Molecular cloning of a cDNA complementary to a UDP-glucose pyrophosphorylase mRNA of dictyostelium discoideum.
Uridine diphosphoglucose pyrophosphorylase (UTP: -alpha-D-glucose-1-phosphate uridyltransferase, EC 2.7.7.9) is an essential enzyme for normal development of Dictyostelium discoideum and its specific activity increases 3- to 10-fold by the later stages of development. Previous experiments have shown that additional forms of the enzyme appear concomitantly with this increase and that two uridine diphosphoglucose pyrophosphorylase (UDPGP) polypeptides are immunoprecipitated from the in vitro translation products of total cellular RNA at any stage of development (B. F. Fishel, R. E. Manrow and R. P. Dottin, 1982, Dev. Biol. 92, 175-187). Using an in vitro translation-immunoprecipitation assay of UDPGP mRNA, we show that an increase in the amount of translatable mRNA is correlated with the accumulation of enzyme during development. A cDNA bank was constructed from a mRNA population that had been enriched for UDPGP mRNA by size fractionation on sucrose gradients containing methylmercuric hydroxide (C. W. Schweinfest, R. W. Kwiatkowski, and R. P. Dottin, 1982, Proc. Natl. Acad. Sci. USA 79, 4997-5000). A 1.8-Kb cDNA complementary to a UDPGP mRNA was identified after screening the bank by hybridization selection and translation. Only the mRNA encoding the higher molecular weight in vitro translation product is hybrid selected by this cDNA. In hybrid-arrested translation experiments, the coding strand of this cDNA selectively inhibits the translation of only one of the two in vitro translation products. Therefore, there are two distinct UDPGP mRNAs.
Fishel
B R
BR
Ragheb
J A
JA
Rajkovic
A
A
Haribabu
B
B
Schweinfest
C W
CW
Dottin
R P
RP
D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004023:Dictyostelium / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000235:genetics*
D012333:RNA, Messenger; D004247:DNA; D009713:Nucleotidyltransferases; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase