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PubMed1990

Identification of a novel human glycophorin, glycophorin E, by isolation of genomic clones and complementary DNA clones utilizing polymerase chain reaction.

In our previous report, we described the structural organizations of glycophorin A and B genes (Kudo, S., and Fukuda, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4619-4623). During the course of isolation of these genomic clones, we also obtained genomic clones encoding a novel glycophorin. This novel glycophorin, termed glycophorin E (GPE), has a similar genomic structure to that of the GPB gene, and its nucleotide sequence is almost identical to that of the GPB gene. These sequences include a region downstream of an Alu repeat sequence, which has been suggested to be a site for homologous recombination in the GPB gene during or after gene duplication. However, the predicted GPE amino acid sequence specifies blood group M, in contrast to GPB which carries blood group N. Polymerase chain reaction was employed to analyze the transcript of this gene, and its cDNA sequence revealed that the novel glycophorin gene encodes 78 amino acids, including a 19-residue leader peptide. Comparison of genomic and complementary DNAs demonstrates that this gene consists of four exons, and point mutations at sites corresponding to the 5'-splicing sites of intron 3 and intron 4 of the GPA gene lead to the joining of the exon 2 to potential exon 5. Interestingly, an insertion of 24 nucleotides coding for eight amino acid residues in-frame was found in exon 5. The predicted amino acid sequence within this exon indicates that it has a hydrophobic character, suggesting the possible expression of GPE as a membrane protein. Northern blot analysis demonstrated that this novel glycophorin gene is expressed in an erythroid-specific manner and coordinately down-regulated together with GPA and GPB genes by a tumor-promoting phorbol ester. During evolution, this gene might have derived from an ancestral gene common to the GPB gene by gene duplication and subsequent nucleotide substitutions, and constitutes a member of a gene family with GPA and GPB genes.

Amino Acid Sequence↗
PubMedDate not supplied

Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-beta.

Inhibin, a specific and potent polypeptide inhibitor of the secretion of follicle-stimulating hormone (FSH), of gonadal origin and thus a potential contraceptive, may constitute a missing link in the mechanism controlling the differential secretion of the pituitary gonadotropins. Inhibin-like bioactivity has been reported in various fluids and extracts of testis and in ovarian follicular fluid. Although there have been several attempts to purify inhibin from seminal plasma, purification from follicular fluid has been more successful (refs 14-16; for review see ref. 17). We have previously isolated two forms (A and B) of inhibin from porcine follicular fluid. Each form comprised two dissimilar subunits of relative molecular mass (Mr) 18,000 (18K, referred to here as the alpha-subunit) and 14K (the beta-subunit), crosslinked by one or more disulphide bridge(s). Forms A and B differ in the N-terminal sequence of their 14K subunit. Preliminary structural characterization of porcine and bovine ovarian inhibins shows that they have similar properties. Here, we have used the N-terminal amino-acid sequence data on the subunits of each inhibin to identify cloned complementary DNAs encoding the biosynthetic precursors and report that inhibins are the product of a gene family that also includes transforming growth factor-beta (TGF-beta) and whose structural organization is similar to that of pituitary and placental glycoprotein hormones.

Amino Acid Sequence↗
PubMed1983

Improved method for cloning DNA complementary to minor mRNAs: preparation of a hybridization probe from purified mRNAs encoding intermediate filament proteins.

A procedure for the rapid fractionation of mRNA has been used to enrich mRNAs encoding a set of intermediate filament proteins in trophoblastoma cells. The procedure involves sucrose-gradient fractionation followed by high-resolution preparative gel electrophoresis. Part of the enriched mRNA preparation has been used to prepare a hybridization probe to screen a trophoblastoma cDNA library in Escherichia coli. A small proportion of the clones hybridized to the probe, and among these a specific clone was identified.

Animals↗
PubMed1990

Cloning and characterization of DNA complementary to human UDP-GalNAc: Fuc alpha 1----2Gal alpha 1----3GalNAc transferase (histo-blood group A transferase) mRNA.

Based on the partial amino acid sequence, the cDNA encoding UDP-GalNAc:Fuc alpha 1----2Gal alpha 1----3GalNAc transferase, the specific primary gene product of histo-blood group A gene (A transferase), was cloned and sequenced. Poly(A)+ RNA from human stomach cancer cell line MKN45, expressing high levels of A antigen, was used for construction of a lambda gt10 cDNA library. Degenerate synthetic oligodeoxynucleotides were used for polymerase chain reactions to detect the presence of the sequence of interest in cDNA (presence test) and to identify the correct clones (identification test) after screening the library with a radiolabeled polymerase chain reaction amplified fragment. Nucleotide sequence analysis revealed a coding region of 1062 base pairs encoding a protein of 41 kDa. Hydrophobicity plot analysis shows the existence of three domains: N-terminal short stretch, transmembranous hydrophobic region, and a long C-terminal domain (a feature common to all glycosyltransferases cloned so far). Southern hybridization analysis has shown that this DNA does not represent a multigene family. No restriction fragment length polymorphism was found to correlate with ABO blood group type. Bands were detected in Northern hybridization of mRNAs from cell lines expressing A, B, AB, or H antigens. These results suggest that sequences of ABO genes are essentially very similar (with minimal differences), and the inability of the O gene to encode A or B transferases is probably due to structural differences rather than A or B transferase expression failure.

