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PubMed2003

[Cloning and sequence analysis of human embryonic nasopharyngeal epithelial CYP2E1 cDNA].

OBJECTIVES: To investigate the role of CYP2E1 gene in chemical carcinogen-induced nasopharyngeal carcinogenesis and to provide new evidence about etiology and pathogenesis of nasopharyngeal carcinoma. METHODS: RT-PCR was used to clone the CYP2E1 gene in human embryonic nasopharyngeal epithelial (HENE) cell, the transformed nasopharyngeal epithelial cell line (7,429), and the nasopharyngeal carcinoma cell line (HNE1). The cloned segments were inserted into pGEM-T Easy vector to sequence by DNA recombination technique. RESULTS: In comparison with HENE-2E1 cDNA, there were two point mutations at positions 846 (A to T) and 901 (A to G) in 7,429-2E1 cDNA as well as only one point mutation at position 901 (A to G) in HNE1-2E1 cDNA. In comparison with human (adult, ethanol-inducible) liver CYP2E1 gene (GenBank NO. J02843), HENE-2E1 cDNA had one point mutation at position 901 (G to A). All these point mutations didn't affect the amino acid sequence. But no base change was found in HNE1-2E1 cDNA. CONCLUSION: There are a few of base substitutions among HENE-2E1 cDNA, 7,429-2E1 cDNA, and HNE1 cDNA sequences. All these point mutations are synonymous mutation. The study reconfirms that the human CYP2E1 gene is relatively well conserved.

Base Sequence↗

PubMed2005

[Construction and preliminary screening of a forward-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state].

OBJECTIVE: To construct and preliminarily screen the forward-subtracted cDNA library of differentially expressed genes in rat liver of prothrombotic state (PTS). METHODS: The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was constructed by suppression subtractive hybridization using cDNAs synthesized from mRNA of PTS rat as Tester and cDNAs from mRNA of control rat as Driver. The products from the last PCR amplification of suppression subtractive hybridization were inserted into a T/A plasmid vectors to transform the Escherichia coli JM109 cells. To produce the library, the transformed cells were incubated at 37 C overnight on a LB agar plate containing ampicillin (50 microg/ml), IPTG and X-gal. Forward-subtracted cDNA probes and reverse-subtracted cDNA probes were prepared by nested PCR amplification, which were labeled with HRP. Positive clones were selected by differential screening in which forward-subtracted and reverse-subtracted cDNA probes were separately hybridized with the membranes slot-blotted by plasmid DNAs amplified and isolated from the library. Inserts in the positive clones were submitted to DNA sequencing. Nucleic acid sequence homology search was performed against the GenBank DNA database (non-redundant, and non-mouse and non-human EST entries) using the Standard nucleotide-nucleotide BLAST [blastn] program via a network connection to the National Center for Biotechnology information. RESULTS: The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was successfully constructed. Two differentially expressed cDNA fragments were found after preliminary screening. CONCLUSION: The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was successfully constructed in the present study.

Animals↗

PubMed1990

Isolation and characterization of cDNA encoding a human nuclear antigen predominantly recognized by autoantibodies from patients with primary biliary cirrhosis.

Autoantibodies to a novel nuclear Ag, Sp100, have recently been described that recognize a nuclear protein with an apparent molecular mass of 95 to 100 kDa and a dot-like distribution within cell nuclei. By immunoscreening of a lambda gt11 cDNA expression library derived from HeLa cells with an anti-Sp100 autoimmune serum a 0.7-kb cDNA (Sp26) coding for a fragment of Sp100 was isolated. Expression of this cDNA and use of the recombinant protein in ELISA revealed that the fragment carries major Sp100 autoepitopes and that anti-Sp100 autoantibodies predominantly occur in patients suffering from primary biliary cirrhosis (50/184). The Sp26 cDNA was used as hybridization probe for isolation of longer cDNA from human liver- and placenta-derived lambda gt10 cDNA libraries. Overlapping fragments were assembled to generate a full length cDNA coding for a protein with a molecular mass of 53 kDa and an isoelectric point of 4.7. The Sp100 autoantigen expressed in vitro from this cDNA and authenticated by a capture immunoblot assay, comigrated in SDS-PAGE with the authentic HeLa autoantigen of 95 to 100 kDa and thus showed an aberrant electrophoretic mobility. Computer based protein sequence analysis of the Sp100 autoantigen revealed regions of striking sequence similarities to the alpha 1 and alpha 2 domains of various human and non-human MHC class I Ag and to several transacting transcriptional regulatory proteins.

