Insulin regulation of a novel WD-40 repeat protein in adipocytes

A 400 bp PCR product generated with degenerate primers derived from the glucagon-like peptide-1 receptor was used to screen a rat skeletal muscle cDNA library. The predicted amino acid sequence of the 978 bp open reading frame has a predicted M(r) of 35 804, an estimated isoelectric point (pI) of 5.31 and contains seven WD-40 repeats, which are common to G-protein beta subunits (Gbeta). Although chemically and structurally similar to Gbeta subunits, the predicted amino acid sequence, when compared with the previously cloned Gbeta isoforms, was found to be only 31-41% similar and thus was named Gbeta-like (GbetaL, 'Gable'). Western blotting of whole-cell lysates and immunoprecipitates of membrane and cytosolic fractions of HEK 293 cells stably overexpressing a carboxy-terminal His-tagged GbetaL indicates that the protein is cytosolic and that it migrates at 42 kDa. A 4 kb transcript was detected in all tissues surveyed by northern blotting; however, an additional 2 kb transcript was detected in testis. Expression of GbetaL mRNA was highest in the brain and testis, followed by lung, heart, kidney, skeletal muscle, spleen and liver. In addition, reverse transcriptase/PCR showed that several other tissues and cell lines express GbetaL. The ubiquitous nature of the tissue expression pattern of GbetaL is similar to that of the insulin receptor, which suggests that insulin may influence GbetaL expression. Indeed, GbetaL protein and mRNA levels, in fully differentiated 3T3-L1 adipocytes, were upregulated by insulin in a concentration-dependent fashion. These changes were highly sensitive to insulin stimulation, being minimally affected by doses as low as 0.1 nM and maximally elevated by 1 nM doses. These data suggest that insulin regulates GbetaL production and imply that some of the actions of insulin may be mediated, in part, by this novel intracellular protein.

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Serine and threonine catabolism in Saccharomyces cerevisiae: the CHA1 polypeptide is homologous with other serine and threonine dehydratases.

Genetics ◽

10.1093/genetics/131.3.531 ◽

1992 ◽

Vol 131 (3) ◽

pp. 531-539 ◽

Cited By ~ 6

Author(s):

C Bornaes ◽

J G Petersen ◽

S Holmberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Amino Acid Sequence ◽

Transcription Initiation ◽

Open Reading Frame ◽

Predicted Amino Acid Sequence ◽

S1 Nuclease ◽

Reading Frame ◽

Threonine Dehydratase ◽

Nuclease Mapping

Abstract The catabolic L-serine (L-threonine) dehydratase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source. Previously we have cloned the CHA1 gene by complementation of a mutant, cha1, lacking the dehydratase activity. Here we present the DNA sequence of a 1,766-bp fragment of the CHA1 region encompassing an open reading frame of 1080 bp. Comparison of the predicted amino acid sequence of the CHA1 polypeptide with that of other serine/threonine dehydratases revealed several blocks of sequence homology. Thus, the amino acid sequence of rat liver serine dehydratase (SDH2) and the CHA1 polypeptide are 44% homologous allowing for conservative substitutions, while 36% similarity is found between the catabolic threonine dehydratase (tdcB) of Escherichia coli and the CHA1 protein. This strongly suggests that CHA1 is the structural gene for the yeast catabolic serine (threonine) dehydratase. S1-nuclease mapping of the CHA1 mRNA ends showed a major transcription initiation site corresponding to an untranslated leader of about 19 nucleotides, while a major polyadenylation site was located about 86 nucleotides downstream from the open reading frame. Furthermore, we have mapped the chromosomal position of the CHA1 gene to less than 0.5 kb centromere proximal to HML on the left arm of chromosome III.

