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PubMed1990

cDNA genes formed after infection with retroviral vector particles lack the hallmarks of natural processed pseudogenes.

Retroviral proteins can encapsidate RNAs without retroviral cis-acting sequences. Such RNAs are reverse transcribed and inserted into the genomes of infected target cells to form cDNA genes. Previous investigations by Southern blot analysis of such cDNA genes suggested that they were truncated at the 3' and the 5' ends (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To analyze such cDNA genes further, we cloned three cDNA genes (derived from a hygromycin B phosphotransferase gene) in lambda vectors and analyzed them by DNA sequencing. We found that they did not correspond to the full-length mRNA: they were truncated at both the 3' and the 5' ends, did not contain a poly(A) tract, and were not flanked by direct repeats. The 3'-end junctions to chromosomal DNA of five more cDNA genes were amplified by polymerase chain reaction, cloned in pUC vectors, and sequenced. All of these cDNA genes had 3'-end truncations, and no poly(A) tracts were found. Further polymerase chain reaction experiments were performed to detect hygromycin B phosphotransferase cDNA genes with a poly(A) tract in DNA extracted from a pool of about 500 colonies of cells containing cDNA genes. No hygromycin B phosphotransferase cDNA gene with a poly(A) tract was found. Investigation of two preintegration sites by Southern analysis revealed that deletions were present in chromosomal DNA at the site of the integration of the cDNA genes. Naturally occurring processed pseudogenes correspond to the full-length mRNA, contain a poly(A) sequence, and are flanked by direct repeats. Our data indicate that cDNA genes formed by infection with retrovirus particles lack the hallmarks or natural processed pseudogenes. Thus, it appears that natural processed pseudogenes were not generated by retrovirus proteins.

Base Sequence↗
PubMed2005

Human immunodeficiency virus type-1 episomal cDNA in semen.

BACKGROUND: Episomal 2-long terminal repeat (LTR) HIV-1 cDNA, a by-product of HIV-1 infection, is used in clinical trials as a marker for ongoing viral replication. It would be useful to employ 2-LTR cDNA to monitor cryptic HIV-1 infection in the genital tract of men on antiretroviral therapy (ART) to predict the evolution of sexually transmissible drug-resistant HIV-1, but studies thus far have failed to detect this marker in semen. The objectives of this study were: 1) to use a technique that maximizes DNA recovery from HIV-1 infected white blood cells in semen to determine if episomal 2-LTR cDNA is detectable in semen of ART-naïve men with other evidence of genital tract HIV-1 infection, and 2) to compare levels of HIV-1 2-LTR cDNA, RNA, and proviral DNA in semen from HIV-1+ men on ART. RESULTS: Using a somatic cell DNA extraction technique, 2-LTR cDNA was detected by PCR/ELISA in 4 out of 8 semen samples from ART-naïve men selected for other signs of seminal HIV-1 infection (positive controls). Southern blot and DNA sequencing confirmed that the amplified sequences were HIV-1 2-LTR cDNA; copy numbers ranged from 55 to 504 copies/sample. Two semen samples from a cohort of 22 HIV-1-infected men on dual nucleoside therapy, one with and one without detectable seminal HIV-1 RNA, were 2-LTR cDNA positive (336 and 8,560 copies/sample). Following addition of indinavir to the therapy regimen, no semen samples from 21 men with controlled peripheral and seminal viral loads were 2-LTR cDNA positive at 1 and 6 month time points, despite the persistence of HIV-1 proviral DNA+ semen cells and seminal cytomegalovirus (CMV) shedding in some cases. However, one individual who failed indinavir therapy and later developed distinct protease inhibitor (PI) drug resistance mutations in semen, maintained elevated levels of HIV-1 RNA and 2-LTR cDNA in semen. CONCLUSION: 2-LTR HIV-1 cDNA is detectable in semen of HIV-1-infected men. Two men on ART had 2-LTR HIV-1 cDNA in semen, suggesting that this marker may prove to be useful to monitor HIV-1 infection in the genital tract of men on ART to predict the evolution of drug resistance mutations in semen.

