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PubMed2001

The HIV-1 repeated sequence R as a robust hot-spot for copy-choice recombination.

Template switching during reverse transcription is crucial for retroviral replication. While strand transfer on the terminal repeated sequence R is essential to achieve reverse transcription, template switching from internal regions of the genome (copy choice) leads to genetic recombination. We have developed an experimental system to study copy-choice recombination in vitro along the HIV-1 genome. We identify here several genomic regions, including the R sequence, where copy choice occurred at high rates. The frequency of copy choice occurring in a given region of template was strongly influenced by the surrounding sequences, an observation that suggests a pivotal role of the folding of template RNA in the process. The sequence R, instead, constituted an exception to this rule since it was a strong hot-spot for copy choice in the different sequence contexts tested. We suggest therefore that the structure of this region has been optimised during viral evolution to ensure efficient template switching independently from the sequences that might surround it.

Genome, Viral↗
PubMed2002

Frequent intracellular clonal expansions of somatic mtDNA mutations: significance and mechanisms.

It has been proposed that age-dependent accumulation of somatic mutations in mtDNA is responsible for some aspects of the aging process. However, most cells contain hundreds to thousands of mtDNA molecules. Any nascent somatic mutant therefore appears as a single copy among a majority of wild-type species. A single mutant molecule is unlikely to influence the physiology of the cell and thus cannot play a role in the aging process. To affect cellular physiology, the nascent somatic mutants must somehow accumulate clonally in the cell to significant levels. The evidence supporting the view that, indeed, clonal expansion of mtDNA mutations is a widespread process in various human tissues, and the mechanisms by which clonal expansions may affect the aging process, are reviewed. Originally, clonal expansion was demonstrated for mtDNA with large deletions in muscle. Cell-by-cell analysis of human cardiomyocytes and buccal epithelial cells revealed that clonal expansion affects point mtDNA mutations as well as deletions. Expansions are not limited to muscle, but likely are present in most tissues, and almost every cell of an aged tissue is likely to be affected by an expansion. While the very existence of clonal expansion is beyond doubt, the mechanisms driving this process are largely controversial. The hypotheses explaining expansion includes random or various selective mechanisms, or both. We show that the spectra of expanded point mutations are drastically different in cardiomyocytes and epithelial cells. This suggests that the mechanisms of expansion in these tissues are different. In particular, we propose random segregation and positive selection models for epithelial and muscle cells, respectively.

Aging↗
PubMed1993

Biosynthesis and posttranslational processing of the neurotensin/neuromedin N precursor in the rat medullary thyroid carcinoma 6-23 cell line. Effect of dexamethasone.

Neurotensin (NT) and Neuromedin N (NN) are two biologically active peptides present in one copy each in the C-terminal region of a 169-residue precursor. Four basic Lys-Arg doublets occur within the precursor and represent putative processing sites. We investigated the effects of dexamethasone on the biosynthesis and the posttranslational processing of the NT/NN precursor in the rat medullary thyroid carcinoma 6-23 cell line (rMTC 6-23). Western blot analysis and RIA coupled to HPLC and arginine-directed tryptic cleavage of precursor forms were performed with antisera specific for precursor sequences adjacent to the four basic doublets. These studies revealed that rMTC 6-23 cells synthesized the NT/NN precursor in response to dexamethasone and had the capability to process this precursor at the three Lys-Arg doublets that flank and separate NT and NN, thus yielding authentic NT, NN, and several larger products. The most N-terminal Lys-Arg doublet was not processed in this system. Dexamethasone increased in a concentration- and time-dependent manner the levels of all the NT/NN precursor-derived products. This increase did not affect the relative proportion of the different products. We also showed by Northern blot analysis that both the 1.1-kilobase and 1.5-kilobase NT/NN precursor messenger RNAs were present in the rMTC 6-23 cell line and that the time course and dose response of dexamethasone-induced messenger RNA synthesis were in good agreement with those observed for dexamethasone-induced increase in processing products. The rMTC 6-23 cell line represents a good model to elucidate the steps involved in the posttranslational processing of the NT/NN precursor.

Amino Acid Sequence↗
PubMed1993

Acetyl-CoA carboxylase from yeast is an essential enzyme and is regulated by factors that control phospholipid metabolism.

