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At least 109 records · Page 6Linked to original sources
PubMed1994

Plant mitochondrial NAD+-dependent malic enzyme. cDNA cloning, deduced primary structure of the 59- and 62-kDa subunits, import, gene complexity and expression analysis.

The 59- and 62-kDa subunits of the mitochondrial NAD+-dependent malic enzyme (EC 1.1.1.39) were purified from Solanum tuberosum L. (potato). NH2-terminal and internal amino acid sequence information was used to identify cDNAs encoding the two subunits. Comparison of the nucleotide sequences revealed that the subunits have 60% identity at the DNA level and 65% identity at the deduced amino acid level, implying that they are derived from a common ancestral gene. The plant NAD+-dependent malic enzymes belong to a family of related enzymes, including cytosolic and chloroplastic NADP+-dependent malic enzymes (EC 1.1.1.40) and bacterial NAD+-dependent malic enzymes (EC 1.1.1.38). The cDNAs were transcribed and translated in vitro and the resultant polypeptides imported into isolated mitochondria and shown to be processed. Southern blot analysis of potato genomic DNA revealed a simple pattern of hybridization for both subunits, indicating a simple gene structure or small number of genes encoding the two subunits. Northern blot analysis of RNA from a range of potato tissues has shown that the steady state levels for the two subunits are equivalent, suggesting that they are coordinately expressed.

Amino Acid Sequence↗
PubMed1994

Isolation and expression analysis of two rice genes encoding the major intrinsic protein.

We isolated two rice cDNAs (rMip1 and rTip1) which are homologous to the genes encoding the major intrinsic protein (Mip) (soybean nod-26 and Arabidopsis gamma-Tip), respectively. Expression of rTip1 in shoots and roots of rice seedlings was enhanced by water stress, salt stress and exogenous ABA. rMip1 was expressed only in shoots. Although mRNA level of rMip1 in shoots was induced to a small extent by exogenous ABA, it did not show any increase under water or salt stress over the course of 12 h. On the basis of the differential expression patterns and evolutional distinctions, it is suggested that the possible channel proteins encoded by rMip1 and rTip1 genes may function in different transport systems.

Abscisic Acid↗
PubMed1996

Structure, chromosomal localization, and expression analysis of the mouse inhibin/activin beta C (Inhbc) gene.

The mouse inhibin/activin beta C gene (Inhbc), a member of the transforming growth factor-beta (TGF-beta) superfamily, was cloned, mapped, and characterized. The gene spans approximately 14 kb, is composed of two exons, and maps to the distal region of mouse chromosome 10, which is syntenic to chromosome 12q13.1, where the human inhibin/activin beta C gene (INHBC) maps. The primary translation product is a preproprotein of 352 amino acids. The mature C-terminal domain of 116 amino acids shares 94% identity with its human homolog. Primer extension analysis shows that transcription starts approximately 130 bp upstream of the translation initiation site, and no TATA box was found in the promoter. Ribonuclease protection analyses reveal that mouse Inhbc is predominantly expressed in adult liver. Embryonic expression is detected beginning from Day 14.5 of gestation.

Activins↗
PubMed2024

Identification and expression analysis of calcium-dependent protein kinase family in oat (Avena sativa L.) and their functions in response to saline-alkali stresses.

Calcium-dependent protein kinases (CDPKs) serve as calcium ion sensors and play crucial roles in all aspects of plant life cycle. While CDPK gene family has been extensively studied in various plants, there is limited information available for CDPK members in oat, an important cereal crop worldwide. Totally, 60 AsCDPK genes were identified in oat genome and were classified into four subfamilies based on their phylogenetic relationship. The members within each subfamily shared similar gene structure and conserved motifs. Collinearity analysis revealed that AsCDPK gene amplification was attributed to segmental duplication events and underwent strong purifying selection. AsCDPK promoters were predicted to contain cis-acting elements associated with hormones, biotic and abiotic stresses. AsCDPK gene expressions were induced by different salt stresses, exhibiting stress-specific under different salt treatments. Moreover, overexpression of AsCDPK26 gene enhanced salt resistance in C. reinhardtii, a single-cell photoautotrophic model plants. Further analysis revealed a significant correlation between AsCDPK26 and Na+/H+ antiporter 1 (p<0.05), suggesting that AsCDPK26 may interact with ion transporter to modulate salt resistance. These results not only provide valuable insights into AsCDPK genes in response to different salt stresses, but also lay the foundation to mine novel candidates for improving salt tolerance in oat and other crops.