Amino Acid Sequence↗

PubMed1990

Characterization of two cDNA clones for pyruvate dehydrogenase E1 beta subunit and its regulation in tricarboxylic acid cycle-deficient fibroblast.

Two distinct types of cDNA clones encoding for the pyruvate dehydrogenase (PDH) E1 beta subunit were isolated from a human liver lambda gt11 cDNA library and characterized. These cDNA clones have identical nucleotide sequences for PDH E1 beta protein coding region but differ in their lengths and in the sequences of their 3'-untranslated regions. The smaller cDNA had an unusual polyadenylation signal within its protein coding region. The cDNA-deduced protein of PDH E1 beta subunit revealed a precursor protein of 359 amino acid residues (Mr 39,223) and a mature protein of 329 residues (Mr 35,894), respectively. Both cDNAs shared high amino acid sequence similarity with that isolated from human foreskin (Koike, K.K., Ohta, S., Urata, Y., Kagawa, Y., and Koike, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 41-45) except for three regions of frameshift mutation. These changes led to dramatic alterations in the local net charges and predicted protein conformation. One of the different sequences in the protein coding region of liver cDNA (nucleotide position 452-752) reported here was confirmed by sequencing the region after amplification of cDNA prepared from human skin fibroblasts by the polymerase chain reaction. Southern blot analysis verified simple patterns of hybridization with E1 beta cDNA, indicating that the PDH E1 beta subunit gene is not a member of a multigene family. The mechanisms of differential expression of the PDH E1 alpha and E1 beta subunits were also studied in established fibroblast cell lines obtained from patients with Leigh's syndrome and other forms of congenital lactic acidosis. In Northern blot analyses for PDH E1 alpha and E1 beta subunits, no apparent differences were observed between two Leigh's syndrome and the control fibroblasts studied: one species of PDH E1 alpha mRNA and three species of E1 beta mRNA were observed in all the cell lines examined. However, in one tricarboxylic acid cycle deficient fibroblast cell line, which has one-tenth of the normal enzyme activity, the levels of immunoreactive PDH E1 alpha and E1 beta subunits were markedly decreased as assessed by immunoblot analyses. These data indicated a regulatory mutation caused by either inefficient translation of E1 alpha and E1 beta mRNAs into protein or rapid degradation of both subunits upon translation. In contrast, the PDH E1 alpha and E1 beta subunits in two fibroblast cell lines from Leigh's syndrome patients appeared to be normal as judged by 1) enzyme activity, 2) mRNA Northern blot, 3) genomic DNA Southern blot, and 4) immunoblot analyses indicating that the lactic acidosis seen in these patients did not result from a single defect in either of these E1 alpha and E1 beta subunits of the PDH complex.

Amino Acid Sequence↗

PubMed1989

Cloning of cDNA from induced K562 cells which can activate globin gene expression.

A cDNA library from induced K562 cells was constructed and differentially screened for the isolation of clones encoding trans-acting factors which increase globin gene expression. The current study assumes that induced K562 cells contain transcriptionally active factors specific for globin genes which are absent or present only at very low levels in uninduced K562 cells. Upon screening the recombinant library, 75 cDNA clones hybridized specifically with cDNA probes from induced K562 cells and hybridized only slightly or not at all with cDNA probes from uninduced K562 cells and HL-60 cells, or from globin genes. Forty-five of the cDNA clones were full length complements to the corresponding RNA. To screen for trans-acting factors which can activate globin gene expression, the cDNA clones were inserted into a eukaryotic expression vector and co-transfected into HeLa cells with another vector containing the epsilon globin promoter 5' to the bacterial CAT (chloramphenicol acetyl transferase) reporter gene. Partial screening of the 45 full length clones revealed one cDNA (clone #17) that was able to increase CAT activity (driven by the epsilon-globin promoter) by about 2.5 times. This cDNA is 522 nucleotides in length and contains a long open reading frame. Examination with known DNA sequences indicates greater than 95% homology with the ferritin heavy chain.

Base Sequence↗

PubMed1989

Cloning of Japanese quail ovalbumin cDNA in E. coli.

