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PubMed1999

Differentiation of chromatin during DNA elimination in Euplotes crassus.

In Euplotes crassus, most of the micronuclear genome is eliminated during formation of a transcriptionally active macronucleus. To understand how this is mediated throughout the genome, we have examined the chromatin structure of the macronucleus-destined sequences and Tec transposons, which are dispersed in 15,000 copies in the micronuclear genome and completely eliminated during formation of the macronuclear genome. Whereas the macronucleus-destined sequences show a typical pattern of nucleosomal repeats in micrococcal nuclease digests, the Tec element chromatin structure digests to a nucleosome-like repeat pattern that is not typical: the minimum digestion products are approximately 300-600 base pairs, or "subnucleosomal," in size. In addition, the excised, circular forms of the Tec elements are exceedingly resistant to nucleases. Nevertheless, an underlying nucleosomal structure of the Tec elements can be demonstrated from the size differences between repeats in partial micrococcal nuclease digests and by trypsin treatment of nuclei, which results in mononucleosome-sized products. Characterization of the most micrococcal nuclease-resistant DNA indicates that micronuclear telomeres are organized into a chromatin structure with digestion properties identical to those of the Tec elements in the developing macronucleus. Thus, these major repetitive sequence components of the micronuclear genome differ in their chromatin structure from the macronuclear-destined sequences during DNA elimination. The potential role of developmental stage-specific histone variants in this chromatin differentiation is discussed.

Animals↗
PubMed2005

A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites.

Apicomplexan parasites constitute one of the most significant groups of pathogens infecting humans and animals. The liver stage sporozoites of Plasmodium spp. and tachyzoites of Toxoplasma gondii, the causative agents of malaria and toxoplasmosis, respectively, use a unique mode of locomotion termed gliding motility to invade host cells and cross cell substrates. This amoeboid-like movement uses a parasite adhesin from the thrombospondin-related anonymous protein (TRAP) family and a set of proteins linking the extracellular adhesin, via an actin-myosin motor, to the inner membrane complex. The Plasmodium blood stage merozoite, however, does not exhibit gliding motility. Here we show that homologues of the key proteins that make up the motor complex, including the recently identified glideosome-associated proteins 45 and 50 (GAP40 and GAP50), are present in P. falciparum merozoites and appear to function in erythrocyte invasion. Furthermore, we identify a merozoite TRAP homologue, termed MTRAP, a micronemal protein that shares key features with TRAP, including a thrombospondin repeat domain, a putative rhomboid-protease cleavage site, and a cytoplasmic tail that, in vitro, binds the actin-binding protein aldolase. Analysis of other parasite genomes shows that the components of this motor complex are conserved across diverse Apicomplexan genera. Conservation of the motor complex suggests that a common molecular mechanism underlies all Apicomplexan motility, which, given its unique properties, highlights a number of novel targets for drug intervention to treat major diseases of humans and livestock.

Actins↗
PubMed1978

Attempted separation of transcriptively active and inactive chromatin by hydroxylapatite thermal chromatography.

Chromatin and purified DNA were fractionated by hydroxylapatite thermal chromatography. Fractions of varying thermal stability were tested for the proportions of transcribed sequences and repetitive sequences relative to the unfractionated genome. The first 80--85% of either total chromatin or purified DNA eluted from hydroxylapatite contained the same proportion of hybridizable sequences as total DNA. The remaining 15--20% of chromatin eluting at the highest temperatures was depleted of transcribed sequences. Analysis of the 20% highest melting fraction of purified DNA showed that, while the first two-thirds of this fraction contained the same proportion of transcribed sequences as unfractionated DNA, the last third, comprising about 6% of total DNA, was depleted of active sequences. Although no major differences were detected in nonrepetitive sequence complexity of chromatin fractions, there was a correlation between relative thermal stability and repetitive sequence content in fractions of both chromatin and DNA separated by thermal chromatography. Fragments eluting at higher temperatures contained a greater proportion of repetitive sequences, as indicated by a rapidly renaturing component. Most likely, the latest eluting fractions from both chromatin and purified DNA were enriched for a nontranscribed, highly reiterated, G+C rich satellite component of the chicken genome.

Animals↗
PubMed2006

Comparative analysis of HOG pathway proteins to generate hypotheses for functional analysis.