Journal Article↗
PubMed1999

[Revisions of the cDNA and primary protein structure of human transcription factor GCF].

GC factor (GCF) was reported as a transcriptional regulator that binds to specific GC-rich sequences in the epidermal growth factor receptor (EGFR) gene promotor, repressing its transcription (Kageyama R. and Pastan I. Cell, 59: 815-825, 1989). In this paper, the author presents revisions of the cDNA and the amino acid sequences of the GCF. 1) 5' rapid amplification of cDNA end (5'RACE) for analysis of RNA of a cancer cell line, A431, was performed, which revealed that the 5' end of GCF cDNA was fused to a 308 bp fragment of other cDNA; simultaneously, the real 5' end cDNA sequence with 31 bp was identified. RNase protection assay presented a main protected band, which was consistent with the result of the RACE analysis. 2) T at the position 787 of the previously reported GCF cDNA was absent from RT-PCR on A431 total RNA. 3) A new sequence with 114 bp was observed on A431 RNA between the positions 851 and 852 of the already reported cDNA by RT-PCR. These observations were confirmed by RT-PCR analyses of RNAs prepared from several other human cell lines, including a non-transformed one (HFL), and white blood cells derived from a normal person. 4) Sequence of genomic GCF DNA was consistent with the new cDNA sequence but not with the previously reported one. 5) The remaining sequence of GCF cDNA was found to be identical to that of the previously reported GCF, based on the results of RT-PCR analyses of RNA prepared from human white blood cells. 6) By the corrections, the GCF cDNA consisted of 2661 bp nucleotides. This revised GCF cDNA (the wild type) encodes a protein of 781 amino acids, including two new sequence regions of 186 amino acids on the N-terminal side of this protein. The revisions eliminated the highly basic region of the amino-terminus of the previously reported GCF, while the other three fourth amino acid sequences of the GCF protein that contained leucine-zipper-like domain had not changed. The revised GCF had no highly homologous protein in the database except the previously reported GCF. 7) The author has developed a specific antibody to human GCF protein. This antibody specifically recognized a protein with a molecular weight of approximately 100 kDa present in the extracts from human cell lines, as confirmed by immunoprecipitation followed by Western blotting. 8) Indirect immunofluorescence of A431 and HeLa cells using the anti-GCF antibody showed that the GCF protein was localized in the nucleus, suggesting that the revised GCF is a nucleoprotein.

Amino Acid Sequence↗
PubMed2005

[Cloning and characterization of p43 cDNA from Trichinella spiralis].

OBJECTIVE: To clone and characterize the p43 cDNA from muscle larvae cDNA library of Trichinella spiralis (Ts) Chinese isolate. METHODS: PCR technique was used to amplify the target cDNA from muscle larvae cDNA library. After cloned in pMD18T vector, it was transformed into E. coli NovaBlue. The positive clones were sequenced and the cDNA was cloned into pET28a expression vector. After induced by IPTG, the inclusion body of the recombinant protein was purified and re-natured. The deoxyribonuclease II (DNase II) activity of the recombinant protein was tested by hydrolyzing XDNA. RESULTS: Open reading frame (ORF) of the p43 cDNA was successfully cloned from the muscle larvae cDNA library of the Chinese Ts, there were mutations of two nucleotides in the ORF of the Chinese Ts p43 cDNA comparing with that from U.S.A. isolate at the positions 210 and 604, namely, C and A in the USA isolate but T and G in the Chinese isolate. Considering that three authors had cloned the same p43 cDNA from the USA isolate and six groups (including this team) had also obtained the same sequence from the Chinese isolate, the mutation of the two nucleotides was considered as the single nucleotide polymorphic (SNP) marker of the Chinese Ts isolate. The DNase II activity of the recombinant protein was successfully detected by hydrolyzing lamdaDNA. CONCLUSION: The p43 cDNA was successfully cloned from the muscle larvae cDNA library of the Ts Chinese isolate. Two SNPs were found in the nucleotide sequence. The DNase II activity was proved.

