Peter Verhasselt

Contribution of mutations in the cytochrome P450 14α-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans

The cytochrome P450 14alpha-demethylase, encoded by the ERG11 (CYP51) gene, is the primary target for the azole class of antifungals. Changes in the azole affinity of this enzyme caused by amino acid substitutions have been reported as a resistance mechanism. Nine Candida albicans strains were used in this study. The ERG11 base sequence of seven isolates, of which only two were azole-sensitive, were determined. The ERG11 base sequences of the other two strains have been published previously. In these seven isolates, 12 different amino acid substitutions were identified, of which six have not been described previously (A149V, D153E, E165Y, S279F, V452A and G4655). In addition, 16 silent mutations were found. Two different biochemical assays, subcellular sterol biosynthesis and CO binding to reduced microsomal fractions, were used to evaluate the sensitivity of the cytochromes for fluconazole and itraconazole. Enzyme preparations from four isolates showed reduced itraconazole susceptibility, whereas more pronounced resistance to fluconazole was observed in five isolates. A three-dimensional model of C. albicans Cyp51p was used to position all 29 reported substitutions, 98 in total identified in 53 sequences. These 29 substitutions were not randomly distributed over the sequence but clustered in three regions from amino acids 105 to 165, from 266 to 287 and from 405 to 488, suggesting the existence of hotspot regions. Of the mutations found in the two N-terminal regions only Y132H was demonstrated to be of importance for azole resistance. In the C-terminal region three mutations are associated with resistance, suggesting that the non-characterized substitutions found in this region should be prioritized for further analysis.

An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans.

Wenonah Vercoutere, Stephen Winters-Hilt, Hugh Olsen, David Deamer, David Haussler, and Mark Akeson Nat. Biotechnol. 19, 248–252 (2001). The URL given for the DNA mfold server in Table 1 (p. 249 ) and in the text (p. 251) is incorrect. The correct URL is http://bioinfo.math.rpi.edu/~mfold/dna/form1.cgiConverting the complete genome sequence of Candida albicans into meaningful biological information will require comprehensive screens for identifying functional classes of genes. Most systems described so far are not applicable to C. albicans because of its difficulty with mating, its diploid nature, and the lack of functional random insertional mutagenesis methods. We examined artificial gene suppression as a means to identify gene products critical for growth of this pathogen; these represent new antifungal drug targets. To achieve gene suppression we combined antisense RNA inhibition and promoter interference. After cloning antisense complementary DNA (cDNA) fragments under control of an inducible GAL1 promoter, we transferred the resulting libraries to C. albicans. Over 2,000 transformant colonies were screened for a promoter-induced diminished-growth phenotype. After recovery of the plasmids, sequence determination of their inserts revealed the messenger RNA (mRNA) they inhibited or the gene they disrupted. Eighty-six genes critical for growth were identified, 45 with unknown function. When used in high-throughput screening for antifungals, the crippled C. albicans strains generated in this study showed enhanced sensitivity to specific drugs.

Structure of the Human Serotonin 5‐HT4 Receptor Gene and Cloning of a Novel 5‐HT4 Splice Variant

Abstract: Several variants of the serotonin 5‐HT4 receptor are known to be produced by alternative splicing. To survey the existence and usage of exons in humans, we cloned the human 5‐HT4 gene. Based on sequence analysis seven C‐terminal variants (a‐g) and one internal splice variant (h) were found. We concentrated in this study on the functional characterization of the novel splice variant h, which leads to the insertion of 14 amino acids into the second extracellular loop of the receptor. The h variant was cloned as a splice combination with the C‐terminal b variant; therefore, we call this receptor 5‐HT4(hb). This novel receptor variant was expressed transiently in COS‐7 cells, and its pharmacological profile was compared with those of the previously cloned 5‐HT4(a) and 5‐HT4(b) isoforms, with the latter being the primary reference for the h variant. In competition binding experiments using reference 5‐HT4 ligands, no significant differences were detected. However, the broadly used 5‐HT4 antagonist GR113808 discriminated functionally among the receptor variants investigated. As expected, it was an antagonist on the 5‐HT4(a) and 5‐HT4(b) variant but showed partial agonistic activity on the 5‐HT4(hb) variant. These data emphasize the importance of variations introduced by splicing for receptor pharmacology and may help in the understanding of conflicting results seen with 5‐HT4 ligands in different model systems.

