PGMD: a comprehensive manually curated pharmacogenomic database.

The PharmacoGenomic Mutation Database (PGMD) is a comprehensive manually curated pharmacogenomics database. Two major sources of PGMD data are peer-reviewed literature and Food and Drug Administration (FDA) and European Medicines Agency (EMA) drug labels. PGMD curators capture information on exact genomic location and sequence changes, on resulting phenotype, drugs administered, patient population, study design, disease context, statistical significance and other properties of reported pharmacogenomic variants. Variants are annotated into functional categories on the basis of their influence on pharmacokinetics, pharmacodynamics, efficacy or clinical outcome. The current release of PGMD includes over 117 000 unique pharmacogenomic observations, covering all 24 disease superclasses and nearly 1400 drugs. Over 2800 genes have associated pharmacogenomic variants, including genes in proximity to intergenic variants. PGMD is optimized for use in annotating next-generation sequencing data by providing genomic coordinates for all covered variants, including Single Nucleotide Polymorphisms (SNPs), insertions, deletions, haplotypes, diplotypes, Variable Number Tandem Repeats (VNTR), copy number variations and structural variations.

Control of filament formation in Candida albicans by the transcriptional repressor TUP1.

The pathogenic yeast Candida albicans regulates its cellular morphology in response to environmental conditions. Ellipsoidal, single cells (blastospores) predominate in rich media, whereas filaments composed of elongated cells that are attached end-to-end form in response to starvation, serum, and other conditions. The TUP1 gene, which encodes a general transcriptional repressor in Saccharomyces cerevisiae, was isolated from C. albicans and disrupted. The resulting tup1 mutant strain of C. albicans grew exclusively as filaments under all conditions tested. TUP1 was epistatic to the transcriptional activator CPH1, previously found to promote filamentous growth. The results suggest a model where TUP1 represses genes responsible for initiating filamentous growth and this repression is lifted under inducing environmental conditions.

YPD, PombePD and WormPD: model organism volumes of the BioKnowledge library, an integrated resource for protein information.

The BioKnowledge Library is a relational database and web site (http://www.proteome.com) composed of protein-specific information collected from the scientific literature. Each Protein Report on the web site summarizes and displays published information about a single protein, including its biochemical function, role in the cell and in the whole organism, localization, mutant phenotype and genetic interactions, regulation, domains and motifs, interactions with other proteins and other relevant data. This report describes four species-specific volumes of the BioKnowledge Library, concerned with the model organisms Saccharomyces cerevisiae (YPD), Schizosaccharomyces pombe (PombePD) and Caenorhabditis elegans (WormPD), and with the fungal pathogen Candida albicans (CalPD). Protein Reports of each species are unified in format, easily searchable and extensively cross-referenced between species. The relevance of these comprehensively curated resources to analysis of proteins in other species is discussed, and is illustrated by a survey of model organism proteins that have similarity to human proteins involved in disease.

Topography of transcription factor complexes on the Saccharomyces cerevisiae 5 S RNA gene.

Locations of component proteins of yeast RNA polymerase III transcription factors (TFIII) A, C and B on a 5 S rRNA gene have been determined by site-specific DNA-protein photo-crosslinking. Comparison with a previously analyzed tRNA gene shows that similar nucleoprotein structures assemble on these two genes despite their differently located internal promoter elements. A principal signature of this homology is the placement of the 95 kDA subunit of TFIIIC, which associates with the box A promoter element of the tRNA gene. On the 5 S rRNA gene, the 95 kDa subunit occupies the same space in the absence of a box A sequence, and despite the presence of a box A-like sequence 30 base-pairs further downstream. A 90 kDa component that was not previously recognized as an integral part of TFIIIC has been specifically located at the 3' end of the 5 S rRNA gene.

TUP1, CPH1 and EFG1 make independent contributions to filamentation in candida albicans.

The common fungal pathogen, Candida albicans, can grow either as single cells or as filaments (hyphae), depending on environmental conditions. Several transcriptional regulators have been identified as having key roles in controlling filamentous growth, including the products of the TUP1, CPH1, and EFG1 genes. We show, through a set of single, double, and triple mutants, that these genes act in an additive fashion to control filamentous growth, suggesting that each gene represents a separate pathway of control. We also show that environmentally induced filamentous growth can occur even in the absence of all three of these genes, providing evidence for a fourth regulatory pathway. Expression of a collection of structural genes associated with filamentous growth, including HYR1, ECE1, HWP1, ALS1, and CHS2, was monitored in strains lacking each combination of TUP1, EFG1, and CPH1. Different patterns of expression were observed among these target genes, supporting the hypothesis that these three regulatory proteins engage in a network of individual connections to downstream genes and arguing against a model whereby the target genes are regulated through a central filamentous growth pathway. The results suggest the existence of several distinct types of filamentous forms of C. albicans, each dependent on a particular set of environmental conditions and each expressing a unique set of surface proteins.

