Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Library preparation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 811 records · Page 45Linked to original sources
PubMed1988

Tissue-specific expression of the rat galanin gene.

We have isolated and characterized cDNAs encoding rat galanin from a cDNA library prepared from rat hypothalamic tissue. Analysis of these clones reveals that rat galanin is synthesized initially as part of a 124-amino acid precursor that includes a signal peptide, galanin (29 amino acids), and a 60-amino acid galanin mRNA-associated peptide. In the precursor, galanin includes a C-terminal glycine and is flanked on each side by dibasic tryptic cleavage sites. The deduced amino acid sequence of rat galanin is 90% similar to porcine galanin, with all three amino acid differences in the C-terminal heptapeptide. The predicted galanin mRNA-associated peptide includes a 35-amino acid sequence that is 78% similar to the previously reported porcine analogue. This sequence is set off by a single basic tryptic cleavage site and includes a 17-amino acid region that is nearly identical to the porcine counterpart. The high interspecies conservation suggests a biological role for this putative peptide. Blot hybridization analysis using rat genomic DNA is consistent with a single galanin-encoding gene. RNA blot analysis of total RNA prepared from rat tissues reveals a single band of hybridizing mRNA that is approximately 900 nucleotides long. Rat galanin mRNA is located predominantly in the central nervous system and gastrointestinal tract. Highest central nervous system concentrations are found in the hypothalamus, with lower levels in the cortex and brainstem. Gastrointestinal rat galanin mRNA is most abundant in the duodenum, with progressively lower concentrations in the stomach, small intestine, and colon.

Amino Acid Sequence↗
PubMed1988

Induction of fibronectin gene transcription and mRNA is a primary response to growth-factor stimulation of AKR-2B cells.

A cDNA library, prepared from poly(A)+ RNA isolated from quiescent AKR-2B cells 4 hr after stimulation with epidermal growth factor in the presence of cycloheximide, was screened to identify RNA transcripts whose abundance is specifically increased as a primary response to growth stimulation. Approximately 40% of the inducible clones detected by this procedure corresponded to either cytoskeletal beta- or gamma-actin genes. One nonactin clone, designated c99, was found to be derived from an 8.5-kilobase RNA whose abundance began to increase as early as 30 min after stimulation. DNA sequencing established the identity of this RNA as fibronectin. Several additional mitogens were then tested and found to efficiently induce fibronectin mRNA. These included fetal calf serum, platelet-derived growth factor, and transforming growth factor type beta. For at least one inducer, fetal calf serum, the increase in mRNA was preceded by an increase in fibronectin gene transcription. This increase was rapid, reaching maximal levels within 10 min, and was accompanied by near-coordinate increases in both c-fos and beta-actin transcription. These results indicate that fibronectin is a member of a class of "early-response" genes, typified by c-fos and including beta-actin, whose rapid expression may be important in mediating cellular responses to peptide growth factors.

Actins↗
PubMed1988

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.

Amino Acid Sequence↗
PubMed1988

The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence.

The primary structure of the beta chain of human glycoprotein Ib (GPIb), the platelet receptor for von Willebrand factor, has been established by a combination of cDNA cloning and amino acid sequence analysis. A lambda phage cDNA expression library prepared from human erythroleukemia cells (HEL cells) was screened with a radiolabeled affinity-purified rabbit polyclonal antibody to the beta chain of GPIb. Eighteen positive clones were isolated and plaque-purified and the nucleotide sequences of three were determined. The composite sequence spanned 968 nucleotides and included a 5' untranslated region of 22 nucleotides, an open reading frame of 618 nucleotides encoding a signal peptide of 28 amino acids and a mature protein of 181 amino acids, a stop codon, and a 3' noncoding region of 307 nucleotides. The 3' noncoding sequence also contained a polyadenylylation signal (AATAAA) 14 nucleotides upstream from the poly(A) tail of 18 nucleotides. Edman degradation of the intact beta chain and of peptides produced by chemical cleavage yielded amino acid sequences spanning 76 residues that were identical to those predicted from the cDNA. The amino-terminal region of the beta chain contains a leucine-rich sequence of 24 amino acids that is similar to a sequence that occurs as seven tandem repeats in the alpha chain of GPIb and nine tandem repeats in leucine-rich alpha 2-glycoprotein. The leucine-rich sequence in the beta chain of GPIb is flanked on both sides by amino acid sequences that are similar to those flanking the leucine-rich tandem repeats of the alpha chain of GPIb and leucine-rich alpha 2-glycoprotein. The amino-terminal region of the beta chain of GPIb is followed by a transmembrane segment of 25 amino acids and an intracellular segment of 34 amino acids at the carboxyl terminus of the protein. The intracellular segment contains an unpaired cysteine and two potential sites for phosphorylation by cAMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1989

