Molecular cloning of the mouse CCK gene: expression in different brain regions and during cortical development.

In this paper we describe experiments that address specific issues concerning the regulation of the mouse cholecystokinin gene in brain and intestine. The mouse cholecystokinin gene was cloned and sequenced. Extensive homology among the mouse, man and rat genes was noted particularly in the three exons and the regions upstream of the RNA start site. RNAse protection assays for each of the three exons were used to demonstrate that CCK is expressed in only a subset of tissues and that the same cap site and splice choices are used in brain, intestine as well as in cerebellum, cortex, midbrain, hypothalamus and hippocampus. CCK RNA was also noted to be detectable in kidney. Thus the same gene using the same promoter is expressed in subsets of cells that differ in their biochemical, morphologic and functional characteristics. The level of expression of CCK was also monitored during mouse cortical development and the appearance of CCK RNA was compared to glutamate decarboxylase (GAD), enkephalin and somatostatin. It was noted that each of these cortical markers was first expressed at different times during cortical development. The appearance of CCK RNA during intestinal development was also measured and found to precede appearance in cortex by several days.     

Molecular gene cloning, expression, and characterization of bovine brain glutamate dehydrogenase

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The Km and Vmax values for NAD+ were 0.1 mM and 1.08 micromol/min/mg, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - 100 microM, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.     

Molecular cloning of cDNA encoding alpha-N-acetylgalactosaminidase from Acremonium sp. and its expression in yeast

Alpha-N-acetylgalactosaminidase (alpha-GalNAc-ase; EC 3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal alpha-linked N-acetylgalactosamine in various sugar chains. The cDNA, nagA, encoding alpha-GalNAc-ase from Acremonium sp. was cloned, sequenced, and expressed in yeast Saccharomyces cerevisiae. The nagA contains an open reading frame which encodes for 547 amino acid residues including 21 residues of a signal peptide in its N-terminal. The calculated molecular mass of mature protein from the deduced amino acid sequence of nagA is 57260 Da, which corresponds to the value obtained from SDS-PAGE of native and recombinant enzymes treated with endo-beta-N-acetylglucosaminidase H. The amino acid sequence of NagA showed significant similarity to those of eukaryotic alpha-GalNAc-ases and alpha-galactosidases (alpha-Gal-ases), particularly alpha-Gal-ase A (AglA) from Aspergillus niger. Phylogenetic analysis revealed that NagA does not belong to the cluster of vertebrate alpha-GalNAc-ase and alpha-Gal-ase but forms another cluster with AglA and yeast alpha-Gal-ases. Thus, the evolutionary origin of the fungal alpha-GalNAc-ase is suggested to be different from that of vertebrate alpha-GalNAc-ase. This is the first report of a microbial alpha-GalNAc-ase gene.     

Molecular cloning and expression of the gene encoding a phospholipase A1 from Aspergillus oryzae.

Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes removal of the acyl group from position 1 of lecithin to form lysolecithin. The genomic DNA and cDNA encoding PLA1 from Aspergillus oryzae were cloned with the mixed deoxyribonucleotide-primed polymerase chain reaction. The PLA1 gene is composed of 1,056 bp and has four exons and three short introns (63, 54, and 51 bp). The deduced amino acid sequence of PLA1 contained the N-terminal sequence of the mature PLA1 analyzed by Edman degradation. PLA1 cDNA has an open reading frame of 885 bp encoding the PLA1 precursor of 295 amino acid residues. The mature PLA1 is composed of 269 amino acid residues, and a prepro-sequence of 26 amino acid residues is at the N-terminal region of the PLA1 precursor. PLA1 has two possible N-glycosylation sites (Asn27 and Asn55). PLA1 has a consensus pentapeptide (-Gly-His-Ser-Xaa-Gly-), which is conserved in lipases. The amino acid sequence of PLA1 showed 47% identity with that of mono- and diacylglycerol lipase from Penicillium camembertii. The PLA1 cDNA was expressed in Saccharomyces cerevisiae KS58-2D, indicating the cloned gene to be functional.     

Evolutionary rate and gene expression across different brain regions

Background

The evolutionary rate of a protein is a basic measure of evolution at the molecular level. Previous studies have shown that genes expressed in the brain have significantly lower evolutionary rates than those expressed in somatic tissues.

