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PubMed1997

DD/AP-PCR: combination of differential display and arbitrarily primed PCR of oligo(dT) cDNA.

In this report we describe the first direct comparison of differential display (DD) and arbitrarily primed PCR (AP-PCR) amplification of oligo(dT)-primed cDNA. Our results indicate that both of these widely used RNA fingerprinting techniques have their respective advantages and limitations. DD produces profiles specific to the anchored oligo(dT) primer used for cDNA synthesis. AP-PCR displays significant redundancy of profiles generated from different oligo(dT) cDNA pools, but is not as biased to the isolation of A/T-rich or 3' sequences. It was found that both techniques can utilize cDNA synthesized using a generic anchored oligo(dT) primer (dT12VN; equimolar amounts of dT12VA, dT12VC, dT12VG, and dT12VT, where V is dA, dC, or dG); this efficiently selects for poly(A)+ sequences from total RNA, and significantly reduces the number of cDNA preparations required per experiment. Using dT12VN cDNA pools generated from rat liver, spleen, and brain, the two approaches (AP-PCR and DD) were used in combination. Several known mRNAs were identified; some were unique to either technique and some were common to both. Since it is the RNA which is usually the limiting resource, maximum utilization may be achieved by generating a single pool of dT12VN-primed cDNA and performing both AP-PCR and DD (DD/AP-PCR).

Animals↗

PubMed1993

Antisense and sense cDNA expression cloning using autonomously replicating vectors and toxic lectin selection.

CHOP2 cells, a subline of the Chinese hamster ovary (CHO) cell glycosylation mutant Lec2 which expresses polyoma virus large T antigen, was used as the host cell line to select cDNAs conferring resistance to the toxic effects of ricin. Glycoconjugates in CHOP2 cells are deficient in sialic acid and therefore the cells are hypersensitive to ricin, a galactose-binding lectin. CHOP2 cells acquiring cDNA that either corrected the Lec2 mutation or created a Lec2/Lec1 phenotype were expected to show a selective growth advantage in ricin-containing medium. After a single cycle of transfection with a lymphoid cDNA library in pCDM8 followed by ricin selection, the the predominant cDNA recovered from the cells was antisense N-acetyl-glucosaminyltransferase I (GlcNAc-TI) which conferred a Lec2/Lec1 phenotype. cDNA encoding GlcNAc-TI in a sense orientation was also enriched by transfecting a cDNA library into CHOP-1 cells, a mutant deficient in this enzyme, and selecting in medium containing ConA lectin. The results show that cell selection with toxic agents can be used to expression-clone both antisense and sense cDNA sequences from bi-directional cDNA libraries in the pCDM8.

Animals↗

PubMed1994

Preparation of a subtractive cDNA library enriched in cDNAs which expressed at a high level in cultured senescent human fibroblasts.

Subtracted cDNA library was prepared by subtracting [cDNA from young growing SV40-transformed human fibroblasts] from [cDNA from growing SV40-transformed fibroblasts in extended lifespan]. Isolated cDNA clones which expressed at high level in life-extended transformed cells also expressed at high level in normal senescent fibroblasts but did at low level in growing and growth-arrested young cells. Neither fibronectin nor procollagen cDNA was isolated. This cDNA library is useful for isolation of senescent-specific cDNA species which express at high level in normal senescent cells but at low level in growing and growth-arrested young cells, avoiding growth-arrest-specific cDNAs.

Animals↗

PubMed1994

Human liver estrogen sulfotransferase: identification by cDNA cloning and expression.