Comparative genomics allows comparison of different proteins that execute presumably identical functions in different organisms. In contrast to paralogues, orthologues per definition perform the same function and interact with the same partners and, consequently, should display conservation in all these properties. We have employed 20 fungal genomes to analyse key components of the high osmolarity glycerol signalling pathway of Saccharomyces cerevisiae. Among the proteins scrutinised are a complete phosphotransfer module, a MAP kinase, two scaffold proteins, one of which is also a MAPKK, and two transcription factors. Sequence alignments, domain structure and size analysis, combined with the rich information available in the literature, allowed us to probe previous structural and functional studies and to generate hypotheses for future experimental studies. Although certain domains are too highly conserved across fungal species for meaningful comparative studies, others, like interaction domains, can be studied in closely related species. Moreover, putative functionally relevant sites for protein modifications can be identified in such comparative studies. We provide several relevant examples and present a number of previously un(der)characterised domains of potential functional significance in osmosensing and signal transduction. We propose that any functional protein analysis in fungi should make use of the unique resource that fungal genome sequences offer.

Basic-Leucine Zipper Transcription Factors↗
PubMed2001

Additional rep-encoding DNAs associated with banana bunchy top virus.

Banana bunchy top nanovirus (BBTV) has a multicomponent circular single-stranded DNA (cssDNA) genome consisting of at least six components. We have cloned, sequenced and analysed two additional cssDNA components, designated BBTV-S1 and S2, associated with a Taiwanese BBTV isolate. The sequences of BBTV-S1 and S2 comprised 1109 and 1095 nucleotides (nt), respectively, and like BBTV DNA-1, potentially encoded replication initiation proteins (Reps). However, the genome organisation of BBTV-S1 and S2 differed from that of BBTV DNA-1 in that (i) the stem sequence of the CR-SL was not conserved, (ii) the internal gene was absent and (iii) the probable TATA boxes were located 5' of the stem-loop. Further, sequence and phylogenetic analysis of the Rep genes indicated that BBTV DNA-S1 and S2 were distinct from BBTV DNA-1. When different geographical isolates of BBTV were tested for the presence of BBTV-S1/S2, these components were detected in various isolates from Vietnam, Taiwan, the Philippines, Tonga and Samoa but were not detected in isolates from Australia, Egypt, Fiji, and India. Based on these results, BBTV-S1 and S2 do not appear to be integral components of the BBTV genome and represent additional Rep-encoding DNAs associated with BBTV.

Amino Acid Sequence↗
PubMed1999

Estimation of P-to-O ratio in Bacillus subtilis and its influence on maximum riboflavin yield.

Simultaneous growth and riboflavin overproduction were investigated using a previously developed stoichiometric model of Bacillus subtilis metabolism. A fit of model predictions to experimental data was used to obtain estimates of fundamental energetic parameters of B. subtilis. Although multiple solutions describe the experimental data, evidence for a P-to-O ratio of about 1(1/3) mole of ATP produced per atom of oxygen consumed in oxidative phosphorylation was provided by genomic analysis of electron transport components, because no homologue of the proton-translocating NADH dehydrogenase I was found in the B. subtilis genome database. These results allow us to devise a rational metabolic engineering strategy to improve riboflavin production. The potential influence of increased energy coupling in oxidative phosphorylation on riboflavin yield is discussed. Higher coupling is most significant under carbon-limiting conditions in slow-growing cells, that is, in fed-batch processes of industrial interest.

Adenosine Triphosphate↗
PubMed1981

Transcription of the simian virus 40 genome in DNA-transformed murine teratocarcinoma stem cells.

To study the molecular basis for lack of expression of the simian virus 40 (SV40) early region genes in murine teratocarcinoma-derived stem cells, we introduced a recombinant plasmid consisting of pBR322 linked to the herpes simplex virus type 1 thymidine kinase gene and SV40 genome into thymidine kinase-deficient F9 stem cells. The resulting stem cell clone, 12-1, and a retinoic acid-induced differentiated daughter cell clone, 12-1a, each contain one copy per cell of the entire recombinant plasmid integrated into the cellular genome through a site on the pBR322 genome. Restriction endonuclease analyses indicate that there is no difference in integration site or organization of the three component parts of the plasmid genome within cellular DNA of stem and differentiated cells; yet the differentiated cells, 12-1a, express SV40 large tumor antigen whereas the stem cells, 12-1, do not. Both stem and differentiated cells produce two size classes of polyadenylylated RNA, 2900 and 2600 bases in length, homologous to the early region of the SV40 genome, detectable by RNA blotting analysis. S1 nuclease analysis of the SV40 transcripts present in stem and differentiated cells indicate that the SV40 mRNAs were identically spliced in the two cell types, in a manner consistent with that observed for spliced large and small tumor antigen mRNAs in SV40-infected monkey kidney cells. Thus, the failure of 12-1 teratocarcinoma stem cells, containing an integrated SV40 genome, to express SV40 tumor antigen is not due to a lack of transcription of the SV40 early region or to an inability to splice primary transcripts.

