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At least 811 records · Page 45Linked to original sources
PubMed1994

Exploring the microtubule-binding region of bovine microtubule-associated protein-2 (MAP-2): cDNA sequencing, bacterial expression, and site-directed mutagenesis.

A 1.1 kilobase fragment of bovine microtubule-associated protein-2 (MAP-2) cDNA coding for bovine MAP-2 microtubule-binding region (MTBR) was sequenced. Relative to mouse, rat, and human MAP-2, we observed striking preservation of primary structure, even beyond the sequence and spacing of the three nonidentical peptide repeats responsible for microtubule-binding interactions. For further analysis of microtubule-MAP interactions using site-directed mutagenesis, we developed a bacterial expression system coding for the MT-binding fragment of MAP-2 starting at the thrombin cleavage site (position 1629) and continuing to the C-terminus. This MT-binding fragment was purified to homogeneity by taking advantage of the unusual heat-stability and isoelectric properties of this cytomatrix component. We found that the MT-binding domain readily promoted tubulin polymerization, and the critical tubulin concentration was reduced in the presence of this recombinant protein. Because a second repeated sequence analogue can promote tubulin polymerization as well as displace the MT-binding region of MAP-2, this study was designed to learn more about the importance of each repeated sequence in MT binding. Accordingly, we mutated the first and third sequences to resemble the second repeated sequence, thereby generating the mutants designed m12-m2-m3, m1-m32, and m12-m2-m32. These recombinant proteins bound with an affinity comparable to or slightly better than equal concentrations of wild-type MT-binding fragment. Likewise, when the first or third sequence was replaced by an exact copy of the second octadecapeptide repeat, there was little, if any, increase in binding affinity, as reflected in the ability of mutant MT-binding fragments to promote tubulin polymerization.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Cloning, expression, and cDNA sequence of surface antigen P22 from Toxoplasma gondii.

Immunoblot, immunofluorescence, and complement-mediated cytolytic assays revealed that two new monoclonal antibodies raised against a membrane-enriched fraction of Toxoplasma gondii tachyzoites recognize protein P22 on the surface of the parasite. Using these monoclonal antibodies to screen a cDNA expression library in lambda gt11, several clones expressing recombinant fusion proteins were isolated. Subsequent screening of the library with a synthetic oligonucleotide derived from the 5' end of one of these cDNAs permitted the isolation of additional nonexpressing clones containing the entire translated sequence. Blots of parasite RNA and DNA suggested that the corresponding gene occurs as a single copy in the tachyzoite genome. The amino acid sequence deduced from the composite cDNA indicates a primary translation product with a theoretical molecular weight of 18,959. As expected for surface protein P22, the putative polypeptide contains a predicted N-terminal signal sequence and a C-terminal hydrophobic region characteristic of proteins attached to the membrane by a glycophospholipid anchor. Recombinant fusion proteins produced by the expressing clones were recognized on immunoblots by IgG antibodies in the sera of humans with acute and chronic T. gondii infection. Antibodies selected by the fusion protein reacted predominantly with a 22-kDa antigen on immunoblots of parasite lysate.

Amino Acid Sequence↗
PubMed1987

cDNA sequences of two apolipoproteins from lamprey.

The messages for two small but abundant apolipoproteins found in lamprey blood plasma were cloned with the aid of oligonucleotide probes based on amino-terminal sequences. In both cases, numerous clones were identified in a lamprey liver cDNA library, consistent with the great abundance of these proteins in lamprey blood. One of the cDNAs (LAL1) has a coding region of 105 amino acids that corresponds to a 21-residue signal peptide, a putative 8-residue propeptide, and the 76-residue mature protein found in blood. The other cDNA (LAL2) codes for a total of 191 residues, the first 23 of which constitute a signal peptide. The two proteins, which occur in the "high-density lipoprotein fraction" of ultracentrifuged plasma, have amino acid compositions similar to those of apolipoproteins found in mammalian blood; computer analysis indicates that the sequences are largely helix-permissive. When the sequences were searched against an amino acid sequence data base, rat apolipoprotein IV was the best matching candidate in both cases. Although a reasonable alignment can be made with that sequence and LAL1, definitive assignment of the two lamprey proteins to typical mammalian classes cannot be made at this point.

Amino Acid Sequence↗
PubMed1994

Complete cDNA sequence of human lysosome-associated membrane protein-2.

