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At least 721 records · Page 40Linked to original sources
PubMed1993

Schistosoma mansoni: determination of the sex of cercariae using a nonradioactively labelled DNA probe.

Cercarial sex determination protocol which utilizes nonradioactive nucleic acid labelling with digoxigenin (DIG)-11-dUTR, offers a rapid "clean" identification of the sex of Schistosoma mansoni cercaria. DIG labelled pW1-DNA (plasmid female-specific genomic DNA clone--W1) hybridized with the native cercarial DNA, and hybridization signals are visualized with Lumigen PPD. Results obtained are comparable with those obtained with the 32P labelled pW1.

Animals↗
PubMed1998

Molecular characterization of the mouse gene encoding cellular retinaldehyde-binding protein.

PURPOSE: To clone and characterize the mouse gene encoding cellular retinaldehyde-binding protein (CRALBP). CRALBP appears to modulate enzymatic generation and processing of 11-cis-retinol and regeneration of visual pigment in the vertebrate visual cycle. Mutations in human CRALBP segregate with autosomal recessive retinitis pigmentosa. METHODS: A genomic clone encompassing the 5' end of the CRALBP gene through exon 6 was isolated from a mouse 129/Sv genomic DNA library. Exons 7 and 8 were PCR amplified from mouse eye cDNA and 129/SvJ genomic DNA. The gene structure was determined by automated DNA sequence analysis. RESULTS: The sequence of 6855 nucleotides was determined, including all 8 exons, 3 introns plus 3932 and 629 bases from the 5'- and 3'-flanking regions, respectively. The lengths of introns 3-6 were determined by PCR amplification. Northern analysis identifies a approximately 2.1 kb transcript in mouse eye; Southern analysis supports a single copy gene. CONCLUSIONS: The mouse CRALBP gene is similar to the human gene; the coding sequence is approximately 87% identical, the non-coding sequence approximately 65% identical. In contrast to the human gene, the mouse gene contains a consensus TATA box. One of two photoreceptor consensus elements important for CRALBP expression in human retinal pigment epithelium is also present in the mouse gene. Additional conserved and species-specific consensus sequences are identified. The mouse CRALBP genomic clones and structure provide valuable tools for developing an in vivo model to study protein function and gene regulation.

Animals↗
PubMed2003

A tool-kit for cDNA microarray and promoter analysis.

We describe two sets of programs for expediting routine tasks in analysis of cDNA microarray data and promoter sequences. The first set permits bad data points to be flagged with respect to a number of parameters and performs normalization in three different ways. It allows combining of result files into comprehensive data sets, evaluation of the quality of both technical and biological replicates and row and/or column standardization of data matrices. The second set supports mapping ESTs in the genome, identifying the corresponding genes and recovering their promoters, analyzing promoters for transcription factor binding sites, and visual representation of the results. The programs are designed primarily for Arabidopsis thaliana researchers, but can be adapted readily for other model systems. Availability and Supplementary information: http://www.personal.psu.edu/nhs109/Programs/

Algorithms↗
PubMed2005

[Splicing site mutation of D19S418 in PRPF-31 gene and its phenotypic characters with autosomal dominant retinitis pigmentosa].

OBJECTIVE: To evaluate the disease-causing gene and phenotypic characters of a large family with autosomal dominant retinitis pigmentosa (adRP). METHODS: Disease status and associated ocular abnormalities of eight patients and six unaffected members who represent different generations of this family were assessed by measurement of visual psychophysics, full-field and multifocal electrophysiology (ERG and mfERG) and funds fluorescent angiography (FFA). The DNA samples of nineteen patients and fifteen unaffected individuals in this family were examined by Genome scanning, linkage analysis and mutation detection to identify coding sequence changes. RESULTS: A case with variable, early onset night blindness before 10 years and visual field loss in their teens was found. Macular dystrophy, progressing to a retinitis pigmentosa phenotype was demonstrated in most adult cases. Both a-wave and b-wave amplitudes of photopic and scotopic full-field ERG were marked reduced and nearly non-detectable, demonstrating severe damage of photoreceptor systems. There were two obligate gene carriers in the family which remained asymptomatic in the clinical. But one of them was found with a minimal RP characteristic and the other was normal by examination of fundus and ERG. An unreported splicing site mutation (IVS5-1G > A) was identified in intron 5- acceptor site of PRPF-31 gene on chromosome 19. ERG and molecular genetic findings were consistent with the reclassification of this disease as an autosomal dominant RP. CONCLUSION: It is a novel splicing site mutation that IVS5-1G > A of D19S418 site in PRFP31, the relative phenotypes by which main displayed type I/diffuse has variable expressivity and complex phenotype.

