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1.
Ann Otol Rhinol Laryngol ; 109(12 Pt 1): 1093-8, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11130817

ABSTRACT

The 28 kd protein extracted from the guinea pig inner ear membranous fraction, which reacted with sera from patients with Meniere's disease, has been subjected to microsequencing. Nineteen amino acids were obtained (IVQQFGFQRRASDDGKLTQ). A protein data bank search showed that this sequence corresponded to residues 41 to 60 of human Raf-1 protein. Sera from 16 of 27 patients with Meniere's disease showed reactivity to the recombinant purified glutathione-S-transferase-Raf-1 protein. These results support the hypothesis that a subgroup of patients who suffer from Meniere's disease, as well as some other kinds of autoimmune inner ear diseases, have an autoantibody against Raf-1 protein.


Subject(s)
Autoantibodies/blood , Autoantibodies/immunology , Autoantigens/immunology , Autoimmune Diseases/blood , Autoimmune Diseases/immunology , Meniere Disease/blood , Meniere Disease/immunology , Proto-Oncogene Proteins c-raf/immunology , Amino Acid Sequence , Animals , Autoantigens/chemistry , Autoantigens/genetics , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Female , Guinea Pigs , Humans , Molecular Sequence Data , Mutation , Proto-Oncogene Mas , Proto-Oncogene Proteins c-raf/chemistry , Proto-Oncogene Proteins c-raf/genetics
2.
Ann Otol Rhinol Laryngol ; 108(3): 255-64, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10086618

ABSTRACT

Myelin protein P0 has been identified as an autoantigen in inner ear diseases. In order to study autoimmune hearing loss, we performed brain stem auditory-evoked potential (BAEP) studies on P0-sensitized mice. Two P0-sensitized mice showed hunched posture, poor coat, loss of body weight, and abnormal walking with a waddling gait. About 25% of the P0-sensitized mice developed hearing loss. In the BAEP study, peak latencies of waves I, III, and V and the interpeak latency I-III were prolonged in the P0-sensitized hearing loss group of mice. Hearing thresholds were elevated in this group of mice in comparison with the control mice. Inflammatory cell infiltration was observed in the cochlear nerve region, and a reduced number of spiral ganglion cells was also detected. These results suggest that P0-sensitized mice are a useful model for studying autoimmune inflammation of the peripheral portion of the auditory system.


Subject(s)
Autoimmune Diseases/physiopathology , Hearing Disorders/physiopathology , Myelin P0 Protein/immunology , Animals , Auditory Threshold , Autoimmune Diseases/etiology , Autoimmune Diseases/metabolism , Autoimmune Diseases/pathology , Cochlea/chemistry , Evoked Potentials, Auditory, Brain Stem , Hearing Disorders/etiology , Hearing Disorders/metabolism , Hearing Disorders/pathology , Immunization , Mice , Mice, Inbred DBA , Myelin P0 Protein/analysis
3.
Ann Otol Rhinol Laryngol ; 107(11 Pt 1): 917-27, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9823840

ABSTRACT

Experimental allergic encephalomyelitis is an animal model of a T cell-mediated autoimmune disease -- for example, multiple sclerosis. We demonstrated that mice with experimental allergic encephalomyelitis developed retrocochlear hearing loss, and that the lesion of the auditory pathway might be related to T cell receptor Vbeta8-expressing T cells. To investigate whether anti-Vbeta8 antibody could prevent hearing loss, we carried out brain stem auditory evoked potential testing, histologic examinations, and flow cytometry in antibody-treated and control myelin basic protein-immunized B10.PL mice. The antibody was administered just before immunization of myelin basic protein. The disease incidence and severity were significantly reduced in the mice injected with the antibody. The results of brain stem auditory evoked potential testing, histologic examinations, and flow cytometry indicated that the depletion of Vbeta8-expressing T cells brings the prevention of hearing loss, as well as prevention of other neurologic deficits. The development of T cell receptor-specific antibody therapy might help treat retrocochlear hearing loss in multiple sclerosis.


