Molecular cloning and chromosomal localization of human 4-beta-galactosyltransferase.

Abstract

A cDNA clone to human 4-beta-galactosyltransferase (EC 2.4.1.38) was isolated from a human liver lambda gt11 expression library by using a monospecific polyclonal antiserum to affinity-purified bovine enzyme. The authenticity of this cDNA clone has been demonstrated by several criteria. Under conditions of chronic treatment with the beta-adrenergic receptor agonist isoproterenol, rat parotid glands show an approximately 10-fold increase in 4-beta-galactosyltransferase activity. The increased enzyme activity was reflected in dot-blot analysis of control and isoproterenol-treated rat parotid RNA by using the human cDNA as probe. Hybrid-selection and in vitro translation identified a protein product with a molecular mass of 47 kDa that was immunoprecipitated with the bovine antiserum. The full-length human cDNA clone was then isolated and the DNA sequence for the NH2-terminal portion of the protein was deduced. Comparison of the NH2-terminal protein sequence from the bovine protein with that of the human cDNA clone confirmed its identity. In addition, the human cDNA clone was used to localize the gene for 4-beta-galactosyltransferase to human chromosome 4 by Southern analysis of a somatic cell hybrid panel.

Increased expression of a 58-kDa protein kinase leads to changes in the CHO cell cycle.

“…Human adult and fetal liver (Clontech) Agtll cDNA libraries were screened using the Ahp58-1 cDNA isolate and sequenced as described (10). A site-directed mutant cDNA encoding [Gly']p58 was made using an oligodeoxynucleotide (5'-AACAGGATCGGGGAGGGCACC-3') and then sequenced to confirm mutagenesis by standard procedures (11).…”

Localization of glycosylation sites in the golgi apparatus using immunolabeling and cytochemistry

“…At present we completely lack information how the array of different functional compartments is established and maintained and what the molecular basis for cell-type related differences in the functional subdivision of the Golgi apparatus is. With the availability of cloned genes for various enzymes involved in glycosylation (Humphreys-Beher et al, 1987;Narimatsu et al, 1986;Shaper et al, 1986Shaper et al, , 1988Weinstein et al, 19871, site-directed mutagenesis experiments to produce mislocalized mutants of glycosyltransferases should enable us to define the molecular basis for the sorting and retention of this class of enzymes. On the other hand, expression of glycosyltransferase cDNA in host cells normally not expressing this particular glycosyltrans-ferase could be used to alter oligosaccharide sequences in a specific way Paulson et al, 1989).…”

Expression of bovine beta-1,4-galactosyltransferase cDNA in COS-7 cells.

“…All these clones show strong homology to each other. On the other hand the 5' sequence of human liver GT cDNA clone isolated by Humphreys-Beher et al (10) does not exhibit homology to any of the reported GT cDNAs (4)(5)(6)(7)(8), in spite of the fact that the deduced partial amino acid sequence of the cDNA clone showed partial homology to the amino-terminal sequence of the bovine GT protein they isolated. This raises the question whether the described clones (4-8) do indeed code for active GT.…”

“…The functional significance of the clone by Humphreys-Beher et al (10) remains to be demonstrated. The constructs and the antibodies reported here, plus the a-lactalbumin constructs prepared earlier in this laboratory (27), should facilitate the delineation of the functional and interacting domains of GT and a-lactalbumin.…”