Molecular cloning of a cDNA that encodes the peptide core of a mouse mast cell secretory granule proteoglycan and comparison with the analogous rat and human cDNA.

A cDNA that encodes a mouse secretory granule proteoglycan peptide core was isolated from a cDNA library prepared from nontransformed mouse bone marrow-derived mast cells (BMMC) using as a probe a 280-base-pair fragment of a rat cDNA that encodes the proteoglycan peptide core of rat basophilic leukemia (RBL)-1 cells. Based on the consensus nucleotide sequence and deduced amino acid sequence of the cDNA, the mouse BMMC proteoglycan peptide core is 16.7 kDa and contains a 21-amino acid glycosaminoglycan attachment region consisting of alternating serine and glycine residues. When the predicted amino acid sequence of the mouse BMMC proteoglycan peptide core was compared with the predicted amino acid sequences of the homologous molecules expressed in RBL-1 cells and in human promyelocytic leukemia HL-60 cells, the mouse-derived sequence was more closely homologous to the rat sequence than the human sequence except for the length of the serine-glycine repeat region. The N terminus was found to be a highly conserved region of the molecule in the three species, suggesting that this region is important for the structure, function, and/or metabolism of this family of proteoglycans. Nucleotide sequences within the 5' and 3' untranslated regions of the mouse, rat, and human proteoglycan cDNA were conserved. That similar sequences were also present in the corresponding regions of a cDNA that encodes a rat mast cell protease suggests that particular nucleotide sequences may be important for regulation of expression of those proteins that are destined to reside in secretory granules.

Aggrecans↗
PubMed1989

Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor.

A cDNA encoding a human macrophage migration inhibitory factor (MIF) was isolated, through functional expression cloning in COS-1 cells, from a cDNA library prepared from a lectin-stimulated T-cell hybridoma, T-CEMB. The 115-amino acid polypeptide encoded by the MIF cDNA (p7-1) was effectively released from the transfected COS-1 cells and yielded readily detectable MIF activity in the culture supernatant despite the apparent lack of a classical protein secretory sequence. Insertional mutational analysis and elution of MIF activity from polyacrylamide gel slices demonstrated that the Mr 12,000 protein with MIF activity released by the COS-1 cells is encoded by p7-1. The p7-1 cDNA hybridized with a 700-base mRNA expressed by Con-A-stimulated lymphocytes but not unstimulated lymphocytes. The availability of the MIF cDNA clone and recombinant MIF will facilitate the analysis of the role of this lymphokine in cell-mediated immunity, immunoregulation, and inflammation.

Amino Acid Sequence↗
PubMed1989

Cloning and expression of the cDNA for the murine interferon gamma receptor.

