Developmental expression of chicken antithrombin III is regulated by increased RNA abundance and intracellular processing.
We isolated and sequenced a 432 bp cDNA to cAT-III, that encoded 115 nucleotides of 5' untranslated sequence, a 17 amino acid long signal peptide and residues 1-88 of the mature protein, and used it to prepare a probe for measuring and correlating the developmental changes of steady-state cAT-III mRNA levels with known changes in antigen levels. Densitometric analysis of nuclease protection (n = 2), Northern blot (n = 4), and slot blots (n = 3) of total RNA from chick livers of 16-day-old embryos to 6-day-old chicks showed a 2.6 +/- 0.5-fold increase in steady-state cAT-III mRNA levels. Assay of functional mRNA levels by in vitro translation of poly(A)+ RNA and specific immunoprecipitation of 35S-Met-labelled cAT-III was comparable to RNA analysis (16-day-old embryos vs. 10-day-old hatchlings). We evaluated whether there were developmental differences in post-translational secretion which may also contribute to the regulation of the circulating level of this protein. Pulse-chase studies of freshly-isolated hepatocytes from 16-day-old embryos and 10-day-old hatchlings maintained in suspension demonstrated a approx. 5.0-5.5-fold increase in cAT-III levels at steady-state secretion. The above findings indicate that changes in circulating cAT-III levels during late embryonic development are primarily due to increased abundance of cAT-III mRNA. In addition, we postulate that post-translational intracellular processing may account for further differences in circulating protein levels.
Amrani
D L
DL
Department of Health Sciences, University of Wisconsin-Milwaukee 53201.
Rosenberg
J
J
Samad
F
F
Bergtrom
G
G
Banfield
D K
DK
D000818:Animals; D000990:Antithrombin III / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D002642:Chick Embryo; D004247:DNA / Q000302:isolation & purification; D005786:Gene Expression Regulation; D007700:Kinetics; D008099:Liver / Q000196:embryology / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis*
D012333:RNA, Messenger; D000990:Antithrombin III; D004247:DNA