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PubMed1996

Isolation of the mRNA-capping enzyme and ferric-reductase-related genes from Candida albicans.

The mRNA-capping enzyme (mRNA 5'-guanylyltransferase) gene was cloned from a Candida albicans genomic DNA library by functional complementation of a Saccharomyces cerevisiae ceg1 delta null mutation. This gene, referred to as CGT1 (C. albicans guanylyltransferase 1), can encode a 52 kDa protein that is highly homologous to S. cerevisiae Ceg1p. CGT1 in a single-copy plasmid complemented the lethality of the S. cerevisiae ceg1 delta null mutation and, like S. cerevisiae Ceg1p, bacterially expressed Cgt1p was able to form a stable complex with the GMP moiety of GTP and to synthesize the cap structure in vitro, demonstrating that CGT1 is the C. albicans mRNA 5'-guanylyltransferase gene. CGT1 seemed to exist as a single copy in the C. albicans genome and was actively transcribed into mRNA. Another ORF was found in an opposite strand very close to the CGT1 locus. This gene shared significant sequence homology with S. cerevisiae FRE1, the gene encoding ferric reductase, and therefore was designated CFL1 (C. albicans ferric-reductase-like gene 1). Despite its sequence homology with S. cerevisiae FRE1, CFL1 mRNA was not induced by iron deprivation, and CFL1 did not complement the slow growth of a S. cerevisiae fre1 delta null mutant in the absence of iron, suggesting that CFL1 is functionally distinct from S. cerevisiae FRE1.

Amino Acid Sequence↗
PubMed2006

A genome-wide distribution of 8-oxoguanine correlates with the preferred regions for recombination and single nucleotide polymorphism in the human genome.

8-Oxoguanine (8-oxoG), a major spontaneous form of oxidative DNA damage, is considered to be a natural cause of genomic diversity in organisms because of its mutagenic potential. The steady-state level of 8-oxoG in the nuclear genome of a human cell has been estimated to be several residues per 10(6) guanines. In the present study, to clarify the genome-wide distribution of 8-oxoG in the steady state, we performed fluorescence in situ detection of 8-oxoG on human metaphase chromosomes using a monoclonal antibody. Multiple dot-like signals were observed on each metaphase chromosome. We then mapped the position of the signal at megabase resolution referring to the cytogenetically identified chromosomal band, and demonstrated that 8-oxoG is unevenly distributed in the normal human genome and that the distribution pattern is conserved among different individuals. Moreover, we found that regions with a high frequency of recombination and single nucleotide polymorphisms (SNPs) are preferentially located within chromosomal regions with a high density of 8-oxoG. Our findings suggest that 8-oxoG is one of the main causes of frequent recombinations and SNPs in the human genome, which largely contribute to the genomic diversity in human beings.

Adult↗
PubMed2003

Twenty thousand ORFan microbial protein families for the biologist?

The genomes of most newly sequenced organisms contain a significant fraction of ORFs (open reading frames) that match no other sequence in the databases. We refer to these singleton ORFs as sequence ORFans. Because little can be learned about ORFans by homology, the origin and functions of ORFans remain a mystery. However, in this era of full genome sequencing, it seems that ORFans have been underemphasized. In this minireview, we draw attention to the increasing number of ORFans and to the consequences of this growth to biological research in the postgenomic era.

Animals↗
PubMed2006

Genotypic analyses of Mycoplasma gallisepticum isolates from songbirds by random amplification of polymorphic DNA and amplified-fragment length polymorphism.

Mycoplasma gallisepticum (MG) conjunctivitis emerged in 1994 as a disease of free-ranging house finches (Carpodacus mexicanus) in North America and has also been isolated from other songbirds with conjunctivitis. Random amplification of polymorphic DNA (RAPD) of house finch and other songbird isolates has suggested that a single 'strain' initiated this outbreak. To explore the possibility of genomic variability among house finch isolates of MG and to evaluate the utility of a second technique for MG genotyping, we selected samples from our archive of reference strains and wild songbird isolates to analyze using both RAPD and amplified-fragment length polymorphism (AFLP); this is a newer technique that has been successfully used to explore the genomic variability of several Mycoplasma species. Both RAPD and AFLP results confirmed previous observations that during the initial stages of the MG epidemic in songbirds, isolates from different geographic locations and songbird species had genotypes that appeared to be highly similar, further supporting a single point source of origin. One 2001 isolate from New York was clearly different from the other songbird samples and clustered together with the vaccine and reference strains, indicating that substantial molecular evolution or a separate introduction has occurred.

