Isolation de pax4

Pages: 6 (1309 mots) Publié le: 1 novembre 2009
Brief Genetics Report Isolation and Characterization of the Human PAX4 Gene
Tsuyoshi Tao, Jon Wasson, Ernesto Bernal-Mizrachi, Philip S. Behn, Susan Chayen, Laura Duprat, Joanne Meyer, Benjamin Glaser, and M. Alan Permutt

AX genes are members of a family of developmental control genes that encode nuclear factors and play critical roles during fetal development (1). Results of recent genetargeting experiments revealed that Pax4 and Pax6 are involved in pancreatic islet development: Pax4 mutant mice lacked - and -cells (2), whereas Pax6 mutant mice lacked -cells (3). Because impaired insulin production by pancreatic islet -cells is a major hallmark of type 2 diabetes (4), genes that regulate pancreatic islet development and insulin biosynthesis might contribute to the pathogenesis ofthis disorder. As the first step to test the hypothesis that PAX4 might be one of these genes, we now report the isolation and characterization of the human PAX4 gene. The Basic Local Alignment Search Tool (BLAST) was used to search public databases with the partial sequence of the mouse Pax4 cDNA (GenBank accession no. Y09584). The search revealed highly homologous sequences in human cosmid cloneg1572c264 (GenBank accession no. AC000359). We found that this human homolog appeared to be present in five separate fragments within a 2.5-kb region of this cosmid clone. Based on the sequence of this human homolog, we designed a pair of oligonucleotide primers (GSP1, 5 -TGGAATGCGGCCCTGTGACAT-3 ; GSP2, 5 -AGCTGCATTTCCCACTTGAGCT3 ). The partial cDNA of human PAX4 was amplified with these primers bypolymerase chain reaction (PCR) with human placenta Marathon-Ready cDNA (Clontech, Palo Alto, CA) used as template. Thermal cycling was accomplished with Advantage cDNA Polymerase Mix (Clontech) at an annealing temperature of 60°C. Reaction products were subcloned and identified as the partial human PAX4 cDNA by sequencing. To isolate the 5 - and 3 -ends of the human PAX4 cDNA, 5 and 3 rapidamplification cDNA ends (RACE) reactions were performed on
From the Division of Metabolism, Endocrinology and Diabetes (T.T., J.W., E.B.M., P.S.B., M.A.P.), Washington University Medical School, St. Louis, Missouri; Millennium Pharmaceuticals (L.D., J.M.), Cambridge, Massachusetts; and the Department of Endocrinology and Metabolism (S.C., B.G.), Hebrew University Hadassah Medical Center, Jerusalem, Israel.Address correspondence and reprint requests to M. Alan Permutt, MD, Metabolism Division, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8127, St. Louis, MO 63110. E-mail: Received for publication 23 February 1998 and accepted in revised form 7 July 1998. BLAST, Basic Local Alignment Search Tool; bp, base pair; HET, heterozygosity; NPL,nonparametric linkage; ORF, open reading frame; PCR, polymerase chain reaction; RACE, rapid amplification cDNA ends; RT, reverse transcription; UTR, untranslated region. 1650


human placental Marathon-Ready cDNA. Gene-specific primers corresponding to the sequence of identified partial human PAX4 cDNA (GSP3 for 5 -RACE, 5 -CCTTAAGGATCCGTGAGATGTCACA-3 ; and GSP4 for 3 -RACE, 5-ATGGCGTCGGCAAGAGAAGCTCAAGT-3 ) were designed. The reaction products were subcloned and sequenced. These experiments yielded three identical clones for 5 RACE and four clones for 3 -RACE, in which one clone was the longest; the other three clones were identical to each other and 516 base pair (bp) shorter than the longest. As a result, we isolated cDNA containing the complete coding region of the human PAX4 gene (Fig. 1).At the 5 -end, there was an in-frame stop codon 190 bp upstream of the first methionine codon. At the 3 -end, a consensus AATAAA polyadenylation signal was located distantly upstream from the poly(A)+ addition site of the longest clone, but 21 bases upstream of the 3 -end of the other three clones. The differences in size among the 3 -RACE clones occurred within the 3 -untranslated region...
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