Abstract
Deficiency of lysosomal beta-mannosidase activity results in a severe neurodegenerative disease in goats and cattle and a relatively milder phenotype in humans. A cDNA coding for the entire beta-mannosidase protein is described. Mixed oligonucleotides derived from bovine beta-mannosidase peptide sequences were used to screen a bovine thyroid cDNA library. Clones covering about 80% of the C-terminal region were recovered. The missing 5'-region was obtained using the technique of 5'-rapid amplification of cDNA ends. The composite cDNA contains 3852 nucleotides, encoding 879 amino acids. The N-terminal methionine is followed by 16 amino acids displaying the characteristics of a typical signal peptide sequence. The deduced amino acid sequence is colinear with all peptide sequences determined by protein microsequencing. Northern blot analysis demonstrates a single 4.2-kilobase transcript in various tissues from both normal and affected goats and calves. The mRNA level is decreased in tissues of affected beta-mannosidosis animals. The gene encoding beta-mannosidase is localized to human chromosome 4 as shown by Southern analysis of rodent/human somatic cell hybrids. This is the first report of cloning of lysosomal beta-mannosidase.
Highlights
Deficiency of lysosomal fJ-mannosidase activity results in a severe neurodegenerative disease in goats and cattle and a relatively milder phenotype in humans
Mixed oligonucleotides derived from bovine fJ-mannosidase peptide sequences were used to screen a bovine thyroid cDNA library
No open reading frame was found before the EcoRI site and the internal sequence homology with the 5' end of clones 46MJ4 and 47MJ4 ceased right at the EcoRI site of clone 46MJ4. These results clearly indicated that the EcoRI sites of these clones were not methylated by EcoRI methylase during the construction of this bovine thyroid cDNA library
Summary
Partial Amino Acid Sequencing-I3-Mannosidase protein was purified from 2.4-kg bovine kidney (Ada Beef Co., Ada, MI) using anti-Smannosidase monoclonal antibody as described [28, 29]. The purified protein was dialyzed against 5 mM ammonium bicarbonate solution, lyophilized, and submitted to the Keck Foundation Biotechnology ResourceLaboratory at Yale University for amino acid sequencing.Approximately740 pmol of purified protein (from a total of 1240 pmol)was subjected to CNBr/ trypsin digestion. Sequence information was obtained from 12 peptides. Regionswith minimal codonredundancy were chosento construct mixedoligonucleotide probes.Guessmers were constructed according to the typical codon usage frequency of human protein [30].
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