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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Immunoglobulin heavy chain mRNA was purified from immunoprecipitated polysomes derived from the mouse myeloma tumor, MOPC-31C. The purified mRNA migrated predominantly as a single band upon polyacrylamide gel electrophoresis in 98% formamide and the molecular weight of this mRNA was calculated to be 700,000. This mRNA was as active as the purified light chain mRNA when it was employed as a template in a cell-free protein synthesizing system from wheat germ. The translation product had a molecular weight of 55,000 daltons, and migrated slightly faster than mature heavy chain upon polyacrylamide gel electrophoresis in sodium dodecylsulfate. The protein synthesized by the direction of this mRNA was shown to yield tryptic peptides corresponding to those derived from the mature heavy chain protein except that one missing peptide was replaced by another additional peptide. DNA complementary to the mRNA was synthesized by RNA-dependent DNA polymerase from avian myeloblastosis virus. Hybridization kinetic analysis between the heavy chain mRNA and its complementary DNA indicated that the RNA was essentially homogenous with rabbit globin mRNA as a standard.
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PMID:Purification of immunoglobulin heavy chain messenger RNA by immunoprecipitation from the mouse myeloma tumor, MOPC-31C. 40 24

We present evidence for RNA transcripts encoding two forms of human epsilon immunoglobulin (Ig) heavy chain that differ significantly from those of other isotypes. We previously demonstrated three human epsilon mRNA species, instead of the two, corresponding to membrane and secreted proteins, seen with other heavy chain transcripts. In human genomic DNA downstream of the C epsilon gene, we identified sequences homologous to the two putative murine exons M1 (encoding a hydrophobic, presumably transmembrane region) and M2 (encoding hydrophilic residues). To determine the structures of epsilon transcripts containing these sequences, we amplified epsilon-related RNAs with the reverse transcriptase polymerase chain reaction. RNA was examined from fresh human B cells stimulated to IgE production by interleukin 4 plus anti-CD40, as well as from the human IgE-producing line AF10. Instead of the single CH4-M1-M2 splice product predicted for murine membrane IgE, we found two other RNA species. One form has the structure CH4-M1'-M2, in which M1' includes the human sequence homologous to the murine M1 as well as a unique segment of 52 codons further upstream in the genomic sequence; this RNA species apparently encodes the IgE expressed on the membrane of IgE-producing lymphocytes. The other RNA has the structure CH4-M2', in which M2' is spliced in an alternative reading frame that includes an additional 109 codons downstream of the termination codon of the CH4-M1'-M2 form. Because the CH4-M2' mRNA form does not encode a hydrophobic segment, its translated product should be secreted. A secreted epsilon protein of approximately the size predicted for this form was identified by Western blotting. This novel IgE protein could play a significant and distinctive role in allergic disorders.
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PMID:Two unusual forms of human immunoglobulin E encoded by alternative RNA splicing of epsilon heavy chain membrane exons. 161 58

The platelet glycoprotein (GP) IIb-IIIa complex figures prominently as an immunogen in autoimmune (idiopathic) thrombocytopenic purpura (ITP). 2E7 is a human monoclonal IgM autoantibody, derived from splenocytes of a patient with ITP, that recognizes a specific octapeptide amino acid sequence, Phe-Asp-Gly-Tyr-Trp-Gly-Tyr-Ser, on the heavy chain of GPIIb. This represents the first precise identification of an epitope on GPIIb-IIIa recognized by a human antibody. In this study, we have isolated total mRNA from 2E7, synthesized the corresponding cDNA using reverse transcriptase, and amplified the immunoglobulin mu and kappa chain cDNA by the Taq 1 polymerase chain reaction (PCR) using specific primers. The 2E7 mu chain variable region is encoded by a VH3 gene segment that is 98% homologous to the germline gene VH1.9III, a D-gene that is not homologous to any of the germline D-genes reported to date, and a JH6 gene segment that is essentially germline. The heavy-chain sequence, save for the unique D-gene, is similar to that of a number of human autoantibodies. The 2E7 kappa variable region is encoded by a Vk1 gene segment linked to a Jk1 gene segment. The Vk1 sequence of 2E7, with the exception of one nucleotide, is identical to that of autoantibody HF2-1/17, a prototype of SLE-associated anti-DNA autoantibodies bearing the 16/6 idiotype. The single base substitution results in a relatively conservative exchange of Asp for Glu at position 70 of the protein sequence. Despite this near identity in sequence, 2E7 does not bind to either single-stranded or double-stranded DNA. From these results, we conclude that specificity of 2E7 is likely to reside in either or both the D-JH region (CDR3) of the mu chain and the Jk region (CDR3) of the kappa chain. In addition to the identification of a novel D-gene, we also provide evidence that the 2E7 VHIII gene is probably a prototype of a VHIII subfamily, that the germline Vk1 gene shared by 2E7 and autoantibodies of the 16/6 idiotype probably represents a separate Vk family, and that this Vk gene cannot itself attribute specificity for DNA.
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PMID:Nucleotide sequence of the human autoantibody 2E7 specific for the platelet integrin IIb heavy chain. 171 98