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Cloning of pig prostaglandin F2alphaFP receptor cDNA and expression of its mRNA in the corpora lutea

Reproduction ◽

10.1530/rep.0.1250053 ◽

2003 ◽

pp. 53-64 ◽

Cited By ~ 16

Author(s):

U Boonyaprakob ◽

JE Gadsby ◽

V Hedgpeth ◽

P Routh ◽

GW Almond

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Oestrous Cycle ◽

Small Cells ◽

Predicted Amino Acid Sequence ◽

Corpora Lutea ◽

Reading Frame ◽

Luteal Tissue ◽

Fp Receptor ◽

Steroidogenic Cells

Changes in the expression and localization of luteal mRNA for PGF(2alpha) (FP) receptors may be critical in determining the luteolytic action of PGF(2alpha) in pig corpora lutea. In this study, a full-length FP receptor (FPr) cDNA was isolated and cloned from pig corpora lutea. This isolate (GenBank accession no. U91520) contains an open reading frame of 1086 bases coding for a protein of 362 amino acids with seven potential transmembrane domains. The predicted amino acid sequence of this isolate was 83% identical to the FPr amino acid sequence of other species including sheep, cattle and humans. Northern blot analysis showed the presence of an FPr message of about 5 kb in mRNA from pig corpora lutea. Relatively weak FPr mRNA expression was detected on day 4 and day 7 of the oestrous cycle. The expression was greater (P < 0.05) on days 10, 13 and 15 than on days 4 and 7. In situ hybridization analysis revealed that mRNA for FPr was expressed predominantly in the steroidogenic large luteal subtype of cell, although there was some expression in small luteal cells, with histological appearance of steroidogenic small cells. Localization of hybridization signals of FPr was observed in luteal tissue at all stages examined. These data demonstrate that FPr is expressed in pig corpora lutea throughout the oestrous cycle and that upregulation of the FPr mRNA occurs when the corpora lutea becomes sensitive to PGF(2alpha). Direct luteal targets of PGF(2alpha) appear to be primarily large steroidogenic cells in this species.

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197 MOLECULAR CLONING AND EXPRESSION OF THE CASHMERE GOAT IZUMO GENE

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab197 ◽

2011 ◽

Vol 23 (1) ◽

pp. 199

Author(s):

R. H. Na ◽

L. Liang ◽

L. Fu

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Transmembrane Protein ◽

Northern Blotting ◽

Cloning And Expression ◽

Immunoglobulin Superfamily ◽

Predicted Amino Acid Sequence ◽

Type I ◽

Cashmere Goat ◽

Calculated Molecular Mass

During the fertilization process, complex events are involved in the fusion between the reacted spermatozoa and the mature oocyte. Fusion implies that many proteins are present on the cell membrane of the gametes. Recently, a new protein, Izumo, has been shown to play a role in the sperm–egg fusion. Izumo was identified through the generation of a monoclonal antibody that inhibits this fusion. This protein belongs to the immunoglobulin superfamily type I transmembrane protein. Izumo can be detected only after acrosome reaction at the spermatozoal surface. The cashmere goat Izumo gene was identified and cloned by 3′ and 5′ rapid amplification of cDNA ends-PCR. The expression of cashmere goat Izumo was examined by RT-PCR and Northern blotting. The full-length cDNA of cashmere goat Izumo contains 1536 bp and an open reading frame of 1035 bp, encoding a polypeptide of 344 amino acids with a calculated molecular mass of 38.76 kDa and a theoretical isoelectric point of 8.18. This predicted amino acid sequence showed 89.82% amino acid identity with the bovine Izumo. The deduced amino acid sequence contained 3 conserved domains of the signal peptide, immunoglobulin-like domain, and transmembrane region. Reverse transcription-PCR and Northern blotting analysis showed that cashmere goat Izumo transcripts were highly expressed in the testis, caput, corpus, cauda epididymis. Cashmere goat Izumo may play a role in the biological process of fertilization. This work was supported by the National Natural Science Foundation (No. 30560103 and No.30740043), China, and the China Postdoctoral Science Foundation.

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Duplication-induced mutation of a new Neurospora gene required for acetate utilization: properties of the mutant and predicted amino acid sequence of the protein product

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2638-2644.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2638-2644

Author(s):

S Marathe ◽

I F Connerton ◽

J R Fincham

Keyword(s):

