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PubMed1992

Cloning and expression of the cDNA sequence encoding the lysosomal glycosidase di-N-acetylchitobiase.

Di-N-acetylchitobiase (chitobiase) is a lysosomal glycosidase involved in the degradation of asparagine-linked glycoproteins. Previous studies have revealed that chitobiase is unique among lysosomal glycosidases in that it may not be expressed universally in mammals. In this study we have isolated full-length cDNA clones for human placenta and rat liver chitobiase. The cDNAs from both species encode a glycosylated polypeptide of approximately 40 kDa that displays chitobiase activity when expressed in COS-1 cells. By using the rat cDNA sequence as a hybridization probe, genomic DNA from several species was analyzed for chitobiase gene sequences. The results from these experiments suggest bovine and dog, two species that are believed to be chitobiase-deficient, maintain the chitobiase gene as part of their genetic load. The first three exons of the bovine chitobiase gene were cloned and found to encode an open reading frame that is 77% identical to both human and rat chitobiase. Northern blotting and amplification of mRNA by the polymerase chain reaction indicate that the chitobiase gene in bovine is functional, however, the level of expression is low. The presence of residual amounts of chitobiase enzyme activity in bovine liver and brain was demonstrated. Congruency of the very low levels of chitobiase enzyme to a similarly low level of chitobiase gene expression in bovine indicates that chitobiase in this species has a minor role in hydrolyzing the reducing end GlcNAc of asparagine-linked glycoproteins within the lysosomes. This is in contrast to a species such as human that express substantial quantities of this glycosidase. Thus, the extreme range of chitobiase gene expression among species explains why either 1 or 2 GlcNAc residues remain intact at the reducing end of stored oligosaccharides when either chitobiase-expressing or chitobiase-deficient species, respectively, suffers from a lysosomal storage disease.

Acetylglucosaminidase↗
PubMed1987

Human tissue factor: cDNA sequence and chromosome localization of the gene.

A human placenta cDNA library in lambda gt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, lambda HTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of lambda HTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of approximately 3.2 kilobases in poly(A)+ RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased severalfold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of lambda HTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(A) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 amino acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

The zebrafish Fgf-3 gene: cDNA sequence, transcript structure and genomic organization.

We report the isolation and characterization of genomic and cDNA clones encoding zebrafish fibroblast growth factor 3 (FGF3). An initial cDNA clone was generated by PCR amplification using degenerate oligo primers corresponding to a conserved region of protein found in the mouse and human homologues. Screening a cDNA library made from 30-33-h-old zebrafish embryos with this PCR product led to the isolation of two cDNA clones. Sequence analysis of the longest cDNA insert (1810 bp) revealed a 256-amino-acid (aa) orf. The central region, composed of approx. 155 aa, shares 78% identity with the analogous region of Xenopus laevis FGF3 and 72% identity with the product of the more distantly related human gene. However, the N-and C-terminal domains of zebrafish FGF3 are very different from those of other known homologues. The cDNA was used as a probe on genomic DNA to create a physical map of the locus and to isolate a genomic clone encompassing the entire coding region and 5' sequences. DNA sequencing and RNase protection analyses indicate that zebrafish Fgf-3 (ZFgf-3) is structurally analogous to the mouse gene and regulated through two different promoters. The transcription start point of the proximal promoter aligns to that of mouse promoter P3 and lies within a conserved region of sequence.

Amino Acid Sequence↗
PubMed1997

mRNA expression and cDNA sequences of beta- and gamma-sarcoglycans are normal in cardiomyopathic hamster heart.

In BIO14.6 cardiomyopathic hamster heart, the dystrophin-glycoprotein complex is disrupted and sarcoglycans are greatly reduced in abundance. We examined whether the gene expression of beta- and gamma-sarcoglycans is indeed defective in this hamster. We found that mRNA expression for these proteins and the cDNA sequences of their coding regions are identical in both normal and myopathic hamster cardiomyocytes. The results strongly suggest that defect in a currently unknown sarcoglycan-associated protein(s) is responsible for the deficiency of the sarcoglycan complex that leads to muscle cell necrosis in the myopathic hamster.

Amino Acid Sequence↗
PubMed1984

Human purine nucleoside phosphorylase cDNA sequence and genomic clone characterization.

