Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “cDNA sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,567 records · Page 87Linked to original sources
PubMed2006

Genomic structure, promoter analysis and expression of the porcine (Sus scrofa) TLR4 gene.

Toll-like receptor 4 (TLR4) is essential for initiating the innate response to lipopolysaccharide (LPS) from Gram-negative bacteria by acting as a signal-transducing receptor. As the pig industry faces a unique array of related pathogens, it is anticipated that the genotype of swine TLR4 could be of crucial importance in future strategies aimed at improving genetic resistance to infectious diseases. In order to help in investigating TLR4 as a candidate disease-resistance gene in pigs, we established its genomic structure and produced sufficient flanking intronic sequences to enable simple PCR amplification of the coding portions of the gene. Expression in different porcine tissues was studied and showed splicing variations in mRNA sequences. The cDNA sequence for poTLR4 contains an open reading frame of 2526bp that codes for 841 aa, 98 and 568bp in the 5'- and 3'-UTRs, respectively. Overall, the general organization of porcine, human, murine, and avian TLR4 genes is quite similar: three exons with the third one very long. A high level of conservation of the size and the sequence, especially for the two last exons and particularly in the sequence corresponding to the LRRs and TIR domain, is observed between species. The important antimicrobial properties of these proteins may account for a conservative selection pressure on these TLR4 coding sequences. Several putative binding sites described in the human and murine promoter of TLR4 genes have been identified in the 5'-flanking region of poTLR4. Conversely, this region lacks a TATA box, consensus initiator sequences, or GC-rich regions. The basic sequence data gathered will allow the establishment of an inventory of naturally occurring variation in porcine TLR4, so that alleles can be tested for disease association studies.

Animals↗
PubMed1992

The primary structure of mouse saposin.

The primary structure of mouse sphingolipid activator protein (saposin) was determined by cDNA sequencing. The amino acid sequence predicted by the cDNA sequence revealed that mouse saposin was highly homologous to human saposin and also to rat sertoli cell glycoprotein. Mouse saposin also has four functional domains, which are structurally similar to each other, and each domain has cysteines, prolines, and a potential glycosylation site at an almost identical position. An amino acid comparison between human and mouse saposins revealed that the similarity was approximately 70%, and human saposin lacks thirty-one amino acids between domains C and D. Heterogeneities of mRNA were found in both the coding and noncoding regions.

Amino Acid Sequence↗
PubMed2003

Redifferentiation of dedifferentiated chondrocytes and chondrogenesis of human bone marrow stromal cells via chondrosphere formation with expression profiling by large-scale cDNA analysis.

Characterization of dedifferentiated chondrocytes (DECs) and mesenchymal stem cells capable of differentiating into chondrocytes is of biological and clinical interest. We isolated DECs and bone marrow stromal cells (BMSCs), H4-1 and H3-4, and demonstrated that the cells started to produce extracellular matrices, such as type II collagen and aggrecan, at an early stage of chondrosphere formation. Furthermore, cDNA sequencing of cDNA libraries constricted by the oligocapping method was performed to analyze difference in mRNA expression profiling between DECs and marrow stromal cells. Upon redifferentiation of DECs, cartilage-related extracellular matrix genes, such as those encoding leucine-rich small proteoglycans, cartilage oligomeric matrix protein, and chitinase 3-like 1 (cartilage glycoprotein-39), were highly expressed. Growth factors such as FGF7 and CTGF were detected at a high frequency in the growth stage of monolayer stromal cultures. By combining the expression profile and flow cytometry, we demonstrated that isolated stromal cells, defined by CD34(-), c-kit(-), and CD140alpha(- or low), have chondrogenic potential. The newly established human mesenchymal cells with expression profiling provide a powerful model for a study of chondrogenic differentiation and further understanding of cartilage regeneration in the means of redifferentiated DECs and BMSCs.

Bone Marrow Cells↗
PubMed2000

Molecular cloning of a gene encoding a 20S proteasome beta subunit from Plasmodium falciparum.

