Acute lymphoblastic leukemia is the most common form of cancer in children. The 10-year event-free survival ranged from 77 to 85% after having achieved complete remission rates of 93% or higher. The ...main cause of treatment failure is relapse arising from outgrowth of residual leukemic cells that are refractory to therapy. An intense effort has been made to develop methods to determine the degree of minimal residual leukemia cells present in patients considered to be in morphological remission. Because of the strong correlation between minimal residual disease (MRD) levels and risk of relapse, monitoring of MRD provides unique information regarding treatment response. The MRD monitoring based on real-time quantitative PCR detection of patient-specific immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements is currently considered to be the most reliable tool for MRD-based diagnosis in ALL. Because the significance of MRD monitoring has been strongly supported by several studies and because it has been implemented in the latest protocols, there has been a significant effort to develop MRD monitoring in the Slovak Republic since 2005. Between October 2006 and December 2009, 50 children with ALL who were treated at three Slovak centers were included in the RQ PCR MRD pilot project. A total of 40 patients with BCP-ALL ( B cell precursor ALL) and 4 patients with T ALL were analyzed for Ig/TCR rearrangement. We identified 106 different rearrangements in the 44 ALL patients analyzed. Based on MRD stratification, we identified 26 patients who were stratified into the HRG ( high risk group) (n = 3; 11.5%), IRG ( intermediate risk group) (n = 14; 54%) and SRG ) standard risk group) (n = 9; 34.5%). Morphology-based risk stratification allows the identification of most HRG patients identified also by MRD-based stratification, but fails to discriminate the IRG assigned to therapy reduction. Patients in the SRG and the IRG could profit from MRD-based risk assignment
Information on the impact of genetic predisposition on metal toxicokinetics in the human body is limited. There is increasing evidence that certain genetic polymorphisms modify lead and mercury ...toxicokinetics. This called for analysis of further candidate genes.
Medical students (
N=324) were examined in order to detect potential associations between lead exposure and polymorphisms in
HFE,
VDR,
ALAD, and
MT genes, as well as between mercury exposure and
GSTT1,
GSTM1,
GSTA1,
GSTP1,
GCLC, and
MT polymorphisms.
The levels of lead and mercury exposure of students were determined by blood, urine, and hair analyses (ICP-MS, CV-AAS). Genotyping of common polymorphisms was examined by MALDI-TOF MS and the TaqMan methodology. Associations between lead and mercury exposures and genetic background were examined by bivariate analysis, and by categorical regression analysis (CATREG) controlled by metal- and matrix-specific variables.
Lead and mercury levels in urine, blood, and hair indicated low exposures.
VDR polymorphism and joint presence of
VDR/ALAD polymorphisms were significantly and independently associated with urine lead concentrations (CATREG
P<0.05). Polymorphisms in
GSTP1-114 and
MT4 genes as well as dual gene combinations including
GSTP1,
GCLC,
GSTT1, and
GSTM1 polymorphisms were independent variables related to mercury body burdens (CATREG
P<0.05).
GSTP1-114/
GSTT1 and
GSTP1-105/
GCLC combinations showed synergistic effects on hair mercury levels compared to single-gene variants.
We found evidence that certain genetic backgrounds were associated with lead and mercury metabolism, suggesting gene–environment and gene–gene–environment interactions. The modes of interaction remain to be evaluated.
Multidrug resistance in yeast results from over-expression of drug efflux transporter genes due to gain-of-function mutations in transcription factors. To suppress multidrug resistance at the level ...of gene expression, we have developed a yeast-based screening system for the detection of compounds down-regulating the major multidrug ABC transporter Pdr5p expressed under the control of Pdr3p transcription factor. Here, we report the construction and properties of the improved set of yeast strains designed along with such screening also for a global analysis of genetic suppressors of multidrug resistance. The basic components of this system, the
P
GAL1-PDR3
and
P
PDR5-pma1(D378N)
fusion genes, were individually or simultaneously integrated into corresponding chromosomes of a hypersensitive
S. cerevisiae strain deleted in the
PDR1 and
PDR3 genes. This resulted in increased mitotic stability of a set of new test strains compared with the original prototrophic strain ZK11-1 developed previously. In addition, some of the strains designed are auxotrophic for leucine, uracil and histidine allowing them to be used in genetic screens for positive selection of multicopy or loss-of-function genetic suppressors of multidrug resistance.
