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J. Biol. Chem., Vol. 263, Issue 15, 7049-7054, May, 1988
JD Paulauskis and HS Sul
Mouse liver mRNA enriched in sequence coding for fatty acid synthase by
sucrose density gradient centrifugation was used as template for cDNA
synthesis. Double-stranded cDNA sequences were inserted into pBR322 and
lambda gt10 and cloned. Clones containing putative cDNA sequences for fatty
acid synthase were identified by differential hybridization with [32P]
cDNAs synthesized from sucrose gradient-purified liver mRNA from mice
fasted or fasted and refed a high carbohydrate diet. Thirteen out of 45
differentially expressed clones were found to contain sequences
complementary to fatty acid synthase mRNA. Northern blot analysis revealed
that, unlike in avian and rat tissues, a single 8.2-kilobase (kb) mRNA
codes for fatty acid synthase in mice. In addition to the fatty acid
synthase cDNA clones, cDNA clones to two specific mRNAs of 5.1 and 7.2 kb
were selected to study nutritional, hormonal, and developmental regulation
at the level of mRNA abundance in mouse liver and in 3T3-L1 cells. The
induction of fatty acid synthase in the livers of previously fasted mice
fed a high carbohydrate diet was controlled pretranslationally by
modulation of the fatty acid synthase mRNA content. The level of the two
mRNAs with sizes of 5.1 and 7.2 kb were also elevated dramatically in the
liver of mice fasted and refed a high carbohydrate diet. A detectable, but
very low level of fatty acid synthase mRNA was found in 3T3-L1
preadipocytes. During the differentiation to adipocytes, both the rate of
synthesis and relative mRNA level for fatty acid synthase increased in a
parallel fashion to a maximum of 17-fold. The levels of 5.1- and 7.2-kb
mRNAs, coding for proteins possibly involved in lipogenesis, increased 45-
and 25-fold, respectively, during differentiation of 3T3-L1 adipocytes.
Treatment of mature 3T3-L1 adipocytes with insulin elicited a 3-fold
increase in both rate of synthesis and mRNA content of fatty acid synthase,
while treatment with dibutyryl cAMP caused a 60% decrease in fatty acid
synthase mRNA and an 80% decrease in the rate of the enzyme synthesis,
indicating pretranslational control of fatty acid synthase expression by
the lipogenic and lipolytic hormones. Similarly, insulin caused a 2- to
3-fold increase in both 7.2- and 5.1-kb mRNAs and dibutyryl cAMP decreased
the levels of 7.2- and 5.1-kb mRNAs to 10 and 20% of control levels,
respectively.
Cloning and expression of mouse fatty acid synthase and other specific mRNAs. Developmental and hormonal regulation in 3T3-L1 cells
Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115.
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