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Biomedical subjects

B Appel

Publications and source records attributed to B Appel.

At least 91 records · Page 5Linked to original sources
PubMed1984

Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing.

Small nuclear ribonucleoprotein particles (snRNPs) from eucaryotic cells can be fractionated on affinity columns prepared with antibodies of high affinity for 2,2,7-trimethyl-guanosine (m3G), which is present in the 5'-terminal caps of the snRNAs. While the snRNPs U1, U2 and U5 are eluted with the nucleoside m3G in the presence of 0.1 M salt, the snRNP species U4 and U6 are only desorbed when the salt concentration is increased. The same fractionation pattern was likewise observed for snRNPs from HeLa or Ehrlich ascites tumor cells. Since U6 RNA lacks the m3G residue and its RNA does not react with anti-m3G, its co-chromatography with U4 RNP on anti-m3G affinity columns suggests either that discrete snRNPs U4 and U6 are intimately associated in nuclear extracts or that both RNAs are organized in one ribonucleoprotein particle. Further evidence for a U4/U6 RNP particle is obtained by sedimentation studies with purified snRNPs in sucrose gradients. Gel fractionation of RNAs shows identical distributions of snRNAs U4 and U6 in the gradient, and the U4/U6 RNP particle sediments faster than the snRNPs U1 or U2. Physical association between snRNPs U4 and U6 during sedimentation is shown by their co-precipitation with anti-m3G IgG from the gradient fractions. Finally, experimental evidence is provided that snRNAs U4 and U6 are associated by intermolecular base pairing in the U4/U6 RNP particle, as demonstrated by our finding that anti-m3G IgG co-precipitates U6 RNA with U4 RNA following phenolization of U4/U6 RNPs at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

5'-Terminal caps of snRNAs are reactive with antibodies specific for 2,2,7-trimethylguanosine in whole cells and nuclear matrices. Double-label immunofluorescent studies with anti-m3G antibodies and with anti-RNP and anti-Sm autoantibodies.

Antibodies specific for 2,2,7-trimethylguanosine (m3G), which do not cross-react with m7G-capped RNA molecules were used to study, by immunofluorescence microscopy, the reactivity of the m3G-containing cap structures of the snRNAs U1 to U5 in situ. In interphase cells, immunofluorescent sites were restricted to the nucleus, whilst nucleoli were free of fluorescence. This indicates that the 5' terminal of most of the nucleoplasmic snRNAs are not protected by an m3G cap-recognizing protein and that the snRNA caps are not necessarily required for the binding of snRNPs to subnuclear structures. The snRNAs in the nucleoplasm appeared as distinct units in the light microscope, and this allowed the comparison of the distribution of snRNP proteins by double label studies with anti-RNP or anti-Sm antibodies within the same cell. The three antibody classes produced superimposable fluorescent patterns. Taking into account that the various IgGs react with antigenic sites on snRNAs or snRNP proteins not shared by all the snRNP species, these data suggest that U1 snRNP particles are distributed in the same way as the other snRNPs in the nucleus. Qualitatively the same results were obtained with DNase-treated nuclear matrices indicating that intact snRNPs are part of the nuclear matrix. Our data are consistent with proposals that the various snRNPs may be involved in processing of hnRNA and that this may take place at the nuclear matrix.

Animals↗
PubMed1984

The 5' terminus of the RNA moiety of U1 small nuclear ribonucleoprotein particles is required for the splicing of messenger RNA precursors.

We have investigated the role of small nuclear ribonucleoprotein particles (snRNPs) in the in vitro splicing of messenger RNA precursors by a variety of procedures. Removal of the U-type snRNPs from the nuclear extracts of HeLa cells with protein A-Sepharose-coupled human autoimmune antibodies leads to complete loss of splicing activity. The inhibition of splicing can be prevented by saturating the coupled antibodies with purified nucleoplasmic U snRNPs prior to incubation with nuclear extract. We further demonstrate that an intact 5' terminus of U1 snRNA is required for the functioning of U1 snRNP in the splicing reaction. Antibodies directed against the trimethylated cap structure of the U snRNAs inhibit splicing. Upon removal of the first eight nucleotides of the U1 snRNA in the particles by site-directed hydrolysis with ribonuclease H in the presence of a synthetic complementary oligodeoxynucleotide splicing is completely abolished. These results are in strong support of current models suggesting that a base-pairing interaction between the 5' terminus of the U1 snRNA and the 5' splice site of a mRNA precursor is a prerequisite for proper splicing.

Antigen-Antibody Complex↗
PubMed1984

Computed tomography in otoneurology.

Modern generation computed axial tomography is of great use in otoneurology with particular advantage coming from its excellent tissue density discrimination, together with its power of simultaneous investigation of both ears and their surrounding intracranial structures and the capability of spatial reformatting. The technique renders outmoded previous techniques including polytomography in most instances of congenital, inflammatory, neoplastic, and traumatic disease. Conventional lateral tomography may identify a small temporal bone fracture, and this examination is recommended in addition to axial and coronal CT scanning in cases of trauma.