Results

We study the evolutionary rates of genes expressed in 21 different human brain regions. We find that genes highly expressed in the more recent cortical regions of the brain have lower evolutionary rates than genes highly expressed in subcortical regions. This may partially result from the observation that genes that are highly expressed in cortical regions tend to be highly expressed in subcortical regions, and thus their evolution faces a richer set of functional constraints. The frequency of mammal-specific and primate-specific genes is higher in the highly expressed gene sets of subcortical brain regions than in those of cortical brain regions. The basic inverse correlation between evolutionary rate and gene expression is significantly stronger in brain versus nonbrain tissues, and in cortical versus subcortical regions. Extending upon this cortical/subcortical trend, this inverse correlation is generally more marked for tissues that are located higher along the cranial vertical axis during development, giving rise to the possibility that these tissues are also more evolutionarily recent.

Conclusions

We find that cortically expressed genes are more conserved than subcortical ones, and that gene expression levels exert stronger constraints on sequence evolution in cortical versus subcortical regions. Taken together, these findings suggest that cortically expressed genes are under stronger selective pressure than subcortically expressed genes.     

Molecular cloning and expression of the hot pepper ERabp1 gene encoding auxin-binding protein

The 22 kDa auxin-binding proteins in higher plants have received considerable attention as candidates for an auxin receptor. A cDNA clone Ca-ERabp1 of hot pepper (Capsicum annum) was isolated using the oligonucleotides as PCR primers. The cDNA codes for a polypeptide related to the major 22 kDa auxin-binding protein from maize and Arabidopsis ERabp1. The deduced amino acid sequence contains an endoplasmic reticulum retention signal, the KDEL sequence located at the C-terminal end, and has two possible auxin-binding sites, HRHSCE and YDDWSVPHTA conserved sequences. Northern hybridization analysis revealed that the Ca-ERabp1 gene is differentially expressed in total RNA isolated from different organs of a pepper plant, showing the highest level of expression in fruits but barely detectable in leaves and roots.     

Molecular cloning and expression of a cDNA encoding the secretin receptor.

Secretin is a 27 amino acid peptide which stimulates the secretion of bicarbonate, enzymes and potassium ion from the pancreas. A complementary DNA encoding the rat secretin receptor was isolated from a CDM8 expression library of NG108-15 cell line. The secretin receptor expressed in COS cells could specifically bind the iodinated secretin with high and low affinities. Co-expression of the secretin receptor with the alpha-subunit of rat Gs protein increased the concentration of the high affinity receptor in the membrane fraction of the transfected COS cells. Secretin could stimulate accumulation of cAMP in COS cells expressing the cloned secretin receptor. The nucleotide sequence analysis of the cDNA has revealed that the secretin receptor consists of 449 amino acids with a calculated Mr of 48,696. The secretin receptor contains seven putative transmembrane segments, and belongs to a family of the G protein-coupled receptor. However, the amino acid sequence of the secretin receptor has no significant similarity with that of other G protein-coupled receptors. A 2.5 kb mRNA coding for the secretin receptor could be detected in NG108-15 cells, and rat heart, stomach and pancreatic tissue.     

Molecular cloning and expression of mouse brain sialidase.

Sialidase (EC 3.2.1.18) catalyzes the release of sialic acid from sialo-oligosaccharides, gangliosides, or sialo-glycoproteins. In this investigation, we cloned a novel cDNA for mouse brain sialidase and expressed the cDNA in COS-7 cells. This 1,699 bp cDNA codes for a 41.6 kDa protein consisting of 372 deduced amino acid residues. In COS-7 cells transiently transfected with the cDNA, a 250-fold increase was observed in specific activity toward 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Similarity searches of the nonredundant GenBank peptide sequence database by the PSI-BLAST program identified rat, hamster, human, and bacterial sialidases homologous to this mouse brain sialidase. Amino acid sequence identities to rat and hamster sialidases (84% and 77%, respectively) suggest that this form of sialidase is conserved in rodents. Sequence identities to human and mouse lysosomal sialidases (30% and 28%, respectively) indicate that the mouse brain sialidase is distinct from the lysosomal enzyme. Mouse brain sialidase has two amino acid sequence motifs common to bacterial sialidases: the 'F/YRIP' motif and the 'Asp-box' motif. The 'F/YRIP' motif is present near the N terminus while two 'Asp-box' motifs are present downstream.     