Amino Acid Sequence↗
PubMed1990

Human alpha-N-acetylgalactosaminidase-molecular cloning, nucleotide sequence, and expression of a full-length cDNA. Homology with human alpha-galactosidase A suggests evolution from a common ancestral gene.

Human alpha-N-acetylgalactosaminidase (alpha-GalNAc, E.C. 3.2.1.49), the lysosomal glycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties from glycoconjugates, is encoded by a gene localized to chromosome 22q13----qter. The deficient activity of alpha-GalNAc is the enzymatic defect in Schindler disease, an inherited neuroaxonal dystrophy. To isolate a full-length cDNA, the enzyme from human lung was purified to homogeneity, 129 non-overlapping amino acids were determined by microsequencing the N terminus and seven tryptic peptides, and four synthetic oligonucleotide mixtures were used to screen a human fibroblast cDNA library. A full-length cDNA, pAGB-3, isolated from a placental lambda gt11 cDNA library, had a 2158-base pair (bp) insert with an open reading frame which predicted an amino acid sequence that was colinear with all 129 microsequenced residues of the purified enzyme. The pAGB-3 insert had a 344-bp 5'-untranslated region, a 1236-bp open reading frame encoding 411 amino acids, a 514-bp 3'-untranslated region, and a 64-bp poly(A) tract. A signal peptide sequence of 17 amino acids as well as six N-glycosylation sites were predicted. The pAGB-3 cDNA was subcloned into the p91023(B) mammalian expression vector and human alpha-GalNAc activity was transiently expressed in COS-1 cells, demonstrating the functional integrity of the full-length cDNA. Northern hybridization analysis of mRNA revealed two transcripts of about 3.6 and 2.2 kilobases (kb), and primer extension studies indicated a cap site at nucleotide -347 for the 2.2-kb transcript. The 3.6-kb cDNA (pAGB-35) was isolated; the 3598-bp pAGB-35 insert was identical to that of the 2.2-kb insert but had additional 5'- and 3'-untranslated sequences including a second downstream polyadenylation signal at nucleotide 3100-3105. Isolation of a genomic clone, gAGB-1, and sequencing the 2048-bp region including pAGB-3 revealed a 1754-bp intron between codons 319 and 320, which also was the site of a 70-bp insertion and a45-bp deletion in other cDNA clones. Notably, the alpha-GalNAc cDNA had remarkable amino acid homology with the human alpha-galactosidase A (alpha-Gal A) cDNA suggesting the evolutionary relatedness of these genes. The alpha-GalNAc cDNA had 46.9-64.7% amino acid identity in sequences (codons 1-319) corresponding to alpha-Gal A exons 1 through 6, while the comparable exon 7 sequence (pAGB-3 codons 320-411) had only 15.8% homology with numerous gaps.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Thiopurine methyltransferase pharmacogenetics. Cloning of human liver cDNA and a processed pseudogene on human chromosome 18q21.1.

Thiopurine methyltransferase (TPMT) is a genetically polymorphic enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. This genetic polymorphism is an important factor responsible for individual variation in thiopurine drug response. A cDNA for TPMT has been cloned from T84 human colon carcinoma cells. Northern blot analysis of multiple human tissues was performed with the T84 human colon carcinoma TPMT cDNA open reading frame (ORF) as a probe as one step toward understanding the molecular basis for the TPMT genetic polymorphism. Three mRNA species (approximately 1.4, 2.0, and 3.6 kb in length) were present in all tissues studied, including liver. However, none of these mRNAs matched the length of the 2.7 kb T84 TPMT cDNA. Therefore, it was important to clone a TPMT cDNA from a human drug-metabolizing organ such as the liver to determine whether its sequence matched that of the cDNA cloned from the T84 cell line. A human liver cDNA library was screened with the T84 TPMT cDNA ORF as a probe, and a 1.8 kb cDNA was isolated with a coding region sequence identical to that of the T84 TPMT cDNA. The TPMT cDNA ORF was then used to screen a human lymphocytes genomic DNA library in an attempt to clone the TPMT gene(s) in humans. Three intronless clones were isolated with identical ORF sequences that were 96% identical to that of the TPMT cDNA, but which contained multiple nucleotide substitutions and one deletion. The 3'- and 5'-flanking regions of one of the genomic DNA clones were sequenced.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1994