Ear↗
PubMed1983

Six Schizosaccharomyces pombe tRNA genes including a gene for a tRNALys with an intervening sequence which cannot base-pair with the anticodon.

We report the sequences of six S. pombe tRNA genes including two genes for tRNAArg, and one gene each for tRNAGlu, tRNAHis, tRNALys and tRNAPhe. All tRNA genes are found independently in the genome and represent individual transcription units. The gene for tRNALys has an 8 bp long intervening sequence which cannot base-pair with the tRNA anticodon. In vitro transcription studies indicate that all genes are faithfully transcribed in a yeast extract. Sequence comparison of the 5' flanking regions of the tRNA genes did not show significant homologies; however, they are very rich in AT base pairs.

Anticodon↗
PubMed1983

5'-terminal caps of snRNAs are accessible for reaction with 2,2,7-trimethylguanosine-specific antibody in intact snRNPs.

Immune precipitation assays with antibodies specific for 2,2,7-trimethylguanosine (m2,2,7(3)G) have been used to study the accessibility of the 5'-terminal m2,2,7(3)G-containing caps of eucaryotic small nuclear RNAs (snRNAs) either as naked RNAs or in intact small nuclear ribonucleoprotein (snRNPs). The antibody selectively precipitates snRNA species U1a, U1b, U2, U4, and U5 from total deproteinized RNA isolated from Ehrlich ascites cells. Binding by the antibody occurs via the m2,2,7(3)G moiety of the snRNAs' caps, since complex formation with the antibody can be completely abolished by excess nucleoside m2,2,7(3)G. The specificity of the antibody is further demonstrated by the complete absence of reaction with deproteinized snRNA species U6, the 5' terminus of which does not contain m2,2,7(3)G. Most importantly, the cap structures of the snRNAs U1a, U1b, U2, U4, and U5 are also accessible for anti-m2,2,7(3)G IgGs when intact snRNPs are reacted with the antibody. In this case, snRNP species U6 is coprecipitated, suggesting that there are intermolecular interactions between this and other snRNPs. Our data demonstrate that the 5'-terminal regions of the above snRNAs are not protected by the snRNP proteins. This finding is of special interest for snRNP species U1, and is discussed in terms of a model which proposes that the 5'-terminal region of U1 participates in the proper alignment of splice junctions in eucaryotic pre-mRNAs (Lerner, M. R., Boyle, J.A., Mount, S.M., Wolin, S.L., and Steitz, J. A. (1980) Nature (Lond.) 283, 220-224).

Animals↗
PubMed1983

Purification of snRNPs U1, U2, U4, U5 and U6 with 2,2,7-trimethylguanosine-specific antibody and definition of their constituent proteins reacting with anti-Sm and anti-(U1)RNP antisera.

Small nuclear ribonucleoprotein particles (snRNPs) of the U-snRNP class from Ehrlich ascites tumor cells were purified in a one-step procedure by affinity chromatography with antibodies specific for 2,2,7-trimethylguanosine (m23.2.7G), which is part of the 5'-terminal cap structure of snRNAs U1-U5. Antibody-bound snRNPs are desorbed from the affinity column by elution with excess nucleoside m23.2.7G; this guarantees maintenance of their native structure. The snRNPs U1, U2, U4, U5 and U6 can be recovered quantitatively from nuclear extracts by this procedure. Co-isolation of U6 snRNP must be due to interactions between this and other snRNPs, as anti-m23.2.7G antibodies do not react with deproteinized U6 snRNA. We have so far defined nine proteins of approximate mol. wts. 10 000, 12 000, 13 000, 16 000, 21 000, 28 000, 32 000, 34 000 and 75 000. Purified snRNPs react with anti-(U1)RNP and with anti-Sm antisera from patients with mixed connective tissue disease and from MRL/l mice. As determined by the protein blotting technique, six of the snRNP polypeptides, characterized by apparent mol. wts. 13 000, 16 000, 21 000, 28 000, 34 000 and 75 000, bear antigenic determinants for one or the other of the above autoantibody classes. This suggests strongly that the U-snRNPs produced by the procedure described here are indeed representative of the snRNPs in the cell. With highly purified snRNPs available, investigation of possible enzymic functions of the particles may now be undertaken.

Animals↗
PubMed1983

[Rendu-Osler-Weber disease. Diagnostic and therapeutic assessment by angiography].

