ABSTRACT
Previously, we have shown that shortening of telomeres by telomerase inhibition sensitized cancer cells to cisplatinum, slowed their migration, increased DNA damage and impaired DNA repair. The mechanism behind these effects is not fully characterized. Its clarification could facilitate novel therapeutics development and may obviate the time consuming process of telomere shortening achieved by telomerase inhibition. Here we aimed to decipher the microRNA and proteomic profiling of cancer cells with shortened telomeres and identify the key mediators in telomere shortening-induced damage to those cells. Of 870 identified proteins, 98 were differentially expressed in shortened-telomere cells. 47 microRNAs were differentially expressed in these cells; some are implicated in growth arrest or act as oncogene repressors. The obtained data was used for a network construction, which provided us with nodal candidates that may mediate the shortened-telomere dependent features. These proteins' expression was experimentally validated, supporting their potential central role in this system.
Subject(s)
MicroRNAs/genetics , Neoplasms/genetics , Neoplasms/metabolism , Proteome/analysis , Telomere Shortening , Gene Expression Regulation, Neoplastic/drug effects , Gene Regulatory Networks , Humans , Oligonucleotides/pharmacology , Proteomics , Tumor Cells, CulturedABSTRACT
Ph.Eur. and BP have introduced a dissolution apparatus for suppositories. Suitability of the apparatus for quality control of paracetamol suppositories was evaluated and the effect of experimental conditions on dissolution profiles was studied. Paracetamol suppositories containing 80-500 mg of the drug, on fatty base, were obtained from four manufacturers (A, B, C, D). The diffusion cell was modified by incorporation of an in-built thermoprobe and large difference (up to 1.7 degrees C) between temperature in the water-bath and in the dissolution chamber was observed. This effect was avoided by increasing the length of tubing immersed in the thermostat at the inlet of the cell. The most reproducible results were observed for A and C suppositories, however from suppository C the total dose of paracetamol was released after 3.5-4.5 h while the release from suppository A was slow with only 40-87% of the total dose liberated during 6 h. Suppositories B did not melt at 37 degrees C and less than 5% of the drug was released. Fast release was observed after melting when the temperature was elevated to 39.5 degrees C. The results demonstrate clearly that essentially complete melting of a suppository in the dissolution chamber is required for an appropriate dissolution of paracetamol in vitro. Disintegration time, softening time, drop point and particle size of the suspended drug were measured and the relevance of these parameters for dissolution behaviour of the preparations was discussed.
Subject(s)
Acetaminophen/pharmacokinetics , Analgesics, Non-Narcotic/pharmacokinetics , Acetaminophen/chemistry , Analgesics, Non-Narcotic/chemistry , Chemistry, Pharmaceutical , Drug Evaluation, Preclinical/methods , Solubility , Suppositories , Technology, Pharmaceutical/methodsABSTRACT
The Saccharomyces cerevisiae genes RAD5, RAD18, and SRS2 are proposed to act in post-replicational repair of DNA damage. We have investigated the genetic interactions between mutations in these genes with respect to cell survival and ectopic gene conversion following treatment of logarithmic and early stationary cells with UV- and gamma-rays. We find that the genetic interaction between the rad5 and rad18 mutations depends on DNA damage type and position in the cell cycle at the time of treatment. Inactivation of SRS2 suppresses damage sensitivity both in rad5 and rad18 mutants, but only when treated in logarithmic phase. When irradiated in stationary phase, the srs2 mutation enhances the sensitivity of rad5 mutants, whereas it has no effect on rad18 mutants. Irrespective of the growth phase, the srs2 mutation reduces the frequency of damage-induced ectopic gene conversion in rad5 and rad18 mutants. In addition, we find that srs2 mutants exhibit reduced spontaneous and UV-induced sister chromatid recombination (SCR), whereas rad5 and rad18 mutants are proficient for SCR. We propose a model in which the Srs2 protein has pro-recombinogenic or anti-recombinogenic activity, depending on the context of the DNA damage.
Subject(s)
Adenosine Triphosphatases , DNA Damage/genetics , Genes, Fungal , Recombination, Genetic/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , DNA Helicases/genetics , DNA Repair , DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Models, Genetic , Radiation, Ionizing , Saccharomyces cerevisiae/radiation effectsABSTRACT
The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.
