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PubMed1998

Identification and cloning of human G-protein gamma 7, down-regulated in pancreatic cancer.

Differentially expressed genes between normal and cancer tissues of the pancreas were investigated using differential display. Consequently, we identified a fragment cDNA that was expressed in the normal tissue but was rarely expressed in the cancer tissue. This cDNA was screened in cDNA library prepared from the normal pancreatic tissue by rapid amplification of cDNA ends (5'RACE). 859 bp of cDNA was cloned and sequenced, and the inferred amino acid sequence was found to encode a G protein gamma subunit with 98% homology to cow G protein gamma 7 and complete homology to human G protein gamma 7. The decreased expression of the G protein gamma 7 was confirmed by Northern blot assay in twelve pancreatic malignancies which included nine duct cell carcinomas, two cystoadenocarcinomas and one blastoma. Reverse transcriptase (RT)-polymerase chain reaction (PCR) assay showed no expression of G protein gamma 7 in five of six pancreatic carcinoma cell lines and two pancreatic cancer tissues. Immunohistochemical analysis also displayed positive staining in the normal tissue but no staining in the cancer tissue. The findings demonstrated that the reduced or suppressed expression of human G-protein gamma 7 may play an important role in pancreatic carcinogenesis.

Amino Acid Sequence↗

PubMed1998

A hVti1 homologue: its expression depends on population doubling levels in both normal and SV40-transformed human fibroblasts.

A cDNA clone was isolated by differential colony hybridization from a cDNA library prepared from life-extended SV40-transformed human fibroblasts. The clone, tentatively named N-10, was 1272 bp in length coding for 232 amino acids. Northern analysis revealed that the expression level of N-10 was increased in normal senescent and life-extended SV40-transformed fibroblasts than in their young counterparts but was not enhanced by growth arrest. The protein fused to GFP (green fluorescent protein) localized in cytoplasmic granule. Enforced expression of N-10 resulted in premature senescence in young fibroblasts. The deduced amino acid sequence of N-10 was identical to the recently reported hVti1 gene except in one amino acid: Asp24(GAC) was ours and Asn24 (AAC) was reported. Additional base differences were found, so we referred to our sequence as the hVti1 homologue. As hVti1 protein was suggested to be involved in the vesicle transport process, the homologue may be concerned with increased secretion of extracellular matrix and various cytokines associated with cellular senescence.

Carrier Proteins↗

PubMed1998

Detection of benzodiazepine receptor ligands in small libraries of flavone derivatives synthesized by solution phase combinatorial chemistry.

Solution phase combinatorial synthesis of flavone derivatives and evaluation of their affinity for the central benzodiazepine receptors is described. The libraries preparation is simple and provides a convenient method for rapid compound generation and screening. Thirty one new compounds were obtained of which the most promising, as high affinity benzodiazepine receptor ligands, were 6-bromo-3'-fluoroflavone; 6,3'-dichloroflavone; 6-bromo-3'-chloroflavone and 6-chloro-3'-bromoflavone.

Animals↗

PubMed1998

cDNA sequence and deduced amino acid sequence of a fungal stress protein induced in Rhizopus nigricans by steroids.

cDNA clone was isolated from lambdagt11 library prepared from Rhizopus nigricans after growing the fungus in the presence of progesterone. Northern blot analysis of total RNA showed that expression of corresponding mRNA was up-regulated in R. nigricans after treatment with different steroids and after exposure of the fungus to heat shock or osmotic stress. Sequence analysis revealed an open reading frame for a 364-amino-acid polypeptide. The predicted amino acid sequence exhibited significant similarity to several sugar epimerases in two domains common to these enzymes. Our results suggest that the analyzed cDNA is coding for a fungal stress inducible protein belonging to sugar epimerases.

Amino Acid Sequence↗

PubMed1999

Identification of ribosomal protein L34 as a novel Cdk5 inhibitor.

