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At least 1,135 records · Page 63Linked to original sources
PubMed1997

Two-dimensional map of Haemophilus influenzae following protein enrichment by heparin chromatography.

Two-dimensional gel electrophoresis separates several hundred protein molecules in one single experiment and is efficiently used to study the products expressed by different genomes. Low-copy-number gene products are invisible on a stained two-dimensional map and must be enriched such that sufficient amounts are present for visualization and identification. We investigated the enrichment of proteins of the bacterium Haemophilus influenzae by chromatography on immobilized heparin which has affinity for growth and protein biosynthesis factors. Total soluble proteins of the microorganism were fractionated on Heparin-Actigel which resulted in enrichment of approximately 160 proteins. The eluates, representing about 40% of the applied proteins, were analyzed by two-dimensional gel electrophoresis and the protein spots were characterized by amino acid composition analysis and matrix-assisted laser desorption ionization mass spectrometry. The proteins enriched by chromatography on the heparin gel were not exclusively low-copy-number gene products and they did not exclusively belong to one single class of proteins. The proteins that bound to the heparin gel are indicated in a two-dimensional protein map which includes more than 110 newly identified proteins.

Bacterial Proteins↗
PubMed1993

Transient and stable gene expression in the fungal maize pathogen Cochliobolus heterostrophus after transformation with the beta-glucuronidase (GUS) gene.

The bacterial GUS (beta-glucuronidase) gene has been used as a reporter gene in plants and bacteria and was recently expressed in filamentous fungi. Here, we report the application of GUS for the establishment of transient and stable gene expression systems in the phytopathogenic fungus Cochliobolus heterostrophus. The utility of the transient expression system is demonstrated in applications involving promoter analysis and in tests of various parameters of a transformation system, for comparing the rates of stable and transient transformation events using GUS as sole screening marker and for comparing different transformation systems using either GUS or a dominant selection marker. For these purposes two plasmids were constructed harbouring the GUS gene and the hph gene of Escherichia coli which confers resistance to the antibiotic hygromycin B (HygB), ligated either to the P1 or GPD1 (glyceraldehyde 3 phosphate dehydrogenase) promoter of C. heterostrophus. In transient expression studies the first appearance of GUS activity was observed within 2 h after transformation and maximal values were obtained after 7 or 10 h, depending on the promoter fused to the GUS gene. At peak activity, the GPD1 promoter was revealed to be five fold stronger than the P1 promoter. The same difference in promoter strength was observed when the vectors were stably integrated in the fungal genome. Using the GUS gene as a colour selection marker in plate assays, it was possible to detect transformants and monitor the process of transient gene expression visually. Blue transformants obtained by screening for the GUS phenotype were mitotically unstable. Transformants obtained by selecting for HygB resistance were mitotically stable and expressed the beta-glucuronidase gene constitutively.(ABSTRACT TRUNCATED AT 250 WORDS)

Ascomycota↗
PubMed2005

Pol III proofreading activity prevents lesion bypass as evidenced by its molecular signature within E.coli cells.

Replication of genomes that contain blocking DNA lesions entails the transient replacement of the replicative DNA polymerase (Pol) by a polymerase specialized in lesion bypass. Here, we isolate and visualize at nucleotide resolution level, replication intermediates formed during lesion bypass of a single N-2-acetylaminofluorene-guanine adduct (G-AAF) in vivo. In a wild-type strain, a ladder of replication intermediates mapping from one to four nucleotides upstream of the lesion site, can be observed. In proofreading-deficient strains (mutD5 or dnaQ49), these replication intermediates disappear, thus assigning the degradation ladder to the polymerase-associated exonuclease activity. Moreover, in mutD5, a new band corresponding to the insertion of a nucleotide opposite to the lesion site is observed, suggesting that the polymerase and exonuclease activities of native Pol III enter a futile insertion-excision cycle that prevents translesion synthesis. The bypass of the G-AAF adduct located within the NarI sequence context requires the induction of the SOS response and involves either Pol V or Pol II in an error-free or a frameshift pathway, respectively. In the frameshift mutation pathway, inactivation of the proofreading activity obviates the need for SOS induction but nonetheless necessitates a functional polB gene, suggesting that, although proofreading-deficient Pol III incorporates a nucleotide opposite G-AAF, further extension still requires Pol II. These data are corroborated using a colony-based bypass assay.