Japanese quail oviduct total cDNA was synthesized from total mRNA by the classical method, using AMV reverse transcriptase, the Klenow fragment of DNA polymerase I and S1 nuclease. The results of the synthesis of total ss-cDNA partially differed from those published for the synthesis of chicken total ss-cDNA. The presumed causes of the differences in the complete reverse transcription of mRNAcon and incomplete reverse transcription of mRNAlys and a large part of mRNAov in our case are discussed. An atypical strategy was used for cloning full-length cDNAov. The total cDNA was dC-tailed, then fractionated by analytical agarose electrophoresis and 4 cDNA fractions of different lengths were isolated from the gel using DEAE cellulose membranes. A cDNA fraction about 1500-2500 bp long containing full-length cDNAov was annealed with dG-tailed PstI-linearized plasmid pBR322 and cloned into competent E. coli DHl cells. Seventy-two clones were screened for the presence of full-length cDNAov, initially by insert size and then by means of hybrid-arrested translation. Four clones containing 1900-1980 bp cDNAov were obtained. The cDNA ends in one of these clones were sequenced. Comparison of these sequences with those of chicken mRNAov indicated that almost full-length cDNAov's had been cloned. They lacked a small number of nucleotides at their 5' ends, which had probably been split off during the degradation of the hairpin loop by S1 nuclease. A sequence of 134 bases from the 5' end of mRNAov is presented and compared with the known sequence of chicken mRNAov. The advantages of the cloning strategy employed, in particular, its cloning efficiency and the possibility of simultaneously identifying clones of also other oviduct cDNA species (in this work: cDNAY and, tentatively, cDNAcon), are discussed.

Animals↗

PubMed1978

Cloning of a double-stranded cDNA that codes for a portion of chicken preproalbumin. A general method for isolating a specific DNA sequence from partially purified mRNA.

A scheme is presented for cloning a double-stranded cDNA molecule that codes for a portion of chicken preproalbumin. This method, which does not require pure mRNA or cDNA, has widespread applicability. Chicken preproalbumin was identified as a Mr = 72,000 polypeptide by immunoprecipitation of proteins synehesized in a wheat germ cell-free translation system from total, guanidine.HCl-extracted, rooster liver RNA. After removal of the bulk of the ribosomal RNA by poly(U)-Sephadex G-10 chromatography, albumin mRNA was enriched approximately 2-fold by centrifugation through low salt, isokinetic sucrose gradients, until it represented about 30% of the mRNA sequences present. Double-stranded cDNA prepared from this mRNA was then inserted into the Pst 1 site of the plasmid PBR322 by the "G-C tailing" technique and the recombinant DNA was used to transform Echerichia coli stran X1776. Transformants containing putative albumin DNA sequences were identified by colony hybridization with a cDNA probe that was highly enriched for albumin cDNA sequences. This probe was isolated by hybridizing the partially purified RNA preparation to its cDNA, under conditions of RNA excess, to a R0t value such that only the most abundant cDNA sequences had hybridized. Unhybridized, less abundant, sequences were destroyed by subsequent S1 nuclease digestion. The identity of clones that hybridized to this abundant class cDNA was established by DNA-mRNA hybrid-arrested cell-free translation. Hybridization of nick-translated, albumin-containing, plasmid DNA to total liver poly(A)+ RNA, that had been separated on methyl mercury agarose gels and transferred to diazobenzyloxymethyl paper, established that avian albumin mRNA has a molecular weight of 850,000. This molecular weight corresponds to approximately 2,600 nucleotides, or 600 nucleotides longer than the size required to code for the preproalbumin polypeptide.

Animals↗

PubMed1994

Human histamine N-methyltransferase pharmacogenetics: cloning and expression of kidney cDNA.

