Measurement and analysis of yeast pre-mRNA sequence contribution to splicing efficiency.
Explore the source record for details and available documents.
Base Sequence↗
Biomedical subjects
P Legrain
Publications and source records attributed to P Legrain.
The yeast PRP6 gene encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein, and the PRP9 gene encodes a protein required for U2 snRNP binding.
PRP6 and PRP9 are two yeast genes involved in pre-mRNA splicing. Incubation at 37 degrees C of strains that carry temperature-sensitive mutations at these loci inhibits splicing, and in vivo experiments suggested that they might be involved in commitment complex formation (P. Legrain and M. Rosbash, Cell 57:573-583, 1989). To examine the specific role that the PRP6 and PRP9 products may play in splicing or pre-mRNA transport to the cytoplasm, we have characterized in vitro splicing and spliceosome assembly in extracts derived from prp6 and prp9 mutant strains. We have also characterized RNAs that are specifically immunoprecipitated with the PRP6 and PRP9 proteins. Both approaches indicate that PRP6 encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein and that the PRP9 protein is required for a stable U2 snRNP-substrate interaction. The results are discussed with reference to the previously observed in vivo phenotypes of these mutants.
Genes, Fungal↗
Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm.
We designed a strategy to identify splicing factors that act by preventing pre-mRNA transport into the cytoplasm. A yeast synthetic intron was inserted into a lacZ gene so that only the pre-mRNA could be translated to produce beta-galactosidase activity. Deletion of either of the 5' splice junction sequence GUAUGU and the branchpoint sequence UACUAAC resulted in a dramatic increase in pre-mRNA translation, indicating its cytoplasmic localization. In rna6 and rna9 mutant strains assayed at the nonpermissive temperature, splicing inhibition occurred simultaneously with a large increase in pre-mRNA translation. Similarly, a point mutation in U1 snRNA decreased splicing efficiency and increased pre-mRNA translation. From these results, we conclude that early acting factors, probably including U1 snRNA, and the RNA6 and RNA9 gene products, interact in vivo with the 5' splice junction and the branchpoint sequence to commit the pre-mRNA to the splicing pathway, thereby preventing its transport to the cytoplasm.
Amino Acid Sequence↗
Antibody-mediated binding of a murine ecotropic Moloney retroviral vector to human cells allows internalization but not the establishment of the proviral state.
We have attempted to introduce a neomycin-resistance gene, recombined in a murine (ecotropic) Moloney retrovirus, into HEp2 human cells which are resistant to infection by this retrovirus. To allow the binding of the retroviral vector on HEp2 cells, we have tested several antibody constructions between two monoclonal antibodies, one (mAb 5E9) directed against the human transferrin receptor and the other (mAb 615) directed against the gp70 envelope viral protein. The crosslinking of mAbs 5E9 and 615 by a sheep anti-murine kappa light chain antibody allowed the binding of virus on HEp2 cells. After incubation at 37 degrees, virus was internalized by HEp2 cells. However, no neomycin-resistant colonies of HEp2 cells have been obtained under various conditions. Our results suggest that binding and internalization of a retrovirus are not sufficient for establishment of the proviral state in cells which do not express a specific receptor.
Antibodies, Monoclonal↗
Early commitment of yeast pre-mRNA to the spliceosome pathway.
Pre-mRNA splicing in vitro is preceded by complex formation (spliceosome assembly). U2 small nuclear RNA (snRNA) is found in the earliest form of the spliceosome detected by native gel electrophoresis, both in Saccharomyces cerevisiae and in metazoan extracts. To examine the requirements for the formation of this early complex (band III) in yeast extracts, we cleaved the U2 snRNA by oligonucleotide-directed RNase H digestion. U2 snRNA depletion by this means inhibits both splicing and band III formation. Using this depleted extract, we were able to design a chase experiment which shows that a pre-mRNA substrate is committed to the spliceosome assembly pathway in the absence of functional U2 snRNP. Interactions occurring during the commitment step are highly resistant to the addition of an excess of unlabeled substrate and require little or no ATP. Sequence requirements for this commitment step have been analyzed by competition experiments with deletion mutants: both the 5' splice site consensus sequence and the branch point TACTAAC box sequence are necessary. These experiments strongly suggest that the initial assembly process requires a trans-acting factor(s) (RNA and/or proteins) that recognizes and stably binds to the two consensus sequences of the pre-mRNA prior to U2 snRNP binding.