Alleles↗
PubMed2002

Clinical features of achromatopsia in Swedish patients with defined genotypes.

PURPOSE: To describe the clinical phenotype, with emphasis on the electrophysiological findings, of patients with autosomal recessive rod monochromacy (RM) and defined mutations in the CNGA3/CNGB3 genes. METHODS: RM patients from eight different families were included in the study. Their genotypes were determined by DNA sequencing and/or RFLP analysis of PCR-amplified genomic segments of the CNGA3 and CNGB3 genes. For comparison, we investigated one patient with blue-cone monochromacy (BCM). The clinical examination included best-corrected visual acuity, fundus examination, and full-field ERG. In six patients, the examination was complemented by multifocal ERG (MERG). RESULTS: Three patients had three different CNG3A genotypes. Five patients were homozygous and one patient compound heterozygous for a 1-bp deletion (1148delC) in the CNGB3 gene. All patients examined presented with a visual acuity of 0.1-0.15. Small residual cone responses were noted in four young RM patients. The oldest patient examined (age 47 years) presented with pigmentary changes in the mid-peripheral retina and concentric constrictions of the visual fields. CONCLUSIONS: Patients with RM and mutations in the CNGA3/CNGB3 genes presented a similar clinical phenotype, confirming the essential function of both the alpha- and beta-subunits of the cGMP-gated cation channel in cone photoreceptor function. Small remaining cone responses in a few of the younger patients and mid-peripheral pigmentary degenerations in the oldest patient examined indicate that there could be some degree of progression in retinal dysfunction in at least some patients with RM.

Adolescent↗
PubMed1996

Nonradioactive arbitrarily primed polymerase chain reaction: a novel technique for detecting genetic defects in skin tumors.

Initiation and progression of melanocytic and non-melanocytic skin tumors are accompanied and probably caused by a variety of genetic defects. In contrast to other human tumors, however, limited amounts of available tissue in skin cancer often hamper extensive genetic studies of native material of early lesions. Therefore, we applied a novel DNA fingerprinting technique based on polymerase chain reaction (PCR), the arbitrarily primed PCR (AP-PCR). This technique enabled us to scan large parts of the genome (about 30 kb/PCR reaction) for somatic mutations starting with minute amounts of tissue. In contrast to previous reports on AP-PCR, we were able to visualize PCR products by a rapid nonradioactive silver-staining technique using a simple device for staining of large polyacrylamide gels. In nine benign and malignant melanocytic skin tumors, the method provided a set of reproducible DNA fingerprints. Genetic defects were detected by comparing the fingerprints of tumor cells and constitutive DNA from blood leukocytes. Since nonradioactive AP-PCR fingerprinting also offers the unique capability to isolate and sequence polymorphic DNA fragments from fingerprint gels, we conclude that this technique seems to be important and practically feasible for elucidating the genetic roots of skin tumors.

Base Sequence↗
PubMed1998

Towards the isolation of a human malignant extragonadal germ cell tumour-associated breakpoint in chromosome 11q13.

In a previous study we have defined a subgroup of human malignant extragonadal germ cell tumours that is characterized by complex translocations involving chromosomes 6 and 11 (Echten et al. 1995). Here we report (i) the use of fluorescent in situ hybridization, pulsed field gel electrophoresis and direct visual hybridization techniques to localize the tumour-associated breakpoint within band 11q13, and (ii) the construction of a phage library enriched for this region to facilitate genomic walks towards the breakpoint. Extensive breakpoint-flanking contigs were generated and within these contigs six candidate genes could be identified.

Adult↗
PubMed2002

Experimental versus in silico fluorescent amplified fragment length polymorphism analysis of Mycobacterium tuberculosis: improved typing with an extended fragment range.