Amino Acid Sequence↗
PubMed1988

Molecular cloning and sequencing of DNA complementary to chicken liver fatty acid synthase mRNA.

The cDNA corresponding to 4.18 kilobases (kb) of the mRNA of chicken liver fatty acid synthase has been cloned and sequenced. The cDNA corresponds to the 3' end of the mRNA and consists of a 1.87-kb noncoding tail and a 2.31-kb region encoding 769 amino acids of the C terminus of the enzyme. The thioesterase at the C terminus, preceded by the acyl carrier protein, can be identified from known amino acid sequences. However, the identity of the enzymes N terminal to the acyl carrier protein could not be ascertained. The partial amino acid sequence of the chicken liver fatty acid synthase shows greater than 70% similarity with the rat mammary gland enzyme.

Amino Acid Sequence↗
PubMed1987

Molecular cloning of DNA complementary to Drosophila melanogaster alpha-amylase mRNA.

Several lambda clones containing cDNAs from Drosophila melanogaster were identified in a lambda cDNA bank using two different approaches: (i) cross-species hybridization using a mouse amylase cDNA probe, and (ii) probing with a differential probe, generated from Drosophila RNA. An amylase cDNA fragment was used, in turn, for the isolation and characterization of amylase genomic clones. The size of the Drosophila amylase mRNA was estimated at 1650 b. This is comparable with the size of the murine amylase messenger that encodes a protein of similar molecular weight. In Drosophila larvae, amylase mRNA can account for as little as 0.01% of the poly(A)+ RNA under conditions of dietary glucose repression or greater than 1% of poly(A)+ RNA under derepressing dietary conditions.

Animals↗
PubMed1985

Molecular cloning and nucleotide sequence of DNA complementary to rat ribosomal protein S26 messenger RNA.

A cDNA clone specific for rat ribosomal protein S26 was isolated by a positive hybridization translation assay from a cDNA library made for 8-9S poly(A)mRNA from regenerating rat liver. The nucleotide sequence of the cDNA was determined. The sequence contains 23 base pairs in the 5' noncoding region, 345 base pairs in the protein coding region and 67 base pairs in the 3' noncoding region besides the poly(A) tail. The primary structure of the protein S26 was deduced from the nucleotide sequence. It consists of 115 amino acids. Its molecular weight is 13,015 and its pI is about 11.1. The calculated amino acid composition is consistent with the reported composition of S26. From the results of Southern blot analysis, the protein S26 appears to have multiple genes.

Amino Acid Sequence↗
PubMed1985

[Synthesis and cloning of DNA complementary to mRNA of the bovine mammary gland].

Poly(A+)mRNA from bovine mammary glands was used to synthesize double-stranded cDNAs that were subsequently inserted into the plasmid vector pBR322 at the Pst1 site by means of oligo(dG)-oligo(dC) tailing. After transfection of Escherichia coli JC5183, recombinant plasmid library containing 5400 clones was screened by serial rounds of colony hybridization in situ to total [23P] poly(A+)mRNA and electrophoretically homogenious [32P]16SmRNA of mammary glands. Then hybrid selection of mRNA and subsequent in vitro translation of selected mRNAs were performed. In this manner, recombinant clones coding for alpha S1- beta-, kappa-casein were identified. cDNA clones range in size from 35% for beta-casein, 65% for alpha S1-casein to about 95% for kappa-casein, in comparison with their respective mRNAs.

Animals↗
PubMed1986

Molecular cloning and analysis of DNA complementary to three mouse Mr = 68,000 heat shock protein mRNAs.

