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M Riva

Publications and source records attributed to M Riva.

At least 55 records · Page 3Linked to original sources
PubMed1994

Arguments against a close relationship between non-phosphorylating and phosphorylating glyceraldehyde-3-phosphate dehydrogenases.

Non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.9) from spinach leaves was purified to homogeneity using an improved purification procedure. Thus, a major contaminant with molecular mass and ion-exchange properties similar to non-phosphorylating GAPDH was eliminated. Using this pure non-phosphorylating GAPDH, cofactor stereospecificity was determined by 1H NMR. Analysis of the NADPH formed from the hydride transfer from glyceraldehyde-3-phosphate to [4-2H]NADP showed that the enzyme belongs to the A-stereospecific dehydrogenase family. This stereospecificity is the same as that described for the aldehyde dehydrogenase (ALDH) superfamily and opposite to that of the phosphorylating GAPDH. Moreover, results from peptide sequencing analysis suggest a similarity in sequence between the non-phosphorylating GAPDH and ALDHs. Thus, the results taken all together strongly suggest that non-phosphorylating GAPDH belongs to the ALDH family and has no close relationship to the phosphorylating GAPDH class.

Amino Acid Sequence↗
PubMed1994

The HMG box-containing nucleolar transcription factor UBF interacts with a specific subunit of RNA polymerase I.

The mammalian transcription activator protein UBF contains five tandemly repeated HMG homology domains which are required for DNA binding. We have used highly purified RNA polymerase I (Pol I) and upstream binding factor (UBF) and investigated whether these two proteins interact in solution. We show by a variety of different experimental approaches, such as immunoprecipitation, glycerol gradient sedimentation, affinity chromatography and protein blotting, that UBF physically associates with Pol I. Mutational analysis reveals that the HMG boxes play an important role in this specific interaction. UBF binds to mouse and yeast Pol I, demonstrating that the interaction of UBF with Pol I has been conserved during evolution. Interestingly, in both species one Pol I-specific subunit (34.5 kDa in yeast and 62 kDa in mouse) was recognized by UBF. No specific interaction was observed with Pol II. Unexpectedly, UBF was found to associate also with a unique subunit of yeast Pol III. This apparent specific interaction of UBF with the two classes of RNA polymerases may reflect functionally important interactions of HMG box-containing transcription factors with the transcriptional apparatus.

Animals↗
PubMed1994

Lithium ratio, phospholipids and the incidence of side effects.

The main aims of our Lithium Centre, which has been operating since 1985, are as follows: 1. the treatment of affective disorders and particularly the recurrent ones; 2. the emphasis of the Li-ratio as a predictive index of therapeutic effects on the one hand, and of the appearance of side effects on the other hand; 3. the prevention and pharmacological control of side effects and research about their mechanism of arising.; 4. organization of a computerized bibliographic service.

Adolescent↗
PubMed1994

[Incidence and treatment of local recurrences after conservative surgical treatment for cancer of the breast].

Long-term survival is comparable after total mastectomy or conservative surgery for early breast cancer. Our purpose was to evaluate the rate and therapy of breast relapse at the Regional Hospital of Varese (Italy) in a 10 years period. From 1/1/1980 to 31/12/1990 a total of 606 patients with early breast cancer (Stage I or II early) were evaluated. They all were submitted to quadrantectomy, axillary dissection and radiotherapy. In case of metastases to the axillary lymph nodes, hormono or chiotherapy were performed. 425 patients were regularly followed for a period of 2.5-13 years. In 15 patients (3.2%) a local recurrence was discovered. They were treated as follows, 8 patients: mastectomy, 4 patients: conservative surgery, 4 patients: no therapy. The surgical approach of the local recurrence, mastectomy or conservative surgery, does not influence long-term survival of patients treated with conservative surgery and radiotherapy for early stage breast cancer. Conservative surgery is often possible in treating local recurrence without interfering with complete removal of the tumor and long-term survival.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1993

Purification, characterization, and cellular localization of the 100-kDa human placental GTPase-activating protein.

