The sheep erythropoietin gene: molecular cloning and effect of hemorrhage on plasma erythropoietin and renal/liver messenger RNA in adult sheep.
Erythropoietin (Epo) production was studied in adult sheep. Nine ewes, body weight (BW) 39 +/- 1.3 kg, were hemorrhaged a volume of blood equivalent to 1.6% BW, and sampled at 0, 2, 4, 6, 24 h. Oxygen content (O2 CT) decreased by 2.7 +/- 0.6 ml/dl at 2 h. Plasma immunoreactive (IR) Epo was only significantly increased at 24 h, from 18.5 +/- 3.5 to 40 +/- 10.7 mU/ml (mean +/- SEM). A further 5 ewes were bled extensively (2793 +/- 82 ml) over 54 h, and killed for Epo mRNA determination. The O2 CT decreased from 12.3 +/- 1.6 to 4.1 +/- 0.6 ml/dl, and plasma Epo increased from 15 +/- 4 to 1675 +/- 287 mU/ml. The sequence of ovine Epo cDNA was derived from the kidney RNA of a severely bled sheep using reverse transcription/polymerase chain reaction (RT/PCR), and from an ovine Epo genomic clone. The cDNA encodes a peptide of 194 amino acids, including a 27 amino acid signal peptide. The deduced amino acid sequence of sheep Epo shows 82%, 78% and 80% homology with mature Epos of human, mouse and monkey, respectively. The gene structure resembles closely those of human and mouse, with 5 exons and 4 introns. The expression of the ovine Epo gene in tissues from normal and hemorrhaged sheep was analysed by a competitive RT/PCR method. Epo mRNA was difficult to detect in liver from normal sheep, but was detectable at 0.01-0.04 amol/microgram total RNA in kidney from normal sheep. In the kidneys of severely bled sheep, the Epo mRNA levels (per micrograms total RNA) increased 400-1500-fold compared to that of normal kidneys, and were approximately 60-fold greater than those in the livers of the hemorrhaged sheep.
Fu
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Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria, Australia.
Evans
B
B
Lim
G B
GB
Moritz
K
K
Wintour
E M
EM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004921:Erythropoietin / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D005796:Genes; D000882:Haplorhini / Q000235:genetics; D006470:Hemorrhage / Q000503:physiopathology*; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D012756:Sheep / Q000235:genetics*; D013045:Species Specificity
D012333:RNA, Messenger; D004921:Erythropoietin; D004247:DNA