Human liver thermolabile phenol sulfotransferase: cDNA cloning, expression and characterization.

The sulfate conjugation of phenolic biogenic amines in humans is catalyzed by the thermolabile (TL) form of phenol sulfotransferase (PST). As a first step toward cloning a cDNA for TL PST, the enzyme was purified from jejunal mucosa, the human tissue with the highest known specific activity, and purified TL PST was subjected to limited proteolysis and amino acid sequencing. The PCR was then performed with human liver cDNA as template and primers designed on the basis of an 18 amino acid stretch of TL PST sequence to obtain a probe for screening cDNA libraries. When cDNA library screening proved unsuccessful, PCR primers were designed based on the nucleotide sequence of a functionally uncharacterized human brain cDNA that had been speculated to represent an aryl sulfotransferase. This brain cDNA encoded the 18 amino acid sequence that we had determined in TL PST. With human liver cDNA as template, these primers amplified a PCR product that contained an 885 nucleotide open reading frame that encoded 295 amino acids--including the 18 amino acids of TL PST sequence. In vitro transcription and translation of this human liver cDNA resulted in the synthesis of a 35.5 kDa protein. The human liver cDNA was also expressed in COS-1 cells, and the biochemical and physical characteristics of the encoded enzyme were identical with those of human liver TL PST. The cloning of a cDNA for human liver TL PST represents an important step toward understanding the molecular basis for the regulation of this enzyme in humans.

Amino Acid Sequence↗
PubMed1994

Isolation and mapping of human chromosome 21 cDNA: progress in constructing a chromosome 21 expression map.

We have isolated 175 cDNA clones from a fetal brain library by direct cDNA selection using genomic DNA isolated from pools of human chromosome 21 (HC21) cosmids. DNA sequences have revealed that 16 of these cDNA clones contain overlapping sequences. Of the other 159 cDNA sequences, 10 match previously identified HC21 genes, and 9 match previously determined cDNA sequences, including the Wilms tumor related transcript (QM), the human testican cDNA, the mammalian calponin cDNA, and 6 anonymous expressed sequence tags. All isolated cDNAs were hybridized to their corresponding cosmids, which suggests that they originated from HC21. We have localized 92 cDNA clones to previously reported HC21q YACs. The remaining unmapped cDNAs contain either sequences not included in the isolated HC21q YACs or sequences that hybridize to yeast DNA. The cDNAs not included in the YACs should be useful in isolating new YACs to bridge the gaps. PCR primers were derived from 4 novel cDNA sequences that had been mapped to the YACs in the suspected Down syndrome region and used in RT-PCR analysis. All 4 primer sequences amplified RNA fragments with the expected sizes, suggesting that these sequences could be used for expression analysis. The construction of a chromosome 21 cDNA map not only is important in the refinement of physical maps, but also will identify a set of genes in the disease regions for detailed characterization.

Base Sequence↗
PubMed1988

Construction and quality of cDNA libraries prepared from cytoplasmic RNA not enriched in poly(A)+RNA.