The isolation of a cDNA clone containing the complete coding region for human purine nucleoside phosphorylase (PNP) has been described previously. In this report we present the nucleotide sequence of this cDNA clone and compare the derived amino acid sequence, encoding a protein of 32 kilodaltons, with the published amino acid composition. Using a fragment of the cDNA clone as a probe, human PNP genomic clones from a bacteriophage lambda library have been isolated and the structural organization of the wild type PNP gene determined.

Base Sequence↗
PubMed1998

A new human selenium-containing protein. Purification, characterization, and cDNA sequence.

Selenium which occurs in proteins as the amino acid, selenocysteine, is essential for numerous biological processes and for human health. A prominent 75Se-labeled protein detected in human T-cells migrated as a 15-kDa band by SDS-polyacrylamide gel electrophoresis. This protein subunit was purified and subjected to tryptic digestion and peptide sequence analyses. Sequences of tryptic peptides derived from the protein corresponded to a human placental gene sequence containing an open reading frame of 162 residues and a readthrough in-frame TGA codon. Three different peptide sequences of the 15-kDa protein corresponded to a nucleotide sequence located downstream of this codon, suggesting that the T-cell 15-kDa selenoprotein contains a selenocysteine residue encoded by TGA. Post-translational processing of the N-terminal portion of the predicted gene product to give the 15-kDa protein was suggested on the basis of molecular mass, amino acid analysis, and immunoblot assays of the purified protein. The 3'-untranslated region (UTR) of the gene encoding the 15-kDa protein contained a sequence that is very similar to the canonical selenocysteine-inserting sequence element. Computer analysis of transcript map data bases indicated that this gene was located on human chromosome 1. Its coding sequence showed no homology to known protein-encoding genes. The 15-kDa protein gene was expressed as mRNA in a wide range of tissues, with increased levels in the thyroid, parathyroid, and prostate-derived cells as evidenced by searches of partial cDNA sequences in public data bases. Genes corresponding to the 15-kDa selenocysteine-containing protein were found in mice and rats, while the corresponding genes in Caenorhabditis elegans and Brugia malayi contained a cysteine codon in place of TGA. The discovery of a new human selenoprotein provides an additional example of the role of selenium in mammalian systems.

Amino Acid Sequence↗
PubMed1999

Insulin-like growth factor-I and -II in the ovary of a bony fish, Oreochromis mossambicus, the tilapia: in situ hybridisation, immunohistochemical localisation, Northern blot and cDNA sequences.

There is accumulating evidence that insulin-like growth factor (IGF)-I and IGF-II are present in the mammalian ovary but comparable studies on bony fish remain scarce. Thus, the present study aims to analyse several parameters of the IGFs in the ovary of a bony fish, the tilapia, (Oreochromis mossambicus). Molecular biological and morphological techniques were applied. The IGF-I and IGF-II cDNA sequences established from the ovary indicate that the same molecules are present in ovary and liver. Northern blot analysis revealed four IGF-I mRNA transcripts (6.0, 3.9, 1.9, 0.5 kb) and three IGF-II mRNA transcripts (5.0, 4.0, 2.0 kb) in ovary and liver. The amounts of IGF-I and IGF-II mRNA in the ovary were considerably high when compared to those in liver (IGF-I: 80.7%; IGF-II: 63.7%). The expression of IGF-I mRNA and IGF-II mRNA in the ovary were studied by in situ hybridisation and the peptides located by immunohistochemistry. The expression of IGF-I varied between the different developmental stages. Both IGF-I mRNA and IGF-I immunoreactivity were present in small oocytes. Moderate IGF-I expression and immunoreactivity occurred in granulosa cells of follicles at the lipid stage. A high IGF-I expression was observed in the granulosa and theca cells surrounding oocytes at the yolk globule stages and mature oocytes but neither IGF-I mRNA nor IGF-I immunoreactivity occurred in oocytes of the later stages. Thus, the IGF-I production seems to change from the young oocyte to the surrounding follicle cells at the later stages. In contrast, IGF-II mRNA and IGF-II-immunoreactivity occurred only in granulosa cells of the late follicle stages. The results suggest that both IGF-I and IGF-II are involved in the maturation of bony fish oocytes and in follicle development in a paracrine/autocrine manner. IGF-I and IGF-II may exert their effects at different stages of development. Furthermore, the intraovarian IGF-I and IGF-II systems seem to have a long phylogenetic history indicating the importance of the IGFs in reproductive biology.

