Overexpression of the heat-shock protein 70 is associated to imatinib resistance in chronic myeloid leukemia.

Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative disorder characterized by the expression of the recombinant oncoprotein Bcr-Abl. In this investigation, we studied an imatinib-resistant cell line (K562-r) generated from the K562 cell line in which none of the previously described mechanisms of resistance had been detected. A threefold increase in the expression of the heat-shock protein 70 (Hsp70) was detected in these cells. This increase was not associated to heat-shock transcription factor-1 (HSF-1) overexpression or activation. RNA silencing of Hsp70 decreased dramatically its expression (90%), and was accompanied by a 34% reduction in cell viability. Overexpression of Hsp70 in the imatinib-sensitive K562 line induced resistance to imatinib as detected by a large reduction in cell death in the presence of 1 muM of imatinib. Hsp70 level was also increased in blast cells of CML patients resistant to imatinib, whereas the level remained low in responding patients. Taken together, the results demonstrate that overexpression of Hsp70 can lead to both in vitro and in vivo resistance to imatinib in CML cells. Moreover, the overexpression of Hsp70 detected in imatinib-resistant CML patients supports this mechanism and identifies potentially a marker and a therapeutic target of CML evolution.

Forest fire indicators and mercury deposition in an intense land use change region in the Brazilian Amazon (Alta Floresta, MT).

Black carbon, charcoal and mercury fluxes were measured from sediment cores taken in an artificial water dam in an intense land use change area in the Alta Floresta district in the Brazilian Amazon, in order to characterize the differences in the evolution of human occupation patterns in the region during the last 18 years. A positive correlation between the black carbon and charcoal particle fluxes and the evolution of the Brazilian gross domestic production (GDP) was observed. Mercury fluxes showed a positive correlation with gold production and exhibited a distinct evolution pattern when compared to in relation to the forest fires indicators and Brazilian GDP. The fluxes of forest fires markers showed an increase in deforestation activities in the region after 1993. Mercury deposition showed a substantial decrease after 1994. The patterns of distribution in both forest fires tracers and gold mining tracers indicate substitution of the regional economic model. It also marked different antropogenic impact type in the ecosystem.

Vegetative incompatibility in filamentous fungi: a roundabout way of understanding the phenomenon.

Vegetative incompatibility limits heterokaryon formation in fungi. It results from genetic differences at specific loci (het loci). Characterization of het genes and, more recently, of incompatibility reaction suppressors has, provided insight into the mechanisms involved. A link between development, vegetative incompatibility and signaling pathways has been established in Podospora anserina.

MOD-D, a Galpha subunit of the fungus Podospora anserina, is involved in both regulation of development and vegetative incompatibility.

Cell death via vegetative incompatibility is widespread in fungi but molecular mechanism and biological function of the process are poorly understood. One way to investigate this phenomenon was to study genes named mod that modified incompatibility reaction. In this study, we cloned the mod-D gene that encodes a Galpha protein. The mod-D mutant strains present developmental defects. Previously, we showed that the mod-E gene encodes an HSP90. The mod-E1 mutation suppresses both vegetative incompatibility and developmental defects due to the mod-D mutation. Moreover, we isolated the PaAC gene, which encodes an adenylate cyclase, as a partial suppressor of the mod-D1 mutation. Our previous results showed that the molecular mechanisms involved in vegetative incompatibility and developmental pathways are connected, suggesting that vegetative incompatibility may result from disorders in some developmental steps. Our new result corroborates the involvement of mod genes in signal transduction pathways. As expected, we showed that an increase in the cAMP level is able to suppress the defects in vegetative growth due to the mod-D1 mutation. However, cAMP increase has no influence on the suppressor effect of the mod-D1 mutation on vegetative incompatibility, suggesting that this suppressor effect is independent of the cAMP pathway.

Use of a linear plasmid containing telomeres as an efficient vector for direct cloning in the filamentous fungus Podospora anserina.

In Podospora anserina a linear plasmid with telomeric ends behaves as an artificial acentric minichromosome. Transformation is at least 100 times more efficient than with integrative vectors. Genomic DNA was inserted in this plasmid in vitro and the mixture used to transform a leu1-1 strain. Many fungal clones containing the leu1 gene as a genomic insert in the linear plasmid were identified. The leu1 gene was rescued as a circular plasmid in Escherichia coli demonstrating that a direct cloning procedure can be applied for the fungus P. anserina. The conservation of telomeric sequences among filamentous fungi suggests that a telomere-based linear plasmid could provide a general cloning vector for filamentous fungi.

A mutation in an HSP90 gene affects the sexual cycle and suppresses vegetative incompatibility in the fungus Podospora anserina.

Vegetative incompatibility is widespread in fungi but its molecular mechanism and biological function are still poorly understood. A way to study vegetative incompatibility is to investigate the function of genes whose mutations suppress this phenomenon. In Podospora anserina, these genes are known as mod genes. In addition to suppressing vegetative incompatibility, mod mutations cause some developmental defects. This suggests that the molecular mechanisms of vegetative incompatibility and development pathway are interconnected. The mod-E1 mutation was isolated as a suppressor of the developmental defects of the mod-D2 strain. We show here that mod-E1 also partially suppresses vegetative incompatibility, strengthening the link between development and vegetative incompatibility. mod-E1 is the first suppressor of vegetative incompatibility characterized at the molecular level. It encodes a member of the Hsp90 family, suggesting that development and vegetative incompatibility use common steps of a signal transduction pathway. The involvement of mod-E in the sexual cycle has also been further investigated.

