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At least 631 records · Page 35Linked to original sources
PubMed1987

Characterization of a cDNA coding for sex steroid-binding protein of human plasma.

A cDNA (912 nucleotides) coding for human plasma sex steroid-binding protein (SBP) was characterized from a phage clone previously isolated by screening a Charon 21A human liver cDNA library with rat androgen binding protein (ABP) cDNA. The deduced amino acid sequence from the cDNA indicated that the insert was a partial clone coding for 281 amino acids starting with residue 92 (glycine) encompassing the alternating leucyl residues and the carboxyl-end 373 (histidine) as previously reported [(1986) Biochemistry 25, 7584]. The potential polyadenylation signal sequence ATTAAA is present as part of the 3'-coding region and the stop codon TAA. Both are followed by a short 20 untranslated nucleotides and a poly(A) tract of 49 nucleotides. Significant homologous sequences (76%) at the DNA level exist between human SBP and rat ABP which might suggest the possibility that both evolved from a common primordial gene. Demonstration of the presence of an SBP cDNA in a human liver cDNA library provides the first evidence that liver is the site of SBP biosynthesis.

Amino Acid Sequence↗
PubMed1989

Molecular cloning of cDNA for cholesterol 7 alpha-hydroxylase from rat liver microsomes. Nucleotide sequence and expression.

A complete cDNA clone encoding cholesterol 7 alpha-hydroxylase was isolated from a rat liver cDNA library by the use of specific antibodies to the enzyme. The isolated cDNA clone was 3.6 kbp long and contained a 1509-bp open reading frame encoding 503 amino acid residues (Mr = 56,880). The identity of the cDNA was confirmed by expression of cholesterol 7 alpha-hydroxylase activity and the immunoreactive protein in COS cells transfected with pSVL expression vector carrying the cDNA insert. The primary structure of cholesterol 7 alpha-hydroxylase deduced from the nucleotide sequence of the cDNA indicated that the enzyme constitutes a novel P-450 family.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and bacterial expression of a cDNA encoding furostanol glycoside 26-O-beta-glucosidase of Costus speciosus.

Furostanol glycoside 26-O-beta-glucosidase (F26G) purified from Costus speciosus rhizomes was digested with endoproteinase, and several internal peptide fragments were obtained. Degenerate oligonucleotide primers based on amino acid sequences of the peptides were used for amplification of F26G cDNA fragments by applying nested polymerase chain reactions to cDNAs from in vitro cultured plantlets of C. speciosus. Using primers based on sequences of the cDNA fragments, the 5'- and 3'-end clones were isolated by rapid amplification of cDNA ends (RACE) methods. Finally, the entire coding portion of F26G cDNA was cloned by using primers designed from sequences of the RACE products. The deduced amino acid sequence of CSF26G1, the protein encoded by the cloned cDNA, consists of 562 amino acids and shows high homology to a widely distributed family of beta-glucosidases (BGA family). Cell-free homogenate of Escherichia coli expressing CSF26G1 cDNA showed beta-glucosidase activity specific for cleavage of the C-26 glucosidic bond of furostanol glycosides.

Amino Acid Sequence↗
PubMed1996

Hybrid selection with cDNA-silica.

A partial length ovalbumin cDNA-silica was produced using primer extension of (dT)18-silica with annealed partial ovalbumin RNA and reverse transcriptase. This cDNA-silica was used to test whether full-length ovalbumin RNA could be selectively purified in the presence of a large excess of other (mouse muscle) RNA. The cDNA-silica synthesized had minimally 60 pmol cDNA per gram silica and had a capacity for full-length ovalbumin RNA of minimally 38 micrograms/g. Even when other RNA was present in greater than 1000-fold excess, ovalbumin RNA was selectively retained by the cDNA-silica and was eluted in yields of 43% with an enrichment which varied over the range of 29-162-fold in various experiments. These results show that even rare RNAs can be selectively purified in high yield using cDNA-silica. The importance of these results to hybrid selection and subtractive library preparation is discussed.

