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At least 1,747 records · Page 97Linked to original sources
PubMed1992

A rabbit reticulocyte ubiquitin carrier protein that supports ubiquitin-dependent proteolysis (E214k) is homologous to the yeast DNA repair gene RAD6.

The two isoforms of the 14-kDa ubiquitin carrier protein (E2(14k)) are unique among rabbit E2s in efficiently supporting ubiquitin-protein ligase (E3)-mediated ubiquitination of proteins destined for degradation. To begin determining the structural basis for this property, we have isolated a cDNA encoding the predominant reticulocyte isoform of the E2 from a rabbit skeletal muscle library. The sequence predicts a protein of 152 amino acids with a molecular weight of 17,293. Expression of the cDNA in Escherichia coli and purification of the recombinant protein revealed an E2 with high affinity for E3 and ubiquitin activating enzyme (E1). The latter high affinity interaction appears to be between the ubiquitin charged form of E1 and the uncharged form of E2 and does not result in a stable complex between these two enzymes. The predicted sequence shows regions of strong homology with other sequenced E2s, suggesting that these regions may be involved in binding to E1 and/or in ubiquitin transfer from E1, functions common to all E2s. Surprisingly, the E2(14k)) sequence is markedly more similar to Saccharomyces cerevisiae RAD6 (69% identity) than to its proposed homologs UBC4/UBC5 (38% identity). The sequence is identical to that recently reported for a human 17-kDa E2 which can complement rad6 mutants thereby identifying rabbit E2(14k) as a RAD6 homologue. The biochemical properties of this previously uncharacterized human 17-kDa E2 are now defined and its misassignment as a homologue of rabbit E2(17k) is corrected. Our findings resolve current confusion regarding relationships among E2s and define yeast RAD6, rabbit E2(14k), and the human 17-kDa E2 as a subclass of E2s which biochemically support E3-mediated conjugation and ubiquitin-dependent proteolysis and physiologically play a role in DNA repair.

Amino Acid Sequence↗
PubMed1992

Characterization of germ-line genes of the VGAM3.8 VH gene family from BALB/c mice.

Five germ-line genes of the VGAM3.8 VH family in BALB/c mice have been isolated from genomic libraries and sequenced. The genes are functional and three are expressed in antibodies of different specificities. Overall nucleotide sequence homologies within the family are greater than 90%, whereas homologies with other VH families are less than 70%. Southern blot hybridization and sequencing indicate a minimum family size of six genes. Differences in the coding regions are mostly confined to CDR, where there is a high replacement/silent substitution ratio, indicative of positive selection for diversification associated with Ag binding. VHVGAM3.8 sequences are highly conserved, and polymorphism in the coding regions appears to be very limited. Evidence is presented that the family has evolved, and been homogenized, by recombinatorial events.

Amino Acid Sequence↗
PubMed2003

[Cloning of a DnaJ homolog chaperon PBP and its subcellular localization].

AIM: To isolate and identify a human DnaJ homolog chaperon, PBP, from a human skeleton cDNA library, and to analyze its expression and distribution in transfected mammalian cells. METHODS: (32)p-dCTP labeled probe hybridization was used to screen the human skeleton cDNA library and sequence of the positive clones were analyzed. Then PBP gene was transfected into COS-7 cells using lipofectamin. PBP expressed in the cells were detected by Western-blot and indirect immunofluorescence staining. RESULTS: A full-length(1.5 kb) cDNA of peripherin-binding protein (PBP) was identified, which is identical with that of mrj. Full length PBP was mainly localized to cytoplasms of COS-7 cells in interphase, and to nuclei in mitosis. CONCLUSION: The results indicate that besides cooperating with DnaK (HSP70), PBP itself plays an important role as a member of DnaJ family. PBP may also be involved in the regulation of cell cycle.

Amino Acid Sequence↗
PubMed2005

Suppression subtractive hybridization analysis of Ms2 near-isogenic lines of wheat reveals genes differentially expressed in spikelets and anthers.

