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M Ares

Publications and source records attributed to M Ares.

At least 55 records · Page 3Linked to original sources
PubMed1991

Efficient association of U2 snRNPs with pre-mRNA requires an essential U2 RNA structural element.

To understand the role of U2 RNA structure in pre-mRNA splicing we have characterized several cold-sensitive mutations in an essential stem-loop of yeast U2. Although mutant U2 is stable in vivo after a shift to restrictive temperature, splicing is rapidly inhibited, suggesting a direct effect on U2 function rather than U2 synthesis or snRNP assembly. Splicing complexes form at 23 degrees C in both mutant and wild-type extracts; however, stable association of mutant U2 snRNPs with pre-mRNA in vitro is inefficient at 15 degrees C, a temperature permissive for spliceosome assembly in wild-type extracts, indicating that the cold-sensitive defect is in U2 snRNP association with the assembling spliceosome. In vivo RNA structure probing reveals that the bulk of U2 RNA is misfolded in the mutants, even at permissive temperature. We propose that U2 stem-loop IIa is recognized by an assembly factor that assists U2 snRNP binding to pre-mRNA and that the cold sensitivity is due to a critical deficiency of correctly folded U2 for spliceosome assembly at low temperatures. Evolutionary conservation of the potential to form an interfering alternative RNA structure suggests the possibility that splicing could be regulated negatively at an early step by control of U2 snRNA conformation.

Base Sequence↗
PubMed1990

Lethal and temperature-sensitive mutations and their suppressors identify an essential structural element in U2 small nuclear RNA.

U2 snRNA is an essential component of the splicing apparatus in eukaryotic cells. Three possible secondary structures for the highly conserved 5' half of U2 snRNA are consistent with U2 phylogenetic sequence variation. To distinguish among these models and to test the function of U2 structural elements, we made greater than 35 mutations in the yeast U2 snRNA gene. Some of the mutations were designed in pairs so that combinations could be made that would restore base-pairing to differentiate helix requirements from primary sequence requirements. The mutations identify an essential stem-and-loop structure adjacent to the branchpoint interaction region. A conserved complementarity to the loop just upstream of the Sm site and an additional conserved stem-loop are dispensable for U2 function, even in the background of a previously identified large internal deletion. Non-Watson-Crick base appositions at the 53-62 base pair in the essential stem lead to a variety of temperature and KCl-sensitive phenotypes, as well as an accumulation of unspliced precursors in vivo. Chemical structure probing of U2 RNA in vivo reveals that the bulk of U2 in a yeast cell adopts a structure in good agreement with that deduced from genetic results. We suggest that this stem-loop is not a binding site for an intrinsic U2 snRNP protein but may interact with other factors during spliceosome assembly or splicing.

Base Sequence↗
PubMed1988

Internal sequences that distinguish yeast from metazoan U2 snRNA are unnecessary for pre-mRNA splicing.

U2 small nuclear RNA is a highly conserved component of the eukaryotic cell nucleus involved in splicing messenger RNA precursors. In the yeast Saccharomyces cerevisiae, U2 RNA interacts with the intron by RNA-RNA pairing between the conserved branchpoint sequence UACUAAC and conserved nucleotides near the 5' end of U2 (ref. 4). Metazoan U2 RNA is less than 200 nucleotides in length, but yeast U2 RNA is 1,175 nucleotides long. The 5' 110 nucleotides of yeast U2 are homologous to the 5' 100 nucleotides of metazoan U2 (ref. 6), and the very 3' end of yeast U2 bears a weak structural resemblance to features near the 3' end of metazoan U2. Internal sequences of yeast U2 share primary sequence homology with metazoan U4, U5 and U6 small nuclear RNA (ref. 6), and have regions of complementarity with yeast U1 (ref. 7). We have investigated the importance of the internal U2 sequences by their deletion. Yeast cells carrying a U2 allele lacking 958 nucleotides of internal U2 sequence produce a U2 small nuclear RNA similar in size to that found in other organisms. Cells carrying only the U2 deletion grow normally, have normal levels of spliced mRNA and do not accumulate unspliced precursor mRNA. We conclude that the internal sequences of yeast U2 carry no essential function. The extra RNA may have a non-essential function in efficient ribonucleoprotein assembly or RNA stability. Variation in amount of RNA in homologous structural RNAs has precedence in ribosomal RNA and RNaseP.

Base Sequence↗
PubMed1987

Distinct factors with Sp1 and NF-A specificities bind to adjacent functional elements of the human U2 snRNA gene enhancer.

The enhancer regions of mammalian and avian U1 and U2 small nuclear RNA (snRNA) genes are unusual in containing the sequence GGGCGG (GC-box) immediately upstream from the sequence ATGCAAAT (octamer). We made point mutations in the human U2 snRNA enhancer and tested them for the ability to direct U2 transcription in HeLa cells, as well as for the ability to form complexes with factors present in HeLa cell nuclear extracts. We show that neither the GC-box nor the octamer alone is sufficient for enhancer activity in vivo. Mutations in the GC-box reduce the ability of enhancer DNA fragments to bind a factor (probably Sp1), whereas mutations in the octamer independently reduce the ability to bind a second factor (probably nuclear factor A, NF-A). The results suggest that adjacent binding of Sp1 and NF-A is an important feature of some U-snRNA gene enhancers.