A novel gene was cloned from Plasmodium falciparum. Database searches indicated this gene to be a member of the 20S proteasome beta-subunit family. Comparison of the gene's genomic DNA sequence with cDNA sequence revealed a 156-bp intron 85 bp downstream from the start codon. The nucleotide sequence of the gene contains one open reading frame encoding 265 amino acids with a predicted molecular mass of 30.9 kDa and a pI of 6.2. Northern blot analysis showed the transcript size to be approximately 1.6 kb indicating that some 800 bp of the transcript is non-coding.

Adenosine Triphosphatases↗
PubMed2000

Cloning and expression of human adenylyl cyclase type VI in normal thyroid tissues.

The adenylyl cyclase type VI gene expressed in human normal thyroid tissue was cloned and sequenced. The cDNA sequence (6463 nt) is susceptible to code for a 1168 aa protein. Northern blots using specific probes showed that the expression of adenylyl cyclase type VI gene was significantly higher in one hyperfunctioning thyroid tumor than in normal thyroid tissue, in one follicular cold adenoma or in one papillary carcinoma.

Adenoma↗
PubMed2003

Characterization of the glutamate dehydrogenase gene and its regulation in a euryhaline copepod.

Glutamate dehydrogenase (GDH) plays a key role in the metabolism of free amino acids (FAA) in crustaceans and other metazoans. Glutamate synthesized by GDH via reductive amination is the amino group donor for alanine synthesis and the precursor required for proline synthesis. Since both proline and alanine are important intracellular osmolytes in many marine invertebrates, GDH has been widely implicated as playing a central role in response to hyperosmotic stress in these organisms. We have isolated the gene encoding a GDH homolog from the euryhaline copepod Tigriopus californicus and examined the regulation of GDH under salinity stress. The gene encodes a protein of 557 residues with 76% amino acid identity with Drosophila melanogaster GDH. The gene encodes an N-terminal mitochondrial signal sequence peptide. Only a single intron of 71 bp was found in the GDH gene in T. californicus when genomic sequences and cDNA sequences were compared. The levels of GDH mRNA do not increase during hyperosmotic stress in this copepod. The effects of salt and hyperosmotic stress on GDH enzyme activity were also investigated. GDH activities decrease with increasing NaCl concentrations in in vitro enzyme assays, while live animals exposed to hyperosmotic stress showed no change in GDH enzyme activities. Combined, these results indicate that GDH transcription and enzyme activity do not appear to function in the regulation of alanine and proline accumulation during hyperosmotic stress in T. californicus. The manner in which this important physiological process is regulated remains unknown.

Amino Acid Sequence↗
PubMed1995

The complete sequence of human lens gamma s-crystallin.

The complete sequence of human gamma s-crystallin has been determined and confirmed using a combination of MS methods, peptide sequencing and cDNA sequencing. Regions 21-35 and 102-107, which were previously assumed to be the same as the bovine sequence, differ from the bovine sequence at residues 22, 28, 31 and 104. An additional six residues were also found to be different from the original sequence determined for Pakastani lenses. Whether these differences represent errors in the original sequence or two different sequences among human lens crystallins is not yet known.

Amino Acid Sequence↗
PubMed1996

Prediction from sequence comparisons of residues of factor H involved in the interaction with complement component C3b.

The amino acid sequence of the region of bovine factor H containing the C3b binding site has been derived from sequencing overlapping cDNA clones. A cDNA sequence encoding 669 amino acids was obtained. Like human and mouse factor H the sequence can be arranged into a number of internally homologous units (CPs), each of which is about 60 amino acids long and is based on a framework of four conserved cysteine residues. Bovine factor H is of the same molecular mass as human and mouse factor H, and is therefore likely to be composed of 20 contiguous CPs. Comparisons with human and mouse factor H indicate that the partial bovine sequence encodes CPs 2-12 inclusive of bovine factor H. Bovine factor H binds to human ammonia-treated C3 (causing thiolester cleavage) [C3(NH3)] and promotes the cleavage of human C3(NH3) in the presence of bovine factor I. Other studies indicate that CPs 2-5 of human factor H encompass the C3b binding and factor I cofactor activity site. Multiple sequence alignments of human factor H, mouse factor H (which also interacts with human C3b) and bovine factor H with CP modules whose structures have been determined experimentally, have been used to predict residues in the hypervariable loops of CPs 2-5 and to identify residues of potential importance in human C3 binding and factor I cofactor activity. Leu-17 and Gly-20 of CP 2, Ser-17, Ala-19, Glu-21, Asp-23 and Glu-25 of CP 3 and Lys-18 of CP 4 are all conserved between the three species. It may be that CPs 3 and 4 interact with C3(NH3) directly, whilst CPs 2 and 5 maintain the correct orientation for CPs 3 and 4 to interact.