Adolescent↗
PubMed1991

Detection of the hepatitis C virus genome in acute and chronic experimental infection in chimpanzees.

In order to gain an understanding of the relationship of various markers of hepatitis C virus (HCV) infection in acute and chronic cases of the disease, serial blood samples obtained from chimpanzees before and after infection with HCV were analyzed for the presence of the HCV genome by using polymerase chain reaction (PCR) amplification of cDNA (cDNA PCR) synthesized from plasma- and serum-derived RNA. In a chimpanzee with acute hepatitis C, signals detectable by cDNA PCR appeared 1 week before characteristic ultrastructural changes visualized by electron microscopy, persisted throughout the peak alanine aminotransferase levels, and diminished with the disappearance of alterations visualized by electron microscopy. This was in contrast to the results obtained from chimpanzees with chronic HCV infection, in which the HCV genome was consistently detectable for up to 10 years after infection. The results indicate the usefulness of detection of HCV RNA by cDNA PCR as a sensitive and semiquantitative method for monitoring the course of HCV infection and as a potential marker for differentiating between chronic and acute cases of disease.

Acute Disease↗
PubMed1991

Integration site preferences of endogenous retroviruses.

Retroviruses have the ability to integrate into the genome of their host, in many cases with little apparent sequence or site specificity. However, relatively few studies have addressed more general features of chromosomal integration. In this study we directly visualized the chromosomal organization of three representative endogenous retroviruses by in situ hybridization. Because there are 50-1000 copies of each of these retroviruses in the genome, it was possible to evaluate repeated integration events. Each retroviral sequence exhibited a unique and markedly different integration pattern. In order to characterize more precisely the chromosomal domains targeted by each retrovirus, later replicating domains were differentially labeled. Additionally, prototypic SINES and LINES (short and long interspersed reiterated sequences), which are inhomogeneously distributed on chromosome arms, were simultaneously detected. Retroviral copies of greater than or equal to 2 kb were found (i) exclusively in a discrete set of later replicating domains, most of which have the staining characteristics of constitutive heterochromatin, (ii) widely represented in disparate types of chromosome domains, or (iii) almost completely confined to CpG Alu-rich regions that are known to be early replicating. Retroviral elements in Alu-rich domains would be expected to be actively transcribed in all cells. Surprisingly, hybridization to blots of brain RNA showed an approximately 25 fold lower level of transcripts from these Alu associated elements than from retroviral sequences restricted to later replicating, heterochromatic domains. Retroviral insertions may subvert more typical transcriptional characteristics of a domain. The present results indicate that there are highly specific integration patterns for each endogenous retrovirus that do not readily relate to their sequence or particle classification. Each host genome may utilize these elements for contrary, and possibly beneficial functions.

Adult↗
PubMed2003

Identification of conserved regulatory elements by comparative genome analysis.

BACKGROUND: For genes that have been successfully delineated within the human genome sequence, most regulatory sequences remain to be elucidated. The annotation and interpretation process requires additional data resources and significant improvements in computational methods for the detection of regulatory regions. One approach of growing popularity is based on the preferential conservation of functional sequences over the course of evolution by selective pressure, termed 'phylogenetic footprinting'. Mutations are more likely to be disruptive if they appear in functional sites, resulting in a measurable difference in evolution rates between functional and non-functional genomic segments. RESULTS: We have devised a flexible suite of methods for the identification and visualization of conserved transcription-factor-binding sites. The system reports those putative transcription-factor-binding sites that are both situated in conserved regions and located as pairs of sites in equivalent positions in alignments between two orthologous sequences. An underlying collection of metazoan transcription-factor-binding profiles was assembled to facilitate the study. This approach results in a significant improvement in the detection of transcription-factor-binding sites because of an increased signal-to-noise ratio, as demonstrated with two sets of promoter sequences. The method is implemented as a graphical web application, ConSite, which is at the disposal of the scientific community at http://www.phylofoot.org/. CONCLUSIONS: Phylogenetic footprinting dramatically improves the predictive selectivity of bioinformatic approaches to the analysis of promoter sequences. ConSite delivers unparalleled performance using a novel database of high-quality binding models for metazoan transcription factors. With a dynamic interface, this bioinformatics tool provides broad access to promoter analysis with phylogenetic footprinting.

Algorithms↗
PubMed2003

PCR-RFLP of the ribosomal DNA internal transcribed spacers (ITS) provides markers for the A and B genomes in Musa L.

