John C. Wootton

Mutations in the P. falciparum Digestive Vacuole Transmembrane Protein PfCRT and Evidence for Their Role in Chloroquine Resistance

The determinant of verapamil-reversible chloroquine resistance (CQR) in a Plasmodium falciparum genetic cross maps to a 36 kb segment of chromosome 7. This segment harbors a 13-exon gene, pfcrt, having point mutations that associate completely with CQR in parasite lines from Asia, Africa, and South America. These data, transfection results, and selection of a CQR line harboring a novel K761 mutation point to a central role for the PfCRT protein in CQR. This transmembrane protein localizes to the parasite digestive vacuole (DV), the site of CQ action, where increased compartment acidification associates with PfCRT point mutations. Mutations in PfCRT may result in altered chloroquine flux or reduced drug binding to hematin through an effect on DV pH.

Analysis of compositionally biased regions in sequence databases.

Publisher Summary This chapter discusses the analysis of compositionally biased region in sequence databases. Programs sequence (SEG) and protein sequence (PSEG) are tuned for amino acid sequences and nucleotide sequence (NSEG) for nucleotide sequences. The programs can be applied to either individual sequences, including whole chromosomes if appropriate, or entire sequence databases. Compositional complexity is based only on residue composition, regardless of the patterns or periodicity of sequence repetitiveness. This contrasts with some alternative methods that use counts of k-grams to define residue patterns and clustering. Complexity, pattern, and periodicity are distinct abstract attributes of simple sequences. For genomic studies, it is essential to view compositional bias in the context of many types of other features, such as recognizable functional sites, transcripts, coding sequences, and homologies. For this purpose, the SEG family of programs is being integrated into software packages, or workbenches, that have graphic multilevel browsing facilities and include zoom functions.

Protein Database Searches Using Compositionally Adjusted Substitution Matrices

Almost all protein database search methods use amino acid substitution matrices for scoring, optimizing, and assessing the statistical significance of sequence alignments. Much care and effort has therefore gone into constructing substitution matrices, and the quality of search results can depend strongly upon the choice of the proper matrix. A long‐standing problem has been the comparison of sequences with biased amino acid compositions, for which standard substitution matrices are not optimal. To address this problem, we have recently developed a general procedure for transforming a standard matrix into one appropriate for the comparison of two sequences with arbitrary, and possibly differing compositions. Such adjusted matrices yield, on average, improved alignments and alignment scores when applied to the comparison of proteins with markedly biased compositions.

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 ∼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.

Statistics of local complexity in amino acid sequences and sequence databases

Protein sequences contain surprisingly many local regions of low compositional complexity. These include different types of residue clusters, some of which contain homopolymers, short period repeats or aperiodic mosaics of a few residue types. Several different formal definitions of local complexity and probability are presented here and are compared for their utility in algorithms for localization of such regions in amino acid sequences and sequence databases. The definitions are:—(1) those derived from enumeration a priori by a treatment analogous to statistical mechanics, (2) a log likelihood definition of complexity analogous to informational entropy, (3) multinomial probabilities of observed compositions, (4) an approximation resembling the χ2 statistic and (5) a modification of the coefficient of divergence. These measures, together with a method based on similarity scores of self-aligned sequences at different offsets, are shown to be broadly similar for first-pass, approximate localization of low-complexity regions in protein sequences, but they give significantly different results when applied in optimal segmentation algorithms. These comparisons underpin the choice of robust optimization heuristics in an algorithm, SEG, designed to segment amino acid sequences fully automatically into subsequences of contrasting complexity. After the abundant low-complexity segments have been partitioned from the Swissprot database, the remaining high-complexity sequence set is adequately approximated by a first-order random model.

Non-globular domains in protein sequences: Automated segmentation using complexity measures☆

Computational methods based on mathematically-defined measures of compositional complexity have been developed to distinguish globular and non-globular regions of protein sequences. Compact globular structures in protein molecules are shown to be determined by amino acid sequences of high informational complexity. Sequences of known crystal structure in the Brookhaven Protein Data Bank differ only slightly from randomly shuffled sequences in the distribution of statistical properties such as local compositional complexity. In contrast, in the much larger body of deduced sequences in the SWISS-PROT database, approximately one quarter of the residues occur in segments of non-randomly low complexity and approximately half of the entries contain at least one such segment. Sequences of proteins with known, physicochemically-defined non-globular regions have been analyzed, including collagens, different classes of coiled-coil proteins, elastins, histones, non-histone proteins, mucins, proteoglycan core proteins and proteins containing long single solvent-exposed alpha-helices. The SEG algorithm provides an effective general method for partitioning the globular and non-globular regions of these sequences fully automatically. This method is also facilitating the discovery of new classes of long, non-globular sequence segments, as illustrated by the example of the human CAN gene product involved in tumor induction.