Chlamydomonas reinhardtii↗
PubMed1994

Tobacco cytochrome b5: cDNA isolation, expression analysis and in vitro protein targeting.

A full-length clone encoding cytochrome b5 has been isolated from a tobacco leaf cDNA library in lambda gt11 by PCR using degenerate primers. This cDNA encodes a protein of 139 residues which exhibits a high degree of homology to other cytochrome b5s, the message for which is expressed predominantly in developing seeds and in pigmented flower tissue. In the developing tobacco seed the mRNA is abundant at very early stages (< 10 days after flowering). Southern analysis indicated that more than one gene encodes cytochrome b5 in the tobacco genome. In vitro transcription and translation studies of the cDNA indicated that the protein inserts into the ER membrane by a non-SRP-mediated pathway and that the C-terminus of the protein is required for targeting and insertion.

Amino Acid Sequence↗
PubMed1991

Cloning and expression analysis of a cDNA encoding fumarylacetoacetate hydrolase: post-transcriptional modulation in rat liver and kidney.

Fumarylacetoacetate hydrolase (FAH) is an enzyme which is deficient in human hereditary tyrosinemia type 1. We have cloned and sequenced a rat liver cDNA encoding FAH. The identity of the clone was ascertained by hybrid-selection experiments and deduced amino acid (aa) sequence homologies with sequenced oligopeptide fragments of the purified rat liver protein. The cDNA codes for a 419-aa protein of 45,946 daltons. We used this cDNA as a probe in conjunction with a specific anti-rat FAH antibody to study the expression pattern of the FAH gene in rat liver and kidney. Northern blot analysis indicates that the kidney contains slightly more FAH mRNA that the liver. Western blotting shows, however, that the liver contains about twice as much FAH protein as the kidney. Primer extension experiments suggest that there are no differences in the 5'-untranslated (UT) ends of the FAH mRNA of both tissues. We conclude that synthesis of the FAH protein is in part regulated at the post-transcriptional level in rats liver and kidney, and that this regulation does not appear to be mediated by the 5'-UT sequence of the FAH mRNA.

Amino Acid Sequence↗
PubMed1994

Regulation of Actinobacillus actinomycetemcomitans leukotoxin expression: analysis of the promoter regions of leukotoxic and minimally leukotoxic strains.

The leukotoxin of Actinobacillus actinomycetemcomitans has been implicated as a virulence determinant in various human infections and is encoded by a multigene operon consisting of four known genes, designated ltxC, ltxA, ltxB, and ltxD. The ltx operon appears to be present in all A. actinomycetemcomitans strains, but levels of toxin expression vary greatly among strains. Thus, to gain a better understanding of the expression and regulation of the ltx operon, we have analyzed the ltx promoters of a highly toxic (JP2) and a minimally toxic (652) strain of A. actinomycetemcomitans. The nucleotide sequence of the JP2 ltx promoter contains -10 and -35 elements situated 350 bases upstream of ltxC, and primer extension of JP2 RNA confirmed that they are functional in vivo. However, a second primer extension product of 40 bases was present, and analysis of a series of truncated JP2 promoters fused to lacZ suggested that the region immediately upstream of ltxC also promotes transcription in Escherichia coli. These results suggest that two promoters may direct ltx expression in JP2. In addition, a small open reading frame capable of encoding a peptide of 78 amino acids was identified upstream of ltxC. Northern blots showed that this open reading frame is transcribed as part of a 4.2-kb mRNA, a transcript not previously identified as being derived from the ltx operon. In contrast, strain 652 expresses low steady-state levels of ltx mRNA, and its intact ltx promoter was inefficient in transcribing lacZ in E. coli. The nucleotide sequence of the 652 promoter is similar to that of the JP2 promoter but contains a region of 530 bp that is not present in JP2. Of 15 additional strains of A. actinomycetemcomitans that were analyzed, 13 contained promoters resembling the 652 sequence and 2 possessed JP2-like promoters. Both strains possessing the JP2-like promoter expressed 10- to 20-fold-higher levels of leukotoxin than did the strains possessing promoters resembling the 652 promoter. These results suggest that high levels of leukotoxin expression may correlate with the presence of the JP2-like promoter.

