197 MOLECULAR CLONING AND EXPRESSION OF THE CASHMERE GOAT IZUMO GENE

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.

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

2001 ◽  
Vol 168 (2) ◽  
pp. 325-332 ◽  
Author(s):  
BD Rodgers ◽  
MA Levine ◽  
M Bernier ◽  
C Montrose-Rafizadeh
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Tissue Expression ◽  
Northern Blotting ◽  
Predicted Amino Acid Sequence ◽  
Mrna Levels ◽  
Degenerate Primers ◽  
Hek 293 ◽  
Reading Frame

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.

Thermus aquaticus ATCC 33923 Amylomaltase Gene Cloning and Expression and Enzyme Characterization: Production of Cycloamylose

1999 ◽  
Vol 65 (3) ◽  
pp. 910-915 ◽  
Author(s):  
Yoshinobu Terada ◽  
Kazutoshi Fujii ◽  
Takeshi Takaha ◽  
Shigetaka Okada
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Minimum Degree ◽  
Cloning And Expression ◽  
Gene Cloning And Expression ◽  
Thermus Aquaticus ◽  
Average Molecular Mass ◽  
Calculated Molecular Mass ◽  
E Coli

ABSTRACT The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of theEscherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75°C in a 10-min reaction with maltotriose and was stable at temperatures up to 85°C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic α-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coliamylomaltase and the plant D-enzyme.

scholarly journals NCU-G1 is a highly glycosylated integral membrane protein of the lysosome

2009 ◽  
Vol 422 (1) ◽  
pp. 83-90 ◽  
Author(s):  
Oliver Schieweck ◽  
Markus Damme ◽  
Bernd Schröder ◽  
Andrej Hasilik ◽  
Bernhard Schmidt ◽  
...  
Keyword(s):  
Amino Acid ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mouse Liver ◽  
Lysosomal Membrane ◽  
Molecular Forms ◽  
Type I ◽  
Calculated Molecular Mass ◽  
Sorting Motif ◽  
Lysosomal Membrane Proteins

Until recently, a modest number of approx. 40 lysosomal membrane proteins had been identified and even fewer were characterized in their function. In a proteomic study, using lysosomal membranes from human placenta we identified several candidate lysosomal membrane proteins and proved the lysosomal localization of two of them. In the present study, we demonstrate the lysosomal localization of the mouse orthologue of the human C1orf85 protein, which has been termed kidney-predominant protein NCU-G1 (GenBank® accession number: AB027141). NCU-G1 encodes a 404 amino acid protein with a calculated molecular mass of 39 kDa. The bioinformatics analysis of its amino acid sequence suggests it is a type I transmembrane protein containing a single tyrosine-based consensus lysosomal sorting motif at position 400 within the 12-residue C-terminal tail. Its lysosomal localization was confirmed using immunofluorescence with a C-terminally His-tagged NCU-G1 and the lysosomal marker LAMP-1 (lysosome-associated membrane protein-1) as a reference, and by subcellular fractionation of mouse liver after a tyloxapol-induced density shift of the lysosomal fraction using an anti-NCU-G1 antiserum. In transiently transfected HT1080 and HeLa cells, the His-tagged NCU-G1 was detected in two molecular forms with apparent protein sizes of 70 and 80 kDa, and in mouse liver the endogenous wild-type NCU-G1 was detected as a 75 kDa protein. The remarkable difference between the apparent and the calculated molecular masses of NCU-G1 was shown, by digesting the protein with N-glycosidase F, to be due to an extensive glycosylation. The lysosomal localization was impaired by mutational replacement of an alanine residue for the tyrosine residue within the putative sorting motif.

Conserved DNA binding and self-association domains of the Drosophila zeste protein

1992 ◽  
Vol 12 (2) ◽  
pp. 598-608
Author(s):  
J D Chen ◽  
C S Chan ◽  
V Pirrotta
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Coiled Coil ◽  
Helical Structure ◽  
Binding Activity ◽  
Binding Domain ◽  
Drosophila Virilis ◽  
Predicted Amino Acid Sequence ◽  
Dna Binding Domain

The zeste gene product is involved in two types of genetic effects dependent on chromosome pairing: transvection and the zeste-white interaction. Comparison of the predicted amino acid sequence with that of the Drosophila virilis gene shows that several blocks of amino acid sequence have been very highly conserved. One of these regions corresponds to the DNA binding domain. Site-directed mutations in this region indicate that a sequence resembling that of the homeodomain DNA recognition helix is essential for DNA binding activity. The integrity of an amphipathic helical region is also essential for binding activity and is likely to be responsible for dimerization of the DNA binding domain. Another very strongly conserved domain of zeste is the C-terminal region, predicted to form a long helical structure with two sets of heptad repeats that constitute two long hydrophobic ridges at opposite ends and on opposite faces of the helix. We show that this domain is responsible for the extensive aggregation properties of zeste that are required for its role in transvection phenomena. A model is proposed according to which the hydrophobic ridges induce the formation of open-ended coiled-coil structures holding together many hundreds of zeste molecules and possibly anchoring these complexes to other nuclear structures.