Adenosine Triphosphate↗
PubMed1992

Screening for mutations in human HPRT cDNA using the polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis (CDGE).

Previously, we reported the modification of denaturing gradient gel electrophoresis called constant denaturant gel electrophoresis (CDGE). CDGE separates mutant fragments in specific melting domains. CDGE seems to be a useful tool in mutation detection. Since the hypoxanthine phosphoribosyltransferase (HPRT) gene is widely used as target locus for mutation studies in vitro and in vivo, we have examined the approach of analyzing human HPRT cDNA by polymerase chain reaction (PCR) and CDGE. All nine HPRT exons are included in a 716-bp cDNA fragment obtained by PCR using HPRT cDNA as template. When the full-length cDNA fragment was examined by CDGE, it was possible to detect mutations only in the last part of exon 8 and exon 9. However, digestion of the cDNA fragment with the restriction enzyme AvaI prior to CDGE enabled us to detect point mutations in most of exon 2, the beginning of exon 3, the last part of exon 8 and exon 9. With the use of two internal primer sets, including a GC-rich clamp on one of the primers in each pair, a region containing most of exon 3 through exon 6 was amplified and we were able to resolve fragments with point mutations in this region from wild-type DNA. The approach described here allows for rapid screening of point mutations in about two thirds of the human HPRT cDNA sequence. In a test of this approach, we were able to resolve 12 of 13 known mutants. The mutant panel included one single-base deletion, one two-base deletion and 11 single-base substitutions.

Base Sequence↗
PubMed1993

Identification and characterization of tox21A: a mite cDNA encoding a paralytic neurotoxin related to TxP-I.

A cDNA library established from polyadenylated RNA isolated from the predatory mite Pyemotes tritici was screened for cDNAs homologous to tox34, a cDNA encoding an insect-selective paralytic neurotoxin known as TxP-I. Most of the cDNA inserts of homologous clones were shorter or of equal length to tox34 but a few were longer. Further investigation into the nature of these longer clones led to the identification, sequencing and expression of a distinct cDNA referred to as tox21A. This cDNA is predicted to encode a polypeptide which shares approximately 83% amino acid identity with TxP-I. Larvae infected with a recombinant baculovirus expressing tox21A are paralyzed during infection in a manner similar to larvae infected with tox34-expressing recombinants. The tox21A cDNA may represent a duplicated and diverged copy of the TxP-I gene.

Amino Acid Sequence↗
PubMed1994

Structure of mouse protein S as determined by PCR amplification and DNA sequencing of cDNA.

The cDNA sequence of mouse protein S was derived by conventional PCR amplification from liver mRNA, initially using primers derived from the human cDNA sequence, followed by direct DNA sequencing. Seven overlapping PCR fragments covering all of the mature protein, part of the propeptide, and the 3' noncoding region were generated and sequenced. In some cases primers based upon the human cDNA sequence were ineffective. Subsequent successful amplification with mouse-derived primers to the same regions and comparison of the mouse and human sequences in these regions suggest that the failure of the human primers was due to insufficient degree of heterospecies identity. The mouse protein S cDNA sequence of the coding region shares 82% identity to human. The 3' noncoding region of mouse protein S cDNA has several small deletions and insertions compared to human protein S cDNA. Mature mouse protein S consists of 634 amino acids in a single polypeptide chain and displays domain organization similar to that for other species. The amino acid sequence of mouse protein S is about 80% identical to that of other species. Eleven glutamic acid residues were found in the amino terminal region and are predicted to be sites of gamma-carboxylation. Amino acid residues #80-244 are defined as four cysteine-rich repeat sequences homologous to epidermal growth factor. The remainder of the molecule is homologous to plasma sex steroid binding protein. The mouse protein S contains two potential N-glycosylation sites at positions #458 and 468 and is lacking the putative glycosylation site at #490 found in human protein S.

Amino Acid Sequence↗
PubMed1993

Cloning and sequence analysis of the gene and cDNA encoding mouse spermidine/spermine N1-acetyltransferase--a gene uniquely regulated by polyamines and their analogs.