Animals↗
PubMed2003

MGD: the Mouse Genome Database.

The Mouse Genome Database (MGD) (http://www.informatics.jax.org) one component of a community database resource for the laboratory mouse, a key model organism for interpreting the human genome and for understanding human biology. MGD strives to provide an extensively integrated information resource with experimental details annotated from both literature and on-line genomic data sources. MGD curates and presents the consensus representation of genotype (sequence) to phenotype information including highly detailed information about genes and gene products. Primary foci of integration are through representations of relationships between genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse. Recent developments include a general implementation of database structures for controlled vocabularies and the integration of a phenotype classification system.

Alleles↗
PubMed1990

Replicating single-stranded adenovirus type 5 DNA molecules accumulate within well-delimited intranuclear areas of lytically infected HeLa cells.

The intranuclear compartments which contain adenovirus type 5 (Ad5) genomes during productive infection of HeLa cells were determined by high resolution when the rate of viral DNA replication is at its maximum. In situ hybridization was performed with and without denaturation of Ad5 DNA to reveal those portions of both double-stranded and single-stranded viral DNA and of single-stranded viral DNA alone, respectively, which were present at the surface of Lowicryl thin sections. Autoradiography after short pulse incorporation of tritiated thymidine revealed sites of active DNA replication whereas a pulse-chase experiment visualized the migration of the synthesized DNA. Immunocytochemistry determined distribution of the Ad5 72 kDa protein. Viral DNA was contained within multiple, well-delimited, compact fibrillar structures and their immediately surrounding, less well-defined, fibrillo-granular areas of the cell nucleoplasm. The compact fibrillar masses were primarily storage sites of single-stranded viral DNA and its accompanying 72 kDa protein, so they were named ssDNA accumulation sites. They contained intermittent viral replicative activity. Conversely, viral ssDNA and 72 kDa protein were rare in the surrounding fibrillo-granular areas in which Ad5 replication DNA is continuous, so we referred to them as peripheral replicative zones. Well-delimited subnuclear structures induced by viruses, therefore, represent accumulations of specific components of their replicating genomes.

Adenoviruses, Human↗
PubMedDate not supplied

[Genome of the pogonophore Siboglinum fiordicum: organizational characteristics and divergence with respect to the genomes of representatives of certain types of invertebrates].

DNA of Pogonophora Siboglinum fiordicum was studied. DNA was isolated from ethanol fixed animals. It contains 40.5% GC according to the melting temperature and 39.3% GC as estimated by cation-exchange chromatography. From the reassociation kinetic data with a [125I] tracer 400 nucleotide long it is clear that 70% of the genome consists of repetitive sequences. The fast component (C0t less than 0.04) constitutes 11% of the genome, the intermediate (C0t 1/2 = 1.5, repetition frequency 1600)--59%. The unique sequences comprise about 30% of the genome. Experiments where [125I]tracers of increasing length were reassociated with driver DNA fragments 300 nucleotides long point out that at least 80% of the genome contains repetitive sequences, part of them being organized in short-period interspersion patterns. The hybridization of Pogonophora [125I]DNA fragments with DNA of invertebrates of other types--Echinodermata, Ascidia, Mollusca, Annelida--did not show any measurable homology between their DNAs neither in repetitive, nor in the unique fraction.

Animals↗
PubMed2005

AUGUSTUS: a web server for gene prediction in eukaryotes that allows user-defined constraints.

We present a WWW server for AUGUSTUS, a software for gene prediction in eukaryotic genomic sequences that is based on a generalized hidden Markov model, a probabilistic model of a sequence and its gene structure. The web server allows the user to impose constraints on the predicted gene structure. A constraint can specify the position of a splice site, a translation initiation site or a stop codon. Furthermore, it is possible to specify the position of known exons and intervals that are known to be exonic or intronic sequence. The number of constraints is arbitrary and constraints can be combined in order to pin down larger parts of the predicted gene structure. The result then is the most likely gene structure that complies with all given user constraints, if such a gene structure exists. The specification of constraints is useful when part of the gene structure is known, e.g. by expressed sequence tag or protein sequence alignments, or if the user wants to change the default prediction. The web interface and the downloadable stand-alone program are available free of charge at http://augustus.gobics.de/submission.

