Molecular cloning of gene sequences for avian fatty acid synthase and evidence for nutritional regulation of fatty acid synthase mRNA concentration

A double-stranded cDNA library was constructed using total poly(A)+ RNA from the goose uropygial gland. Clones containing sequences complementary to fatty acid synthase mRNA were initially identified by colony hybridization with a 32P-labeled cDNA transcribed from RNA enriched for fatty acid synthase mRNA. Identity of the fatty acid synthase clones was confirmed by hybrid-selected translation. Mature fatty acid synthase mRNA is approximately 16 kilobases in length. When unfed neonatal goslings were fed for 24 hr, relative synthesis of hepatic fatty acid synthase increased more than 42-fold. Concomitantly, hepatic fatty acid synthase mRNA levels increased 70-fold. Thus, nutritional regulation of the synthesis of hepatic fatty acid synthase probably occurs at the pretranslational level. The availability of a specific probe for fatty acid synthase mRNA should allow us to analyze the regulation of expression of this gene during development, by nutrition and by hormones in both liver and uropygial gland.     

Cloning and expression of mouse fatty acid synthase and other specific mRNAs. Developmental and hormonal regulation in 3T3-L1 cells

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.     

Sequence of a cDNA encoding mouse F1F0-ATP synthase g subunit.

A pregnant-induced clone was identified by differential screening from a cDNA library of mouse mammary gland. The clone was identified as a full-length cDNA encoding the F1F0-ATP synthase g subunit. Comparison of the deduced amino acid sequences of mouse ATP synthase g subunit with those of bovine species showed 86% identity. The high levels of ATP synthase g subunit mRNA were detected in heart and uterine tissues.     

Pumpkin malate synthase. Cloning and sequencing of the cDNA and northern blot analysis.

A cDNA clone encoding the glyoxysomal malate synthase (EC 4.1.3.2) was identified by immunoscreening of a cDNA expression library constructed from poly(A)-rich RNA of etiolated pumpkin cotyledons. Determination of the DNA sequence of the 1979-nucleotide cDNA revealed a 1698-nucleotide open reading frame that encodes a polypeptide of 64632 Da. The identification of the cDNA for malate synthase was confirmed by matching three sequences obtained by peptide-sequence analyses of fragments generated by acid treatment of the purified enzyme. Northern blot analysis revealed that the probe hybridized to a single 2.3-kb species of mRNA species from etiolated pumpkin cotyledons which was not present in green pumpkin cotyledons. In a comparison of deduced amino acid sequences, pumpkin malate synthase was found to exhibit 83% and 48% similarity to the malate synthases from rape and Escherichia coli, respectively. Based on the amino acid sequence similarity and the hydropathy profiles of these three malate synthases, the signal for targeting the enzyme to microbodies is discussed.     

Structure of mouse fatty acid synthase mRNA. Identification of the two NADPH binding sites

Overlapping cDNA clones corresponding to 3.3 kb covering the carboxy-half and 3' non-coding regions of the single 8.2 kb mouse fatty acid synthase mRNA were isolated and sequenced. The sequence coded for 838 amino acid residues, followed by termination codon TAG, 771 nucleotides of 3' untranslated sequence and a poly A tail. For the first time, the two putative components of the NADPH binding sites of fatty acid synthase were identified, thereby making it possible to assign the enoyl reductase and beta-ketoacyl reductase domains of the multifunctional fatty acid synthase. Overall, the deduced amino acid sequence provides the domains for enoyl reductase, beta-ketoacyl reductase, acyl carrier protein and thioesterase of the mouse fatty acid synthase.     

Preparation of functional acetyl-CoA carboxylase mRNA from rat mammary gland

Poly(A)+ RNA from lactating rat mammary glands was fractionated according to size by isokinetic sucrose gradient centrifugation to obtain a fraction enriched for acetyl-CoA carboxylase. In vitro translation of this RNA preparation yielded apparent full-length acetyl-CoA carboxylase with a molecular weight of 260,000. The synthesized protein was identified as acetyl-CoA carboxylase by specific immunoprecipitation. Tests with antiserum to fatty acid synthetase, revealed that the fractions containing acetyl-CoA carboxylase mRNA also contained mRNA for fatty acid synthetase; both of these mRNAs were approximately 10 kb. Fatty acid synthetase with a molecular weight of 250,000 was synthesized. Using an in vitro rabbit reticulocyte lysate translation system, we have shown that the amount of translatable acetyl-CoA carboxylase mRNA increases during lactation. On the fifth day postpartum the level of translatable acetyl-CoA carboxylase mRNA increased to a peak level seven times that on the day of parturition.     

Diurnal variations of lipogenic enzyme mRNA quantities in rat liver.

The diurnal variations in mRNA quantities of lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, malic enzyme and glucose-6-phosphate dehydrogenase) in rat livers were detected. When the rats began feeding actively after lights out at 1900 h, the mRNA quantities were high from 0500 h to 0900 h in the morning. The variation in fatty acid synthase mRNA quantities was the most dramatic. However, no measurable variation in any enzyme levels including fatty acid synthase was detected. It may be because the half-lives of the enzymes are too long to be effected by the mRNAs which were high for several hours.     

Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing, and regulation of cholesterol 7 alpha-hydroxylase mRNA.

Monospecific antibody against purified rat liver cholesterol 7 alpha-hydroxylase cytochrome P-450 was used to screen a lambda gt11 cDNA library constructed from immuno-enriched polysomal RNA of cholestyramine-treated female rat liver. Two types of cDNA clones differing in the length of the 3'-untranslated region were identified, and DNA sequences were determined. The full length clone contains 3561 base pairs plus a long poly(A) tail. The amino acid sequence deduced from the open reading frame revealed a unique P-450 protein containing 503 amino acid residues which belonged to a new gene family designated family VII or CYP7. Southern blot hybridization experiments indicated that the minimal size of P-450 VII gene was 11 kilobase pairs (kb), and there was probably only one gene in this new family. Northern blot hybridization using specific cDNA probes revealed at least two major mRNA species of about 4.0 kb and 2.1 kb, respectively. These two mRNA species may be derived from the use of different polyadenylation signals and reverse-transcribed to two types of cDNA clones. Cholesterol 7 alpha-hydroxylase mRNAs were induced 2- to 3-fold in rat liver by cholestyramine treatment. The mRNA level was rapidly reduced upon the removal of the inducer. Similarly, cholesterol feeding induced enzyme activity, protein, and mRNA levels in the rat by 2-fold, suggesting that cholesterol is an important regulator of cholesterol 7 alpha-hydroxylase in the liver. On the other hand, dexamethasone and pregnenolone-16 alpha-carbonitrile drastically reduced the activity, protein, and mRNA levels. These experiments suggest that the induction of cholesterol 7 alpha-hydroxylase activity by cholestyramine or cholesterol and inhibition of cholesterol 7 alpha-hydroxylase activity by bile acid feedback are results of the rapid turnover of cholesterol 7 alpha-hydroxylase enzyme and mRNA levels.     

Rat apolipoprotein E mRNA. Cloning and sequencing of double-stranded cDNA

A 900-base pair clone corresponding to rat liver apolipoprotein E (apo-E) mRNA, and containing a 3'-terminal poly(A) segment, was identified from a library of rat liver cDNA clones in the plasmid pBR322 by specific hybrid selection and translation of mRNA. A restriction endonuclease DNA fragment from this recombinant plasmid was used to clone the 5'-terminal region of the apo-E mRNA by primed synthesis of cDNA. A portion of the double-stranded cDNA corresponding to the 3'-terminal region of apo-E mRNA was subcloned into the bacteriophage M13mp7 and employed as a template for the synthesis of a radioactively labeled, cDNA hybridization probe. This cDNA probe was used in a RNA-blot hybridization assay that showed the length of the apo-E mRNA to be about 1200 nucleotides. The hybridization assay also demonstrated that apo-E mRNA is present in rat intestine, but at about a 100-fold lower level than that of the rat liver. The nucleotide sequence of rat liver apo-E mRNA was determined from the cloned, double-stranded cDNAs. The amino acid sequence of rat liver apo-E was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 311 amino acids. A comparison to the NH2-terminal amino acid sequence of rat plasma apo-E indicated that the first 18 amino acids of the primary translation product are not present in the mature protein and are probably removed during co-translational processing. The coding region was flanked by a 3'-untranslated region of 109 nucleotides, which contained a characteristic AAUAAA sequence that ended 13 nucleotides from a 3'-terminal poly(A) segment. At the 5'-terminal region of the mRNA, 23 nucleotides of an untranslated region were also determined. The inferred amino acid sequence of mature rat apo-E, which contains 293 amino acids, was compared to the amino acid sequence of human apo-E, which contains 299 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall, 69% of the amino acid positions are identical in both proteins. The amino acid identities are clustered in two broad domains separated by a short region of nonhomology, an NH2-terminal domain of 173 residues where 80% are identical, and a COOH-terminal domain of 84 residues where 70% are identical. These two domains may be associated with specific functional roles in the protein.     

Bovine lactoferrin mRNA: sequence, analysis, and expression in the mammary gland.

The mRNA sequence for bovine lactoferrin expressed in the mammary gland was determined by sequencing three over lapping cDNA clones and by direct sequencing of the mRNA. The mRNA (2351 bases) codes for a 708 amino acid protein with a 19 amino acid signal peptide immediately preceding a sequence identical to the N-terminal 40 amino acids reported for bovine lactoferrin. A putative destabilizing sequence (AUUUA) was identified in the 3'-untranslated region. The nucleic acid sequence and deduced amino acid sequence are highly homologous with other transferrin family members. Lactoferrin mRNA concentrations in bovine mammary tissue were quite low two days before parturition and during lactation but were high three days after the cessation of milking, a sharp contrast from the pattern of regulation of the other milk proteins.     