The dominant male sterility gene Ms2 in wheat has been widely used in recurrent selection and variety improvement. Identification of genes associated with the male sterility in Ms2-carrying wheat will help us understand how Ms2 functions. Using a pair of isogenic lines of Ms2, subtractive hybridization was conducted with cDNA from bulked spikelets at meiophase of sterile plants as the tester and cDNA from the same tissues of fertile plants as the driver. Two major bands at 270 bp and 450 bp were obtained by suppression PCR (polymerase chain reaction) of the subtractive cDNA. A total of 882 recombinants from PCR product cloning were isolated for reverse Northern analysis. The results demonstrated that up to 90% of the inserts in the library were up-regulated in the sterile spikelets. Twenty-one unique inserts from this library were sequenced. Similarity search showed that eighteen of them were homologous to ESTs (expression sequence tags) derived from spike or anther tissues at meiophase. The chromosome locations of nine of the ESTs were determined using C.S. (Chinese spring) nulli-tetrasomic lines, one of which was assigned to chromosome group 4 that includes chromosome 4D where Ms2 is located. In addition, four additional ESTs could also be assigned to this group according to their homology to BACs (bacterial artificial chromosomes) or PAC (P1 artificial chromosomes) of rice chromosome 3. The expression patterns of eight of the inserts examined displayed increased expression in spikelets and anthers of the sterile plants.

Blotting, Northern↗
PubMed1992

Cloning, expression, and characterization of the unique bovine A1 adenosine receptor. Studies on the ligand binding site by site-directed mutagenesis.

The bovine brain A1 adenosine receptor (A1AR) is distinct from other A1ARs in that it displays the unique agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) greater than N6-S-phenylisopropyladenosine (S-PIA) greater than 5'-N-ethylcarboxamidoadenosine and has a 5-10-fold higher affinity for both agonists and antagonists. The cDNA for this receptor has been cloned from a size-selected (2-4-kb) bovine brain library and sequenced. The 2.0-kb cDNA encodes a protein of 326 amino acid residues with a molecular mass of 36,570 daltons. The amino acid sequence fits well into the seven-transmembrane domain motif typical of G protein-coupled receptors. Northern analysis in bovine tissue using the full length cDNA demonstrates mRNAs of 3.4 and 5.7 kb with a tissue distribution consistent with A1AR binding. Subcloning of the cDNA in a pCMV5 expression vector with subsequent transfection into both COS7 and Chinese hamster ovary cells revealed a fully functional A1AR which could inhibit adenylylcyclase and retained the unique pharmacologic properties of the bovine brain A1AR. The A1AR was found to have a single histidine residue in each of transmembrane domains 6 and 7. Histidine residues have been postulated by biochemical studies to be important for ligand binding. Mutation of His-278 to Leu-278 (seventh transmembrane domain) dramatically decreased both agonist and antagonist binding by greater than 90%. In contrast, mutation of His-251 to Leu-251 decreased antagonist affinity and the number of receptors recognized by an antagonist radioligand. In contrast, agonist affinity was not perturbed but the number of receptors detected by an agonist radioligand was also reduced. These data suggest that both histidines are important for both agonist and antagonist binding, but His-278 appears critical for ligand binding to occur.

Adenylyl Cyclase Inhibitors↗
PubMed2005

[Cloning and functional characterization of the gacS gene of the biocontrol strain Pseudomonas fluorescens 2P24].

Pseudomonas fluorescens 2P24, a biocontrol agent for soil-borne diseases has been isolated from the wheat take-all decline soil, and was characterized with efficient production of antifungal compounds. In this study, the gacS gene was cloned by PCR from the P. fluorescens 2P24 genomic library. Nucleotide sequencing indicated that the gacS gene contains 2754 bp, and is predicted to encode a peptide of 917 amino acids with molecular mass of 101 kD. The deduced amino acid sequence shares 91% identity to that of the sensor kinase GacS of P. chlororaphis and 89% identity to GacS of P. fluorescens CHAO. Further research revealed that the gacS-knock-out mutant was unable to produce anti-fungal metabolites, such as 2,4-DAPG, HCN and proteinase. Moreover, it lost the ability to inhibit the growth of Gaeumannomyces graminis in Petri dish and to control the development of wheat take-all disease in greenhouse. The complemented strain containing a plasmid-bearing gacS gene could restore all of the lost phenotypes. The results indicated that GacS in an important regulatory factor and directly controls the synthesis of key biocontrol factors and the biocontrol efficacy of P. fluorescens 2P24.