Actinobacillus Infections↗
PubMed1994

Human cathepsin O. Molecular cloning from a breast carcinoma, production of the active enzyme in Escherichia coli, and expression analysis in human tissues.

A cDNA encoding a novel member of the cysteine proteinase family of proteins has been cloned from a human breast carcinoma cDNA library, by using a polymerase chain reaction-based cloning strategy. The isolated cDNA contains an open reading frame coding for a polypeptide of 321 amino acids that has been tentatively called cathepsin O. This protein presents all the structural features characteristic of the different cysteine proteinases identified to date, including the active site cysteine residue that is involved in covalent intermediate formation during peptide hydrolysis. The cathepsin O cDNA was expressed in Escherichia coli, and after purification and refolding, the recombinant protein was able to degrade the synthetic peptides benzyloxycarbonyl-Phe-Arg-7-amido-4- methylcoumarin and benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin widely used as substrates for cysteine proteinases. Cathepsin O proteolytic activity was abolished by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), an inhibitor of this subclass of proteolytic enzymes, thus providing additional evidence that the isolated cDNA codes for an authentic cysteine proteinase. Northern blot analysis of poly(A)+ RNAs isolated from a variety of human tissues demonstrated that cathepsin O is expressed in all examined tissues, which is consistent with a putative role of this protein as a proteolytic enzyme involved in normal cellular protein degradation and turnover.

Amino Acid Sequence↗
PubMed1995

Cloning and expression analysis of a novel human serine hydrolase with sequence similarity to prokaryotic enzymes involved in the degradation of aromatic compounds.

A full-length cDNA coding for a novel human serine hydrolase has been cloned from a breast carcinoma cDNA library. Nucleotide sequence analysis has shown that the isolated cDNA contains an open reading frame coding for a polypeptide of 274 amino acids and a complete Alu repetitive sequence within its 3'-untranslated region. The predicted amino acid sequence contains the Gly-X-Ser-X-Gly motif characteristic of serine hydrolases and displays extensive similarity to several prokaryotic hydrolases involved in the degradation of aromatic compounds. The highest degree of identities was detected with four serine hydrolases encoded by the bphD genes of different strains of Pseudomonas with the ability to degrade biphenyl derivatives. On the basis of these sequence similarities, this novel human enzyme has been tentatively called Biphenyl hydrolase-related protein (Bph-rp). The Bph-rp cDNA was expressed in Escherichia coli, and after purification, the recombinant protein was able to degrade p-nitrophenylbutyrate, a water-soluble substrate commonly used for assaying serine hydrolases. This hydrolytic activity was abolished by diisopropyl fluorophosphate, a covalent inhibitor of serine hydrolases, providing additional evidence that the isolated cDNA encodes a member of this protein superfamily. Northern blot analysis of poly(A)+ RNAs isolated from a variety of human tissues revealed that Bph-rp is mainly expressed in liver and kidney, which was also confirmed at the protein level by Western blot analysis with antibodies raised against purified recombinant Bph-rp. According to structural characteristics, hydrolytic activity and tissue distribution of Bph-rp, a potential role of this enzyme in detoxification processes is proposed.

