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11.
Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins. 总被引:63,自引:17,他引:46 下载免费PDF全文
In the eucaryotic nucleus, heterogeneous nuclear RNAs exist in a complex with a specific set of proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs). The C proteins, C1 and C2, are major constituents of hnRNPs and appear to play a role in RNA splicing as suggested by antibody inhibition and immunodepletion experiments. With the use of a previously described partial cDNA clone as a hybridization probe, full-length cDNAs for the human C proteins were isolated. All of the cDNAs isolated hybridized to two poly(A)+ RNAs of 1.9 and 1.4 kilobases (kb). DNA sequencing of a cDNA clone for the 1.9-kb mRNA (pHC12) revealed a single open reading frame of 290 amino acids coding for a protein of 31,931 daltons and two polyadenylation signals, AAUAAA, approximately 400 base pairs apart in the 3' untranslated region of the mRNA. DNA sequencing of a clone corresponding to the 1.4-kb mRNA (pHC5) indicated that the sequence of this mRNA is identical to that of the 1.9-kb mRNA up to the first polyadenylation signal which it uses. Both mRNAs therefore have the same coding capacity and are probably transcribed from a single gene. Translation in vitro of the 1.9-kb mRNA selected by hybridization with a 3'-end subfragment of pHC12 demonstrated that it by itself can direct the synthesis of both C1 and C2. The difference between the C1 and C2 proteins which results in their electrophoretic separation is not known, but most likely one of them is generated from the other posttranslationally. Since several hnRNP proteins appeared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as multiple antigenically related polypeptides, this raises the possibility that some of these other groups of hnRNP proteins are also each produced from a single mRNA. The predicted amino acid sequence of the protein indicates that it is composed of two distinct domains: an amino terminus that contains what we have recently described as a RNP consensus sequence, which is the putative RNA-binding site, and a carboxy terminus that is very negatively charged, contains no aromatic amino acids or prolines, and contains a putative nucleoside triphosphate-binding fold, as well as a phosphorylation site for casein kinase type II. The RNP consensus sequence was also found in the yeast poly(A)-binding protein (PABP), the heterogeneous nuclear RNA-binding proteins A1 and A2, and the pre-rRNA binding protein C23. All of these proteins are also composed of at least two distinct domains: an amino terminus, which possesses one or more RNP consensus sequences, and a carboxy terminus, which is unique to each protein, being very acidic in the C proteins and rich in glycine in A1, and C23 and rich in proline in the poly(A)-binding protein. These findings suggest that the amino terminus of these proteins possesses a highly conserved RNA-binding domain, whereas the carboxy terminus contains a region essential to the unique function and interactions of each of the RNA-binding proteins. 相似文献
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Site-directed mutagenesis to fine-tune enzyme specificity 总被引:1,自引:0,他引:1
We have used a combination of a genetic selection and oligonucleotide-directed mutagenesis to introduce a series of amino acid replacements for a single residue into Escherichia coli glutaminyl-tRNA synthetase. The mutant enzymes mischarge supF tRNA(Tyr), with glutamine, to varying degrees depending on the polarity of the side chain introduced but apparently not depending on the size or shape of the side chain. These results indicate that repulsive charge-charge interactions may be important for specific recognition of nucleic acids by proteins and illustrate how a mutant, derived from genetic selection, may be further modified in activity by oligonucleotide-directed mutagenesis. 相似文献
14.
Two types of antibodies raised against T-2 toxin, namely anti-T-2-HS-BSA and anti-3 -Ac -NEOS-HS -BSA, showed good cross-reactivity with deepoxy T-2 toxin. Our results indicate that the epoxide is not an important epitope for the production of antibody against T-2 toxin 相似文献
15.
F Di Virgilio T H Steinberg J A Swanson S C Silverstein 《Journal of immunology (Baltimore, Md. : 1950)》1988,140(3):915-920
Fura-2, loaded into J774.2 macrophages as the acetoxymethyl ester, is sequestered into intracellular vacuoles within 90 min after the beginning of the loading at 37 degrees C. The dye is also efficiently secreted from the cells. Sequestration and secretion of fura-2 reduce the accuracy of measurements of cytosolic free Ca2+ concentration in this cell line. Fura-2 is also sequestered and secreted by J774.2 when the dye is loaded into the cytoplasm as the pentapotassium salt by reversible permeabilization of the plasma membrane. Regardless of the mechanism by which fura-2 is loaded into the cytoplasm, both sequestration and secretion are prevented by 2.5 mM probenecid, a blocker of organic anion transport. Probenecid has no effect on resting or stimulated cytosolic free Ca2+ levels or on FcR-mediated phagocytosis. These findings suggest that macrophages express a transport mechanism for the anionic form of fura-2. This transport system is responsible for the clearance of fura-2 from the cytoplasm of this cell type. Furthermore we suggest that use of probenecid to block secretion and intracellular sequestration of fura-2 may overcome problems arising in the application of this Ca2+ indicator to macrophages and perhaps to other cell types. 相似文献
16.