Poly(A)+RNA and cytoplasmic RNA of Ehrlich ascites tumor cells grown in vivo were used to study the quality and efficiency of cDNA synthesis. It was found that the rates of oligo(dT)-primed and unprimed reverse transcription were very similar in both cases. The size distributions of the cDNA strands prepared from unfractionated RNA reflected the size of cytoplasmic mRNA populations including a significant fraction of long molecules up to 6 kb. The fraction of cDNAs primed on rRNAs by oligo(dT) was found to be as low as 2-3%. Following second-strand synthesis by means of RNase H-induced nick translation by DNA polymerase I the overall yields in double-stranded cDNA were slightly higher when unfractionated cytoplasmic RNA was used as starting template. In repeated experiments we obtained an average yield of 2.2 micrograms of double-stranded cDNA when 70 micrograms of unfractionated cytoplasmic RNA was used as starting material. This amount of cDNA synthesized in one assay was sufficient to construct representative cDNA libraries in different vectors. Southern hybridizations of DNA isolated from cDNA libraries with various radiolabelled probes show that the libraries constructed from cDNA synthesized from cytoplasmic RNA not enriched in poly(A)+RNA contain a high ratio of full-length cDNA clones. The results suggest that representative cDNA libraries of high quality can be constructed without pre-isolation of poly(A)+RNA fractions.

Animals↗
PubMed2005

A modified cDNA subtraction to identify differentially expressed genes from plants with universal application to other eukaryotes.

We have designed a simple and efficient polymerase chain reaction (PCR)-based cDNA subtraction protocol for high-throughput cloning of differentially expressed genes from plants that can be applied to any experimental system and as an alternative to DNA chip technology. Sequence-independent PCR-amplifiable first-strand cDNA population was synthesized by priming oligo-dT primer with a defined 5' heel sequence and ligating another specified single-stranded oligonucleotide primer on the 3' ends of first-strand cDNAs by T4 RNA ligase. A biotin label was introduced into the sense strands of cDNA that must be subtracted by using 5' biotinylated forward primer during PCR amplification to immobilize the sense strand onto the streptavidin-linked paramagnetic beads. The unamplified first strand (antisense) of the interrogating cDNA population was hybridized with a large excess of amplified sense strands of control cDNA. We used magnetic bead technology for the efficient removal of common cDNA population after hybridization to reduce the complexity of the cDNA prior to PCR amplification for the enrichment and sequence abundance normalization of differentially expressed genes. Construction of a subtracted and normalized cDNA library efficiently eliminates common abundant cDNA messages and also increases the probability of identifying clones differentially expressed in low-abundance cDNA messages. We used this method to successfully isolate differentially expressed genes from Pennisetum seedlings in response to salinity stress. Sequence analysis of the selected clones showed homologies to genes that were reported previously and shown to be involved in plant stress adaptation.

Base Sequence↗
PubMed1990

Cloning of rat aorta lysyl oxidase cDNA: complete codons and predicted amino acid sequence.

Lysyl oxidase cDNA clones were identified by their reactivity with anti-bovine lysyl oxidase in a neonatal rat aorta cDNA lambda gt11 expression library. A 500-bp cDNA sequence encoding four of six peptides derived from proteolytic digests of bovine aorta lysyl oxidase was found from the overlapping cDNA sequences of two positive clones. The library was rescreened with a radiolabeled cDNA probe made from one of these clones, thus identifying an additional 13 positive clones. Sequencing of the largest two of these overlapping clones resulted in 2672 bp of cDNA sequence containing partial 5'- and 3'-untranslated sequences of 286 and 1159 nucleotides, respectively, and a complete open reading frame of 1227 bp encoding a polypeptide of 409 amino acids (46 kDa), consistent with the 48 +/- 3 kDa cell-free translation product of rat smooth muscle cell RNA that was immunoprecipitated by anti-bovine lysyl oxidase. The rat aorta cDNA-derived amino acid sequence contains the sequence of each of the six peptides isolated and sequenced from the 32-kDa bovine aorta enzyme, including the C-terminal peptide with sequence identity of 96%. Northern blots screened with lysyl oxidase cDNA probes identified hybridizing species of 5.8 and 4.5 kb in mRNA of rat aorta and lung, while dot blot analyses were negative for lysyl oxidase mRNA in preparations of rat brain, liver, kidney, and heart. A 258-bp segment of the 3'-untranslated region of lysyl oxidase cDNA is 93% identical with a highly conserved region of the 3'-untranslated sequence of rat elastin cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1999

The cDNA-derived amino acid sequence of hemoglobin I from Lucina pectinata.