Amino Acid Sequence↗
PubMed1993

Expression analysis of the mixed function oxidase system in rat brain by the polymerase chain reaction.

Metabolism of therapeutic drugs in the body by the mixed function oxidase system is an important consideration in the analysis of a drug's effectiveness. P450-dependent metabolism within the brain of a neuro-specific drug may affect the drug's course of action. To determine whether cytochrome P450 was expressed in brain, RNA was isolated from the whole brains of rats treated with a variety of known hepatic P450 inducers, including amitriptyline, imipramine, isosafrole, phenobarbital, and beta-naphthoflavone. The RNA was analyzed for the presence of P450 isozymes by the PCR technique. Differential expression of P450IA1, P450IIB1, P450IIB2, P450IID, and P450IIE1 was detected in the brain samples, depending on the treatment. Cytochrome P450 reductase expression was also detected in the brain samples, giving strong evidence that the brain contains a competent mixed function oxidase system under all conditions studied.

Amitriptyline↗
PubMed1990

Expression analysis of a pseudogene in transgenic tobacco: a frameshift mutation prevents mRNA accumulation.

Seeds of the Pinto cultivar of the common bean, Phaseolus vulgaris, are deficient in phytohemagglutinin (PHA), a lectin normally composed of two different polypeptides (PHA-E and PHA-L). In Pinto seeds, there is no PHA-E and only small amounts of PHA-L. The gene coding for the Pinto PHA-E, Pdlec1, is a pseudogene as a result of a single base pair deletion in codon 11, causing a frameshift and premature termination of translation. This mutation explains the absence of the PHA-E polypeptide but not the several-hundredfold reduction of the cytoplasmic Pdlec1 mRNA in developing seeds when compared with a normal PHA-E gene. To find the cause for this reduction in mRNA levels, we swapped gene fragments of Pdlec1 with the homologous parts of a normal PHA gene from the cultivar Greensleeves and introduced these fusions into tobacco. Analysis of the transgenic seeds showed that the Pdlec1 promoter is fully functional. We also repaired the Pdlec1 coding frame in vitro and inserted the repaired and unrepaired versions into a PHA gene expression cassette. In transgenic tobacco, both constructs showed Pdlec1 transcript accumulation in the second half of seed maturation. The single-base frame repair boosted the peak transcript levels by a factor of 40 and resulted in the synthesis of PHA-E at normal levels. We propose that the premature translational stop caused by the frameshift leads to a faster breakdown of the Pdlec1 mRNA, thereby preventing this transcript from accumulating to high levels.

Cloning, Molecular↗
PubMed1996

Cloning and developmental expression analysis of the murine homolog of the spinocerebellar ataxia type 1 gene (Sca1).

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat which encodes glutamine in the novel protein ataxin-1. In order to characterize the developmental expression pattern of SCA1 and to identify putative functional domains in ataxin-1, the murine homolog (Sca1) was isolated. Cloning and characterization of the murine Sca1 gene revealed that the gene organization is similar to that of the human gene. The murine and human ataxin-1 are highly homologous but the CAG repeat is virtually absent in the mouse sequence suggesting that the polyglutamine stretch is not essential for the normal function of ataxin-1 in mice. Cellular and developmental expression of the murine homolog was examined using RNA in situ hybridization. During cerebellar development, there is a transient burst of Sca1 expression at postnatal day 14 when the murine cerebellar cortex becomes physiologically functional. There is also marked expression of Sca1 in mesenchymal cells of the intervertebral discs during development of the spinal column. These results suggest that the normal Sca1 gene, has a role at specific stages of both cerebellar and vertebral column development.

Amino Acid Sequence↗
PubMed1993

Human aminoacylase-1. Cloning, sequence, and expression analysis of a chromosome 3p21 gene inactivated in small cell lung cancer.

