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PubMed2001

[Construction of chicken hypothalamus cDNA library and primary analysis of ESTs sequences from partial clones].

Using hypothalamus as material and lambda gt10 as vector, chicken hypothalamus cDNA library has been constructed. The results showed that the titration of the cDNA library we constructed was 3.8 x 10(6), and the fragment length of inserted DNA ranged mainly from 1.0-3.0 kb. The ESTs was sequenced and primarily analyzed for ten clones randomly selected from above library. After analysis with BLASTn and FASTA3.1, it was found that four ESTs developed matched homologous sequence in chicken, four also had similar sequences in human and other species, have other two sequences were function-unknown gene. The sequences of ten ESTs we studied have been loaded into GenBank.

Animals↗

PubMed1999

Production and characterization of human monoclonal antibody Fab fragments to vaccinia virus from a phage-display combinatorial library.

A combinatorial, phage-display library of human Fab antibody fragments was generated from IgG heavy chain (HC) and light chain (LC) genes cloned from the lymphocytes of a vaccinia virus (VACV)-immune donor. To ascertain the complexity of the library, nucleotide sequences of the variable regions of the HC and LC genes were determined. Fourteen distinct HC and 18 distinct LC (7 kappa and 11 lambda) that formed a combinatorial library of 22 Fabs were identified. Immune-precipitation of radiolabeled VACV revealed that at least six different VACV proteins were recognized by the antibodies. Plaque-reduction neutralization demonstrated that six of the Fabs neutralized VACV in the presence of anti-human antibody. ELISA studies indicated that 15 of the Fabs were cross-reactive with monkeypox virus.

Amino Acid Sequence↗

PubMed1989

Cloning of the cDNA and gene for a human D2 dopamine receptor.

A clone encoding a human D2 dopamine receptor was isolated from a pituitary cDNA library and sequenced. The deduced protein sequence is 96% identical with that of the cloned rat receptor with one major difference: the human receptor contains an additional 29 amino acids in its putative third cytoplasmic loop. Southern blotting demonstrated the presence of only one human D2 receptor gene. Two overlapping phage containing the gene were isolated and characterized. DNA sequence analysis of these clones showed that the coding sequence is interrupted by six introns and that the additional amino acids present in the human pituitary receptor are encoded by a single exon of 87 base pairs. The involvement of this sequence in alternative splicing and its biological significance are discussed.

Amino Acid Sequence↗

PubMed1996

Amplification of genes, single transcripts and cDNA libraries from one cell and direct sequence analysis of amplified products derived from one molecule.

We report a procedure to generate and amplify cDNA libraries and to amplify and sequence genes and single RNA transcript molecules from the same cell without cloning. An absence of cloning steps minimizes potential sources of contamination, which can be especially problematic when working at the single cell level. Potential contamination is further reduced by an absence of any purification step prior to PCR amplification. Amplifications are designed to minimize the production of aberrant molecules in favor of full-length products, which is especially advantageous when generating cDNA libraries. Genes are amplified from isolated single nuclei, which are segregated from cytoplasmic lysates by microcentrifugation. Specific cDNA, total cDNA or both are synthesized from aliquots of the cytoplasmic lysate, and single cDNA molecules are isolated from others of the same species by limiting dilution prior to PCR amplification. In this way, the frequency of amplified products provides for a direct calculation of cDNA copy number by a Poisson analysis. Incorporation errors by Taq DNA polymerase occur at a low frequency and can be eliminated by sequencing independently amplified cDNA molecules from the same cell. Single molecule amplifications provide sufficient material for numerous (approximately 150) direct DNA sequencing reactions. The limiting dilution approach also permits sequence information to be obtained from a single cDNA, when highly related transcripts derived from distinct genes are present in the same cell and simultaneously amplified with the same primers. In sum, this method provides for a maximum amount of nucleic acid information to be extracted from one cell. It has a wide range of applications to studies of the immune system where, to a first approximation, each lymphocyte has a unique receptor identity, where specific states of differentiation may be difficult to assess in a mixed cell population, and where cell immortalization procedures are not always possible nor practical.

Animals↗

PubMed1983

A new troponin T and cDNA clones for 13 different muscle proteins, found by shotgun sequencing.