Amino Acid Sequence↗
PubMed1992

Identification and localization of multiple forms of serine hydroxymethyltransferase in pea (Pisum sativum) and characterization of a cDNA encoding a mitochondrial isoform.

Serine hydroxymethyltransferase (SHMT) has been purified from the mitochondria of green pea leaves. Activity can be fractionated into two distinct peaks by ion exchange chromatography. While these two forms of the enzyme are immunologically indistinguishable, immunoinhibition experiments show the presence of a distinct non-mitochondrial third form of the enzyme to also be present in green pea leaves. While this mitochondrial form of SHMT is abundant in leaves it is absent from roots, although the two tissues have comparable SHMT activity. An antibody raised to purified mitochondrial SHMT was used to screen a cDNA expression library. The sequence of one of the isolated positive clones contained an open reading frame, which encoded a sequence that matched the amino acid sequence determined from the N terminus of the mature protein. The open reading frame encodes a mature protein of 487 amino acids with a M(r) of 54,000, together with a 27-31 amino acid serine-rich leader sequence, presumably required for mitochondrial targeting. The cDNA hybridizes to a small multigene family of 2-3 genes, which appear to be expressed predominantly in leaves. Comparison of the deduced amino acid sequence with the amino acid sequences of the rabbit mitochondrial and cytoplasmic SHMT, show that pea mitochondrial SHMT is equally similar to both of these enzymes. In addition, the rabbit sequences are more like one another than they are to the pea sequence, suggesting an interesting evolutionary relationship for these proteins.

Amino Acid Sequence↗
PubMed1991

Primary structure of bovine endothelin ETB receptor and identification of signal peptidase and metal proteinase cleavage sites.

A cDNA clone corresponding to the entire coding region of the bovine ETB endothelin receptor mRNA was isolated from a lung cDNA library and sequenced. The cDNA encodes 441 amino acids: 26 constituting an NH2-terminal signal peptide and 415 constituting the mature receptor. The signal peptidase cleavage site was determined by direct amino acid sequencing of purified receptor. A comparison of the predicted amino acid sequence with the available bovine ETA and rat ETB endothelin receptor sequences revealed 63 and 85% homology, respectively. Endothelin receptors of various species are known to be very sensitive to a certain metal proteinase(s) and have been shown to be converted to a lower Mr form in the absence of EDTA. The metal proteinase cleavage site was also determined by direct protein sequencing of the proteolysis product. The amino acid sequence (Ala-Gly-X-Pro-Pro-Arg) surrounding the cleavage site (between Ala-79 and Gly-80) is conserved among the ETB endothelin receptors, explaining the above mentioned proteolytic conversion from the higher to lower Mr forms observed in various species.

Amino Acid Sequence↗
PubMed1990

Alternative splicing generates two forms of mRNA coding for human heparin-binding growth factor 1.

Human class 1 heparin-binding growth factor (HBGF-1), also known as acidic fibroblast growth factor, is a mitogen for a variety of mesoderm- and neutroectoderm-derived cells in vitro as well as an angiogenic factor in vivo. Several oncogenes and growth factors have been shown to be homologous to HBGF-1. Four cDNA clones coding for HBGF-1 have been isolated from a human brain stem cDNA library. Nucleotide sequence analysis revealed that alternative splicing generated at least two different forms of HBGF-1 mRNA. Because the difference occurs in the 5'-untranslated regions, these transcripts may result from the usage of alternative promoters. One of the cDNA clones contains the polyadenylation signal, AATAAA, and a poly(A) tail, representing the 3'-end of an HBGF-1 mRNA. RNAase protection assays suggested this cDNA clone corresponds to a minor transcript, and the majority of the HBGF-1 mRNA terminates at 3.1 kbp downstream from the translation termination codon. The biological significance of this unusually long 3'-untranslated sequence is not known. To study the HBGF-1 gene structure, we have isolated 50 kbp of contiguous genomic DNA coding for the HBGF-1 protein. Both restriction enzyme mapping and nucleotide sequencing established that the distance between the first and second protein-coding exons is 13.6 kbp while that between the second and third is 5.3 kbp. By using the HBGF-1 cDNA as a probe, we showed that human fetal heart expresses high levels of HBGF-1 mRNA. Thus, HBGF-1 may be involved in mediating processes such as embryonic development and vascular growth in the heart.