Reactivity in vegetative incompatibility of the HET-E protein of the fungus Podospora anserina is dependent on GTP-binding activity and a WD40 repeated domain.

The het-e gene of the filamentous fungus Podospora anserina is involved in vegetative incompatibility. Co-expression of antagonistic alleles of the unlinked loci het-e and het-c triggers a cell death reaction that prevents the formation of viable heterokaryons between strains that contain incompatible combinations of het-c and het-e alleles. The het-elA gene encodes a polypeptide that contains a putative GTP-binding site and WD40 repeats. The role of these two domains in the reactivity of the HET-E protein in incompatibility was analyzed. An in vitro assay confirmed that the first domain is functional and can bind GTP and not ATP, suggesting that GTP-binding is essential for triggering the incompatibility reaction. The relationship between the number of WD40 repeats and the reactivity of the protein in incompatibility was investigated by estimating this number in different wild-type and mutant het-e alleles. It was deduced that reactive alleles contain a minimal number of ten WD40 repeats. These results demonstrate that the reactivity of the HET-E protein depends on two functional elements, a GTP-binding domain and several WD40 repeats. These motifs are present in separate polypeptides in trimeric G proteins, suggesting that HET-E polypeptides are also involved in signal transduction. Disruption of the het-e locus does not impair the phenotype of strains but DNA hybridization analyses revealed that het-e may belong to a multigenic family.

An additional copy of the adenylate cyclase-encoding gene relieves developmental defects produced by a mutation in a vegetative incompatibility-controlling gene in Podospora anserina.

To identify cellular functions involved in vegetative incompatibility in filamentous fungi, we have initiated the cloning of Podospora anserina (Pa) mod genes. These genes interfere with the lethal reaction triggered by interaction between incompatible het genes. A gene (Pa AC) has been cloned by complementation of developmental defects caused by a mutation in the mod-D gene. This gene encodes a protein of 2145 amino acids (aa)that exhibits strong similarities with many adenylate cyclases (AC). About 65% aa identity has been found between the sequence of the polypeptide encoded by this Pa AC gene and the AC of Neurospora crassa. The organization of peptidic domains in the polypeptide encoded by Pa AC is closely related to that of Saccharomyces cerevisiae CYR1. Restriction-fragment-length polymorphism (RFLP) and genetic analysis have shown that Pa AC and mod-D are distinct genes.

A gene responsible for vegetative incompatibility in the fungus Podospora anserina encodes a protein with a GTP-binding motif and G beta homologous domain.

The het-e-1 gene of the fungus Podospora anserina is responsible for vegetative incompatibility through specific interactions with different alleles of the unlinked gene, het-c. Coexpression of two incompatible genes triggers a cell death reaction that prevents heterokaryon formation. The het-e1 allele has been cloned to get information on the function of the locus. It encodes a putative 1356-amino-acid polypeptide that displays two sequence motifs that have not yet been reported to be present on a single polypeptide. They are a GTP-binding domain and a repeated region that shares similarity with that of the beta-transducin. Contrary to other members of the beta-transducin family, sequence conservation between the repeated units is very strong and the number of repeats is different in wild-type het-e alleles.

Sequence diversity and unusual variability at the het-c locus involved in vegetative incompatibility in the fungus Podospora anserina.

The het-c locus of the filamentous fungus Podospora anserina controls heterokaryon formation through genetic interaction with alleles of the unlinked loci het-e and het-d. We have isolated four wild-type and two mutant alleles of the het-c locus. A comparison of the predicted proteins encoded by the different wild-type alleles revealed an unusual high level of amino-acid replacements compared to silent polymorphisms but only one amino-acid difference is sufficient to modify the specificity of het-c alleles. Chimeric genes constructed in vitro may exhibit a new specificity different from that of any known wild-type allele.

Vegetative incompatibility in filamentous fungi: het genes begin to talk.

Somatic or vegetative incompatibility is widespread in filamentous fungi. It prevents the coexistence of genetically different nuclei within a common cytoplasm. Cloning the het genes that control this process has been achieved in several species. This has provided essential information on the function of the genes in the biology of fungi and has also led to the formulation of models that may explain similar phenomena in other organisms.

Inactivation of the Podospora anserina vegetative incompatibility locus het-c, whose product resembles a glycolipid transfer protein, drastically impairs ascospore production.

The het-c locus contains different alleles that elicit nonallelic vegetative incompatibility through specific interactions with alleles of the unlinked loci het-e and het-d. The het-c2 allele has been cloned. It encodes a 208-amino acid polypeptide that is similar to a glycolipid transfer protein purified from pig brain. Disruption of this gene drastically impairs ascospore production in homozygous crosses, and some mutants exhibit abnormal branching of apical hyphae. The protein encoded by het-c2 is essential in the biology of the fungus and may be involved in cell-wall biosynthesis.