The construction and isolation of three recombinant DNAs complementary to different mouse L-cell Mr = 68,000 heat shock protein (hsp68) mRNAs is described. cDNA libraries derived from heat-shocked mouse L-cell poly(A)+ RNA by the vector-linked primer strategy of cDNA synthesis and cloning of Okayama and Berg (Okayama, H., and Berg, P. (1982) Mol. Cell. Biol. 2, 161-170) were screened first with a Drosophila hsp70 heterologous probe and subsequently with a cDNA probe isolated from the first screening. Positive clones were assigned to one of three sets based on their restriction map, and the largest member of each group was chosen for further analysis. All three cDNAs hybrid-select mRNA for the mouse major heat shock protein (hsp68) as assayed by in vitro translation and hybridize preferentially to two heat shock-induced hsp68 mRNAs on Northern blots. The coding regions of the cDNAs are almost identical and closely resemble other HSP70 genes but the 3' untranslated regions diverge considerably. Differences in the lengths of the untranslated regions are responsible for the two different sized induced hsp68 mRNAs in mouse L-cells. The physical maps of these cDNA clones and the limited number of mouse genomic DNA fragments detected on Southern blots suggest that there are at least three closely related heat shock-inducible members of the mouse HSP70 gene family. None of the cloned cDNAs are derived from the two related cognate genes known to be present in the mouse genome.

Animals↗
PubMedDate not supplied

[Enzymatic synthesis and characterization of DNA complementary to ceruloplasmin mRNA from rat liver].

Poly(A) containing rat liver 21S RNA homogeneous in polyacrylamide gel electrophoresis under denaturing conditions and stimulating the synthesis of ceruloplasmin in a cell-free proteinsynthesizing system, was used as a template for reverse transcription in the presence of T10 primer and highly purified reverse transcriptase from avian myeloblastosis virus. The cDNA made this way was characterized by means of hybridization kinetics with mRNA, by melting of the hybrids formed and by chain length measurements. To increase the degree of representativity, the ceruloplasmin mRNA was fragmented by mild alkaline treatment, enzymatically polyadenylated and transcribed. The cDNA made was fully characterized and the kinetic complexity measured by hybridization with the mRNA was found to be equal to 2300 nucleotides as compared with the value of 3000 nucleotides is expected from gel electrophoresis data. The observed difference may indicate the presence of repeated sequences in the given mRNA. The sufficient representativitness of the synthesized cDNA and its specificity with respect to ceruloplasmin mRNA allows to use it as a molecular probe to study the ceruloplasmin gene structure.

Animals↗
PubMed1985

Molecular cloning of DNA complementary to rat alpha 2-macroglobulin mRNA.

Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase protein synthesized in the liver. Using an in vitro translation system coupled with solid-phase radioimmunoassay, alpha 2M mRNA activity was found to rise to a maximum level in 16-24 h after turpentine injection. Poly(A)+ RNA from turpentine-injected rat liver was converted to cDNA by the method of Okayama-Berg, and about 50,000 transformants were obtained. From these transformants, clones containing alpha 2M cDNA were selected using the following criteria: 1) alpha 2M cDNA should hybridize with synthetic oligonucleotides encoding portions of the alpha 2M amino acid sequence, 2) alpha 2M cDNA should hybridize preferentially with RNA which increases during inflammation, 3) mRNA which hybridizes with alpha 2M cDNA should encode a polypeptide which specifically reacts with antibody against alpha 2M, and 4) the cDNA should contain the nucleotide sequences encoding the amino acid sequences of alpha 2M. We found clones which fulfilled these criteria. Using the cDNA clone as a probe, we demonstrated that the level of alpha 2M mRNA in the liver of inflamed animal markedly increased up to 1000-fold. The size of the alpha 2M mRNA was about 4800 nucleotides in length by Northern analysis.

Animals↗
PubMed1982

Molecular cloning of DNA complementary to mRNA of the baculovirus Autographa californica nuclear polyhedrosis virus: location and gene products of RNA transcripts found late in infection.

DNAs complementary to late Autographa californica nuclear polyhedrosis virus (AcNPV) mRNA were synthesized by reverse transcription and cloned in Escherichia coli by using pBR322 as a vector. Eleven different cDNAs were distinguished in our screening of 45 AcNPV-homologous clones. Location of the regions of cDNA homology with respect to the AcNPV physical map showed that the 11 cDNAs were dispersed throughout the genome. The most abundant cDNA insertion, representing approximately one-third of the late viral mRNAs, was homologous to the AcNPV HindIII-P,Q and EcoRI-P fragments. The direction of transcription in this region was from left to right on a linearized AcNPV physical map. Hybridization selection followed by in vitro translation showed that this region encoded a 7,200-dalton (7.2K) protein which comigrated with a minor protein found in the extracellular nonoccluded form of the virus (NOV). Similarly, the gene for polyhedrin, the major structural protein of the occluded virus form, was located, at least in part, in the HindIII-V/EcoRI-I region of the AcNPV map. The polyhedrin transcript represented approximately one-quarter of the viral polyadenylic acid-containing RNAs at 27 h postinfection. Another relatively abundant cDNA was homologous to the HindIII-A/EcoRI-C/SstI-G region, and RNA selected by this cDNA directed the synthesis of two proteins (31K and 30K). The protein products of five other cDNA-selected RNAs were identified. The HindIII-D/EcoRI-O, HindIII-C/EcoRI-D, HindIII-B1/EcoRI-E, and HindIII-B2/EcoRI-H regions of the AcNPV L-1 genome were homologous to RNAs which directed the synthesis of a 57K protein, a 25K protein, a 61K protein, and a 37K protein (plus a minor 26K protein), respectively. Late mRNA selected by a cDNA homologous to the HindIII-P/EcoRI-B region of the AcNPV map directed the synthesis of 31K and 30K proteins which comigrated with the 31K and 30K proteins translated from RNA selected by the HindIII-A/EcoRI-C/SstI-G cDNA. Three other cDNAs have not been correlated yet with specific protein products.

