Glioblastoma-Specific Protein Interaction Network Identifies PP1A and CSK21 as Connecting Molecules between Cell Cycle-Associated Genes

Glioblastoma (GBM; grade IV astrocytoma) is a very aggressive form of brain cancer with a poor survival and few qualified predictive markers. This study integrates experimentally validated genes that showed specific upregulation in GBM along with their protein-protein interaction information. A system level analysis was used to construct GBM-specific network. Computation of topological parameters of networks showed scale-free pattern and hierarchical organization. From the large network involving 1,447 proteins, we synthesized subnetworks and annotated them with highly enriched biological processes. A careful dissection of the functional modules, important nodes, and their connections identified two novel intermediary molecules CSK21 and protein phosphatase 1 alpha (PP1A) connecting the two subnetworks CDC2-PTEN-TOP2A-CAV1-P53 and CDC2-CAV1-RB-P53-PTEN, respectively. Real-time quantitative reverse transcription-PCR analysis revealed CSK21 to be moderately upregulated and PP1A to be overexpressed by 20-fold in GBM tumor samples. Immunohistochemical staining revealed nuclear expression of PP1A only in GBM samples. Thus, CSK21 and PP1A, whose functions are intimately associated with cell cycle regulation, might play key role in gliomagenesis. Cancer Res; 70(16); 6437-47. (C)2010 AACR.

Studies on Causes of Multiple Sclerosis : From Genes to Transcriptome

Multiple sclerosis (MS) is the most common cause of neurological disability in young adults, affecting more than two million people worldwide. It manifests as a chronic inflammation in the central nervous system (CNS) and causes demyelination and neurodegeneration. Depending on the location of the demyelinated plaques and axonal loss, a variety of symptoms can be observed including deficits in vision, coordination, balance and movement. With a typical age of onset at 20-40 years, the social and economic impacts of MS on lives of the patients and their families are considerable. Unfortunately the current treatments are relatively inefficient and the development of more effective treatments has been impeded by our limited understanding of the causes and pathogenesis of MS. Risk of MS is higher in biological relatives of MS patients than in the general population. Twin and adoption studies have shown that familial clustering of MS is explained by shared genetic factors rather than by shared familial environment. While the involvement of the human leukocyte antigen (HLA) genes was first discovered four decades ago, additional genetic risk factors have only recently been identified through genome-wide association studies (GWAS). Current evidence suggests that MS is a highly polygenic disease with perhaps hundreds of common variants with relatively modest effects contributing to susceptibility. Despite extensive research, the majority of these risk factors still remain to be identified. In this thesis the aim was to identify novel genes and pathways involved in MS. Using genome-wide microarray technology, gene expression levels in peripheral blood mononuclear cells (PBMC) from 12 MS patients and 15 controls were profiled and more than 600 genes with altered expression in MS were identified. Three of five selected findings, DEFA1A3, LILRA4 and TNFRSF25, were successfully replicated in an independent sample. Increased expression of DEFA1A3 in MS is a particularly interesting observation, because its elevated levels have previously been reported also in several other autoimmune diseases. A systematic review of seven microarray studies was then performed leading to identification of 229 genes, in which either decreased or increased expression in MS had been reported in at least two studies. In general there was relatively little overlap across the experiments: 11 of the 229 genes had been reported in three studies and only HSPA1A in four studies. Nevertheless, these 229 genes were associated with several immunological pathways including interleukin pathways related to type 2 and type 17 helper T cells and regulatory T cells. However, whether these pathways are involved in causing MS or related to secondary processes activated after disease onset remains to be investigated. The 229 genes were also compared with loci identified in published MS GWASs. Single nucleotide polymorphisms (SNP) in 17 of the 229 loci had been reported to be associated with MS with P-value less than 0.0001 including variants in CXCR4 and SAPS2, which were the only loci where evidence for correlation between the associated variant and gene expression was found. The CXCR4 variant was further tested for association with MS in a large case-control sample and the previously reported suggestive association was replicated (P-value is 0.0004). Finally, common genetic variants in candidate genes, which had been selected on the basis of showing association with other autoimmune diseases (MYO9B) or showing differential expression in MS in our study (DEFA1A3, LILRA4 and TNFRSF25), were tested for association with MS, but no evidence of association was found. In conclusion, through a systematic review of genome-wide expression studies in MS we have identified several promising candidate genes and pathways for future studies. In addition, we have replicated a previously suggested association of a SNP variant upstream of CXCR4 with MS. Keywords: autoimmune disease, common variant, CXCR4, DEFA1A3, HSPA1A,gene expression, genetic association, GWAS, MS, multiple sclerosis, systematic review