We demonstrate a genomewide approach to determine the physiological role of a putative transcription factor, Ylr266, identified through yeast genome sequencing program. We constructed activated forms ...of the zinc finger (Zn(2)Cys(6)) protein Ylr266, and we analyzed the corresponding transcriptomes with DNA microarrays to characterize the up-regulated genes. The direct target genes of Ylr266 were further identified by in vivo chromatin immunoprecipitation procedure. The functions of the genes directly controlled by YLR266c are in agreement with the observed drug-resistance phenotype of the cell expressing an activated form of Ylr266. These target genes code for ATP-binding cassette or major facilitator superfamily transporters such as PDR15, YOR1, or AZR1 or for other proteins such as SNG1, YJL216c, or YLL056c which are already known to be involved in the yeast pleiotropic drug resistance (PDR) phenomenon. YLR266c could thus be named PDR8. Overlaps with the other PDR networks argue in favor of a new specific role for PDR8 in connection with the well known PDR regulators PDR1/PDR3 and YRR1. This strategy to identify the regulatory properties of an anonymous transcription factor is likely to be generalized to all the Zn(2)Cys(6) transcription factors from Saccharomyces cerevisiae and related yeasts.
We analysed the genome‐wide regulatory properties of an artificial transcription activator in which the DNA‐binding domain of the yeast transcription factor, Pdr1, was fused to the activation domain ...of Gal4 (Pdr1*GAD). This Pdr1*GAD chimera was put under the control of the inducible GAL1 promoter. DNA microarray analyses showed that all the target genes upregulated by the well‐studied native gain‐of‐function Pdr1‐3 mutant were similarly activated by the chimerical factor Pdr1*GAD upon galactose induction. Additionally, this kinetic approach led us not only to confirm previously published targets, but also to define a hierarchy among members of the Pdr1 regulon. Our observations prove, for the first time at the complete genome level, that the DNA‐binding domain of Pdr1 is sufficient to guide its specificity. We propose that this approach could be useful for the study of new transcription factors identified in silico from sequenced organisms. Complete data are available at www.biologie.ens.fr/yeast‐publi.html.
We have isolated a respiration-deficient nuclear mutant of the yeastKluyveromyces lactis that exhibited diminished levels of all cytochromes and did not grow on glycerol and other nonfermentable ...carbon sources. The mutant namedcyc1 was transformed with aK. lactis genomic library and the DNA fragment conferring its wild-type properties was isolated and sequenced. The sequence of the isolated gene showed extensive homology with other eukaryotic cytochrome-c genes. The highest level of homology, based on the deduced amino acid sequences, was observed between the gene products ofK. lactis andHansenula anomala.
We have isolated a respiration-deficient nuclear mutant of the yeast Kluyveromyces lactis that exhibited diminished levels of all cytochromes and did not grow on glycerol and other nonfermentable ...carbon sources. The mutant named cyc1 was transformed with a K. lactis genomic library and the DNA fragment conferring its wild-type properties was isolated and sequenced. The sequence of the isolated gene showed extensive homology with other eukaryotic cytochrome-c genes. The highest level of homology, based on the deduced amino acid sequences, was observed between the gene products of K. lactis and Hansenula anomala.
Sequence analysis of a DNA fragment containing the KlCOX18 gene originating from chromosome II of the yeast Kluyveromyces lactis revealed the presence of an adjacent open reading frame (ORF) for a ...protein exhibiting 78.4% identity with the Saccharomyces cerevisiae Spt4p. Based on the identical length (102 aa) and the conservation of the zinc-finger motif found in Spt4p we named this ORF KlSPT4. When expressed in S. cerevisiae the KlSPT4 gene complemented all spt4 mutant phenotypes. It is proposed that KlSpt4p, like its S. cerevisiae counterpart is a protein involved in the establishment or maintenance of the chromatin structure that influences the expression of many yeast genes.
We describe the isolation and initial characterization of KlCOX18, a gene that is essential for the assembly of a functional cytochrome oxidase in the yeast Kluyveromyces lactis. Cells carrying a ...recessive nuclear mutation in this gene are respiratory deficient and contain reduced levels of cytochromes a and a3. The KlCOX18 gene has been cloned by complementation of the respective nuclear mutation, sequenced, and disrupted. KlCOX18 is located on chromosome II and contains an open reading frame of 939 base pairs. The corresponding protein exhibits 70.4% similarity to the Cox18p of Saccharomyces cerevisiae. It contains three possible membrane-spanning domains and a putative amino-terminal mitochondrial import sequence. The strain carrying a null mutation in KlCOX18 does not grow on non-fermentable carbon sources and is deficient in both cytochrome c oxidase and respiratory activity. It is proposed that KlCox18p, like its S. cerevisiae counterpart, provides an important function at a later step of the cytochrome oxidase assembly pathway.
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