Amino Acid Sequence↗
PubMed2006

Bacterial diversity in the human saliva from different ages.

To obtain primary idea on oral bacterium species that are generally present in periodotally healthy Koreans, the oral bacterial flora in the saliva of four periodontally healthy Koreans at different ages (5, 32, 35, 65) was investigated in this study. For this investigation, 16SrRNA gene clone libraries were generated from the saliva of the four healthy Koreans, and 50 clones were randomly selected from each saliva clone library and sequenced. Totally, 37 different kinds of bacterial 16S rRNA gene sequences were identified based on sequence homology search through GenBank database. The 37 kinds of saliva clone sequences were classified to 14 genera and 2 uncultured and 1 unidentified bacteria. Among the 14 identified genera, Streptococcus, Prevotella, and Veillonella were common genera, and Streptococcus was dominant genus that accounted for 7 different species. Among the seven Streptococcus species, S. salivarius appeared as the most common species. More numbers of species belonging to the genera Streptococcus and Prevotella was present in saliva from ages 32 and 35. While saliva from ages 5 and 65 showed more numbers of species belonging to the genera Rothia, including potential pathogenic species. Overall, saliva of a young child and a senior showed higher bacterial diversity than that of young adults.

Adult↗
PubMed1991

Molecular cloning and characterization of ovine IL-1 alpha and IL-1 beta.

Interleukin-1 (IL-1) is a cytokine with a wide range of effects on a variety of cell types. By hybridization with human IL-1 alpha and IL-1 beta cDNA probes, the corresponding ovine cDNAs were isolated from a stimulated alveolar macrophage cDNA library. The sequences of these cDNAs showed that ovine IL-1 alpha and IL-1 beta encode proteins of 268 and 266 amino acids, respectively, with both the nucleotide and amino acid sequences showing a high degree of homology with their human, mouse and bovine equivalents. In a mammalian COS cell-expression system these cDNAs produced biologically active IL-1. Further experiments demonstrated the importance of sequences within the 3' untranslated portion of the cDNAs in determining the level of expression of these molecules. The analysis of expression of IL-1 alpha- and IL-1 beta-specific mRNA in response to endotoxin, phorbol myristic acid (PMA) or PMA plus ionomycin revealed a distinct pattern of differential regulation of the two genes. From genomic analysis both IL-1 alpha and IL-1 beta appear to exist as single copies in the ovine genome.

Amino Acid Sequence↗
PubMed1991

Complementary DNA encoding human colon cancer manganese superoxide dismutase and the expression of its gene in human cells.

Manganese superoxide dismutase (MnSOD) is a member of a family of metalloenzymes that catalyze the dismutation of the superoxide anion to H2O2. It has been shown that MnSOD activity in tumor cells is lower than that in their normal counterparts. To investigate the molecular basis for the reduced level of MnSOD activity in human tumor cells, the primary structure of human MnSOD has been determined from complementary DNA (cDNA) isolated from a human colon carcinoma (HT-29) cDNA library. The sequence of the mature protein is composed of 198 amino acids preceded by a 24-amino acid leader peptide. DNA sequence analysis revealed that the translated region of the human tumor MnSOD is virtually identical to the MnSOD sequence isolated from normal human sources but exhibits differences in both the 5'- and 3'-untranslated regions. DNA blot analysis of genomic DNA isolated from HT-29, simian virus-transformed human lung fibroblast (SV-40/WI-38), and parental human lung fibroblast (WI-38) cells showed an identical pattern of hybridization to that of MnSOD cDNA. RNA blot analysis revealed that tumor cells have lower levels of MnSOD mRNA. However, the half-life of the mRNA was the same (approximately 10 h) in tumor and normal cells. Immunological measurement of the level of MnSOD in both normal and tumor cells also showed a reduced level of MnSOD protein in the tumor cells. These results suggest that the reduced level of MnSOD activity observed in human tumor cells is not due to a defect in the primary structure of the MnSOD protein, a change in the dosage of the MnSOD gene, or a decrease in the stability of MnSOD mRNA in tumor cells but rather is due to a defect or defects in the expression of the gene.