Histamine N-methyltransferase (HNMT) catalyzes the NT-methylation of histamine. The level of HNMT activity in human red blood cells is controlled by a common genetic polymorphism. We set out to clone and express a cDNA for HNMT from human tissue as a first step toward a determination of the molecular basis for this genetic polymorphism. The cloning strategy was based on possible sequence homology between rat and human kidney HNMT. Human kidney cDNA libraries were screened with the 885-nucleotide open reading frame of rat kidney HNMT cDNA. A 1.4-kilobase cDNA clone was isolated that contained two potential translation initiation codons, both in the same reading frame. The longest open reading frame of the human kidney cDNA clone contained 876 nucleotides and encoded a protein 292 amino acids in length. The amino acid sequence of this protein was 84% identical to that of rat kidney HNMT. The human kidney cDNA clone was transcribed in vitro and translated in a rabbit reticulocyte lystate system to yield a protein with an apparent molecular mass of 33 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The human kidney cDNA was also subcloned into the eukaryotic expression vector p91023(B). Partially purified HNMT isolated from the cytosol of GOS-1 cells transfected with this expression construct had biochemical properties similar to those of human kidney HNMT. Human renal cortical HNMT, partially purified human renal cortical HNMT, and partially purified transfected COS-1 cell HNMT had Km values for histamine and S-adenosyl-L-methionine, the two cosubstrates for the enzyme reaction, of 20, 13, and 14 microM and 2.0, 3.0, and 6.2 microM, respectively. IC50 values for the HNMT inhibitor amodiaquine were 0.50, 0.48, and 0.40 microM, respectively, for enzyme from these same three sources. Northern blot analyses performed with poly(A)+ RNA from a series of human tissues including kidney demonstrated three transcripts, approximately 1.3, 3.8, and 4.0 kilobases in length. Cloning of a cDNA for HNMT may now make it possible to determine the molecular basis for the HNMT genetic polymorphism in humans.

Amino Acid Sequence↗

PubMed1994

Human liver nicotinamide N-methyltransferase. cDNA cloning, expression, and biochemical characterization.

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide and other pyridines. Human liver NNMT activity has a bimodal frequency distribution, an observation which raises the possibility that this enzyme activity might be regulated by a genetic polymorphism, a polymorphism that could have functional implications for individual differences in drug and xenobiotic toxicity. As a first step toward testing that hypothesis, we set out to clone and express a cDNA for human liver NNMT. Human liver NNMT was partially purified, photoaffinity-labeled, subjected to limited proteolysis, and partial amino acid sequence information was obtained. The polymerase chain reaction was then used to amplify a 550-nucleotide sequence with human liver cDNA as template and primers designed on the basis of the NNMT amino acid sequence. The 5'- and 3'-ends of a human liver NNMT cDNA were obtained by use of the rapid amplification of cDNA ends. The combined use of these approaches resulted in the isolation of a human liver NNMT cDNA that was 969 nucleotides in length, with a 792-nucleotide open reading frame that encoded a 264-amino acid protein with a calculated molecular mass of 29.6 kDa. The human liver NNMT cDNA was transcribed in vitro and translated with a reticulocyte lysate system to yield a protein with a molecular mass of approximately 29 kDa that comigrated during SDS-polyacrylamide gel electrophoresis with photoaffinity-labeled human liver NNMT. The NNMT cDNA was also subcloned into the eukaryotic expression vector p91023(B). COS-1 cells transfected with this construct expressed a high level of NNMT enzymatic activity, and the biochemical properties of this activity were similar to those of human liver NNMT. Human liver NNMT and transfected COS-1 cell NNMT had apparent Km values for the two cosubstrates for the reaction, nicotinamide and S-adenosyl-L-methionine, of 0.43 and 0.38 mM and of 1.8 and 2.2 microM, respectively. IC50 values for the inhibition of NNMT by N1-methylnicotinamide were 60 and 30 microns for human liver and COS-1 cell-expressed NNMT, respectively. Cloning of a cDNA for human liver NNMT will help make it possible to test the hypothesis that inheritance may play a role in the regulation of individual differences in human liver NNMT activity.

Amino Acid Sequence↗

PubMed1993

Molecular cloning and expression of cDNA encoding rat skeletal muscle cytosolic sialidase.

We have isolated a cDNA clone encoding the cytosolic sialidase of rat skeletal muscle. Degenerate oligonucleotides, based on amino acid sequence data for the purified enzyme, were used as primers to amplify fragments of the gene from rat skeletal muscle cDNA by the polymerase chain reaction. The amplified cDNA fragment was then applied as probe to screen a rat skeletal muscle cDNA library. The longest cDNA clone thus isolated was incomplete at the 5'-end, and therefore an amplified cDNA from the 5'-end portion of the gene was further generated by polymerase chain reaction. These two cDNAs were used to construct a cDNA encoding the entire sequence of rat sialidase. The composite sequence encodes an open reading frame of 379 amino acids that include all sequenced peptides. Although the deduced amino acid sequence is not largely similar to those of bacterial and parasite sialidases, it contains two Asp blocks, the conserved sequence of the sialidases from these microorganisms. When the cDNA was inserted into an expression vector followed by transformation in Escherichia coli, sialidase activity appeared in the cell extract. The sialidase could be completely immunoprecipitated by antiserum against the cytosolic sialidase of rat skeletal muscle.

