Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Of the six complementarity-determining regions (CDR) forming the structure of the Ab combining site, CDR3 of heavy chain is the most variable in length and sequence. Diversity of this loop is determined by the number of gene segments involved, extent of addition to or deletion from the joining genes, and imprecision of the site of recombination. In neonatal mice and Xenopus tadpoles, the last two factors occur less frequently than in adults, which in tadpoles result in low affinity Ab responses that do not mature. In contrast, adult urodele amphibians make larval-like responses and are notorious for lifelong poor immunocompetence. The mechanism for this is not known, and in this study we cloned germline VH genes from the axolotl and obtained rearrangements to these VH gene segments by reverse-transcriptase PCR. These sequences were analyzed for heavy chain junctional diversity and found to be even less variable than that in newborn mouse or Xenopus tadpoles, although for different reasons. Only 29% of the CDR3 loop in the axolotl consisted of somatically generated sequences, compared with 44% in tadpole, 39% in newborn mice, and 57% in both adult mice and Xenopus. This distinguishing feature of axolotl CDR3 results not only from shorter junctional sequences, but also unusually extensive integration of germline JH sequence. As the CDR3 loop is the most important portion of the Ig sequence for determining Ab combining site diversity, our data provide the molecular basis for a contributing factor in the deficient urodele amphibian Ab responses.
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PMID:Abbreviated junctional sequences impoverish antibody diversity in urodele amphibians. 931 38

The rates of transcription of several protein coding genes during Acanthamoeba differentiation have been examined by nuclear run-on and RNase protection assays. During early encystment, transcription by RNA polymerase II increases approximately 4-fold, whereas transcription by RNA polymerases I and III is decreased, as previously described. The rates of transcription from a wide variety of individual genes are only slightly affected during the first 16 h of encystment, although profilin gene expression is markedly increased. The levels of mRNAs encoding TPBF, TATA binding protein, cyclin-dependent kinase, protein disulfide isomerase, profilin, myosin II heavy chain, ubiquitin and extendin are stable during mature cyst formation, whereas mRNAs encoding actin, S-adenosyl methionine synthase and tubulin are substantially decreased in abundance within 16 h of starvation-induced encystment. We conclude that in contrast to the negative regulation of large rRNA and 5S rRNA synthesis during differentiation, the RNA polymerase II transcription apparatus is not negatively regulated. Control of Acanthamoeba differentiation is likely to be mediated by positive regulation of genes necessary for cyst maturation.
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PMID:Transcription by RNA polymerase II during Acanthamoeba differentiation. 987 98

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) functions at multiple stages of transcription and is involved in the coupling of transcription to pre-mRNA processing. We have used site-specific protein-DNA photocross-linking to determine the position of the CTD along promoter DNA in the transcriptional pre-initiation complex. Comparison of the promoter contacts made by RNAP II with or without the CTD indicate that the CTD approaches promoter DNA downstream of the transcriptional initiation site between positions +16 and +26. Incubation of pre-assembled initiation complexes with antibodies to the CTD prior to UV irradiation led to specific photocross-linking of the IgG heavy chain to nucleotide +17, indicating that the CTD is accessible for protein-protein interactions in a complex containing RNAP II and the general initiation factors. In conjunction with previously published observations, our structural data are fully compatible with the notion that DNA wrapping around RNAP II places the CTD and the RNA exit channel into juxtaposition and provide a rationale for contacts between the SRB-mediator complex and core RNAP II observed in the RNAP II holoenzyme.
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PMID:Topological localization of the carboxyl-terminal domain of RNA polymerase II in the initiation complex. 1039 32

A bacterially expressed single chain antibody (scFv215) directed against the largest subunit of drosophila RNA polymerase II was analysed. Structure and function of the antigen binding site in scFv215 were probed by chain shuffling and by site-specific mutagenesis. The entire variable region of either the heavy or light chain was replaced by an unrelated heavy or light chain. Both replacements resulted in a total loss of binding activity suggesting that the antigen binding site is contributed by both chains. The functional contributions of each complementarity determining region (CDR) were investigated by site specific mutagenesis of each CDR separately. Mutations in two of the CDRs, CDR1 of light chain and CDR2 of heavy chain, reduced the binding activity significantly. Each of the amino acids in these two CDRs was replaced individually by alanine (alanine walking). Seven amino acid substitutions in the two CDRs were found to reduce the binding activity by more than 50%. The data support a computer model of scFv215 which fits an epitope model based on a mutational analysis of the epitope suggesting an alpha-helical structure for the main contact area.
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PMID:Fine mapping of the antigen-antibody interaction of scFv215, a recombinant antibody inhibiting RNA polymerase II from Drosophila melanogaster. 1039 1