Human aminoacylase-1 (N-acyl-L-amino-acid amidohydrolase, EC 3.5.1.14; ACY1) is a homodimeric zinc-binding enzyme that catalyzes the hydrolysis of N alpha-acylated amino acids. ACY1 has been assigned to chromosome 3p21.1, a region reduced to homozygosity in small cell lung cancer (SCLC), and has been reported to exhibit reduced or absent expression in SCLC cell lines and tumors. Two human cDNA libraries and one human genomic DNA library were screened with a previously isolated partial ACY1 cDNA to isolate a full-length transcript. Sequence analysis of clones from each of these libraries resulted in an ACY1 cDNA of 1438 base pairs with an open reading frame of 1224-base pairs coding for a putative protein of 408 amino acids with a predicted molecular mass of 45,882 Da. Sequence analysis revealed no homologies to previously reported cDNA or protein sequences and establishes ACY1 as the first member of a new family of zinc-binding enzymes to be so characterized. The subcellular location of ACY1 has been established as cytosolic by flow cytometry. Southern and northern analyses of ACY1 in SCLC cell lines failed to demonstrate any gross abnormalities of the ACY1 structural gene or instances of absent or aberrantly sized mRNA, respectively.

Amidohydrolases↗
PubMed1984

Cloning and expression analysis of full length mouse cDNA sequences encoding the transformation associated protein p53.

We have cloned and sequenced overlapping cDNA fragments which together encode the entire mouse protein p53. Using these cDNA's we have reconstructed the full length coding region for the protein, and have analysed its coding potential by expression in vitro, both as a full length sequence and as a subfragment contained in a fusion protein. The predicted amino acid sequence contains no obvious homologies to any known oncogenes but includes a possible tyrosine kinase acceptor site.

Amino Acid Sequence↗
PubMed1995

Genetic mapping and expression analysis of the murine DNA ligase I gene.

We mapped the murine DNA ligase I gene (Lig1) in the mouse genome by using a mapping panel from an interspecific cross. Lig1 mapped to a centromeric part of chromosome 7, a region homologous to human chromosome 19q, where the human homologue LIG1 was localized. In addition, Lig1 expression was analyzed during the course of mouse liver-cell regeneration induced by partial hepatectomy, necrogenic doses of carbon tetrachloride, or the mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. The results demonstrate that Lig1 is expressed in the liver during active cell proliferation.

Animals↗
PubMedDate not supplied

Expression analysis of EGF-R and TGFa in human ovarian carcinomas.

Differences in the tumor biology of ovarian carcinomas probably influence operability and response to chemotherapy which are the most relevant prognostic factors. The phenotype of different malignant epithelial tumors including ovarian carcinomas is obviously associated with an activation of the EGF/TGFa signal pathway. When we analysed the expression of EGF-R and TGFa with biochemical, molecular-chemical and immunohistochemical methods in 29 different ovarian carcinomas, we found a correlation between the mRNA and protein levels of EGF-R as well as TGFa for tumors with low or high expressing rates. However, the concentration of measurable free EGF-Rs seems to depend on the amount of TGFa expression by the tumors. The EGF-R binding ligand TGFa is produced by the tumor cells; stromal cells are TGFa negative as shown by immunohistochemistry. By the use of an immunostaining index the TGFa protein concentration was measured semiquantitatively, classifying tumors according to their TGFa production rate. The comparison of TGFa mRNA amounts and staining index supports the hypothesis that TGFa is modified posttranslationally. EGF-R or TGFa expressing ovarian carcinomas had a high response rate to chemotherapy, whereas the EGF-R or TGFa negative tumors mostly exhibit a no change or progressive disease behaviour. These findings are the basis for our assumption that ovarian carcinomas with the basis for our assumption that ovarian carcinomas with an activated EGF-TGFa system are tumor biologically different compared to the EGF-R/TGFa negative tumors.

Antineoplastic Combined Chemotherapy Protocols↗