Musa acuminata Colla (AA genomes) and Musa balbisiana Colla (BB genomes) are the diploid ancestors of modern bananas that are mostly diploid or triploid cultivars with various combinations of the A and B genomes, including AA, AAA, BB, AAB and ABB. The objective of this study was to identify molecular markers that will facilitate discrimination of the A and B genomes, based on restriction-site variations in the internal transcribed spacers (ITS) of the nuclear ribosomal RNA genes. The ITS regions of seven M. acuminata and five M. balbisiana accessions were each amplified by PCR using specific primers. All accessions produced a 700-bp fragment that is equivalent in size to the ITS of most plants. This fragment was then digested with ten restriction enzymes ( AluI, CfoI, DdeI, HaeIII, HinfI, HpaII, MspI, RsaI, Sau3AI and TaqI) and fractionated in 2% agarose gels, stained with ethidium bromide and visualized under UV light. The RsaI digest revealed a single 530-bp fragment unique to the A genome and two fragments of 350-bp and 180-bp that were specific to the B genome. A further 56 accessions representing AA, AAA, AAB, AB and ABB cultivars, and synthetic hybrids, were amplified and screened with RsaI. All accessions with an exclusively A genome showed only the 530-bp fragment, while accessions having only the B-genome lacked the 530-bp fragment but had the 350-bp and 180-bp fragments. Interspecific cultivars possessed all three fragments. The staining intensity of the B-genome markers increased with the number of B-genome complements. These markers can be used to determine the genome constitution of Musa accessions and hybrids at the nursery stage, and, therefore, greatly facilitate genome classification in Musa breeding.

Chimera↗
PubMed1998

Cluster analysis and data visualization of large-scale gene expression data.

The discovery of any new gene requires an analysis of the expression context for that gene. Now that the cDNA and genomic sequencing projects are progressing at such a rapid rate, high throughput gene expression screening approaches are beginning to appear to take advantage of that data. We present a strategy for the analysis for large-scale quantitative gene expression measurement data from time course experiments. Our approach takes advantage of cluster analysis and graphical visualization methods to reveal correlated patterns of gene expression from time series data. The coherence of these patterns suggests an order that conforms to a notion of shared pathways and control processes that can be experimentally verified.

Base Sequence↗
PubMed2005

Functional genome annotation through phylogenomic mapping.

Accurate determination of functional interactions among proteins at the genome level remains a challenge for genomic research. Here we introduce a genome-scale approach to functional protein annotation--phylogenomic mapping--that requires only sequence data, can be applied equally well to both finished and unfinished genomes, and can be extended beyond single genomes to annotate multiple genomes simultaneously. We have developed and applied it to more than 200 sequenced bacterial genomes. Proteins with similar evolutionary histories were grouped together, placed on a three dimensional map and visualized as a topographical landscape. The resulting phylogenomic maps display thousands of proteins clustered in mountains on the basis of coinheritance, a strong indicator of shared function. In addition to systematic computational validation, we have experimentally confirmed the ability of phylogenomic maps to predict both mutant phenotype and gene function in the delta proteobacterium Myxococcus xanthus.

Bacterial Proteins↗
PubMed1974

Viable deletion mutants of simian virus 40: selective isolation by means of a restriction endonuclease from Hemophilus parainfluenzae.

Resistance of simian virus 40 (SV40) DNA to cleavage by Hemophilus parainfluenzae II (HpaII) restriction endonuclease has been used as a positive, in vitro selection for mutants lacking the one HpaII endonuclease-cleavage site of wild-type SV40 DNA. Each of 10 viable mutants isolated by this procedure multiplies significantly more slowly than wild-type virus and contains a small deletion (80 to 190 base pairs in size) of the region of the genome that includes the HpaII endonuclease-recognition sequence. These well-defined mutants, having a selective disadvantage for growth, would not have been readily obtained by conventional methods used to screen for viral mutants. Therefore, in certain circumstances, restriction endonucleases are effective reagents for the selection of new classes of mutants. Because these small deletions can be visualized in heteroduplexes, these mutants provide internal markers for mapping other alterations or features of the simian virus 40 genome.

Animals↗
PubMed2005

SVC: structured visualization of evolutionary sequence conservation.

We have developed a web application for the detailed analysis and visualization of evolutionary sequence conservation in complex vertebrate genes. Given a pair of orthologous genes, the protein-coding sequences are aligned. When these sequences are mapped back onto their encoding exons in the genomes, a scaffold of the conserved gene structure naturally emerges. Sequence similarity between exons and introns is analysed and embedded into the gene structure scaffold. The visualization on the SVC server provides detailed information about evolutionarily conserved features of these genes. It further allows concise representation of complex splice patterns in the context of evolutionary conservation. A particular application of our tool arises from the fact that around mRNA editing sites both exonic and intronic sequences are highly conserved. This aids in delineation of these sites. SVC is available at http://svc.molgen.mpg.de.

Animals↗
PubMed2026

Protocol to predict gene expression from transcriptomic data using PREDICT.