The isolation and sequencing of 15 independent human lysosome-associated membrane protein-2 (h-lamp-2) recombinants from a primary human liver cDNA library has resulted in the determination of a transcript sequence significantly longer than previously reported and reveals the utilization of each of the four potential polyadenylation signals (AATAAA) present in the 3' untranslated region. The most 5' extending cDNA clone initiates upstream of the proposed transcription initiation site. A number of differences with published sequences for the h-lamp-2 transcript were observed, some of which result in amino acid changes in the predicted primary structure of the h-lamp-2 protein, and two of which give rise to restriction fragment length polymorphisms. The knowledge of these sequence alterations and polymorphisms is an important consideration for the further analysis of the h-lamp-2 locus with regard to the delineation of function and association with human inherited disorders.

Amino Acid Sequence↗
PubMed1987

cDNA sequence, protein structure, and chromosomal location of the human gene for poly(ADP-ribose) polymerase.

Recently we described a full-length cDNA for the human nuclear enzyme poly(ADP-ribose) polymerase. Here, we report the chromosomal localization and partial map of the human gene for this enzyme as well as the complete coding sequence for this protein. The nucleotide sequence reveals a single 3042-base open reading frame encoding a protein with a predicted Mr of 113,135. A comparison of this deduced amino acid sequence with the amino acid sequence of three peptides derived from human poly(ADP-ribose) polymerase revealed a match of 27 amino acid residues. A computer-derived structural analysis of the enzyme and a search for similarities with other proteins confirmed that the polymerase belongs to a subfamily of DNA/NAD-binding proteins and DNA-repair proteins. Possible Zn2+-binding "fingers," a nucleotide-binding fold, and a nuclear transport signal were noted. Additionally, chromosomal mapping has identified polymerase-hybridizing sequences on human chromosomes 1 (the active gene), 13, and 14 (processed pseudogenes). Using the polymerase cDNA as a probe, we also have detected several DNA restriction fragment length polymorphisms in normal humans.

Amino Acid Sequence↗
PubMed1986

Link protein cDNA sequence reveals a tandemly repeated protein structure.

Link protein stabilizes the cartilage proteoglycan/hyaluronic acid aggregate by binding to both components. We screened a cDNA library prepared from rat chondrosarcoma mRNA in the lambda gt11 expression vector with monoclonal antibodies and polyclonal antisera to link protein. We obtained a clone for two-thirds of the link protein cDNA and identified it based on its deduced amino acid sequence. There are four RNA transcripts for link protein, ranging from 1.5 to 5.5 kilobases in size. The deduced amino acid sequence for link protein shows two domains of 100 residues each, which share 44% homology; within each domain is a 19-residue stretch 74% homologous with its counterpart. This structure indicates that link protein may have been formed by gene duplication.

Amino Acid Sequence↗
PubMed1996

Constitutive secretion of beta-trace protein by cultivated porcine choroid plexus epithelial cells: elucidation of its complete amino acid and cDNA sequences.

Primary porcine choroid plexus epithelial cells cultivated in chemically defined medium maintain their epithelial characteristics and form confluent monolayers. They produce a fluid the composition of which resembles cerebrospinal fluid. The present study demonstrates constitutive secretion of large amounts of beta-trace protein. This intrathecally synthesized protein is a prominent polypeptide constituent of natural cerebrospinal fluid. According to the identity of amino acid sequences it has previously been tentatively identified as a prostaglandin-D synthase and as a member of the lipocalin protein family. beta-Trace was purified from cell culture supernatants and was subjected to tryptic digestion and amino acid sequencing of the resulting peptides. The complete primary structure of the protein was obtained by additional isolation of the cDNA from cultured epithelial cells. The porcine 163-amino acid polypeptide showed 69% identity with the human beta-trace and contained two N-glycosylation sites occupied by complex-type oligosaccharides as is the case for the human protein. The amino acid sequences around the N-glycosylation sites of mammalian beta-trace proteins (porcine, human, murine, and rat) were highly conserved. The nucleotide sequence was found to be less conserved; the porcine cDNA had a strikingly high GC-content (67%). The constitutive secretion of beta-trace protein from the in vitro cultivated porcine choroid plexus epithelial cells demonstrates that the cells have retained their major in vivo physiological properties: secretion of cerebrospinal fluid proteins. Therefore, this in vitro culture system may be used as a versatile tool for studying the regulation of the formation of cerebrospinal fluid.

Amino Acid Sequence↗
PubMed1985

Presence of hormonogenic and repetitive domains in the first 930 amino acids of bovine thyroglobulin as deduced from the cDNA sequence.