Complete amino acid sequences have been established for 19 muscle-related proteins and these proteins are each sufficiently abundant to suggest that their mRNA levels are about 0.4% or higher. Based on these considerations, a simple theoretical analysis shows that clones for most of these proteins can be identified within a complementary DNA library by sequencing cDNA inserts from 150-200 randomly selected clones. This procedure should not only rigorously identify specific clones, but it could also uncover amino acid sequence variants of major muscle proteins such as the troponins. We have determined sequences for about 20,000 nucleotides within 178 randomly selected clones of a rabbit muscle cDNA library, and report here that in addition to finding sequences encoding the two known skeletal muscle isotypes of troponin C, we have discovered sequences encoding two forms of troponin T. Over the region of nucleotide sequence overlap in the troponin T clones, the new isotype diverges significantly from its counterpart. Altogether, clones for 13 of the 19 known muscle-specific proteins were identified, in addition to the clone for the new troponin T isotype.

Amino Acid Sequence↗

PubMed1994

A complete cDNA sequence for core I protein subunit of human ubiquinol-cytochrome c reductase.

Core I protein is the largest subunit of ubiquinol-cytochrome c reductase. We have isolated a complete cDNA clone of 1575 bp encoding the precursor to this protein by screening a human fibroblast cDNA library. Nucleotide sequence comparison showed that the human core I protein cDNA is about 85% homologous with the reported bovine counter part but with a large difference in the length of coding region which consists of 480 amino acids in human and 362 amino acids in bovine. Human core I protein is presumed to contain a presequence of 34 amino acids. Amino acid sequence alignment study showed that the predicted human core I protein has a significant homology with other members of matrix processing peptidase (MPP) and processing enhancing protein (PEP) family.

Amino Acid Sequence↗

PubMed1999

Molecular cloning and characterization of porcine cDNA encoding a 90-kDa heat shock protein and its expression following hyperthermia.

We have isolated and sequenced cDNA clones encoding a 90-kDa heat shock protein (HSP90) from a porcine brain cDNA library. The sequence of the 2202-nucleotide coding region showed 88.6% homology with that of the human homologue. Moreover, the deduced amino acid sequence of the porcine hsp90 cDNA was 99.7% identical to that of the human counterpart, with a difference of only three amino acids in a total of 733 residues. Expression of the gene was greatly increased in cultured cells during recovery from heat shock treatment at 45 degrees C for 60 min. Three major transcripts 2.2, 3.0, and 4.1kb in size were detected by Northern blot hybridization. These transcripts were further identified in a whole-pig hyperthermia experiment. These three hsp90 transcripts were constitutively expressed in porcine tissues including kidney, liver, brain, and heart, and their levels were markedly enhanced during recovery from 30-min hyperthermia treatment at 43 degrees C. Furthermore, we found that HSP90 was preferentially expressed in pituitary gland, brain, adrenal gland, and testis, in comparison to the other tissues.

Amino Acid Sequence↗

PubMed2003

Pathogen discovery from human tissue by sequence-based computational subtraction.

We have recently reported a new pathogen discovery approach, "computational subtraction". With this approach, non-human transcripts are detected by sequencing cDNA libraries from infected tissue and eliminating those transcripts that match the human genome. We show now that this method is experimentally feasible. We generated a cDNA library from a tissue sample of post-transplant lymphoproliferative disorder (PTLD). 27,840 independent cDNA sequences were filtered by computational subtraction against the known human sequence to identify 32 nonmatching transcripts. Of these, 22 (0.1%) were found to be amplifiable from both infected and noninfected samples and were inferred to be human DNA not yet contained in the available human genome sequence. The remaining 10 sequences could be amplified only from Epstein-Barr virus (EBV)-infected tissues. All 10 corresponded to the known EBV sequence. This proof-of-principle experiment demonstrates that computational subtraction can detect pathogenic microbes in primary human-diseased tissue.

DNA, Complementary↗

PubMed1993

A recombination-based assay demonstrates that the fragile X sequence is transcribed widely during development.

To identify transcribed sequences rapidly and efficiently, we have developed a recombination-based assay to screen bacteriophage lambda libraries for sequences that share homology with a given probe. This strategy determines analytically whether a given probe is transcribed in a given tissue at a given time of development, and may also be used to isolate preparatively the transcribed sequence free of the screening probe. We illustrate this technology for the fragile X sequence, demonstrating that it is transcribed ubiquitously in an 11 week fetus, in a variety of 20 week human fetal tissues, including brain, spinal cord, eye, liver, kidney and skeletal muscle, and in adult jejunum.

Adult↗

PubMed1991

Domain structure and sequence distribution in dentin phosphophoryn.

Phosphophoryn (PP) is a protein unique to the mineralized matrix of dentin. It also has a unique composition, with aspartic acid and phosphoserine comprising greater than 85% of all amino acid residues. Because of this unique composition and high content of phosphoserine, it has been difficult to apply direct peptide sequencing procedures effectively. However, to understand its function, and to prepare suitable probes for screening cDNA libraries, some sequence distribution information is required. To this end, using bovine (b) and rat incisor (ri) PPs, partial mild acid hydrolysis has been used to cleave at the aspartic acid residues and generate free amino acids and small peptides. The nature of the released amino acids and peptides has been determined. Peptides have also been generated by limited digestion with trypsin. Some of the peptides have been purified by h.p.l.c. techniques and sequenced. About 90% of the bPP and riPP were resistant to trypsin, and the large resistant fragment was sharply depleted of the non-aspartic acid and non-phosphoserine [(P)Ser] residues. All peptides isolated were acidic, but the remaining residues (other than aspartic acid and serine) appeared to be collected in regions flanking the trypsin-resistant core. These data show directly the presence of regions [Asp]n, [(P)Ser]m and [Asp-(P)Ser-Asp]k as prominent sequence features. A domain structure model is proposed.

Acetates↗

PubMed2000

Isolation and functional expression of human COQ3, a gene encoding a methyltransferase required for ubiquinone biosynthesis.

The COQ3 gene in Saccharomyces cerevisiae encodes an O-methyltransferase required for two steps in the biosynthetic pathway of ubiquinone (coenzyme Q, or Q). This enzyme methylates an early Q intermediate, 3,4-dihydroxy-5-polyprenylbenzoic acid, as well as the final intermediate in the pathway, converting demethyl-Q to Q. This enzyme is also capable of methylating the distinct prokaryotic early intermediate 2-hydroxy-6-polyprenyl phenol. A full-length cDNA encoding the human homologue of COQ3 was isolated from a human heart cDNA library by sequence homology to rat Coq3. The clone contained a 933-base pair open reading frame that encoded a polypeptide with a great deal of sequence identity to a variety of eukaryotic and prokaryotic Coq3 homologues. In the region between amino acids 89 and 255 in the human sequence, the rat and human homologues are 87% identical, whereas human and yeast are 35% identical. When expressed in multicopy, the human construct rescued the growth of a yeast coq3 null mutant on a nonfermentable carbon source and restored coenzyme Q biosynthesis, although at lower levels than that of wild type yeast. In vitro methyltransferase assays using farnesylated analogues of intermediates in the coenzyme Q biosynthetic pathway as substrates showed that the human enzyme is active with all three substrates tested.

Aging↗

PubMed1986

Molecular cloning of a human prion protein cDNA.

Creutzfeldt-Jakob disease (CJD) of humans and scrapie of animals are degenerative, transmissible neurologic diseases caused by prions. The only known macromolecules within prions are prion proteins (PrP). The cDNA encoding the hamster prion protein (PrP 27-30) has been cloned and sequenced (Oesch et al., 1985). Using that hamster PrP cDNA, we screened a human retina cDNA library and sequenced the cDNA clone with the longest hybridizing insert. This insert was found to contain a long open reading frame (ORF) encoding the human prion protein. Northern transfer analysis showed that a related poly(A)+RNA measuring approximately 2.5 kb is expressed in a variety of human neuroectodermal cell lines. Human PrP differed from hamster PrP at 27 of 253 amino acids and at 98 of 759 ORF nucleotides. Conservation of PrP amino acid sequence between hamster and human is nearly 90%, reflecting similar structural features and shared antigenicity of the two proteins (Bockman et al., 1985). The human PrP sequence contained a presumptive amino-terminal signal peptide of 22 amino acids, two hydrophobic segments of sufficient length to span membranes, and two possible sites for N-glycosylation. The conservation between the hamster and human prion proteins suggests that they may have an important role in cellular metabolism and may explain the similarities between scrapie and CJD.

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of microsatellites from the canine genome.

Microsatellite sequences comprising (dC-dA)n.(dG-dT)n repeats have been isolated from canine libraries and sequenced. Oligonucleotide primers have been synthesized to the microsatellite flanking sequences and used in the polymerase chain reaction to amplify those loci from genomic DNA. The degree of polymorphism of each microsatellite was estimated in a set of unrelated dogs. It is concluded that of the 10 loci studied, nine are sufficiently polymorphic to be useful in genetic studies.

Animals↗

PubMed1996

Developmentally regulated synthesis of an unusually small, basic peptide by Coxiella burnetii.