Sulfation is an important pathway in the biotransformation of steroid hormones such as estrogens. Human liver contains three well-characterized sulfotransferase (ST) enzymes, dehydroepiandrosterone (DHEA) ST and two forms of phenol sulfotransferase (PST). Although two of these enzymes, DHEA ST and one form of PST, can catalyze the sulfate conjugation of estrogens, our goal was to test the hypothesis that human liver might also contain a distinct estrogen ST (EST). We used the PCR to clone a human liver EST cDNA by utilizing degenerate oligonucleotide primers designed on the basis of highly conserved EST sequences in non-human species to amplify a 512 nucleotide portion of the open reading frame (ORF) with single-stranded human liver cDNA as a template. Rapid amplification of cDNA ends (RACE) was then used to obtain the 5'- and 3'-ends of the EST cDNA. The ORF of this cDNA consisted of 882 nucleotides and encoded 294 amino acids. The protein encoded by the human liver EST cDNA was 81%, 73%, and 72% identical to the amino acid sequences of guinea pig adrenocortical, bovine placental and rat liver ESTs, respectively. Human liver EST transiently expressed in COS-1 cells was capable of catalyzing the sulfation of estrone, DHEA and 4-nitrophenol, but not that of dopamine. The expressed human liver EST was also characterized with regard to thermal stability, inhibition by 2,6-dichloro-4-nitrophenol (DCNP) and response to NaCl. Identification of human liver EST by cloning and expression of its cDNA should enhance understanding of the enzymology and regulation of the biotransformation of estrogens and other steroids in humans.

Amino Acid Sequence↗

PubMed2001

Molecular cloning of newt prolactin (PRL) cDNA: effect of temperature on PRL mRNA expression.

A partial prolactin (PRL) cDNA was specifically PCR amplified from a cDNA library constructed from pituitary mRNAs of the newt (Cynops pyrrhogaster) and cloned into plasmid vectors. One clone thus obtained contained a 739-bp insert encoding the C-terminal amino acid sequence of the mature hormone molecule. Using this clone as a probe, the full-length newt PRL cDNA was screened from the cDNA library. The PRL cDNA clone thus obtained consisted of 1024 bp encoding the entire sequence of the mature PRL molecule in addition to its signal peptide. The amino acid sequence of newt PRL deduced from its nucleotide sequence showed higher homologies with those PRL sequences of tetrapod animals than with those of teleosts. Northern blot analysis revealed the newt PRL mRNA size to be approximately 1 kb. In situ hybridization using the newt PRL cDNA as a probe revealed that the pituitary region expressing PRL mRNA corresponded to that immunoreactive with antiserum against PRL. PRL mRNA levels in the pituitary of newts subjected to room and low temperatures were determined by Northern analysis employing the PRL cDNA as a probe. PRL mRNA levels were significantly higher in the pituitaries of newts subjected to 10 degrees than in those of newts kept at 23 degrees. Likewise, immunoassayable plasma PRL levels were higher in animals subjected to 10 degrees than in those kept at 23 degrees.

Amino Acid Sequence↗

PubMed1997

Human sulfotransferase SULT1C1: cDNA cloning, tissue-specific expression, and chromosomal localization.

We have isolated and sequenced a cDNA that encodes an apparent human orthologue of a rat sulfotransferase (ST) cDNA that has been referred to as "ST1C1"-although it was recently recommended that sulfotransferase proteins and cDNAs be abbreviated "SULT." The new human cDNA was cloned from a fetal liver-spleen cDNA library and had an 888-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 62% identical with that encoded by the rat ST1C1 cDNA and included signature sequences that are conserved in all cytosolic SULT enzymes. Dot blot analysis of mRNA from 50 human tissues indicated that the cDNA was expressed in adult human stomach, kidney, and thyroid, as well as fetal kidney and liver. Northern blot analyses demonstrated that the major SULT1C1 mRNA in those same tissues was 1.4 kb in length. We next determined the partial human SULT1C1 gene sequence for a portion of the 5'-terminus of one intron. That sequence was used to design SULT1C1 gene-specific primers that were used to perform the PCR with DNA from human/rodent somatic cell hybrids to demonstrate that the gene was located on chromosome 2. PCR amplifications performed with human chromosome 2/rodent hybrid cell DNA as template sublocalized SULT1C1 to a region between bands 2q11.1 and 2q11.2.

Adult↗

PubMed1999

Use of silent mutations in cDNA encoding human glutathione transferase M2-2 for optimized expression in Escherichia coli.

