scholarly journals Molecular cloning of cDNA species for rat and mouse liver α-methylacyl-CoA racemases

1997 ◽  
Vol 326 (3) ◽  
pp. 883-889 ◽  
Author(s):  
Werner SCHMITZ ◽  
Heli M. HELANDER ◽  
J. Kalervo HILTUNEN ◽  
Ernst CONZELMANN
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Rat Liver ◽  
Mouse Liver ◽  
Amino Acid Sequences ◽  
Cdna Libraries ◽  
Cdna Expression Library ◽  
Coding Region ◽  
Expression Library ◽  
Cdna Expression

cDNA species coding for α-methylacyl-CoA racemase were cloned from rat and mouse liver cDNA libraries and characterized. The rat liver λgt11 cDNA expression library was screened with anti-racemase IgG [Schmitz, Albers, Fingerhut and Conzelmann (1995) Eur. J. Biochem. 231, 815–822]. Several full-length clones were obtained that contained an open reading frame of 1083 bp, coding for a protein of 361 amino acid residues with a predicted molecular mass of 39679 Da. The sequences of three peptides that were isolated by HPLC from a tryptic digest of purified rat liver racemase fully matched the cDNA-derived amino acid sequence. The cDNA coding for mouse racemase was cloned from a mouse liver λZAP cDNA expression library and sequenced. The coding region of 1080 bp codes for a 360-residue protein (molecular mass 39558 Da) that shares 89.7% similarity with the rat protein. Expression of the rat racemase as a recombinant protein in Escherichia coli with the pTrcHisB-expression vector yielded enzymically active protein. The amino acid sequences of α-methylacyl-CoA racemases do not resemble any known sequence of β-oxidation or auxiliary enzymes, supporting the view of a highly diverse evolutionary origin of enzymes acting on fatty acyl-CoA S-esters.

scholarly journals Expression of rat liver ketohexokinase in yeast results in fructose intolerance

1993 ◽  
Vol 291 (1) ◽  
pp. 179-186 ◽  
Author(s):  
I A Donaldson ◽  
T C Doyle ◽  
N Matas
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Molecular Mass ◽  
Rat Liver ◽  
Amino Acid Sequences ◽  
Growth Media ◽  
Predicted Amino Acid Sequence ◽  
Sequence Information ◽  
Coding Region ◽  
Pcr Product

Rat liver ketohexokinase (ATP:D-fructose 1-phosphotransferase; EC 2.7.1.3) was purified to homogeneity and the molecular mass of the protein was found by mass spectrometry to be 32,800 Da. The enzyme was cleaved and the amino acid sequences of seven peptides, comprising 24% of the total sequence, were determined. This sequence information was used to design oligonucleotide primers for a PCR using rat liver single-stranded cDNA as a template. The 224 bp PCR product was used as a probe to screen a rat liver cDNA library. A cDNA sequence of 1342 bp was obtained from three positive clones. This contained the entire coding region for ketohexokinase, and all seven peptides were identified in the predicted amino acid sequence. When ketohexokinase was expressed in Saccharomyces cerevisiae using the yeast expression vector pMA91, the cells became intolerant of the presence of fructose in their growth media. The growth of an exponential-phase culture was completely arrested within 90 min by the addition of fructose to a final concentration as low as 0.1% (w/v). This response is associated with an accumulation of fructose 1-phosphate. The cDNA for ketohexokinase encodes a protein composed of 299 amino acids with a combined molecular mass of 32,728 Da. This is in close agreement with the value for the isolated protein determined by mass spectrometry. The primary structure does not show any significant homology with those of other eukaryotic hexokinases, but it contains a highly conserved region that is present in three prokaryotic phosphotransferases that have furanose substrates.

scholarly journals Structural features of the low-molecular-mass human salivary mucin

1992 ◽  
Vol 287 (2) ◽  
pp. 639-643 ◽  
Author(s):  
M S Reddy ◽  
L A Bobek ◽  
G G Haraszthy ◽  
A R Biesbrock ◽  
M J Levine
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Structural Features ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Mrna Species ◽  
Salivary Mucin