Animals↗
PubMed2000

Genomic organization of the human interleukin-12 receptor beta2-chain gene.

The interleukin-12 receptor (IL-12R) is composed of two subunits, referred to as beta1 and beta2. Both chains are necessary for high-affinity IL-12 binding and signalling, although only the IL-12Rbeta2 chain contains the intracellular tyrosine residues responsible for STAT4 activation. This study presents the intron-exon organization of the human IL-12Rbeta2-chain gene. Polymerase chain reaction (PCR) primers designed across the cDNA (U46198) were used to trace introns, by comparing PCR product sizes obtained using cDNA and genomic DNA as templates. PCR products spanning introns were sequenced to determine the exact splice sites and flanking regions. The coding region of the gene was found to consist of 15 exons and 14 introns. All intron-exon boundaries are consistent with the consensus sequence for splice junctions (5' GT/AG 3'). Comparison of the intron-exon organization with the human GCSFR gene indicated a remarkably well conserved genomic organization between these two class I cytokine receptors. Interestingly, we identified an alternatively spliced mRNA, encoding a putative, truncated protein, lacking all signalling potential.

Alternative Splicing↗
PubMed2004

Molecular identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms.

Fifty strains belonging to Vibrio harveyi, Vibrio campbellii, and the recently described Vibrio rotiferianus, were analysed using phenotypic and genomic techniques with the aim of analysing the usefulness of the different techniques for the identification of V. harveyi-related species. The species V. harveyi and V. campbellii were phenotypically indistinguishable by more than 100 phenotypic features. Thirty-nine experimental strains were phenotypically identified as V. harveyi, but FAFLP, REP-PCR, IGS-PCR and DNA-DNA hybridization proved that they in fact belong to the species V. campbellii. Similar groupings were found among all fingerprinting methodologies (except IGS-PCR). Thirty-two experimental strains clustered with the V. campbellii type and one reference strain; seven strains clustered with the V. harveyi type and three reference strains; and the type and four reference strains of V. rotiferianus grouped together. The correlations between DNA-DNA hybridization and the genomic fingerprinting by FAFLP and (GTG)(5)-PCR were found to be above 0.68 and statistically significant, suggesting the value of the latter techniques for the reliable identification of V. harveyi-related species. The results presented indicate that strains phenotypically identified as V. harveyi are in fact V. campbellii; these findings position V. campbellii as an important species involved in diseases of reared aquatic organisms.

Animals↗
PubMed2000

Complementing genomics with proteomics: the membrane subproteome of Pseudomonas aeruginosa PAO1.

With the completion of many genome projects, a shift is now occurring from the acquisition of gene sequence to understanding the role and context of gene products within the genome. The opportunistic pathogen Pseudomonas aeruginosa is one organism for which a genome sequence is now available, including the annotation of open reading frames (ORFs). However, approximately one third of the ORFs are as yet undefined in function. Proteomics can complement genomics, by characterising gene products and their response to a variety of biological and environmental influences. In this study we have established the first two-dimensional gel electrophoresis reference map of proteins from the membrane fraction of P. aeruginosa strain PA01. A total of 189 proteins have been identified and correlated with 104 genes from the P. aeruginosa genome. Annotated membrane proteins could be grouped into three distinct categories: (i) those with functions previously characterised in P. aeruginosa (38%); (ii) those with significant sequence similarity to proteins with assigned function or hypothetical proteins in other organisms (46%); and (iii) those with unknown function (16%). Transmembrane prediction algorithms showed that each identified protein sequence contained at least one membrane-spanning region. Furthermore, the current methodology used to isolate the membrane fraction was shown to be highly specific since no contaminating cytosolic proteins were characterised. Preliminary analysis showed that at least 15 gel spots may be glycosylated in vivo, including three proteins that have not previously been functionally characterised. The reference map of membrane proteins from this organism is now the basis for determining surface molecules associated with antibiotic resistance and efflux, cell-cell signalling and pathogen-host interactions in a variety of P. aeruginosa strains.