Heterologous expression of human glutathione transferase M2-2 (GST M2-2) using Escherichia coli was improved 140-fold by mutating the cDNA expressing the enzyme. Expression of GST M2-2 from this cDNA clone, pKHXhGM2, generated approximately 190 mg protein per liter of bacterial culture, corresponding to approximately 12% of the total amount of soluble protein. The high-level-expressing cDNA was generated by oligonucleotide-directed mutagenesis introducing alternative silent mutations into the third nucleotide of codons 2, 4-7, and 10-14 in the 5' end of the cDNA coding region. The choice of alternative codons was restricted to those naturally occurring in highly biased genes in E. coli. Furthermore, the wild-type TAG stop codon at the 3' end was replaced with the two stop codons TAA and TGA in tandem to increase translation termination efficiency. The resulting partially randomized cDNA library was assayed for high-level expression using immunoscreening. Sequence similarities between the constructed high-level-expressing GST M2-2 cDNA and a similarly designed cDNA encoding the closely related human GST M1-1 suggest that the codons in the region immediately following the start codon are influential in achieving high-level expression. Pyrimidines seem to be more favorable than purines in the third position of codons in optimizing the expression of these enzymes in E. coli.

Amino Acid Sequence↗

PubMed1994

Construction of a recombinant cDNA of echovirus 6 that established a persistent in vitro infection.

cDNA clones of lytic acute and nonlytic persistent strains of echovirus 6 were used to construct a recombinant cDNA. The 3' region of the infectious wild-type cDNA genome, which extended from VPg to the end of the noncoding region, was exchanged with the cDNA fragment representing the same region of the persistent viral genome. Sequence analyses indicated that there was one mutation in the 3C protease and eight mutations in the 3D polymerase. Transfection of the recombinant cDNA into WISH cells resulted in cellular survival and synthesis of viral RNA. The viral RNA was retained in the transfected cell line after cultivation for 7 months. Supernates, collected from cell cultures at 1, 3, and 7 months after transfection with the recombinant cDNA, transmitted the viral RNA to uninfected cells. The results indicated that the recombinant cDNA established a persistent echovirus 6 infection that was transmissible by nonlytic virus particles.

Base Sequence↗

PubMed1992

Human glial fibrillary acidic protein (GFAP): molecular cloning of the complete cDNA sequence and chromosomal localization (chromosome 17) of the GFAP gene.

We isolated three glial fibrillary acidic protein (GFAP) cDNA clones from a glioma cell line, U-251 MG. One clone isolated from a U-251 MG cDNA library was long, but lacked both ends. Using poly(A)+ RNA and primers synthesized according to the sequence of this clone, we used the polymerase chain reaction-assisted rapid amplification of cDNA ends (PCR-RACE) method, which is a strategy to isolate cDNA ends, and obtained cDNA clones for the 5' and 3' ends. From the sequences of these overlapping clones, the complete nucleotide sequence of human GFAP cDNA was established. The start (ATG) and the stop (TGA) signals were seen at nucleotide positions 15 and 1311, respectively, and divided the entire sequence of 3027 bp into 14 bp of 5' non-coding, 1296 bp of coding and 1717 bp of 3' non-coding regions. Using cDNA probes made from both the coding and the 3' non-coding regions, Northern blot hybridization was performed with two different stringencies on RNAs from human and rodent brains and human GFAP-positive and -negative cells. It was shown that the 3' non-coding region probe was more specific for human GFAP than the coding region probe which was specific only under higher stringency conditions. This was also suggested by homology analysis of the sequence with those of various intermediate filament proteins. Based on these findings, we performed spot blot hybridization of sorted human chromosomes and Southern blot hybridization of PCR-amplified DNAs of a panel of hamster-human somatic cell hybrids and localized the human GFAP gene to chromosome 17.

Amino Acid Sequence↗

PubMed1990

Cloning and sequence analysis of a cDNA for cellulase (FI-CMCase) from Aspergillus aculeatus.