Whole-genome fingerprinting fluorescent amplified fragment length polymorphism (FAFLP) data were compared with in silico data for the sequenced strains of Mycobacterium tuberculosis (H37Rv and CDC1551). For this G+C-rich genome, many predicted fragments were not detected experimentally. For H37Rv, only 108 (66%) of the 163 predicted EcoRI-MseI fragments between 100 and 500 bp were visualized in vitro. FAFLP was also used to identify polymorphism in 10 clinical isolates of M. tuberculosis characterized previously by IS6110 typing, examining fragments of up to 1,000 bp in size rather than up to 500 bp as was done previously. Five isolates had unique IS6110 profiles and were not known to be epidemiologically related, two isolates were the same single-band IS6110 type but were not known to be epidemiologically related, and the remaining three isolates were epidemiologically related with identical IS6110 profiles. Analysis of fragments in the 500- to 1,000-bp range using nonselective primers differentiated better between strains than analysis of fragments in the 50- to 500-bp range using a set of four selective primers. Seventeen polymorphic fragments were identified between 500 and 1,000 bp in size compared with nine polymorphic fragments between 50 and 500 bp. Using the 500- to 1,000-bp analysis, a level of discrimination similar to that of IS6110 typing was achieved which, unlike the IS6110 typing, was able to differentiate the two M. tuberculosis strains, each of which had only a single copy of IS6110.

Bacterial Typing Techniques↗
PubMed2006

Visualizing gene determinants of disease in drug discovery.

Target discovery, subphenotype detection and the detection of human heterogeneity are major challenges in drug discovery and development on which genetic markers can have an impact. Visualizing gene determinants of traits in case-control study individuals during drug discovery using contrasts of empirically-derived log Bayes factors (LBFs) from whole-genome scan single nucleotide polymorphism (SNP) data is presented to aid this. Examples of the use of eigen analysis, covariate overlays and individualized aggregation to ontologies are included from disease research studies. Displays of individuals, or exposures of biological features of interest, can encompass unlimited numbers of markers in a single multivariate analysis without multiple testing. This filtering approach is aimed at nonspecialists who find themselves asked to undertake work such as that performed by the authors.

Animals↗
PubMed1994

[Chromosomal in situ hybridization and interphase cytogenetics in single cell and tissue section preparations: new methods in tumor diagnosis and clinical cytogenetics].

To overcome the shortcomings encountered in classical cytogenetics, a variety of in situ hybridization techniques have been developed enabling metaphase and interphase cytogenetics on routinely processed tissues and cells. These techniques comprise the detection of numerical chromosome aberrations, the delineation or "painting" of whole chromosomes or certain chromosome regions and the visualization of structural chromosome rearrangements in interphase nuclei. Some of these new methods can reliably be applied also on paraffin-embedded tissues, among them the comparative genomic in situ hybridization (CGH) technique suitable for providing a survey of over- or underrepresented genetic material on the chromosomal level even if only tumour DNA is available.

Chromosome Aberrations↗
PubMed1997

Advances in the generation of transgenic pigs via embryo-derived and primordial germ cell-derived cells.

The development of new technologies that would increase the efficiency for generation of transgenic livestock and would overcome some of the problems associated with random insertion of the transgene will greatly benefit animal agriculture. A potential alternative technology to pronuclear injection for the generation of transgenic pigs involves the isolation, culture and genetic manipulation of cell lines that can be reintroduced into the embryo for participation in the formation of the germ cells. We have isolated and cultured pig primordial germ cells (PGC) while maintaining them in an undifferentiated state as determined by morphology and alkaline phosphatase (AP) activity. More importantly, PGC-derived cells were stably transformed with the green fluorescent protein marker driven by the cytomegalovirus promoter. After visual identification of transgenic colonies, the pluripotential characteristics of the transgenic PGC-derived cells were tested by chimaera formation and to date we have identified, by genomic Southern blots, two chimaeric fetuses that contain tissues with the transgene incorporated into their chromosomes. To our knowledge, this is the first report of a chimaeric transgenic pig fetus obtained via a cultured cell line.

Animals↗
PubMed1994

Human immunodeficiency viruses regulated by alternative trans-activators: genetic evidence for a novel non-transcriptional function of Tat in virion infectivity.

