Download 145 The ovine stearyl-CoA desaturase gene: Cloning and

Biochemical Society Transactions (1997) 25 S673
145
The ovine stearyl-CoA desaturase gene: Cloning and
determination of gene number within the ovine genome.
Richard J Ward*t, Maureen T Travers*, Richard G Vernon*,
Andrew M Saltert, Peter J Butteryt and Michael C Barber*
*Hannah Research Institute, Ayr, Ayrshire, KA6 5HL, UK
tDepartment of Applied Biochemistry and Food Science,
Section of Biochemistry and Nutrition, University of
Nottingham, Sutton Bonington Campus, Loughborough,
Leicestershire, LE12 5RD, UK.
It has been observed that high levels of saturated fatty acid
in the diet increase the plasma low density lipoprotein (LPL)
concentration which is associated with an increased risk of
premature atherosclerotic vascular disease [ 11. Unsaturated
fatty acids, however, have the effect of lowering the plasma
LDL [ I ] and, in the case of monounsaturated as opposed to
polyunsaturated fatty acids, the lowering occurs without a
concomitant decrease in the high density lipoprotein (HDL)
which is thought to have a protective effect [2]. Since meat
products, particularly those of ruminants, account for around
a quarter of the fat intake of the UK diet [3] it may be
advantageous to increase the proportion of monounsaturated
fatty acids in the ruminant carcass. To this end, we are
investigating the possibility of increasing the proportion of
oleic acid in ovine tissue by manipulating the expression of
the enzyme stearyl-CoA desaturase (SCD). This enzyme
converts stearyl-CoA into oleyl-CoA by the introduction of a
double bond at the A9 position. SCD homologues have been
cloned and sequenced from a number of species including
human [4], rat [5] and mouse [6] In the case of rodents two
highly homologous, differentially expressed, genes (SCD 1
and SCDZ) have been identified [6,7].
An ovine adipose tissue cDNA library was screened (2x105
plaques) using a 1.2Kb rat SCDl probe which resulted in the
isolation of twelve positives, nine of which were purified to
homogeneity. The cDNA inserts were subcloned from the
hgtl 1 recombinants into pGEM 7zf and were found, by
restriction digestion and sequence analysis, to be of
transcripts of the same gene, containing either all or part of a
single open reading frame (figure 1). This ORF encoded a
protein which was found to be homologous to the previously
published SCD sequences, (89%-93% with mouse rat and
human sequences). The coding sequence has 1077
nucleotides which encode a protein of 359 amino acids,
which has a calculated molecular weight of 41742.
IOOObp
Obp
PAHNc
E
ZOOObp
A
I
4900b
IR ~ ~ ~ C D I
XoAS
E.2
PAHNc
L
10 (1986bp)
ZB (463bp)
ZD (801bp)
PAHNc
E
PAHNc
E
XoAS
II I
PXaNs
II I
3 D (-3 4Kb)
PAHNc
E
XoAS
PXaNs
I I
4C (-2.5Kb)
u
A
3000bp
3 A (1306bp)
Figure 1 . Restriction maps of representative ovine SCD
clones shown in relation to the rat SCDl message, coding
sequence shown as a shaded box. (P=Pstl, A=Apul,
H=Hzndl 11, Nc=Nco 1, E=EcoRV, Xo=Xho 1, S=Suc 1,
Xa=Xbul and Ns=Nsil).
The largest clone isolated (3D, 3.4Kb) is significantly
smaller then the predicted message size from northern
blotting (approximately 5Kb) and does not possess a poly-A
tail or a polyadenylation site at the 3’ terminus, in common
with the other clones. This suggests that, since the clones
were isolated from an oligo-dT primed library, they are the
result of non-poly-A tail priming of the message.
Since all the clones were demonstrably from the same gene
and of similar homology to both the mouse SCDl and 2
genes (89%), the question of SCD gene number within the
ovine genome was raised and answered using evidence from a
number of different experiments as follows; Southern blotting
of ovine genomic DNA fragmented with a variety of
restriction endonucleases and hybridised to an ovine SCD
probe (clone 3A, figurel) showed that a much less complex
pattern of bands is produced then that seen for murine
genomic DNA cut with the same enzymes and hybridised to a
murine SCD probe [6,7]. An ovine genomic DNA cosmid
library was screened ( 4 ~ 1 colonies)
0~
using an ovine SCD
probe (clone 3A) which resulted in the isolation of three
clones. These were shown by restriction and sequence
analysis to be overlapping clones of the same gene These
were Southern blotted, after digestion with the same enzymes
as the genomic DNA and hybridised to a similar SCD probe
This resulted in a similar pattern of bands to that seen for
genomic DNA. We conclude from these observations that the
ovine genome has one SCD gene.
This work was hnded by a LINK grant from the
Biotechnology and Biological Sciences Research Council and
the Scottish Office Agriculture and Environment Department
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