Subject(s)
Cell Cycle Proteins/physiology , Cell Cycle/genetics , DNA-Binding Proteins , Fungal Proteins/physiology , Genes, Fungal , RNA Helicases , RNA Precursors/metabolism , RNA Splicing/genetics , RNA, Fungal/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Amino Acid Motifs , Amino Acid Sequence , Animals , Caenorhabditis elegans/genetics , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , DEAD-box RNA Helicases , Fungal Proteins/chemistry , Fungal Proteins/genetics , G2 Phase/genetics , Humans , Molecular Sequence Data , RNA Splicing Factors , RNA, Small Nuclear/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/physiology , Sequence Alignment , Sequence Homology, Amino Acid , Spliceosomes/geneticsABSTRACT
Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty). Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing. In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition. Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes. We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression.
Subject(s)
Cell Cycle/physiology , DNA-Binding Proteins , Fungal Proteins/metabolism , RNA Precursors/genetics , RNA Splicing , RNA-Binding Proteins , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Base Sequence , Cell Cycle Proteins/metabolism , DNA Primers , Molecular Sequence Data , Oligodeoxyribonucleotides , Plasmids , RNA Splicing Factors , RNA, Fungal/geneticsABSTRACT
Homologous recombination is an important DNA repair mechanism in vegetative cells. During the repair of double-strand breaks, genetic information is transferred between the interacting DNA sequences, thus creating a gene-conversion event. Gene conversion of a functional member of a gene family, which uses an inactive member (such as a pseudogene) as a template, might have deleterious consequences. It is therefore important for the cell to prevent recombination between divergent sequences. We have studied the repair of a double-strand break by recombination in a haploid yeast strain carrying 99% identical alleles located on different chromosomes. The fate of the broken chromosome was followed in the whole cell population without imposing selective constraints. Our results show that all the cells were able to repair the broken chromosome by gene conversion. During the repair, the cells arrest in the cell cycle with a "dumbbell" configuration characteristic of G2/M-arrested cells. Surprisingly, although all the cells repaired the broken chromosome, 60% of them were unable to resume growth and to form colonies after the repair was completed. The low level of cell recovery was due to the 1% divergence between the alleles, but was not dependent on the function of the mismatch-repair system. Cell death, however, could be prevented by the presence of an alternative source of perfect homology located on a different chromosome.
Subject(s)
Base Pair Mismatch , Cell Cycle/physiology , DNA Repair , Recombination, Genetic , Saccharomyces cerevisiae/genetics , Gene Conversion , Genes, Fungal , Restriction Mapping , Saccharomyces cerevisiae/cytologyABSTRACT
Prokaryotic and eukaryotic cells have developed a network of DNA repair systems that restore genomic integrity following DNA damage from endogenous and exogenous genotoxic sources. One of the mechanisms used to repair damaged chromosomes is genetic recombination, in which information present as a second chromosomal copy is used to repair a damaged region of the genome. In this review, I summarized what is known about the molecular and cellular mechanisms by which various DNA-damaging agents induce recombination in yeast. The yeast Saccharomyces cerevisiae has served as an excellent model organism to study the induction of recombination. It has helped to define the basic phenomenology and to isolate the genes involved in the process. Given the evolutionary conservation of the various DNA repair systems in eukaryotes, it is likely that the knowledge gathered about induced recombination in yeast is applicable to mammalian cells and thus to humans. Many carcinogens are known to induce recombination and to cause chromosomal rearrangements. An understanding of the mechanisms, by which genotoxic agents cause increased levels of recombination will have important consequences for the treatment of cancer, and for the assessment of risks arising from exposure to genotoxic agents in humans.
Subject(s)
Cell Cycle/genetics , DNA Damage/genetics , Recombination, Genetic , Alkylating Agents/pharmacology , Cross-Linking Reagents/pharmacology , DNA Damage/drug effects , DNA Damage/radiation effects , Humans , Transcription, Genetic , Ultraviolet RaysABSTRACT
Homologous recombination can result in the transfer of genetic information from one DNA molecule to another (gene conversion). These events are often accompanied by a reciprocal exchange between the interacting molecules (termed "crossing over"). This association suggests that the two types of events could be mechanistically related. We have analyzed the repair, by homologous recombination, of a broken chromosome in yeast. We show that gene conversion can be uncoupled from crossing over when the length of homology of the interacting substrates is below a certain threshold. In addition, a minimal length of homology on each broken chromosomal arm is needed for crossing over. We also show that the coupling between gene conversion and crossing over is affected by the mismatch repair system; mutations in the MSH2 or MSH6 genes cause an increase in the crossing over observed for short alleles. Our results provide a mechanism to explain how chromosomal recombinational repair can take place without altering the stability of the genome.
Subject(s)
Base Pair Mismatch , Chromosomes , Crossing Over, Genetic , DNA Repair , Gene Conversion , Recombination, Genetic , Saccharomyces cerevisiae Proteins , Alleles , Blotting, Southern , DNA Restriction Enzymes/metabolism , DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Kinetics , Models, Genetic , MutS Homolog 2 Protein , Mutagenesis, Site-Directed , Mutation , Plasmids/metabolism , Saccharomyces cerevisiae/genetics , Time FactorsABSTRACT
Biochemical and genetic experiments have shown that the PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that plays a role during the second catalytic step of the splicing reaction. It was found recently that PRP17 is identical to the cell division cycle CDC40 gene. cdc40 mutants arrest at the restrictive temperature after the completion of DNA replication. Although the PRP17/CDC40 gene product is essential only at elevated temperatures, splicing intermediates accumulate in prp17 mutants even at the permissive temperature. In this report we describe extensive genetic interactions between PRP17/CDC40 and the PRP8 gene. PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and for both catalytic steps of the splicing reaction. We show that mutations in the PRP8 gene are able to suppress the temperature-sensitive growth phenotype and the splicing defect conferred by the absence of the Prp17 protein. In addition, these mutations are capable of suppressing certain alterations in the conserved PyAG trinucleotide at the 3' splice junction, as detected by an ACT1-CUP1 splicing reporter system. Moreover, other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p and show a reduced ability to splice an intron bearing an altered 3' splice junction. On the basis of these findings, we propose a model for the mode of interaction between the Prp8 and Prp17 proteins during the second catalytic step of the splicing reaction.
Subject(s)
Cell Cycle/genetics , DNA-Binding Proteins , Genes, Fungal , RNA Precursors/genetics , RNA Splicing , RNA, Messenger/genetics , RNA-Binding Proteins , Saccharomyces cerevisiae Proteins , Alleles , Base Sequence , Cell Cycle Proteins/genetics , Dosage Compensation, Genetic , Fungal Proteins/genetics , Mutagenesis , Phenotype , RNA , RNA Splicing Factors , Ribonucleoprotein, U4-U6 Small Nuclear , Ribonucleoprotein, U5 Small Nuclear , Saccharomyces cerevisiae/geneticsABSTRACT
The RAD52 group of genes in the yeast Saccharomyces cerevisiae controls the repair of DNA damage by a recombinational mechanism. Despite the growing evidence for physical and biochemical interactions between the proteins of this repair group, mutations in individual genes show very different effects on various types of recombination. The RAD59 gene encodes a protein with similarity to Rad52p which plays a role in the repair of damage caused by ionizing radiation. In the present study we have examined the role played by the Rad59 protein in mitotic ectopic recombination and analyzed the genetic interactions with other members of the repair group. We found that Rad59p plays a role in ectopic gene conversion that depends on the presence of Rad52p but is independent of the function of the RecA homologue Rad51p and of the Rad57 protein. The RAD59 gene product also participates in the RAD1-dependent pathway of recombination between direct repeats. We propose that Rad59p may act in a salvage mechanism that operates when the Rad51 filament is not functional.