Molecular cloning and heterologous expression of a Klebsiella pneumoniae gene encoding alginate lyase

The alginate lyase (Aly; guluronate specific)-coding gene of Klebsiella pneumoniae was cloned using the cosmid vector pMMB33, transduced into Escherichia coli and expressed in this host. Four Aly-positive clones with unstable phenotypes were identified out of 700 kanamycin-resistant transductants. A stable derivative of one of the clones was studied further and contained 12.1-kb of insert DNA. The Aly-coding gene (aly), still partially under the control of its native promoter, was localised within a 1.95-kb HindIII fragment by transposon gamma delta mutagenesis and sub-cloning. Most of the Aly produced was secreted into the medium by both the original K. pneumoniae strain (71.7%) and the E. coli recombinant clones (85.1%). The enzyme from both K. pneumoniae and the E. coli clones had a pI of 8.9 and comprised a single 28-kDa polypeptide chain. Other minor bands were also observed on isoelectric focusing and these were attributed to processing intermediates of a single gene product. It is concluded that E. coli can recognise and process the signal peptide of Aly to produce a mature polypeptide that is identical to that synthesised by K. pneumoniae.     

Molecular cloning of human plasma glutathione peroxidase gene and its expression in the kidney.

A genomic clone containing the human plasma glutathione peroxidase (GSH-Px) gene has been isolated using a rat plasma GSH-Px cDNA as a probe. The partial nucleotide sequence of the clone completely matched the sequence of the human plasma GSH-Px cDNA. The results of Southern blot hybridization indicate that the human plasma GSH-Px gene consists of at least 4 exons and 3 introns, and spans about 12 kb. RNA blot analysis demonstrated that the human plasma GSH-Px gene is expressed in the kidney.     

Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacteriumAnabaena sp. strain PCC 7120

Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacteriumAnabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of theAnabaena ADPGlc PPase gene and its expression inEscherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48347 Da which is in agreement with the molecular mass determined by SDS-PAGE for theAnabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and theE. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in theAnabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in anE. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the nativeAnabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be theAnabaena enzyme.     

Molecular cloning, nucleotide sequence and expression of the gene encoding prepro-polygalacturonaseII of Aspergillus niger

PolygalacturonaseII of Aspergillus niger was fragmented using CNBr and the NH2-terminal fragment and another fragment were partially sequenced. The polygalacturonaseII (pgaII) gene was then isolated by using an oligonucleotide mixture based on the internal amino acid sequence as a probe. The nucleotide sequence of the pgaII structural gene was determined. It was found that polygalacturonaseII is synthesized as a precursor having an NH2-terminal prepro-sequence of 27 amino acids. The cloned gene was used to construct polygalacturonaseII over-producing A. niger strains. PolygalacturonaseII was isolated from one such strain and was determined to be correctly processed and to be fully active.     

Molecular cloning,sequence identification,and gene expression analysis of bovine ADCY2 gene

Adenylyl cyclase 2 (ADCY2), a class B member of adenylyl cyclases, is important in accelerating phosphor-acidification as well as glycogen synthesis and breakdown. Given its distinct role in flesh tenderization after butchering, we cloned and sequenced the ADCY2 gene from Yanbian cattle and assessed its expression in bovine tissues. A 2947 bp nucleotide sequence representing the full-length cDNA of bovine ADCY2 gene was obtained by 5′ and 3′ remote analysis computations for gene expression. Analyses of the putative protein sequence showed that ADCY2 had high homology among species, except with the non-mammal Oreochromis niloticus. Gene structural domain analyses in humans and rats indicated that the ADCY2 protein had no flaw; only the transmembrane domain was reduced and the CYCc structure domain was shortened. Assessment of ADCY2 expression in bovine tissues by real-time PCR showed that the highest expression was in the testes, followed by the longissimus dorsi, tensor fasciae latae, and latissimus dorsi. These data will serve as a foundation for further insight into the cattle ADCY2 gene.