The low-molecular-mass human salivary mucin has at least two isoforms, MG2a and MG2b, that differ primarily in their sialic acid and fucose content. In this study, we characterize further these isoforms, particularly their peptide moieties. Trypsin digests of MG2a and MG2b yielded high- and low-molecular-mass glycopeptides following gel filtration on Sephacryl S-300. The larger glycopeptides from MG2a and MG2b had similar amino acid compositions and identical N-terminal sequences, suggesting common structural features between their peptides. An oligonucleotide probe generated from the amino acid sequence of the smaller glycopeptide from MG2a was employed in Northern-blot analysis. This probe specifically hybridized to two mRNA species from human submandibular and sublingual glands. A cDNA clone selected from a human submandibular gland cDNA expression library with antibody generated against deglycosylated MG2a also hybridized to these two mRNA species. In both cases, the larger mRNA was polydisperse, and the hybridization signal was more intense in the sublingual gland. In addition, the N-terminal amino acid sequence of the larger glycopeptide was found to be part of one of the selected MG2 cDNA clones.

A member of the HSP90 family from ovineBabesiain China: molecular characterization, phylogenetic analysis and antigenicity

Parasitology ◽  
2015 ◽  
Vol 142 (11) ◽  
pp. 1387-1397 ◽  
Author(s):  
GUIQUAN GUAN ◽  
JUNLONG LIU ◽  
AIHONG LIU ◽  
YOUQUAN LI ◽  
QINGLI NIU ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Structural Characteristics ◽  
Heat Shock Protein 90 ◽  
Cellular Transformation ◽  
Amino Acid Sequences ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Reading Frame

SUMMARYHeat shock protein 90 (HSP90) is a key component of the molecular chaperone complex essential for activating many signalling proteins involved in the development and progression of pathogenic cellular transformation. AHsp90gene (BQHsp90) was cloned and characterized fromBabesiasp. BQ1 (Lintan), an ovineBabesiaisolate belonging toBabesia motasi-like group, by screening a cDNA expression library and performing rapid amplification of cDNA ends. The full-length cDNA ofBQHsp90is 2399 bp with an open reading frame of 2154 bp encoding a predicted 83 kDa polypeptide with 717 amino acid residues. It shows significant homology and similar structural characteristics toHsp90of other apicomplex organisms. Phylogenetic analysis, based on the HSP90 amino acid sequences, showed that theBabesiagenus is clearly separated from other apicomplexa genera. Five Chinese ovineBabesiaisolates were divided into 2 phylogenetic clusters, namelyBabesiasp. Xinjiang (previously designated a new species) cluster andB. motasi-like cluster which could be further divided into 2 subclusters (Babesiasp. BQ1 (Lintan)/Babesiasp. Tianzhu andBabesiasp. BQ1 (Ningxian)/Babesiasp. Hebei). Finally, the antigenicity of rBQHSP90 protein from prokaryotic expression was also evaluated using western blot and enzyme-linked immunosorbent assay (ELISA).

Molecular cloning and nucleotide sequence analysis of complementary DNA for bullfrog prolactin

1990 ◽  
Vol 5 (3) ◽  
pp. 281-287 ◽  
Author(s):  
N. Takahashi ◽  
K. Yoshihama ◽  
S. Kikuyama ◽  
K. Yamamoto ◽  
K. Wakabayashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rana Catesbeiana ◽  
Amino Acid Sequences ◽  
Protein Sequencing ◽  
Nucleotide Sequence Analysis ◽  
Cdna Expression Library ◽  
Mrna Levels ◽  
Expression Library ◽  
Untranslated Sequence ◽  
Prolactin Mrna