A Secreted Serine-Threonine Kinase Determines Virulence in the Eukaryotic Pathogen Toxoplasma gondii

Toxoplasma gondii strains differ dramatically in virulence despite being genetically very similar. Genetic mapping revealed two closely adjacent quantitative trait loci on parasite chromosome VIIa that control the extreme virulence of the type I lineage. Positional cloning identified the candidate virulence gene ROP18, a highly polymorphic serine-threonine kinase that was secreted into the host cell during parasite invasion. Transfection of the virulent ROP18 allele into a nonpathogenic type III strain increased growth and enhanced mortality by 4 to 5 logs. These attributes of ROP18 required kinase activity, which revealed that secretion of effectors is a major component of parasite virulence.

Inducible erythromycin resistance in staphlyococci is encoded by a member of the ATP‐binding transport super‐gene family

A Staphylococcus epidermidis plasmid conferring inducible resistance to 14‐membered ring macrolides and type B streptogramins has been analysed and the DNA sequence of the gene responsible for resistance determined. A single open reading frame of 1.464kbp, preceded by a complex control region containing a promoter and two ribosomal binding sites, was identified. The deduced sequence of the 488‐amino‐acid protein (MsrA) revealed the presence of two ATP‐binding motifs homologous to those of a family of transport‐related proteins from Gram‐negative bacteria and eukaryotic cells, including the P‐glycoprotein responsible for multidrug resistance. In MsrA, but not these other proteins, the two potential ATP‐binding domains are separated by a Q‐linker of exceptional length. Q‐linkers comprise a class of flexible inter‐domain fusion junctions that are typically rich in glutamine and other hydrophilic amino acids and have a characteristic spacing of hydrophobic amino acids, as found in the MsrA sequence. Unlike the other transport‐related proteins, which act in concert with one or more hydrophobic membrane proteins, MsrA appears to function independently when cloned in a heterologous host (Staphylococcus aureus RN4220). MsrA might, therefore, interact with and confer antibiotic specificity upon other transmembrane efflux complexes of staphylococcal cells. The active efflux of [14C]‐erythromycin from cells of S. aureus RN4220 containing msrA has been demonstrated.

Multiple transporters associated with malaria parasite responses to chloroquine and quinine

Mutations and/or overexpression of various transporters are known to confer drug resistance in a variety of organisms. In the malaria parasite Plasmodium falciparum, a homologue of P‐glycoprotein, PfMDR1, has been implicated in responses to chloroquine (CQ), quinine (QN) and other drugs, and a putative transporter, PfCRT, was recently demonstrated to be the key molecule in CQ resistance. However, other unknown molecules are probably involved, as different parasite clones carrying the same pfcrt and pfmdr1 alleles show a wide range of quantitative responses to CQ and QN. Such molecules may contribute to increasing incidences of QN treatment failure, the molecular basis of which is not understood. To identify additional genes involved in parasite CQ and QN responses, we assayed the in vitro susceptibilities of 97 culture‐adapted cloned isolates to CQ and QN and searched for single nucleotide polymorphisms (SNPs) in DNA encoding 49 putative transporters (total 113 kb) and in 39 housekeeping genes that acted as negative controls. SNPs in 11 of the putative transporter genes, including pfcrt and pfmdr1, showed significant associations with decreased sensitivity to CQ and/or QN in P. falciparum. Significant linkage disequilibria within and between these genes were also detected, suggesting interactions among the transporter genes. This study provides specific leads for better understanding of complex drug resistances in malaria parasites.

Genetic and molecular analysis of a cluster of rpf genes involved in positive regulation of synthesis of extracellular enzymes and polysaccharide in Xanthomonas campestris pathovar campestris

SummaryThe cosmid clone. pIJ3020 containing DNA from the plant pathogenic bacterium Xanthomonas campestris pathovar campestris has previously been shown to complement a non-pathogenic mutant defective in synthesis of extracellular enzymes. The DNA cloned in pIJ3020 was analysed by mutagenesis with Tn5 and Tn5lac and by nucleotide sequencing. The results indicate that this region of the genome contains a cluster of genes, mutation in any of which results in failure of the enzymes and extracellular polysaccharide to be synthesized. The designation rpf (regulation of pathogenicity factors) is proposed for these genes. The nucleotide sequence of one gene (rpfC) predicts a protein product with homology to conserved domains of both sensor and regulator proteins of prokaryotic two-component regulatory systems, which are usually involved in regulating gene expression in response to environmental stimuli.