Amino Acid Sequence↗
PubMed2004

The lysozyme of the starfish Asterias rubens. A paradygmatic type i lysozyme.

On the basis of a partial N-terminal sequence, Jollès and Jollès previously proposed that the lysozyme from the starfish Asterias rubens represents a new form of lysozyme, called type i (invertebrate) lysozyme. Indeed, it differed from both the types c (chicken) and g (goose) known in other animals, as well as from plant and phage lysozymes. Recently, several proteins belonging to the same family have been isolated from protostomes. Here we report the complete mature protein sequence and cDNA sequence of the lysozyme from Asterias. These sequences vindicate the previously proposed homology between the starfish, a deuterostome, and protostome lysozymes. In addition, we present a structural analysis that allows us to postulate upon the function of several conserved residues.

Amino Acid Sequence↗
PubMed1996

Characterization of serum amyloid A protein mRNA expression and secondary amyloidosis in the domestic duck.

Secondary amyloidosis is a common disease of water fowl and is characterized by the deposition of extracellular fibrils of amyloid A (AA) protein in the liver and certain other organs. Neither the normal role of serum amyloid A (SAA), a major acute phase response protein, nor the causes of secondary amyloidosis are well understood. To investigate a possible genetic contribution to disease susceptibility, we cloned and sequenced SAA cDNA derived from livers of domestic ducks. This revealed that the three C-terminal amino acids of SAA are removed during conversion to insoluble AA fibrils. Analysis of SAA cDNA sequences from several animals identified a distinct genetic dimorphism that may be relevant to susceptibility to secondary amyloid disease. The duck genome contained a single copy of the SAA gene that was expressed in liver and lung tissue of ducklings, even in the absence of induction of acute phase response. Genetic analysis of heterozygotes indicated that only one SAA allele is expressed in livers of adult birds. Immunofluorescence staining of livers from adult ducks displaying early symptoms of amyloidosis revealed what appear to be amyloid deposits within hepatocytes that are expressing unusually high amounts of SAA protein. This observation suggests that intracellular deposition of AA may represent an early event during development of secondary amyloidosis in older birds.

Amino Acid Sequence↗
PubMed2001

Identification of a 350-kDa ClpP protease complex with 10 different Clp isoforms in chloroplasts of Arabidopsis thaliana.

A 350-kDa ClpP protease complex with 10 different subunits was identified in chloroplast of Arabidopsis thaliana, using Blue-Native gel electrophoresis, followed by matrix-assisted laser desorption ionization time-of-flight and nano-electrospray tandem mass spectrometry. The complex was copurified with the thylakoid membranes, and all identified Clp subunits show chloroplast targeting signals, supporting that this complex is indeed localized in the chloroplast. The complex contains chloroplast-encoded pClpP and six nuclear-encoded proteins nCpP1-6, as well as two unassigned Clp homologues (nClpP7, nClpP8). An additional Clp protein was identified in this complex; it does not belong to any of the known Clp genes families and is here assigned ClpS1. Expression and accumulation of several of these Clp proteins have never been shown earlier. Sequence and phylogenetic tree analysis suggests that nClpP5, nClpP2, and nClpP8 are not catalytically active and form a new group of Clp higher plant proteins, orthologous to the cyanobacterial ClpR protein, and are renamed ClpR1, -2, and -3, respectively. We speculate that ClpR1, -2, and -3 are part of the heptameric rings, whereas ClpS1 is a regulatory subunit positioned at the axial opening of the ClpP/R core. Several truncations and errors in intron and exon prediction of the annotated Clp genes were corrected using mass spectrometry data and by matching genomic sequences with cDNA sequences. This strategy will be widely applicable for the much needed verification of protein prediction from genomic sequence. The extreme complexity of the chloroplast Clp complex is discussed.