Subject(s)
DNA-Binding Proteins/metabolism , Genes, Fungal/physiology , Recombination, Genetic/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , DNA Repair/genetics , DNA Repair Enzymes , DNA-Binding Proteins/genetics , Endonucleases/genetics , Endonucleases/metabolism , Epistasis, Genetic , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Conversion/genetics , Gene Conversion/radiation effects , Genes, Fungal/genetics , Mitosis/genetics , Mutation , Rad51 Recombinase , Rad52 DNA Repair and Recombination Protein , Recombination, Genetic/radiation effects , Repetitive Sequences, Nucleic Acid/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/radiation effects , Ultraviolet RaysABSTRACT
Homologous recombination is an important DNA repair mechanism in vegetative cells. During the repair of double-strand breaks, genetic information is transferred between the interacting DNA sequences (gene conversion). This event is often accompanied by a reciprocal exchange between the homologous molecules, resulting in crossing over. The repair of DNA damage by homologous recombination with repeated sequences dispersed throughout the genome might result in chromosomal aberrations or in the inactivation of genes. It is therefore important to understand how the suitable homologous partner for recombination is chosen. We have developed a system in the yeast Saccharomyces cerevisiae that can monitor the fate of a chromosomal double-strand break without the need to select for recombinants. The broken chromosome is efficiently repaired by recombination with one of two potential partners located elsewhere in the genome. One of the partners has homology to the broken ends of the chromosome, whereas the other is homologous to sequences distant from the break. Surprisingly, a large proportion of the repair is carried out by recombination involving the sequences distant from the broken ends. This repair is very efficient, despite the fact that it requires the processing of a large chromosomal region flanking the break. Our results imply that the homology search involves extensive regions of the broken chromosome and is not carried out exclusively by sequences adjacent to the double-strand break. We show that the mechanism that governs the choice of homologous partners is affected by the length and sequence divergence of the interacting partners, as well as by mutations in the mismatch repair genes. We present a model to explain how the suitable homologous partner is chosen during recombinational repair. The model provides a mechanism that may guard the integrity of the genome by preventing recombination between dispersed repeated sequences.
Subject(s)
DNA/physiology , Nucleic Acid Heteroduplexes/physiology , Recombination, Genetic/physiology , Saccharomyces cerevisiae/genetics , Aldehyde Oxidoreductases/genetics , Alleles , Base Pair Mismatch/genetics , Fungal Proteins/genetics , L-Aminoadipate-Semialdehyde Dehydrogenase , Models, Genetic , Time FactorsABSTRACT
The argH gene encoding argininosuccinate lyase (ASL) of Methanococcus maripaludis was cloned on a 4.7-kb HindIII genomic fragment. The gene is preceded by a short open reading frame (ORF149), which encodes a polypeptide with an unknown function. The two genes are co-transcribed. The ASL of M. maripaludis shares a high amino acid identity with ASLs from both bacterial and eukaryal origins and was able to complement both an argH Escherichia coli mutant and an arg4 yeast mutant, showing its extraordinary evolutionary conservation. Attempts to create an argH auxotroph of M. maripaludis by disrupting the genomic allele were unsuccessful: although a knockout allele of argH was integrated into the M. maripaludis chromosome by homologous recombination, the intact copy was not excluded, suggesting that the argH gene is essential.
Subject(s)
Argininosuccinate Lyase/genetics , Argininosuccinate Lyase/metabolism , Escherichia coli/genetics , Methanococcus/genetics , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Argininosuccinate Lyase/chemistry , Conserved Sequence , DNA, Archaeal , Escherichia coli/enzymology , Genetic Complementation Test , Methanococcus/enzymology , Molecular Sequence Data , Plasmids/genetics , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/enzymology , Sequence Alignment , Transformation, GeneticABSTRACT
We have identified a novel splicing factor, Isy1p, through two-hybrid screens for interacting proteins involved in nuclear pre-mRNA splicing. Isy1p was tagged and demonstrated to be part of the splicing machinery, associated with spliceosomes throughout the splicing reactions. At least a portion of the Isy1 protein population is associated with snRNAs; low levels of U5 and U6 snRNAs are coimmunoprecipitated specifically with Isy1p. When the ISY1 gene was knocked out, no defect in vegetative growth was observed. Using a sensitive in vivo splicing assay, however, we observed lower splicing efficiency in the isy1 null mutant compared to wild-type, indicating that Isy1 p is important in the optimization of splicing.