Base Sequence↗
PubMed1986

U2 RNA from yeast is unexpectedly large and contains homology to vertebrate U4, U5, and U6 small nuclear RNAs.

I have determined the structure of the gene from Saccharomyces cerevisiae coding for the yeast homolog of vertebrate U2 snRNA. Surprisingly, the RNA is 1175 nucleotides long, six times larger than U2 RNAs from other organisms, including Schizosaccharomyces pombe. Nearly 100 nucleotides of the large RNA share sequence homology and potential secondary structure with metazoan U2. The large RNA also contains homology to vertebrate U4, U5, and U6 snRNAs, implying a "poly-snRNP" structure for the RNP containing the large RNA. The gene LSR1, encoding the large RNA, is essential for growth, suggesting that the yeast spliceosome can be dissected using genetic approaches. The different organization of spliceosomal RNA may underlie differences in splicing between yeast and metazoans.

Animals↗
PubMed1986

Human U2 small nuclear RNA genes contain an upstream enhancer.

The human U1 and U2 snRNA genes lack an obvious TATA box, but are extremely powerful RNA polymerase II transcription units capable of accurately initiating at least one transcript per gene every 2-4 s. We have investigated the location of cis-acting regulatory elements within the flanking sequences of human U2 and U1 genes. By introducing marked human U2 genes into HeLa cells on SV40- and pUC13-based vectors, we found that transient expression of the marked U2 gene did not require the SV40 enhancer. The U2 promoter element responsible for SV40 enhancer-independent U2 expression was localized within the 5'-flanking sequence of the gene, and shown to stimulate transcription from the U2 basal promoter in an orientation- and position-independent fashion. In addition, the U2 element could be functionally replaced by either the SV40 enhancer or by distal sequences from the human U1 promoter. We conclude that the human U2 and U1 genes contain functionally equivalent enhancer elements. Moreover, since the human U2 enhancer sequences resemble the Xenopus U2 enhancer-like element, enhancers appear to be a general feature of vertebrate snRNA promoter structure.

Base Sequence↗
PubMed1985

U1 small nuclear RNA genes are subject to dosage compensation in mouse cells.

Multiple copies of a gene that encodes human U1 small nuclear RNA were introduced into mouse C127 cells with bovine papilloma virus as the vector. For some recombinant constructions, the human U1 gene copies were maintained extrachromosomally on the viral episome in an unrearranged fashion. The relative abundance of human and mouse U1 small nuclear RNA varied from one cell line to another, but in some lines human U1 RNA accounted for as much as one-third of the total U1. Regardless of the level of human U1 expression, the total amount of U1 RNA (both mouse and human) in each cell line was nearly the same relative to endogenous mouse 5S or U2 RNA. This result was obtained whether measurements were made of total cellular U1 or of only the U1 in small nuclear ribonucleoprotein particles that could be precipitated with antibody directed against the Sm antigen. The data suggest that the multigene families encoding mammalian U1 RNA are subject to some form of dosage compensation.

Animals↗
PubMed1985

Sequences required for 3' end formation of human U2 small nuclear RNA.

Xenopus oocytes injected with human U2 snRNA genes synthesize mature U2 as well as a U2 precursor with about 10 extra 3' nucleotides (human pre-U2 RNA). Formation of the pre-U2 3' end requires a downstream element located between position +16 and +37 in the U2 3'-flanking sequence. The distance between this element and the U2 coding region can be increased without affecting formation of the pre-U2 3' end. When the natural sequence surrounding the pre-U2 3' end is changed, novel 3' ends are still generated within a narrow range upstream from the element. The 3' terminal stem-loop of U2 snRNA is not required for pre-U2 3' end formation. A sequence within the 3' element (GTTTN0-3AAAPuNNAGA) is conserved among snRNA genes transcribed by RNA polymerase II. Our results suggest that the 3' ends of pre-U2 RNA and histone mRNA may be generated by related but distinct RNA processing mechanisms.

Animals↗
PubMed1985

Orientation-dependent transcriptional activator upstream of a human U2 snRNA gene.

We examined the structure of the promoter for the human U2 snRNA gene, a strong RNA polymerase II transcription unit without an obvious TATA box. A set of 5' deletions was constructed and assayed for the ability to direct initiation of U2 snRNA after microinjection into Xenopus oocytes. Sequences between positions -295 and -218 contain an activator element which stimulates accurate initiation by 20- to 50-fold, although as few as 62 base pairs of 5' flanking sequence are sufficient to direct the accurate initiation of U2 RNA. When the activator was recloned in the proper orientation at either of two different upstream locations, the use of the normal U2 start site was stimulated. Inversion of the element destroyed the stimulation of accurate U2 initiation, but initiation at aberrant upstream start sites was enhanced by the element in both orientations. A 4-base-pair deletion that destroyed the activity of the element lies within a sequence (region III) which is highly conserved among U2 genes from different organisms. Mutations in the activator also affected the ability of the U2 template to compete with a wild-type U1 gene in coinjection experiments. We propose that the element enhances the efficiency of transcription in part by facilitating the association of a limiting factor with transcription complexes. Human U1 snRNA genes possess a region homologous to U2 region III, and we suggest that upstream activator elements may be a general feature of snRNA promoters.