A murine interferon gamma (IFN-gamma) receptor cDNA was isolated by screening a murine T-cell hybridoma library prepared in lambda gt10 with probes prepared from a human IFN-gamma receptor cDNA. The 2.1-kilobase (kb) cDNA encoded a serine-rich polypeptide of 477 amino acids that was 52% identical to the human protein. Southern and Northern (RNA) blot analyses indicated the presence of a single receptor gene and a single predominant 2.3-kb receptor transcript. Human embryonic kidney fibroblasts, stably transfected with the murine IFN-gamma receptor cDNA, expressed murine IFN-gamma receptors as detected by flow cytometry with either ligand or a receptor-specific monoclonal antibody. Nontransfected cells bound neither ligand nor antibody. Radioligand-binding analysis demonstrated that the transfectants expressed 530,000 murine IFN-gamma receptors per cell and bound murine IFN-gamma with a Ka of 1 x 10(9) M-1. However, despite high-level expression of murine IFN-gamma receptors, the transfected human cells responded only to human and not to murine IFN-gamma as detected by enhancement of major histocompatibility class I antigen expression and induction of antiviral activity. These results thus document the isolation and expression of a full-length murine IFN-gamma receptor cDNA and suggest that additional species-specific components may be necessary to form a biologically active IFN-gamma receptor.

Amino Acid Sequence↗
PubMed1991

Complementation of snf1, a mutation affecting global regulation of carbon metabolism in yeast, by a plant protein kinase cDNA.

A cDNA, cRKIN1, encoding a putative homologue of the yeast (Saccharomyces cerevisiae) SNF1-encoded protein-serine/threonine kinase, has been isolated from a library prepared from rye endosperm mRNA. Northern blot analysis demonstrated the presence of cRKIN1-related transcripts in developing endosperms but not in shoots, and Southern blot analysis showed the presence of a small gene family. SNF1 plays a central role in carbon catabolite repression in yeast and expression of the RKIN1 sequence in yeast snf1 mutants restored SNF1 function. This suggests that the RKIN1 protein has a role in the control of carbon metabolism in endosperms of rye.

Amino Acid Sequence↗
PubMed1993

Identification of the gene associated with the recurring chromosomal translocations t(3;14)(q27;q32) and t(3;22)(q27;q11) in B-cell lymphomas.

Chromosomal translocations involving chromosome 3, band q27, are among the most common rearrangements in B-cell non-Hodgkin lymphoma. From a bacteriophage lambda library prepared from a lymphoma characterized by a t(3;14)(q27;q32), genomic clones were isolated using a probe from the immunoglobulin heavy chain locus (IGH) joining region. In addition to clones containing an apparently normal IGH rearrangement, others were found to contain one of the translocation breakpoint junctions. Normal chromosome 3 sequences and the reciprocal breakpoint junction were subsequently isolated. DNA probes on each side of the chromosome 3 breakpoint hybridized at high stringency to the DNA of various mammalian species, demonstrating evolutionary conservation. One such probe from the presumptive der(3) chromosome detected an 11-kilobase transcript when hybridized to RNA of B- and T-cell lines. A probe made from partial cDNA clones isolated from a T-cell line hybridized with genomic DNA from both sides of the chromosome 3 breakpoint, indicating that the t(3;14) is associated with a break within the gene on chromosome 3. In situ chromosomal hybridization revealed that the same gene is involved in the t(3;22)(q27;q11). Preliminary nucleotide sequencing shows no identity of the cDNA to gene sequences in available data banks. We propose the name BCL6 (B-cell lymphoma 6) for this gene, since it is likely to play a role in the pathogenesis of certain B-cell lymphomas.

Animals↗
PubMed1993

Molecular cloning and sequencing of a canine tracheobronchial mucin cDNA containing a cysteine-rich domain.