scholarly journals Isolation and Characterization of a Type I Gene Encoding a Light-harvesting Chlorophyll a/b-binding Protein of Photosystem II in Peach

1998 ◽  
Vol 123 (4) ◽  
pp. 493-499 ◽  
Author(s):  
Kyu H. Chung ◽  
Dennis E. Buetow ◽  
Schuyler S. Korban
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Photosystem Ii ◽  
Amino Acid Sequence ◽  
Woody Plants ◽  
Light Harvesting ◽  
Binding Protein ◽  
Transit Peptide ◽  
Type I ◽  
Polyadenylation Sites

A nuclear gene, Lhcb1*Pp1, encoding a light-harvesting chlorophyll a/b-binding protein of photosystem II has been isolated from peach [Prunus persica (L.) Batsch. `Stark Earliglo'] leaf genomic DNA, cloned, and sequenced. This gene encodes a precursor polypeptide of 267 amino acids with a transit peptide of 34 and a type I mature protein of 233 amino acids. The amino acid sequence of the mature polypeptide is 89% to 94% and 80% to 94% similar to those encoded by type I Lhcb genes of annual and other woody plants, respectively. In contrast, the amino acid sequence of the peach transit peptide is less conserved being 47% to 69% similar to those of annual plants and only 17% to 22% similar to those of other woody plants. The peach gene was used as a probe for Lhcb gene expression. Lhcb mRNA is detected in leaves of field-grown trees during June to October. Lhcb mRNA is detected at a high level in leaves of peach shoots grown in tissue culture in the light, but only at a trace level in leaves grown in the dark. Some Lhcb genes appear to be light-modulated in stems. Lhcb1*Ppl contains four potential polyadenylation sites. S1 nuclease analysis detected transcripts of the sizes expected from each of the four polyadenylation sites. All four are found in leaves of light-grown shoots and of field-grown trees throughout the growing season. In contrast, only three are detected in stems of light-grown shoots.

Primary structure and microtubule-interacting domain of the SP-H antigen: a mitotic MAP located at the spindle pole and characterized as a homologous protein to NuMA

1993 ◽  
Vol 105 (2) ◽  
pp. 589-600 ◽  
Author(s):  
T. Maekawa ◽  
R. Kuriyama
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Functional Organization ◽  
Spindle Pole ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Globular Domain ◽  
Calculated Molecular Mass ◽  
Mammalian Cell Lines ◽  
Cell Biol

Using a human autoantibody, SP-H, we identified a 200–230 kDa mitotic MAP in a variety of mammalian cell lines which shows affinity for the minus end of microtubules and also becomes associated with the spindle pole during mitosis. To examine the detailed structure and functional organization of the protein, the gene coding for the end-specific MAP was isolated and characterized by screening a human placenta lambda gt11 expression library using SP-H as a probe. Overlapping cDNA clones, which covered the entire length of the coding region of the SP-H antigen, were obtained. Polyclonal antibodies raised against fusion proteins generated from non-overlapping cDNA fragments stained the HeLa SP-H antigen in interphase and mitotic cells, and recognized a single 215 kDa band on immunoblots, as did the original SP-H antibody. Analysis of the nucleotide sequence revealed a 7,091 nucleotide sequence with an open reading frame of 6,345 nucleotides encoding a 2,115 amino acid polypeptide with a calculated molecular mass of 238,376 Da. The predicted amino acid sequence showed the protein to be composed of an alpha-helical domain, flanked by globular domains located at the amino and carboxy termini. The sequence contained five repeats of the hypothetical leucine zipper motif: one is in the N-terminal globular domain, and four are in the central alpha-helical stalk. Comparison with other sequences in the database shows that the SP-H antigen is identical to the NuMA protein reported by Yang et al. (1992) J. Cell Biol. 116, 1303–1317, but there are differences between the SP-H antigen and NuMA sequence reported by Compton et al. (1992) J. Cell Biol. 116, 1395–1408. cDNA inserts of the truncated SP-H antigen were expressed in both insect Sf9 cells and in cultured mammalian cells. The recombinant protein corresponding to the C-terminal half of the protein was restricted to the nucleus, whereas the N-terminal half of the protein was localized in the cytoplasm, suggesting the presence of a nuclear translocation signal(s) in the C-terminal domain. The C-terminal polypeptide expressed in mitotic COS cells was shown to specifically localize at the spindle pole. Microtubule-binding assays using in vitro transcribed/translated polypeptide products from different domains of the SP-H antigen further suggested that the SP-H antigen interacts with microtubules through the globular domain at the C-terminus.

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