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Identification, Isolation, and Cloning of Growth Hormone (GH)-Inducible Interscapular Brown Adipose Complementary Deoxyribonucleic Acid from GH Antagonist Mice¹

Edison Biotechnology Institute (Y.L., B.K., J.J.K.), Molecular and Cellular Biology Program, Department of Biological Sciences (Y.L.), and Department of Biomedical Sciences (J.J.K.), Ohio University College of Osteopathic Medicine, Athens, Ohio 45701

Address all correspondence and requests for reprints to: John J. Kopchick, Ph.D., Konneker 206A, Edison Biotechnology Institute, Ohio University, Athens, Ohio 45701-2979. E-mail: kopchick{at}ohiou.edu

In a dwarf mouse line that expresses a GH antagonist, we found that the interscapular brown adipose tissue (iBAT) mass is significantly greater than that in nontransgenic littermates. We proposed that gene expression in iBAT may be up- or down-regulated by GH. To identify these genes, we employed the PCR-select subtraction approach to construct subtractive libraries from iBAT total RNAs. We have generated forward and reverse subtractive libraries. Clones were screened by differential hybridization and identified by BLAST similarity to expressed sequence tags and complementary DNA sequences. Four novel expressed sequence tags were isolated from the reverse subtractive library. Of them, clone 42, was further analyzed. It encodes a 2475-bp messenger RNA with an open reading frame of 346 amino acids. Northern blot analysis demonstrated two RNA isoforms (2.5 and 1.3 kb) in various tissues. Differential expression of both isoforms was verified in GH antagonist and nontransgenic mouse iBAT. BLAST searches suggested that clone 42 is highly homologous to a gene found in a human female fetal brain and a related gene found in a human pituitary tumor.

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