Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$      

Appointments and awards

Elliot Meyerowitz the Biology Department, Caltech, and a member of the Board of Directors of the ISPMB, has been named Fellow of the American Academy of Arts and Science.     

The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments

Here, a complete study is described of all the genes and isoenzymes for aspartate aminotransferase (AspAT) present in Arabidopsis thaliana . Four classes of cDNAs representing four distinct AspAT genes ( ASP1—ASP4 ) have been cloned from Arabidopsis . Sequence analysis of the cDNAs suggests that the encoded proteins are targeted to different subcellular compartments. ASP1 encodes a mitochondrial form of AspAT, ASP3 encodes a chloroplastic/plastidic form of AspAT, whereas ASP2 and ASP4 each encode cytosolic forms of AspAT. Three distinct AspAT holoenzymes (AAT1—AAT3) were resolved by activity gel analysis. Organelle isolation reveals that AAT1 is mitochondrial-localized, AAT3 is plastid-localized, and AAT2 is cytosolic. Gene-specific Northern analysis reveals that each Asp mRNA accumulates differentially with respect to organ-type. However, the individual Asp mRNAs show no dramatic fluctuations in response to environmental stimuli such as light. Southern analysis reveals that four distinct nuclear genes probably represent the entire AspAT gene family in Arabidopsis . These molecular studies shed light on the subcellular synthesis of aspartate in Arabidopsis and suggest that some of the AspAT isoenzymes may play overlapping roles in plant nitrogen metabolism.     

Transfer RNA genes in the cap-oxil region of yeast mitochondrial DNA.

A cytoplasmic "petite" (rho-) clone of Saccharomyces cerevisiae has been isolated and found through DNA sequencing to contain the genes for cysteine, histidine, leucine, glutamine, lysine, arginine, and glycine tRNAs. This clone, designated DS502, has a tandemly repeated 3.5 kb segment of the wild type genome from 0.7 to 5.6 units. All the tRNA genes are transcribed from the same strand of DNA in the direction cap to oxil. The mitochondrial DNA segment of DS502 fills a sequence gap that existed between the histidine and lysine tRNAs. The new sequence data has made it possible to assign accurate map positions to all the tRNA genes in the cap-oxil span of the yeast mitochondrial genome. A detailed restriction map of the region from 0 to 17 map units along with the locations of 16 tRNA genes have been determined. The secondary structures of the leucine and glutamine tRNAs have been deduced from their gene sequences. The leucine tRNA exhibits 64% sequence homology to an E. coli leucine tRNA.     

Assembly of the Mitochondrial Membrane System: Nuclear Suppression of a Cytochrome b Mutation in Yeast Mitochondrial DNA

In a previous study, a mitochondrial mutant expressing a specific enzymatic deficiency in co-enzyme QH₂-cytochrome c reductase was described (Tzagoloff, Foury and Akai 1976). Analysis of the mitochondrially translated proteins revealed the absence in the mutant of the mitochondrial product corresponding to cytochrome b and the presence of a new low molecular weight product. The premature chain-termination mutant was used to obtain suppressor mutants with wild-type properties. One such revertant strain was analyzed genetically and biochemically. The revertant was determined to have a second mutation in a nuclear gene that is capable of partially suppressing the original mitochondrial cytochrome b mutation. Genetic data indicate that the nuclear mutation is recessive and is probably in a gene coding for a protein involved in the mitochondrial translation machinery.     

Assembly of the mitochondrial membrane system: mutations in the pho2 locus of the mitochondrial genome of Saccharomyces cerevisiae.

Two mutants of Saccharomyces cerevisiae which show a loss of mitochondrial rutamycin-sensitive ATPase activity are described. Although phenotypically similar to mutants of the mitochondrial locus pho1 [F. Foury and A. Tzagoloff (1976) Eur. J. Biochem. 68, 113-119], these mutants define a second ATPase locus on the mitochondrial DNA (designated pho2), which is genetically unlinked to pho1. Analysis of recombination in crosses involving multiple antibiotic resistance markers indicates that the locus is in the segment of the genome between ery1 and oli2, very close to oli1. In fact it is proposed that the oli1 and pho2 mutations are in the same gene. Supporting evidence for this proposal includes: 1. The analysis of marker retention in petite mutants shows that the oli1 and pho2 loci were either retained or lost together in all cases. 2. Recombination frequencies of 0.05% or less are observed in crosses between the oli1 and pho2 loci. 3. When rho+ revertants are isolated from the pho2 mutants they frequently are oligomycin resistant. 4. pho2 mutants have an altered subunit 9 of the ATPase complex.