The cell cycle is regulated by sequential activation, inactivation of cyclin dependent kinases (Cdk-s). Like all other Cdk-s, the catalytic subunit of Cdk5 is present in cycling cells. However, its highest concentration is found in differentiated neurons, and the only known protein that activates Cdk5 (i.e., p35) is expressed solely in the brain. Active Cdk5 is thought to be involved in the in vivo phosphorylation of the neurofilament proteins and tau which are hyperphosphorylated in neurodegenerative diseases. Recent reports suggest that Cdk5 may also contribute to cellular differentiation. Therefore, it would not be unusual to surmise that there exist specific proteins that regulate Cdk5 activity in cycling cells. In order to find if this was true, a cDNA library prepared from HeLa cells was screened using the yeast-two-hybrid system. The 60S ribosomal protein, L34, was identified as a Cdk5-interacting protein. Biochemical analyses reveal that L34 cannot activate Cdk5 but potently inhibits the p35-activated kinase. L34 also interacts with Cdk4 and, in parallel, inhibits the Cdk4/cyclin D1 activity. Interestingly, L34 does not interact with Cdk2 in the two-hybrid assay nor does it inhibit the Cdk2/cyclin A enzyme. The fact that a ribosomal protein inhibits Cdk5 and Cdk4 may suggest that these two kinases have a cellular role in translational regulation.

Cell Cycle↗

PubMed1999

Characterization of a novel member of the FGF family, XFGF-20, in Xenopus laevis.

The cDNA for a novel member of the FGF family (XFGF-20) was isolated from a Xenopus cDNA library prepared at the tailbud stage using as a probe the product of degenerate PCR performed with primers based on mammalian FGF-9s. This cDNA was 1860 bp long, and contained a single open reading frame that encoded 208 amino acid residues. The deduced amino acid sequence contained a motif characteristic of the FGF family and it was similar (73.1% overall homology) to XFGF-9 but differed from XFGF-9 in its amino-terminal region (33.3% homology). XFGF-20 mRNA was expressed only zygotically in embryos at and after the blastula stage, but it was also specifically expressed in the stomach and testis of adults. By contrast, XFGF-9 mRNA was expressed maternally in eggs and in many adult tissues. When XFGF-20 mRNA was overexpressed in early embryos, gastrulation was abnormal and development of anterior structures was suppressed. In such embryos, the expression of the Xbra transcript was suppressed during gastrulation while the expression of the transcripts of cerberus, Siamois, dkk-1, chordin, and Xotx-2 genes was normal. These results suggest that correct expression of XFGF-20 during gastrulation is required for the formation of normal head structures in Xenopus laevis during embryogenesis and that expression of the Xbra gene mediates this phenomenon.

Amino Acid Sequence↗

PubMed1999

Induction of hsp70 in the fungus Rhizopus nigricans.

We characterised two positive clones, RnH20/2 and RnH2/3, isolated from the cDNA library prepared from the fungus Rhizopus nigricans previously exposed to heat shock. Nucleotide sequences of both cDNA clones contained open reading frames for polypeptides with a significant amino acid identity to each other and to cytoplasmic members of Hsp70s from different eukaryotic organisms. Northern blot analysis, using a fragment of RnH2/3 as a probe, revealed that in the fungus R. nigricans the Hsp70 transcripts were inducible with deoxycorticosterone and testosterone as well as with heat stress, ethanol, CuSO(4), and H(2)O(2). This is the first report on isolation of cDNAs encoding Hsp70s from the fungal phylum Zygomycota.

Amino Acid Sequence↗

PubMed2000

Characterization of a novel member of the FGFR family, HrFGFR, in Halocynthia roretzi.

The cDNA for a novel member of the FGFR family, named HrFGFR, was isolated from a Halocynthia roretzi cDNA library prepared at the mid-tailbud stage. This cDNA was 3507b long, and the deduced amino acid sequence contained a motif characteristic of the vertebrate FGFRs. The existence of a single copy of the FGFR homologue gene in H. roretzi was suggested by restriction site analysis of multiple clones. HrFGFR mRNA was expressed strongly in the posterior region in the epidermis from the middle neurula stage. By contrast, Xenopus FGFR homologues are expressed in the anterior region and are known to induce anterior neural formation. A transition of the region expressing FGFR might have induced the more complicated brain or head formation characteristic of vertebrates.

Amino Acid Sequence↗

PubMed2000

Rice (Oryza sativa L.) OsPR1b gene is phytohormonally regulated in close interaction with light signals.