The monoclonal antibody Jel42 is specific for the Escherichia coli histidine-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. The binding domain (Fv) has been produced as a single chain Fv (scFv). The scFv gene was synthesized in vitro and coded for pelB leader peptide-heavy chain-linker-light chain-(His)(5) tail. The linker is three repeats from the C-terminal repetitive sequence of eukaryotic RNA polymerase II. This linker acts as a tag; it is the antigen for the monoclonal antibody Jel352. The codon usage was maximized for E.coli expression, and many unique restriction endonuclease sites were incorporated. The scFv gene incorporated into pT7-7 was highly expressed, yielding 10-30% of the cell protein as the scFv, which was found in inclusion bodies with the leader peptide cleaved. Jel42 scFv was purified by denaturation/renaturation yielding preparations with K(d) values from 20 to 175 nM. However, based upon an assessment of the amount of active refolded scFv, the binding dissociation constant was estimated to be 2.7 +/- 2.0 nM compared with 2.8 +/- 1.6 and 3.7 +/- 0.3 nM previously determined for the Jel42 antibody and Fab fragment respectively. The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment. It was concluded that the small percentage ( approximately 6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv cannot be detected in the binding assay.
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PMID:Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs. 1043 89

We describe the establishment and characterization of a new multiple myeloma (MM) cell line, KYdelta-1, which expressed delta/kappa type immunoglobulin (Ig). The patient was a 65-year-old woman with MM, who presented extramedullary dissemination, lymphadenopathy and short survival. The KYdelta-1 cell line was derived from the pleural fluid obtained in the terminal phase of the disease. The cells expressed delta/kappa Ig in the cytoplasm, and CD10, CD29, CD33, CD38, CD44, CD54, and HLA-DR antigens on the cell surface. Chromosomal analysis revealed two independent translocations, t(3;14)(p21;q32) and t(3;11)(p21;q13), which were confirmed by fluorescence in situ hybridization using chromosome painting probes. Reverse transcriptase-mediated polymerase chain reaction (PCR) and Northern blot analyses demonstrated overexpression of the CCND1 gene, suggesting alteration of the BCL1-CCND1 locus. We thus performed long-distance inverse PCR using nested primers for the Calpha constant region of immunoglobulin heavy chain gene (IGH) and obtained a clone that encompassed the 11q13/IGH fusion. Nucleotide sequencing determined that the fusion occurred at the Salpha2 switch region and at the centromeric side of the major translocation cluster of BCL1. The other IGH allele consisted of a VDJ complex that was adjacent to the Cdelta constant gene, indicating that a class switch-like mechanism from the C(mu) to Cdelta was involved in the production of the Ig delta heavy chain. Point mutations within the P53 and N-RAS genes were presumably related to the rapidly progressive disease in this particular MM patient.
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PMID:Establishment and characterization of a new human myeloma cell line, KYdelta-1, producing the delta/kappa type immunoglobulin. 1167 73

Biological polyadenylation, first recognized as an enzymatic activity, remained an orphan enzyme until poly A sequences were found on the 3' ends of eukarvotic mRNAs. Their presence in bacteria viruses and later in archeae (ref. 338) established their universality. The lack of compelling evidence for a specific function limited attention to their cellular formation. Eventually the newer techniques of molecular biology and development of accurate nuclear processing extracts showed 3' end formation to be a two-step process. Pre-mRNA was first cleaved endonucleolytically at a specific site that was followed by sequential addition of AMPs from ATP to the 3' hydroxyl group at the end of mRNA. The site of cleavage was specified by a conserved hexanucleotide, AAUAAA, from 10 to 30 nt upstream of this 3' end. Extensive purification of these two activities showed that more than 10 polypeptides were needed for mRNA 3' end formation. Most of these were in complexes involved in the cleavage step. Two of the best characterized are CstF and CPSF, while two other remain partially purified but essential. Oddly, the specific proteins involved in phosphodiester bond hydrolysis have yet to be identified. The polyadenylation step occurs within the complex of poly A polymerase and poly A-binding protein, PABII, that controls poly A length. That the cleavage complex, CPSF, is also required for this step attests to a tight coupling of the two steps of 3' and formation. The reaction reconstituted from these RNA-free purified factors correctly processes pre-mRNAs. Meaningful analysis of the role of poly A in mRNA metabolism or function was possible once quantities of these proteins most often over-expressed from cDNA clones became available. The large number needed for two simple reactions of an endonuclease, a polymerase and a sequence recognition factor, pointed to 3' end formation as a regulated process. Polyadenylation itself had appeared to require regulation in cases where two poly A sites were alternatively processed to produce mRNA coding for two different proteins. The 64-KDa subunit of CstF is now known to be a regulator of poly A site choice between two sites in the immunoglobulin heavy chain of B cells. In resting cells the site used favors the mRNA for a membrane-bound protein. Upon differentiation to plasma cells, an upstream site is used the produce a secreted form of the heavy chain. Poly A site choice in the calcitonin pre-mRNA involves splicing factors at a pseudo splice site in an intron downstream of the active poly site that interacts with cleavage factors for most tissues. The molecular basis for choice of the alternate site in neuronal tissue is unknown. Proteins needed for mRNA 3' end formation also participate in other RNA-processing reactions: cleavage factors bind to the C-terminal domain of RNA polymerase during transcription; splicing of 3' terminal exons is stimulated port of by cleavage factors that bind to splicing factors at 3' splice sites. nuclear ex mRNAs is linked to cleavage factors and requires the poly A II-binding protein. Most striking is the long-sought evidence for a role for poly A in translation in yeast where it provides the surface on which the poly A-binding protein assembles the factors needed for the initiation of translation. This adaptability of eukaryotic cells to use a sequence of low information content extends to bacteria where poly A serves as a site for assembly of an mRNA degradation complex in E. coli. Vaccinia virus creates mRNA poly A tails by a streamlined mechanism independent of cleavage that requires only two proteins that recognize unique poly A signals. Thus, in spite of 40 years of study of poly A sequences, this growing multiplicity of uses and even mechanisms of formation seem destined to continue.
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PMID:A history of poly A sequences: from formation to factors to function. 1210 57

