Chloroquine resistance not linked to mdr-like genes in a Plasmodium falciparum cross

Chloroquine is thought to act against falciparum malaria by accumulating in the acid vesicles of the parasite and interfering with their function. Parasites resistant to chloroquine expel the drug rapidly in an unaltered form, thereby reducing levels of accumulation in the vesicles. The discovery that verapamil partially reverses chloroquine resistance in vitro led to the proposal that efflux may involve an ATP-driven P-glycoprotein pump similar to that in mammalian multidrug-resistant (mdr) tumor cell lines. Indeed, Plasmodium falciparum contains at least two mdr-like genes, one of which has been suggested to confer the chloroquine resistant (CQR) phenotype. To determine if either of these genes is linked to chloroquine resistance, we performed a genetic cross between CQR and chloroquine-susceptible (CQS) clones of P. falciparum. Examination of 16 independent recombinant progeny indicated that the rapid efflux phenotype is controlled by a single gene or a closely linked group of genes. But, there was no linkage between the rapid efflux, CQR phenotype and either of the mdr-like P. falciparum genes or amplification of those genes. These data indicate that the genetic locus governing chloroquine efflux and resistance is independent of the known mdr-like genes.     

Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum

Widespread use of antimalarial agents can profoundly influence the evolution of the human malaria parasite Plasmodium falciparum. Recent selective sweeps for drug-resistant genotypes may have restricted the genetic diversity of this parasite, resembling effects attributed in current debates to a historic population bottleneck. Chloroquine-resistant (CQR) parasites were initially reported about 45 years ago from two foci in southeast Asia and South America, but the number of CQR founder mutations and the impact of chlorquine on parasite genomes worldwide have been difficult to evaluate. Using 342 highly polymorphic microsatellite markers from a genetic map, here we show that the level of genetic diversity varies substantially among different regions of the parasite genome, revealing extensive linkage disequilibrium surrounding the key CQR gene pfcrt and at least four CQR founder events. This disequilibrium and its decay rate in the pfcrt-flanking region are consistent with strong directional selective sweeps occurring over only approximately 20-80 sexual generations, especially a single resistant pfcrt haplotype spreading to very high frequencies throughout most of Asia and Africa. The presence of linkage disequilibrium provides a basis for mapping genes under drug selection in P. falciparum.     

The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein

Mammalian tumours displaying multidrug resistance overexpress a plasma membrane protein (P-glycoprotein), which is encoded by the MDR1 gene and apparently functions as an energy-dependent drug efflux pump. Tissue-specific expression of MDR1 and other members of the MDR gene family has been observed in normal cells, suggesting a role for P-glycoproteins in secretion. We have isolated a gene from the yeast Saccharomyces cerevisiae that encodes a protein very similar to mammalian P-glycoproteins. Deletion of this gene resulted in sterility of MATa, but not of MAT alpha cells. Subsequent analysis revealed that the yeast P-glycoprotein is the product of the STE6 gene, a locus previously shown to be required in MATa cells for production of a-factor pheromone. Our findings suggest that the STE6 protein functions to export the hydrophobic a-factor lipopeptide in a manner analogous to the efflux of hydrophobic cytotoxic drugs catalysed by the related mammalian P-glycoprotein. Thus, the evolutionarily conserved family of MDR-like genes, including the hlyB gene of Escherichia coli and the STE6 gene of S. cerevisiae, encodes components of secretory pathways distinct from the classical, signal sequence-dependent protein translocation system.     

重症监护病房多重耐药铜绿假单胞菌Ⅰ型整合子的检测及鉴定

 整合子是介导细胞多重耐药的重要机制之一,鉴定了2008年某医院重症监护病房分离的23株多重耐药铜绿假单胞菌1型整合子,并应用脉冲场电泳分析其同源性。1型整合子的阳性率达成60.9%。3种1型整合子基因盒被鉴定,其中整合子blaOXA 10 acc6 Ⅱ cmlA8为首次发现报道。基因盒主要编码氨基糖苷类耐药基因,包括 aacA4, aadA2, aadB, aac6 Ⅱ。脉冲电泳结果表明,23株多重耐药铜绿假单胞菌分为5个基因型,A 型(n=5)、B型 (n=6)、C型 (n=4)、D型(n=2)和E型(n=2)。研究表明编码1型整合子是多重耐药铜绿单胞菌较为普遍的特征,且1型整合子与多重耐药表型存相关。研究结果同时也表明防止多重耐药铜绿单胞菌交叉感染仍然是ICU的一项挑战性工作。     

Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum

Throughout the latter half of this century, the development and spread of resistance to most front-line antimalarial compounds used in the prevention and treatment of the most severe form of human malaria has given cause for grave clinical concern. Polymorphisms in pfmdr1, the gene encoding the P-glycoprotein homologue 1 (Pgh1) protein of Plasmodium falciparum, have been linked to chloroquine resistance; Pgh1 has also been implicated in resistance to mefloquine and halofantrine. However, conclusive evidence of a direct causal association between pfmdr1 and resistance to these antimalarials has remained elusive, and a single genetic cross has suggested that Pgh1 is not involved in resistance to chloroquine and mefloquine. Here we provide direct proof that mutations in Pgh1 can confer resistance to mefloquine, quinine and halofantrine. The same mutations influence parasite resistance towards chloroquine in a strain-specific manner and the level of sensitivity to the structurally unrelated compound, artemisinin. This has important implications for the development and efficacy of future antimalarial agents.     

Prediction of multiple drug resistance phenotype in cancer cell lines using gene expression profiles and phylogenetic trees

When microarray gene expression data are used to predict multiple drug resistance (MDR) phenotypes for anticancer drugs, the normalization strategy and the quality of the selected signature genes are usually the main causes of inconsistency among different experiments. A stable statistical drug response prediction model is urgently required in oncology. In this study, the microarray gene expression data of multiple cancer cell lines with MDR was analyzed. For each probe-set, the expression value was defined as present/absent (1/0) and was classified into a gene set defined with protein domain organization (PDO). After employing the gene content method of phylogenetic analysis, a phylogenetic model (cell tree) for MDR phenotype prediction was built at the PDO gene set level. The results indicate that classification of cancer cell lines is predominantly affected by both the histopathological features and the MDR phenotype (paclitaxel and vinblastine). When applying this model to predict the MDR phenotype of independent samples, the phylogenetic model performs better than signature gene models. Although the utility of our procedure is limited due to sample heterogeneity, it still has potential application in MDR research, especially for hematological tumors or established cell lines.     

多药耐药基因与肿瘤

介绍多药耐药（MDR）基因结构和表达、在肿瘤组织中的表达、检测方法及MDR逆转。指出研究MDR基因与肿瘤相互关系,除了具有理论意义外,还可为临床提高肿瘤化疗效果、正确选择治疗方案提供新思路。     

食品源单增李斯特菌喹诺酮类抗生素耐药机制研究

以945株食品源单核细胞增生李斯特菌(Listeria monoocytogens,LM)作为研究对象,分析其对喹诺酮类抗生素耐药的分子机制。采用KB法筛选出喹诺酮类耐药的LM,利用PCR检测喹诺酮耐药决定区(quinolone resistance-determining region,QRDR)的基因突变以及质粒介导喹诺酮耐药(plasmid-mediated quinolone resistance,PMQR)基因的分布情况,结合最小抑菌浓度(MIC)值和外排泵抑制剂利血平分析其外排泵的作用,多位点序列分型(multilocus sequence typing, MLST)结合血清组对耐药菌株进行遗传多样性分析。结果表明:32株耐药菌株中gyrA、gyrB、parC与parE的QRDR均未发生氨基酸突变,且未检测到PMQR相关基因;而7株fepR基因发现新的氨基酸突变位点,其作用机制有待阐明;外排泵基因lde在耐药LM中皆有检出,78.1%(25/32)LM加入利血平后MIC值降低至原MIC的1/2以下,lde可能是导致LM对喹诺酮耐药的重要因素。血清组分析发现32株耐药菌株分属血清组I.1、I.2、II.1、II.2和III,MLST共分为10种ST型,其中ST87、ST9和ST8为优势株,具有一定的致病能力。结果表明,lde是LM产生喹诺酮耐药的重要因素,但LM潜在喹诺酮耐药分子机制仍有待阐明,同时其潜在的致病性应引起重视。     

Detecting selection using a single genome sequence of M. tuberculosis and P. falciparum