Subject(s)
RNA Splicing/genetics , RNA-Binding Proteins/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Cloning, Molecular , Fungal Proteins/chemistry , Fungal Proteins/genetics , Gene Deletion , Genes, Reporter/genetics , Molecular Sequence Data , Mutation , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Precipitin Tests , RNA Precursors/genetics , RNA, Messenger/analysis , RNA-Binding Proteins/chemistry , Ribonucleoproteins, Small Nuclear/genetics , Sequence Alignment , Spliceosomes/geneticsABSTRACT
The S. cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is essential only at elevated temperatures. Cells carrying mutations in this gene arrest with a large bud and a single nucleus with duplicated DNA content. Cdc40p is also required for spindle establishment or maintenance. Sequence analysis reveals that CDC40 is identical to PRP17, a gene involved in pre-mRNA splicing. In this paper, we show that Cdc40p is required at all temperatures for efficient entry into S-phase and that cell cycle arrest associated with cdc40 mutations is independent of all the known checkpoint mechanisms. Using immunofluorescence, we show that Cdc40p is localized to the nuclear membrane, weakly associated with the nuclear pore. Our results point to a link between cell cycle progression, pre-mRNA splicing, and mRNA export.
Subject(s)
Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA-Binding Proteins , RNA Splicing , RNA-Binding Proteins , S Phase/genetics , Saccharomyces cerevisiae/genetics , Cell Cycle Proteins/drug effects , Cell Membrane , Cell Nucleus , Fluorescent Antibody Technique , G1 Phase/genetics , Methyl Methanesulfonate/pharmacology , RNA Splicing Factors , RNA, Messenger , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , TemperatureABSTRACT
The PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that participates in the second step of the splicing reaction. It was found recently that the yeast PRP17 gene is identical to the cell division cycle CDC40 gene. The PRP17/CDC40 gene codes for a protein with several copies of the WD repeat, a motif found in a large family of proteins that play important roles in signal transduction, cell cycle progression, splicing, transcription, and development. In this report, we describe the identification of human, nematode, and fission yeast homologues of the PRP17/CDC40 gene of S. cerevisiae. The newly identified proteins share homology with the budding yeast protein throughout their entire sequence, with the similarity being greatest in the C-terminal two thirds that includes the conserved WD repeats. We show that a yeast-human chimera, carrying the C-terminal two thirds of the hPRP17 protein, is able to complement the cell cycle and splicing defects of a yeast prp17 mutant. Moreover, the yeast and yeast-human chimeric proteins co-precipitate the intron-exon 2 lariat intermediate and the intron lariat product, providing evidence that these proteins are spliceosome-associated. These results show the functional conservation of the Prp17 proteins in evolution and suggest that the second step of splicing takes place by a similar mechanism throughout eukaryotes.
Subject(s)
Cell Cycle Proteins/genetics , Cell Cycle/genetics , DNA-Binding Proteins , RNA Splicing/genetics , RNA-Binding Proteins , Sequence Homology, Amino Acid , Animals , Caenorhabditis elegans/genetics , Cell Cycle Proteins/metabolism , Gene Expression , Genes/genetics , Genetic Complementation Test , Humans , Molecular Sequence Data , RNA Splicing Factors , RNA, Messenger/analysis , Recombinant Fusion Proteins , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/genetics , Sequence Alignment , Spliceosomes/metabolismABSTRACT
We have created an isogenic series of yeast strains that carry genetic systems to monitor different types of recombination and mutation [B. Liefshitz, A. Parket, R. Maya, M. Kupiec, The role of DNA repair genes in recombination between repeated sequences in yeast, Genetics 140 (1995) 1199-1211.]. In the present study we characterize the effect of mutations in genes of the 'error-prone' or postreplicative repair group on recombination and mutation. We show that rad5 and rad18 strains have elevated levels of spontaneous recombination, both of ectopic gene conversion and of recombination between direct repeats. The increase in recombination levels is similar in both mutants and in the rad5 rad18 double mutant, suggesting that the RAD5 and RAD18 gene products act together with respect to spontaneous recombination. In contrast, RAD5 and RAD18 play alternative roles in mutagenic repair: mutations in each of these genes elevate spontaneous forward mutation at the CAN1 locus, but when both genes are deleted, a low level of spontaneous mutagenesis is seen. The RAD5/RAD18 pathway of mutagenic repair is dependent on the REV3-encoded translesion polymerase. We analyze the interactions between the RAD5 and RAD18 gene products and other repair genes. The high recombination levels seen in rad5 and rad18 mutants is dependent on the RAD1, RAD51, RAD52, and RAD57 genes. The Srs2 helicase plays an important role in creating the recombinogenic substrate(s) processed by the RAD5 and RAD18 gene products.