Total cellular Poly A+ RNA from TEPC15 myeloma and murine lymphoid tissues was analyzed by denaturing agarose gel electrophoresis and Northern blot hybridization to specific heavy chain constant region cDNAs; mu, gamma 2b and alpha RNA species were identified in each of the tissues and in the IgA producing TEPC15 myeloma. In total Poly A+ RNA from TEPC15 myeloma, alpha-cDNA hybridized predominantly to a 2.3 kb RNA species; 11.5 and 4.1 kb RNA species were evident as well. Successive hybridization of the same RNA to mu- and gamma 2b-specific cDNAs demonstrated the presence of both mu and gamma 2b specific RNA species with electrophoretic mobilities apparently identical to the 11.5 and 2.3 kb RNA species identified by alpha-specific hybridization. These data establish the presence of Poly A+ RNA species containing alpha, mu, and gamma 2b sequences in TEPC15 cells and suggest the transcription of RNA from both CH-containing chromosomes in TEPC15 myeloma (one chromosome in the TEPC15 cell line contains all the CH genes while the other chromosome has deleted all CH genes except alpha). In total Poly A+ RNA from normal mouse tissues (Peyer's patch and spleen) all three cDNA probes hybridized predominantly to an 11.5 kb RNA species. Primer extension experiments demonstrated that alpha cDNA could prime for the synthesis by reverse transcriptase of gamma 2b DNA when Peyer's patch Poly A+ RNA was used as the template. This suggests the existence of a single transcript containing alpha and gamma 2b sequences. Murine lymphoid tissues contained putative mRNAs for mu, gamma 2b and alpha heavy chain proteins, whereas TEPC15 myeloma polysomal Poly A+ RNA contained only alpha mRNA.
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PMID:Identification of the major immunoglobulin heavy chain poly A+ RNA in murine lymphoid tissue. 242 42

The complete sequence of a cDNA clone (pmu3) encoding a secreted IgM heavy chain from a rabbit of VHa2 allotype has been determined. Comparison of the nucleic acid and amino acid sequences with a comparable mouse mu-chain shows unusually high homologies of the sequences of the fourth domains and secretory portions of the molecules (80 to 90% vs 56 to 73% for the other domains). This high degree of homology is also seen with the sequences of the fourth domain and secreted terminus of human mu-chain. The DNA sequence of a second cDNA clone encoding the membrane form of rabbit IgM is approximately 90% homologous with the mouse and human coding sequences for the membrane terminus, and there is 98 and 97% amino acid sequence homology. We found an unusually long (156 base pair) 5' untranslated region in clone pmu3 and discovered that 105 bases at the 5' end were complementary to the terminal portion of the CH4 domain of our mu sequence. Southern blotting analyses and the finding of a 13 base pair segment in rabbit genomic V region DNA that is homologous to the complementary strand of a segment of the CH4 of rabbit mu strongly suggests that this extended sequence resulted from a reverse transcriptase "hooking error". We have also obtained and sequenced another cDNA clone from a VHa1 cDNA library. Comparison of the V region of clone pmu3 from VHa2 with the 117 available positions of a1 DNA sequence shows 25 differences, nine of which are in allotype-associated codons. There is also a two-codon deletion in the VHa1 compared to VHa2 in the third framework region that may contribute to allotypic differences. Development of VHa allotype-specific probes, now in progress, is complicated by the fact that most of the allotype-associated amino acids of a1, a2, and a3 can be derived from each other by single base changes.
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PMID:Complete sequence of a cloned cDNA encoding rabbit secreted mu-chain of VHa2 allotype: comparisons with VHa1 and membrane mu sequences. 641 3