Strategies evolved by plants to counteract a variety of biotic/abiotic stresses include induction of genes encoding pathogenesis-related (PR) protein, in particular the PR class 1 (PR1) gene family, widely used in stress response studies. In spite of its immense importance as a PR family member, and an accepted gene marker in plant disease/defense in dicots, little is known about rice PR1 genes. Recently, we cloned and characterized the first OsPR1a (rice acidic PR1) gene (Agrawal et al. (2000) Biochem. Biophys. Res. Commun. 274, 157-165). Here, we report characterization of a rice basic PR1 (OsPR1b) gene, identified from screening a cDNA library prepared from jasmonic acid (JA)-treated rice seedling leaf, providing detailed and valuable insights into rice PR1 gene expression. The deduced amino acid sequence of OsPR1b reveals only 63.1% homology with the OsPR1a protein, whereas Southern blot analyses indicate that OsPR1b is a multigene family. The JA-inducible OsPR1b gene was also up-regulated by salicylic acid (SA), abscisic acid (ABA), and kinetin (KN). Furthermore, protein phosphatase inhibitors, cantharidin (CN) and endothall (EN) strongly induced the OsPR1b transcript. However, OsPR1b was not cut-responsive, diagrammatically opposite to cut inducibility of OsPR1a. This induction was light-, time-, and dose-dependent, as demonstrated by using, JA, CN, and EN, and completely inhibited by cycloheximide, but not by tetracycline. The simultaneous application of SA, and ABA, with JA, respectively, showed almost complete inhibition of the JA-induced OsPR1b transcript by 200 microM SA or ABA, but not by 100 microM concentrated solutions, suggesting a potential interaction among JA, SA, and ABA, whereas KN dramatically enhanced JA-induced OsPR1b transcript upon simultaneous application. Moreover, a simultaneous application of staurosporine enhances JA-, CN-, and EN-induced OsPR1b transcript, in particular with CN. Finally, a comparative analysis with the OsPR1a gene gives us insight into the differential regulation of the PR1 gene family, while proposing OsPR1 genes as important gene markers in rice, with potential use(s) in analyzing plant defense responses.

Amino Acid Sequence↗

PubMed2000

A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif.

Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

Amino Acid Motifs↗

PubMed2001

Mutation of human molybdenum cofactor sulfurase gene is responsible for classical xanthinuria type II.

Drosophila ma-l gene was suggested to encode an enzyme for sulfuration of the desulfo molybdenum cofactor for xanthine dehydrogenase (XDH) and aldehyde oxidase (AO). The human molybdenum cofactor sulfurase (HMCS) gene, the human ma-l homologue, is therefore a candidate gene responsible for classical xanthinuria type II, which involves both XDH and AO deficiencies. However, HMCS has not been identified as yet. In this study, we cloned the HMCS gene from a cDNA library prepared from liver. In two independent patients with classical xanthinuria type II, we identified a C to T base substitution at nucleotide 1255 in the HMCS gene that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 419. A classical xanthinuria type I patient and healthy volunteers lacked this mutation. These results indicate that a functional defect of the HMCS gene is responsible for classical xanthinuria type II, and that HMCS protein functions to provide a sulfur atom for the molybdenum cofactor of XDH and AO.

Aged↗

PubMed2000

Gene up-regulation in heart during mammalian hibernation.

A cDNA library prepared from heart of hibernating golden-mantled ground squirrels, Spermophilus lateralis, was differentially screened to clone genes that were up-regulated during hibernation. Two differentially expressed clones were found after three rounds of screening and were confirmed as up-regulated by Northern blotting. Clone Ang6 encoded a polypeptide with 116 amino acids that was identified as the ventricular isoform of myosin light chain 1 (MLC1(v)). Clone Ang19 coded for 274 amino acid residues of the mitochondrially encoded protein subunit 2 of NADH-ubiquinone oxidoreductase (ND2). Both proteins showed high amino acid sequence identity with their human counterparts, 97.5% for MLC1(v) and 66% for ND2. Northern blot hybridization revealed differential expression of these genes in multiple organs during hibernation. Transcript levels of both were approximately twofold higher in heart and three- to fourfold higher in skeletal muscle of hibernating, versus euthermic, animals. ND2 was also up-regulated in hibernator liver. Hibernation-induced up-regulation of MLC1(v) suggests that a restructuring of myosin subunit composition could contribute to changes in muscle contractility needed for hypothermic function, whereas changes in ND subunit composition may affect the function of the electron transport chain during hibernation.

Amino Acid Sequence↗

PubMed1995

A maternal RNA localized in the yellow crescent is segregated to the larval muscle cells during ascidian development.