Bacterial Proteins↗
PubMed2002

Genetic requirements for the episomal maintenance of oncogenic herpesvirus genomes.

Herpesviruses are large double-stranded DNA viruses that are characterized by lifelong latency. Epstein-Barr virus (EBV), the recently discovered Kaposi's sarcoma associated herpesvirus (KSHV), also referred to as human herpesvirus-8 (HHV-8), and the simian Herpesvirus saimiri (HVS) are associated with malignant lymphoproliferative diseases. These viruses establish latent infection in lymphoid cells. During latency only a few viral genes are expressed and the viral genome persists as a multicopy circular episome. The episome contains repetitive sequences that serve as multiple cooperative binding sites for the viral DNA binding proteins Epstein-Barr virus nuclear antigen 1 (EBNA-1) of EBV and latency-associated nuclear antigen (LANA1) of KSHV and HVS, which are expressed during latency. The oligomerized proteins associate with the viral genome and tether it to host chromosomes, assuring continual lifelong persistence of the virus.

Antigens, Viral↗
PubMed2006

Transcript mapping with high-density oligonucleotide tiling arrays.

MOTIVATION: High-density DNA tiling microarrays are a powerful tool for the characterization of complete transcriptomes. The two major analytical challenges are the segmentation of the hybridization signal along genomic coordinates to accurately determine transcript boundaries and the adjustment of the sequence-dependent response of the oligonucleotide probes to achieve quantitative comparability of the signal between different probes. RESULTS: We describe a dynamic programming algorithm for finding a globally optimal fit of a piecewise constant expression profile along genomic coordinates. We developed a probe-specific background correction and scaling method that employs empirical probe response parameters determined from reference hybridizations with no need for paired mismatch probes. This combined analysis approach allows the accurate determination of dynamical changes in transcription architectures from hybridization data and will help to study the biological significance of complex transcriptional phenomena in eukaryotic genomes. AVAILABILITY: R package tilingArray at http://www.bioconductor.org.

Algorithms↗
PubMed2001

Heavy metals of relevance to human health induce genomic instability.

Heavy metals used in medical prostheses or present in water supplies or tobacco can build up in tissues and blood and are well known to produce toxic effects. Normally, legislative controls on the levels of these substances are determined by reference to the acute toxicity data. This paper shows that cadmium and nickel can produce delayed effects in human cells in vitro, which are characteristic of genomic instability. The effects occur even at levels where no acute toxic effects can be demonstrated. Genomic instability can be demonstrated by persistent induction of cytogenetic abnormalities and delayed cell death in progeny of cells many generations after exposure. Formerly, this syndrome has only been definitively proven to occur following irradiation, but in these experiments cell populations exposed for only 1 or 24 hours were expanded over several months, involving eight passages, and the yield of chromosomal aberrations and cell loss due to lethal mutations did not decrease. The consequences of this genomic instability are not yet known but it is possible that many of the systemic symptoms associated with exposure to low concentrations of these metals could involve delayed expression of cellular damage. It is also clear that these effects cannot be predicted from acute toxicity data.

Cadmium↗
PubMed2004

Megabase deletions of gene deserts result in viable mice.

The functional importance of the roughly 98% of mammalian genomes not corresponding to protein coding sequences remains largely undetermined. Here we show that some large-scale deletions of the non-coding DNA referred to as gene deserts can be well tolerated by an organism. We deleted two large non-coding intervals, 1,511 kilobases and 845 kilobases in length, from the mouse genome. Viable mice homozygous for the deletions were generated and were indistinguishable from wild-type littermates with regard to morphology, reproductive fitness, growth, longevity and a variety of parameters assaying general homeostasis. Further detailed analysis of the expression of multiple genes bracketing the deletions revealed only minor expression differences in homozygous deletion and wild-type mice. Together, the two deleted segments harbour 1,243 non-coding sequences conserved between humans and rodents (more than 100 base pairs, 70% identity). Some of the deleted sequences might encode for functions unidentified in our screen; nonetheless, these studies further support the existence of potentially 'disposable DNA' in the genomes of mammals.