Subject(s)
Adenosine Triphosphatases , DNA Repair/genetics , Genes, Fungal/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , DNA Helicases , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Fungal Proteins/genetics , Fungal Proteins/physiology , Mutagenesis/genetics , Mutagenesis/physiology , Mutation/genetics , Recombination, Genetic/genetics , Research DesignABSTRACT
This report describes the results of applying the computer-assisted image analysis system for the measurement of some cytological parameters of LPS-stimulated and nonstimulated human monocytes. The experiments were carried out by means of the digital cell image analysis of haematoxilyn stained monocytes. Five different parameters describing the morphology of monocytes and their nuclei were selected to quantitate the differences between control and activated cells area, perimeter, elongation, dispersion, and extension of images of cell projections. The results suggest that all of the analysed parameters can be used to discriminate stimulated from nonstimulated monocytes which permits detailed monitoring of the changes in cell morphology during monocyte activation.
Subject(s)
Image Processing, Computer-Assisted , Lipopolysaccharides/pharmacology , Monocytes/cytology , Biometry , Humans , Lymphocyte ActivationABSTRACT
Ty elements belong to the family of LTR-containing retrotransposons. Ty RNA is reverse transcribed by Ty-encoded proteins. The cDNA then transposes to new locations in the genome by a process that involves the integrase protein encoded by the element. We have previously shown that the Ty cDNA molecule can participate in recombination events with genomic Tys. In this study we have analyzed the role of the integrase protein in cDNA-mediated Ty recombination. We found that this process involves the integrase protein in a temperature-dependent manner. In addition we have investigated whether double-stranded DNA is the only molecule that can participate in cDNA-mediated Ty recombination. We have shown that mutations in the polypurine tract that abolish plus-strand synthesis do not prevent cDNA-mediated recombination, implying that other types of intermediates from the reverse-transcription process (e.g. single-stranded DNA or a hybrid RNA-cDNA molecule) can participate in cDNA-mediated Ty recombination.
Subject(s)
DNA, Complementary/genetics , Integrases/physiology , Recombination, Genetic/genetics , Retroelements/genetics , DNA, Complementary/biosynthesis , DNA, Fungal/biosynthesis , DNA, Fungal/genetics , Integrases/genetics , Saccharomyces cerevisiae/geneticsABSTRACT
Mitotic recombination in the yeast Saccharomyces cerevisiae is induced when cells are irradiated with UV or X-rays, reflecting the efficient repair of damage by recombinational repair mechanisms. We have used multiply marked haploid strains that allow the simultaneous detection of several types of ectopic recombination events. We show that inter-chromosomal ectopic conversion of lys2 heteroalleles and, to a lesser extent, direct repeat recombination (DRR) between non-tandem repeats, are increased by DNA-damaging agents; in contrast, ectopic recombination of the naturally occurring Ty element is not induced. We have tested several hypotheses that could explain the preferential lack of induction of Ty recombination by DNA-damaging agents. We have found that the lack of induction cannot be explained by a cell cycle control or by an effect of the mating-type genes. We also found no role for the flanking long terminal repeats (LTRs) of the Ty in preventing the induction. Ectopic conversion, DRR, and forward mutation of artificial repeats show different kinetics of induction at various positions of the cell cycle, reflecting different mechanisms of recombination. We discuss the mechanistic and evolutionary aspects of these results.
Subject(s)
DNA Damage , DNA Repair , Recombination, Genetic , Repetitive Sequences, Nucleic Acid , Saccharomyces cerevisiae/genetics , Cell Cycle , DNA Transposable Elements , Genes, Fungal , Genes, Mating Type, Fungal , Mitosis , Ultraviolet Rays , X-RaysABSTRACT
In the yeast Saccharomyces cerevisiae ectopic recombination has been shown to occur at high frequencies for artificially created repeats, but at relatively low frequencies for a natural family of repeated sequences, the Ty family. Little is known about the mechanism(s) that prevent recombination between repeated sequences. We have previously shown that nonreciprocal recombination (gene conversion) of a genetically marked Ty can be induced either by the presence of high levels of Ty cDNA or by transcription of the marked Ty from a GAL1 promoter. These two kinds of induction act in a synergistic manner. To further characterize these two kinds of Ty recombination, we have investigated the role played by the RAD52 and RAD1 genes. We have found that the RAD52 and RAD1 gene products are essential to carry out transcription-induced Ty conversion whereas cDNA-mediated conversion can take place in their absence.