Expression of the Volvox gene encoding nitrate reductase: mutation-dependent activation of cryptic splice sites and intron-enhanced gene expression from a cDNA.
Use of the nitrate reductase encoding gene (nitA) as selection marker has facilitated the successful nuclear transformation of Volvox carteri. The Volvox nitA gene contains 10 introns. A stable nitA mutation in the Volvox recipient strain 153-81 resides in a G-to-A transition of the first nucleotide in the 5' splice site of nitA intron 2. This mutation resulted in at least three non-functional splice variants, namely: (1) intron 2 was not spliced at all; (2) a cryptic 5' splice site 60 nt upstream or (3) a cryptic 5' splice site 16 nt downstream of the mutation were activated and used for splicing. When we used nitA cDNA (pVcNR13) for transformation of V. carteri 153-81, a low efficiency of about 5 x 10(-5) transformants per reproductive cell was observed. Re-integration of either intron 1 (pVcNR15) or introns 9 and 10 (pVcNR16) in the transforming cDNA increased transformation rates to 5 x 10(-4). In parallel, pVcNR15-transformed Volvox exhibited growth rates that were 100-fold increased over the pVcNR13-transformed alga. This intron-enhancement of nitA gene expression appears to be associated with post-transcriptional processing and 'channelling' of the message. These data suggest an important role of splicing for gene expression in V. carteri.
Gruber
H
H
Lehrstuhl für Genetik, Universität Regensburg, Germany.
Kirzinger
S H
SH
Schmitt
R
R
D001483:Base Sequence; D000460:Chlorophyta / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D007438:Introns; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics*; D012326:RNA Splicing; D014170:Transformation, Genetic
D018076:DNA, Complementary; D009565:Nitrate Reductases; D050901:Nitrate Reductase