The polyamine catabolizing enzyme, spermidine/spermine N1-acetyltransferase (SSAT), has been implicated as a critical determinant of polyamine pool maintenance. SSAT has recently been shown to be positively regulated in human cell lines by polyamines and their analogs at the level of mRNA accumulation. Mouse LA-4 lung adenoma cells treated with either spermine or the spermine analog, N1,N12-bis(ethyl)spermine, produced a 2.3 and 6.5-fold increase, respectively, in SSAT mRNA. Prior evidence for transcriptional control of the enzyme prompted investigation of SSAT gene structure and its regulatory elements. The mouse SSAT gene was isolated as a 3650 bp EcoRI fragment from a lambda-J1 Mus saxicola genomic library by hybridization with human SSAT cDNA. An additional 431 bp downstream from the 3' EcoRI site were sequenced from a BamHI fragment (total gene sequence, 4066 bp). The gene contains six exons and five introns. Sequence analysis of the 774 bp of the 5' non-coding region revealed the absence of TATAA or CCAAT sequence motifs and the presence of a number of binding motifs in the 5' region of the gene with consensus binding sequences for transcription factors SP1, AP1, E2F, AP2, PEA-3 and others. The deduced amino acid sequence of the coding region differs from that of the human SSAT cDNA by five amino acids. The 527 bp of the 3' non-coding region contains four possible polyadenylation signal sites of which only one displays a typical consensus sequence. A 940 bp SSAT cDNA was isolated from Mus domesticus (BALB-C) liver lambda gt11 cDNA library. It contains a 5' untranslated region 89 bp in length and a 3' untranslated region 376 bp in length. The amino acid sequence deduced from Mus domesticus differs from that of Mus saxicola by one amino acid, from the hamster cDNA, by four amino acids and from the human cDNA by six amino acids. Further elucidation of the structural features of the SSAT gene may reveal how it is positively regulated by polyamines and their analogs.

Acetyltransferases↗
PubMed1995

The 3'-untranslated region of rat lysyl oxidase cDNA.

The cloning of the 3'-untranslated region of rat lysyl oxidase cDNA was completed. cDNA clones were generated by reverse transcriptase PCR from neonatal rat aorta smooth muscle cell RNA, and sequenced. Several polyadenylated clones were obtained, providing 2.1 kb of new sequence. Clones were polyadenylated at three different positions. The cDNA clones were verified by PCR-cloning and sequencing of genomic DNA, and by Northern blotting studies. Evidence is presented that the polyadenylation patterns of rat lysyl oxidase mRNAs are similar, but not identical to mouse or human transcripts. Interestingly, the nonconsensus polyadenylations in rat did not occur at the same positions as was found in mouse lysyl oxidase cDNAs. Multiple transcription initiation sites were found by primer extension mapping. Thus, the complex pattern of rat lysyl oxidase mRNAs on Northern blots is principally due to differential use of polyadenylation signals, and to the occurrence of multiple transcription initiation sites. All clones lacked a previously reported 258 bp segment nearly identical to a conserved segment of the 3'-untranslated region of elastin cDNA. We conclude that the elastin-like sequence previously reported in rat lysyl oxidase cDNA is not a species-specific sequence, and most probably resulted from spurious ligation reactions during construction of the cDNA library.

Animals↗
PubMed1996

Heterogeneity of rat type I 5 alpha-reductase cDNA: cloning, expression and regulation by pituitary implants and dihydrotestosterone.

