Functional exploration of the yeast (Saccharomyces cerevisiae) genome: use of a mini-Mu transposon to analyse randomly cloned DNA sequences.
The development of mega-sequencing techniques requires new methods for global functional analysis of cloned DNA fragments. We have developed a mini-Mu transposon adapted to yeast cloned DNA fragment analysis. This transposon allows us to do the following in a single construction: (i) to probe yeast cells for the presence of expressed open reading frames (ORFs) in the cloned DNA fragment; (ii) to localize these ORFs in the fragment and determine their transcription orientation; (iii) to use beta-galactosidase protein fusions to study regulation of these ORFs; and (iv) to disrupt the corresponding chromosomal genes. On a 5-kb yeast DNA sequence, we have verified the reliability of this new tool by comparing the data obtained with the mini-Mu transposon to those obtained by classical methods. This transposon should be of immediate use in the yeast genome sequencing programme.
Daignan-Fornier
B
B
Laboratoire de Génétique Moléculaire, Université Paris-Sud, Orsay, France.
Bolotin-Fukuhara
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004271:DNA, Fungal / Q000235:genetics*; D005786:Gene Expression Regulation; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012331:RNA, Fungal / Q000032:analysis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014158:Transcription, Genetic
D004251:DNA Transposable Elements; D004271:DNA, Fungal; D012331:RNA, Fungal