Amino Acid Sequence↗

PubMed1998

CLoning of equine interleukin 1 alpha and equine interleukin 1 beta and determination of their full-length cDNA sequences.

OBJECTIVES: To clone equine interleukin 1 alpha (IL-1 alpha) and equine interleukin 1 beta (IL-1 beta) and determine their full-length cDNA sequences. PROCEDURES: The mRNA isolated from lipopolysaccharide-stimulated cultured equine monocytes was reverse transcribed, and a cDNA library was constructed in a lambda phage. The cDNA library was screened by means of plaque hybridization with radiolabeled human IL-1 alpha and IL-1 beta cDNA probes. The cDNA nucleotide sequences for equine IL-1 alpha and equine IL-1 beta were determined by use of the dideoxy chain termination technique. The cDNA sequences were analyzed, using computer software, for sequence characteristics and compared with sequences reported for other species. RESULTS: The cDNA for equine IL-1 alpha was 1,728 base pairs in length with an ORF encoding a peptide of 270 amino acids with a predicted molecular mass of 30.823 kd. The cDNA for equine IL-1 beta was 1,473 base pairs in length with an ORF encoding a peptide of 268 amino acids with a predicted molecular mass of 30.342 kd. Similarity between amino acid sequence of equine IL-1 alpha and sequences for IL-1 alpha of other species ranged from 62.5 to 82.2%; similarity between amino acid sequence of equine IL-1 beta and sequences for IL-1 beta of other species ranged from 62.5 to 82.2%; similarity between amino acid sequences of equine IL-1 alpha and equine IL-1 beta was 26%. CONCLUSIONS AND CLINICAL RELEVANCE: Results establish a basis for studying the roles of interleukin 1 in healthy and diseased joints in horses.

Amino Acid Sequence↗

PubMed1998

[A rapid method for subtractive screening of hepatoma apoptotic cells cDNA library].

OBJECTIVE: To expedite the subtractive screening of human hepatoma apoptotic cells cDNA library. METHODS: The method of subtractive hybridization combined with dot blot hybridization was adopted. First, minus cDNA probe was used to screen the cDNA library, the minus clones that did not hybridize with the minus cDNA probe were picked up as the source of the second grade dot blot hybridization. Secondly, two probes of the plus and minus cDNA were used to screen the clones;the clones that only hybridize with the plus cDNA probe were picked up as the source of the third grade dot blot hybridization. RESULTS: Four clones were obtained and the lengths of the inserted cDNA fragments were about 1.5kb long. CONCLUSION: The results demonstrate this is a feasible, simple and quick method for subtractive screening of the cDNA library.

Apoptosis↗

PubMed1997

Identification of genes with specific expression in pancreatic cancer by cDNA representational difference analysis.

cDNA representational difference analysis (cDNA-RDA) is a polymerase-chain-reaction-coupled subtractive and kinetic enrichment procedure for the isolation of differentially expressed genes. In this study, the technique was used to isolate novel genes specifically expressed in pancreatic cancer. cDNA-RDA was done on cDNA reverse transcribed from a poly(A)+ mRNA pool made from 10 cancer tissues (tester) by using as a driver a cDNA from a poly(A)+ mRNA pool made from a combination of 10 tissues of chronic pancreatitis and 10 healthy pancreatic tissues. The use of chronic pancreatitis in addition to healthy pancreas mRNA in the driver preparation eliminated the influence of stromal tissue components present as contamination in the cancer-specific preparations. Such cDNA-RDA led to the isolation of 16 distinct, cancer-specific gene fragments. These were confirmed to be overexpressed in pancreatic cancer tissues by Northern blot analysis. Sequence analysis revealed homologies to five genes previously implicated in the carcinogenesis of the pancreas or other tissues. Eleven fragments had no significant homology to any known gene and thus represent novel candidate disease genes. The experiments demonstrate that cDNA-RDA is a reproducible and highly efficient method for the identification of novel genes with cancer-specific expression.