Adenosine Triphosphatases↗
PubMed2003

Nucleotide sequence and expression of a cDNA encoding canine carbonic anhydrase VI (CA-VI).

A full-length cDNA clone of a canine carbonic anhydrase VI (CA-VI) was generated from the canine parotid gland by using a reverse transcription-polymerase chain reaction (RT-PCR) technique with degenerate primers designed from conserved regions of the same locus in humans and bovines employing RACE (rapid amplification of cDNA ends) techniques. The cDNA sequence was 1351 base pairs (bp) long and was predicted to encode a 320-amino-acid polypeptide containing a putative signal peptide of 17 amino acids. The deduced amino acid sequence of mature CA-VI showed the highest similarity of 74% to that of human CA-VI. RT-PCR analysis with primers specific to the canine CA-VI demonstrated strong signals in the major salivary glands and weak signals in the minor salivary glands and esophagus of a healthy dog. No CA-VI mRNA was detected in the pancreas, liver or the digestive tract except the esophagus.

Animals↗
PubMed1988

Sequence of contactin, a 130-kD glycoprotein concentrated in areas of interneuronal contact, defines a new member of the immunoglobulin supergene family in the nervous system.

The primary amino acid sequence of contactin, a neuronal cell surface glycoprotein of 130 kD that is isolated in association with components of the cytoskeleton (Ranscht, B., D. J. Moss, and C. Thomas. 1984. J. Cell Biol. 99:1803-1813), was deduced from the nucleotide sequence of cDNA clones and is reported here. The cDNA sequence contains an open reading frame for a 1,071-amino acid transmembrane protein with 962 extracellular and 89 cytoplasmic amino acids. In its extracellular portion, the polypeptide features six type 1 and two type 2 repeats. The six amino-terminal type 1 repeats (I-VI) each consist of 81-99 amino acids and contain two cysteine residues that are in the right context to form globular domains as described for molecules with immunoglobulin structure. Within the proposed globular region, contactin shares 31% identical amino acids with the neural cell adhesion molecule NCAM. The two type 2 repeats (I-II) are each composed of 100 amino acids and lack cysteine residues. They are 20-31% identical to fibronectin type III repeats. Both the structural similarity of contactin to molecules of the immunoglobulin supergene family, in particular the amino acid sequence resemblance to NCAM, and its relationship to fibronectin indicate that contactin could be involved in some aspect of cellular adhesion. This suggestion is further strengthened by its localization in neuropil containing axon fascicles and synapses.

Amino Acid Sequence↗
PubMed2006

Automated classification of alternative splicing and transcriptional initiation and construction of visual database of classified patterns.

MOTIVATION: Large-scale detection and classification of alternative splicing and transcriptional initiation (ASTI) is the first step towards detailed studies of the functional implication and mechanisms of these phenomena. RESULTS: We have developed an algorithm that classifies all observed units of ASTI into an extendable set of distinct types (e.g. cassette type) by converting a collection of alignments between a genomic DNA sequence and cDNA sequences into binary description. This description system can uniquely and compactly encode not only typical patterns but also any rare patterns that are usually collectively assigned to 'others.' More than 150 distinct ASTI types were found when this system was applied to genome-wide detection of ASTI units in human and five other eukaryotes. AVAILABILITY: The data detected by this system are available through ASTRA (http://alterna.cbrc.jp/), a database equipped with a Java-based browser that can interactively reorganize the order of displayed splicing patterns on demand.

Algorithms↗
PubMed1986

Primary structure of a proteinase inhibitor II gene from potato (Solanum tuberosum).