A cDNA library prepared from one-cell zygotes of the ascidian Styela clava was screened with probes from isolated cellular fractions to identify clones encoding RNAs localized in the yellow crescent or myoplasm, a cytoskeletal domain with multiple developmental roles. The differential screen yielded five overlapping cDNA (Styela clava yellow crescent or ScYC) clones encoding a 1.2-kb polyadenylated RNA (yellow crescent or YC RNA) which is present throughout embryonic development. In situ hybridization confirmed that YC RNA is localized in the yellow crescent. Antisense probes containing the 3' region of YC RNA hybridize with multiple maternal and zygotic RNAs, suggesting sequence homologies with other transcripts. YC RNA was first detected during oogenesis when transcripts accumulate in the perinuclear region of vitellogenic oocytes and are gradually translocated to the cortex. The YC transcripts are localized in the cortex of unfertilized eggs but after fertilization segregate with the myoplasm to the yellow crescent. During cleavage most YC transcripts enter the primary muscle cell lineage. YC RNA is also present in the secondary muscle cells. The YC transcripts are retained in the myoplasm of oocytes and eggs extracted with the non-ionic detergent Triton X-100, suggesting that they are associated with the cytoskeleton. The nucleotide sequence of the longest ScYC clone contains a short open reading frame (ORF). The YC ORF would encode a putative polypeptide of 49 amino acids, which shows no significant homology to known proteins. Several features of the YC RNA, however, suggest that it functions as an RNA rather than as a protein coding molecule. We conclude that the myoplasm contains a novel maternal RNA which is associated with the cytoskeleton and segregated to the muscle cells during ascidian embryogenesis. The YC RNA may be a new member of a growing family of noncoding RNAs that play important roles in growth and development.

Amino Acid Sequence↗

PubMed1998

Molecular cloning and characterization of meichroacidin (male meiotic metaphase chromosome-associated acidic protein).

We have isolated a cDNA clone encoding a germ cell specific protein from an expression cDNA library prepared from the mouse testis, using testis-specific polyclonal antibodies. Sequence analysis of the cDNA revealed that the deduced amino acid sequence consisted of 284 residues, including a nominal repeat structure in the N-terminal region. Northern blot analysis revealed the presence of a transcript of 1.3 kb exclusively expressed in the testis and ovary, but at relatively low levels in the ovary. In contrast, no other tissues and organs expressed significant levels of the transcript. Expression of the mRNA in the testis was first detected on day 14 in postnatal development. Western blot analysis showed the presence of the protein with a molecular weight of approximately 40 kDa and an isoelectric point of 4.9. The protein was exclusively found in the testis and ovary, but in a far lesser amount in the ovary as was the case with the transcript. Immunohistochemical examination revealed that the protein was predominantly present in the cytoplasm in pachytene spermatocytes through to round spermatids. However, during the disappearance of the nuclear envelope at both the first and second meiotic divisions, the protein was localized around the metaphase chromosomes and spindles. Because of this, the name meichroacidin which stands for male meiotic metaphase chromosome-associated acidic protein is proposed for this antigen. The highly regulated stage-specific expression of meichroacidin and its specific association with the metaphase chromosomes and spindles suggest that the protein plays important roles in male meiosis.

Amino Acid Sequence↗

PubMed2000

Keratin 9 is a component of the perinuclear ring of the manchette of rat spermatids.

Previous work in our laboratory has shown that a 62- to 64-kDa protein was a major component of the perinuclear ring of manchettes fractionated from rat spermatids. Mass spectrometry analysis of this protein indicated the presence of a glycine-rich domain homologous to human keratin 9 (K9). Several antibodies to K9, raised against synthetic peptides of human K9, recognized the 64- to 62-kDa protein in the perinuclear ring of the manchette as well as in keratinocytes of the suprabasal layer of the rat and human footpad/sole epidermis in both immunoblotting and immunocytochemical experiments. Based on these data, human-derived K9 primers were used to clone rat K9 cDNA from epidermis by RT-PCR. Rat-specific K9 primers were then used to perform a two-step (nested) PCR to amplify the K9-specific rat testicular RNA and to obtain cDNA to demonstrate K9 gene expression in rat testis. The deduced amino acid sequence of rat K9 cDNA contains 618 amino acids with an estimated molecular mass of 63,020 Da, in agreement with that obtained by electrophoretic fractionation of rat manchette and epidermis footpad proteins. The deduced protein structure correlates with the recognizable pattern of keratins: a rod domain of 304 amino acids with well-conserved initiation and termination sequences (MQNLNSRLASY and EIETYRKLLEG, respectively), flanked by glycine/serine-rich head and tail domains of 141 and 173 amino acids, respectively. A high content of phenylalanine was detected in the head domain and a repetitive motif (SGGSYGGGS) in the tail domain. A comparison with human keratin 9 showed an overall nucleotide and amino acid similarity of 75%. An increased level of K9 transcripts was detected in a cDNA library prepared from fractionated round spermatids. Results of this study show that rat testis expresses K9 and that this protein is a component the perinuclear ring of the manchette of rat spermatids.