Animals↗
PubMed1990

Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin.

ADP-ribosylation factors (ARFs) are approximately 20-kDa proteins that act as GTP-dependent allosteric activators of cholera toxin. With deoxyinosine-containing degenerate oligonucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences. Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis. One amplified DNA contained no introns and had a sequence different from those of known ARFs. Based on this sequence, selective oligonucleotide probes were prepared and used to isolate clone psi ARF 4, a putative ARF pseudogene, from a human genomic library in lambda phage EMBL3. Reverse transcription-PCR was then used to clone from human poly(A)+ RNA the cDNA corresponding to the expressed homolog of psi ARF 4, referred to as human ARF 4. It appears that psi ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues. The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.

ADP-Ribosylation Factors↗
PubMed1999

The three copies of the ATP1 gene are arranged in tandem on chromosome II of Saccharomyces cerevisiae S288C.

In the yeast Saccharomyces cerevisiae there are three copies of the F(1)F(0)-ATPase alpha-subunit gene ATP1 on chromosome II (Takeda et al., 1995). However, after genome analysis using S. cerevisiae strain S288C, only one ATP1 gene sequence was observed (Feldman et al., 1994; Obermaier et al., 1995). To check whether the number of copies of ATP1 is strain-dependent or not, we carried out three different experiments: (a) long-PCR analyses of total DNAs isolated from several reference strains, carried out by preparing 29-mer oligonucleotides based on the 5'- and 3'- up- and downstream regions of the ATP1 nucleotide sequence using the data from the genome project to synthesize primers; (b) restriction analyses of chromosome II from the reference strains with SplI; and (c) long-PCR analyses of prime clones 70113 and 70804, both of which contained two ATP1 gene copies, ATP1a and ATP1b, and ATP1b and ATP1c, respectively, using 30 nucleotides just inside the 3'-end (sense) and 5'-end (antisense) of the ATP1-coding region as primers. In the case of the long-PCR experiments, the reference strains DC5, SEY2102, W303-1A, W303-1B, LL20 and DBY746, as well as strain S288C, generated a DNA fragment of approximately 32 kb, which hybridized with ATP1. During SplI digestion, a DNA fragment of more than 50 kb which hybridized with ATP1, was obtained from all reference strains. In the case of prime clone analyses using the long-PCR experiments, the distance between ATP1a and ATP1b or ATP1b and ATP1c was approximately 10 kb or 7 kb, respectively. The S288C strain generated these two DNA fragments, as do the other strains. These results showed that all these strains contained three copies of ATP1 on chromosome II.

Base Sequence↗
PubMed2002

Targeting critical regions in genomic DNA with AT-specific anticancer drugs.

Cellular DNA is not a uniform target for DNA-reactive drugs. At the nucleotide level, drugs recognize and bind short motifs of a few base pairs. The location of drug adducts at the genomic level depends on how these short motifs are distributed in larger domains. This aspect, referred to as region specificity, may be critical for the biological outcome of drug action. Recent studies demonstrated that certain minor groove binding (MGB) drugs, such as bizelesin, produce region-specific lesions in cellular DNA. Bizelesin binds mainly T(A/T)(4)A sites, which are on average scarce, but occasionally cluster in distinct minisatellite regions (200-1000 bp of approximately 85-100% AT), herein referred to as AT islands. Bizelesin-targeted AT islands are likely to function as strong matrix attachment regions (MARs), domains that organize DNA loops on the nuclear matrix. Distortion of MAR-like AT islands may be a basis for the observed inhibition of new replicon initiation and the extreme lethality of bizelesin adducts (<10 adducts/cell for cell growth inhibition). Hence, long AT-islands represent a novel class of critical targets for anticancer drugs. The AT island paradigm illustrates the potential of the concept of regional targeting as an essential component of the rational design of new sequence-specific DNA-reactive drugs.