Primer extension analysis reveals the presence of different forms of mRNA species for rat type I 5 alpha-reductase. Using a 5 alpha-reductase cDNA probe to screen the rat liver lambda gt11 cDNA library, we isolated cDNA clones that have 4 additional amino acids in the NH2-terminal region as compared with the previously reported sequence for rat type I 5 alpha-reductase. These four additional amino acids elongate the rat type I 5 alpha-reductase amino acid sequence to 259 amino acids, the same number as in human type I 5 alpha-reductase, with which it shares 60% identity. Expression of the long and short rat type I 5 alpha-reductase by transfection in human adrenal adenocarcinoma cells, SW-13 cells, indicated that the long cDNA encoded a protein with a higher affinity for the substrate than the short cDNA. To determine the effect of pituitary hormones and dihydrotestosterone (DHT), the mRNA levels in the livers of rats treated with pituitary implants, hypophysectomized, castrated, and castrated coupled with DHT treatment were quantified by dot-blot hybridization assay using rat type I 5 alpha-reductase cDNA as probes. The results demonstrated that rat type I 5 alpha-reductase mRNA is stimulated by pituitary hormones and castration but is decreased by DHT and hypophysectomy.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of a library of cDNA clones that are preferentially expressed in the embryonic telencephalon.

In order to isolate genes involved in development of the mammalian telencephalon we employed an efficient cDNA library procedure. By subtracting an adult mouse telencephalic cDNA library from an embryonic day 15 (E15) mouse telencephalic cDNA library we generated two subtracted libraries (ES1 and ES2). We estimate that ES1 contains between 200 and 600 different cDNA clones, which approximates the number of genes that are preferentially expressed in the E15 telencephalon, compared to the adult telencephalon. Northern analysis of 20 different cDNA clones shows that 14 of these are expressed at least 5-fold more in the E15 telencephalon than the adult telencephalon. Limited sequencing of the 14 differentially expressed clones reveals that 10 have no significant identity to sequences in GenBank and EMBL databases, whereas the other 4 have significant sequence identity to vimentin, histone 3.3, topoisomerase I and the B2 repeat element. In situ hybridization using one of the differentially expressed cDNAs, TES-1, demonstrates that it is transiently expressed in the anlage of the basal ganglia. In situ hybridization with another differentially expressed cDNA clone, TES-4, shows that it is specifically expressed in differentiating cells of the neural axis with a distinctive rostral-caudal temporal pattern. These findings, and the methods that we have developed, provide a framework for future investigations of the genetic control of telencephalon development.

Animals↗
PubMed1988

FOSP-1 (frog oviduct-specific protein-1) gene: cloning of cDNA and induction by estrogen in primary cultures of Xenopus oviduct cells.

We describe the isolation of a cloned cDNA from a cDNA library of oviduct of estrogen-treated adult Xenopus. Although the protein encoded by this cDNA is not known, it is designated as FOSP-1 (frog oviduct-specific protein-1). A partial restriction map of FOSP-1 cDNA, which is 1.5 kb in size, is presented. Northern hybridization analysis showed that FOSP-1 cDNA codes for a single species of mRNA of 2.6 kb which is exclusively expressed in Xenopus oviduct. Southern blot analysis showed that the gene was present in only one or two copies. Sequencing of partial FOSP-1 cDNA did not reveal homology with any protein in the sequence data bank. Measurement of steady-state levels of FOSP-1 mRNA in primary cultures of Xenopus oviduct cells by a technique of quantitative slot-blot analysis showed that both 17 beta and 17 alpha stereoisomers of estradiol caused a rapid 5-fold enhancement of accumulation of the mRNA with maximum values obtained at 5 X 10(-8) M estrogen. Progesterone caused only a small increase in FOSP-1 mRNA concentration. This hormone-specific induction of mRNA makes FOSP-1 a valuable candidate for exploring tissue specificity of regulation by estrogen of gene expression.

Amino Acid Sequence↗
PubMed1990

The structure of cDNA clones encoding the aromatase P-450 isolated from a rat Leydig cell tumor line demonstrates differential processing of aromatase mRNA in rat ovary and a neoplastic cell line.