Human placenta contains, in addition to the ubiquitous p120-GTPase-activating protein (GAP), another isoform of 100 kDa, which is specific to this organ. We have established a method for purifying this placental p100-GAP to near homogeneity. The purified p100-GAP allowed the preparation of polyclonal and monoclonal anti Ras-GAP antibodies. Two monoclonal antibodies were selected for a two-site enzyme immunoassay. This simple and accurate assay in turn facilitated the detection of the GAPs during purification. The purified p100-GAP has a specific activity identical to and catalytic properties similar to those of native p120-GAP. Sequence analysis of p100-GAP revealed almost total identity to the known corresponding sequences predicted by the cDNA. The purified p100-GAP kept its activity for 1 year when stored at -80 degrees C. Our immunometric assay showed GAP to be present in human placental extracts at the exceptional abundance of about 0.1% of the total protein content. Quantitative assays showed p100-GAP to be up to 10 times more abundant than p120-GAP. Use of our antibodies allowed the specific localization of placental GAPs to cytotrophoblasts and in the syncytiotrophoblast barrier. Hence p100-GAP is shown to be found only in trophoblasts. The large quantity of p100-GAP in trophoblasts suggests that it may play a regulatory role in the proliferation or the differentiation of this cell type.

Amino Acid Sequence↗
PubMed1993

The TFIIIB-assembling subunit of yeast transcription factor TFIIIC has both tetratricopeptide repeats and basic helix-loop-helix motifs.

The multisubunit yeast transcription factor IIIC (TFIIIC; also called tau) can undergo considerable conformational changes upon binding to the A and B blocks of tRNA genes. After binding to DNA encoding tRNA (tDNA), TFIIIC acts as an assembly factor to recruit an initiation factor, TFIIIB, via its tau 131 subunit. We have cloned the gene encoding the tau 131 subunit and named it TFC4. This gene is unique, essential for cell viability, and encodes a M(r) 120,153 protein. Epitope-tagging and mobility-shift assays indicated the presence of a single tau 131 subunit in TFIIIC-tDNA complexes. tau 131 contains two sequence motifs, accounting for nearly one-half of the protein mass, that may provide a molecular explanation for the properties of TFIIIC-tDNA complex. A series of 11 copies of the tetratricopeptide repeat motif may account for the flexibility and interaction properties of TFIIIC. A motif akin to the basic helix-loop-helix motif of MyoD suggests the direct involvement of tau 131 in promoting DNA binding of TFIIIB.

Amino Acid Sequence↗
PubMed1993

Three-dimensional model of yeast RNA polymerase I determined by electron microscopy of two-dimensional crystals.

Two-dimensional crystals of yeast RNA polymerase I dimers were obtained upon interaction with positively charged lipid layers. A three-dimensional surface model of the enzyme was determined by analyzing tilted crystalline areas and by taking advantage of the non-crystallographic internal symmetry of the dimer to correct for the missing viewing directions. The structure shows, at approximately 3 nm resolution, an irregularly shaped molecule 11 nm x 11 nm x 15 nm in size characterized by a 3 nm wide and 10 nm long groove which constitutes a putative DNA binding site. The overall structure is similar to the Escherichia coli holo enzyme and the yeast RNA polymerase II delta 4/7 structures. The most remarkable structural feature is a finger-shaped stalk which partially occludes the entrance of the groove and forms a 2.5 nm wide channel. We discuss the possible location of the catalytic centre and of the carboxy-terminal region of the beta-like subunit in the channel. The interference of different DNA fragments with RNA polymerase dimerization and crystallization indicates the orientation of the template in the putative DNA binding groove.

Amino Acid Sequence↗
PubMed1992

TFC3: gene encoding the B-block binding subunit of the yeast transcription factor IIIC.

Yeast transcription factor IIIC (TFIIIC) is a multisubunit protein complex that interacts with two control elements of class III promoters called the A and B blocks. Here we describe the gene encoding the 138-kDa subunit (tau 138), which is involved in B-block binding. From the DNA sequence, the open reading frame, interrupted by an intron with an unusual 3' splice acceptor site, is in agreement with all the microsequencing data for peptides within tau 138. TFC3 is a single-copy gene located on chromosome I; it is essential for cell viability as shown by a gene disruption experiment. Epitope-tagging of the TFC3 gene product and DNA binding experiments are consistent with the presence of one copy of tau 138 in TFIIIC-DNA complexes.

Amino Acid Sequence↗
PubMed1992

An essential and specific subunit of RNA polymerase III (C) is encoded by gene RPC34 in Saccharomyces cerevisiae.