Amino Acid ◽

Carbon Source ◽

Amino Acid Sequence ◽

Genomic Sequence ◽

Protein Product ◽

Predicted Amino Acid Sequence ◽

Wild Type ◽

Reading Frame ◽

Acetate Utilization ◽

Group 2

A cloned Neurospora crassa genomic sequence, selected as preferentially transcribed when acetate was the sole carbon source, was introduced in extra copies at ectopic loci by transformation. Sexual crossing of transformants yielded acetate nonutilizing mutants with methylation and restriction site changes within both the ectopic DNA and the normally located gene. Such changes are typical of the duplication-induced premeiotic disruption (the RIP effect) first described by Selker et al. (E. U. Selker, E. B. Cambareri, B. C. Jensen, and K. R. Haack, Cell 51:741-752, 1987). The mutants had the unusual phenotype of growth on ethanol but not on acetate as the carbon source. In a cross to the wild type of a mutant strain in which the original ectopic gene sequence had been removed by segregation, the acetate nonutilizing phenotype invariably segregated together with a RIP-induced EcoRI site at the normal locus. This mutant was transformed to the ability to use acetate by the cloned sequence. The locus of the mutation, designated acu-8, was mapped between trp-3 and un-15 on linkage group 2. The transcribed portion of the clone, identified by probing with cDNA, was sequenced, and a putative 525-codon open reading frame with two introns was identified. The codon usage was found to be strongly biased in a way typical of most Neurospora genes sequenced so far. The predicted amino acid sequence shows no significant resemblance to anything previously recorded. These results provide a first example of the use of the RIP effect to obtain a mutant phenotype for a gene previously known only as a transcribed wild-type DNA sequence.

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Duplication-induced mutation of a new Neurospora gene required for acetate utilization: properties of the mutant and predicted amino acid sequence of the protein product.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2638 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2638-2644 ◽

Cited By ~ 21

Author(s):

S Marathe ◽

I F Connerton ◽

J R Fincham

Keyword(s):

Amino Acid ◽

Carbon Source ◽

Amino Acid Sequence ◽

Genomic Sequence ◽

Protein Product ◽

Predicted Amino Acid Sequence ◽

Wild Type ◽

Reading Frame ◽

Acetate Utilization ◽

Group 2

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Cloning and characterization of a novel nuclease from shrimp hepatopancreas, and prediction of its active site

Biochemical Journal ◽

10.1042/bj3460799 ◽

2000 ◽

Vol 346 (3) ◽

pp. 799-804 ◽

Cited By ~ 3

Author(s):

Wen-Yi WANG ◽

Shwu-Huey LIAW ◽

Ta-Hsiu LIAO

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Active Site ◽

Sequence Similarity ◽

Distinct Group ◽

Structural Data ◽

Hydrolytic Activity ◽

Degenerate Primers ◽

Homologous Proteins ◽

Reading Frame

Approximately 95% of the amino acid sequence of a shrimp (Penaeus japonicus) nuclease was derived from protease-digested peptides. A 1461-base cDNA for the nuclease was amplified and sequenced with degenerate primers based on the amino acid sequence and then specific primers by 3ʹ and 5ʹ RACE (rapid amplification of cDNA ends). It contains an open reading frame encoding a putative 21-residue signal peptide and a 381-residue mature protein. The N-terminus of the enzyme is pyroglutamate, deduced from composition and matrix-assisted laser desorption ionization-time-of-flight MS analyses, and confirmed by a glutamine residue in the cDNA sequence. The enzyme has 11 Cys residues, forming five intramolecular disulphides. The eleventh Cys residue was linked to a thiol compound with an estimated molecular mass of between 500 and 700 Da. A sequence similarity search revealed no homologous proteins but residues 205-255 shared a conserved active-site motif within a distinct group of nucleases. His211 in this conserved motif was shown to be very important in catalysis by site-specific modification with 14C-labelled iodoacetate. The shrimp nuclease, previously designated DNase I, does indeed possess a low level of hydrolytic activity towards RNA in the presence of Mg2+ and Ca2+. The conservation of functionally important residues during distant evolution might imply that the catalytic mechanisms are similar in these nucleases, which should be classified in one subfamily. Finally, an active-site structure for shrimp nuclease was proposed on the basis of published structural data and the results of mutational and biochemical analyses of Serratia nuclease.

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Peroxisomal multifunctional enzyme of β-oxidation metabolizing d-3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and characterization

Biochemical Journal ◽

10.1042/bj3210021 ◽

1997 ◽

Vol 321 (1) ◽

pp. 21-28 ◽

Cited By ~ 61

Author(s):

Yong-Mei QIN ◽

Matti H. POUTANEN ◽

Heli M. HELANDER ◽

Ari-Pekka KVIST ◽

Kirsi M. SIIVARI ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Rat Liver ◽

Predicted Amino Acid Sequence ◽

Peptide Fragments ◽

Reading Frame ◽

Multifunctional Enzyme ◽

Type Iv ◽

Liver Peroxisomes ◽

Enoyl Coa Hydratase

In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an Mr of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578–21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted Mr of 79331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17β-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal β-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and d-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters. The results showed that in addition to an earlier described multifunctional isomerase–hydratase–dehydrogenase enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes. They both catalyse sequential hydratase and dehydrogenase reactions of β-oxidation but through reciprocal stereochemical courses.

