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Title: MOLECULAR CLONING, CHARACTERIZATION AND EXPRESSION OF CHICKEN AMP-ACTIVATED PROTEIN KINASE SUBUNIT GENES

Author
item Proszkowiec-Weglarz, Monika
item Richards, Mark
item Poch, Stephen

Submitted to: BARC Poster Day
Publication Type: Abstract Only
Publication Acceptance Date: 4/6/2006
Publication Date: 4/26/2006
Citation: Proszkowiec-Weglarz, M., Richards, M.P., Poch, S.M. 2006. Molecular cloning, characterization and expression of chicken AMP-activated protein kinase subunit genes [abstract]. Seventeenth Annual BARC Poster Day. p. 40, Abstract Number 32.

Interpretive Summary:

Technical Abstract: AMP-activated protein kinase (AMPK) is a trimeric enzyme complex that consists of one catalytic (alpha) and two regulatory (beta and gamma) subunits. AMPK is involved in the regulation of cellular energy homeostasis and, on the whole animal level, in regulation of energy balance and food intake. There are two known isoforms for the alpha and the beta subunits and three for the gamma subunit. Each of the subunit isoforms is encoded by a separate gene. AMPK is activated through phosphorylation by an upstream kinase (LKB1 or CaMKK). Active AMPK, in turn, phosphorylates a variety of downstream protein targets that affect carbohydrate, protein, and lipid metabolism. Because so little is known about chicken AMPK subunit genes, the present study was designed to clone, sequence and characterize the expression of the seven gene homologues. A molecular cloning strategy involving primer-directed reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (5’-RACE) was developed to sequence cDNAs corresponding to complete coding regions and portions of the 5’- and 3’-untranslated regions (UTRs) of chicken AMPK subunit mRNA transcripts. AMPK alpha-1, alpha-2, beta-1 and beta-2 genes code for predicted proteins containing 560, 552, 273 and 272 amino acids, respectively. The homology among the alpha and beta subunit isoform proteins was 74 and 75%, respectively. The AMPK beta-2 gene produces multiple transcript variants that differ at the 5’ ends (5’-UTRs); however, all code for the same predicted amino acid sequence. The presence or absence of a single coding exon by alternative splicing of gamma-1 gene transcripts resulted in two variants that code for predicted proteins containing 298 or 276 amino acids. Use of an alternate transcription initiation site and promoter and/or alternative splicing of gamma-2 gene transcripts resulted in four different variants that code for predicted proteins containing 567, 452, 328 and 158 amino acids. Alternative splicing of exon 3 in the gamma-3 gene resulted in a shift of the open reading frame and the production of ‘long’ and ‘short’ transcript variants that code for predicted proteins of 382 and 378 amino acids. Each of the AMPK subunit genes exhibited a unique tissue-specific expression pattern. In general, AMPK subunit genes displayed similar structures and high sequence homology in the chicken as compared to corresponding mammalian homologues. Understanding chicken AMPK subunit gene structure and expression patterns provides new insight into the role of the AMPK pathway in metabolic regulation in poultry (USDA ARS CRIS Project No. 1265-3100-087-00).