The tropical clam Lucina pectinata contains a unique hemoglobin (HbI) which serves to transport H2S to autotrophic bacteria. The cDNA-derived amino acid sequence was obtained from overlapping clones containing the cDNA that codes for HbI. The reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods were employed to synthesize the cDNA fragments. An initial 354-bp cDNA clone encoding 118 amino acid residues of HbI was amplified from total RNA by RT-PCR using degenerate oligonucleotides. Gene-specific primers derived from the HbI-partial cDNA sequence were used for obtaining the 5' and 3' ends of the cDNA by RACE. The length of the HbI cDNA, estimated from sequence analysis of overlapping clones, was 1322 bp for the full-length cDNA. The coding region of the full-length cDNA codes for 143 amino acid residues. The most conserved amino acid residues in HbI from Lucina pectinata were identified by a multiple alignment with nonvertebrate globin sequences.

Amino Acid Sequence↗
PubMed1998

Sequence of the cDNA of a human dihydrodiol dehydrogenase isoform (AKR1C2) and tissue distribution of its mRNA.

Human liver contains three isoforms (DD1, DD2 and DD4) of dihydrodiol dehydrogenase with 20alpha- or 3alpha-hydroxysteroid dehydrogenase activity; the dehydrogenases belong to the aldo-oxo reductase (AKR) superfamily. cDNA species encoding DD1 and DD4 have been identified. However, four cDNA species with more than 99% sequence identity have been cloned and are compatible with a partial amino acid sequence of DD2. In this study we have isolated a cDNA clone encoding DD2, which was confirmed by comparison of the properties of the recombinant and hepatic enzymes. This cDNA showed differences of one, two, four and five nucleotides from the previously reported four cDNA species for a dehydrogenase of human colon carcinoma HT29 cells, human prostatic 3alpha-hydroxysteroid dehydrogenase, a human liver 3alpha-hydroxysteroid dehydrogenase-like protein and chlordecone reductase-like protein respectively. Expression of mRNA species for the five similar cDNA species in 20 liver samples and 10 other different tissue samples was examined by reverse transcriptase-mediated PCR with specific primers followed by diagnostic restriction with endonucleases. All the tissues expressed only one mRNA species corresponding to the newly identified cDNA for DD2: mRNA transcripts corresponding to the other cDNA species were not detected. We suggest that the new cDNA is derived from the principal gene for DD2, which has been named AKR1C2 by a new nomenclature for the AKR superfamily. It is possible that some of the other cDNA species previously reported are rare allelic variants of this gene.

Base Sequence↗
PubMed1982

Isolation of a cDNA clone for the human HLA-DR antigen alpha chain by using a synthetic oligonucleotide as a hybridization probe.