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Molecular Characterization of the β-N-Acetylglucosaminidase of Escherichia coliand Its Role in Cell Wall Recycling

Journal of Bacteriology ◽

10.1128/jb.182.17.4836-4840.2000 ◽

2000 ◽

Vol 182 (17) ◽

pp. 4836-4840 ◽

Cited By ~ 115

Author(s):

Qiaomei Cheng ◽

Hongshan Li ◽

Keith Merdek ◽

James T. Park

Keyword(s):

Cell Wall ◽

Amino Acid ◽

Amino Acid Sequence ◽

Sequence Data ◽

Degradation Products ◽

Predicted Amino Acid Sequence ◽

The Novel ◽

Sequence Comparisons ◽

Reading Frame ◽

Vibrio Furnissii

ABSTRACT The β-N-acetylglucosaminidase of Escherichia coli was found to have a novel specificity and to be encoded by a gene (nagZ) that maps at 25.1 min. It corresponds to an open reading frame, ycfO, whose predicted amino acid sequence is 57% identical to that of Vibrio furnissiiExoII. NagZ hydrolyzes the β-1,4 glycosidic bond betweenN-acetylglucosamine and anhydro-N-acetylmuramic acid in cell wall degradation products following their importation into the cell during the process for recycling cell wall muropeptides. From amino acid sequence comparisons, the novel β-N-acetylglucosaminidase appears to be conserved in all 12 gram-negative bacteria whose complete or partial genome sequence data are available.

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Yeast NOP2 encodes an essential nucleolar protein with homology to a human proliferation marker.

The Journal of Cell Biology ◽

10.1083/jcb.127.6.1799 ◽

1994 ◽

Vol 127 (6) ◽

pp. 1799-1813 ◽

Cited By ~ 49

Author(s):

E de Beus ◽

J S Brockenbrough ◽

B Hong ◽

J P Aris

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Ribosomal Protein ◽

Nucleolar Protein ◽

Mrna Levels ◽

Multicopy Plasmid ◽

Yeast Saccharomyces Cerevisiae ◽

Protein Levels ◽

Significant Amino Acid Sequence ◽

Ribosomal Protein L3

We have isolated a gene (NOP2) encoding a nucleolar protein during a search for previously unidentified nuclear proteins in the yeast Saccharomyces cerevisiae. The protein encoded by NOP2 (Nop2p) has a predicted molecular mass of 70 kD, migrates at 90 kD by SDS-PAGE, and is essential for cell viability. Nop2p shows significant amino acid sequence homology to a human proliferation-associated nucleolar protein, p120. Approximately half of Nop2p exhibits 67% amino acid sequence identity to p120. Analysis of subcellular fractions indicates that Nop2p is located primarily in the nucleus, and nuclear fractionation studies suggest that Nop2p is associated with the nucleolus. Indirect immunofluorescence localization of Nop2p shows a nucleolar-staining pattern, which is heterogeneous in appearance, and a faint staining of the cytoplasm. The expression of NOP2 during the transition from stationary phase growth arrest to rapid growth was measured, and compared to the expression of TCM1, which encodes the ribosomal protein L3. Nop2p protein levels are markedly upregulated during the onset of growth, compared to the levels of ribosomal protein L3, which remain relatively constant. NOP2 mRNA levels also increase during the onset of growth, accompanied by a similar increase in the levels of TCM1 mRNA. The consequences of overexpressing NOP2 from the GAL10 promoter on a multicopy plasmid were investigated. Although NOP2 overexpression produced no discernible growth phenotype and had no effect on ribosome subunit synthesis, overexpression was found to influence the morphology of the nucleolus, as judged by electron microscopy. Overexpression caused the nucleolus to become detached from the nuclear envelope and to become more rounded and/or fragmented in appearance. These findings suggest roles for NOP2 in nucleolar function during the onset of growth, and in the maintenance of nucleolar structure.

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