To date the complete sequence of only one mammalian mucin cDNA, MUC1, has been reported, although several mucin proteins have been partially characterized. Here we report the nucleotide sequence of a canine tracheal mucin cDNA containing two potential translation initiation codons, one translation termination codon and a poly(A) tail. A lambda gt11 cDNA library prepared from canine tracheal epithelial cells was screened with polyclonal anti-apo-canine tracheal mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. Antibody-positive clones containing overlapping inserts of various lengths were purified and used for nucleotide sequencing. Based on the sequencing data, synthetic oligonucleotide primers were constructed and both ends (5' and 3') of the cDNA were determined. The complete sequence was 3.7 kb and included an open reading frame with coding capacity for 1118 aa, two translation initiation ATG codons in context with Kozak consensus sequences, one polyadenylylation site, and a poly(A) stretch. The protein was rich in Thr, Pro, Ser, Gly, and Ala and poor in Tyr, Phe, and Trp. Although tandem repeats of amino acids were absent in the deduced canine tracheal mucin sequence, motifs TPTPTP and TTTTPV appeared 13 and 19 times, respectively. The C-terminal region contained a Cys-rich domain (although a few Cys residues were also present in the middle of the protein) as has been reported for bovine submaxillary mucin, porcine submaxillary mucin, rat intestinal mucin, human intestinal mucin, and frog skin mucin. This suggested that a broad group of mucins contain such a Cys-rich domain whose functional significance is yet to be understood. Three potential N-glycosylation sites were present in canine tracheal mucin and the amino acid sequence showed homology with both human tracheal and intestinal mucins. The N-terminal domain showed more flexibility (probably due to a high number of Pro residues in this region) when analyzed by the University of Wisconsin Genetics Computer Group program package to determine the predicted secondary structure. Evaluation of the transcripts using the canine mucin cDNA as a probe indicated a polydisperse message with total RNA.

Amino Acid Sequence↗
PubMed1994

Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection.

The cultured T-cell line TIL1200, established from the tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an antigen on most HLA-A2+ melanomas and on all HLA-A2+ cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an antigen recognized by TIL1200 was isolated by screening an HLA-A2+ breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2+ melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pmel17 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types of tumor tested. The cell line transfected with this cDNA also expressed antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible tumor rejection antigen and may be useful for the development of immunotherapies for patients with melanoma.

Adult↗
PubMed1995

Rat skeletal muscle selenoprotein W: cDNA clone and mRNA modulation by dietary selenium.

Rat skeletal muscle selenoprotein W cDNA was isolated and sequenced. The isolation strategy involved design of degenerate PCR primers from reverse translation of a partial peptide sequence. A reverse transcription-coupled PCR product from rat muscle mRNA was used to screen a muscle cDNA library prepared from selenium-supplemented rats. The cDNA sequence confirmed the known protein primary sequence, including a selenocysteine residue encoded by TGA, and identified residues needed to complete the protein sequence. RNA folding algorithms predict a stem-loop structure in the 3' untranslated region of the selenoprotein W mRNA that resembles selenocysteine insertion sequence (SE-CIS) elements identified in other selenocysteine coding cDNAs. Dietary regulation of selenoprotein W mRNA was examined in rat muscle. Dietary selenium at 0.1 ppm as selenite increased muscle mRNA 4-fold relative to a selenium-deficient diet. Higher dietary selenium produced no further increase in mRNA levels.

Amino Acid Sequence↗
PubMed1999

Biosynthetic origin of conjugated double bonds: production of fatty acid components of high-value drying oils in transgenic soybean embryos.

Vegetable oils that contain fatty acids with conjugated double bonds, such as tung oil, are valuable drying agents in paints, varnishes, and inks. Although several reaction mechanisms have been proposed, little is known of the biosynthetic origin of conjugated double bonds in plant fatty acids. An expressed sequence tag (EST) approach was undertaken to characterize the enzymatic basis for the formation of the conjugated double bonds of alpha-eleostearic (18:3Delta(9cis, 11trans,13trans)) and alpha-parinaric (18:4Delta(9cis,11trans, 13trans,15cis)) acids. Approximately 3,000 ESTs were generated from cDNA libraries prepared from developing seeds of Momordica charantia and Impatiens balsamina, tissues that accumulate large amounts of alpha-eleostearic and alpha-parinaric acids, respectively. From ESTs of both species, a class of cDNAs encoding a diverged form of the Delta(12)-oleic acid desaturase was identified. Expression of full-length cDNAs for the Momordica (MomoFadX) and Impatiens (ImpFadX) enzymes in somatic soybean embryos resulted in the accumulation of alpha-eleostearic and alpha-parinaric acids, neither of which is present in untransformed soybean embryos. alpha-Eleostearic and alpha-parinaric acids together accounted for as much as 17% (wt/wt) of the total fatty acids of embryos expressing MomoFadX. These results demonstrate the ability to produce fatty acid components of high-value drying oils in transgenic plants. These findings also demonstrate a previously uncharacterized activity for Delta(12)-oleic acid desaturase-type enzymes that we have termed "conjugase."