The many ways to delimit species: hairs, genes and surface chemistry

Species identification forms the basis for understanding the diversity of the living world, but it is also a prerequisite for understanding many evolutionary patterns and processes. The most promising approach for correctly delimiting and identifying species is to integrate many types of information in the same study. Our aim was to test how cuticular hydro- carbons, traditional morphometrics, genetic polymorphisms in nuclear markers (allozymes and DNA microsatellites) and DNA barcoding (partial mitochondrial COI gene) perform in delimiting species. As an example, we used two closely related Formica ants, F. fusca and F. lemani, sampled from a sympatric population in the northern part of their distribu- tion. Morphological characters vary and overlap in different parts of their distribution areas, but cuticular hydrocarbons include a strong taxonomic signal and our aim is to test the degree to which morphological and genetic data correspond to the chemical data. In the morphological analysis, species were best separated by the combined number of hairs on pro- notum and mesonotum, but individual workers overlapped in hair numbers, as previously noted by several authors. Nests of the two species were separated but not clustered according to species in a Principal Component Analysis made on nuclear genetic data. However, model-based Bayesian clustering resulted in perfect separation of the species and gave no indication of hybridization. Furthermore, F. lemani and F. fusca did not share any mitochondrial haplotypes, and the species were perfectly separated in a phylogenetic tree. We conclude that F. fusca and F. lemani are valid species that can be separated in our study area relatively well with all methods employed. However, the unusually small genetic differen- tiation in nuclear markers (FST = 0.12) shows that they are closely related, and occasional hybridization between F. fusca and F. lemani cannot be ruled out.

Identification of Mxr1p-binding sites in the promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic yeast Pichia pastoris

Expression of genes involved in methanol metabolism of Pichia pastoris is regulated by Mxr1p, a zinc finger transcription factor. In this study, we studied the target gene specificity of Mxr1p by examining its ability to bind to promoters of genes encoding dihydroxyacetone synthase (DHAS) and peroxin 8 (PEX8), since methanol-inducible expression of these genes is abrogated in mxr1-null mutant strains of P. pastoris. Different regions of DHAS and PEX8 promoter were isolated from P. pastoris genomic DNA and their ability to bind to a recombinant Mxr1p protein containing the N-terminal 150 amino acids, including the zinc finger DNA-binding domain, was examined. These studies reveal that Mxr1p specifically binds to promoter regions containing multiple 5'-CYCC-3' sequences, although all DNA sequences containing the 5'-CYCC-3' motif do not qualify as Mxr1p-binding sites. Key DNA-binding determinants are present outside 5'-CYCC-3' motif and Mxr1p preferably binds to DNA sequences containing 5'-CYCCNY-3' than those containing 5'-CYCCNR-3' sequences. This study provides new insights into the molecular determinants of target gene specificity of Mxr1p, and the methodology described here can be used for mapping Mxr1p-binding sites in other methanol-inducible promoters of P. pastoris. Copyright (C) 2010 John Wiley & Sons, Ltd.

Crucial contribution of the multiple copies of the initiator tRNA genes in the fidelity of tRNA(fMet) selection on the ribosomal P-site in Escherichia coli

The accuracy of the initiator tRNA (tRNA(fMet)) selection in the ribosomal P-site is central to the fidelity of protein synthesis. A highly conserved occurrence of three consecutive G-C base pairs in the anticodon stem of tRNA(fMet) contributes to its preferential selection in the P-site. In a genetic screen, using a plasmid borne copy of an inactive tRNA(fMet) mutant wherein the three G-C base pairs were changed, we isolated Escherichia coli strains that allow efficient initiation with the tRNA(fMet) mutant. Here, extensive characterization of two such strains revealed novel mutations in the metZWV promoter severely compromising tRNA(fMet) levels. Low cellular abundance of the chromosomally encoded tRNA(fMet) allows efficient initiation with the tRNA(fMet) mutant and an elongator tRNA(Gln), revealing that a high abundance of the cellular tRNA(fMet) is crucial for the fidelity of initiator tRNA selection on the ribosomal P-site in E. coli. We discuss possible implications of the changes in the cellular tRNA(fMet) abundance in proteome remodeling.

Cloning and characterization of a cluster of genes coding for 5S rRNA and three tRNAs from Azospirillum lipoferum

A genomic library was constructed from a HindIII digest of Azospirillum lipoferum chromosomal DNA in the HindIII site of pUC19. From the library, a clone, pALH64, which showed strong hybridization with 3' end labeled A. lipoferum total tRNAs and which contains a 2.9 kb insert was isolated and restriction map of the insert established. The nucleotide sequence of a 490 bp HindIII-HincII subfragment containing a cluster of genes coding for 5S rRNA, tRNA(Val)(UAC), tRNA(Thr)(UGA) and tRNA(Lys)(UUU) has been determined. The gene organization is 5S rRNA (115 bp), spacer (10 bp), tRNA(Val) (76 bp), spacer (3 bp), tRNA(Thr) (76 bp), spacer (7 bp) and tRNA(Lys) (76 bp). Hybridization experiments using A. lipoferum total tRNAs and 5S rRNA with the cloned DNA probes revealed that all three tRNA genes and the 5S rRNA gene are expressed in vivo in the bacterial cells.