Animals↗
PubMed2006

Porcine SPARC: isolation from dentin, cDNA sequence, and computer model.

Genes encoding the major enamel matrix proteins and non-collagenous proteins of bone and dentin are members of the secretory calcium-binding phosphoprotein (SCPP) family, which originated from ancestral SPARC (secreted protein, acidic and rich in cysteine; BM-40/osteonectin). To better understand the role of SPARC in mineralizing systems, we isolated SPARC from developing pig teeth, deduced its primary structure from the cDNA sequence, and determined its quaternary structure by homology modelling with reference to human SPARC crystal structures. The guanidine/EDTA extract from porcine dentin was fractionated by anion-exchange and size-exclusion chromatography. Stains-all positive bands at 38 and 35 kDa gave the N-terminal sequences APQQEALPDETEV and DFEKNYNMYIFPV, which corresponded to the SPARC N terminus and an internal region of the protein. Porcine SPARC contains 300 amino acids, including the 17-amino acid signal peptide, and shares 96.2% amino acid sequence identity with human SPARC. Without post-translational modifications, the 283-amino acid secreted protein has a molecular mass of 32.3 kDa. The three-dimensional model revealed that porcine SPARC contains a single N-linked glycosylation at N113, seven intramolecular disulfide bridges, and assembles into dimers. SPARC is composed of three structural/functional domains: an acidic Ca2+-binding, a follistatin-like, and an extracellular calcium-binding domain.

Amino Acid Sequence↗
PubMed1990

Detection of hepatitis C virus cDNA sequence by the polymerase chain reaction in hepatocellular carcinoma tissues.

We found the presence of hepatitis C virus (HCV) infection in liver tissues of hepatocellular carcinoma (HCC) patients who had antibodies to HCV but no serological markers for hepatitis B virus infection by the sensitive reverse transcription/polymerase chain reaction (R/PCR) method. The primers used were derived from the non-structural (NS) 3 and/or the structural (C/E) region. Amplified cDNA sequences of HCV were detected in either cancerous or non-cancerous portion of liver tissues from four out of eight HCC patients with primers of NS3 region. Similar but less efficient results were obtained with primers of C/E region. These results indicate that HCV persists in the liver tissue of HCC. A possible role of persistent infection of HCV for the development of HCC is discussed.

Base Sequence↗
PubMed1998

Identification of posttranslational modifications and cDNA sequencing errors in the rat S100 proteins MRP8 and 14 using electrospray ionization mass spectrometry.

MRP8 and 14 are S100 proteins expressed by myeloid cells and are predicted to have important functions in inflammation. The proteins were isolated from spleens from three rat strains. Electrospray ionization mass spectrometry indicated masses of 10,149 +/- 2 Da for MRP8 and 13,069 +/- 2 Da for MRP14 compared to masses calculated from proteins derived from their cDNA sequences of 10,211 and 13,214 Da, respectively, indicating posttranslational modifications and/or errors in the derived protein sequences. Several endoprotease digest peptides did not correspond to any theoretical digest products after comparison of ESI masses with those derived from the theoretical digest. Both proteins were N-terminally acetylated after deletion of the initiator Met, reducing the theoretical masses by 89 Da. A peptide with mass 28 Da greater than the theoretical was isolated from the Asp N digestion of MRP8. N-terminal sequencing indicated translated Val instead of the predicted Ala at position 72 of MRP8. A peptide 56 Da less than the theoretical was isolated from the chymotryptic digestion of MRP14, and the carboxyamidomethylated form was N-terminally sequenced and found to have translated Ser instead of the predicted Arg at position 105. In addition, His106 was methylated. The corrected theoretical masses, incorporating the posttranslational modifications and sequencing errors, are 10,149.4 and 13,069.9 Da for MRP8 and 14, respectively, in good agreement with the experimental masses.

Amino Acid Sequence↗
PubMed2002

Identification and characterisation of a cDNA sequence encoding a glutamic acid-rich protein specifically transcribed in Trichinella spiralis newborn larvae and recognised by infected swine serum.