Thirteen genetically altered HIV-1 proviruses were created. These various genomes can be segregated into three groups: (i) a set of tat(-) viruses that have a functional HTLV-I Tax inserted in nef; (ii) a set of tat(-) viruses with Gal4 binding sites inserted in U3 and a Gal4-VP16 cDNA inserted in nef; and (iii) a set of tat(+) HIV genomes that are 5' and 3' TAR(-) and are Gal4-binding-site(+) in U3 and Gal4-VP16(+) in nef. We found that viruses in groups (i) and (ii), although tat(-), were fully complemented for viral gene expression based on quantitative measurements of viral protein synthesis and on the visualization by electron microscopy of the proper assembly of morphologically correct virions. Interestingly, group (i) and (ii) virions were defective in a spreading cytopathic infection when assayed in T-lymphocytes. Group (iii) viruses, although capable of producing intact Tat protein, also could not use Tat for transcription/gene expression because of the TAR(-) genotype. However, this class of viral genomes produced viruses that were highly infectious and cytopathic in primary and in continuously propagated T-lymphocytes. These three groups of viruses are all transcriptionally Tat-TAR independent. Their distinct differences in infectivity/cytopathicity provide genetic evidence that Tat provides a transcriptionally independent function in determining infectivity and cytopathicity in the setting of a spreading viral infection. Given that all HIV virions normally contain four intact copies of TAR RNA, our findings suggest a re-examination of whether Tat could be a virion-TAR-associated protein and the possible implications of this for virus infectivity/cytopathicity.

Base Sequence↗
PubMed2000

Evaluation of the myosin VIIA gene and visual function in patients with Usher syndrome type I.

Usher syndrome type I (USH1) is a recessively-inherited disorder consisting of retinitis pigmentosa, profound congenital deafness, and vestibular ataxia. It can be caused by mutations in at least six different loci (USH1A-1F). The gene encoding human myosin VIIA (MYO7A) is the USH1B locus. In this study, 66 unrelated patients with USH1 were evaluated for defects in MYO7A using single-strand conformation polymorphism analysis and direct genomic sequencing. Twenty-nine per cent of cases were found to have likely pathogenic MYO7A mutations. A total of 22 likely pathogenic changes were identified, 18 of which were novel. Cosegregation analysis of mutations in five available families showed that the MYO7A changes segregated with the disease in an autosomal recessive fashion. Average visual function as measured by visual acuity, visual field area, and ERG amplitude was not significantly different between the group of patients with likely pathogenic MYO7A changes and the group in which no likely pathogenic MYO7A changes were detected.

Adult↗
PubMed2005

ProGenGrid: a grid-enabled platform for bioinformatics.

In this paper we describe the ProGenGrid (Proteomics and Genomics Grid) system, developed at the CACT/ISUFI of the University of Lecce which aims at providing a virtual laboratory where e-scientists can simulate biological experiments, composing existing analysis and visualization tools, monitoring their execution, storing the intermediate and final output and finally, if needed, saving the model of the experiment for updating or reproducing it. The tools that we are considering are software components wrapped as Web Services and composed through a workflow. Since bioinformatics applications need to use high performance machines or a high number of workstations to reduce the computational time, we are exploiting a Grid infrastructure for interconnecting wide-spread tools and hardware resources. As an example, we are considering some algorithms and tools needed for drug design, providing them as services, through easy to use interfaces such as the Web and Web service interfaces built using the open source gSOAP Toolkit, whereas as Grid middleware we are using the Globus Toolkit 3.2, exploiting some protocols such as GSI and GridFTP.

Computational Biology↗
PubMed2006

Molecular analysis of transgene and vector backbone integration into the barley genome following Agrobacterium-mediated transformation.

We report a large-scale study on the frequency of transgene and T-DNA backbone integration following Agrobacterium-mediated transformation of immature barley embryos. One hundred and ninety-one plant lines were regenerated after hygromycin selection and visual selection for GFP expression at the callus stage. Southern blotting performed on a subset of 53 lines that were PCR positive for the GFP gene documented the integration of the GFP gene in 27 of the lines. Twenty-three of these lines expressed GFP in T(1) plantlets. Southern blotting with a vector backbone probe revealed that 13 of the 27 lines possessed one or more vector backbone fragments illustrating the regular occurrence of vector backbone integration following Agrobacterium infection of barley immature embryos.