We have isolated a 1.2-kilobase pair cDNA fragment in a screening for yeast genes regulated at the level of transcription by soluble lipid precursors, inositol and choline. Sequence analysis and comparison of the deduced amino acid sequence to protein databases unveiled 68% similarity of a 374-amino acid peptide fragment to published C termini of chicken and rat acetyl-CoA carboxylase and almost 100% identity to the product of the FAS3 gene from yeast. Several lines of evidence confirm that the cloned gene represents the yeast structural gene ACC1 encoding acetyl-CoA carboxylase. Overexpression of the ACC1 gene from a high copy number plasmid resulted in overexpression of a 250-kDa biotin-enzyme and enzymatic activity of acetyl-CoA carboxylase. Disruption of one ACC1 allele in a diploid wild-type strain resulted in 50% reduction of ACC1-specific mRNA and acetyl-CoA carboxylase specific activity and a marked decrease of biotin associated with a 250-kDa protein, compared to wild-type. After sporulation of diploid disruptants, spores containing the disrupted acc1 allele failed to enter vegetative growth, despite fatty acid supplementation, suggesting that acetyl-CoA carboxylase activity is essential for a process other than de novo fatty acid synthesis and that only a single functional copy of the ACC1 gene exists. ACC1 transcription was repressed 3-fold by lipid precursors, inositol and choline, and was also controlled by regulatory factors Ino2p, Ino4p, and Opi1p, providing evidence that the key step of fatty acid synthesis is regulated in conjunction with phospholipid synthesis at the level of gene expression. The 5'-untranslated region of the ACC1 gene contains a sequence reminiscent of an inositol/choline-responsive element identified in genes encoding phospholipid biosynthetic enzymes.

Acetyl-CoA Carboxylase↗
PubMed1987

HRAS1-selected, chromosome mediated gene transfer; in situ hybridization with combined biotin and tritium label localizes the oncogene and reveals duplications of the human transgenome.

Chromosomes isolated from a human bladder carcinoma cell line which contains the actively transforming oncogene HRAS1 on chromosome 11 can be used to transform mouse cells. We have analyzed these chromosome mediated gene transformants by in situ hybridization techniques using biotinylated human DNA and a double antibody detection system to visualize the whole of the transgenome in a number of cell lines. In some transformants, where the amount of the transgenome was below the level of detection by the simple biotin system, we used a gold-silver enhancement technique. We have developed a combined in situ hybridization procedure using biotinylated human DNA plus antibodies and 3H-labeled HRAS1 DNA plus autoradiography to locate the actively transforming oncogene within the human transgenome in a selection of these transformants. In each of these there were complex insertions of the transgenome, either at multiple sites or with duplicated inserts at a single site. Each insertion contained a copy of HRAS1. The double in situ hybridization analysis helps define the types of arrangement and rearrangement which can accompany the chromosome mediated gene transfer process and, consequently, the potentials and limitations of the technique as a somatic cell and molecular genetic tool. Our analysis also suggests that multiple copies of the HRAS1 gene may be needed for stable transformation.

Animals↗
PubMed1995

Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease.

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1+ gene as a multi-copy suppressor of snm1. The pac1+ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1+ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1+ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.

Amino Acid Sequence↗
PubMed2001

Substitution of Asp114 or Arg116 in the fingers domain of moloney murine leukemia virus reverse transcriptase affects interactions with the template-primer resulting in decreased processivity.

Reverse transcriptase, an essential retroviral DNA polymerase, replicates the single-stranded RNA genome of the retrovirus, producing a double-stranded DNA copy, which is subsequently integrated into the host's genome. Substitution of Ala for either Asp114 or Arg116, two highly conserved residues in the fingers domain of Moloney murine leukemia virus reverse transcriptase, results in enzymes (D114A or R116A) with significant defects in their abilities to processively synthesize DNA using RNA or DNA as a template. D114A and R116A enzymes also bind more weakly to template-primer in the presence of added deoxyribonucleotides, as seen by gel-shift analysis, but retain the ability to strand transfer and accumulate smaller RNase H cleavage products when compared to the wild-type enzyme. In addition, mutant proviruses, including D114A and R116A substitutions in Moloney murine leukemia virus reverse transcriptase, are not viable despite the presence of processed reverse transcriptase in the viral particles. A potential mechanistic role in processive synthesis for D114 and R116 is discussed in the context of our results, related studies on HIV-1 reverse transcriptase, and previous structural studies.

Amino Acid Substitution↗
PubMed2002

Recombinant adeno-associated virus type 2 mediates highly efficient gene transfer in regenerating rat skeletal muscle.

