Expression of the Volvox gene encoding nitrate reductase: mutation-dependent activation of cryptic splice sites and intron-enhanced gene expression from a cDNA

Plant Mol Biol. 1996 Apr;31(1):1-12. doi: 10.1007/BF00020601.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Chlorophyta / enzymology
  • Chlorophyta / genetics*
  • Cloning, Molecular
  • DNA, Complementary
  • Gene Expression Regulation, Enzymologic*
  • Gene Expression Regulation, Plant*
  • Introns*
  • Molecular Sequence Data
  • Nitrate Reductase
  • Nitrate Reductases / genetics*
  • RNA Splicing
  • Transformation, Genetic

Substances

  • DNA, Complementary
  • Nitrate Reductases
  • Nitrate Reductase

Associated data

  • GENBANK/J02459