Amino Acid Sequence↗

PubMed1996

Habrec1, a novel serine/threonine kinase TGF-beta type I-like receptor, has a specific cellular expression suggesting function in the developing organism and adult brain.

Members of the TGF-beta superfamily signal through a dual receptor system consisting of a type II receptor protein kinase that binds the ligand, after which this complex associates with a type I receptor to mediate intracellular signaling. In mammals, six type I and five type II receptors mediating responses to different TGF-beta family members have been identified to date. Using primers from conserved regions of the protein kinase domain of the serine/threonine kinase receptors in a low-stringency polymerase chain reaction-based screening procedure, and deselecting known receptors with colony hybridization, we now report cloning a novel receptor member. The novel receptor was found in a cDNA library prepared from the habenular nucleus area and was designated Habrec1. Although only a partial sequence is available, it fits the criteria for a TGF-beta type I serine/threonine kinase receptor. In situ hybridization of Habrec1 reveals mRNA expression in several distinct areas of the developing central nervous system, including cortex cerebri, cerebellum, hippocampus, striatum, and thalamic nuclei. Expression is also seen in the anterior pituitary. In the periphery, strong expression prenatally includes brown fat, the gastrointestinal tract, liver, pancreas, thymus, and nasal cavity epithelium. In the adult brain Habrec1 mRNA is prominently found in cerebellum, cortex cerebri, and striatum, but at lower levels in several additional areas. We conclude that Habrec1 is a member of the TGF-beta type I receptor family with expression patterns in the developing animal, suggesting specific functions in and outside the nervous system, and in the adult CNS, suggesting roles in both cortical and subcortical brain circuitry.

Adipose Tissue, Brown↗

PubMed1995

cDNA cloning of thyroid hormone receptor beta for the Japanese flounder.

cDNA encoding the beta type of thyroid hormone receptor (THR) was cloned from a lambda gt10 library prepared from the whole bodies of metamorphosing flounder (Paralichthys olivaceus). The deduced amino acid sequence of the flounder THR beta (fTHR beta 1) showed higher homologies to the known THR beta s of other vertebrate animals than to THR alpha s, including flounder THR alpha s. Unlike any other THR, fTHR beta 1 possessed an insertion sequence composed of nine amino acids in the region prior to the hormone-binding domain. PCR analyses suggested the presence of transcripts for another THR beta (fTHR beta 2) that had a longer insertion sequence than fTHR beta 1. The analyses further suggested that the cDNA sequences of the two flounder THR beta s most likely shared a constant sequence except for a 60-base additional sequence found only in fTHR beta 2. The flounder genome possessed a single gene for both fTHR beta 1 and beta 2, suggesting that the two THR beta variants are produced from the same gene through an alternative splicing system.

Amino Acid Sequence↗

PubMed2002

Mycobacterium bovis BCG cell wall and lipopolysaccharide induce a novel gene, BIGM103, encoding a 7-TM protein: identification of a new protein family having Zn-transporter and Zn-metalloprotease signatures.

To identify novel genes induced during innate immune activation, we screened a cDNA library prepared from monocytes stimulated with Mycobacterium bovis BCG cell wall. A novel transcript with three-protein coding potential was identified, and the expressed proteins from individual frames showed distinct intracellular localization. Live and heat-killed Mycobacterium, bacterial cell wall, and inflammatory cytokines like TNFalpha were found to be potent inducers of the transcript. Expression of this gene is very low or undetectable in unstimulated monocytes, while a steady expression level was observed during differentiation of monocytes to dendritic cells and macrophages. The entire gene consisted of eight major exons and was localized on chromosome 4q22-q24, spanning approximately 84 kb. The main open reading frame of the transcript encoded a putative seven-transmembrane (TM) protein that showed homology with a number of functionally unknown proteins in the database. Further analysis revealed that all of these proteins have detectable similarity with the ZIP family of metal transporters. In fact, increased accumulation of intracellular Zn(2+) was observed due to the expression of BIGM103 in CHO cells. However, the identified proteins are structurally unique compared to known ZIP members and they also possess the hallmark of Zn-metalloproteases, suggesting a new class of multi-TM protein with dual features. Here we present a collection of these proteins and discuss the functional aspects of BIGM103, based on our results and current findings on two members of the family, Drosophila Catsup and Arabidopsis IAR1.

Amino Acid Sequence↗