Annexin A3↗

PubMed1998

Cloning and sequencing of cDNA encoding for a human sperm antigen involved in fertilization.

A cDNA encoding for a sperm antigen, designated NZ-2, was cloned and sequenced from human testis cDNA-lambda gt11 expression library by using antibodies to human sperm surface antigens belonging to 14-18 kD molecular regions. These sperm antigens are involved in binding to zona pellucida of the human oocyte. Computer generated translation analysis of 963-bp cDNA yielded an open reading frame (ORF) of 163 amino acids (aa) with first ATG, Met start codon at nucleotide (nt) 335 and the stop codon TAA at nt 824. The NZ-2 cDNA has 335-bp 5' and 139-bp 3' noncoding regions. The translated protein has a calculated molecular weight of approximately 19 kD, and has two casein kinase II (CK-2) sites at aa 94-97 and 149-152, respectively. Extensive computer search in the GenBank, National Biomedical Research Foundation (NBRF), and Swiss database indicates it to be a novel protein, having 99.5% nt sequence similarity, except for the first 40-bp, only with the human bacterial artificial chromosome (BAC) containing cloned human sperm DNA, at position 76935-76009. The in vitro translated product of T3 RNA polymerase by using NZ-2 cDNA digested with XhoI yielded a protein band of approximately 20 kD, indicating it to be sense strand. The in vitro translated product of T7 RNA polymerase by using NZ-2 cDNA digested with NotI did not yield any protein band, indicating it to be antisense strand. The approximately 20 kD protein was recognized specifically by the antisperm IgG, not by the control IgG in the Western blot procedure. Neither antisperm IgG nor control IgG recognized any protein band in the in vitro translation products of the antisense strand. The human genomic DNAs from three different cells/tissues namely, sperm, kidney, and testis when cut by HindIII, and then hybridized with the NZ-2 cDNA probe in the Southern blot procedure, showed restriction fragment length polymorphism (RFLP). The recombinant human sperm NZ-2 antigen may find applications in the development of a contraceptive vaccine, and diagnosis and treatment of infertility in humans.

Amino Acid Sequence↗

PubMed1994

Molecular cloning and expression of a cDNA encoding a protein detected by the K1 antibody from an ovarian carcinoma (OVCAR-3) cell line.

MAb KI recognizes a cell-surface glycoprotein (MW approximately 40 kDa) present in ovarian carcinomas, malignant mesotheliomas, squamous-cell carcinomas and normal mesothelial cells. In this study, expression screening was used to isolate cDNA clones encoding an antigen recognized by MAb KI from a cDNA library made from a human ovarian carcinoma cell line (OVCAR-3). Subsequently, other clones were isolated by DNA hybridization using a cDNA probe derived from one of the initial clones. The sequence of all the clones was similar. The longest cDNA contains 2,444 base pairs, and encodes a polypeptide of 263 amino acids with a calculated molecular weight of 30,511 daltons. The nucleotide sequence and deduced amino-acid sequence of the protein show no homology to other sequences in current data bases. In vitro translation of RNA transcripts from the cDNA inserts yielded polypeptides of 29 and 30 kDa. Similar-sized proteins were obtained upon expression of the cDNA in Escherichia coli, and these proteins were reactive with MAb KI. The protein(s) expressed in E. coli were purified and used to make rabbit or mouse antisera. These antisera reacted strongly with a soluble cytosolic protein in OVCAR-3 cells, but not with the membrane-bound antigen. Soluble cytosolic proteins of a similar size, recognized with MAb KI, were found in OVCAR-3 and N87 (gastric cancer) cells but not in 10 other cancer cell lines. These data indicate that the cloned cDNA encodes a cytosolic protein that reacted with MAb KI. This soluble protein is expressed only in cells containing the CAKI surface glycoprotein, suggesting that the 2 proteins could be structurally related.

Amino Acid Sequence↗

PubMed1988

Isolation and characterization of cDNA clones encoding epitopes shared with Alzheimer neurofibrillary tangles.

