Isolation de pax4

Isolation de pax4

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 gene targeting 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 of this disorder. As the ? rst 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 clone g1572c264 (GenBank accession no. AC000359). We found that this human homolog appeared to be present in ?

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ve 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 ampli? ed with these primers by polymerase 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 identi? ed as the partial human PAX4 cDNA by sequencing. To isolate the 5 – and 3 -ends of the human PAX4 cDNA, 5 and 3 rapid ampli? cation 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: [email protected] wustl. edu. 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 ampli? ation cDNA ends; RT, reverse transcription; UTR, untranslated region. 1650 P human placental Marathon-Ready cDNA. Gene-speci? c primers corresponding to the sequence of identi? ed 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 ? rst 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 (UTR); thus, the open reading frames (ORFs) were identical. This ORF encoded a predicted protein of 343 amino acids.

Interestingly, the isolated human PAX4 cDNA had relatively low homology to mouse Pax4 (76. 4% at amino acid level) compared with that between human and mouse for other members of the PAX gene family (>95% at amino acid level). These results originally suggested that our isolated cDNA was not PAX4, but perhaps a new PAX gene. To examine this hypothesis, human genomic DNA was subjected to Southern blot analyses with fragments of the human placental PAX4 cDNA and the mouse Pax4 cDNA as probes under conditions of low stringency. Mouse Pax4 cDNA was ampli? d with reverse transcription (RT)-PCR from total RNA of transfected mouse insulinoma cell line ( TC6-F7, supplied by S. Efrat, Albert Einstein Medical School) using a standard protocol. Southern blot analysis using a fragment of human PAX4 cDNA (88. 8% identical to mouse Pax4 cDNA and 95% with a total genetic risk of 5- to 10-fold, with a single major gene contributing (8). These results do not exclude the possibility of a minor role for the PAX4 gene in a polygenic model, however. It is also possible that PAX4 contributes to type 2 diabetes in other racial or ethnic groups and that the markers de? ed here will be useful for these analyses. ACKNOWLEDGMENTS This work was supported in part by National Institutes of Health Grants DK-16746 (M. A. P. ), DK-49583 (M. A. P. , S. C. , L. D. , J. M. , B. G. ), and DK-07120 (P. S. B. ). T. T. was the recipient of a Mentor-Based Fellowship Award from the American Diabetes Association. Oligonucleotides and human islets were provided by the Protein Chemistry and Islet Cores of the Diabetes Research and Training Center (National Institutes of Health Grant 2P60-DK-20579), respectively. The authors would also like to thank Jeannie Wokurka for preparation of the manuscript.

We also wish to acknowledge Roche Pharmaceuticals for their support in collection of families. REFERENCES 1. Dahl E, Koseki H, Balling R: Pax genes and organogenesis. Bioessays 19:755–765, 1997 2. Sosa-Pineda B, Chowdhury K, Torres M, Oliver G, Gruss P: The Pax4 gene is essential for differentiation of insulin-producing -cells in the mammalian pancreas. Nature 386:399–402, 1997 3. St-Onge L, Sosa-Pineda B, Chowdhury K, Mansouri A, Gruss P: Pax6 is required for differentiation of glucagon-producing -cells in mouse pancreas. Nature 387:406–409, 1997 4.

Porte DJ: -Cell in type II diabetes mellitus. Diabetes 40:166–180, 1991 5. Matsushita T, Yamaoka T, Otsuka S, Moritani M, Matsumoto T, Itakura M: Molecular cloning of mouse paired-box-containing gene (Pax)-4 from an islet cell line and deduced sequence of human Pax-4. Biochem Biophys Res Commun 242:176–180, 1998 6. Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW: Automated method for isolation of human pancreatic islets. Diabetes 37:413–420, 1988 7. Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES: Parametric and nonparametric linkage analysis: a uni? d multipoint approach. Am J Hum Genet 58:1347–1363, 1996 8. Risch N: Linkage strategies for genetically complex traits: II. The power of affected relative pairs: Am J Hum Genet 46:229–241, 1990 DIABETES, VOL. 47, OCTOBER 1998 1653 Author Queries (please see Q in margin and underlined text) Q1: In sentence beginning “This encoded a predicted protein… ,” please add word(s) after “This” to clarify its meaning. Thanks. Q2: Figure 2 legend—OK to change “of mRNA (2 µg polyA+)” to “2 µg of poly(A)+ mRNA,” as given in text?

Q3: Please provide ? rst name for Dr. Mohanakumar. Thanks. Q4: Were the 6 alleles for AF047018 (as well as the 11 alleles for AF047019) observed among Ashkenazi Jews? Current punctuation of sentence indicates that they were not. Q5: Please provide city and state in which Research Genetics, Inc. is located. Thanks. Q6: Please provide expansion of the abbreviation CEPH. Thanks. Q7: Is “centiRays (cR)”. Fig. 3 legend—Is “radiation hybrid” the correct expansion of “RH,” shown in part A? >