Chromosome Deletion↗
Increase of retroviral infection in vitro by the binding of antiretroviral antibodies.
Monoclonal antiretrovirus antibodies were assayed for their ability to influence retrovirus infection in vitro. Some antibodies increased murine cell infection by an ecotropic virus and both murine and human cell infection by an amphotropic virus. The stability of these viruses was not modified, suggesting that antibody-virus complexes may be more infectious than free virus particles.
Animals↗
The expression of a private idiotope requires pretreatment with noncomplementary anti-idiotypic antibodies.
The levan-binding ABPC48 myeloma protein is characterized by 3 idiotopes, (Ids), defined by 3 syngeneic monoclonal anti-idiotypic antibodies (IDA 10, IDA 16 and IDA 17). When BALB/c mice are immunized with levan, they produce anti-levan antibodies, some of which carry the Id 10 and Id 16 but not the Id 17 determinants. In the present study, we attempted to induce the synthesis of Id 17 positive anti-levan molecules. We found that immunization with IDA 17 antibodies alone was ineffective in inducing an Id 17 positive anti-levan response. By contrast, successive immunizations with IDA 10, IDA 16 and IDA 17 antibodies resulted in the synthesis of Id 17 positive anti-levan immunoglobulins. The synthesis of these molecules was concommitant with the induction of Id 10 and Id 16 positive anti-levan antibodies. Thus our data suggest that the Id 17 determinant on anti-levan antibodies is coexpressed with Id 10 and Id 16, and that successive anti-idiotypic treatment may result in the selective expansion of rare ABPC48 cross-reactive idiotype B-cell clone precursors.
Animals↗
A single VH-gene associated with a variety of D- and J-segments encodes for a large family of ABPC48-related antibodies induced by antiidiotypic immunization.
Two series of monoclonal antibodies have been obtained from BALB/c mice immunized against two antiABPC48 antiidiotypic antibodies. They are divided into two serologically different classes. Class I antibodies bind only the immunizing antibody; class II antibodies display a broad binding capacity to various antiidiotypic antibodies, and some bind levan, as does ABPC48. Northern blot analyses and partial mRNA sequencing show that all class II antibodies express the VH-gene coding for ABPC48 and UPC10 antilevan antibodies associated with a variety of D- and J-segments. The third hypervariable region of the sequenced antibody with antilevan activity is structurally related to that of ABPC48 and UPC10 antibodies but has a different genetic origin. This study indicates that the identification of idiotype-related antibodies arising from antiidiotypic immunization may be misleading, if based on their antigen-binding properties; and it stresses the importance of structural approaches for the analysis of regulatory mechanisms ruling immune responses.
Amino Acids↗
Immune response induced by a single or several syngeneic monoclonal antiABPC48 antiidiotypic antibodies: no predominant coexpression of ABPC48 idiotopes.
BALB/c mice were immunized with monoclonal BALB/c antibodies IDA10, IDA16 and IDA17 raised against the BALB/c ABPC48 myeloma protein. Several procedures of immunization--copolymers with lipopolysaccharide or keyhole limpet hemocyanin, simultaneous or sequential injections of different IDAs--were performed in an attempt to orient the immune response towards the production of ABPC48-like idiotypes. We used a binding assay which identifies two idiotopes on the same molecule to measure the population of antibodies induced in these responses. The expression of ABPC48 cross-reactive idiotypes in immune sera was analyzed. The results show that, with all immunization protocols, immune responses to different monoclonal antiidiotypic antibodies are mostly independent of each other: the coexpression of ABPC48 idiotopes, either private or recurrent on the induced antibodies, is rarely found; it makes it difficult to discriminate by a serological approach between cross-reactive idiotypes and anti-antiidiotypic antibodies. We discuss the interest of combining molecular and serological approaches to identify these two populations of antibodies.