The conversion of androgens to estrogens is catalyzed by a complex of enzymes that includes a specific cytochrome P-450 aromatase (P-450arom). In this paper we describe the high level expression of aromatase activity in the rat Leydig cell tumor line, R2C. We also report the isolation of cDNA clones encoding the rat aromatase P-450arom from a cDNA library prepared from this cell line. Analysis of these cDNA clones predicts a protein sequence with a high degree of sequence conservation when compared to the chicken and human P-450arom enzymes. Notably, four of the cDNA clones were found to lack the last coding exon that contains the heme-binding domain, a structural feature essential for aromatase activity. These clones were found to contain instead a segment of genomic DNA derived from an unspliced intron. Northern analysis using a fragment of the coding region of the rat P-450arom cDNA as probe revealed that three species of P-450arom mRNa are expressed in rat ovary that are similar to those identified in RNA samples prepared from the rat R2C cell line. Analysis of the same samples of RNA using a probe derived from the 3' terminal intron segment of the rat aromatase cDNA clones that lack the heme-binding domain indicates that two of the species of aromatase mRNA transcripts present in both rat ovary and R2C cell lack the heme-binding domain and thus must encode a nonfunctional aromatase protein. These findings have important implications for the measurement of aromatase mRNA and appear to explain why three sizes of rat P-450arom mRNA exist on Northern analysis and why previous studies failed to demonstrate a clear relationship between aromatase mRNA, protein, and enzymatic activity in the rat ovary.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of cDNA for the B beta subunit of Xenopus fibrinogen, the product of a coordinately-regulated gene family.

Fibrinogen, the principal blood-clotting protein, is made up of three different subunits synthesized in the liver. In vitro administration of glucocorticoids to liver cells from the frog Xenopus laevis causes a dramatic increase in fibrinogen synthesis. Investigations of molecular mechanisms underlying this hormonal stimulation at the mRNA level require cDNA clones complementary to the mRNAs coding for the three fibrinogen subunits, called A alpha, B beta, and gamma. We describe here the isolation and characterization of cDNA clones for the B beta subunit of Xenopus fibrinogen. cDNA libraries in both plasmid (pBR322) and phage (lambda gt10) cloning vectors were constructed from frog liver mRNA and screened with a rat B beta cDNA. Clones thus isolated hybridized to two Xenopus liver mRNAs 2500 and 1800 bases long, the previously-determined sizes for B beta mRNAs. The identity of the plasmid clone B beta-27 was confirmed by hybridization-selection of complementary mRNA which translated in vitro into the B beta polypeptide, as determined by size and susceptibility to thrombin cleavage. lambda/B beta 10, a clone representing nearly all of the 2500-base B beta mRNA, was isolated from the phage cDNA library. The 3'-end of this clone includes a polyadenylation signal about 20 residues upstream of a stretch of 34 adenosine residues, which probably represents the 3'-poly(A) tail of the messenger RNA. lambda/B beta 10 lacks only 20 nucleotides of full-length B beta mRNA at the 5'-end and there is one major start site of transcription. The 2500-base B beta mRNA has a 700-base extension at the 3'-end that is not present in the 1800-base mRNA. The Xenopus laevis genome contains two or three genes for the B beta fibrinogen subunit. Using the cDNA clone as a probe, B beta mRNA was shown to be induced at least 20-fold by glucocorticoid treatment of purified parenchymal cells of Xenopus liver maintained in primary culture.

Animals↗
PubMed1984

Identification of bacterial clones encoding bovine caseins by direct immunological screening of the cDNA library.

A sensitive immunoassay was used to identify recombinant plasmids carrying cDNA fragments of bovine caseins in the cDNA library from bovine mammary gland mRNA. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated cellulose filter paper. Antigens covalently bound to CNBr-activated paper or bound to nitrocellulose filters were detected by reaction with antiserum to caseins, followed by 125I-labelled Staphylococcus aureus protein A and autoradiography. Six clones were found positive among 5400 of the cDNA library: 3-A1, 3-B2, 3-B5, 3-H7, 2-A5 and 2-C9. The molecular weights of chimeric pre-beta-lactamase: casein proteins synthesized in Escherichia coli were estimated by immunoblotting. Colony hybridization and nucleotide sequence analysis showed that clone 3-B5 contained a cDNA fragment of bovine chi-casein, clone 3-H7 contained a cDNA fragment of beta-casein, while clones 2-A5 and 2-C9 carried cDNA fragments of alpha s1-casein.