The recent identification of genes responsible for several muscle diseases, particularly inherited myopathies, has made gene transfer to pathologic muscle tissue an attractive research field. As early pathologic changes in myopathic muscle involve repeated necrosis-regeneration cycles, leading to the coexistence of myofibers at different stages of maturity, a delivery system for efficient, durable gene therapy of inherited muscle diseases should allow gene transfer into myofibers at any stage of maturity. Experiments with rat skeletal muscles showed that recombinant adeno-associated virus (rAAV) type 2 can be highly efficient and even improve gene transfer in regenerating as compared with mature muscle, provided that vector injection is performed during the myotube growth period of the regenerative process. At this early period of muscle regeneration, young regenerating myotubes strongly express heparan sulfate proteoglycan AAV type 2 receptor. Improvement was associated with a greater number of transduced myofibers in muscle samples and an increase in viral genomic copies in transduced muscle. No significant deleterious effects on muscle phenotype or any evident alterations in the regenerative process were observed in transduced muscles. Unlike other available viral vectors, whose transduction efficiencies are highly maturation-dependent, rAAV type 2-based vectors provide efficient in vivo gene transfer in myofibers at various stages of maturity, making AAV a promising delivery system for pathological muscle tissue.

Animals↗
PubMed2005

Multiplex ligation-dependent probe amplification for the detection of chromosomal gains and losses in formalin-fixed tissue.

Molecular analysis on formalin-fixed paraffin-embedded tissue is of increasing importance in diagnostic histopathology and tumor research. Multiplex ligation-dependent probe amplification (MLPA) is a technique that can be used for detection of copy number alterations of up to 45 different DNA sequences in one experiment. It can be performed on partially degraded DNA, which makes this technique very suitable for analysis of formalin-fixed lesions. We tested the reliability of MLPA by analyzing DNA isolated from formalin-fixed melanomas that were previously characterized by comparative genomic hybridization (CGH), and additionally the applicability of MLPA was tested by analyzing 29 routinely processed melanocytic lesions. MLPA appears to be a reliable and efficient method to evaluate DNA copy number changes as 86% of the loci tested revealed concordant CGH results. Discordance mainly involved alterations that were detected by MLPA and not by CGH probably due to a combination of lower resolution of CGH and occasionally false positive MLPA results. For application of MLPA in a diagnostic setting, different probes on a specific region of interest should be used to prevent false positive MLPA results. In a research setting as well as in a diagnostic setting, MLPA is a fast technique to screen large numbers of formalin-fixed lesions for DNA gains and losses.

Aneuploidy↗
PubMed1994

Post-translational processing of the neurotensin/neuromedin N precursor in the central nervous system of the rat--I. Biochemical characterization of maturation products.

Neurotensin and neuromedin N are two biologically active related peptides which are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide containing in its C-terminal region one copy each of neurotensin and neuromedin N. Four Lys-Arg sequences which are thought to represent putative processing sites occur in the precursor molecule. Of these sites, the three that are closest to the C-terminus flank and separate neurotensin and neuromedin N. The fourth precedes a neuromedin N-like sequence. The present studies were aimed at determining the extent to which each of these four dibasic sites is cleaved and at identifying and quantifying the intermediate and mature products to which this cleavage gives rise in extracts from whole rat brain, hippocampus and globus pallidus. This was achieved by means of radioimmunoassays specific for sequences of the neurotensin/neuromedin N precursor that are adjacent to the dibasic processing sites used in combination with high pressure liquid chromatography and arginine-directed trypsin digestion of tissue extracts. In all tissue extracts, it was found that the three most C-terminal dibasic processing sites in the neurotensin/neuromedin N precursor are processed to a similar extent, whereas the dibasic site that precedes the neuromedin N-like sequence is processed to a lesser extent. As reported previously, the globus pallidus was shown to contain proportionally lower levels of neuromedin N than other brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Primary structure of the precursor for the anthozoan neuropeptide antho-RFamide from Renilla köllikeri: evidence for unusual processing enzymes.

Neuropeptides containing the C-terminal sequence Arg-Phe-NH2 are an important group of hormones mediating or modulating neuronal communication. Arg-Phe-NH2 peptides are abundant in evolutionarily "old" nervous systems such as those of coelenterates, the lowest animal group having a nervous system. Here, we have cloned the precursor protein for the anthozoan neuropeptide Antho-RFamide (< Glu-Gly-Arg-Phe-NH2) from the sea pansy Renilla köllikeri. This precursor contains 36 copies of immature Antho-RFamide (Gln-Gly-Arg-Phe-Gly) and two additional putative neuropeptide sequences, which are regularly distributed over the precursor protein. Of the 36 Antho-RFamide sequences, 29 copies are separated by the five amino acid spacer sequence Arg-Glu/Gly-Asn/Ser/Asp-Glu/Lys-Glu. This implicates processing at single Arg and single Glu residues. Endoproteolytic cleavage at the C-terminal side of paired or single basic residues is a well known initial step in the maturation of precursor proteins. Cleavage at the C-terminal side of acidic residues, however, is unusual and must be catalyzed by a new type of processing enzyme. This processing enzyme is most likely to be an endoprotease, because the simplest way to generate Antho-RFamide is by endoproteolytic cleavage at the C-terminal side of Glu residues. The enzyme could also be an aminopeptidase, but in this case other proteases must be involved. As a possible alternative, one single "unspecific" aminopeptidase could cleave at Glu, Asp, Gly, Asn, Ser, and possibly also at other residues, and thus liberate all Antho-RFamide sequences. The processing of one precursor molecule probably yields 38 neuropeptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2005