The sequence of the first 2831 nucleotides of bovine thyroglobulin mRNA has been determined from the analysis of a cDNA clone. Following a 41-nucleotide 5' untranslated sequence, a single open-reading frame encoding 930 amino acids was observed. This corresponds to the aminoterminal third of thyroglobulin, preceded by a putative signal peptide of 19 amino acids. The protein sequence was found to be essentially made of the sevenfold repetition of a 60-amino-acid-long building unit, interrupted at fixed positions by unrelated segments of variable length. The presence of an internal homology within the repetitive unit itself suggests that the 5' region of the thyroglobulin gene has evolved from the initial duplication of a relatively short sequence, followed by the serial duplication of the resulting unit. The tyrosine residue at position five has been assigned an important hormonogenic function [Mercken, L., Simons, M.-J. and Vassart, G. (1982) FEBS Lett. 149, 285-287]. This residue is flanked by sequence elements related to the repeated unit, suggesting that the hormonogenic domain evolved also from the basic ancestor sequence.

Amino Acids↗
PubMed1997

Genomic and cDNA sequence analysis of the cell matrix adhesion regulator gene.

The cell matrix adhesion regulator (CMAR) gene has been suggested to be a signal transduction molecule influencing cell adhesion to collagen and, through this, possibly involved in tumor suppression. The originally reported CMAR cDNA was 464 bp long with a tyrosine phosphorylation site at the extreme 3' end, which mutagenesis studies had shown to be central to the function of this gene. Since the discovery of a 4-bp insertion polymorphism within the originally reported coding region, further sequence information has been obtained. The cDNA has been extended 5' by approximately 2 kb revealing a 559-bp region showing strong homology to the proposed 5' untranslated sequence of a murine protein kinase receptor family member, variant in kinase (vik). CMAR genomic sequencing has shown the presence of an intron, the intron/exon boundary lying within this region of homology. An RNA transcript for CMAR of approximately 2.5 kb has also been identified. The data suggest complex mechanisms for control of expression of two closely associated genes, CMAR and the vik- associated sequence.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1991

Characterization of human deoxycytidine kinase. Correlation with cDNA sequences.

Existing data on the structure of human deoxycytidine kinase (dCK) diverge. A monomeric 60 kDa form has been isolated and the cloning of a cDNA coding for 626 amino acids corresponding to a 71 kDa protein has been reported. However, pure dCK isolated from leukemic spleen is a dimer of 30 kDa subunits. Amino acid sequences of peptides from digests of this protein are now presented. None of the peptide structures obtained correspond to the cDNA for the 71 kDa protein, but to a cDNA for a 30.5 kDa dCK recently cloned. Furthermore, homology of the peptide sequences od dCK to parts of thymidine kinases and protein-tyrosine kinases are detected.

Amino Acid Sequence↗
PubMed1994

cDNA sequencing: a means of understanding cellular physiology.

High-throughput automated sequencing has enabled researchers to examine large numbers of clones from a cDNA library as a measure of the steady-state levels of mRNA species. The past year has witnessed many new applications of this technique to allow the qualitative and quantitative comparison of the changes in transcript levels from multiple genes.

Animals↗
PubMed1989

Cytovillin, a microvillar Mr 75,000 protein. cDNA sequence, prokaryotic expression, and chromosomal localization.

Cytovillin is a microvillar cytoplasmic peripheral membrane protein, with prominent expression in vivo in placental syncytiotrophoblasts and certain human tumors. Cytovillin cDNA was cloned from a human placental lambda gt11 library using affinity purified antibodies. The identity of cytovillin cDNA clones was confirmed by expression of cytovillin in Escherichia coli and using antibodies raised against the expressed fusion protein in comparison with antibodies against cytovillin purified from cultured human choriocarcinoma cells. In these cells Northern blotting analysis identified a major 3.5-kilobase cytovillin mRNA. The cDNA encodes a protein of 575 amino acids corresponding to a molecular weight of 68,084. According to secondary structure prediction, cytovillin is a hydrophilic protein with an extensive internal alpha-helical region ending in a sequence of 7 consecutive prolines. The predicted alpha-helical region showed limited homology to alpha-helical regions of cytoskeletal proteins and certain other proteins, but no extensive homologies were found in the cytovillin cDNA or the deduced amino acid sequence to other registered DNA or protein sequences. Southern blot analysis of a DNA panel of human mouse somatic cell hybrids localized the cytovillin gene to the end of the long arm of chromosome 6 (6q22-q27). Our results show that cytovillin is representative of a novel class of microvillar proteins.

Amino Acid Sequence↗
PubMed2002

Identification of mammalian-type transglutaminase in Physarum polycephalum. Evidence from the cDNA sequence and involvement of GTP in the regulation of transamidating activity.