We have used a synthetic 20-nucleotide hybridization probe to isolate a cDNA clone encoding the alpha chain of the HLA-DR antigen from a cDNA library constructed from membrane-bound poly(A)+ mRNA. A set of synthetic 11-nucleotide fragments, potentially complementary to the codons for amino acids 11-14 of the HLA-DR alpha chain, were used to prime a cDNA synthesis reaction on various poly(A)+ mRNA templates. Extension of the primers in the presence of a single dideoxynucleotide triphosphate resulted in an 18-nucleotide cDNA product whose sequence corresponded to the NH2-terminal amino acids of the HLA-DR alpha chain. An oligonucleotide was synthesized based on this sequence information and its specificity for HLA-DR alpha mRNA was confirmed by primer extension and blot analysis. The cDNA library made from mRNA from the lymphoblastoid cell line CA-SC was probed with 32P-labeled cDNA synthesized on poly(A)+ mRNA from a B-cell line (CA-SC) or from a T-cell line (Molt-4) to enrich for B-cell-specific clones. A set of cDNA clones that hybridized preferentially with the B-cell probe was screened with the 32P-labeled 20-nucleotide probe. The cDNA clone isolated by this procedure is 1,100 nucleotides long; the nucleotide sequence of the 5' end of the cDNA insert corresponds to the amino acid sequence of the HLA-DR alpha chain. Hybridization of this cDNA clone to genomic blots suggests that the HLA-DR alpha chain is encoded by a single-copy gene. One of the restriction endonucleases used in genomic DNA digests reveals a restriction fragment polymorphism.

Amino Acid Sequence↗
PubMed1986

cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts.

The entire genome of tobacco mosaic virus (TMV) was copied into a series of subgenomic cDNA clones. cDNA sequences of the 5' and 3' ends of TMV were cloned separately. A synthetic oligonucleotide primer was used to generate a Pst I site at the 5' terminus, whereas a different primer was used to generate an Nde I site at the 3' terminus. This strategy permitted removal of non-TMV sequences from cloned cDNA inserts by treatment with exonuclease VII following restriction endonuclease cleavage. Pst I linkers were added to TMV 3' terminal cDNAs. Subgenomic cDNA fragments were ligated together into several independent full-genomic constructions from which TMV cDNA sequences could be cleanly excised as a single fragment by Pst I digestion. Full-genomic TMV cDNA was ligated immediately downstream from the lambda phage promoter from pPM1 and transcribed in vitro with Escherichia coli RNA polymerase. RNA transcripts from three of four full-genomic cDNA constructions were infectious, even though they contained 6 non-TMV nucleotides at the 3' end. Transcripts from a construction with 6 extra nucleotides at the 5' end also were infectious. Progeny virus from plants infected with cDNA transcripts appeared identical to the parental virus. Restriction maps of independent cDNA clones of the same regions of the genome were identical to each other and as predicted from the reported nucleotide sequence of TMV. Also, sequences of the 200 nucleotides proximal to the 5' termini of four independent cDNA clones were identical to each other and to published sequences, suggesting that independent isolates of TMV may have remarkably similar sequences.

Journal Article↗
PubMed1997

Cloning and expression of bovine corneal antigen cDNA.

PURPOSE: A cornea-associated antigen (CO-Ag) has been found to be the target for autoantibodies in patients with Mooren's ulcer. The study goals were to isolate a full-length clone encoding CO-Ag from a bovine corneal cDNA library and to express this clone in Escherichia coli (E. coli). METHODS: A DNA fragment of CO-Ag was generated, using unique oligonucleotide primers and reverse transcription polymerase chain reaction. This fragment was used as a probe to obtain cDNA clones from a bovine corneal cDNA library. The clone with the longest cDNA insert was selected for sequence analysis. Expression of the CO-Ag protein in E. coli was induced by isopropyl beta-D-thiogalactopyranoside (IPTG). The bacterially-produced CO-Ag was partially purified by calcium (Ca2+)-dependent hydrophobic interaction chromatography. RESULTS: The cDNA insert sequence was 273 nucleotides in length for the entire mRNA coding region, 212 nucleotides in the 5' untranslated region, 83 nucleotides in the 3' untranslated region and a poly(A) tail. The DNA base sequence of this clone also contained a standard initiation codon, termination codon, and the polyadenylation signal. This cDNA predicts a protein which contains 91 amino acids with a molecular weight of 10,584 daltons. The cDNA and deduced amino acid sequence of CO-Ag are completely identical to a S-100 protein, bovine calgranulin C. The cDNA was expressed in E. coli as a fusion protein consisting of 583 N-terminal amino acids of beta-galactosidase (beta-gal), 91 amino acids of CO-Ag, and possibly a number of additional N-terminal and C-terminal residues. The bacterially produced CO-Ag was fully functional with respect to hydrophobic interaction with phenyl-Sepharose matrix for its isolation. The fusion protein was recognized by antiserum raised against bovine CO-Ag protein on Western blots. CONCLUSIONS: The isolation and analysis of a cDNA clone containing the complete coding sequence of the CO-Ag protein and the expression of the CO-Ag protein in E. coli is reported. The availability of a CO-Ag cDNA probe and larger quantities of the CO-Ag protein should aid in elucidating the possible pathogenic role of CO-Ag in Mooren's ulcer.

