Showing papers by "Michael Snyder published in 1986"

Bent DNA at a yeast autonomously replicating sequence

Abstract: DNA fragments that show retarded electrophoretic mobility through poly aery lamide gels have been found in both prokaryotes and eukaryotes1–7. In the case of kinetoplast DNA, evidence has been presented that the DNA is curved or ‘bent’8,9. Bent DNA has previously been found at the λ and simian virus 40 (SV40) DNA replication origins6,7. Here we show the existence of bent DNA at a yeast autonomously replicating sequence (ARS1), a putative replication origin. The bent DNA has been localized to a 40–55 base pair (bp) segment and contains six (A)3–5 stretches (that is, six poly(A) stretches, three to five nucleotides in length) phased approximately every 10.5 bp. This region contains a DNA binding site for a yeast protein factor. This site lies at the 3′ end of the TRP1 gene, in a region devoid of nucleosomes, and is positioned 80 bp away from the ARS consensus sequence; removal of this region impairs ARS function in vivo. The bent DNA may be involved in transcription termination or the prevention of nucleosome assembly in this region.

Rapid mapping of antigenic coding regions and constructing insertion mutations in yeast genes by mini-Tn10 "transplason" mutagenesis

Abstract: A "transplason" mutagenesis procedure was developed for the dual purposes of low resolution mapping of antigenic coding regions (using transposons) and constructing insertion mutations in yeast genes (by transplacement). Mini-Tn10 transposon derivatives containing both Escherichia coli and yeast selectable markers have been constructed. These elements are used to mutagenize lambda gt11 clones that express foreign antigens in E. coli. The transposition events are first selected in E. coli, and the effect of these insertions on antigen expression is used to locate the antigenic coding regions on the cloned DNA. Insertion mutations located within a desired yeast sequence are then substituted for the genomic copies by one-step gene transplacement. This provides a powerful method for rapidly mapping antigenic coding sequences of cloned genes and inactivating these genes in yeast to help determine their function. Several examples using this technique are presented.