Presently, little is known of the mechanism by which Trichinella penetrates and modulates reprogramming of muscle cells. In light of evidence demonstrating strong protective characteristics of antigens derived from this stage, understanding this process may shed light on potential targets for effective abatement of infection. To this end, a PCR-derived cDNA expression library was constructed using 0.5 micro g of total RNA from Trichinella spiralis newborn larvae. The library consisted of >125000 insert-containing clones. Approximately 40-50 x 10(3) clones were screened immunologically using sera from pigs experimentally infected with 7000 Trichinella L1. Multiple clones reacting positively with the swine infection serum and encoding portions of a glutamic acid-rich protein were identified. Northern and Southern blots indicated at least two distinct genes that encoded the glutamic acid-rich proteins and that these genes were transcribed specifically in the newborn larvae stage. cDNA sequence data predicted open reading frames of 1497 and 1,716 bp generating proteins of 498 amino acids and 571 amino acids, respectively. Both sequences consisted of approximately 39% glutamic acid and 16% serine residues, and differed by the presence of a 219 bp fragment present in the 1716 bp sequence that was absent from the 1497 bp sequence. PCR data indicated that additional isoforms exist within this gene family that are different in length from those described above. In addition, it was found that more than one isoform can exist within a single worm and that this pattern can vary between individual worms within a population. Mouse antibodies to recombinant antigen localised the glutamic acid-rich proteins to the periphery of the developing stichocyte cells within the newborn larvae consistent with the hypothesis that the newborn larval antigens are secreted.

Amino Acid Sequence↗
PubMed1991

The transthyretin cDNA sequence is normal in transthyretin-derived senile systemic amyloidosis.

A variety of mutations leading to amino acid substitutions have been described in the transthyretin gene in association with different familial amyloidoses and have been implicated to be involved in the pathogenesis of amyloid deposits. However, there has been disagreement whether or not a transthyretin mutation is present in the most common form of transthyretin-derived amyloid, namely senile systemic amyloidosis. Therefore, the cDNA sequence of liver transthyretin was determined in a 91-year-old patient with typical senile systemic amyloidosis. This sequence was completely normal and lacked any variation. We conclude that in senile systemic amyloidosis factors other than the presence of a sequentially variant transthyretin must determine the amyloid fibril formation.

Aged↗
PubMed1989

cDNA sequence of the long terminal repeat of a PrB strain of the Rous sarcoma virus.

The cDNA library of the polyA+ RNA from Prague B strain of RSV infected cells was constructed by the use of oligo(dT) primer and the reverse transcription method. The clones containing the LTR sequences were studied. The DNA sequences of several clones were studied and compared with the cDNA sequence of the long terminal repeat (LTR) region of a PrB strain of Rous sarcoma virus (RSV) which had previously been determined. By the analyses of several different cDNA clones, mutations were detected in the R region as well as in the U3 region of the LTR in two different cDNA clones. By comparing the DNA sequences of these cDNA clones with the original viral LTR, the error-prone hypothesis of the RSV reverse transcriptase was confirmed.

Avian Sarcoma Viruses↗
PubMed1988

The complete amino acid sequence of the human erythrocyte membrane anion-transport protein deduced from the cDNA sequence.

1. We have isolated cDNA clones corresponding to the red cell membrane anion-transport protein (Band 3). 2. The cDNA clones cover 3475 bases of the mRNA and contain the entire protein-coding region, 150 bases of the 5' untranslated region and part of the 3' non-coding region, but do not extend to the 3' end of the mRNA. 3. The translated protein sequence predicts that the human red cell anion transporter contains 911 amino acids. 4. The availability of the amino acid sequence allows the interpretation of some of the many studies on the chemical and proteolytic modification of the human protein aimed at examining the structure and mechanism of this membrane transport protein.

Amino Acid Sequence↗
PubMed1992

An S5 self-incompatibility allele-specific cDNA sequence from Brassica oleracea shows high homology to the SLR2 gene.