We have cloned and characterized the cDNA coding for a major component of cellulase, endoglucanase (FI-CMCase), produced by Aspergillus aculeatus. The cDNA was isolated from a A. aculeatus cDNA library using synthetic oligonucleotide mixtures that correspond to the internal amino acid sequence of the mature FI-CMCase protein. Nucleotide sequence analysis of the cloned cDNA insert revealed a 711 bp open reading frame that encoded a protein of 237 amino acid residues. The primary structure of FI-CMCase deduced from the nucleotide sequence of cDNA agreed with that found by amino acid sequencing of peptide fragments obtained by digestion with several proteinases and cyanogen bromide cleavage. There may be a signal peptide sequence of 16 amino acid residues at the N-terminus. The molecular mass of the mature protein calculated from the cDNA is 24002 daltons, which compares favorably with molecular mass estimates of purified FI-CMCase obtained from SDS-PAGE (25000 Da). No distinct homology was found between the amino acid sequence of FI-CMCase and known cellulase sequences of other microorganisms. This study is the first example of cDNA cloning of an endoglucanase from the genus Aspergillus.

Amino Acid Sequence↗

PubMed1984

Sequence of a cDNA coding for a rat class II A alpha chain: extensive DNA and protein sequence identity to H-2 A alpha and HLA-DC1 alpha chains.

The rat major histocompatibility complex (RT1-B region) codes for two sets of class II molecules (Ia antigens) referred to as A and E. Each class II molecule is composed of two glycoprotein chains called the A alpha and A beta or E alpha and E beta. Two cDNA clones encoding rat A alpha chains were identified from cDNA derived from rat spleen mRNA using a combination of mRNA selection and colony hybridization techniques. The complete nucleotide sequence of the cDNA insert of one of these cDNA clones, pRIa.2, was determined. This sequence codes for the carboxy-terminal 129 amino acids of the rat A alpha chain and 293 nucleotides of 3' untranslated sequence. The rat A alpha chain was shown to be highly homologous in terms of both protein and DNA sequence identities to HLA-DC1 alpha and H-2 A alpha chains. Comparison between the coding regions of the cDNA insert of pRIa.2 and the corresponding region of a cDNA insert encoding an HLA-DC1 alpha chain showed sequence identities of 85% and 81% at the protein and DNA levels, respectively. Comparison between pRIa.2 and cDNA encoding an H-2 A alpha chain sequence showed identities of 91% for both protein and DNA. Results are discussed which strongly suggest that the class II A and E primordial genes arose by gene duplication prior to the evolutionary divergence of the mammals.

Amino Acid Sequence↗

PubMed1988

cDNA cloning, expression and mapping of human laminin B2 gene to chromosome 1q31.

A laminin B2 chain cDNA clone was isolated from a human lung cDNA library by screening with antibody against mouse laminin. The authenticity of the human cDNA clone was established by comparison of the nucleotide and deduced amino acid sequences of the cDNA insert with those of the previously reported mouse laminin cDNA clones. The human clone (LC7) contained an insert of 0.75 kb (kilobase pair) that corresponded to the last 232 amino acid residues in the carboxyl terminus of the B2 chain. Northern blot analyses with the LC7 probe detected two mRNA transcripts of 8.2 and 5.6 kb in both normal human skin fibroblasts and three human tumor cell lines. The cDNA probe was also used in Southern blot analysis of DNA from human rodent somatic cell hybrids to localize the gene to human chromosome 1. In situ hybridization of the cDNA with metaphase chromosome spreads confirmed the assignment and further mapped the human laminin B2 chain gene to the long arm of chromosome 1 in the band q31.

Amino Acid Sequence↗

PubMed1989

Paradoxical regulation of human argininosuccinate synthetase cDNA minigene in opposition to endogenous gene: evidence for intragenic control sequences.