Cloning and characterization of human histone deacetylase 8

To date, seven different human histone deacetylases (HDACs) have been identified, which fall into two distinct classes. We have isolated and characterized a cDNA encoding a novel human HDAC, which we name HDAC8. HDAC8 shows a high degree of sequence similarity to HDAC1 and HDAC2 and thus belongs to the class I of HDACs. HDAC8 is expressed in a variety of tissues. Human cells overexpressing HDAC8 localize the protein in sub‐nuclear compartments whereas HDAC1 shows an even nuclear distribution. In addition, the HDAC8 gene is localized on the X chromosome at position q13, which is close to the XIST gene and chromosomal breakpoints associated with preleukemia.

In Vitro Antimycobacterial Spectrum of a Diarylquinoline ATP Synthase Inhibitor

ABSTRACT The diarylquinoline R207910 is in clinical development for tuberculosis treatment. The MIC50 for 41 drug-susceptible and 44 multidrug-resistant Mycobacterium tuberculosis clinical isolates was 0.032 μg/ml. Out of 20 additional mycobacterial species, three were found to be naturally resistant to R207910 and were shown to exhibit a polymorphism in their atpE genes.

A HUMAN HOMOLOGUE OF THE SCHIZOSACCHAROMYCES POMBE RAD1+ CHECKPOINT GENE ENCODES AN EXONUCLEASE

In the fission yeast Schizosaccharomyces pombe the rad1 + gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints. We have identified a human homologue of the S. pombe rad1 + gene, designated Hrad1, as well as a mouse homologue: Mrad1. Two Hrad1alternative splice variants with different open reading frames have been identified; one codes for a long form, Hrad1A, and the other encodes a short form because of N-terminal truncation, Hrad1B. Hrad1A has 60% identity to the S. pombe rad1 +sequence at the DNA level and 49% identity and 72% similarity at the amino acid level. Northern blot analysis indicates elevated levels of expression in testis and cancer cell lines. Chromosomal localization by fluorescence in situ hybridization indicates thatHrad1 is located on chromosome 5p13.2–13.3. This region is subject to loss of heterozygosity in several human cancers. Hrad1 also shares homology with the Saccharomyces cerevisiaeRAD17 and Ustilago maydis REC1 proteins. REC1 has previously been characterized as a 3′ → 5′ exonuclease with a C-terminal domain essential for cell cycle checkpoint function. We have expressed and purified polyhistidine-tagged fusions of Hrad1A and Hrad1B and show that HisHrad1A has 3′ → 5′ exonuclease activity, whereas HisHrad1B lacks such activity. The biological functions of the two proteins remain to be determined.

Characterization of an orphan G protein-coupled receptor localized in the dorsal root ganglia reveals adenine as a signaling molecule

The cloning of novel G protein-coupled receptors and the search for their natural ligands, a process called reverse pharmacology, is an excellent opportunity to discover novel hormones and neurotransmitters. Based on a degenerate primer approach we have cloned a G protein-coupled receptor whose mRNA expression profile indicates highest expression in the dorsal root ganglia, specifically in the subset of small neurons, suggesting a role in nociception. In addition, moderate expression was found in lung, hypothalamus, peripheral blood leukocytes, and ovaries. Guided by a receptor-activation bioassay, we identified adenine as the endogenous ligand, which activated the receptor potently and with high structural stringency. Therefore, we propose to name this receptor as the adenine receptor. Hormonal functions have already been demonstrated for adenine derivatives like 6-benzylaminopurine in plants and 1-methyladenine in lower animals. Here, we demonstrate that adenine functions as a signaling molecule in mammals. This finding adds a third family besides P1 and P2 receptors to the class of purinergic receptors.