Animals↗
The VK gene expressed by BALB/c ABPC48 cross-reactive idiotypes induced by anti-idiotypic immunization is identical to that of BALB/c anti-oxazolone and A/J anti-arsonate antibodies.
Anti-idiotypic immunization triggers the production of antibodies that are structurally related to the idiotype. We have shown that the heavy chain variable regions of antibodies produced after anti-ABPC48 (A48) anti-idiotypic immunization of BALB/c mice are homologous to that of A48, except for the third hypervariable region. We present here partial light chain sequences of A48 and of antibodies induced by anti-idiotypic immunization. Nearly perfect homology is found, suggesting that these chains are the products of genes derived from a unique VK germ-line gene. These observations indicate that the H and L hypervariable regions contribute to define the structure of A48 idiotopes. Remarkably, the VK sequence we identify is the same as that described for anti-arsonate and anti-oxazolone antibodies. We discuss the relative importance of particular amino acids for idiotype expression and antigen-binding activity.
Animals↗
ABPC 48 cross-reactive idiotopes in BALB/c mice. Natural and levan-induced expression.
Using monoclonal antiidiotypic antibodies, we developed a sensitive binding assay that detects molecules with one or with two idiotopes of the ABPC48 idiotype. ABPC48 cross-reactive idiotypes were thus shown to be present in substantial amounts in sera of nonimmunized mice. Levan binding sites are found on these idiotypes. During the life time of the mice, the natural anti-levan titer increases while ABPC48 idiotypic expression remains constant, suggesting different controls for these two activities. On the other hand, ABPC48 cross-reactive idiotypes participate--as minor components--in the response that follows a deliberate immunization with bacterial levan. This induction process is likely to reflect the selection of idiotopes expressed by the B cell clones preactivated in sera of nonimmunized mice rather than the activation of silent clones. We suggest that a similar situation might explain the reported emergence of ABPC48 idiotypes in animals primed with antiidiotypic antibodies and subsequently stimulated with levan.
Animals↗
Rosette-forming cell assay for detection of antibody-synthesizing hybridomas.
Explore the source record for details and available documents.
Animals↗
Monoclonal antibodies to rabbit progesterone receptor: crossreaction with other mammalian progesterone receptors.
A mouse was immunized with purified rabbit uterine cytosolic progesterone receptor (specific activity: 3 nmol of steroid bound per mg of protein). After fusion of its spleen cells with Sp2-OAg myeloma cells, supernatants of 11 hybrid cultures were found to react in both an immunoenzymatic test and a double-immunoprecipitation test with the progesterone receptor. Clones were obtained from the five hybrid cells that gave the strongest response in both tests. Antibodies from cell culture supernatants and ascitic fluids were characterized. Three are of the IgG1 and two of the IgG2a isotype. Their apparent affinity for the progesterone receptor was measured by immunoprecipitation in physiological salt conditions. The equilibrium dissociation constants were between 0.1 and 4 nM. All five monoclonal antibodies crossreacted with the rabbit nuclear receptor, the human cytosolic receptor, and other mammalian (rat, guinea pig) but not avian (chicken) cytosolic progesterone receptors. There was no interaction with the glucocorticoid receptor and corticosteroid binding globulin.
Animals↗
The level of expression and the molecular distribution of ABPC 48 idiotopes in levan- or anti-idiotope-primed BALB/c mice.
Explore the source record for details and available documents.
Animals↗
[Production of anti-L-histidine decarboxylase monoclonal antibodies].