Human somatic cell variants resistant to the arginine analog, canavanine, express 200-fold increased levels of argininosuccinate synthetase (AS) mRNA as compared to parental cells. In this study we examined whether AS cDNA sequences contain cis-acting regulatory elements that are involved in the induction of AS mRNA in canavanine-resistant cells. Minigene constructs containing AS cDNA sequences under the transcriptional control of a viral promoter were stably transfected into the human squamous cell carcinoma line, RPMI 2650. Upon conversion of cells to canavanine-resistance, expression of the endogenous AS gene increased by two orders of magnitude as expected. Surprisingly, however, expression of AS cDNA minigenes decreased 10- to 15-fold in canavanine-resistant cell variants. The observed down-modulation of AS cDNA minigene expression was dependent upon a concomitant induction of the endogenous AS gene and not simply expression of the canavanine-resistant phenotype. This paradoxical regulation was specific for AS gene sequences since a minigene containing the neomycin-resistance gene in place of AS cDNA sequences failed to regulate. Furthermore, minigenes lacking a substantial portion of the AS cDNA also failed to exhibit the down-modulation. These findings suggest that expression of the human AS gene is regulated by a specific and limiting, positively-acting, trans-acting mechanism in canavanine-resistant cells and that exogenous AS cDNA (mRNA) sequences can compete for this mechanism.

Argininosuccinate Synthase↗

PubMed2004

Construction and significance of directional expression cDNA library from human NB4 cells.

Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and second strands of cDNA were synthesized by reverse transcription. After blunting, the cDNA fragments were ligated with EcoR I adapters. Then the cDNAs were digested with Xho I, and less than 400 bp cDNA fragment was removed by Sephacryl-S400 spin column, the remaining were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E. coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the NB4 cell line cDNA library consisting of 1.65 x 10(6) recombinant bacteriophages was constructed with the recombinant ratio of 99.6%. The average length of the recombinant exogenous inserts was about 1.7 kb. It was concluded that the constructed cDNA library are deserved to screen target clones.

Bacteriophages↗

PubMed1999

Murine phospholipid hydroperoxide glutathione peroxidase: cDNA sequence, tissue expression, and mapping.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), also known as glutathione peroxidase 4 (GPX4), is a 19-kDa, monomeric enzyme that protects cells from lipid peroxide-mediated damage by catalyzing the reduction of lipid peroxides. PHGPx is synthesized in two forms, as a 194-amino acid peptide that predominates in gonadal tissue and localizes to mitochondria, and as a 170-amino acid protein that predominates in most somatic tissues and localizes to the cytoplasm. With the rapid amplification of cDNA ends (RACE) procedure, an 876-bp PHGPx cDNA was amplified from mouse testis, and a 767-bp PHGPx cDNA was amplified from mouse heart. The cDNA sequences were identical except that the testis cDNA contained an additional 109 bp at its 5' end. With a partial cDNA with complete homology to both the testis and myocardial PHGPx cDNAs, the murine tissue distribution of PHGPx mRNA expression was determined by Northern blotting. Highest level of PHGPx expression was found in the testis, followed by the kidney, heart and skeletal muscle, liver, brain, lung, and spleen. Northern blotting performed with a cDNA specific for the longer PHGPx transcript demonstrated that this longer PHGPx transcript was present only in the testis. A 1.4-kb PHGPx genomic fragment was amplified from murine kidney DNA and used to map the PHGPx gene by linkage analysis of restriction fragment length variants (RFLVs). The murine PHGPx gene (Gpx4) was mapped to a region of murine Chromosome (Chr) 10, located 43 cM from the centromere, that is syntenic with the human locus, which is located at the terminus of the short arm of human Chr 19. This information may be valuable in characterizing the role of PHGPx in modulating susceptibility to lipid peroxide-mediated injury in inbred murine strains and for targeted disruption of the gene.

Amino Acid Sequence↗

PubMed2006

Transduction of beta3 integrin subunit cDNA confers on human keratinocytes the ability to adhere to gelatin.