The isolation and characterization of a genomic clone encoding proteinase inhibitor II of potato (Solanum tuberosum) is described. The structure of this gene was determined by sequencing a genomic fragment of about 2 kb containing the entire RNA coding as well as about 900 nucleotides of the 5'-upstream and 250 nucleotides of the 3'-downstream region. The transcription start site was determined by RNase protection experiments. The comparison of the genomic sequence with cDNA sequences reveals the presence of one intron with a length of 117 nucleotides. The genomic clone contains an open reading frame of 462 nucleotides allowing for a protein of 154 amino acids. The proteinase inhibitor II gene displays typical features of eucaryotic genes. The sequence TATAAA is found 26 nucleotides upstream of the transcription initiation site and the sequence CAAAT at position--103. In the 3'-region the sequence AATAA is found 33 nucleotides in front of the poly-A addition site.

Amino Acid Sequence↗
PubMed1981

Reiterated sequences within the intron of an immediate-early gene of herpes simplex virus type 1.

We describe the nucleotide sequence of a herpes simplex virus type 1 DNA fragment containing the intron of the immediate-early mRNA-5 (IE mRNA-5) gene. The location of the intron within this fragment was determined by a Berk & Sharp nuclease S1 protection analysis, and by cloning and sequencing cDNA containing sequences overlapping t he IE mRNA-5 splice point. We found that the 149 base pair (bp) intron contained four copies of an identical 23 bp GC rich tandem repeat followed by a further reiteration consisting of the first 15 bp only.

Cloning, Molecular↗
PubMed1986

Complete sequence of the major nucleocapsid protein gene of human parainfluenza type 3 virus: comparison with other negative strand viruses.

The sequence of the major nucleocapsid protein (NP) mRNA and its encoded protein were deduced by sequencing a cDNA clone representing the complete mRNA. The cDNA sequence was confirmed by dideoxynucleotide sequencing of purified viral genomic RNA by primer extension using synthetic oligonucleotides. The NP mRNA contains 1,641 nucleotides exclusive of poly(A) and encodes an NP protein of 515 amino acids. Alignment of the human parainfluenza type 3 virus (PF3) NP protein sequence with that of Sendai virus showed that the two proteins shared considerable sequence identity (58.8%). Additional comparisons provided highly significant statistical evidence that the PF3 NP protein sequence is related to those of measles and canine distemper viruses, but there was no evidence of relatedness with the nucleocapsid proteins of respiratory syncytial virus, influenza B virus, or vesicular stomatitis virus.

Amino Acid Sequence↗
PubMed1990

Two amino acids, Phe 16 and Ala 776, on the polyprotein are most likely to be responsible for the diabetogenicity of encephalomyocarditis virus.

The diabetogenic D variant of encephalomyocarditis virus (EMC-D) was previously shown to differ from the non-diabetogenic B variant (EMC-B) by 14 nucleotides out of 7829 bases. Similar approaches with a new nondiabetogenic variant, EMC-DV1, obtained by plaque purification of the EMC-D variant stock pool, enabled us to narrow down further the possible genomic area responsible for the diabetogenicity of EMC virus. EMC-DV1 does not induce interferon in vitro, differing from the highly interferon-inducing EMC-B. The complete nucleotide sequence of EMC-DV1 was determined by RNA-dependent DNA sequencing and cDNA sequencing. The genomic size and organization of EMC-DV1 are similar to those of EMC-D and EMC-B, with a long open reading frame encoding a polyprotein of 2292 amino acids. Comparative analyses of sequence information as well as biological activities of EMC-DV1 with EMC-D and EMC-B suggest that (i) the diabetogenicity is apparently distinct from the ability to induce interferon, which is probably due to the single U base insertion at position 765 in EMC-B, and (ii) the diabetogenicity of EMC virus is most probably controlled by one or both of two amino acids, Phe 16 (on the leader peptide) and Ala 776 (152nd amino acid on the VP1) on the polyprotein.

Alanine↗