Coxiella burnetii undergoes a poorly defined developmental cycle within phagolysosomes of eukaryotic host cells. Two distinct developmental forms are part of this cycle: a small-cell variant (SCV) and large-cell variant (LCV). Ultrastructurally, the SCV is distinguished from the LCV by its smaller size and condensed chromatin. At a molecular level, little is known about morphogenesis in C. burnetii, and no proteins specific to the SCV have been identified. Preparative isoelectric focusing was conducted to purify basic proteins possibly involved in SCV chromatin structure. A predominant protein of low M(r) was present in the most basic fraction, eluting with a pH of approx. 11. Degenerate deoxyoligonucleotides corresponding to the N-terminal sequence of this protein were used to recover a cosmid clone from a C. burnetii genomic library. Nucleotide sequencing of insert DNA revealed an open reading frame designated scvA (Small-Cell-variant protein A) with coding potential for a 30 amino acid protein (ScvA) with a predicted M(r) of 3610. ScvA is 46% arginine plus 46% glutamine with a predicted pl of 12.6. SDS-PAGE and silver staining of lysates of SCV and LCV purified by caesium chloride-equilibrium density centrifugation revealed a number of proteins unique to each cell type. Immunoblot analysis with ScvA antiserum demonstrated the presence of ScvA only in the SCV. By Immunoelectron microscopy, ScvA antiserum labelled only the SCV, with the label concentrated on the condensed nucleoid. In addition, ScvA bound double-stranded DNA in gel mobility-shift assays. A 66% reduction in the mean number of gold particles per Coxiella call was observed at 12 h post-infection when compared with the starting inoculum. Collectively, these data suggest that synthesis of ScvA is developmentally regulated, and that the protein may serve a structural or functional role as an integral component of the SCV chromatin. Moreover, degradation of this protein may be a necessary prerequisite for morphogenesis from SCV to LCV.

Amino Acid Sequence↗

PubMed2006

Cloning and expression of a xylitol-4-dehydrogenase gene from Pantoea ananatis.

The Pantoea ananatis ATCC 43072 mutant strain is capable of growing with xylitol as the sole carbon source. The xylitol-4-dehydrogenase (XDH) catalyzing the oxidation of xylitol to L-xylulose was isolated from the cell extract of this strain. The N-terminal amino acid sequence of the purified protein was determined, and an oligonucleotide deduced from this peptide sequence was used to isolate the xylitol-4-dehydrogenase gene (xdh) from a P. ananatis gene library. Nucleotide sequence analysis revealed an open reading frame of 795 bp, encoding the xylitol-4-dehydrogenase, followed by a 5' region of another open reading frame encoding an unknown protein. Results from a Northern analysis of total RNA isolated from P. ananatis ATCC 43072 suggested that xdh is transcribed as part of a polycistronic mRNA. Reverse transcription-PCR analysis of the transcript confirmed the operon structure and suggested that xdh was the first gene of the operon. Homology searches revealed that the predicted amino acid sequence of the P. ananatis XDH shared significant identity (38 to 51%) with members of the short-chain dehydrogenase/reductase family. The P. ananatis xdh gene was successfully overexpressed in Escherichia coli, XDH was purified to homogeneity, and some of its enzymatic properties were determined. The enzyme had a preference for NAD+ as the cosubstrate, and in contrast to previous reports, the enzyme also showed a side activity for the D-form of xylulose. Xylitol was converted to L-xylulose with a high yield (>80%) by the resting recombinant cells, and the L-xylulose was secreted into the medium. No evidence of D-xylulose being synthesized by the recombinant cells was found.

Amino Acid Sequence↗

PubMed2006

[The effects of angiotensin II on gene expression of hepatic stellate cells].