Cloning, Molecular↗
PubMed2005

Signaling networks in cutaneous melanoma metastasis identified by complementary DNA microarrays.

BACKGROUND: Melanoma is a complex multigenic disease, susceptibility to which is determined by several parallel and stepwise progressive pathways affecting growth control, differentiation, cell adhesion, and survival. Melanoma and human cancers in general undergo a continuous development from benign to malignant states, as most thoroughly documented in the multistep mole-to-melanoma transition. OBJECTIVE: To examine how high-throughput microarrays are being used in expression profiling to identify regulated genes, patterns, and pathways that may lead to functional characterization and tumor subclassification. DESIGN: Ten melanoma metastases were analyzed by DNA array technology for important regulated candidate genes, with subsequent confirmation by real-time reverse transcription polymerase chain reaction. RESULTS: Hepatocyte growth factor receptor c-met, growth factor receptor-bound protein 10, B-raf proto-oncogene, and several mitogen-activated protein kinase kinase genes were significantly up-regulated in melanoma metastases and several melanoma cell lines relative to normal human melanocytes (P = .03). Among the up-regulated genes, phosphorylated growth factor receptor-bound protein 10 is known to serve a molecular switch turning on the mitogen-activated protein kinase pathway in response to hepatocyte growth factor receptor binding. CONCLUSIONS: As suggested by the DNA arrays, we found the mitogen-activated protein kinase kinase/extracellular-regulated kinase pathway to be activated in most of the cutaneous melanoma metastasis specimens. These findings are in the context of the current microarray technology in melanoma research. Additional steps are needed to gain insights into the pluralistic signaling milieu of this malignancy as we enter the postgenomic era.

Biomarkers, Tumor↗
PubMed1991

Transfection of human CD59 complementary DNA into rat cells confers resistance to human complement.

We have examined the role of the human CD59 antigen in inhibiting complement-mediated lysis by transfer and expression of a CD59 cDNA in rat cells. A cDNA encoding CD59 was subcloned into the expression vector pSFSVneo and stably transfected into the rat T cell line NB2-6TG. Indirect immunofluorescence staining using the anti-CD59 monoclonal antibody YTH53.1 demonstrated the presence of human CD59 antigen on transfected cells and its attachment to the cell surface by a rat glycolipid anchor. Transfected cells were found to contain a single 3.3-kb species of CD59 mRNA by Northern blot hybridization. Immunoblotting revealed that this encoded a protein band of the same size as that observed in human erythrocytes. To determine the biological effect of expression of human CD59 in rat cells, an assay was devised which measured the relative lysis of transfected cells compared to untransfected cells in the presence of human complement and a lytic monoclonal antibody. It was observed that CD59-transfected rat cells are less susceptible to lysis by human complement and that this effect was blocked by a F(ab')2 fragment of YTH53.1. These experiments provide a direct demonstration that CD59 can function as an homologous complement restriction factor for nucleated cells.

Animals↗
PubMed1987

Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA.

Neutral endopeptidase (EC 3.4.24.11) is a major constituent of kidney brush border membranes. It is also present in the brain where it has been shown to be involved in the inactivation of opioid peptides, methionine- and leucine-enkephalins. For this reason this enzyme is often called 'enkephalinase'. In order to characterize the primary structure of the enzyme, oligonucleotide probes were designed from partial amino acid sequences and used to isolate clones from kidney cDNA libraries. Sequencing of the cDNA inserts revealed the complete primary structure of the enzyme. Neutral endopeptidase consists of 750 amino acids. It contains a short N-terminal cytoplasmic domain (27 amino acids), a single membrane-spanning segment (23 amino acids) and an extracellular domain that comprises most of the protein mass. The comparison of the primary structure of neutral endopeptidase with that of thermolysin, a bacterial Zn-metallopeptidase, indicates that most of the amino acid residues involved in Zn coordination and catalytic activity in thermolysin are found within highly honmologous sequences in neutral endopeptidase.

Amino Acid Sequence↗