Microsatellite instability in Drosophila spellchecker1 (MutS homolog) mutants

Abstract

We have cloned a mutS homolog from Drosophila melanogaster called spellchecker1 (spel1) and have constructed spel1 mutant f lies. MutS proteins promote the correction of DNA mismatches and serve important roles in DNA replication, recombination, and repair. The spel1 gene belongs to a subfamily of mutS first characterized by the MSH2 gene of yeast and which also includes hMSH2, one of the two major hereditary nonpolyposis colon cancer loci of humans. Like msh2 mutants in other species, we find that f lies lacking the spel1 gene suffer a highly increased rate of instability in long runs of dinucleotide repeats when analyzed after 10-12 f ly generations. Using a new assay, we have also discovered that mutations in spel1 decrease the stability of a dinucleotide repeat when it is copied into the site of a double-strand break during gene conversion. Contrary to the case in mammalian cells, spel1 deficiency does not affect tolerance of f lies to a methylating agent nor does it affect resistance to ␥-irradiation.

Functional Conservation of Subfamilies of Putative UDP-N-acetylgalactosamine:Polypeptide N-Acetylgalactosaminyltransferases inDrosophila, Caenorhabditis elegans, and Mammals

“…Further support is found in analysis of sequence polymorphisms in the region that shows high conservation of the dGalNAc-T1 reading frame, whereas a complementary putative reading frame had multiple nonconservative polymorphisms and even deletion of the potential start ATG codon (Table IV). In summary, these data provide strong evidence that the 5-kb rescue construct used by Flores and Engels (25) indeed, as proposed by these authors, rescued dGalNAc-T1 deficiencies. Further proof may be obtained by ongoing complementation studies with smaller constructs containing the intact dGalNAc-T1 open reading frame as well as constructs where the catalytic unit or the lectin domain of dGalNAc-T1 has been mutated.…”

“…Thus, the first well characterized example of a GalNAc-transferase gene knock-out in eukaryotes is the l(2)35Aa gene encoding dGalNAc-T1. Deletion mutations in the l(2)35Aa gene were recessive lethal, and the phenotype was reversible by complementation when the intact gene was expressed ectopically (25).…”

“…Recently, a GalNAc-transferase homologous gene in the parasite Toxoplasma gondii was also determined to encode a GalNAc-transferase with GalNAc glycopeptide substrate specificity (41). (25) searched for the candidate gene of the lethal phenotype associated with l (2)35Aa and demonstrated that a 5-kb rescue fragment contained an open reading frame encoding a protein with similarity to polypeptide GalNAc-transferases. Analysis of the 5-kb rescue fragment reveals only one conventional open reading frame that predicts a protein with similarity to known proteins and from which ESTs have been mapped (Fig.…”

“…A developmental disorder resulting in rotated abdomen of adult flies is assigned to a defect in the putative polypeptide O-mannosyltransferase encoded by the rt gene (23,24). Flores and Engels (25) have reported that a gene homologous to mammalian polypeptide GalNAc-transferases, the l(2)35Aa gene, is likely to be essential for development of D. melanogaster. Transgenic flies expressing an ectopic 5-kb fragment containing the l(2)35Aa gene were found to be viable and fertile (25).…”

“…Flores and Engels (25) have reported that a gene homologous to mammalian polypeptide GalNAc-transferases, the l(2)35Aa gene, is likely to be essential for development of D. melanogaster. Transgenic flies expressing an ectopic 5-kb fragment containing the l(2)35Aa gene were found to be viable and fertile (25). The 5-kb rescue fragment contains essentially only one predicted intact open reading frame, and this encodes a protein with homology to GalNAc-transferases.…”