Animals↗
PubMed1984

Methods for cDNA cloning and sequencing tobacco mosaic virus RNA.

DNA complementary to tobacco mosaic virus (TMV) RNA (cDNA) was prepared by priming reverse transcription with synthetic oligonucleotides. The cDNAs terminated prematurely at many specific sites and no transcripts longer than about 2000 nucleotides were obtained. However, the entire 6395 nucleotide long TMV RNA could be copied into cDNA by specific priming with a series of 13 to 17 residue long oligonucleotides or by non-specific priming with short, 4 to 7 residue, oligonucleotides. A number of different priming methods were used to convert the cDNA into double-stranded DNA (dsDNA). The double-stranded cDNA was recovered by shotgun cloning into M13 and analysed by sequencing. The frequency at which cDNA clones were recovered has been used to compare various cDNA cloning strategies.

Base Sequence↗
PubMed1987

Expression of antibody cDNA in murine myeloma cells: possible involvement of additional regulatory elements in transcription of immunoglobulin genes.

Expression vectors for cDNA of the kappa and gamma 1 chains of a monoclonal antibody directed against creatine kinase were introduced into murine myeloma cells. Kappa and gamma 1 cDNA were either under the control of the SV40 early promoter or of the cognate promoters and enhancers of the light- and heavy-chain genes. Secretion of immuno-reactive kappa and gamma 1 chains into the culture medium was demonstrated with the SV40 promoter as well as with the cognate promoters. Expression of gamma 1 cDNA with the SV40 early promoter was about twice as high as with the heavy-chain promoter and enhancer. Expression of kappa cDNA under the control of the SV40 early promoter was about 17 times higher than with the light-chain promoter and enhancer. These expression levels were compared to those of a genomic immunoglobulin (Ig) kappa determinant, including introns. Such an entire kappa gene led to expression of the light chain at levels double those with the kappa cDNA construction using the SV40 promoter and about 35 times as high when using kappa cDNA and the cognate promoter and enhancer. This result might indicate that, besides the cognate promoter and enhancer elements, other intragenic elements are involved in the regulation of Ig expression. However, the SV40 early promoter seems to be able to compensate for the absence of these postulated regulatory elements probably located in the introns.

Animals↗
PubMed1988

The baboon beta-myosin heavy-chain gene: construction and characterization of cDNA clones and gene expression in cardiac tissues.

We have constructed a cDNA library from baboon ventricle and have used a rabbit beta-myosin heavy chain (beta-MHC) cDNA probe to isolate cross-hybridizing clones. The nucleotide sequence of one such clone, lambda BMHC beta 14, contains a portion of the coding region of the light meromyosin (LMM) region and the 3'-untranslated region of the baboon ventricular MHC. This cDNA clone is identified as containing beta-MHC sequences on the basis of similarity with the 3'-untranslated regions of beta-MHC genes from man (96% homologous) and rat (71% homologous), and dissimilarity with the 3'-untranslated region of the rat alpha-MHC gene (25% homologous). Alignment and comparison of the baboon cDNA nucleotide sequence with a human cDNA sequence reveal two amino acid substitutions in the LMM region that cause differences in their hydrophilicity profiles. These differences may alter MHC functions such as filament assembly. We have used baboon beta-MHC cDNA clones to construct probes for S1 nuclease protection studies to detect and to distinguish cardiac MHC gene transcripts in baboon ventricle, atrium, and diaphragm. As in human tissues, beta-MHC gene transcripts are detected in RNA from baboon ventricle and diaphragm. In baboon atrium, we detect beta-MHC gene transcripts as well as transcripts that may represent expression of the alpha-MHC gene. This study represents the first examination of cardiac MHC genes and gene expression in tissues from a large mammal that is closely related to man.

Amino Acid Sequence↗