Sequence analysis of zein cDNAs obtained by an efficient mRNA cloning method

Abstract

A cDNA library was generated from mRNA Isolated from the developing endosperm of W22 maize inbred. cDNA clones for zeln, the maize storage protein family, were isolated and analyzed by DNA sequencing. The DNA sequences of four clones containing cDNA coples of mRNAs belonging to one zeln subfamily were determined. The data support the following conclusions: a) genes encoding the larger of the two zeln species contain eleven instead of nine repeat units within the coding sequence of the gene; b) transcription can be terminated at either of the two polyadenlatlon signals and c) transcription starts 31 basepairs downstream from the first T In the TATA box. To facilitate this analysis a new method for the construction of cDNA libraries was developed. The mRNA was annealed to linearized and oligo-dT tailed pUC9 plasmld DNA, which then primed synthesis of the first strand of the cDNA. Oligo-dG tails were added to the cDNA-plasmld molecules, which were then centrlfuged through an alkaline sucrose gradient. The gradient step removed small molecules and separated ttie two cONAs which were formerly attached to the same double stranded plasmld molecule. An excess of ollgo-dG tailed denatured pUC9 DNA was added and the DNA was renatured under conditions that favor the circularlzatlon of monomers by the ollgo-dC and ollgo-dG tails. The oligo-dC tall served as primer for the synthesis of the second strand of the cDNA. The library was screened by colony hybridization using ³²P-labelled cDNA and DNA from genomic zeln clones as probes. We obtained 20,000 clones hybridizing total cDNA starting with 1 µg of plasmld DNA and 1 µg of mRNA.