Daniel R. Vlock Beth Arnold Jeannette Humpierres Donald R. Schwartz Shan R. Baker Charles J. Krause Neil Swanson Thomas E. Carey 《Cancer immunology, immunotherapy : CII》1992,34(5):329-336
Summary In previous studies we evaluated the incidence and specificity of autologous antibody reactivity against squamous cell carcinoma of the head and neck (SCCHN). We were able to demonstrate that autologous antibody reactivity is present in native sera but was usually of too low a titer to allow further analysis. Dissociation of immune complexes by acidification and ultrafiltration of serum augmented autologous antibody reactivity in nine out of nine autologous systems tested. Native antibody and antibody derived from immune complexes produced by the host and reactive with autologous tumor cells may be directed against physiologically relevant antigens. Therefore, correlations of antibody titers with clinical course may provide insight into the nature of the host response to cancer. In the present analysis, serological studies of six patients with SCCHN were performed with serum samples obtained over many months. Results of serial serological assays were correlated to tumor progression and clinical course. Fluctuations in autologous antibody reactivity were noted over time. In four cases, rises in autologous antibody titers preceded the clinical diagnosis of recurrence by several months. Drops in autologous antibody reactivity were noted in two cases following surgery or radiation therapy. In two cases of long-term survivors, no correlation between antibody reactivity and clinical course was noted. Specificity analysis of the six autologous systems demonstrated reactivity against autologous and allogeneic SCCHN as well as melanoma cell lines. These sera did not react with glioma, neuroblastoma, renal cell, breast, bladder and colon carcinoma cell lines nor with fetal calf serum, pooled lymphocytes, red blood cells and platelets. Autologous serial serological studies may provide a means by which to evaluate the host/tumor relationship in patients with SCCHN. 相似文献
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19.
Human neutrophil response to recombinant neisserial Opa proteins 总被引:13,自引:0,他引:13
Interactions of human neutrophils with recombinant Escherichia coli expressing gonococcal outer membrane Opa proteins were examined using chemiluminescent and biological assays. Seven opa loci from Neisseria gonorrhoeae MS11 4.8 were expressed as beta-lactamase-Opa fusion proteins that contained all but the mature N-terminal amino acid of the full-length Opa protein fused to three N-terminal amino acids derived from the mature beta-lactamase. The Opa fusion proteins were exported and assembled in the outer membrane of E. coli in a manner similar to that of Opa in N. gonorrhoeae, as evaluated by antibody binding and in situ proteolytic cleavage. All fusion proteins exhibited the characteristic heat-modifiable migration in SDS-polyacrylamide gel electrophoresis that typifies Opa proteins of neisseriae. Opa fusion proteins conferred on E. coli the ability to stimulate a chemiluminescent response from human neutrophils in the absence of antibody or complement. The nature of the response in terms of chemiluminescence, phagocytosis, and killing was in all cases analogous to that seen using N. gonorrhoeae expressing the equivalent Opa protein. Neither E. coli nor gonococci expressing OpaA elicited a response from neutrophils. Use of E. coli expressing Opa fusions should be useful in defining their biological activities and pathogenic roles. 相似文献
20.
Identification and characterization of an {alpha}-mannosidase from Trypanosoma cruzi 总被引:1,自引:0,他引:1
Swanson Paul M.; Carter Clint E.; Hager Cindy; Kim Wan Joon; Obermeier Sarah; Oeltmann Thomas N. 《Glycobiology》1992,2(6):563-569
In this report we describe the first purification and characterizationof the acid -mannosidase from the human parasite Trypanosomacruzi. The purified enzyme exhibited a native mol. wt of 240000 Da and is apparently composed of four identical subunitsof mol. wt 58 000 Da. Each of the four subunits contains oneN-linked high-mannose-type oligosaccharide. The -mannosidaseexhibited a pH optimum of 3.5 and a pI of 5.9. This low pH optimumand the ability of swainsonine to inhibit its activity suggestthat the -mannosidase is a lysosomal enzyme. Antibodies againstthe T.cruzi enzyme did not react with mammalian lysosomal -mannosidaseand, conversely, antibody against a rat lysosomal -mannosidasedid not react with the T.cruzi enzyme. Thus, the T.cruzi enzymeappears to be distinct from its mammalian counterpart. -mannosidase lysosomal enzyme Trypanosoma cruzi 相似文献