Amino Acid Sequence↗
PubMedDate not supplied

Complete sequence of cytochrome P450 3c cDNA and presence of two mRNA species with 3' untranslated regions of different lengths.

Two cDNAs (pLM3c 4.1 and pLM3c 6.1) coding for rabbit cytochrome P450 3c were sequenced. cDNA 4.1 (1768 bp) exhibits an open reading frame from nucleotides 74 to 1576 encoding the 501 amino acid residues of the entire protein. cDNA 6.1 (189 bp) appears to encode the last 24 amino acids. Comparative amino acid sequence analysis indicated that P450 PCN1, PCN2, and HLp from rat and man, were 70, 67, and 73% homologous, respectively, to P450 3c. According to the cytochrome P450 nomenclature, the P450 3c gene is termed P450IIIA4. Comparison of the nucleotide sequences indicated that cDNA 6.1 was 100% homologous to cDNA 4.1. However, whereas a poly(A) tract started 23 nucleotides after the AATAAA consensus sequence in cDNA 6.1, cDNA 4.1 had a 3' untranslated region extending 101 bp beyond the polyadenylation signal, which lacked poly(A). This observation is consistent with the previous finding that both cDNA 4.1 and 6.1 hybridized with two distinct species of poly(A)RNA (1700 and 1850 bases) from rabbit liver. The extreme 3'-end 79-bp fragment of cDNA 4.1 therefore was isolated by subcloning in pUC12 (clone p18-Rsa I) and used to probe Northern blots of poly(A)RNA from control and rifampicin-treated rabbit liver. In contrast to cDNA 4.1 and 6.1, p18-Rsa I cDNA hybridized only with the largest (1850 bases) mRNA species. We conclude that rabbit liver contains two P450 3c mRNA species differing in the length of their 3' untranslated region.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of a cDNA encoding a chicken beta thyroid hormone receptor.

We have isolated and characterized a cDNA encoding a chicken beta homolog of c-erbA, or thyroid hormone receptor (TR). Chicken liver cDNA libraries were screened with a rat TR beta-1 cDNA probe, and several cDNA inserts were isolated and characterized. The sequence of one cDNA predicts a 369-amino-acid open reading frame (ORF), with a protein sequence that possesses 96% identity with that of rat TR beta-1, but only 88% identity with chicken TR alpha. These data indicate that the cDNA likely encodes a beta form of TR that has the expected putative DNA and T3 binding domains. The chicken TR beta (chTR beta) in vitro translated protein binds T3 with high affinity, and binds both the thyroid hormone response element (TRE) from the rat growth hormone gene and the Xenopus vitellogenin A2 gene estrogen response element (ERE), similarly to that of the rat TR beta-1. Northern blot analysis revealed the expression of a 7.0-kb RNA in several tissues including cerebellum, pituitary, kidney, and liver. This chicken liver TR beta cDNA sequence varies in both the 5' and 3' untranslated regions from the chicken kidney TR beta cDNA sequence recently reported (Forrest et al., 1990). The 5' untranslated cDNA sequence divergence occurs near a potential splice site junction of the human TR beta gene, suggesting that this chicken liver cDNA may represent an alternatively spliced RNA product of the chicken TR beta gene.

Amino Acid Sequence↗