his3-delta200 and PET56

Karen Sirum-connolly KSIRUM at deimos.oit.umass.edu
Mon Aug 7 11:15:06 EST 1995


Dear Colin, Ed, Mike, and anyone else using the his3-delta200 allele,
	The PET56 gene encodes a ribose methyltransferase that specifically 
modifies a universally conserved guanine in the peptidyl transferase center of
the yeast mitochondrial large subunit ribosomal RNA.  Null alleles of pet56
convert to rho- or rho0 cytoplasmic petites, strains that have lost portions
or all of their mitochondrial genomes. This loss of mitochondrial DNA is a
hallmark of loss-of-function mutations in genes essential for the formation of
a functional mitochondrial translation apparatus. 
	Struhl (1985, PNAS 82:8419-8423), in his studies on the promoter
region of HIS3, made the observation that strains harboring the his3-delta200
allele and expressing only 10-20% of wild type levels of PET56, resemble
petites in that there is a higher proportion of small (petite) clonies when
the cells are grown at 30 degrees on a fermentable carbon source (glucose),
hence the name _PET_56.
	The first indication of a connection between PET56 function and the
mitochondrial ribosome came from our observation that the his3-delta200 allele
markedly enhanced the leaky phenotype of a null allele of MRP49, the nuclear
gene for a protein of the large subunit of the mitochondrial ribosome.  We
found that his3-delta200 mutants are respiratory deficient when grown at
18 degrees (and they convert to rho- or rho0 at a high frequency), and when
grown at 30 degrees, these cells contain near wild type levels of the
mitochondrial small subunit but have only 25% of wild type levels of the
mitochondrial large subunit (Sirum-Connolly and Mason, 1993, Science
262:1886-1889). We also showed that other his3- alleles did not enhance the
leaky mrp49 phenotype.
	I have not tried to sporulate a his3-delta200 homozygous diploid,
but I have observed that haploid his3-delta200 cells convert to rho- at a
relatively high frequency when the cells are left in the refrigerator too 
long or when they are left on my bench in a saturated culture for too long. 
Colin, providing PET56 function on a plasmid should solve your sporulation 
problem and I would be happy to provide you with a suitable plasmid (let me 
know what you need in terms of markers). Since others have been able to 
sporulate his3-delta200 diploids, maybe the gene you are trying to disrupt
is involved in mitochondrial biogenesis (ribosomes?) and one copy of the gene
in the diploid is not enough when the Pet56 methylase is present at reduced
levels.
	Last year in this newsgroup there was a discussion about other
his3 deletion strains and plasmids available that apparently don't have this
problem. I am interested in hearing from anyone else who has a his3-delta200
story to tell: I may learn something new about one of my favorite
genes, PET56.

Sincerely,

Karen Sirum-Connolly
Department of Biochemistry and Molecular Biology
University of Massachusetts
Amherst, MA 01003-4505
phone: (413)545-0469
fax: (413)545-3291
email: ksirum at biochem.umass.edu




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