Transcriptional regulators of the Schizosaccharomyces pombe fbp1 gene include two redundant Tup1p-like corepressors and the CCAAT binding factor activation complex.

The Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK). To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity. Two such clones express amino-terminally truncated forms of the S. pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor. These clones appear to act as dominant negative alleles. Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription. In strains lacking tup11 and tup12, the atf1-pcr1 transcriptional activator continues to play a central role in fbp1-lacZ expression; however, spc1 MAPK phosphorylation of atf1 is no longer essential for its activation. We discuss possible models for the role of tup11- and tup12-mediated repression with respect to signaling from the MAPK and PKA pathways. A third clone identified in our screen expresses the php5 protein subunit of the CCAAT-binding factor (CBF). Deletion of php5 reduces fbp1 expression under both repressed and derepressed conditions. The CBF appears to act in parallel to atf1-pcr1, although it is unclear whether or not CBF activity is regulated by PKA.

Identification and characterization of TUP1-regulated genes in Candida albicans.

TUP1 encodes a transcriptional repressor that negatively controls filamentous growth in Candida albicans. Using subtractive hybridization, we identified six genes, termed repressed by TUP1 (RBT), whose expression is regulated by TUP1. One of the genes (HWP1) has previously been characterized, and a seventh TUP1-repressed gene (WAP1) was recovered due to its high similarity to RBT5. These genes all encode secreted or cell surface proteins, and four out of the seven (HWP1, RBT1, RBT5, and WAP1) encode putatively GPI-modified cell wall proteins. The remaining three, RBT2, RBT4, and RBT7, encode, respectively, an apparent ferric reductase, a plant pathogenesis-related protein (PR-1), and a putative secreted RNase T2. The expression of RBT1, RBT4, RBT5, HWP1, and WAP1 was induced in wild-type cells during the switch from the yeast form to filamentous growth, indicating the importance of TUP1 in regulating this process and implicating the RBTs in hyphal-specific functions. We produced knockout strains in C. albicans for RBT1, RBT2, RBT4, RBT5, and WAP1 and detected no phenotypes on several laboratory media. However, two animal models for C. albicans infection, a rabbit cornea model and a mouse systemic infection model, revealed that rbt1Delta and rbt4Delta strains had significantly reduced virulence. TUP1 appears, therefore, to regulate many genes in C. albicans, a significant fraction of which are induced during filamentous growth, and some of which participate in pathogenesis.

Bending of the Saccharomyces cerevisiae 5S rRNA gene in transcription factor complexes.

Bending of the yeast 5S rRNA gene by its transcription factors (TF) IIIA, IIIC, and IIIB has been investigated by two electrophoretic methods that exploit the anomalous mobility of bent DNA in tight gel networks. A minor bend is induced by TFIIIA, and a very strong bend, centered at its upstream DNA-binding site, is induced by TFIIIB. Despite binding to different DNA sequences on the 5S rRNA gene and the previously analyzed tRNA(Glu) gene, TFIIIB generates nearly identical bends in each site. Fully assembled transcription factor complexes bend the 5S rRNA gene in the same net direction as does TFIIIB alone.

NRG1, a repressor of filamentous growth in C.albicans, is down-regulated during filament induction.

In response to a variety of external signals, the fungal pathogen Candida albicans undergoes a transition between ellipsoidal single cells (blastospores) and filaments composed of elongated cells attached end-to-end. Here we identify a DNA-binding protein, Nrg1, that represses filamentous growth in Candida probably by acting through the co-repressor Tup1. nrg1 mutant cells are predominantly filamentous under non-filament-inducing conditions and their colony morphology resembles that of tup1 mutants. We also identify two filament-specific genes, ECE1 and HWP1, whose transcription is repressed by Nrg1 under non-inducing conditions. These genes constitute a subset of those under Tup1 control, providing further evidence that Nrg1 acts by recruiting Tup1 to target genes. We show that growth in serum at 37 degrees C, a potent inducer of filamentous growth, causes a reduction of NRG1 mRNA, suggesting that filamentous growth is induced by the down-regulation of NRG1. Consistent with this idea, expression of NRG1 from a non-regulated promoter partially blocks the induction of filamentous growth.