Animals↗
PubMed1983

Analysis of transcription during the cell cycle in toluenized Chlamydomonas reinhardi cells.

A toluene-permeabilized cell system was established to examine the transcription of certain RNAs regulated during the cell cycle in Chlamydomonas reinhardi. The incorporation of [alpha-32P]UTP into RNA which hybridizes to specific cloned cDNA, such as beta-tubulin, indicates that the cell cycle pattern of RNA accumulation may be controlled, in part, by differential transcription.

Cell Cycle↗
PubMed1982

Cell cycle stage-specific accumulation of mRNAs encoding tubulin and other polypeptides in Chlamydomonas.

The accumulation pattern of a number of mRNAs during the cell cycle of Chlamydomonas was examined by two-dimensional gel analysis of in vitro translation products and by RNA blot hybridization analysis. Two-dimensional gel analysis revealed that 10-15% of the 300 most abundant translation products are differentially synthesized from RNA obtained at various cell cycle stages. RNAs that direct the synthesis of alpha- and beta-tubulins and that hybridize to cloned alpha- and beta-tubulin probes accumulate coordinately during the predivision period of the cell cycle, reaching peak levels before or during division. Other RNAs represented by selected cloned cDNA probes show a number of different cell cycle patterns of accumulation. The accumulation patterns of these RNAs are not directly influenced by ongoing illumination conditions, even though alternating light-dark illumination cycles are used to synchronize Chlamydomonas cells. The results suggest that there may be a complex program of gene expression correlated with cell cycle progression in Chlamydomonas.

Cell Cycle↗
PubMed1978

Cycloheximide resistance can be mediated through either ribosomal subunit.

Two cycloheximide-resistant mutants of Tetrahymena thermophila were analyzed to determine the site of their cycloheximide resistance. The mutations in both strains had been previously shown to be genetically dominant and located at separate loci (denoted Chx-A and Chx-B). Strains carrying these mutations were readily distinguished by the extent to which they were resistant to the drug. The homozygous double mutant was more resistant than either single mutant. Cell-free extracts of wild type and of the three mutant strains, assayed for protein synthetic activity by both runoff of natural mRNA and poly(U)-dependent phenylalanine polymerization, demonstrated that in vitro the mutants were all more resistant than the wild type. Further fractionation of the cell-free systems into ribosomes and supernates localized cycloheximide resistance to the ribosome for both Chx-A and Chx-B homozygotes. Ribosome dissociation and pairwise subunit mixing in the in vitro system indicated that ribosome resistance was conferred by the 60S subunit from one strain whereas resistance in the other strain was mediated through the 40S subunit. This was further confirmed by reconstruction of all four cycloheximide-resistance "phenotypes" by mixing ribosomal subunits from appropriate strains. This finding suggests that the mechanisms by which these mutations confer resistance to cycloheximide are different.

Animals↗
PubMed1978

Isolation and genetic characterization of a mutation affecting ribosomal resistance to cycloheximide in Tetrahymena.

A dominant mutation at a new locus affecting resistance to cycloheximide has been isolated by exploiting a synergistic relationship with a previously known mutation for cycloheximide resistance in Tetrahymena. The new mutation (ChxB) was induced in a line homozygous for ChxA and was recovered from that background by a new technique termed interrupted genomic exclusion. Segregation data from the interrupted genomic exclusion suggest that ChxA and ChxB are separate, linked loci showing 30% recombination. Minimal lethal doses of cycloheximide for the four possible combinations of the wild-type and mutant alleles of these two genes are: wild type 6 microgram/ml, ChxA 125 microgram/ml, ChxB 10 microgram/ml, ChxA-ChxB 175 microgram/ml.

Animals↗
PubMedDate not supplied

Molecular tracking of infections by Leishmania infantum.

Leishmania infantum is a major opportunistic parasite in patients with acquired immune deficiency syndrome and is very variable in these subjects. Isoenzyme characterization is not able to explain this variability, since half of the stocks isolated from patients co-infected with human immunodeficiency virus and Leishmania belong to zymodeme MON-1. Amplification of L. infantum minicircles by the polymerase chain reaction (PCR) and digestion of the amplified product to reveal restriction fragment length polymorphisms (RFLP) has proved very useful in distinguishing between relapses and reinfections in co-infected, treated patients. We have confirmed the existence of a leishmaniasis outbreak among intravenous drug users in north-east Spain, previously detected by isoenzymatic analysis. We have documented persistence of the same strain of Leishmania in 2 treated co-infected patients throughout several years, regardless of the theoretical rapid evolution ascribed to kinetoplast deoxyribonucleic acid minicircle sequences. We suggest using this PCR-RFLP technique to detect reinfections in treated co-infected subjects.

AIDS-Related Opportunistic Infections↗