Agrobacterium tumefaciens↗
PubMed1997

Sensitive chemiluminescence in situ hybridization for the detection of human papillomavirus genomes in biopsy specimens.

We developed a sensitive chemiluminescence in situ hybridization assay for detection of human papillomavirus (HPV) DNA for objective and semiquantitative evaluation of the results. The hybridization reaction was performed using either digoxigenin-, biotin-, or fluorescein-labeled probes, visualized with alkaline phosphatase as the revealing enzyme and a highly sensitive 1,2 dioxetane phosphate as chemiluminescent substrate. The light emitted from the hybridized probes was detected, analyzed, and measured using a high-performance, low light-level imaging luminograph connected to an optical microscope and to a personal computer for quantification of the photon fluxes and for image analysis. The system operated in consecutive steps: First, hybridized specimens were recorded in transmitted light. Then the net luminescent signal was recorded, and then an overlay of the two images provided by the transmitted light and by the luminescent signal allowed the spatial distribution of the target DNA to be localized, measured, and evaluated. Biopsy specimens from different pathological conditions associated with HPV, which had previously been proved positive for HPV DNA with the polymerase chain reaction (PCR), were analysed. The chemiluminescence in situ hybridization proved sensitive and specific with digoxigenin-, biotin-, or fluorescein-labeled probes, and provided an objective evaluation of the results. The results obtained with chemiluminescence in situ hybridization were also compared with results obtained with in situ hybridization with colorimetric detection, with good concordance of the data. Chemiluminescence in situ hybridization therefore offers the possibility of detecting HPV DNA with great sensitivity in biopsy specimens. Moreover, the images of the samples, stored in the computer, are a permanent record of the reaction and can also be sent for evaluation or comparison to other laboratories using computer networks.

Biotin↗
PubMed2006

Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays.

Localized accessibility of critical DNA sequences to the regulatory machinery is a key requirement for regulation of human genes. Here we describe a high-resolution, genome-scale approach for quantifying chromatin accessibility by measuring DNase I sensitivity as a continuous function of genome position using tiling DNA microarrays (DNase-array). We demonstrate this approach across 1% ( approximately 30 Mb) of the human genome, wherein we localized 2,690 classical DNase I hypersensitive sites with high sensitivity and specificity, and also mapped larger-scale patterns of chromatin architecture. DNase I hypersensitive sites exhibit marked aggregation around transcriptional start sites (TSSs), though the majority mark nonpromoter functional elements. We also developed a computational approach for visualizing higher-order features of chromatin structure. This revealed that human chromatin organization is dominated by large (100-500 kb) 'superclusters' of DNase I hypersensitive sites, which encompass both gene-rich and gene-poor regions. DNase-array is a powerful and straightforward approach for systematic exposition of the cis-regulatory architecture of complex genomes.

Chromatin↗
PubMed1999

Retrotransposon BARE-1: expression of encoded proteins and formation of virus-like particles in barley cells

Retrotransposons are ubiquitous and major components of plant genomes, and are characteristically retroviral-like in their genomic structure and in the major proteins encoded. Nevertheless, few have been directly demonstrated to be transcribed or reverse transcribed. The BARE-1 retrotransposon family of barley (Hordeum vulgare) is highly prevalent, actively transcribed, and contains well conserved functional regions. Insertion sites for BARE-1 are highly polymorphic in the barley genome. Here we show that BARE-1 is translated and the capsid protein (GAG) and integrase (IN) components of the predicted polyprotein are processed into polypeptides of expected size. Some of the GAG sediments as virus-like particles together with IN and with BARE-1 cDNA. Reverse transcriptase activity is also present in gradient fractions containing BARE-1 translation products. Virus-like particles have also been visualized in fractions containing BARE-1 components. Thus BARE-1 components necessary for carrying out the life cycle of an active retrotransposon appear to be present in vivo, and to assemble. This would suggest that post-translational mechanisms may be at work to prevent rapid genome inflation through unrestricted integration.

Journal Article↗