Base Sequence↗
PubMed1991

Structure of the human lipoprotein-associated coagulation inhibitor gene. Intro/exon gene organization and localization of the gene to chromosome 2.

Lipoprotein-associated coagulation inhibitor (LACI) is a multivalent, Kunitz-type proteinase inhibitor which appears to play an important role in the regulation of hemostasis. LACI directly inhibits factor Xa, and, in a Xa-dependent fashion, also inhibits the factor VIIa-tissue factor catalytic complex. Hybridization of a LACI cDNA probe to DNA isolated from a panel of human-mouse somatic cell hybrids containing different human chromosomes localized the human LACI gene to chromosome 2. In situ hybridization to metaphase chromosomes further mapped the gene to the region 2q31----2q32.1. Exons of the human LACI gene were cloned from genomic or chromosome 2-specific phage libraries and sequenced, including approximately 500 base pairs of 5' upstream DNA. The 5' DNA did not contain a prototypical TATAA box or CCAAT sequence, and attempts to identify a unique site for the initiation of transcription were unsuccessful in that primer extension and S1 nuclease protection analysis indicate multiple transcription initiation sites for LACI messages. Comparing the gene sequence with LACI cDNA sequences indicates that the gene contains nine exons and that alternative splicing can occur, resulting in the absence of exon 2 in the 5' untranslated region of some messages. The three Kunitz domains in LACI are encoded on separate exons. Introns which interrupt coding sequences all occur in the same codon phase interrupting the first and second bases of the codon triplets. The data are consistent with LACI evolving by a combination of gene segment duplications and exon shuffling.

Amino Acid Sequence↗
PubMed1991

The cDNA for rat selenoprotein P contains 10 TGA codons in the open reading frame.

Selenoprotein P is a plasma protein recently purified and characterized as containing 7.5 +/- 1.0 selenium atoms/molecule as selenocysteine. In rats maintained on a defined diet containing nutritionally adequate amounts of selenate as the sole selenium source, over half the selenium in plasma is accounted for by selenoprotein P. Its cDNA has been cloned from a rat liver library and sequenced. The sequence is highly unusual, containing 10 TGA codons in its open reading frame prior to the TAA termination codon. TGA designates selenocysteine in other selenoproteins, and limited peptide sequencing that included the amino acids encoded by two of the TGA codons verified that they correspond to selenocysteine. The deduced 366-amino acid sequence is histidine- and cysteine-rich and contains 9 of its selenocysteines in the terminal 122 amino acids. Comparison of the deduced amino acid sequence of selenoprotein P with those of other selenoprotein reveals no significant similarities. Selenoprotein P represents a new class of selenoproteins and is the first protein described with more than 1 selenocysteine in a single polypeptide chain. The primary structure of selenoprotein P suggests that it might be responsible for some of the antioxidant properties of selenium.

Amino Acid Sequence↗
PubMed1990

Characterization of the mRNAs encoding the gene 2 sarcoplasmic/endoplasmic-reticulum Ca2+ pump in pig smooth muscle.