Subject(s)
Antibodies/therapeutic use , Encephalomyelitis, Autoimmune, Experimental/therapy , Hearing Disorders/prevention & control , Receptors, Antigen, T-Cell, alpha-beta/immunology , Retrocochlear Diseases/prevention & control , Animals , Antibody Specificity , Auditory Threshold/physiology , Cochlear Nucleus/pathology , Encephalomyelitis, Autoimmune, Experimental/physiopathology , Evoked Potentials, Auditory, Brain Stem/physiology , Female , Flow Cytometry , Male , Mice , Mice, Inbred Strains
4.
J Allergy Clin Immunol ; 101(1 Pt 1): 51-9, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9449501

ABSTRACT

Sensitivity induced by house dust mite (HDM) extract in mice was investigated in this study. Sensitized B10.RIII mice (H-2r background) had T-cell proliferative responses to HDM extract in vitro and an HDM-specific IgE response. When mice were immunized by injection and intranasal inhalation with HDM extract, a histologic study showed eosinophils and mononuclear cell infiltration in the lung tissue and bronchial wall. Tcr alphabeta-positive cells were also found in the cell infiltration area of the lung lesions. In the control mice that were immunized by injection or intranasal inhalation (but not both), we did not observe cell accumulation in the lung tissue or in the bronchial wall. Epitope studies suggest that T cells recognize multiple epitopes. Molecular analysis of these HDM-specific T-cell hybridoma clones suggest that T-cell receptor use is restricted to members of the V alpha 8 and Vbeta 6 subfamilies.


Subject(s)
Dust/adverse effects , Hypersensitivity/etiology , Mites/immunology , Allergens/toxicity , Amino Acid Sequence , Animals , Base Sequence , DNA Primers/genetics , Disease Models, Animal , Hybridomas , Hypersensitivity/immunology , Hypersensitivity/pathology , Immunization , Immunoglobulin E/biosynthesis , In Vitro Techniques , Lung/immunology , Lung/pathology , Lymphocyte Activation , Mice , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , T-Lymphocyte Subsets/immunology , Trachea/immunology , Trachea/pathology
5.
J Neuroimmunol ; 80(1-2): 13-22, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9413255

ABSTRACT

We analyzed myelin basic protein (MBP) specific T cell hybridoma clones from (B10.PL x PL/J)F1 mice. MBP-reacting T cell hybridomas from F1 mice preferentially expressed B10.PL TcraV2.3 (53%) and B10.PL TcraV4.2 (13%) with minor expression of TcraV4.4 (13%) gene segments. A dominant expression of TcrbV8.2 (73%) accompanying with TcrbV8.1 (20%) and TcrbV13 (7%) gene segments have been identified in these MBP-reacting T cell hybridomas from F1 mice. There was less restrictive but non-random usage of the TcraJ and TcrbJ gene segments. Overall, the MBP-reacting T cell hybridomas from (B10.PL x PL/J)F1 mice were dominated by the MBP-reacting T cell pattern seen in B10.PL mice.


Subject(s)
Gene Rearrangement, T-Lymphocyte/immunology , Genes, T-Cell Receptor/immunology , Myelin Basic Protein/genetics , T-Lymphocytes/metabolism , Amino Acid Sequence , Animals , Base Sequence , Crosses, Genetic , Flow Cytometry , Gene Expression Regulation/immunology , Hybridomas , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Multigene Family , Myelin Basic Protein/immunology , Myelin Basic Protein/metabolism , Rats , Receptors, Antigen, T-Cell, alpha-beta/biosynthesis , Receptors, Antigen, T-Cell, alpha-beta/genetics , Species Specificity
6.
Article in English | MEDLINE | ID: mdl-9253025

ABSTRACT

A type II collagen autoimmune response results in arthritis, auricular chondritis and tympanosclerosis in humans and animals. The purpose of this study is to further define the molecular and pathogenic events involved in these lesions in rodents. Type II collagen fragment CB11-specific monoclonal antibodies induced lesions in joints, ear lobes and tympanic membranes. In immunized mice, the thickness of tympanic membranes increased to two- to fourfold normal size. Electron micrography showed that the arrangement of collagen fibers is irregular in both radial and auricular layers, containing fibroblasts, a homogeneous material resembling low-density cholesterol crystals and cell infiltration. The mice with auricular chondritis had lymphocytes expressing V beta-8 T cell receptor (TCR) in arthritic joints and lymphocytes expressing V beta-6 TCR in ear lobe lesions. A monoclonal antibody specific to the TCR V beta-8 subfamily suppressed the onset of arthritis. Sequence analyses of the V beta structure of TCR involved in the lesions confirm the immunohistologic study.