Misregulation of 2 microm circle copy number in a SUMO pathway mutant.

Attachment of the ubiquitin-like protein SUMO to other proteins is an essential process in Saccharomyces cerevisiae. However, yeast mutants lacking the SUMO ligases Siz1 and Siz2/Nfi1 are viable, even though they show dramatically reduced levels of SUMO conjugation. This siz1Delta siz2Delta double mutant is cold sensitive and has an unusual phenotype in that it forms irregularly shaped colonies that contain sectors of wild-type-appearing cells as well as sectors of enlarged cells that are arrested in G(2)/M. We have found that these phenotypes result from misregulation of the copy number of the endogenous yeast plasmid, the 2 microm circle. siz1Delta siz2Delta mutants have up to 40-fold-higher levels of 2 microm than do wild-type strains. Furthermore, 2 microm is responsible for the siz1Delta siz2Delta mutant's obvious growth defects, as siz1Delta siz2Delta [cir(0)] strains, which lack 2 microm, are no longer heterogeneous and show growth characteristics similar to those of the wild type. Possible mechanisms for SUMO's effect on 2 microm are suggested by the finding that both Flp1 recombinase and Rep2, two of the four proteins encoded by 2 microm, are covalently modified by SUMO. Our data suggest that SUMO attachment negatively regulates Flp1 levels, which may partially account for the increased 2 microm copy number in the siz1Delta siz2Delta strain.

Cell Cycle↗
PubMed1997

Bleomycin hydrolase (Blh1p), a multi-sited thiol protease in search of a distinct physiological role.

Bleomycin hydrolase, Blh1p, from yeast was co-purified with Gce1p, a cAMP-binding ectoprotein, anchored to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Blh1p is a hydrophilic thiol protease lacking transmembrane domains. We have used polyclonal antibodies to study the topology of the over-expressed protein in yeast and have found that it is amphitropic. Part of Blh1p is associated with plasma membranes, and most of the rest occurs in the cytosol. Both the growth conditions and calcium were found to have minor influences on the topology of Blh1p, in that glucose and the earth-alkali ion slightly enhanced recruitment to the membrane. We have examined the possibility that co-purification of Blh1p with Gce1p has a functional basis, and have observed that over-expression of BLH1 in yeast leads to an acceleration of the glucose-induced amphiphilic to hydrophilic conversion of Gce1p, wherein Blh1p could either directly catalyse the proteolytic removal of the polar head-group of the GPI anchor subsequent to an initial lipolytic cleavage by a GPI-specific phospholipase C or indirectly modulate the reaction. The data show that a thiol protease is involved, but point to an indirect role of Blh1p in GPI processing. Proteases with similar or overlapping substrate specificity are likely to exist, since deletion of BLH1 neither entails a growth-defect on any carbon source tested, nor the loss of proteolytic processing of the GPI anchor of Gce1p. Reduced proteolytic GPI processing is, however, observed in the blh1 mutant and the corresponding acceleration in the respective BLH1 multi-copy transformant.

Calcium↗
PubMed2026

Chromosome-scale genome remodeling in tumor evolution: Copy number alterations and structural variants as two sides of the same coin.

Chromosome-scale genomic rearrangements are a dominant force in tumor evolution. Copy-number alterations (CNAs) and structural variants (SVs) constitute two complementary axes of this process. Although detection technologies now deliver near-comprehensive catalogs, technical resolution has outpaced conceptual integration. In this review, we frame CNAs and SVs as inextricable facets of chromosomal aberrations. They reshape cancer genomes through altered gene dosage and three-dimensional regulatory rewiring. CNAs quantify the gene-dosage imbalance, yet arise through mechanistically distinct routes. Segmental CNAs typically require chromosomal breakage, and therefore often coincide with SV junctions. By contrast, whole-chromosome aneuploidy and whole-genome doubling (WGD) primarily reflect mitotic or cytokinetic failure and can occur without local breakpoints, while nevertheless reshaping the karyotypic landscape and seeding subsequent structural complexity. SVs, in turn, range from unbalanced events that alter copy number to ostensibly balanced exchanges that predominantly rewire regulatory architecture. Despite their diverse and sometimes catastrophic architectures, SVs are ultimately rooted in double-strand break formation and error-prone resolution. By integrating CNAs and SVs within a unified mechanistic and functional framework, we aim to convert catalogs into concepts and distill the organizing principles that govern tumor genome evolution.