The gene 2 sarcoplasmic/endoplasmic-reticulum (SR/ER) Ca2+ pump is expressed in slow skeletal and cardiac muscle, smooth muscle and non-muscle tissues. We have analysed the gene 2 Ca2+ pump mRNAs using a panel of anti-sense RNA probes which recognize either the muscle (class 1) or the non-muscle (class 2) transcript, or both. In pig smooth muscle, we confirmed the presence of the class 1 and class 2 mRNAs of 4.4 kb length and we also detected a third mRNA of 8.0 kb which reacted with both the class 1 and class 2 riboprobes. A 4.2 kb cDNA corresponding to the 3' part of the 8.0 kb mRNA was cloned from a pig gastric smooth muscle cDNA library. Nucleotide sequence analysis of this clone revealed that the 8.0 kb mRNA (class 3 transcript) contained both the non-muscle-specific and the muscle-specific exons separated by a 2.4 kb intron which has not been removed. The class 3-mRNA-encoded SR/ER Ca2+ pump is identical to the class 2-encoded non-muscle isoform. Northern blot analysis demonstrated that, in cardiac muscle, the class 1 mRNA (encoding the muscle isoform) is the predominant messenger, whereas in non-muscle tissues the class 2 and 3 mRNAs (encoding the non-muscle isoform) predominate. In smooth muscle all three mRNA types are present. The tissue distribution of the mRNA types suggests a tissue-dependent processing of the primary transcript of the sarcoplasmic/endoplasmic reticulum Ca2+ pump gene 2.

Animals↗
PubMed1990

Alternative splicing of the human c-myb gene.

Two cDNA clones of the human c-myb gene have been isolated from a CCRF-CEM leukemia cell cDNA library and sequenced in their entirety. These sequences, when compared with those previously reported for the human c-myb gene, reveal an alternative splicing process that generates at least four forms of the c-myb message. Three of these forms co-migrate on Northern blots and are co-expressed in several human hematopoietic cell types. Data on sequence comparisons with mouse and chicken homologues of c-myb coupled with oligonucleotide hybridization to genomic clones of the human c-myb gene indicate that this alternative splicing process utilizes three closely spaced splice donor sites and two unique exons present between viral defined exons 5 and 6. In one clone, the alternative splicing would generate a predicted myb protein with a three amino acid deletion in the region involved in transcription activation. In the other clone, incorporation of a new exon leads to introduction of a translation stop codon leading to loss of the entire carboxy terminus of the protein. This includes loss of a portion of the region involved in transcription activation as well as a separate highly conserved domain. The effect of these changes on protein function is currently unknown.

Base Sequence↗
PubMed1990

Sorting of peroxisomal membrane protein PMP47 from Candida boidinii into peroxisomal membranes of Saccharomyces cerevisiae.

A gene encoding PMP47, a peroxisomal integral membrane protein of the methylotrophic yeast Candida boidinii, was isolated from a genomic library. DNA sequencing of PMP47 revealed an open reading frame of 1269 base pairs capable of encoding a protein of 46,873 Da. At least two membrane-spanning regions in the protein are predicted from the sequence. Since the 3 amino acids at the carboxyl terminus are -AKE, PMP47 lacks a typical peroxisomal sorting signal. No significant similarities in primary structure between PMP47 and known proteins were observed, including PMP70, a rat peroxisomal membrane protein whose sequence has recently been reported (Kamijo, K., Taketani, S., Yokota, S., Osumi, T., and Hashimoto, T. (1990). J. Biol. Chem. 265, 4534-4540). In order to study the import and assembly of PMP47 into peroxisomes by genetic approaches, the gene was expressed in the yeast Saccharomyces cerevisiae. When PMP47 was expressed in cells grown on oleic acid to induce peroxisomes, the protein was observed exclusively in peroxisomes as determined by marker enzyme analysis of organelle fractions. Most of the PMP47 co-purified with the endogenous peroxisomal membrane proteins on isopycnic sucrose gradients. Either in the native host or when expressed in S. cerevisiae, PMP47 was not extractable from peroxisomal membranes by sodium carbonate at pH 11, indicating an integral membrane association. These results indicate that PMP47 is competent for sorting to and assembling into peroxisomal membranes in S. cerevisiae.

Amino Acid Sequence↗