A pool of ten monoclonal antibodies to SDS-insoluble epitopes of Alzheimer neurofibrillary tangles (NFT) was used to screen an adult human brain cDNA expression library. Fourteen clones were isolated, two of which are described. The largest of the clones encodes 80 kD, or approximately 600 amino acids, of microtubule-associated protein 2 (MAP 2). The MAP 2 region encoded by the clone shares at least two epitopes with human tau, another microtubule-associated protein which cross-reacts with NFT. In rat brain mRNA, the MAP 2 cDNA hybridizes to a single transcript of 9.5 kb. In human neuroblastoma mRNA, the MAP 2 cDNA hybridizes, at high stringency, to two transcripts of 9.5 kb and 6 kb. The 6-kb transcript comigrates with the transcript for tau, as detected by a human tau cDNA. The properties of the MAP 2 cDNA suggest that, in humans, MAP 2 and tau have a common domain which may play a role in NFT formation. Another clone isolated with the anti-NFT antibodies shares epitopes, but not nucleic acid homology, with the MAP 2 cDNA. This clone detects a single abundant transcript of 1 kb present in RNA from human neuroblastoma and from several non-neuronal human cell lines. The properties of this cDNA suggest that it encodes a protein other than those previously reported to cross-react with NFT.

Adult↗

PubMed1991

Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17 beta-estradiol and 2-aminofluorene in V79 Chinese hamster cells.

In continuation of our work toward the establishment of a working cell bank for metabolic and toxicological studies, V79 Chinese hamster cells were genetically engineered for stable expression of rat cytochrome P450IA2. Full-length cDNA encoding rat P450IA2 was obtained by searching a cDNA library made from Aroclor 1254-induced rat liver mRNA and by joining a small 5'-end fragment to a fragment containing the rest of the cDNA. The sequence of the cDNA was confirmed by DNA sequencing and comparison to a previously published cDNA sequence. The reconstructed full-length cDNA was inserted into a simian virus 40 early promoter-containing eukaryotic expression vector and cotransferred with the neomycin phosphotransferase gene as a selective marker into V79 cells by the calcium/phosphate-coprecipitation technique. G418-resistant V79 cell clones were checked for chromosomal integration of the cDNA by Southern blotting, for expression of authentic mRNA and protein by northern and western blotting, and for P450IA2-specific enzymatic activities such as hydroxylation of 17 beta-estradiol and 2-aminofluorene.

Amino Acid Sequence↗

PubMed1995

cDNA encoding a functional feline liver/bone/kidney-type alkaline phosphatase.

Feline alkaline phosphatase (FeALP) was copurified with the putative 70-kDa feline leukemia virus subgroup-A (FeLV-A) receptor protein from feline T-lymphocyte cells (FeT) by two-dimensional gel electrophoresis. The sequence of the N-terminal 17 amino acids and five other internal tryptic peptides revealed that it is homologous to the liver/bone/kidney (L/B/K)-type alkaline phosphatase of other mammalian species. Corresponding oligonucleotides were synthesized and used for amplification of a 1.2-kb segment of the FeALP gene by polymerase chain reaction, using phage DNA from a FeT cell cDNA library as template. The 1.2-kb FeALP gene fragment generated was then used as a probe to isolate a 2127-bp L/B/K-type FeALP cDNA clone from the same library harboring a large, intact open reading frame. This cDNA possessed an open reading frame encoding a 524-amino-acid protein including a putative signal peptide of 17 amino acids followed by 14-amino-acid residues identical to the N-terminal sequence determined from the purified protein. Sequences closely related to five tryptic peptides from the purified protein were also contained within the cDNA-encoded protein. Homology with the human, bovine, rat and mouse L/B/K-type ALP was found to be 88-90% at both the nucleotide and the amino acid levels. The cDNA was transferred into a eukaryotic expression vector and expressed following transfection into murine and mink lung fibroblast cell lines. High levels of enzymatically active ALP were detected, along with a 70-kDa protein reactive in immunoblot assay using a polyclonal antibody against the original crude FeALP preparation. FeALP was specifically released from intact cells by treatment with phosphoinositol-specific phospholipase-C. By Northern blot analysis, only one species of mRNA was detected using a 32P-labeled cDNA probe. These results indicate that the 2127-bp cDNA encodes a functional feline L/B/K-type ALP expressed on cell surfaces via phosphatidylinositol-glycan linkage. Despite electrophoretic comigration in two dimensions and following deglycosylation in a third dimension, FeALP failed to function as an FeLV receptor since its expression failed to provide for attachment or entry of virus into cells.

Alkaline Phosphatase↗