Characterization of a bovine cDNA encoding citrate synthase, and presence of citrate synthase mRNA during bovine pre-attachment development.
Citrate synthase is a key regulatory metabolic enzyme that catalyzes the first step in the tricarboxylic acid (TCA) cycle, the synthesis of citrate from acetyl coenzyme A and oxaloacetate. Aerobic metabolism via the TCA cycle is high in bovine embryos at the 4-cell stage then decreases until the compact morula stage before increasing at the expanded blastocyst stage. This study characterizes the presence of citrate synthase mRNA in bovine pre-attachment embryos to determine if a variation in mRNA transcript expression patterns is associated with previous reports of the patterns of TCA cycle activity. The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect citrate synthase mRNA from the 1-cell to blastocyst stage of bovine embryo development, and in embryos cultured under either an atmosphere of 5% CO(2) in air or 5% CO(2)/5% O(2)/90%N(2). The nucleotide sequence encoding citrate synthase was determined from bovine heart cDNA by the rapid amplification of cDNA ends (RACE) technique. This 1455-bp nucleotide fragment contained an open reading frame that encoded a deduced protein of 466 amino acids. The bovine nucleotide sequence was 92.1% and 93.8% identical to the human and porcine coding sequence, respectively. The amino acid sequence predicted from the bovine sequence is 95.1% identical to the human sequence and 96.3% identical to the porcine sequence. The porcine sequence contains a stop codon that results in a peptide truncated by 2 amino acids. The detection of citrate synthase transcripts from the 1-cell to blastocyst stage demonstrates that the decrease in TCA cycle activity observed following the 4-cell stage is not associated with an absence of citrate synthase mRNA.
Winger
Q A
QA
Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843-4466, USA.
Hill
J R
JR
Watson
A J
AJ
Westhusin
M E
ME
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000378:metabolism; D002417:Cattle; D002950:Citrate (si)-Synthase / Q000096:biosynthesis* / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D004622:Embryo, Mammalian / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013552:Swine
D018076:DNA, Complementary; D012333:RNA, Messenger; D002950:Citrate (si)-Synthase