ABSTRACT A prolactin cDNA was cloned from a cDNA expression library constructed from total RNA of bullfrog (Rana catesbeiana) adenohypophyses by immunoscreening with antiserum against bullfrog prolactin. The cDNA clone thus obtained contained a 249 bp insert. Using this clone as a probe, plaque hybridizations were performed and two additional clones obtained. These clones had a polyadenylation site different from that of the first obtained clone, suggesting that the 3′-untranslated sequence was heterogeneous in length. The longest clone contained 830 bp, which encoded part of the signal peptide and the entire sequence of mature prolactin. The deduced amino acid sequence was in good accord with that determined by direct protein sequencing of purified bullfrog prolactin. The length of the bullfrog prolactin mRNA was estimated by Northern blot analysis to be about 1·0 kb. Homologies of prolactin nucleotide and amino acid sequences between bullfrog and other vertebrates were 64 and 65% for man, 66 and 68% for pig, 61 and 52% for rat, 69 and 74% for chicken, and 50 and 35% for salmon respectively. Highly conserved regions reported for mammalian prolactins also existed in bullfrog prolactin. Homologies of nucleotide and amino acid sequences between prolactin and GH of bullfrog origin were 49 and 25% respectively. Using the cDNA, the content of prolactin mRNA in the pituitary glands of metamorphosing tadpoles was measured. Prolactin mRNA levels rose at the mid-climax stage, suggesting that the increase in plasma and pituitary prolactin levels known to occur at the climax stage accompanies the increase in prolactin synthesis.

Molecular Cloning of Bovine Amelogenin cDna

1987 ◽  
Vol 1 (2) ◽  
pp. 293-297 ◽  
Author(s):  
H. Shimokawa ◽  
Y. Ogata ◽  
S. Sasaki ◽  
M.E. Sobel ◽  
C.I. Mcquillan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Cloning ◽  
Peptide Sequence ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Coding Region ◽  
Expression Library ◽  
Amino Acid Homology ◽  
Signal Peptide Sequence ◽  
Cloned Cdna

Molecular cloning of a bovine amelogenin cDNA was accomplished by construction of a cDNA expression library (λgt11 cDNA library) from the bovine ameloblast mRNA and then screening of the library with antibodies to bovine amelogenins. The complete primary structure of an amelogenin was deduced from cloned cDNA. One of the cDNA clones isolated from a bovine ameloblast phage λgt11 library had an 864-base-pair-long insert that encoded a protein with 216 amino acid residues. This cDNA clone appears to represent the complete coding region of amelogenin mRNA, including a putative AUG initiation codon and a signal peptide sequence. The predicted bovine amelogenin sequence has 87% amino acid homology with murine amelogenin.

scholarly journals 42Sp48 in previtellogenic Xenopus oocytes is structurally homologous to EF-1 alpha and may be a stage-specific elongation factor.

1991 ◽  
Vol 112 (2) ◽  
pp. 237-243 ◽  
Author(s):  
N J Coppard ◽  
K Poulsen ◽  
H O Madsen ◽  
J Frydenberg ◽  
B F Clark
Keyword(s):  
Amino Acid ◽  
Three Dimensional ◽  
Elongation Factor ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
Cdna Libraries ◽  
Coding Region ◽  
Functional Homology ◽  
Tailbud Stage ◽  
Specific Elongation

We have isolated the cDNA for 42Sp48 and EF-1 alpha from mixed stage oocytes and tailbud (stage 22) Xenopus laevis cDNA libraries by use of the cDNA for human elongation factor-1 alpha (EF-1 alpha) as probe. The nucleotide and deduced amino acid sequences of the entire coding region of 42Sp48 and EF-1 alpha cDNA were established. The proposed functional homology of the proteins is reflected in highly conserved amino acid sequences (91% identity), while the large number of silent mutations at the gene level may serve to prevent recombination at their loci. 42Sp48 is apparently encoded by two genes in Xenopus, while no sequences corresponding to 42Sp48 could be found in murine or human genomic DNA. 42Sp48 has been proposed to act as a stage-specific elongation factor in Xenopus. Comparison of the deduced amino acid sequences of 42Sp48 and EF-1 alpha with that of elongation factor Tu from E. coli, for which the three-dimensional structure including that of the GTP binding sites have been determined, supports this hypothesis.

