The expression of a gene for eukaryotic elongation factor Tu in Artemia during development. Translation of poly(A)+ RNA and the use of a synthetic oligonucleotide to detect the presence of eukaryotic elongation factor Tu-specific mRNA.
Total in vivo proteins from Artemia embryos at different developmental stages were examined by two-dimensional gel electrophoresis. A variety of peptides change during development, with one of them, the eukaryotic elongation factor Tu (eEF-Tu), presenting a dramatic increase from dormant embryos to nauplii. When poly(A)+ RNA is translated in vitro, the same relative increase is seen for eEF-Tu during development. Based on the amino acid sequence for Artemia eEF-Tu (Amons, R., Pluijms, W., Roobol, K., and Möller, W. (1983) FEBS Lett. 153, 37-42), a synthetic oligodeoxynucleotide was prepared and used to prime the synthesis of cDNA with poly(A)+ RNA from 12-h developing embryos as template. Direct sequence analysis of the 900-base primary cDNA product shows it to be specific for the 5' end of Artemia eEF-Tu mRNA. Hybridization of a "Northern" blot of denatured (poly(A)+ RNA from different developmental stages with this cDNA reveals a major band migrating at about 1800 bases, which increase in intensity as development proceeds, paralleling the increase in eEF-Tu seen by in vitro translation. When poly(A)+ RNA is separated on a nondenaturing gel, blotted to poly(U) paper, and hybridized with the eEF-Tu cDNA, a single band is observed migrating faster than 18 S. Elution and in vitro translation of this band results in a major product migrating with eEF-Tu in a dodecyl sulfate-polyacrylamide gel and which is precipitable with eEF-Tu-specific antibodies.
H A
HA
Daum
P W
PW
Bragg
D B
DB
Sittman
J N
JN
Dholakia
C L
CL
Woodley
A J
AJ
Wahba
D000595:Amino Acid Sequence; D000818:Animals; D001156:Artemia / Q000196:embryology*; D004591:Electrophoresis, Polyacrylamide Gel; D004625:Embryo, Nonmammalian; D005796:Genes; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D010444:Peptide Elongation Factor Tu / Q000235:genetics*; D011061:Poly A / Q000235:genetics*; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D010444:Peptide Elongation Factor Tu