Isolation of a rat liver Golgi mannosidase II clone by mixed oligonucleotide-primed amplification of cDNA.
A clone encoding Golgi mannosidase II (MII; GlcNAc-transferase I-dependent alpha 1,3(alpha 1,6) mannosidase), an enzyme involved in asparagine-linked oligosaccharide processing, was isolated from a rat liver lambda gt11 cDNA library by a method that employs a modification of the polymerase chain reaction. Specific oligonucleotide primers were designed from two regions of protein sequence and were combined in an amplification reaction with a single-stranded cDNA preparation derived from rat liver poly(A)+ RNA. Based upon mapping of the protein sequences 42 kDa apart on the MII polypeptide, the procedure was expected to generate an approximately 1150-base-pair amplification product representing a segment of the MII gene between the two primer regions. The size of the amplified product (1170 base pairs) was in close agreement with this predicted fragment size. The authenticity of the amplified fragment was confirmed by the agreement of the DNA sequence with additional protein sequence data. A 1474-base-pair clone was isolated from a cDNA library by plaque hybridization using the amplification fragment as a radiolabeled probe. The nucleotide sequence of this clone predicts a single continuous open reading frame and, based upon a polypeptide molecular mass of 117 kDa for the enzyme subunit, is consistent with the clone representing approximately 50% of the coding sequence of MII. Both the clone and the amplification product hybridized to a rat liver mRNA of approximately 8 kilobases, a message size approximately 4.7 kilobases larger than the size of the predicted open reading frame. This extensive non-coding information on the MII message is a feature common to two other Golgi processing enzymes, both of which contain most of the non-coding information on the 3' end of their messages. The function of these disproportionately large untranslated regions is not clear.
Moremen
K W
KW
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D005784:Gene Amplification; D005796:Genes; D006056:Golgi Apparatus / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008361:Mannosidases / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014158:Transcription, Genetic
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D004247:DNA; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase