A cDNA clone from Zea mays endosperm sucrose synthetase mRNA

A cDNA clone for maize endosperm sucrose synthetase of 62o nucleotide pairs length was obtained by cloning double stranded DNA obtained from the total maize endosperm poly(A) RNA in pBR322, and identifying the appropriate clone by hybrid-promoter translation. In Southern blotting to genomic BamHI-digested DNA, a single band only of approximately 20 Kb lights up, indicating that the sucrose synthetase gene is unique, or that closely linked copies are located on this DNA fragment.     

Selection of a rat glutamine synthetase cDNA clone

We have selected a glutamine synthetase clone (pGSRK-1) from a rat kidney cDNA library. A partial restriction map has been constructed for the 1.65 kilobase pair (kbp) glutamine synthetase cDNA. Northern hybridization analysis indicates that 1) GS-specific RNA increases many-fold during adipocyte differentiation and 2) dexamethasone increases and insulin decreases GS-specific RNA in 3T3-L1 adipocytes.     

Characterization of the fatty acid synthetase system of Curtobacterium pusillum

Curtobacterium pusillum contains 11-cyclohexylundecanoic acid as a major component of cellular fatty acids. A trace amount of 13-cyclohexyltridecanoic acid is also present. Fatty acids other than omega-cyclohexyl fatty acids present are 13-methyltetradecanoic, 12-methyltetradecanoic, n-pentadecanoic, 14-methylpentadecanoic, 13-methylpentadecanoic, n-hexadecanoic, 15-methylhexadecanoic, 14-methylhexadecanoic, and n-heptadecanoic acids. The fatty acid synthetase system of this bacterium was studied. Various 14C-labeled precursors were added to the growth medium and the incorporation of radioactivity into cellular fatty acids was analyzed. Sodium [14C]acetate and [14C]glucose were incorporated into almost all species of cellular fatty acids, the incorporation into 11-cyclohexylundecanoic acid being predominant. [14C]Isoleucine was incorporated into 12-methyltetradecanoic and 14-methylhexadecanoic acids: [14C]leucine into 13-methyltetradecanoic and 15-methylhexadecanoic acids; and [14C]valine into 14-methylpentadecanoic acid. [14C]-Shikimic acid was incorporated almost exclusively into omega-cyclohexyl fatty acids. The fatty acid synthetase activity of the crude enzyme preparation of C. pusillum was reconstituted on the addition of acyl carrier protein. This synthetase system required NADPH and preferentially utilized cyclohexanecarbonyl-CoA as a primer. The system was also able to use branched- and straight-chain acyl-CoAs with 4 to 6 carbon atoms effectively as primers but was unable to use acetyl-CoA. However, if acetyl acyl carrier protein was used as the priming substrate, the system produced straight-chain fatty acids. The results imply that the specificity of the initial acyl-CoA:acyl carrier protein acyltransferase dictates the structure of fatty acids synthesized and that the enzymes catalyzing the subsequent chain-elongation reactions do not have the same specificity restriction.     

Characterization of a pollen-specific cDNA clone from Zea mays and its expression.

A pollen-specific cDNA clone, Zmc13, has been isolated from a cDNA library constructed to poly(A) RNA from mature maize pollen. The cDNA as shown by primer extension analysis is a full-length copy of the mRNA. The cDNA has been sequenced and is 929 nucleotides in length plus a 47-nucleotide poly(A) tail. Putative polyadenylation signals are identifiable in the 3'-nontranslated region. The mRNA codes for a predicted polypeptide containing 170 amino acid residues and with a molecular mass of 18.3 kilodaltons. The hydropathy profile suggests a possible signal sequence on the amino terminus. A comparison of the nucleotide and deduced amino acid sequence with sequences in data banks has not shown homology to known molecules. In situ hybridizations using RNA probes show that the mRNA is located in the cytoplasm of the vegetative cell of the pollen grain and after germination is distributed throughout the pollen tube cytoplasm.     

Cell-free translation and partial purification of rat liver fatty acid synthetase mRNA.

The cell-free synthesis of rat liver fatty acid synthetase has been demonstrated in a modified reticulocyte lysate translation system. The mRNA was partially purified from membrane-free polysomes by oligo (dT)-cellulose chromatography and subsequent sucrose density gradient centrifugation.     

Characterization of a cDNA clone for a rare mRNA modulated by ovariectomy in mammary carcinomas

A complementary DNA (cDNA) clone (p13) for a rare mRNA was isolated from a cDNA library generated from total polyA+ RNA of 14-day lactating rat mammary gland. In vitro translation of the positively selected mRNA from p13 cDNA revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) a polypeptide of 24 kDa. The p13 cDNA clone hybridized on northern blots predominantly to approximately 1100 base size RNA and weakly to approximately 3800 base size RNA from lactating mammary gland. It hybridized only to approximately 3800 base size RNA from rat liver. Southern blot analysis of genomic DNA showed differences in gene organization in mammary gland and liver. The mRNA level for the 24 kDa polypeptide was higher in 7-12 DMBA-induced tumor and lower in the MTW9 carcinoma as compared to lactating mammary gland. After ovariectomy, the mRNA level in mid pregnant gland increased but was reduced in the 7-12 DMBA tumors.     

Characterization of a maize beta-amylase cDNA clone and its expression during seed germination.

A maize (Zea mays L.) cDNA clone (pZMB2) encoding beta-amylase was isolated from a cDNA library prepared from the aleurone RNA of germinating kernels. The cDNA encodes a predicted product of 488 amino acids with significant similarity to known beta-amylases from barley (Hordeum vulgare), rye (Secale cereale), and rice (Oryza sativa). Glycine-rich repeats found in the carboxyl terminus of the endosperm-specific beta-amylase of barley and rye are absent from the maize gene product. The N-terminal sequence of the first 20 amino acids of a beta-amylase peptide derived from purified protein is identical to the 5th through 24th amino acids of the predicted cDNA product, indicating the absence of a conventional signal peptide in the maize protein. Recombinant inbred mapping data indicate that the cDNA clone is single-copy gene that maps to chromosome 7L at position 83 centimorgans. Northern blot analysis and in vitro translation-immunoprecipitation data indicate that the maize beta-amylase is synthesized de novo in the aleurone cells but not in the scutellum during seed germination.     

Characterization and sequence of a cDNA clone of gamma-glutamyltranspeptidase.

We have isolated several cDNA's complementary to gamma-glutamyltranspeptidase (GGT) mRNA by screening a rat kidney library constructed in lambda gtll with antibodies specifically reactive to the enzyme protein. The clone selected an mRNA that was translated into a 62 Kd peptide, corresponding to the GGT precursor. The longest clone isolated was 1842 bp long with an open reading frame coding for 565 amino acids. The length of the mRNA coding for GGT was estimated to be 2.2 kb long. The amino acid sequence derived from the nucleotide sequence matched the short sequences determined by us as well as by other authors.     

Characterization of a cDNA clone encoding the complete amino acid sequence of cotton isocitrate lyase

A cDNA clone encoding the glyoxysomal enzyme isocitrate lyase (ICL) (EC 4.1.3.1) was isolated from a library prepared from cotton (Gossypium hirsutum L.) cotyledon poly(A)+ RNA. The clone is 1893 basepairs (bp) in length and contains a 1728 bp open reading frame encoding a polypeptide of 576 residues (Mr = 64,741). The deduced amino acid sequence of cotton ICL is 85.2%, 90.3% and 41.1% identical to ICL from rapeseed, castor bean and E. coli, respectively. Cotton ICL has a C-terminal tripeptide of A-R-M which is a putative trafficking signal for peroxisome (glyoxysome) proteins.     

Characterization of a Drosophila cDNA clone that encodes a 252-amino acid non-muscle tropomyosin isoform

We report here the isolation and DNA sequence of a cDNA clone encoding a 252-amino acid non-muscle or cytoskeletal tropomyosin (cTm) isoform from Drosophila. The Drosophila cTm shows considerable homology with vertebrate cTm throughout the middle portion of the molecule. The amino-terminal end of the molecule, however, shows less homology and contains five more amino acids than the equine platelet and human tropomyosins. There is also a proline at position 6 in the Drosophila protein. The carboxyl-terminal 27 amino acids also show little homology with vertebrate non-muscle tropomyosins. This is a region of the molecule that shows considerably diversity among other Drosophila tropomyosins and vertebrate tropomyosins. A comparison of the DNA sequence of the cTm cDNA and a previously reported muscle tropomyosin II cDNA sequence shows regions of identical DNA sequence alternating with regions of nonidentical sequence, suggesting that both mRNAs are produced by alternate splicing of the same gene.     

Characterization of a genomic and cDNA clone coding for the thioesterase domain and 3' noncoding region of the chicken liver fatty acid synthase gene

The fatty acid synthase (FAS) of animal tissue is a dimer of two identical subunits, each with a Mr of 260,000. The subunit is a single multifunctional protein having seven catalytic activities and a site for binding of the prosthetic group 4'-phosphopantetheine. The mRNA coding for the subunit has an estimated size of 10-16 kb, which is about twice the number of nucleotides needed to code for the estimated 2300 amino acids. We have isolated a positive clone, lambda CFAS, containing FAS gene sequences by screening a chicken genomic library with a segment of a 3' untranslated region of goose fatty acid synthase cDNA clone, pGFAS3, as a hybridization probe. The DNA insert in lambda CFAS hybridizes with synthetic oligonucleotide probes prepared according to the known amino acid sequence of the thioesterase component of the chicken liver fatty acid synthase [Yang, C.-Y., Huang, W.-Y., Chirala, S., & Wakil, S.J. (1988) Biochemistry (preceding paper in this issue)]. Further characterization of the DNA insert shows that the lambda CFAS clone contains about a 4.7-kbp segment from the 3' end of the chicken FAS gene that codes for a portion of the thioesterase domain. Complete sequence analyses of this segment including S1 nuclease mapping, showed that the lambda CFAS clone contains the entire 3' untranslated region of the chicken FAS gene and three exons that code for 162 amino acids of the thioesterase domain from the COOH-terminal end of the fatty acid synthase. Using the exon region of the genomic clone, we were able to isolate a cDNA clone that codes for the entire thioesterase domain of chicken liver fatty acid synthase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mammalian fatty acid synthetase. III. Characterization of human liver synthetase products and kinetics of methylmalonyl-CoA inhibition.

When propionyl-CoA was substituted for either acetyl-CoA or butyryl-CoA in the presence of [14C]malonyl-CoA and NADPH, the pure human liver fatty acids synthetase complex synthesized only straight-chain, saturated, 15- and 17-carbon radioactive fatty acids. At optimal concentrations, propionyl-CoA was a better primer of fatty acid synthesis than acetyl-CoA. Methylmalonyl-CoA inhibited the synthetase competitively with respect to malonyl-CoA. The Ki was calculated to be 8.4 muM. These findings provide an in vitro model and offer a direct explanation at the molecular level for some of the abnormal manifestations observed in diseases characterized by increased cellular concentrations of propionyl-CoA and methylmalonyl-CoA.     

Molecular cloning and sequencing of a cDNA encoding the thioesterase domain of the rat fatty acid synthetase

A cloned cDNA containing the entire coding sequence for the long-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase I) component as well as the 3'-noncoding region of the fatty acid synthetase has been isolated using an expression vector and domain-specific antibodies. The coding region was assigned to the thioesterase I domain by identification of sequences coding for characterized peptide fragments, amino-terminal analysis of the isolated thioesterase I domain and the presence of the serine esterase active-site sequence motif. The thioesterase I domain is 306 amino acids long with a calculated molecular mass of 33,476 daltons; its DNA is flanked at the 5'-end by a region coding for the acyl carrier protein domain and at the 3'-end by a 1,537-base pairs-long noncoding sequence with a poly(A) tail. The thioesterase I domain exhibits a low, albeit discernible, homology with the discrete medium-chain S-acyl fatty acid synthetase thioester hydrolases (thioesterase II) from rat mammary gland and duck uropygial gland, suggesting a distant but common evolutionary ancestry for these proteins.     

Glutamine synthetase in Lupinus luteus. Identification and preliminary characterization of nodule-specific cDNA clone

Two glutamine synthetase (GS) cDNA clones from L. luteus were identified and characterized. The nucleotide sequence analysis proved that they represent highly homologous but distinct mRNA species. Northern blot hybridization revealed that pc LINGS encodes the nodule-specific subunit of the GS while pcLIGS1 represents the nonspecific one present in nodule tissue as well as in uninfected roots.