Identification and characterization of a new plasmid carrying genes for degradation of 2,4-dichlorophenoxyacetate from Pseudomonas cepacia CSV90

Pseudomonas cepacia CSV90 is able to utilize 2,4-dichlorophenoxyacetate (2,4-D) and 2-methyl-4-chlorophenoxyacetate as sole sources of carbon and energy. Mutants of the strain CSV90 which had lost this ability appeared spontaneously on a nonselective medium. The wild-type strain harbored a 90-kb plasmid, pMAB1, whereas 2,4-D-negative mutants either lost the plasmid or had a 70-kb plasmid, pMAB2. The plasmid pMAB2 was found to have undergone a deletion Of a 20-kb fragment of pMAB1. The plasmid-free mutants regained the ability to degrade 2,4-D after introduction of purified pMAB1 by electroporation. Cloning in Escherichia coli of a 10-kb BamHI fragment from pMAB1, the region absent in pMAB2, resulted in the expression of the gene tfdC encoding 3,5-dichlorocatechol 1,2-dioxygenase. After subcloning, the tfdC gene was located in a 1.6-kb HindIII fragment. The nucleotide sequence of the tfdC gene and the restriction map of its contiguous region are identical to those of the well-characterized 2,4-D-degradative plasmid pJP4 of Alcaligenes eutrophus, whereas the overall restriction maps of the two plasmids are different. The N-terminal 44-amino-acid sequence of the enzyme purified from the strain CSV90 confirmed the reading frame in the DNA sequence for tfdC and indicated that the initiation codon GUG is read as methionine instead of valine.

Detection of nif genes in nitrogen-fixing bacterial isolate from tomato (Lycopersicon esculentum Mill cv Pusa ruby)

Nitrogen-fixing bacterial isolate from the intercellular spaces of tomato root cortical cells was studied for the location of nif genes on the chromosomal or plasmid DNA. The bacterial isolate showed two plasmids of approximate molecular sizes of 220 and 120 kb. Klebsiella pneumoniae nif HDK probe hybridized with the chromosomal DNA and not with the plasmid DNA thereby showing that nif genes are localised on the chromosomal DNA.

Mismatch repair genes of eukaryotes

Mismatches that arise during replication or genetic recombination or owing to damage to DNA by chemical agents are recognized by mismatch repair systems. The pathway has been characterized in detail in Escherichia coli. Several homologues of the genes encoding the proteins of this pathway have been identified in the yeast Saccharomyces cerevisiae and in human cells. Mutations in the human genes hMSH2, hMLH1, hPMS1 and hPMS2 have been linked to hereditary nonpolyposis colon cancer (HNPCC) and to some sporadic tumours. Mismatch repair also plays an antirecombinogenic role and is implicated in speciation.

Design and structural analysis of hairpin-TFO for transcriptional activation of genes in S-cerevisiae

Triplex forming oligonucleotides (TFOs) have the potential to modulate gene expression. While most of the experiments are directed towards triplex mediated inhibition of gene expression the strategy potentially could be used for gene specific activation. In an attempt to design a strategy for gene specific activation in vivo applicable to a large number of genes we have designed a TFO based activator-target system which may be utilized in Saccharomyces cerevisiae or any other system where Gal4 protein is ectopically expressed. The total genome sequence of Saccharomyces cerevisiae and expression profiles were used to select the target genes with upstream poly (pu/py) sequences. We have utilized the paradigm of Gal4 protein and its binding site. We describe here the selection of target genes and design of hairpin-TFO including the targeting sequences containing polypurine stretch found in the upstream promoter regions of weakly expressed genes. We demonstrate, the formation of hairpin-TFO, its binding to Gal4 protein, its ability to form triplex with the target duplex in vitro, the effect of polyethylenimine on complex formation and discuss the implication on in vivo transcription activation.