Animals↗
PubMed2005

Genome-wide search for genes affecting serum uric acid levels: the Framingham Heart Study.

Serum uric acid levels are associated with hypertension, cardiovascular disease, and renal disease. Uric acid has been shown to be heritable; however, genome-wide linkage analyses have not been reported. Genome-wide multipoint variance components linkage analyses with 401 markers spaced at approximately 10 centimorgan (cM) were conducted on 1258 subjects of the Framingham Heart Study, using the average of two serum uric acid measurements obtained in examinations 1 and 2 around 1971 and 1979. Covariates in fully adjusted model included sex, age, body mass index (BMI), serum creatinine, alcohol consumption, diabetes, diuretic treatment, and triglycerides. To investigate possible pleiotropic effects between uric acid and covariates that may have a genetic component, bivariate linkage analyses of uric acid with BMI, triglycerides, and glucose were conducted at the uric acid linkage regions. The heritability of uric acid was 0.63. The highest multipoint log-of-the-odds (LOD) score was 3.3 at 50 cM on chromosome 15 for age-sex-adjusted uric acid, but decreased to 1.5 after multivariable adjustment. Additional evidence of linkage was seen on chromosomes 2 (LOD score 1.1 at 4 cM) and 8 (LOD score 1.7 at 6 cM) for multivariable-adjusted uric acid. Pleiotropic effects were only found between uric acid and glucose and BMI at chromosomes 8 and 15 linkage locations, respectively. We have identified several novel loci linked to uric acid. We found possible pleiotropic effects between uric acid and BMI and glucose. Further research is necessary to identify the genes involved in uric acid metabolism and their roles in hypertension, cardiovascular disease, and renal disease.

Adult↗
PubMed2026

The Rise of Plant Pan-Genomes: From Genome Variation to Predictive Breeding.

Plant pan-genomics is entering a new phase beyond genome variation discovery, requiring a shift from cataloguing genomic diversity toward understanding how variation generates biological function and breeding value. Here, we propose that the future of plant pan-genomics will be shaped by three conceptual transitions. First, structural variation (SV), presence-absence variation (PAV), and haplotype diversity should be interpreted not merely as genomic differences, but as regulatory components that influence gene networks, chromatin organization, and complex traits. Second, the expansion from species-level pan-genomes to genus-level super pan-genomes provides an evolutionary framework for uncovering adaptive genetic modules preserved in wild relatives and overlooked during domestication. Third, integrating pan-genomes with pan-omics, three-dimensional genome analyses, and artificial intelligence will enable the transformation of genomic variation into predictive models for crop improvement. We further propose that the ultimate value of pan-genomes lies not in generating increasingly complete genome collections, but in establishing a mechanistic bridge between genome diversity, biological function, and breeding decisions. This transition will move crop improvement from empirical selection toward rational genome design, where evolutionary diversity can be systematically interpreted, predicted, and engineered.

Journal Article↗
PubMed2006

Marine cyanophages and light.

In contrast to the phages of heterotrophic hosts, light can play a key role in all aspects of the life cycle of phages infecting ecologically important marine unicellular cyanobacteria of the genera Synechococcus and Prochlorococcus. Phage adsorption, replication, modulation of the host cell metabolism, and survival in the environment following lysis, all exhibit light-dependent components. The analysis of cyanophage genomes has revealed the acquisition of key photosynthetic genes during the course of evolution, such as those encoding central components of the light harvesting apparatus. These discoveries are beginning to reveal novel features of the interactions between parasite and host that shape the biology of both.

Bacteriophages↗
PubMed2006

Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey.

BACKGROUND: Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. AIMS: In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC) library for the nurse shark, Ginglymostoma cirratum. RESULTS: The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 x 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6-28 primary positive clones per probe of which 50-90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible. CONCLUSION: We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.

Animals↗
PubMed2006

Genomewide scan for real-word reading subphenotypes of dyslexia: novel chromosome 13 locus and genetic complexity.