Hybridomas secreting monoclonal antibodies directed against Rat gastric L-histidine decarboxylase (H.D.) have been obtained from a partially purified preparation and screened by immunoprecipitation of the enzyme activity. They cross react with Rat brain H.D., indicating at least that these two enzymes share common antigenic determinants.
Animals↗
Idiotype-anti-idiotype interactions and the control of the anti-beta (2 leads to 6) polyfructosan response in the mouse: specificity and idiotypy of anti-ABPC48 anti-idiotypic monoclonal antibodies.
Seventeen hybridomas, secreting monoclonal anti-idiotypic antibodies (IDA) directed against the BALB/c ABPC48 idiotype, were isolated from one immunized BALB/c mouse. Several IDA also bind another Balb/c idiotype: UPC10. ABPC48 and UPC10 are both myeloma proteins with a beta (2 leads to 6)-polyfructosan (levan) specificity. The binding of every IDA to the ABPC48 idiotype can be completely inhibited by levan molecules, but at different concentrations. Mutual inhibition assays between the IDA made it possible to define six groups of IDA which bind at least three different idiotopes of ABPC48. Sixteen IDA have been studied by means of mouse anti-anti-idiotypic antibodies (Ab3) directed against two of them, IDA3 and IDA10. Anti-IDA3 Ab3 recognize idiotopes particular to IDA3 which are not found on other monoclonal anti-idiotypic antibodies (Ab2). Anti-IDA10 AB3 cross-reacts with several monoclonal Ab2, including Ab2 with different spectrotypes belonging or not to the same isotype and Ab2 with different specificities for the ABPC48 idiotype. Some IDA10 idiotopes are present in the polyclonal anti-ABPC48 antibody response of BALB/c, A/J and CBA mice showing that they are recurrent and that their expression is not linked to a particular Igh-C haplotype. In contrast, IDA3 idiotopes are not detected in the same anti-ABPC48 antisera.
Animals↗
Cross-linking of surface immunoglobulins and endocytosis of antigen are not sufficient to suppress antibody production of two hybridoma cell lines.
The two cell lines Lev 1-3 and AS3, which secrete monoclonal anti-levan antibody and express surface membrane immunoglobulins that are capable of binding the levan, were cultured for 24 h or 48 h with levan of various molecular weights (5 x 10(4), 2 x 10(5) or 2 x 10(7) daltons). The production of antibody was measured by a plaque-forming cell assay for AS3 cells and by biosynthetic labelling for Lev 1-3 cells. Surface immunoglobulins were detected by a rosette-forming cell assay. By using immunoenzymatic procedures, we observed that levan of 5 x 10(4) and 2 x 10(5) daltons was intensely endocytosed by the cells and localized mainly in the cellular center, whereas levan of 2 x 10(7) daltons remained mostly associated with the plasma membrane and formed surface aggregates. After transfer of cells in levan-free medium, it was shown that internalized levan could persist several days inside the cells without being degraded. Viability, growth, antibody synthesis and secretion were not modified in cells cultured with levan, whatever the molecular weight of the antigen used. Our results show that cross-linking of surface immunoglobulins by multivalent antigen or endocytosis of antigen do not appear to be sufficient to induce a suppression of antibody production or membrane immunoglobulin expression.
Animals↗
Search for ribosomal mutants in Podospora anserina: genetic analysis of mutants resistant to paromomycin.
It has recently been shown that paromomycin, an antibiotic of the aminoglycoside family, is also active on eukaryotic cytoplasmic ribosomes. In the fungus Podospora anserina, genetic analysis of ten mutants resistant to high doses of paromomycin shows that this resistance is caused by mutations in two different nuclear genes. These mutants display pleiotropic phenotypes (cold sensitivity, mycelium and spore appearance and coloration, cross-resistance to other antibiotics). Double mutants are either lethal or very altered and unstable. Moreover, the cytochrome spectra of these mutants seem to indicate that cytoplasmic protein synthesis is affected. The mutants also display a slight suppressor effect. We can therefore assume that these mutations affect cytoplasmic ribosomes.
Ascomycota↗