Selective pressures on proteins are usually measured by comparing nucleotide sequences. Here we introduce a method to detect selection on the basis of a single genome sequence. We catalogue the relative strength of selection on each gene in the entire genomes of Mycobacterium tuberculosis and Plasmodium falciparum. Our analysis confirms that most antigens are under strong selection for amino-acid substitutions, particularly the PE/PPE family of putative surface proteins in M. tuberculosis and the EMP1 family of cytoadhering surface proteins in P. falciparum. We also identify many uncharacterized proteins that are under strong selection in each pathogen. We provide a genome-wide analysis of natural selection acting on different stages of an organism's life cycle: genes expressed in the ring stage of P. falciparum are under stronger positive selection than those expressed in other stages of the parasite's life cycle. Our method of estimating selective pressures requires far fewer data than comparative sequence analysis, and it measures selection across an entire genome; the method can readily be applied to a large range of sequenced organisms.     

Somatic variants of murine immunoglobulin lambda light chains

Studies of the murine lambda light chains produced by myeloma cells provided the first evidence for somatic point mutation of germ-line variable (V) region genes. An examination of the variable regions of 19 lambda 1 chains revealed seven which differed from a common sequence by one to three amino acid substitutions. Subsequently, one of these presumed somatic variants of the single lambda 1 V gene was characterized by DNA sequence analysis of the rearranged functional gene. The predicted DNA sequence alteration was observed and no silent mutation was evident. These studies of lambda chain variants suggested that the hypervariable, complementarity-determining regions (CDRs) ht be a preferred site of somatic mutation because all seven characterized variants contained substitutions only in these regions. By contrast, comparisons of closely related kappa chain variable region amino acid sequences, and more recently VK and VH genes, have suggested that somatic mutation probably occurs in codons for both framework and CDR residues. To examine this apparent discrepancy between the sites of somatic mutations in lambda and kappa genes, we have determined the nucleotide sequence of two lambda 1 gene from hybridomas and a lambda 2 gene from a myeloma. These sequences demonstrate that somatic mutation in lambda genes can occur in both the framework and CDR residues.     

华南地区汉族人群MDR1基因单核苷酸多态性研究

 多药耐药基因1（MDR1）单核苷酸多态性（SNP）与疾病的易感性、药物治疗效果以及患者的生存复发等预后密切相关。采用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）和聚合酶链反应-单链构象多态性（PCR-SSCP）方法,对华南地区无亲缘关系的汉族人群MDR1基因编码区及部分启动子区进行SNP筛查,并比较不同人种间等位基因频率的差异。结果共检测出5个多态位点：T-2410C、T-129C、C1236T、G2677T/A和C3435T,其等位基因频率-2410C为4.65%,-129C为3.11%,1236T为63.31%,2677T为44.66%,2677A为14.47%,3435T为41.18%。除T-2410C位点因报道的太少无法比较外,其余位点等位基因频率在东亚、高加索以及非洲人群中的分布存在显著差异。该研究为进一步研究华南地区汉族人群MDR1基因SNP与药物效果、药物毒副作用以及疾病易感性之间的相关性提供依据。     

肠球菌临床耐药性分析及耐药基因vanM在屎肠球菌中的检测

目的了解肠球菌临床分离株耐药性及vanM基因在屎肠球菌的分布情况,为临床合理用药及探讨耐药机制提供依据.方法采用VITEK60全自动微生物分析仪对肠球菌进行鉴定及药敏试验,按NCCLS/CLSI标准判断并统计分析结果;PCR法检测耐万古霉素、替考拉宁菌株中的耐药基因vanM,并对阳性基因进行测序,分析耐药基因与耐药性的关系.结果临床分离肠球菌共506株,其中屎肠球菌277株,粪肠球菌229株.尿液标本来源比例最高,为167株（33%）;粪便标本114株（22.5%）;痰液标本78株（15.4%）;胆汁标本57株（11.3%）;脓液标本54株（10.7%）;其他来源36株（7.1%）.药敏结果显示：肠球菌对大部分临床抗菌药高度耐药,屎肠球菌对β-内酰胺类抗菌药物（氨苄西林）、喹诺酮类（左氧氟沙星）、糖肽类（万古霉素、替考拉宁）的耐药率明显高于粪肠球菌（P〈0.05）.vanM基因在耐万古霉素菌株中的检出率为100%,耐替考拉宁菌株检出率为90%.结论屎肠球菌可引起临床各类感染,屎肠球菌对大多数抗生素的耐药率明显高于粪肠球菌,且呈多重耐药趋势.vanM基因可能在糖肽类药物万古霉素、替考拉宁耐药机制中发挥重要作用.     