The RPC34 gene of Saccharomyces cerevisiae was cloned by immunological screening, using antibodies raised against the C34 polypeptide of the RNA polymerase III (C). This single copy gene was located near the centromere of chromosome XIV. It included a coding sequence of 317 amino acids that strictly matched two internal oligopeptides of C34. This polypeptide is a specific component of RNA polymerase III, with no significant homology to any other RNA polymerase subunit known so far. It is an essential subunit, since inactivation by deletion or nonsense mutations led to a recessive lethal phenotype. Moreover, a partially blocked mutant, rpc34-F297, had a reduced tRNA synthesis in vivo but no detectable effect on 5 S RNA synthesis. The latter phenotype was observed for all conditionally defective RNA polymerase III mutants isolated so far.

Amino Acid Sequence↗
PubMed1992

Determination of lysine residues affinity labeled in the active site of yeast RNA polymerase II(B) by mutagenesis.

In a previous study, yeast RNA polymerase II(B) was affinity labeled with two nucleotide derivatives (III and VIII) (1). In both cases, the labeled site was localized to the C-terminal part of the B150 subunit. The potential target lysyl residues of derivative III were mapped to the conserved domain H, between Asn946 and Met999. In the present work, we have mutagenized to arginine the five lysines present in domain H. Three lysines can be replaced, individually or simultaneously, without affecting cell growth, and each mutated enzyme can still be affinity labeled. Hence one or both of the other two lysyl residues, Lys979 and Lys987, is the target of the affinity reagent. These two lysines were each found to be essential for cell viability. Derivative VIII labeled another domain in addition to domain H. Supported by analogous results obtained for E. coli RNA polymerase using derivative VIII (2), we hypothesized that the second domain labeled by this derivative in the B150 subunit was domain I. Mutagenesis of the unique lysine present in domain I demonstrated that Lys 1102 was the target of derivative VIII. These results indicate that in both prokaryotic and eukaryotic RNA polymerases, domains H and I are in close proximity and participate to the active site.

Affinity Labels↗
PubMed1992

RPC82 encodes the highly conserved, third-largest subunit of RNA polymerase C (III) from Saccharomyces cerevisiae.

RNA polymerase C (III) promotes the transcription of tRNA and 5S RNA genes. In Saccharomyces cerevisiae, the enzyme is composed of 15 subunits, ranging from 160 to about 10 kDa. Here we report the cloning of the gene encoding the 82-kDa subunit, RPC82. It maps as a single-copy gene on chromosome XVI. The UCR2 gene was found in the opposite orientation only 340 bp upstream of the RPC82 start codon, and the end of the SKI3 coding sequence was found only 117 bp downstream of the RPC82 stop codon. The RPC82 gene encodes a protein with a predicted M(r) of 73,984, having no strong sequence similarity to other known proteins. Disruption of the RPC82 gene was lethal. An rpc82 temperature-sensitive mutant, constructed by in vitro mutagenesis of the gene, showed a deficient rate of tRNA relative to rRNA synthesis. Of eight RNA polymerase C genes tested, only the RPC31 gene on a multicopy plasmid was capable of suppressing the rpc82(Ts) defect, suggesting an interaction between the polymerase C 82-kDa and 31-kDa subunits. A group of RNA polymerase C-specific subunits are proposed to form a substructure of the enzyme.

Amino Acid Sequence↗
PubMed1992

RPC10 encodes a new mini subunit shared by yeast nuclear RNA polymerases.

Yeast RNA polymerases A, B, and C share five small subunits, two of which, ABC10 alpha and ABC10 beta, comigrate on SDS polyacrylamide gels. The gene encoding ABC10 alpha, RPC10, was isolated based on microsequence data. RPC10 is a single copy gene localized on chromosome VIII. It codes for a very basic protein of only 70 amino acids, which contains a zinc binding domain of the form CX2CX13CX2C. Deletion of its gene indicated that, despite its very small size, the ABC10 alpha subunit is essential for yeast cell viability. ABC10 alpha and ABC10 beta have little sequence similarity.

Amino Acid Sequence↗
PubMed1991

Two additional common subunits, ABC10 alpha and ABC10 beta, are shared by yeast RNA polymerases.