A protein required for RNA processing and splicing in Neurospora mitochondria is related to gene products involved in cell cycle protein phosphatase functions.

The Neurospora crassa cyt-4 mutants have pleiotropic defects in mitochondrial RNA splicing, 5' and 3' end processing, and RNA turnover. Here, we show that the cyt-4+ gene encodes a 120-kDa protein with significant similarity to the SSD1/SRK1 protein of Saccharomyces cerevisiae and the DIS3 protein of Schizosaccharomyces pombe, which have been implicated in protein phosphatase functions that regulate cell cycle and mitotic chromosome segregation. The CYT-4 protein is present in mitochondria and is truncated or deficient in two cyt-4 mutants. Assuming that the CYT-4 protein functions in a manner similar to the SSD1/SRK1 and DIS3 proteins, we infer that the mitochondrial RNA splicing and processing reactions defective in the cyt-4 mutants are regulated by protein phosphorylation and that the defects in the cyt-4 mutants result from failure to normally regulate this process. Our results provide evidence that RNA splicing and processing reactions may be regulated by protein phosphorylation.

The Neurospora crassa cyt-20 gene encodes cytosolic and mitochondrial valyl-tRNA synthetases and may have a second function in addition to protein synthesis.

The cyt-20-1 mutant of Neurospora crassa is a temperature-sensitive, cytochrome b- and aa3-deficient strain that is severely deficient in both mitochondrial and cytosolic protein synthesis (R.A. Collins, H. Bertrand, R.J. LaPolla, and A.M. Lambowitz, Mol. Gen. Genet. 177:73-84, 1979). We cloned the cyt-20+ gene by complementation of the cyt-20-1 mutation and found that it contains a 1,093-amino-acid open reading frame (ORF) that encodes both the cytosolic and mitochondrial valyl-tRNA synthetases (vaIRSs). A second mutation, un-3, which is allelic with cyt-20-1, also results in temperature-sensitive growth, but not in gross deficiencies in cytochromes b and aa3 or protein synthesis. The un-3 mutant had also been reported to have pleiotropic defects in cellular transport process, resulting in resistance to amino acid analogs (M.S. Kappy and R.L. Metzenberg, J. Bacteriol. 94:1629-1637, 1967), but this resistance phenotype is separable from the temperature sensitivity in crosses and may result from a mutation in a different gene. The 1,093-amino-acid ORF encoding vaIRSs is the site of missense mutations resulting in temperature sensitivity in both cyt-20-1 and un-3 and is required for the transformation of both mutants. The opposite strand of the cyt-20 gene encodes an overlapping ORF of 532 amino acids, which may also be functional but is not required for transformation of either mutant. The cyt-20-1 mutation in the vaIRS ORF results in severe deficiencies of both mitochondrial and cytosolic vaIRS activities, whereas the un-3 mutation does not appear to result in a deficiency of these activities or of mitochondrial or cytosolic protein synthesis sufficient to account for its temperature-sensitive growth. The phenotype of the un-3 mutant raises the possibility that the vaIRS ORF has a second function in addition to protein synthesis.

Two allelic genes responsible for vegetative incompatibility in the fungus Podospora anserina are not essential for cell viability.

Vegetative incompatibility is a lethal reaction that destroys the heterokaryotic cells formed by the fusion of hyphae of non-isogenic strains in many fungi. That incompatibility is genetically determined is well known but the function of the genes triggering this rapid cell death is not. The two allelic incompatibility genes, s and S, of the fungus Podospora anserina were characterized. Both encode 30 kDa polypeptides, which differ by 14 amino acids between the two genes. These two proteins are responsible for the incompatibility reaction that results when cells containing s and S genes fuse. Inactivation of the s or S gene by disruption suppresses incompatibility but does not affect the growth or the sexual cycle of the mutant strains. This suggests that these incompatibility genes have no essential function in the life cycle of the fungus.

repa, a repetitive and dispersed DNA sequence of the filamentous fungus Podospora anserina.

The sequences of homologous DNA regions of two wild-type strains of the fungus Podospora anserina, revealed in one strain the presence of a 349bp insertion leading to a RFLP. This DNA sequence is repeated in the genome and some of its locations are different in various wild-type strains. This DNA element exhibits structural similarities with the yeast solo delta, sigma or tau elements.

The ura5 gene of the filamentous fungus Podospora anserina: nucleotide sequence and expression in transformed strains.

We have sequenced the ura5 gene of the filamentous fungus Podospora anserina. The deduced sequence for the orotidylic acid pyrophosphorylase (OMPppase) has been compared with the Escherichia coli enzyme which is the only known sequence for this enzyme. This comparison shows extensive blocks of homology. The expression of the ura5 gene has been studied in a ura5 mutant which has been transformed by a recombinant plasmid carrying the ura5 gene. We observed that strains carrying integrated multicopies of the transforming vector exhibit higher specific activity for OMPppase than wild type (wt). By recombination we have constructed a strain in which the level of this enzyme is 32 times higher than in the wt strain.