Antineoplastic Agents↗
PubMed2006

Mapping the proteome of poplar and application to the discovery of drought-stress responsive proteins.

Poplar is the first forest tree genome to be decoded. As an initial step to the comprehensive analysis of poplar proteome, we described reference 2-D-maps for eight tissues/organs of the plant, and the functional characterization of some proteins. A total of 398 proteins were excised from the gels. About 91.2% were identified by nanospray LC-MS/MS, based on comparison with 260,000 Populus sp. ESTs. In comparison, reliable PMFs were obtained for only 51% of the spots by MALDI-TOF-MS, from which 43% (83 spots) positively matched gene models of the Populus trichocarpa genome sequence. Among these 83 spots, 58% matched with the same proteins as identified by LC-MS/MS, 21.7% with unknown function proteins and 19.3% with completely different functions. In the second phase, we studied the effect of drought stress on poplar root and leaf proteomes. The function of up- and down-regulated proteins is discussed with respect to the physiological response of the plants and compared with transcriptomic data. Some important clues regarding the way poplar copes with water deficit were revealed.

Databases, Protein↗
PubMed2004

Survey of the number of two-component response regulator genes in the complete and annotated genome sequences of prokaryotes.

The numbers of potential response regulator genes were determined from the complete and annotated genome sequences of Archaea and Bacteria. The numbers of each class of response regulators are shown for each organism, determined principally from BLASTP searches, but with reference to the gene category lists where available. The survey shows that for Bacteria there is a link between the total number of potential response regulator genes and both the genome complexity (number of potential protein-coding genes) and the organism's lifestyle/habitat. Increasingly complex lifestyles and genome complexities are matched by an increase in the average number of potential response regulator genes per genome, indicating that a higher degree of complexity requires a higher level of control of gene expression and cellular activity. Detailed results of this study are available online at and.

Data Collection↗
PubMed2004

Decreasing the effects of horizontal gene transfer on bacterial phylogeny: the Escherichia coli case study.

Phylogenetic reconstructions of bacterial species from DNA sequences are hampered by the existence of horizontal gene transfer. One possible way to overcome the confounding influence of such movement of genes is to identify and remove sequences which are responsible for significant character incongruence when compared to a reference dataset free of horizontal transfer (e.g., multilocus enzyme electrophoresis, restriction fragment length polymorphism, or random amplified polymorphic DNA) using the incongruence length difference (ILD) test of Farris et al. [Cladistics 10 (1995) 315]. As obtaining this "whole genome dataset" prior to the reconstruction of a phylogeny is clearly troublesome, we have tested alternative approaches allowing the release from such reference dataset, designed for a species with modest level of horizontal gene transfer, i.e., Escherichia coli. Eleven different genes available or sequenced in this work were studied in a set of 30 E. coli reference (ECOR) strains. Either using ILD to test incongruence between each gene against the all remaining (in this case 10) genes in order to remove sequences responsible for significant incongruence, or using just a simultaneous analysis without removals, gave robust phylogenies with slight topological differences. The use of the ILD test remains a suitable method for estimating the level of horizontal gene transfer in bacterial species. Supertrees also had suitable properties to extract the phylogeny of strains, because the way they summarize taxonomic congruence clearly limits the impact of individual gene transfers on the global topology. Furthermore, this work allowed a significant improvement of the accuracy of the phylogeny within E. coli.

DNA, Bacterial↗
PubMed1994

Two genomic variations in the E1 region of canine adenovirus type 2 strains.

We amplified the E1 region of canine adenovirus type 2 genomes by the polymerase chain reaction (PCR) and analyzed the PCR products by using eight restriction endonucleases. Restriction patterns of the E1 region cleaved with HaeIII and RsaI revealed two genomic variations among the canine adenovirus type 2 strains. Although the clinical significance of two distinct genotypes among the canine adenovirus type 2 strains is currently unknown, these genomic variations are well conserved among different strains in each genotype and suggest that the Japanese field strains, with reference to the E1 region, are different from the non-Japanese strains examined.

Adenoviruses, Canine↗