Yeast nuclear RNA polymerases are multisubunit enzymes that contain in common some small subunits. We show that the smallest, a 10-kDa component of three enzymes (A10, B10, and C10), is heterogeneous. In each case, it can be resolved into two distinct polypeptides (alpha and beta) by reverse-phase chromatography. A10 alpha, B10 alpha, and C10 alpha were indistinguishable on the basis of their electrophoretic and chromatographic behaviors, characteristic silver staining, and tryptic peptide analysis. All three polypeptides are blocked at their amino termini. By the same criteria, A10 beta, B10 beta, and C10 beta were also indistinguishable. The amino-terminal sequence of A10 beta and C10 beta corresponded to that of subunit B10 recently cloned by Woychik and Young (Woychik, N. A., and Young, R. A. (1990) J. Biol. Chem. 265, 17816-17819). Thus, the three forms of RNA polymerase share two additional and distinct polypeptides, ABC10 alpha and ABC10 beta, that therefore can be considered bona fide subunits of these enzymes. Interestingly, these two subunits bind zinc.

Chromatography, High Pressure Liquid↗
PubMed1991

Zinc-binding subunits of yeast RNA polymerases.

The zinc-binding subunits of yeast RNA polymerase A(I) and B(II) have been identified by a zinc-blotting technique. The two largest subunits of each enzyme (A190, A135, B220, and B150), as well as A12.2, A10, B44.5, B12.6, and B10, bind 65Zn(II). Predicted zinc-binding motifs have been noted in the NH2-terminal part of B220 and the COOH-terminal region of B150 subunits. Subdomains encompassing these motifs have been overproduced as MalE-fusion proteins and shown to retain zinc binding activity. Site-directed mutagenesis in the predicted metal-binding domain of B150 demonstrated its role in zinc binding. Mutations of cysteine residues C1163, C1166, C1182, and C1185 affected 65Zn2+ binding in vitro and caused a lethal or thermosensitive phenotype for growth. The ability to bind zinc is not sufficient for function since mutations in vicinal residues not affecting zinc binding were either lethal or thermosensitive. The role of zinc in RNA polymerase structure and function is discussed in the light of the present results.

Base Sequence↗
PubMed1991

RPC19, the gene for a subunit common to yeast RNA polymerases A (I) and C (III).

Yeast RNA polymerases A (I) and C (III) share a subunit called AC19. The gene encoding AC19 has been isolated from yeast genomic DNA using oligonucleotide probes deduced from peptide sequences of the isolated subunit. This gene (RPC19) contains an intron-free open reading frame of 143 amino acid residues. RPC19 is a single copy gene that maps on chromosome II and is essential for cell viability. The amino acid sequence contains a sequence motif common to the Escherichia coli RNA polymerase alpha subunit, the Saccharomyces cerevisiae AC40 and B44.5 subunits, the human hRPB33 product, and the CnjC conjugation-specific gene product of Tetrahymena. The 5'-upstream region contains a sequence element, the PAC box, that has been conserved in at least 10 genes encoding subunits of RNA polymerases A and C.

Amino Acid Sequence↗
PubMed1991

Identification of the genes coding for the second-largest subunits of RNA polymerases I and III of Drosophila melanogaster.

We have isolated cDNA and genomic clones of Drosophila melanogaster by cross-hybridization with a 658 bp fragment of the yeast gene coding for the second-largest subunit of RNA polymerase III (RET1). Determination of the sequence by comparison of genomic and cDNA regions reveals an ORF of 3405 nucleotides which is interrupted in the genomic sequence by an intron of 48 bp. The deduced polypeptide consists of 1135 amino acids with a calculated molecular weight of 128 kDa. The protein sequence shows the same conserved regions of homology as those observed for all the second-largest subunits of RNA polymerases cloned so far. The gene (DmRP128) obviously codes for a second-largest subunit of an RNA polymerase which is different from DmRP140 and DmRP135. We have purified three distinct RNA polymerase activities from D. melanogaster. By using specific RNA polymerase inhibitors in enzyme assays and by comparing their subunit composition we were able to distinguish between RNA polymerase I, II, and III. RNA polymerase preparations of D. melanogaster were blotted and the second-largest subunits were identified with antibodies raised against polypeptides expressed from DmRP128 and DmRP135. Anti-DmRP135 antibodies react strongly with the second-largest subunit of RNA polymerase I but do not react with the respective subunits of RNA polymerase II and III. The second-largest subunit of RNA polymerase III is only recognized by anti-DmRP128. Previously, we have claimed that DmRP135 codes for the second-largest subunit of RNA polymerase III.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