The subunit structure of Saccharomyces cerevisiae transcription factor IIIC probed with a novel photocrosslinking reagent.

A photocrosslinking nucleotide, 5-[N-(p-azidobenzoyl)-3-aminoallyl]-deoxyuridine monophosphate (N3Rd-UMP), has been used to identify four polypeptides that are associated with the large Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIC, and to map the locations of these subunits along DNA when TFIIIC binds to the S.cerevisiae SUP4 tRNA(Tyr) gene. The 145 kd subunit of TFIIIC is primarily accessible to photocrosslinking from the vicinity of the box B + internal promoter element; 95 and 55 kd subunits are located on opposite sides of the DNA helix in the vicinity of the box A internal promoter element; a 135 kd subunit is less strongly crosslinked to the box A region and to a DNA segment between boxes B and A. DNA probes containing more than one N3RdUMP residue can form crosslinks between polypeptide chains. The specific circumstances of formation and the apparent mol. wts of two of these products lead to the tentative suggestion that a protomer of TFIIIC may contain two 95 kd subunits.

S. cerevisiae TFIIIB is the transcription initiation factor proper of RNA polymerase III, while TFIIIA and TFIIIC are assembly factors.

The S. cerevisiae RNA polymerase III (pol III) transcription factor TFIIIB binds to DNA upstream of the transcription start site of the SUP4 tRNA(Tyr) gene in a TFIIIC-dependent reaction and to the major 5S rRNA gene in a reaction requiring TFIIIC and TFIIIA. It is shown here that TFIIIB alone correctly positions pol III for repeated cycles of transcription on both genes, with the same efficiency as fully assembled transcription complexes. Thus, TFIIIB is the sole transcription initiation factor of S. cerevisiae pol III; TFIIIC and TFIIIA are assembly factors for TFIIIB. The TFIIIB-dependent binding of pol III to the SUP4 tRNA and 5S rRNA genes has been analyzed in binary (protein and DNA only) and precisely arrested ternary (protein, DNA, and RNA) transcription complexes. Pol III unwinds at least 14 bp of DNA at the SUP4 transcription start in a temperature-dependent process. The unwound DNA segment moves downstream with nascent RNA as a transcription bubble of approximately the same size.

Probing close DNA contacts of RNA polymerase III transcription complexes with the photoactive nucleoside 4-thiodeoxythymidine.

Photoactive 4-thiodeoxythymidine 5'-triphosphate (4-S-dTTP) has been synthesized and used to enzymatically incorporate the corresponding nucleotide, 4-thiodeoxythymidine 5'-phosphate (4-S-dTMP), at specific positions of the Saccharomyces cerevisiae 5 S rRNA and SUP4 tRNA(Tyr) genes. RNA polymerase III transcription complexes have been assembled on this DNA and analyzed by photocrosslinking for proteins making close contact with DNA. Comparison DNA probes with a long-tether photoactive nucleotide 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP (N3RdUMP) incorporated at the same positions have also been analyzed, in order to compare the properties of these two crosslinking reagents. At least 10 of the 16 different S. cerevisiae polymerase III subunits make direct contact with DNA. The 120-kDa subunit of transcription factor (TF)IIIC, which is thought to play the key role in positioning TFIIIB upstream of the transcriptional start site, also contacts DNA near the transcriptional start site in TFIII(C+B) complexes with a SUP4 tRNA(Tyr) gene. The photocrosslinking patterns generated by 4-S-dTMP and N3RdUMP are distinctive, implying that these two reagents can yield complementary information about the structures of complex protein assemblies on DNA. Surprisingly, some subunits of the S. cerevisiae RNA polymerase III are crosslinked by 4-S-dTMP but not by N3RdUMP.

Multiple states of protein-DNA interaction in the assembly of transcription complexes on Saccharomyces cerevisiae 5S ribosomal RNA genes.

Multiple stages of protein-DNA interaction in the assembly of RNA polymerase III transcription complexes on a Saccharomyces cerevisiae 5S rRNA gene have been distinguished by DNase I "footprinting" and gel retardation. Transcription factor IIIA interacts with approximately 35 base pairs of the internal promoter region. Transcription factors IIIC and IIIB incrementally extend the interaction along the 5S gene, if, and only if, transcription factor IIIA is also bound. Complexes assembled from the complete set of purified transcription factors or from a complete transcription system extend over the entire transcription unit together with almost 50 base pairs of 5' flanking sequence.