Isolation and expression of novel human glutamate carboxypeptidases with N-acetylated alpha-linked acidic dipeptidase and dipeptidyl peptidase IV activity.

Hydrolysis of the neuropeptideN-acetyl-l-aspartyl-l-glutamate (NAAG) by N-acetylated α-linked acidic dipeptidase (NAALADase) to release glutamate may be important in a number of neurodegenerative disorders in which excitotoxic mechanisms are implicated. The gene coding for human prostate-specific membrane antigen, a marker of prostatic carcinomas, and its rat homologue glutamate carboxypeptidase II have recently been shown to possess such NAALADase activity. In contrast, a closely related member of this gene family, rat ileal 100-kDa protein, possesses a dipeptidyl peptidase IV activity. Here, we describe the cloning of human ileal 100-kDa protein, which we have called a NAALADase- “like” (NAALADase L) peptidase based on its sequence similarity to other members of this gene family, and its inability to hydrolyze NAAG in transient transfection experiments. Furthermore, we describe the cloning of a third novel member of this gene family, NAALADase II, which codes for a type II integral membrane protein and which we have localized to chromosome 11 by fluorescent in situ hybridization analysis. Transient transfection of NAALADase II cDNA confers both NAALADase and dipeptidyl peptidase IV activity to COS cells. Expression studies using reverse transcription-polymerase chain reaction and Northern blot hybridization show that NAALADase II is highly expressed in ovary and testis as well as within discrete brain areas.

Identification of a human homologue of the Schizosaccharomyces pombe rad17+ checkpoint gene

In the fission yeast Schizosaccharomyces pombe the rad17 + gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints. We have identified a human cDNA homologue of the S. pombe rad17 + checkpoint gene, designatedHrad17. Hrad17 has 49% identity to theS. pombe rad17 + sequence at the DNA level and 49% identity and 72% similarity at the amino acid level. Northern blot analysis indicates elevated levels of expression in testis and in cancer cell lines. Chromosomal localization by fluorescence in situ hybridization indicates that Hrad17 is located on chromosome 4q13.3–21.2. This region is subject to loss of heterozygosity in several human cancers. To begin to understand the protein-protein interactions of the human checkpoint machinery, we have used the yeast two-hybrid system to examine potential interactions between Hrad1, Hrad9, and Hrad17. We demonstrate a physical interaction between Hrad17 and Hrad1 but no interaction with Hrad9.

Minor variant detection in amplicons using 454 massive parallel pyrosequencing: experiences and considerations for successful applications

Ultra-deep sequencing (UDS) of amplicons is a major application for next-generation sequencing technologies, even more so for the 454 Genome Sequencer FLX. Especially for this application, errors that might be introduced during any of the sample processing or data analysis steps should be avoided or at least recognized, as they might lead to aberrant sequence variant calling. Since 454 pyrosequencing relies on PCR-driven target amplification, it is key to differentiate errors introduced during the amplification step from genuine minority variants. Thereto, optimal primer design is imperative because primer selection, primer dimer formation, and nonspecific binding may all affect the quality and outcome of amplicon-based deep sequencing. Also, other intrinsic PCR characteristics including amplification drift and the formation of secondary structures may influence sequencing data quality. We illustrate these phenomena using real life case studies and propose experimental and analytical evidence-based solutions for effective practice. Furthermore, because accuracy of the DNA polymerase is vital for reliable UDS results, a comparative analysis of error profiles from seven different DNA polymerases was performed and experimentally assessed in parallel by 454 sequencing. Finally, intra and interrun variability evaluation of the 454 sequencing protocol revealed highly reproducible results in amplicon-based UDS.