scholarly journals Isolation and sequence analysis of a cDNA encoding rat liver α-l-fucosidase

1989 ◽  
Vol 264 (3) ◽  
pp. 695-701 ◽  
Author(s):  
K J Fisher ◽  
N N Aronson
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Signal Peptide ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Expression Library ◽  
Cdna Insert ◽  
Untranslated Sequence ◽  
N Terminus

cDNA clones for alpha-L-fucosidase were isolated from a rat liver lambda gt11 expression library by using both monospecific polyclonal antibodies against the affinity-purified enzyme and biotinylated rat liver fucosidase cDNA sequences as probes. The largest clone, lambda FC9, contained a 1522 bp full-length cDNA insert (FC9) that encoded the 434-amino acid-residue subunit (Mr 50439) of rat liver alpha-L-fucosidase. A putative signal peptide 28 amino acid residues in length preceded the sequence for the mature protein. In addition, FC9 specified for 11 nucleotide residues of 5′ untranslated sequence, 78 nucleotide residues of 3′ untranslated sequence and a poly(A) tail. The deduced amino acid sequence from FC9 in conjunction with the experimentally determined N-terminus of the mature enzyme suggested that rat liver fucosidase did not contain a pro-segment. However, there was the possibility of limited N-terminal processing (one to five amino acid residues) having occurred after removal of the predicted signal peptide. Amino acid sequences deduced from FC9 were co-linear with amino acid sequences measured at the N-terminus of purified fucosidase and on two of its CNBr-cleavage peptides. An unusual aspect of rat liver alpha-L-fucosidase protein structure obtained from the FC9 data was its high content of tryptophan (6%). The coding sequence from FC9 showed 82% sequence identity with that from a previously reported incomplete human fucosidase sequence [O'Brien, Willems, Fukushima, de Wet, Darby, DiCioccio, Fowler & Shows, (1987) Enzyme 38, 45-53].

scholarly journals ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin

1998 ◽  
Vol 141 (1) ◽  
pp. 199-208 ◽  
Author(s):  
Julie Haskins ◽  
Lijie Gu ◽  
Erika S. Wittchen ◽  
Jennifer Hibbard ◽  
Bruce R. Stevenson
Keyword(s):  
Amino Acid ◽  
Tight Junction ◽  
Molecular Mass ◽  
Mdck Cells ◽  
Cdna Libraries ◽  
Pdz Domains ◽  
Large Tumor ◽  
Junction Protein ◽  
Discs Large

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19–amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of ∼130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.

scholarly journals Murine erythropoietin gene: cloning, expression, and human gene homology.

1986 ◽  
Vol 6 (3) ◽  
pp. 849-858 ◽  
Author(s):  
C B Shoemaker ◽  
L D Mitsock
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Cdna Probe ◽  
Amino Acid Sequences ◽  
Nucleotide Sequence Analysis ◽  
Coding Region ◽  
Transcription Control ◽  
Erythroid Progenitor ◽  
Human Genes ◽  
Cell Expression

The gene for murine erythropoietin (EPO) was isolated from a mouse genomic library with a human EPO cDNA probe. Nucleotide sequence analysis permitted the identification of the murine EPO coding sequence and the prediction of the encoded amino acid sequence based on sequence conservation between the mouse and human EPO genes. Both the coding DNA and the amino acid sequences were 80% conserved between the two species. Transformation of COS-1 cells with a mammalian cell expression vector containing the murine EPO coding region resulted in secretion of murine EPO with biological activity on both murine and human erythroid progenitor cells. The transcription start site for the murine EPO gene in kidneys was determined. This permitted tentative identification of the transcription control region. The region included 140 base pairs upstream of the cap site which was over 90% conserved between the murine and human genes. Surprisingly, the first intron and much of the 5'- and 3'-untranslated sequences were also substantially conserved between the genes of the two species.

Sign in / Sign up

Export Citation Format

Share Document