Transglutaminase (TGase) catalyses the post-translational modification of proteins by transamidation of available glutamine residues. While several TGase genes of fish and arthropods have been cloned and appear to have similar structures to those of mammals, no homologous gene has been found in lower eukaryotes. We have cloned the acellular slime mold Physarum polycephalum TGase cDNA using RT-PCR with degenerated primers, based on the partial amino acid sequence of the purified enzyme. The cDNA contained a 2565-bp ORF encoding a 855-residue polypeptide. By Northern blotting, an mRNA of approximately 2600 bases was detected. In comparison with primary sequences of mammalian TGases, surprisingly, significant similarity was observed including catalytic triad residues (Cys, His, Asn) and a GTP-binding region. The alignment of sequences and a phylogenetic tree also demonstrated that the structure of P. polycephalum TGase is similar to that of TGases of vertebrates. Furthermore, we observed that the purified TGase had GTP-hydrolysing activity and that GTP inhibited its transamidating activity, as in the case of mammalian tissue-type TGase (TGase 2).

Amino Acid Motifs↗
PubMedDate not supplied

Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells.

The complete 1,210-amino acid sequence of the human epidermal growth factor (EGF) receptor precursor, deduced from cDNA clones derived from placental and A431 carcinoma cells, reveals close similarity between the entire predicted v-erb-B mRNA oncogene product and the receptor transmembrane and cytoplasmic domains. A single transmembrane region of 23 amino acids separates the extracellular EGF binding and cytoplasmic domains. The receptor gene is amplified and apparently rearranged in A431 cells, generating a truncated 2.8-kilobase mRNA which encodes only the extracellular EGF binding domain.

Amino Acid Sequence↗
PubMed1990

Cloning and expression of eukaryotic initiation factor 4B cDNA: sequence determination identifies a common RNA recognition motif.

Eukaryotic protein synthesis initiation factor 4B (eIF-4B) is an 80,000 dalton polypeptide which is essential for the binding of mRNA to ribosomes. A highly purified preparation of eIF-4B from HeLa cells was subjected to enzymatic cleavage and amino-terminal amino acid sequence analysis. Degenerate oligonucleotide probes were used to isolate a 3851 bp cDNA encoding eIF-4B from a human cDNA library. The DNA encodes a protein comprising 611 residues with a mass of 69,843 daltons. The amino-terminal domain of eIF-4B contains a consensus RNA binding domain present in a number of other RNA binding proteins. Expression of eIF-4B in transfected COS-1 cells yielded a polypeptide which reacted with anti-eIF-4B antiserum and comigrated with purified eIF-4B. Expression of eIF-4B in COS-1 cells resulted in a general inhibition of translation, possibly due to a 50-fold eIF-4B overproduction.

Amino Acid Sequence↗
PubMed2002

Complete cDNA sequence of chitin deacetylase from Gongronella butleri and its phylogenetic analysis revealed clusters corresponding to taxonomic classification of fungi.

A cDNA library containing a chitin deacetylase (CDA) gene from a zygomycete Gongronella butleri was constructed and the complete gene was sequenced. The complete gene contains an open reading frame of 1290 nucleotides which encodes a sequence of 430 amino acid residues. The gene sequence consists of nucleotides encoding a polysaccharide deacetylase domain located in the middle, covering 34% of the entire sequence. This domain shares high sequence similarities with CDAs of fungi and other deacetylases including the nodb-like protein from Sinorhizobium meliloti and Bacillus subtilis, xylanase D from Cellulomonas fimi, and acetylxylan esterase A from Streptomyces lividans. The deduced amino acid sequence shows homology with CDAs from Mucor rouxii (48% identity) and Phycomyces blakesleeanus (30% identity). Phylogenetic analysis reveals that CDAs are clustered in accordance with the taxonomic classification of the fungi.

Journal Article↗
PubMed1992

The cDNA sequence, expression pattern and protein characteristics of mouse protein kinase C-zeta.

A 2199-bp complementary DNA (cDNA) that encodes protein kinase C-zeta (PKC-zeta) has been isolated from mouse brain by a combination of reverse transcription and primer extension. The predicted PKC-zeta protein consists of 592 amino acids which are 99% identical to those of rat PKC-zeta. Northern blots that were probed with this cDNA revealed abundant 2200-nucleotide (nt) and 4200-nt PKC-zeta mRNAs in mouse brain in roughly equal amounts. PKC-zeta mRNA was also abundant in normal lung, kidney, and testes, and in several hemopoietic tumor lines. In all other mouse tissues and cell lines that were examined, at least faint levels of PKC-zeta mRNAs could also be detected. In tissues other than brain, the amount of PKC-zeta mRNA was less, and the smaller species generally predominated. Furthermore, in these tissues, both PKC-zeta mRNAs appear to be approximately 200 nt longer than the two mRNAs found in the brain. When the cDNA is expressed in insect cells via a baculovirus expression vector, a 75-kDa protein is synthesized which, unlike other PKC isoforms, does not bind phorbol ester, even at very high concentrations.

Amino Acid Sequence↗