Amino Acid Sequence↗
PubMed1995

Identification and characterization of a novel cytokine-inducible nuclear protein from human endothelial cells.

Vascular endothelial cells undergo profound changes upon cellular activation including expression of a spectrum of cell activation-associated genes. These changes play important roles in many physiological and pathological events. By differential screening of a cDNA library prepared from interleukin-1 alpha and tumor necrosis factor-alpha-stimulated human dermal microvascular endothelial cells, we have identified a novel cytokine-inducible gene, designated as C-193. The compiled cDNA sequence of C-193 is 1901 base pairs long and shows no significant homology with any known gene sequence. Genomic DNA analysis revealed that C-193 is encoded by a single gene, which is conserved in different mammalian species. The C-193 gene was localized to human chromosome 10 by Southern blot analysis of somatic cell hybrids. Multiple AT-rich mRNA decay elements were identified in the 3'-untranslated region. C-193 mRNA expression was rapidly and transiently induced by treatment with interleukin-1 alpha or tumor necrosis factor-alpha, reached a peak of expression about 16 h post tumor necrosis factor-alpha stimulation, and the induction of C-193 was protein synthesis independent. Lipopolysaccharide and cycloheximide were also potent inducers of C-193 mRNA. Therefore, C-193 represents a new addition to the primary response gene family. In vitro translation of C-193 yielded a 36-kDa protein product, consistent with the predicted open reading frame of 318 amino acids and a calculated molecular mass of 36 kDa for C-193 protein. The predicted protein sequence contains a basic amino acid cluster similar to a nuclear localization signal, four tandem repeats of ankyrin-like sequence, and multiple consensus protein phosphorylation sites. C-193 was engineered with a FLAG tag at its carboxyl terminus and transiently expressed in COS cells. Consistent with the presence of a putative nuclear localization signal, the C-193-FLAG protein was localized to the nucleus of transfected COS cells by indirect immunofluorescence microscopy. C-193-FLAG prepared in vitro was capable of binding DNA cellulose. These results indicate that C-193 protein may play an important role in endothelial cell activation.

Amino Acid Sequence↗
PubMed1995

A novel plasminogen activator from snake venom. Purification, characterization, and molecular cloning.

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native Glu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg561-Val562. Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-glycosylation site at Asn161. The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Agkistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Amides↗
PubMed1995

Molecular cloning of a new interferon-induced factor that represses human immunodeficiency virus type 1 long terminal repeat expression.

Transcriptional induction of genes is an essential part of the cellular response to interferons. To isolate yet unidentified IFN-regulated genes we have performed a differential screening on a cDNA library prepared from human lymphoblastoid Daudi cells treated for 16 h with human alpha/beta interferon (Hu-alpha/beta IFN). In the course of these studies we have isolated a human cDNA which codes for a protein sharing homology with the mouse Rpt-1 gene; it will be referred as Staf-50 for Stimulated Trans-Acting Factor of 50 kDa. Amino acid sequence analysis revealed that Staf-50 is a member of the Ring finger family and contains all the features of a transcriptional regulator able to initiate a second cascade of gene induction (secondary response). Staf-50 is induced by both type I and type II IFN in various cell lines and down-regulates the transcription directed by the long terminal repeat promoter region of human immunodeficiency virus type 1 in transfected cells. These data are consistent with a role of Staf-50 in the mechanism of transduction of the IFN antiviral action.