We determined the molecular sequence of monoclonal antibodies (mAbs) to serogroups B and C capsular polysaccharides (PS) of Neisseria meningitidis. N. meningitidis infections are a leading cause of bacterial septicemia and meningitis in humans. Antibodies to PS are fundamental to host defense and diagnostics. The polysaccharide capsule of group B N. meningitidis is poorly immunogenic and thus is an important model for studying pathogen-host co-evolution through understanding the molecular basis of the host immune response. We used a modified reverse-transcriptase PCR to amplify and sequence the V-genes of murine hybridomas produced against types B and C capsular PS. Databank analysis of the sequences encoding the V-genes of type C capsular PS mAb, 4-2-C, reveal that heavy chain alleles are recurrently used to encode this specificity in mice. Interestingly, a V-gene from the same germline family also encodes the V-domain of mAbs 2-2-B, which targets the antigenically distinct serogroup B capsular PS. Somatic mutation, junctional diversity and alternative light chains collectively impart the specificity for these serologically distinct epitopes. Knowledge of the specific immunoglobulin genes used to target common bacterial virulence factors may lead to insights on pathogen-host co-evolution, and the potential use of this information in pre-symptomatic diagnosis is discussed.
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PMID:Molecular analysis of monoclonal antibodies to group variant capsular polysaccharide of Neisseria meningitidis: recurrent heavy chains and alternative light chain partners. 1558 22

We describe the cytogenetic, fluorescence in situ hybridization (FISH), and molecular findings in a patient who developed a typical chronic lymphocytic leukemia (CLL) 20 months after the diagnosis of a Philadelphia (Ph)-positive chronic myeloid leukemia. Unstimulated bone marrow culture showed a 46,XX,t(9;22)(q34;q11) karyotype, and interphase FISH detected the presence of a BCR/ABL fusion signal in 13% of cells. On stimulated bone marrow culture, a normal karyotype and a 13q14 deletion by interphase FISH with D13S319 probe in 14% of the cells were found. Molecular studies detected the chimeric BCR/ABL messengers by nested reverse-transcriptase polymerase chain reaction. The B-cellular clone was documented by the presence of a clonal heavy chain immunoglobulin rearrangement. The coexistence of these two hematologic malignancies leads to questions about their cell(s) of origin. We provide evidence that CLL arose in a Ph-negative clone. The implications of these findings are discussed.
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PMID:Chronic lymphocytic leukemia developing in a patient with chronic myeloid leukemia: evidence of distinct lineage-associated genomic events. 1608 Sep 61

A previously described polyol-responsive monoclonal antibody (PR-mAb) was converted to a single-chain variable fragment (scFv). This antibody, PR-mAb NT73, reacts with the beta' subunit of Escherichia coli RNA polymerase and has been used for the immunoaffinity purification of polymerase. mRNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were used as the template for cDNA synthesis. The sequences were joined by the addition of a "linker" sequence and then cloned into several expression vectors. A variety of expression plasmids and E. coli hosts were used to determine the optimal expression system. Expression was highest with the pET22b(+) vector and the Rosetta(DE3)pLysS host strain, which produced approximately 60 mg purified His-tagged scFv per liter of culture (3.3 g wet weight cells). Although the production of soluble scFv was preferred, overproduced scFv formed inclusion bodies under every expression condition. Therefore, inclusion bodies had to be isolated, washed, solubilized, and refolded. The FoldIt protein refolding kit and enzyme-linked immunosorbent assay were sequentially used to determine the optimal refolding conditions that would produce active His-tagged scFv. Immobilized metal affinity chromatography was used for the final purification of the refolded active scFv. The polyol-responsiveness of the scFv was determined by an ELISA-elution assay. Although the scFv loses considerable affinity for its antigen, it maintains similar polyol-responsiveness as the parent monoclonal antibody, PR-mAb NT73.
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PMID:Expression and purification of a single-chain variable fragment antibody derived from a polyol-responsive monoclonal antibody. 1621 25


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