland. Sequencing clones at random, 8696 high quality 5′ end expressed sequenced tags (ESTs) were obtained and analyzed. The initial sequences were assembled into 2855 clusters. Among which, one of the clusters (Agkihagin) consisting of 74 ESTs was identified as a novel metalloprtoteinase based on RT-PCR and sequence analysis. RESULTSThe titers of library were 2.048×106. The novel metalloproteinase belonged to PⅢ type metalloproteinase. Its open reading frame was composed of 1827 nucleotides and coded a pre-zymogen of 608 amino acid with zinc-binding domain for metalloproteinase and Asp-Glu-Cys-Asp(DECD) domain for disintegrin. CONCLUSION The capacity of cDNA library of venom gland is above the general level of cDNA library. It would be a helpful platform to construct a catalog for transcripts in the venom gland of the Agkistrodon acutus. The sequence analysis indicates that the deduced amino acid sequence of the identified gene for metalloproteinase share the highest 87% identity with the metalloproteinase genes of other snakes in the GenBank. It lays a good foundation for the study of structure-function relationships of snake venom metalloproteinases.
2.Polymorphisms in genes involved in innate immunity and susceptibility to benzene-induced hematotoxicity.
Min SHEN ; Luoping ZHANG ; Kyoung Mu LEE ; Roel VERMEULEN ; H Dean HOSGOOD ; Guilan LI ; Songnian YIN ; Nathaniel ROTHMAN ; Stephen CHANOCK ; Martyn T SMITH ; Qing LAN
Experimental & Molecular Medicine 2011;43(6):374-378
Benzene, a recognized hematotoxicant and carcinogen, can damage the human immune system. We studied the association between single nucleotide polymorphisms (SNPs) in genes involved in innate immunity and benzene hematotoxicity in a cross-sectional study of workers exposed to benzene (250 workers and 140 controls). A total of 1,236 tag SNPs in 149 gene regions of six pathways were included in the analysis. Six gene regions were significant for their association with white blood cell (WBC) counts (MBP, VCAM1, ALOX5, MPO, RAC2, and CRP) based on gene-region (P < 0.05) and SNP analyses (FDR < 0.05). VCAM1 rs3176867, ALOX5 rs7099684, and MPO rs2071409 were the three most significant SNPs. They showed similar effects on WBC subtypes, especially granulocytes, lymphocytes, and monocytes. A 3-SNP block in ALOXE3 (rs7215658, rs9892383, and rs3027208) showed a global association (omnibus P = 0.0008) with WBCs even though the three SNPs were not significant individually. Our study suggests that polymorphisms in innate immunity genes may play a role in benzene-induced hematotoxicity; however, independent replication is necessary.
Adult
;
Arachidonate 5-Lipoxygenase/genetics/*metabolism
;
Benzene/toxicity
;
Cell Count
;
Cross-Sectional Studies
;
Female
;
Genetic Association Studies
;
Genetic Predisposition to Disease
;
Hematologic Diseases/chemically induced/genetics/*metabolism/pathology
;
Humans
;
Immunity, Innate/genetics
;
Leukocytes/*drug effects/metabolism/pathology
;
Male
;
Occupational Exposure/adverse effects
;
Peroxidase/genetics/*metabolism
;
Polymorphism, Single Nucleotide
;
Vascular Cell Adhesion Molecule-1/genetics/*metabolism