Dyslexia is a common learning disability exhibited as a delay in acquiring reading skills despite adequate intelligence and instruction. Reading single real words (real-word reading, RWR) is especially impaired in many dyslexics. We performed a genome scan, using variance components (VC) linkage analysis and Bayesian Markov chain Monte Carlo (MCMC) joint segregation and linkage analysis, for three quantitative measures of RWR in 108 multigenerational families, with follow up of the strongest signals with parametric LOD score analyses. We used single-word reading efficiency (SWE) to assess speed and accuracy of RWR, and word identification (WID) to assess accuracy alone. Adjusting SWE for WID provided a third measure of RWR efficiency. All three methods of analysis identified a strong linkage signal for SWE on chromosome 13q. Based on multipoint analysis with 13 markers we obtained a MCMC intensity ratio (IR) of 53.2 (chromosome-wide P < 0.004), a VC LOD score of 2.29, and a parametric LOD score of 2.94, based on a quantitative-trait model from MCMC segregation analysis (SA). A weaker signal for SWE on chromosome 2q occurred in the same location as a significant linkage peak seen previously in a scan for phonological decoding. MCMC oligogenic SA identified three models of transmission for WID, which could be assigned to two distinct linkage peaks on chromosomes 12 and 15. Taken together, these results indicate a locus for efficiency and accuracy of RWR on chromosome 13, and a complex model for inheritance of RWR accuracy with loci on chromosomes 12 and 15.

Adolescent↗
PubMed2005

Genome-wide linkage scans for prediabetes phenotypes in response to 20 weeks of endurance exercise training in non-diabetic whites and blacks: the HERITAGE Family Study.

AIMS/HYPOTHESIS: Impaired insulin secretion, insulin action, insulin-independent glucose effectiveness, glucose tolerance and the associated abnormalities in insulin and glucose metabolism phenotypes are precursors of type 2 diabetes. Genome-wide multipoint variance component linkage scans were carried out using 654 markers to identify quantitative trait loci for insulin sensitivity, acute insulin response to glucose, disposition index and glucose effectiveness training responses in whites and blacks in the HERITAGE Family Study. METHODS: These phenotypes were obtained from an IVGTT with the minimal model. The distributions of insulin sensitivity, acute insulin response to glucose and disposition index training responses (post-training minus baseline) were approximately normalised using a square-root transformation. All phenotypes were adjusted for the effects of age, BMI and their respective baseline values within sex and generation by race prior to linkage scans. RESULTS: In blacks, a promising linkage with a maximum lod score of 3.1 on 19q (54-62 Mb) for glucose effectiveness training response was found. Six interesting linkages with lod scores of at least 1.0 were found for disposition index training response in whites. They included 1p (30 Mb), 3q (152 Mb), 6p (23-42 Mb), 7q (95-96 Mb), 10p (15 Mb) and 12q (119-126 Mb). CONCLUSIONS/INTERPRETATION: Quantitative trait loci for 20 weeks of endurance exercise training responses in insulin action and glucose metabolism phenotypes were found on chromosome 19q as well as 6p and 7q, with nominal (6p, 7q) but consistent (6p) linkages across the races.

Adolescent↗
PubMed2006

Role of the beta1 subunit in the function and stability of the 20S proteasome in the hyperthermophilic archaeon Pyrococcus furiosus.

The hyperthermophilic archaeon Pyrococcus furiosus genome encodes three proteasome component proteins: one alpha protein (PF1571) and two beta proteins (beta1-PF1404 and beta2-PF0159), as well as an ATPase (PF0115), referred to as proteasome-activating nucleotidase. Transcriptional analysis of the P. furiosus dynamic heat shock response (shift from 90 to 105 degrees C) showed that the beta1 gene was up-regulated over twofold within 5 minutes, suggesting a specific role during thermal stress. Consistent with transcriptional data, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that incorporation of the beta1 protein relative to beta2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the beta2/beta1 ratio varied linearly with temperature from 3.8, when assembled at 80 degrees C, to 0.9 at 105 degrees C. The recombinant alpha+beta1+beta2 CP assembled at 105 degrees C was more thermostable than either the alpha+beta1+beta2 version assembled at 90 degrees C or the alpha+beta2 version assembled at either 90 degrees C or 105 degrees C, based on melting temperature and the biocatalytic inactivation rate at 115 degrees C. The recombinant CP assembled at 105 degrees C was also found to have different catalytic rates and specificity for peptide hydrolysis, compared to the 90 degrees C assembly (measured at 95 degrees C). Combination of the alpha and beta1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the beta1 subunit in the P. furiosus 20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that beta subunit composition is a factor in archaeal proteasome function during thermal stress, when polypeptide turnover is essential to cell survival.

Archaeal Proteins↗