Characterization of two mRNA species encoding human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17

Using two 33-mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of estradiol 17 beta-dehydrogenase (E2DH) and polyclonal antibodies raised against the enzyme purified from human placenta, clones were isolated from a lambda gt11 human placental cDNA library. A 327-amino acid sequence was deduced from cDNA sequencing. Two mRNA species have been identified in poly(A)+ RNA from human placenta, a major species migrating at 1.3 kb while a minor one is found at approx. 2.2 kb. Primer extension and S1 nuclease analysis indicate that the major mRNA species starts 9-10 nucleotides while the minor mRNA starts 971 nucleotides upstream from the ATG initiating codon, respectively. Sequence analysis of the longest cDNA clone (2092 bp) shows that it possesses identical coding and non-coding sequences in the regions of overlap with the shorter cDNA clones. The 32P-labeled 5' non-coding fragment hybridizes only to the 2.2 kb band, thus providing evidence for the existence of two distinct mRNA species which differ in their 5' noncoding regions. Using hp E2DH-36 cDNA as a probe for in situ hybridization of translocated chromosomes, the human E2DH gene was localized to the q11-q12 region of chromosome 17.     

Molecular cloning of an amino acid-regulated mRNA (amino acid starvation-induced) in rat hepatoma cells

Using the combination of a subtracted library and differential hybridization, a 409-base pair cDNA was identified that corresponds to a mRNA that is induced 2-3-fold when rat Fao hepatoma cells are subjected to amino acid starvation for 12 h. While this mRNA species was induced during starvation, others such as beta-actin, Cu-Zn superoxide dismutase, glyceraldehyde-3-P, and histone H4 were decreased in abundance to 25-50% of their original levels. The induction of the amino acid starvation-induced (ASI) mRNA was repressed when starved cells were returned to a medium supplemented with amino acids. Tissue distribution analysis showed the ASI mRNA, approximately 650 base pairs in length, to be present in every rat tissue tested. The cDNA clone has been sequenced and appears to correspond to the 3'-most end of the mRNA. The cDNA sequence includes the poly(A) tail, two potential polyadenylation signal sequences, and an open reading frame that we presume to be a portion of the coding sequence. The ASI cDNA will be used to investigate the molecular mechanisms for amino acid-dependent regulation of protein expression by mammalian cells.     

Rat mammary gland fatty acid synthase: localization of the constituent domains and two functional polyadenylation/termination signals in the cDNA.

The rat fatty acid synthase (FAS) is active only as a dimer, although the eight component functions are contained in a single polypeptide chain. Using mRNA from lactating rat mammary glands a cDNA expression library was established. With the overlapping immunologically positive clones we have an 8.9kb cDNA sequence for rat FAS. In the 3'-nontranslated region of the rat FAS cDNA we find a prototype polyadenylation/termination signal and 779 nucleotides upstream, a mutated one. Both of these polyadenylation/termination signals are used and give rise to two equally abundant mRNA species which are coordinately regulated. In the derived amino acid sequence we could locate six of the eight component functions; their order is NH2- beta-ketoacyl synthase - acetyl/malonyl transferases -enoyl reductase - acyl carrier protein - thioesterase -COOH. Comparison of FAS from different sources shows that the primary sequence is conserved only for the active residues and the amino acids in their immediate vicinity.     

Use of a cDNA library for the study of mRNA changes during muscle differentiation

A cDNA library was used to measure changes in many individual mRNAs during muscle differentiation in culture. A library of 1000 clones was constructed from total myofiber poly(A) RNA. About 23% of these clones gave a detectable colony hybridization signal using end-labeled myofiber mRNA, the remainder containing muscle sequences too rare to be detected with this assay. The 230 positive clones were grouped into four classes based on relative visual intensity. Reconstruction experiments using pure globin mRNA enable us to determine the approximate percentage of total RNA made up by each mRNA hybridizing to a cDNA clone. Those clones containing sequences complementary to developmentally regulated mRNAs were identified by a differential hybridization procedure. The cDNA library was screened with end-labeled mRNA from both undifferentiated myoblasts and differentiated myofibers. Although the bulk of the clones hybridized essentially the same with both RNA populations, several dozen were found which hybridized differentially. Some clones contained sequences which were not present at all in myoblasts and present in very high quantities in myofibers. Others contained sequences found in both myoblasts and myofibers but in increased quantities in the differentiated cells. Still others contained sequences which decreased in quantity during muscle differentiation. The clones in the first group were chosen for immediate analysis since they likely contain contractile protein mRNA sequences. However, all the characterized cDNA clones can now be used as probes to study the chromosomal organization and developmental expression of genes active during muscle differentiation.