Subject(s)
Arthritis, Experimental/immunology , Autoimmune Diseases/immunology , Cartilage Diseases/immunology , Collagen/immunology , Ear Cartilage/immunology , Tympanic Membrane/immunology , Animals , Antibodies, Monoclonal/therapeutic use , Collagen/ultrastructure , Female , Male , Mice , Mice, Inbred DBA , Mice, Inbred Strains , Receptors, Antigen, T-Cell, alpha-beta/immunology
7.
Article in English | MEDLINE | ID: mdl-9104744

ABSTRACT

Sera from patients with various inner-ear diseases, especially Ménière's disease, were investigated by Western blot against guinea pig inner-ear proteins. Of 45 patients, 24 (53%) with various inner-ear diseases had antibodies against inner-ear proteins, compared with 0 of 10 (0%) in control subjects without inner-ear diseases. Of the 10 proteins that showed a positive reaction with patient sera, the 28-kD band was unique in that it appeared only in the membranous fraction of the inner ear and was highly positive (28%) in reaction with Ménière's disease patient sera. The results in the present study with proteins extracted from guinea pig inner ear were consistent with our previous study using proteins from human inner ear, suggesting that the 28-kD protein may be a candidate for detecting autoimmune inner-ear disease.


Subject(s)
Autoantibodies/blood , Ear, Inner/immunology , Labyrinth Diseases/immunology , Proteins/immunology , Animals , Autoimmune Diseases/diagnosis , Blotting, Western , Case-Control Studies , Electrophoresis, Polyacrylamide Gel , Guinea Pigs , Hearing Disorders/immunology , Hearing Loss, Sensorineural/immunology , Humans , Male , Meniere Disease/immunology , Otosclerosis/immunology , Proteins/analysis , Proteins/classification , Sodium Dodecyl Sulfate , Tinnitus/immunology
9.
Ann Otol Rhinol Laryngol ; 105(11): 905-15, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8916868

ABSTRACT

Experimental allergic encephalomyelitis (EAE) is an experimental model for multiple sclerosis. In order to study autoimmune retrocochlear hearing loss, we performed brain stem auditory-evoked potential (BAEP) studies on EAE mice. The EAE was induced in B10.PL and (PL/J x SJL)F1 mice. In the BAEP study, all of the peak and interpeak latencies were prolonged significantly in the diseased mice. Hearing thresholds were slightly elevated in the immunized mice during the acute phase. Inflammatory and phagocytic cell infiltration, demyelination, and V beta 8.1, 8.2 T-cell receptor-positive cells were observed in the cochlear nerves or their proximity by histologic study. It is suggested that immunologic reactions identified with EAE also occurred in the cochlear nerve, and that these reactions were responsible for the hearing problems. It appears that EAE is a useful model system for studying autoimmune insults on the neural portion of the auditory system.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/physiopathology , Evoked Potentials, Auditory, Brain Stem/physiology , Hearing Disorders/physiopathology , Animals , Cochlear Nerve/metabolism , Cochlear Nerve/pathology , Encephalomyelitis, Autoimmune, Experimental/etiology , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Hearing Disorders/metabolism , Hearing Disorders/pathology , Hearing Tests , Immunoenzyme Techniques , Male , Mice , Myelin Basic Protein , Phagocytes/pathology , Receptors, Antigen, T-Cell, alpha-beta/metabolism
10.
Clin Immunol Immunopathol ; 78(3): 263-75, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8605702

ABSTRACT

Collagen-induced arthritis is mediated by autoantibodies to type II collagen (CII). This experimental model has proven useful in determining the molecular and cellular mechanisms responsible for autoimmune arthritis. We have shown that polyarthritis can be transferred to normal mice by administering combinations of three or four complement-fixing monoclonal antibodies (mAbs) which recognize cross-reactive epitopes on the alpha 1(II)-CB11 region of chick and mouse CII. Currently, the light- and heavy-chain variable-region structures on a panel of alpha 1 (II)-CB11-specific mAbs that cross-react with chick and mouse CII, or react solely with chick CII, have been analyzed. The results indicate biased usage of VK19 and VK21 families of light-chain variable-region genes but random VH gene usage. Interestingly, two mAbs derived from different mice recognized identical epitopes on mouse CII and had nearly identical light- and heavy-chain variable-region structure including junctionally derived sequence.