The beta-glucoside genes of Klebsiella aerogenes: conservation and divergence in relation to the cryptic bgl genes of Escherichia coli

The ability to metabolize aromatic beta-glucosides such as salicin and arbutin varies among members of the Enterobacteriaceae. The ability of Escherichia coli to degrade salicin and arbutin appears to be cryptic, subject to activation of the bgl genes, whereas many members of the Klebsiella genus can metabolize these sugars. We have examined the genetic basis for beta-glucoside utilization in Klebsiella aerogenes. The Klebsiella equivalents of bglG, bglB and bglR have been cloned using the genome sequence database of Klebsiella pneumoniae. Nucleotide sequencing shows that the K. aerogenes bgl genes show substantial similarities to the E. coli counterparts. The K. aerogenes bgl genes in multiple copies can also complement E. coli mutants deficient in bglG encoding the antiterminator and bglB encoding the phospho-beta-glucosidase, suggesting that they are functional homologues. The regulatory region bglR of K aerogenes shows a high degree of similarity of the sequences involved in BglG-mediated regulation. Interestingly, the regions corresponding to the negative elements present in the E. coli regulatory region show substantial divergence in K aerogenes. The possible evolutionary implications of the results are discussed. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.v. All rights reserved.

Features of a unique intronless cluster of class I small heat shock protein genes in tandem with box C/D snoRNA genes on chromosome 6 in tomato (Solanum lycopersicum)

Physical clustering of genes has been shown in plants; however, little is known about gene clusters that have different functions, particularly those expressed in the tomato fruit. A class I 17.6 small heat shock protein (Sl17.6 shsp) gene was cloned and used as a probe to screen a tomato (Solanum lycopersicum) genomic library. An 8.3-kb genomic fragment was isolated and its DNA sequence determined. Analysis of the genomic fragment identified intronless open reading frames of three class I shsp genes (Sl17.6, Sl20.0, and Sl20.1), the Sl17.6 gene flanked by Sl20.1 and Sl20.0, with complete 5' and 3' UTRs. Upstream of the Sl20.0 shsp, and within the shsp gene cluster, resides a box C/D snoRNA cluster made of SlsnoR12.1 and SlU24a. Characteristic C and D, and C' and D', boxes are conserved in SlsnoR12.1 and SlU24a while the upstream flanking region of SlsnoR12.1 carries TATA box 1, homol-E and homol-D box-like cis sequences, TM6 promoter, and an uncharacterized tomato EST. Molecular phylogenetic analysis revealed that this particular arrangement of shsps is conserved in tomato genome but is distinct from other species. The intronless genomic sequence is decorated with cis elements previously shown to be responsive to cues from plant hormones, dehydration, cold, heat, and MYC/MYB and WRKY71 transcription factors. Chromosomal mapping localized the tomato genomic sequence on the short arm of chromosome 6 in the introgression line (IL) 6-3. Quantitative polymerase chain reaction analysis of gene cluster members revealed differential expression during ripening of tomato fruit, and relatively different abundances in other plant parts.

Regulation of lipid biosynthesis by phosphatidylinositol-specific phospholipase C through the transcriptional repression of upstream activating sequence inositol containing genes

The regulation of phospholipid biosynthesis in Saccharomyces cerevisiae through cis-acting upstream activating sequence inositol (UAS(ino)) and trans-acting elements, such as the INO2-INO4 complex and OPI1 by inositol supplementation in growth is thoroughly studied. In this study, we provide evidence for the regulation of lipid biosynthesis by phosphatidylinositol-specific phospholipase C (PLC) through UAS(ino) and the trans-acting elements. Gene expression analysis and radiolabelling experiments demonstrated that the overexpression of rice PLC in yeast cells altered phospholipid biosynthesis at the levels of transcriptional and enzyme activity. This is the first report implicating PLC in the direct regulation of lipid biosynthesis. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

An analysis of substitution, deletion and insertion mutations in cancer genes

Cancer-associated mutations in cancer genes constitute a diverse set of mutations associated with the disease. To gain insight into features of the set, substitution, deletion and insertion mutations were analysed at the nucleotide level, from the COSMIC database. The most frequent substitutions were c -> t, g -> a, g -> t, and the most frequent codon changes were to termination codons. Deletions more than insertions, FS (frameshift) indels more than I-F (in-frame) ones, and single-nucleotide indels, were frequent. FS indels cause loss of significant fractions of proteins. The 5'-cut in FS deletions, and 5'-ligation in FS insertions, often occur between pairs of identical bases. Interestingly, the cut-site and 3'-ligation in insertions, and 3'-cut and join-pair in deletions, were each found to be the same significantly often (p < 0.001). It is suggested that these features aid the incorporation of indel mutations. Tumor suppressors undergo larger numbers of mutations, especially disruptive ones, over the entire protein length, to inactivate two alleles. Proto-oncogenes undergo fewer, less-disruptive mutations, in selected protein regions, to activate a single allele. Finally, catalogues, in ranked order, of genes mutated in each cancer, and cancers in which each gene is mutated, were created. The study highlights the nucleotide level preferences and disruptive nature of cancer mutations.