Amino Acid Sequence↗
PubMed1995

Isolation of human cDNAs that restore methotrexate sensitivity and reduced folate carrier activity in methotrexate transport-defective Chinese hamster ovary cells.

This report describes the isolation, nucleotide sequencing, and functional expression of human cDNAs that restore reduced folate carrier activity in transport-defective cells. Based on homology to a partial murine cDNA probe, two functional cDNAs were isolated from a lambda gt11 library prepared from methotrexate transport upregulated K562 cells (K562.4CF). A 2.8-kilobase (kb) clone, KS43, contained a 1776-base pair open reading frame. The 2.5-kb clone, KS32, contained an internal deletion (626 base pairs) resulting a shortened open reading frame and 3'-untranslated region. KS43 and KS32 encoded proteins with multiple hydrophobic domains, one consensus N-glycosylation site, and predicted molecular masses of 65 and 58 kDa, respectively. The deduced amino acid sequence of KS43 is 79% and 80% homologous to the mouse and hamster sequences, respectively (Dixon, K. H., Lanpher, B. C., Chiu, J., Kelley, K., and Cowan, K. H. (1994) J. Biol. Chem. 269, 17-20; Williams, F. M. R., Murray R. C., Underhill, T. M., and Flintoff, W. F. (1994) J. Biol. Chem. 269, 5810-5816). Northern blots identified one primary transcript at 3.1 kb in parental K562, K562.4CF, and transport-impaired K500E cells; transcript levels varied by 7-fold. The expression of both KS43 and KS32 in methotrexate transport-defective Chinese hamster ovary cells restored methotrexate sensitivity and transport. Certain transport characteristics of the transfected cells resembled both the wild type human (K562) and hamster "classical" reduced folate carriers, suggesting the expression of a hybrid system. For instance, based on Ki values, up to a 4-fold increased affinity for 1843U89 over wild type hamster cells (typical of human cells), and a 19-fold increased affinity for methotrexate over K562 cells (typical of hamster cells) was observed. Further, a photoaffinity probe with high specificity for the reduced folate carrier labeled 94-kDa proteins in K562 cells and the transfectant containing the full-length KS43, and a 85-kDa protein in the transfectant containing the 3'-truncated KS32. No specifically labeled proteins were detected in wild type or mock-transfected hamster cells. Collectively, our results suggest that the KS43/KS32 cDNAs encode the human reduced folate carrier; however, additional modulatory/regulatory factors may be required to manifest the full spectrum of transport substrate activities typical of this system.

Amino Acid Sequence↗
PubMed1995

Transcription factors Stat3 and Stat5b are present in rat liver nuclei late in an acute phase response and bind interleukin-6 response elements.

Proteins binding at the interleukin-6 response element of the rat alpha 2 macroglobulin gene were purified by a combination of chromatographic procedures including binding site-specific DNA-affinity chromatography as the principal step. Three polypeptides of 92, 91, and 86 kDa were enriched approximately 6,300-fold from nuclei of rat livers excised 12 h after the induction of an experimental acute phase response. Amino acid sequence analysis identified the 86- and 91-kDa species as two forms of the transcription factor Stat3 and the 92-kDa species as the factor Stat5b. This identification was confirmed by gel mobility shift-supershift experiments using specific antisera for Stat3 and Stat5. Unexpectedly, activated Stat5 was also detected in the nuclei of untreated control rats. cDNA clones representing Stat3 and two isoforms of Stat5b were isolated from a cDNA library prepared with mRNA from rat livers excised at the peak of an experimental acute phase response. Full-length Stat5b, predicted from cDNA, consisted of 786 amino acids, while the variant Stat5b delta 40C lacked 41 amino acids at the COOH terminus. The amino acid sequence of rat Stat5b showed 26.7% overall identity with rat Stat3, 87.3% with sheep Stat5a, 92.5% with murine Stat5a, and 98.7% with murine Stat5b.

Acute-Phase Proteins↗