Subject(s)
Arthritis/immunology , Autoantibodies/genetics , Collagen/immunology , Immunoglobulin Variable Region/genetics , Amino Acid Sequence , Animals , Antibody Specificity , Base Sequence , Cross Reactions , Epitopes , Hybridomas , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Isotypes/genetics , Immunoglobulin Light Chains/genetics , Mice , Mice, Inbred DBA , Molecular Sequence Data , Sequence Analysis, DNA , Species Specificity
12.
Cell Immunol ; 157(1): 249-62, 1994 Aug.
Article in English | MEDLINE | ID: mdl-7518752

ABSTRACT

An immune response directed against type II collagen (CII) has been reported in several autoimmune diseases including the animal models of collagen-induced arthritis (CIA) and collagen-induced autoimmune ear disease (CIAED). In this communication, we have found that T cells from type II collagen-immunized DBA/1-lac could transfer auricular chondritis to naive mice. The T cells from type II collagen-immunized H-2r and H-2q mice recognize different epitopes from the CB11 peptide of CII. The CII-specific T cells from H-2q background mice recognize peptide residues p121-147 (P1) but do not respond to residues p211-247 (P2). The T cells of H-2r mice immunized with CII respond better to P2 rather than P1. By altering certain amino acids within these epitopes, the response of CII-specific TCR to antigen has been increased or abolished. Our results suggest that the lysine residues at positions 129, 141, and 147 in P1, the arginine residue at position 227, and glutamic acid at position 230 in P2 might play an important role in the trimolecular interaction. Ten clonally distinct T cell hybridomas specific for CII have been established from H-2r B10.RIII mice and the beta chains of their TCR have been analyzed. Three subfamilies, V beta 1, V beta 6, and V beta 8, were utilized with dominant expression of V beta 8 (60%). This is quite similar to the pattern found in type II collagen-induced arthritis in H-2q mice. This preferential use of V beta 8 in CIAED implies that an immunotherapy may make it possible to control this autoimmune disease, even in a MHC-diverse situation.


Subject(s)
Autoimmune Diseases/immunology , Collagen/immunology , Ear Diseases/immunology , Epitopes/immunology , Receptors, Antigen, T-Cell, alpha-beta/genetics , Amino Acid Sequence , Animals , Autoimmune Diseases/genetics , Base Sequence , Cartilage Diseases/immunology , Cartilage Diseases/pathology , Ear Diseases/pathology , Hybridomas , Lymphocyte Activation , Mice , Mice, Inbred DBA , Mice, Inbred Strains , Molecular Sequence Data , T-Lymphocytes/physiology , T-Lymphocytes/transplantation
13.
Genomics ; 13(2): 319-31, 1992 Jun.
Article in English | MEDLINE | ID: mdl-1612591

ABSTRACT

The six members of the human prothymosin alpha gene family have been cloned and sequenced. One gene (PTMA) contains introns and appears to be the source of all isolated prothymosin alpha cDNAs. The remaining five genes are processed pseudogenes. Four of them have consensus TATA elements upstream of sequences nearly identical to the transcriptional start region of the intron-containing gene. Those four genes also contain open reading frames coding for proteins closely related to prothymosin alpha. In two of the pseudogenes, PTMAP2 and 5, the encoded proteins differ from the product of the parental gene at only two and four locations, respectively. The fifth pseudogene (PTMAP1) encodes a different protein owing to an upstream translational initiation start site and multiple deletions and insertions. Because the potential for expression exists in this system, a search for pseudogenomic transcripts was undertaken using the polymerase chain reaction to amplify reverse transcripts of mRNAs from many human tissues and bulk DNA from several human cDNA libraries. Evidence for pseudogenomic transcripts was not obtained. Therefore, we conclude that the human prothymosin alpha gene family contains only one functional gene.


Subject(s)
Multigene Family , Protein Precursors/genetics , Pseudogenes , Thymosin/analogs & derivatives , Amino Acid Sequence , Base Sequence , Consensus Sequence , DNA/genetics , Gene Library , Genes , Humans , Introns , Molecular Sequence Data , Open Reading Frames , Sequence Alignment , TATA Box , Thymosin/genetics , Transcription, Genetic
14.
Biochemistry ; 30(44): 10614-23, 1991 Nov 05.
Article in English | MEDLINE | ID: mdl-1718423

