Isolation of null alleles of the Caenorhabditis elegans gly-12, gly-13 and gly-14 genes, all of which encode UDP-GIcNAc:: α-3-D-mannoside β1,2-N-acetylglucosaminyltransferase I activity

被引:11
作者
Chen, SH
Spence, AM
Schachter, H
机构
[1] Hosp Sick Children, Dept Struct Biol & Biochem, Toronto, ON M5G 1X8, Canada
[2] Univ Toronto, Dept Biochem, Toronto, ON M5S 1A8, Canada
[3] Univ Toronto, Dept Mol & Med Genet, Toronto, ON M5S 1A8, Canada
[4] Burnham Inst, La Jolla, CA 92037 USA
关键词
C; elegans; null mutations; N-acetylglucosaminyltransferase; N-glycan synthesis; developmental biology;
D O I
10.1016/S0300-9084(03)00009-9
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
UDP-GlcNAc: alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I (GnT I) is a Golgi-resident enzyme which transfers a GlcNAc residue in beta1,2 linkage to the Manalpha1,3Manbeta-terminus of (Manalpha1,6(Manalpha1,3)Manalpha1,6)(Manalpha1,3)Manbeta1,4GlcNAcbeta1,4GlcNAc-Asn-protein, thereby initiating the synthesis of hybrid N-glycans. Three Caenorhabditis elegans genes homologous to mammalian GnT I (designated gly-12, gly-13 and gly-14) have been cloned. All three cDNAs encode proteins with GnT I enzyme activity. We report in this paper the preparation by ultra-violet (UV) light irradiation in the presence of trimethylpsoralen, of mutants lacking either gly-12, gly-13 or gly-14. A double null mutation in the gly-12 and gly-14 genes (gly-14; gly-12) has also been prepared. These mutations are intragene deletions, removing large portions of the GnT I catalytic domain, and are therefore, all molecular nulls. The gly-12 and gly-14 mutants as well as the gly-14; gly-12 double mutant all displayed wild-type phenotypes, indicating that neither gly-12 nor gly-14 is necessary for worm development under standard laboratory conditions. In contrast, about 60% of the mutants lacking the gly-13 gene arrested as L1 larvae at 20 degreesC and the remaining 40% homozygous worms grew to adulthood but displayed severe morphological and behavioral defects despite the presence of the other two GnT I genes, gly-12 and gly-14. Attempts to rescue the gly-13 null phenotype with the wild type transgene were not successful. However, lethality co-segregated with the gly-13 deletion within 0.02 map units (mu) in genetic mapping experiments, suggesting that the gly-13 mutation is responsible for the phenotype. (C) 2003 Editions scientifiques et medicales Elsevier SAS and Societe francaise de biochimie et biologie moleculaire. All rights reserved.
引用
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页码:391 / 401
页数:11
相关论文
共 26 条
[1]  
Ausubel FM., 1993, Current Protocols in Molecular Biology
[2]  
BARTON MK, 1990, GENETICS, V125, P29
[3]  
BRENNER S, 1974, GENETICS, V77, P71
[4]   Expression of three Caenorhabditis elegans N-acetylglucosaminyltransferase I genes during development [J].
Chen, SH ;
Zhou, SH ;
Sarkar, M ;
Spence, AM ;
Schachter, H .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (01) :288-297
[5]   UDP-N-acetylglucosamine:: α-3-D-mannoside β1,2-N-acetylglucosaminyltransferase I and UDP-N-acetylglucosamine:: α-6-D-mannoside β-1,2-N-acetylglucosaminyltransferase II in Caenorhabditis elegans [J].
Chen, SH ;
Tan, J ;
Reinhold, VN ;
Spence, AM ;
Schachter, H .
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 2002, 1573 (03) :271-279
[6]  
Edgley ML, 1995, METHOD CELL BIOL, V48, P147
[7]  
Epstein HF, 1995, CAENORHABDITIS ELEGA, pxxi659
[8]   Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J].
Fire, A ;
Xu, SQ ;
Montgomery, MK ;
Kostas, SA ;
Driver, SE ;
Mello, CC .
NATURE, 1998, 391 (6669) :806-811
[9]   Characterization of mutations induced by ethyl methanesulfonate, UV, and trimethylpsoralen in the nematode Caenorhabditis elegans [J].
Gengyo-Ando, K ;
Mitani, S .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2000, 269 (01) :64-69
[10]   PAR-1, A GENE REQUIRED FOR ESTABLISHING POLARITY IN C-ELEGANS EMBRYOS, ENCODES A PUTATIVE SER/THR KINASE THAT IS ASYMMETRICALLY DISTRIBUTED [J].
GUO, S ;
KEMPHUES, KJ .
CELL, 1995, 81 (04) :611-620