Differential translatability of antifreeze protein mRNAs in a transgenic host.
The expression of fusion gene constructs containing Drosophila regulatory sequences and the structural portions of fish antifreeze protein genes have been examined by transfer into Drosophila melanogaster using P elements. A fusion gene, containing the enhancer, promoter, and cap site of the yolk polypeptide 1 gene, joined in the 5'-untranslated region to the structural portion of the winter flounder type I antifreeze gene, was transcribed in mature female transformants to give an mRNA of the predicted size, but no antifreeze protein was detected by Western blotting. When the same antifreeze protein gene was fused to a Drosophila hsp 70 gene regulatory region and placed downstream of the yolk polypeptide gene enhancer, appropriate expression of mRNA was directed by both gene regulatory elements. However, a translation product from this mRNA was only observed under heat shock conditions and was present at low levels. It is suggested that type I antifreeze mRNA, with its high content of alanine codons and their grouping into clusters of up to seven in a row, is poorly translated when in competition with other host mRNAs. In agreement with this hypothesis, a fusion gene construct between the yolk protein gene regulatory region and two type III antifreeze protein genes produced sub-mmolar concentrations of antifreeze protein in mature females from each of several transgenic lines analysed. The type III antifreeze protein does not have an imbalanced amino acid composition or sequence irregularities, and may be an appropriate choice for conferring freeze protection to frost-susceptible hosts by gene transfer.
Rancourt
D E
DE
Department of Biochemistry, Queen's University Kingston, Ontario, Canada.
Davies
P L
PL
Walker
V K
VK
D000818:Animals; D030801:Animals, Genetically Modified; D021301:Antifreeze Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D005260:Female; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D014170:Transformation, Genetic
D021301:Antifreeze Proteins; D006023:Glycoproteins