ABSTRACT

The binding of substrates to recombinant reverse transcriptase from human immunodeficiency virus (HIV) and the natural enzyme from avian myeloblastosis virus (AMV) has been examined by analyzing both the ribonuclease H and the RNA-dependent DNA polymerase activities. With 3'-end-labeled globin mRNA hybridized to (dT)15 as the substrate in the ribonuclease H reaction, the enzymes partially deadenylated the mRNA in a distributive manner. Under these conditions, there was a rapid initial burst followed by a prolonged, but much slower, steady-state rate. The biphasic reaction made possible determinations of kinetic constants as follows: values for Km, KD, and kcat were, respectively, 27 nM, 11 nM, and 5 x 10(-3) s-1 for the HIV enzyme and 30 nM, 9 nM, and 5 x 10(-3) s-1, respectively, for the avian enzyme. These constants were used to derive other parameters: The rate of association of the template-primer with reverse transcriptase was approximately 2 x 10(5) M-1 s-1, and the rate of dissociation was approximately 2 x 10(-3) s-1, regardless of the source of the enzyme. The rate of release of the product was essentially equivalent to the value of kcat indicated above for each of the enzymes. The polymerase reaction was evaluated under processive conditions of synthesis; values of Km and kcat of approximately 6 nM and approximately 2.5 s-1, respectively, for the human enzyme, and approximately 10 nM and approximately 2 s-1, respectively, for the avian enzyme were observed. The interaction of substrates with HIV reverse transcriptase was characterized further with the aid of ribonucleoside-vanadyl complexes. These complexes inhibited the polymerase and ribonuclease H activities of the enzyme competitively with respect to globin mRNA.(dT)15. Values of Ki ranging from 1 to 3 mM were obtained. With respect to deoxyribonucleoside triphosphate substrates in the polymerase reaction, mixed inhibition was observed. Deoxyribonucleoside triphosphates had no effect on kinetic parameters governing the ribonuclease H activity of the HIV enzyme but apparently facilitated the formation of active enzyme. These data fit a model in which one template-primer binding site serves both the polymerase and the ribonuclease H catalytic sites.


Subject(s)
HIV/enzymology , RNA-Directed DNA Polymerase/metabolism , Avian Myeloblastosis Virus/enzymology , Binding Sites , Catalysis , DNA/metabolism , Globins/genetics , Hydrolysis , Kinetics , Nucleic Acid Hybridization , Oligodeoxyribonucleotides/metabolism , RNA/metabolism , RNA, Messenger/metabolism , RNA-Directed DNA Polymerase/chemistry , Reverse Transcriptase Inhibitors , Ribonuclease H/metabolism , Vanadates/metabolism
15.
Anal Biochem ; 183(1): 64-73, 1989 Nov 15.
Article in English | MEDLINE | ID: mdl-2559629

ABSTRACT

A direct method for mapping introns has been devised. The technique makes use of a radioactive synthetic RNA transcript of the gene and a complementary, single-stranded DNA copy of mRNA-derived sequences. Upon hybridization of the cDNA to RNA and cleavage with ribonuclease H, only exonic RNA sequences are degraded. The surviving RNA fragments are the introns. Electrophoretic analysis in denaturing agarose gels reveals the number and size of the introns. The order of the introns is determined separately using unlabeled RNA transcripts; surviving RNA fragments are transferred to a solid support and the blot is probed sequentially with a nested set of genomic RNA transcripts of the opposite strand. Using the human prothymosin alpha gene as an example, four introns were identified which from 5' to 3' were 2.6, 0.47, 0.47, and 0.28 kb in size. From mapping and sequencing experiments the sizes are 2.6, 0.465, 0.459, and 0.295 kb, respectively. Similarly, the presence of two 300-bp insertions in a human prothymosin alpha pseudogene was established; the inserts were later identified as 295-bp Alu repetitive elements.


Subject(s)
Chromosome Mapping , Exons , Introns , Blotting, Northern , DNA/genetics , DNA Transposable Elements , Humans , Methods , Protein Precursors/genetics , RNA, Messenger/genetics , Thymosin/analogs & derivatives , Thymosin/genetics , Transcription, Genetic
16.
Proc Natl Acad Sci U S A ; 86(10): 3539-43, 1989 May.
Article in English | MEDLINE | ID: mdl-2471188