OBJECTIVE: Our previous investigation demonstrated that angiotensin II could induce proliferation and differentiation of hepatic stellate cells, and also could up-regulate its extracellular matrix synthesis. The objective of this study was to determine the effects of 10(-5) mol/L angiotensin II on gene expression of hepatic stellate cells. METHODS: After incubation with 10(-5) mol/L angiotensin II for 48 hours, the cultured hepatic stellate cells were collected. The mRNA and total protein were obtained from cell lysate and then suppression subtractive hybridization (SSH), 2D-gel electrophoresis and MALDI-TOF mass spectrometry were used to identify these cDNAs and proteins. RESULTS: A total of 36 clones from the subtracted cDNA library were sequenced and compared to sequences in the GenBank using BLAST. Of the 36 differentially expressed gene fragments from the subtracted library, 13 differentially expressed gene fragments showed significant homology to other known proteins, such as ribosomal protein, beta-actin FE-3, leucyl-tRNA synthetase, CD147, pyruvate kinase, peroxiredoxin 1, and BAT3, while 2 other gene fragments encoding protein BC097361 and BC057380 and their functions were not disclosed. About 1110 and 1008 protein spots were detected by employing the ImageMaster 2D Platinum 4.9 proteome image analysis system in angiotensin-treated hepatic stellate cells and control cells separately. Among these spots 108 proteins were up-regulated while the other 153 proteins were down-regulated. Several up-regulated proteins were chosen to be excised and in-gel digest MALDI-TOF-MS and Database analysis showed that among the high expression proteins, there were prohibitin, RBL-NDP kinase 1.8x10(4) subunit, electron transfer flavoprotein alpha-subunit, guanine nucleotide binding protein, alpha 15, and heat shock 7.0x10(4) protein 5. CONCLUSION: Our results suggest that the up-regulation of hepatic stellate cell mRNA influences proliferation, differentiation and apoptosis of those cells. The proteins of signal transduction, metabolizing regulation, apoptosis suppression, and fibrogenesis regulation of hepatic stellate cells were up-regulated.

Angiotensin II↗

PubMed1987

Molecular analysis of human and rat calmodulin complementary DNA clones. Evidence for additional active genes in these species.

A cDNA clone, lambda rCB1, encoding calmodulin was isolated from a rat brain expression library. The sequence was determined and compared to the structures of the previously described rat genes, lambda SC4 and lambda SC8 (Nojima, H., and Sokabe, H. (1986) J. Mol. Biol. 190, 391-400). Faithful sequence conservation is observed in the coding regions of lambda rCB1 and lambda SC4, the bona fide gene. Both cDNAs encode identical amino acid sequence. Very limited sequence homology, however, is noted in the 3'-untranslated segments of these clones. Surprisingly, when the lambda rCB1 nucleotide structure is compared to the processed intronless gene, lambda SC8, extensive sequence homology is found both in the coding and noncoding regions. The inferred protein sequences of lambda SC8 and lambda rCB1, however, are divergent. Using a fragment of lambda rCB1 to screen an expression library derived from a human embryonic cell line, a calmodulin cDNA clone, lambda hCE1, was isolated and characterized. Comparison of the sequence of lambda hCE1 to the cDNA from human liver, hCWP (Wawrzynczak, E. J., and Perham, R. N. (1984) Biochem. Int. 9, 177-185), reveals substantial structural divergence. Strikingly poor homology is seen in the 5'- and 3'-noncoding segments, but the coding regions were 85% homologous. Both lambda hCE1 and hCWP encode proteins of identical primary structure which is equivalent to the protein sequence deduced from lambda rCB1 and lambda SC4. Taken together these results suggest the existence of an additional actively transcribed calmodulin gene, not previously identified, in each of the human and rat genomes. Transcripts of lambda rCB1 and lambda hCE1 were observed in all tissues examined indicating the absence of tissue-specific expression. Calmodulin gene polymorphisms were detected using TaqI, HindIII, and MspI.

Amino Acid Sequence↗

PubMed1992

The primary structure of porcine aminoacylase 1 deduced from cDNA sequence.

A cDNA encoding the complete amino acid sequence of aminoacylase 1 (N-acylamino acid aminohydrolase, ACY-1) [EC 3.5.1.14], a dimeric metalloprotein having two Zn2+ in the molecule, which catalyzes the deacylation of N-acylated L-amino acids except L-aspartic acid, has been isolated from porcine kidney lambda gt10 cDNA library and sequenced. From sequence analysis of the cDNA and the N- and C-terminal amino acid analyses of the purified protein, it is deduced that porcine kidney ACY-1 consists of two identical subunits (M(r) 45,260), each of which consists of a single chain of 406 amino acids with acetylalanine at the N-terminus. A cDNA encoding porcine liver ACY-1 was also cloned. The amino acid sequence deduced from the nucleotide sequence of the cDNA from porcine liver was identical to that deduced for porcine kidney ACY-1. Northern blot analysis suggested that ACY-1 is more highly expressed in kidney than in liver. Comparison of the amino acid sequence of porcine ACY-1 with those of other Zn2+-binding metalloenzymes showed no significant homologies in either the overall sequence or the consensus sequences for the metal binding sites. This indicates that ACY-1 is a new type of metalloprotein.

Amidohydrolases↗