The principal manifestation of Rendu-Osler-Weber disease is epistaxis, associated multiple mucocutaneous localizations generally being asymptomatic. Full advantage does not appear to have been taken of the various possible treatments of epistaxis: though they are all palliative, certain of them provide aleatory effects in relation to others (surgery or radiotherapy and embolization). In consequence, embolization should be the initial procedure, and should be repeated whenever necessary and possible, for all epistaxis requiring hospitalization for transfusion. Results of embolization are improved when no previous therapy has been applied, when the disease has not advanced, and when few localizations, even asymptomatic, exist in the head and neck region. Results are independent of the sex, the duration of the disease, the chronicity of the bleeding episodes, and their frequency and severity. The most effective solution in the majority of cases is probably the association of treatment of the hemorrhagic syndrome with basic estrogen therapy of the disease process.

Aged↗
PubMed1982

Isolation and characterization of rabbit anti-m3 2,2,7G antibodies.

Antibodies specific for intact 2,2,7-trimethylguanosine (m3 2,2,7G) were induced by immunization of rabbits with a nucleoside-human serum albumen (HSA) conjugate. Competition radioimmunoassay showed that the antibody distinguishes well between intact m3 2,2,7G and its alkali-hydrolysed form (m3 2,2,7G*). Antibody specificity is largely dependent on the presence of all three methyl groups in m3 2,2,7G: none of the less extensively methylated nucleosides m7G, m2G and m2 2,2G is able to compete efficiently with the homologous hapten. Little or no competition was observed with m1G, m1A, m6A, m5U and each of the four unmodified ribonucleosides. Binding studies with nucleoplasmic RNAs from Ehrlich ascites cells suggest that the antibody reacts specifically with the m3 2,2,7G-containing cap structure of the small nuclear U-RNAs (U-snRNAs). Thus the antibody should be a valuable tool for studying the role of the 5'-terminal regions of the U-snRNAs of eucaryotic cells.

Animals↗
PubMed1982

The 5S RNA genes of Schizosaccharomyces pombe.

The genomic arrangement and sequences of S. pombe 5S RNA genes are reported here. The 5S gene sequences appear to be dispersed within the genome, and are found independently of other rRNA genes. The sequences of two 5S genes examined show identical coding regions of 119 base pairs but have widely varying flanking sequences. A tRNAAsp gene is found in the 3' flanking region of one of the 5S genes. The tRNAAsp gene is faithfully transcribed in an X. laevis in vitro system, while the 5S genes are not transcribed in this system. The phylogenetic position of S. pombe is examined through comparison of 5S RNA sequences.

Animals↗
PubMed1982

Post-transcriptional nucleotide addition is responsible for the formation of the 5' terminus of histidine tRNA.

All sequenced histidine tRNAs have one additional nucleotide at the 5' end when compared to other tRNA species. Sequence analysis of histidine tRNA genes from Drosophila melanogaster and Schizosaccharomyces pombe showed that the terminal guanylate residue of the mature tRNAs is not encoded by the genes. Analysis of the products from in vitro transcription of these genes in extracts from Drosophila Kc cells demonstrated that the 5'-terminal nucleotide present in the mature tRNA is added post-transcriptionally. The addition reaction requires ATP. A portion of the mature tRNAs are then modified at the 5'-terminal pG. Analysis of the RNA species formed during the in vitro maturation of the Drosophila histidine tRNA primary transcript uncovered the following maturation scheme: (i) the primary transcript is processed by RNase P at the 5' end to form an intermediate precursor; (ii) the 3'-flanking sequence is endonucleolytically removed, and a guanylate moiety is added to the 5' end to form mature-sized histidine tRNA; and (iii) a fraction of the 5'-terminal guanylate residues then undergoes modification. In contrast to the capping of eukaryotic mRNA, the guanylate addition to histidine tRNA results in the formation of a (3'-5')-phosphodiester bond. There are no precedents for the post-transcriptional addition of nucleotides (in phosphodiester linkage) to the 5' end of RNA precursors.

Animals↗
PubMed1981

Determination of base pairing in yeast 5S and 5.8S RNA infrared spectroscopy.

Infrared Spectroscopy was used to determine the numbers of base pairs for yeast 5S RNA and 5.8S RNA. The spectra were recorded at 20 degrees C and 50 degrees C, where tertiary interactions are assumed to be of less importance. It may be concluded that the structure of both RNAs is highly ordered and that there are large contributions of tertiary interactions. The results are compared with data derived from structural models that were proposed in the literature as well as with data previously published for prokaryotic 5S RNAs.

Base Composition↗
PubMed1979

Determination of base pairing in Escherichia coli and Bacillus stearothermophilus 5S RNAs by infrared spectroscopy.

The extent of base pairing in Escherichia coli and Bacillus stearothermophilus 5S RNAs was determined by infrared spectroscopy. From the infrared spectra taken at 20 degrees and 52 degrees C it is concluded that E. coli and B. stearothermophlius 5S RNAs possess a large number of base pairs (Table I). Comparison of our results with those previously published using other methods leads to the conclusion that the structures of prokaryotic 5S RNAs involve a large number of tertiary interactions, in which the base pairing is not necessarily solely of the Watson-Crick type.

Base Composition↗