ABSTRACT

A series of test substrates have been synthesized to establish the effect of termini on the putative exoribonuclease H activity of reverse transcriptase. Recombinant reverse transcriptase from human immunodeficiency virus, natural enzyme from avian myeloblastosis virus, and a known endonuclease, Escherichia coli ribonuclease H, cleaved relaxed, circular, covalently closed plasmids in which 770 consecutive residues of one strand were ribonucleotides. The avian enzyme also deadenylated capped globin mRNA with a covalently attached oligo(dT) tail at the 3' end. These results resolve a long-standing controversy--that the viral enzymes are obligatory exonucleases in vitro, based on their failure to cleave certain substrates for E. coli ribonuclease H, including circular poly(A).linear poly(T) and ribonucleotide-substituted supercoiled plasmids, but resemble endonucleases in vivo, based on their ability to degrade RNA in complex DNA.RNA hybrids. The data strongly suggest that the viral enzymes are endonucleases with exquisite sensitivity to the conformation of heteroduplexes. Inhibition of viral, but not cellular, ribonuclease H with ribonucleoside-vanadyl complexes further distinguishes these enzymes.


Subject(s)
Endoribonucleases/metabolism , RNA-Directed DNA Polymerase/metabolism , Avian Myeloblastosis Virus/enzymology , Endoribonucleases/antagonists & inhibitors , Escherichia coli/enzymology , HIV/enzymology , RNA, Messenger/metabolism , Reverse Transcriptase Inhibitors , Ribonuclease H , Structure-Activity Relationship , Substrate Specificity
17.
J Biol Chem ; 264(13): 7546-55, 1989 May 05.
Article in English | MEDLINE | ID: mdl-2708378

ABSTRACT

Prothymosin alpha and thymosin alpha 1 are believed to be thymus-derived, hormone-like materials with immunomodulatory functions performed outside the cell. These functions are inconsistent with the existence of a full length cDNA clone that does not encode an amino-terminal signal peptide or several consecutive hydrophobic residues. A study of the prothymosin alpha mRNAs and genes was undertaken in search of evidence for secreted forms of the protein. Prothymosin alpha mRNA was localized exclusively on free, rather than membrane-bound, polysomes. Upon screening cosmid and plasmid libraries totaling 2 X 10(6) clones, a gene family consisting of six members was identified. Sequence information from the 5'-ends of all the genes indicated that none encodes an amino-terminal signal peptide. One of the genes, apparently by means of alternate splicing, gives rise to two prothymosin alpha mRNAs, one of which has an additional internal glutamic acid codon with respect to the other. Comparison of the translated nucleic acid sequences of the five remaining genes with those encoded in the mRNAs revealed 30-98% homology in the first 50 amino acids. These five genes appear to be processed genes and/or pseudogenes. The localization of prothymosin alpha mRNAs on free polysomes, together with the partial nucleotide sequences of the genes, strongly suggest an intracellular function for prothymosin alpha. Therefore, the possibility must be raised that prothymosin alpha and its peptide derivatives act as xenobiotics when introduced into assays of immune function.


Subject(s)
Protein Precursors/genetics , Thymosin/analogs & derivatives , Amino Acid Sequence , Base Sequence , Cell Compartmentation , Cloning, Molecular , DNA/genetics , Exons , Humans , Molecular Sequence Data , Multigene Family , Polyribosomes/metabolism , Protein Precursors/metabolism , RNA Splicing , RNA, Messenger/metabolism , Regulatory Sequences, Nucleic Acid , Restriction Mapping , Sequence Homology, Nucleic Acid , Thymosin/genetics , Thymosin/metabolism , Tumor Cells, Cultured
20.
Anal Biochem ; 153(2): 315-23, 1986 Mar.
Article in English | MEDLINE | ID: mdl-2422979

ABSTRACT

A method has been developed for measuring the molar concentration of RNA and the mole fraction of polyadenylated RNA. Using known mixtures of globin mRNA and rRNA composed of 20 to 85% rRNA, the molar concentration of globin mRNA, a polyadenylated species, was determined in 45 min, with the consumption of less than 100 ng of total RNA. The technique is particularly well suited for determining the molar concentration of poly(A)+ RNA after chromatographic enrichment in columns of oligo(dT)-cellulose or poly(U)-Sepharose. The method makes possible the adoption of a molar standard.


Subject(s)
Microchemistry/methods , Poly A/analysis , RNA, Ribosomal/analysis , RNA/analysis , Animals , Cattle , Endoribonucleases , Globins , Kinetics , Phosphorus Radioisotopes , Polynucleotide Adenylyltransferase , RNA, Messenger , Rabbits , Ribonuclease H , Spectrophotometry, Ultraviolet
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