The human immunoglobulin (Ig) heavy chain VH6 gene promoter contains an imperfect octamer (AgGCAAAT) and is not dependent on the Ig heavy chain enhancer for activity; reporter constructs containing this promoter are very active in non-B cells. In experiments designed to characterize regions upstream of the transcriptional start site that are important for promoter function, we produced a series of deletion constructs, including one containing sequences between -74 and -146. Surprisingly, this fragment had promoter activity in both orientations. Inspection of the VH6 promoter sequence indicated that there was a possible TATA box in the proper orientation upstream of the imperfect octamer. The -74 to -146 fragment functioned as a promoter in the reverse orientation in three B cell lines and in non-B (HeLa) cells, with a much higher level of activity seen in the HeLa cells. To determine if the promoter could work in both directions simultaneously, reporter genes were positioned up- and downstream of a VH6 promoter fragment. Reporter gene activity was found for both genes in B cells and HeLa cells. Using a reverse transcriptase-polymerase chain reaction procedure (RT-PCR), we found a transcript corresponding to sequences upstream of the VH6 promoter in RNA from both the lymphoblastoid cell line ML-1, which actively transcribes the VH6 promoter, and the REH cell line, which does not. No transcripts were found in the KB epithelial cell line. Two or three mRNA 5' ends were found that mapped between -137 to -143 from the authentic VH6 transcription site, 31-37 nucleotides upstream of the putative TATA box. Inspection of the sequence upstream of the VH6 promoter demonstrated the presence of an open reading frame capable of coding for 96 amino acids. The VH6 promoter represents the second Ig promoter with bidirectional activity.
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PMID:Bidirectional transcription from the human immunoglobulin VH6 gene promoter. 754 16

Neurons have evolved strategies to evade immune surveillance that include an inability to synthesize the heavy chain of the class I major histocompatibility complex (MHC), proteins that are necessary for cytotoxic T lymphocyte (CTL) recognition of target cells. Multiple viruses have taken advantage of the lack of CTL-mediated recognition and killing of neurons by establishing persistent neuronal infections and thereby escaping attack by antiviral CTL. We have expressed a class I MHC molecule (Db) in neurons of transgenic mice using the neuron-specific enolase (NSE) promoter to determine the pathogenic consequences of CTL recognition of virally infected, MHC-expressing central nervous system (CNS) neurons. The NSE-Db transgene was expressed in H-2b founder mice, and transgene-derived messenger RNA was detected by reverse transcriptase-polymerase chain reaction in transgenic brains from several lines. Purified primary neurons from transgenic but not from nontransgenic mice adhered to coverslips coated with a conformation-dependent monoclonal antibody directed against the Dv molecule and presented viral peptide to CTL in an MHC-restricted manner, indicating that the Db molecule was expressed on transgenic neurons in a functional form. Transgenic mice infected with the neurotropic lymphocytic choriomeningitis virus (LCMV) and given anti-LCMV, MHC-restricted CTL displayed a high morbidity and mortality when compared with controls receiving MHC-mismatched CTL or expressing alternative transgenes. After CTL transfer, transgenic brains showed an increased number of CD8+ cells compared with nontransgenic controls as well as an increased rate of clearance of infectious virus from the CNS. Additionally, an increase in blood-brain barrier permeability was detected during viral clearance in NSE-Db transgenic mice and lasted several months after clearance of virus from neurons. In contrast, LCMV-infected, nontransgenic littermates and mice expressing other gene products from the NSE promoter showed no CNS disease, no increased intraparenchymal CTL, and no blood-brain barrier damage after the adoptive transfer of antiviral CTL. Our study indicates that viral infections and CTL-CNS interactions may induce blood-brain barrier disruptions and neurologic disease by a "hit-and-run" mechanism, triggering a cascade of pathogenic events that proceeds in the absence of continual viral stimulation.
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PMID:Consequences of cytotoxic T lymphocyte interaction with major histocompatibility complex class I-expressing neurons in vivo. 759 91