alphavbeta3 is a multiligand integrin receptor that interacts with fibrinogen (FG), fibrin (FB), fibronectin (FN), vitronectin (VN), and denatured collagen. We previously reported that cultured normal human keratinocytes, like in vivo keratinocytes, do not express alphavbeta3 on the cell surface, and do not adhere to and migrate on FG and FB. Furthermore, we reported that human keratinocytes transduced with beta3 integrin subunit cDNA by a retrovirus-mediated transduction method express alphavbeta3 on the cell surface and adhere to FG, FB, FN, and VN significantly compared with beta-galactosidase (beta-gal) cDNA-transduced keratinocytes (control). In this study, we determined whether these beta3 integrin subunit cDNA-transduced keratinocytes or normal human keratinocytes adhere to denatured collagen (gelatin) using a 1 h cell adhesion assay. beta3 cDNA-transduced keratinocytes adhered to gelatin, whereas no significant adhesion was observed with the control cells (beta-gal cDNA-transduced keratinocytes and normal human keratinocytes). The adhesion to gelatin was inhibited by LM609, a monoclonal antibody to alphavbeta3, and RGD peptides but not by normal mouse IgG1 nor RGE peptides. Thus, transduction of beta3 integrin subunit cDNA confers on human keratinocytes the ability to adhere to denatured collagen (gelatin) as well as to FG, FB, VN, and FN. Otherwise, normal human keratinocytes do not adhere to gelatin. These data support the idea that beta3 cDNA-transduced human keratinocytes can be a good material for cultured epithelium to achieve better take rate with acute or chronic wounds, in which FG, FB, and denatured collagen are abundantly present.

Antibodies, Monoclonal↗

PubMed1997

Isolation of cDNA clones for genes showing enhanced expression in barley leaves during dark-induced senescence as well as during senescence under field conditions.

Senescence of barley (Hordeum vulgare L. cv. Carina) primary foliage leaves was induced by transfer of the plants into darkness for 2 d. Under these conditions senescence was characterized by a light-reversible decline in the efficiency of photosystem II, and in chlorophyll and protein contents. To isolate senescence-associated genes a differential display of cDNA fragments amplified from reversely transcribed RNA was employed. By this method, gene expression in leaves of control plants collected at the onset of the dark period was compared with gene expression in senescing leaves collected at the end of the extended dark period. The expression of the genes represented by various differentially displayed cDNA fragments was examined by Northern blot hybridizations with RNA derived from primary foliage leaves before and after induction of senescence by darkness. In order to test whether these genes with enhanced expression during dark-induced senescence also show enhanced expression during natural senescence, Northern blot hybridizations were carried out with RNA samples prepared from flag leaves of barley plants during maturation and senescence under field conditions. Five of the cDNA fragments representing transcripts associated with dark-induced senescence, as well as with natural senescence, were selected as probes for screening a cDNA library from senescent flag leaves. With one probe a larger cDNA including a complete open reading frame with homology to the sequence of a known proteinase inhibitor was found. Another cDNA isolated by this means showed high sequence similarity with a gene coding for a 4-hydroxyphenylpyruvate dioxygenase. The other three larger cDNA clones isolated by this procedure so far do not show significant homologies with known sequences.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMed1999

The argininosuccinate lyase gene of Chlamydomonas reinhardtii: cloning of the cDNA and its characterization as a selectable shuttle marker.

We describe the cDNA sequence for ARG7, the gene that encodes argininosuccinate lyase--a selectable nuclear marker--in Chlamydomonas reinhardtii. The 5' end of the cDNA contains one more exon and the organisation of the mRNA is different from that predicted from the genomic sequence. When expressed under the control of the endogenous RbcS2 promoter, the 2.22-kb cDNA complements the arg7 mutation as well as the genomic DNA. A linear cDNA fragment lacking promoter sequences is also able to complement, suggesting that it could be used in promoter-trapping experiments. Despite the presence of a sequence encoding a potential chloroplast transit peptide in the cDNA the protein is not targeted to the chloroplast, nor can it complement the arg7 mutation when expressed there. By inserting a T7 bacteriophage promoter into the plasmid, a version of the cDNA which is able to complement both the C. reinhardtii arg7 mutant and the Escherichia coli argH mutant has been created. This modified Arg7 cDNA provides two advantages over the genomic DNA currently in use for gene tagging: it is shorter (6.2 kb versus 11.9 kb for pARG7.8phi3), and the selectable marker used in C. reinhardtii is the same as that used in E. coli, making plasmid rescue of the tag much more likely to succeed.

Algal Proteins↗