The sequence is reported of a cDNA molecule homologous to an mRNA from stigma tissue of Brassica oleracea plants homozygous for the S5 self-incompatibility allele. This cDNA is closely related to a previously published sequence designated SLR2, which was obtained from the same stigma cDNA library and is also related to the SLR1 gene, a cDNA for which has also been obtained from this library. Various B. oleracea lines differing in S alleles and of different varieties have been screened for the presence of particular S gene family sequences using a method involving hybridization of sequence-specific oligonucleotide probes to PCR products. The results indicate that a gene homologous to the sequence presented here is absent from a line lacking the S5 allele, though present in other lines containing the S5 allele, regardless of their genetic background. This finding suggests that the sequence represents a transcript of the SLG (S locus glycoprotein) gene. A similar approach has confirmed that a cDNA derived from a Brassica line containing the S29 allele is also S allele-specific. The predicted amino acid sequences derived from a number of S gene family sequences are compared using numerical methods and possible evolutionary relationships between them are discussed.

Alleles↗
PubMed1998

Porcine spleen deoxyribonuclease II. Covalent structure, cDNA sequence, molecular cloning, and gene expression.

Porcine spleen DNase II, a lysosomal acid hydrolase, is a noncovalently linked alpha.beta heterodimer (Liao, T.-H. (1985) J. Biol. Chem. 260, 10708-10713). The alpha subunit, after disulfide cleavage, yields two chains, alpha1 and alpha2. The complete amino acid sequences of the alpha1, beta, and alpha2 chains were elucidated by protein sequencing, and the pairings of one interchain disulfide between alpha1 and alpha2 and of three intrachain disulfides in alpha2 were assigned. Six carbohydrate attachment sites, two in beta and four in alpha2, were detected by sugar analyses. The cDNA of DNase II was amplified using primers synthesized on the basis of the amino acid sequences determined. The amplified fragments shown to be a cDNA sequence of 1,292 bases. This cDNA sequence has an open reading frame encoding a 364-amino acid polypeptide containing a putative transmembrane peptide at the NH2-end, two small connecting peptides in the middle, and a peptide at the COOH terminus. These are evidently removed to form mature DNase II. Thus, all three chains in the sequence alpha1, beta, and alpha2 are coded by the same cDNA. When Chinese hamster ovary cells were transfected with a cloned plasmid with an inserted cDNA fragment encoding the entire reading frame, the expressed protein was released into the growth medium as an active form of DNase II.

Amino Acid Sequence↗
PubMed1987

The cDNA sequence and gene analysis of the human pim oncogene.

The putative oncogene pim-1 is frequently activated by provirus insertion in MuLV-induced T cell lymphomas in mice. By analogy with other cellular oncogenes that are similarly activated (e.g., myc, myb, erbB) it is possible that pim-1 may also be involved in some human tumors. To study human pim-1 we cloned and sequenced human pim-1 cDNA clones. The human pim-1 codes for a protein of 313 amino acids (aa) which is highly homologous (94%) to the deduced amino acid sequence of mouse pim-1. All the mouse pim-1 residues which are homologous to protein kinases are conserved in the human pim-1. We also isolated the human pim-1 gene and mapped it relative to the cDNA. The RNA transcript of human pim-1 is approx. 3.0 kb and it is highly expressed in the erythroleukemia cell line K562.

Animals↗
PubMed1995

cDNA sequence and gene locus of the human retinal phosphoinositide-specific phospholipase-C beta 4 (PLCB4).

Defects in the Drosophila norpA (no receptor potential A) gene encoding a phosphoinositide-specific phospholipase C (PLC) block invertebrate phototransduction and lead to retinal degeneration. The mammalian homolog, PLCB4, is expressed in rat brain, bovine cerebellum, and the bovine retina in several splice variants. To determine a possible role of PLCB4 gene defects in human disease, we isolated several overlapping cDNA clones from a human retina library. The composite cDNA sequence predicts a human PLC beta 4 polypeptide of 1022 amino acid residues (MW 117,000). This PLC beta 4 variant lacks a 165-amino-acid N-terminal domain characteristic for the rat brain isoforms, but has a distinct putative exon 1 unique for human and bovine retina isoforms. A PLC beta 4 monospecific antibody detected a major (130 kDa) and a minor (160 kDa) isoform in retina homogenates. Somatic cell hybrids and deletion panels were used to localize the PCLB4 gene to the short arm of chromosome 20. The gene was further sublocalized to 20p12 by fluorescence in situ hybridization.

Amino Acid Sequence↗