Mutation rates differ among regions of the mammalian genome

In the traditional view of molecular evolution, the rate of point mutation is uniform over the genome of an organism and variation in the rate of nucleotide substitution among DNA regions reflects differential selective constraints. Here we provide evidence for significant variation in mutation rate among regions in the mammalian genome. We show first that substitutions at silent (degenerate) sites in protein-coding genes in mammals seem to be effectively neutral (or nearly so) as they do not occur significantly less frequently than substitutions in pseudogenes. We then show that the rate of silent substitution varies among genes and is correlated with the base composition of genes and their flanking DNA. This implies that the variation in both silent substitution rate and base composition can be attributed to systematic differences in the rate and pattern of mutation over regions of the genome. We propose that the differences arise because mutation patterns vary with the timing of replication of different chromosomal regions in the germline. This hypothesis can account for both the origin of isochores in mammalian genomes and the observation that silent nucleotide substitutions in different mammalian genes do not have the same molecular clock.     

Size variation in chromosomes from independent cultured isolates of Plasmodium falciparum

The complexity of the life cycle of the protozoan malaria parasite Plasmodium falciparum has hindered genetic analysis; even the number of chromosomes in P. falciparum is uncertain. The blood stages of rodent malaria parasites are haploid and hybridization with cloned complementary DNAs similarly suggests a haploid genome in P. falciparum blood stages (ref. 4 and our unpublished results). A novel approach to karyoptic and linkage analysis in P. falciparum has been provided recently by the technique of pulsed-field gradient (PFG) gel electrophoresis, which allows the fractionation of DNA molecules of 30-3,000 kilobases (kb), a range including the sizes of intact chromosomal DNA molecules from eukaryotes such as yeast and trypanosomatids. We describe here the fractionation by PFG electrophoresis of chromosomal DNA molecules from P. falciparum into at least seven discrete species which vary in size by up to 20% between different isolates. Several genes for P. faciparum antigens which contain repetitive sequences are located on different chromosomes. Surprisingly, two of the chromosomes seem to contain the same sequences.     

Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum

Persistent and recurrent infections by Plasmodium falciparum malaria parasites result from the ability of the parasite to undergo antigenic variation and evade host immune attack. P. falciparum parasites generate high levels of variability in gene families that comprise virulence determinants of cytoadherence and antigenic variation, such as the var genes. These genes encode the major variable parasite protein (PfEMP-1), and are expressed in a mutually exclusive manner at the surface of the erythrocyte infected by P. falciparum. Here we identify a mechanism by which var gene sequences undergo recombination at frequencies much higher than those expected from homologous crossover events alone. These recombination events occur between subtelomeric regions of heterologous chromosomes, which associate in clusters near the nuclear periphery in asexual blood-stage parasites or in bouquet-like configurations near one pole of the elongated nuclei in sexual parasite forms. We propose that the alignment of var genes in heterologous chromosomes facilitates gene conversion and promotes the diversity of antigenic and adhesive phenotypes. The association of virulence factors with a specific nuclear subcompartment may also have implications for variation during mitotic recombination in asexual blood stages.     

Systematic identification of genomic markers of drug sensitivity in cancer cells

Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers for responses to targeted agents. Here, to uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines--which represent much of the tissue-type and genetic diversity of human cancers--with 130 drugs under clinical and preclinical investigation. In aggregate, we found that mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing's sarcoma cells harbouring the EWS (also known as EWSR1)-FLI1 gene translocation to poly(ADP-ribose) polymerase (PARP) inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.     

Sequence of Plasmodium falciparum chromosomes 1, 3-9 and 13

Since the sequencing of the first two chromosomes of the malaria parasite, Plasmodium falciparum, there has been a concerted effort to sequence and assemble the entire genome of this organism. Here we report the sequence of chromosomes 1, 3-9 and 13 of P. falciparum clone 3D7--these chromosomes account for approximately 55% of the total genome. We describe the methods used to map, sequence and annotate these chromosomes. By comparing our assemblies with the optical map, we indicate the completeness of the resulting sequence. During annotation, we assign Gene Ontology terms to the predicted gene products, and observe clustering of some malaria-specific terms to specific chromosomes. We identify a highly conserved sequence element found in the intergenic region of internal var genes that is not associated with their telomeric counterparts.