Humans↗
PubMed2002

pH reduction as a trigger for dissociation of herpes simplex virus type 1 scaffolds.

Assembly of the infectious herpes simplex virus type 1 virion is a complex, multistage process that begins with the production of a procapsid, which is formed by the condensation of capsid shell proteins around an internal scaffold fashioned from multiple copies of the scaffolding protein, pre-VP22a. The ability of pre-VP22a to interact with itself is an essential feature of this process. However, this self-interaction must subsequently be reversed to allow the scaffolding proteins to exit from the capsid to make room for the viral genome to be packaged. The nature of the process by which dissociation of the scaffold is accomplished is unknown. Therefore, to investigate this process, the properties of isolated scaffold particles were investigated. Electron microscopy and gradient sedimentation studies showed that the particles could be dissociated by low concentrations of chaotropic agents and by moderate reductions in pH (from 7.2 to 5.5). Fluorescence spectroscopy and circular dichroism analyses revealed that there was relatively little change in tertiary and secondary structures under these conditions, indicating that major structural transformations are not required for the dissociation process. We suggest the possibility that dissociation of the scaffold may be triggered by a reduction in pH brought about by the entry of the viral DNA into the capsid.

Animals↗
PubMed2003

Evidence for a mechanism of recombination during reverse transcription dependent on the structure of the acceptor RNA.

Genetic recombination is a major force driving retroviral evolution. In retroviruses, recombination proceeds mostly through copy choice during reverse transcription. Using a reconstituted in vitro system, we have studied the mechanism of strand transfer on a major recombination hot spot we previously identified within the genome of HIV-1. We show that on this model sequence the frequency of copy choice is strongly influenced by the folding of the RNA template, namely by the presence of a stable hairpin. This structure must be specifically present on the acceptor template. We previously proposed that strand transfer follows a two-step process: docking of the nascent DNA onto the acceptor RNA and strand invasion. The frequency of recombination under copy choice conditions was not dependent on the concentration of the acceptor RNA, in contrast with strand transfer occurring at strong arrests of reverse transcription. During copy choice strand transfer, the docking step is not rate limiting. We propose that the hairpin present on the acceptor RNA could mediate strand transfer following a mechanism reminiscent of branch migration during DNA recombination.

HIV-1↗
PubMedDate not supplied

[Psychiatry reform in Brazil from the 1980's to present days: its history and concepts].

This article presents a brief history of the Brazilian psychiatry reform from the 1980's and 1990's to April 2001, when the Mental Health Law was finally signed. The author discusses the basic concepts that characterize the present condition of the reform as opposed to those of previous initiatives. He also presents a general view of the preceding events to the present process; the concepts and values that characterize the Brazilian reform; the first experiments in São Paulo; the legislative initiative with an analysis of the new law; the different public policies in relation to mental health during the period analyzed with a copy of the Ministry of Health Ordinances 224/92 and 106/2000; the present condition of the de-institutionalization process in the country and the restructuring of the assistance system in the municipality of Rio de Janeiro.

Brazil↗
PubMed1993

Small nucleolar RNAs encoded by introns of the human cell cycle regulatory gene RCC1.

Eukaryotic cells contain a large number of U small nuclear RNAs (U-snRNAs) involved in various RNA processing reactions in the nucleoplasm and nucleous. Most of the U-snRNAs have 5'-terminal caps added to the end of the primary transcript. Here we describe two variants of a snRNA, called U17, identified in human HeLa cells. U17 RNA may be involved in ribosome biogenesis since it is found in the nucleolus and sediments with 40S structures possibly representing nascent ribosomal subunits. U17 RNAs contain no cap but have a monophosphate at the 5'-terminus indicating that they are processed from longer precursors. The U17 RNAs are encoded within introns 1 and 2 of the single copy gene RCC1 which codes for an important cell cycle regulatory protein. In HeLa cell S-100 extract, U17 RNA is faithfully excised from a longer RNA transcript derived from the intron yielding 5'-monophosphorylated RNA. These data suggest that U17 RNAs are not independently transcribed but are processed out of the RCC1 pre-mRNA or out of the spliced introns.

Base Sequence↗