A mutant approach and molecular strategy to study fungal cell walls.
The current study describes recombinant plasmids which complement the hypersusceptibility to killing bleomycin of blm1-1 mutant cells of Saccharomyces cerevisiae, and a strategy developed and used to recover active clones from a stable yeast genomic library. The resistance of a spontaneous revertant isolated from the original blm1-1 mutant strain and of mutant cells transformed with each of several recombinant plasmids which complemented the recessive blm1-1 mutation was comparable to the resistance of the parental (non-mutant) strain from which the original blm1-1 mutant was derived. The strategy for cloning S. cerevisiae DNA was based on complementation and in situ hybridization. This strategy employed 32P-labelled 6.6-kb BamHI and 3.8-kb BamHI-ClaI probes from a cloned DNA fragment to recover clones which either fully or partially complemented the hypersensitivity of mutant cells to killing by bleomycin. This method considerably reduced the time and effort required to recover biologically active clones from a genomic library.
Pramanik
A
A
Department of Microbiology and Immunology, City University of New York Medical School, NY 10031, USA.
Martinez
M
M
McKoy
J F
JF
Ospina
R
R
Daly
J
J
Moore
C W
CW
D001761:Bleomycin / Q000737:chemistry / Q000494:pharmacology*; D002473:Cell Wall / Q000737:chemistry / Q000187:drug effects*; D004271:DNA, Fungal / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli; D005656:Fungal Proteins / Q000378:metabolism; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008562:Membrane Glycoproteins / Q000378:metabolism; D015394:Molecular Structure; D016296:Mutagenesis; D010084:Oxidation-Reduction; D010692:Phleomycins / Q000737:chemistry / Q000494:pharmacology*; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D014170:Transformation, Genetic
D004271:DNA, Fungal; D004274:DNA, Recombinant; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D010692:Phleomycins; C025363:mannoproteins; D001761:Bleomycin