Cloning, characterization, and expression of human ceramide galactosyltransferase cDNA

Dmitri Kapitonov, Robert K Yu

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Galactosylceramide (galactocerebroside, GalC) and its sulfated derivative, sulfatide, are major lipid components of the central and peripheral nervous system : myelin sheath, The enzyme UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) catalyzes the final step of galactosylceramide synthesis, In this report we describe isolation of the complete copy of human CGT cDNA. Total RNA from N-370 FG cells, a human fetal glioma cell line, was reverse-transcribed and dG-tailed. Degenerate primers synthesized based on rat CGT cDNA sequence were used in 5'- and 3'- rapid amplification of cDNA ends reaction (RACE). The obtained sequence was used to synthesize the primers for the complete coding region to be amplified and cloned into a pCR 3.1 expression vector. Following transfection of the CHOP cells with the resulting vector, the cell homogenate was assayed for the galactosyltransferase activity. Northern blot hybridization was used to determine the length of CGT mRNA and Southern blot hybridization was used to determine the number of homologous genes. Our results indicate that human CGT retains all conservative features of rat and mouse CGT. It is a single copy gene with mRNA transcript of about 4 kb.

Original languageEnglish (US)
Pages (from-to)449-453
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume232
Issue number2
DOIs
StatePublished - Mar 17 1997
Externally publishedYes

Fingerprint

N-Acylsphingosine Galactosyltransferase
Cloning
Galactosylceramides
Organism Cloning
Complementary DNA
Ganglioside Galactosyltransferase
Rats
Genes
Sulfoglycosphingolipids
Galactosyltransferases
Messenger RNA
Peripheral Nervous System
Neurology
Myelin Sheath
Southern Blotting
Glioma
Northern Blotting
Transfection
Amplification
Central Nervous System

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Cloning, characterization, and expression of human ceramide galactosyltransferase cDNA. / Kapitonov, Dmitri; Yu, Robert K.

In: Biochemical and Biophysical Research Communications, Vol. 232, No. 2, 17.03.1997, p. 449-453.

Research output: Contribution to journalArticle

@article{a2558c00bc454845b02e25a63bb9ddb4,
title = "Cloning, characterization, and expression of human ceramide galactosyltransferase cDNA",
abstract = "Galactosylceramide (galactocerebroside, GalC) and its sulfated derivative, sulfatide, are major lipid components of the central and peripheral nervous system : myelin sheath, The enzyme UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) catalyzes the final step of galactosylceramide synthesis, In this report we describe isolation of the complete copy of human CGT cDNA. Total RNA from N-370 FG cells, a human fetal glioma cell line, was reverse-transcribed and dG-tailed. Degenerate primers synthesized based on rat CGT cDNA sequence were used in 5'- and 3'- rapid amplification of cDNA ends reaction (RACE). The obtained sequence was used to synthesize the primers for the complete coding region to be amplified and cloned into a pCR 3.1 expression vector. Following transfection of the CHOP cells with the resulting vector, the cell homogenate was assayed for the galactosyltransferase activity. Northern blot hybridization was used to determine the length of CGT mRNA and Southern blot hybridization was used to determine the number of homologous genes. Our results indicate that human CGT retains all conservative features of rat and mouse CGT. It is a single copy gene with mRNA transcript of about 4 kb.",
author = "Dmitri Kapitonov and Yu, {Robert K}",
year = "1997",
month = "3",
day = "17",
doi = "10.1006/bbrc.1997.6240",
language = "English (US)",
volume = "232",
pages = "449--453",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Cloning, characterization, and expression of human ceramide galactosyltransferase cDNA

AU - Kapitonov, Dmitri

AU - Yu, Robert K

PY - 1997/3/17

Y1 - 1997/3/17

N2 - Galactosylceramide (galactocerebroside, GalC) and its sulfated derivative, sulfatide, are major lipid components of the central and peripheral nervous system : myelin sheath, The enzyme UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) catalyzes the final step of galactosylceramide synthesis, In this report we describe isolation of the complete copy of human CGT cDNA. Total RNA from N-370 FG cells, a human fetal glioma cell line, was reverse-transcribed and dG-tailed. Degenerate primers synthesized based on rat CGT cDNA sequence were used in 5'- and 3'- rapid amplification of cDNA ends reaction (RACE). The obtained sequence was used to synthesize the primers for the complete coding region to be amplified and cloned into a pCR 3.1 expression vector. Following transfection of the CHOP cells with the resulting vector, the cell homogenate was assayed for the galactosyltransferase activity. Northern blot hybridization was used to determine the length of CGT mRNA and Southern blot hybridization was used to determine the number of homologous genes. Our results indicate that human CGT retains all conservative features of rat and mouse CGT. It is a single copy gene with mRNA transcript of about 4 kb.

AB - Galactosylceramide (galactocerebroside, GalC) and its sulfated derivative, sulfatide, are major lipid components of the central and peripheral nervous system : myelin sheath, The enzyme UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) catalyzes the final step of galactosylceramide synthesis, In this report we describe isolation of the complete copy of human CGT cDNA. Total RNA from N-370 FG cells, a human fetal glioma cell line, was reverse-transcribed and dG-tailed. Degenerate primers synthesized based on rat CGT cDNA sequence were used in 5'- and 3'- rapid amplification of cDNA ends reaction (RACE). The obtained sequence was used to synthesize the primers for the complete coding region to be amplified and cloned into a pCR 3.1 expression vector. Following transfection of the CHOP cells with the resulting vector, the cell homogenate was assayed for the galactosyltransferase activity. Northern blot hybridization was used to determine the length of CGT mRNA and Southern blot hybridization was used to determine the number of homologous genes. Our results indicate that human CGT retains all conservative features of rat and mouse CGT. It is a single copy gene with mRNA transcript of about 4 kb.

UR - http://www.scopus.com/inward/record.url?scp=0031575817&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031575817&partnerID=8YFLogxK

U2 - 10.1006/bbrc.1997.6240

DO - 10.1006/bbrc.1997.6240

M3 - Article

C2 - 9125199

AN - SCOPUS:0031575817

VL - 232

SP - 449

EP - 453

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 2

ER -