Whether neurons express major histocompatibility complex (MHC) class I genes has not been firmly established. The techniques of confocal laser microscopy, patch clamp electrophysiology, and reverse transcriptase-polymerase chain reaction were combined here to directly examine the inducibility of MHC class I genes in individual cultured rat hippocampal neurons. Transcription of MHC class I genes was very rare in neurons with spontaneous action potentials. In electrically silent neurons, transcription was noted, with expression of beta 2-microglobulin under tighter control than in class I heavy chain molecules. Surface expression of class I molecules occurred only in electrically silent neurons treated with interferon gamma. Immunosurveillance by cytotoxic T cells may be focused on functionally impaired neurons.
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PMID:Induction of MHC class I genes in neurons. 748 56

An early step in immunoglobulin isotype switching involves the initiation of active transcription of downstream heavy chain loci while they are still in germ-line configuration. This results in the production of 'sterile' germ-line RNA transcripts that do not contain VDJ sequences, the role of which in isotype switching is under intense scrutiny. We investigated whether such human epsilon germ-line transcripts employ the full complement of complex alternative 3' RNA splicing, splicing we have recently reported occurring with productive epsilon mRNA transcripts. Using a reverse transcriptase polymerase chain reaction (RT-PCR) strategy, in which the 5' primer was located in the I region of the epsilon gene, a region expressed in germ-line but not productive (VDJ containing) epsilon transcripts, we showed that the full range of alternative 3' epsilon splices occur with germ-line transcripts. These results demonstrate that epsilon 3' splicing events are independent of 5' isotype DNA switching. Additionally, we showed that, just as with mature epsilon mRNA, the relative production of the various epsilon germ-line mRNA isoforms was responsive to modulation by stimuli such as interleukin-10 (IL-10). Thus B cells, when stimulated to produce epsilon germ-line transcripts, generate a family of germ-line mRNA that differ not only in their initiation sites but, more importantly, also differ in their 3' sequences, sequences that could be important in regulation of the parent gene itself. Furthermore, by discontinuous or trans-splicing, cells could utilize these various epsilon germ-line transcripts to produce the full range of mature IgE proteins prior to undergoing deletional recombination of isotype switching.
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PMID:Germ-line human epsilon heavy chain gene RNA transcripts utilize the full range of alternative 3' splicing seen in productive epsilon mRNA. 764 11

We demonstrate that in addition to possessing binding sites for intact factor V (FV), unstimulated peripheral blood monocytes also express activated factor V (FVa) on their surfaces. FVa was identified on the monocyte surface by monoclonal antibody B38 recognizing FVa light chain and by human oligoclonal antibodies H1 (to FVa light chain) and H2 (to FVa heavy chain) using immunofluorescence microscopy and flow cytometry. On Western blots, partially cleaved FV could be identified as a 220 kDa band in lysates of monocytes. In addition to surface expression of FVa, monocytes also contain intracellular FV as detected only after permeabilization by Triton X-100 by monoclonal antibody B10 directed specifically to the Cl domain not present in FVa. We sought to determine whether the presence of FV in peripheral blood monocytes is a result of de novo synthesis. Using in situ hybridization, no FV mRNA could be detected in monocytes, while in parallel control studies, factor V mRNA was detectable in Hep G2 cells and CD18 mRNA in monocytes. In addition, using reverse transcriptase and the polymerase chain reaction, no FV mRNA was detected in mononuclear cells or in U937 cells, but mRNA for factor V was present in Hep G2 cells using the same techniques. These data suggest that FV is present in human monocytes, presumably acquired by binding of plasma FV, and that the presence of this critical coagulation factor is not due to de novo synthesis.
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PMID:Characterization of monocyte-associated factor V. 769 89


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