Linking DNA sequence variation to context-specific transcriptional programs is a critical challenge in regulatory genomics, especially for non-model organisms. Here, we present PREDICT, a modular Python package for discovering cis-regulatory elements and transcription factor binding motifs. We describe steps to identify enriched k-mers from differentially expressed genes, map them to known motifs, quantify their impact on gene expression, and visualize motif co-occurrences. PREDICT provides a robust, k-mer-based approach to uncover regulatory logic in diverse genomic systems. For complete details on the use and execution of this protocol, please refer to Yen et al. and Liu et al.1,2.

Gene Expression Profiling↗
PubMed1992

Cloning of repetitive DNA sequences from Toxoplasma gondii and their usefulness for parasite detection.

Genomic DNA of Toxoplasma gondii was digested with the restriction endonuclease Hpa II and the resulting repetitive DNA sequences were visualized after electrophoresis on agarose gels and staining with ethidium bromide. Three repetitive DNA sequences were isolated and cloned in the plasmid pUC19. The recombinant plasmids (pTg8, pTg4 and pTg1) had inserts of 840, 440, and 180 basepairs, respectively. The estimated copy number of these cloned sequences in the T. gondii genome was approximately 800-1,000 for pTg4, 150-200 for pTg8, and 30-40 for pTg1. In dot-blot hybridization tests, pTg4 was able to detect as little as 80 pg of purified T. gondii DNA or 1,000 parasites in the presence or absence of 1.5 x 10(6) human or mouse leukocytes. No cross-hybridization was detected with 10 micrograms of either human and mouse DNA or 100 ng of DNA from other parasites (Eimeria tenella, E. acervularia, Trypanosoma cruzi, and Leishmania donovani), or among the three DNA probes. Each probe identified T. gondii tachyzoites in tissue (liver and spleen) obtained from experimentally infected mice in which histologic damage was detected. In addition, early detection of T. gondii in brain tissue and blood samples was possible with the pTg4 probe.

Animals↗
PubMed2004

Topography of genetic loci in the nuclei of cells of colorectal carcinoma and adjacent tissue of colonic epithelium.

To determine the influence of increased gene expression and amplification in colorectal carcinoma on chromatin structure, the nuclear distances between pairs of bacterial artificial chromosome (BAC) clones with genomic separation from 800 to 29,000 kb were measured and compared between the tumor and parallel epithelial cells of six patients. The nuclear distances were measured between the loci in chromosomal bands 7p22.3-7p21.3; 7q35-7q36.3; 11p15.5-11p15.4; 20p13; 20p12.2; 20q11.21 and 20q12 where increased expression had been found in all types of colorectal carcinoma. The loci were visualized by three-dimensional fluorescence in situ hybridization using 22 BAC clones. Our results show that for short genomic separations, mean nuclear distance increases linearly with increased genomic separation. The results for some pairs of loci fell outside this linear slope, indicating the existence of different levels of chromatin folding. For the same genomic separations the nuclear distances were frequently shorter for tumor as compared with epithelial cells. Above the initial growing phase of the nuclear distances, a plateau phase was observed in both cell types where the increase in genomic separation was not accompanied by an increase in nuclear distance. The ratio of the mean nuclear distances between the corresponding loci in tumor and epithelium cells decreases with increasing amplification of loci. Our results further show that the large-scale chromatin folding might differ for specific regions of chromosomes and that it is basically preserved in tumor cells in spite of the amplification of many loci.

Adult↗
PubMed1997

Complementation of a potato virus X mutant mediated by bombardment of plant tissues with cloned viral movement protein genes.

Microprojectile bombardment was used to examine the transport function of the 25 kDa movement protein (MP) encoded in the triple gene block of potato virus X (PVX). A 25 kDa MP-defective full-length cloned PVX genome carrying a beta-glucuronidase (GUS) reporter gene was co-bombarded with 35S promoter constructs containing either the 25 kDa MP gene of wild-type PVX, the MP gene of either of two tobamoviruses (tomato mosaic virus or crucifer tobamovirus), red clover necrotic mosaic dianthovirus (RCNMV) or brome mosaic bromovirus (BMV). When inoculated alone, the MP-defective PVX was unable to move out of the inoculated cell, as visualized by in situ staining for GUS activity. However, cell-to-cell movement of the mutant PVX genome was restored by co-inoculation with 35S constructs containing the MP cDNA of PVX, either tobamovirus or RCNMV. The BMV MP construct did not complement movement of the defective PVX. These results show that co-bombardment of cDNA of an MP-defective virus with plasmids designed to express MP of other viruses could be used as a fast and simple method for transcomplementation experiments.

Brassica↗
PubMed1999

Polymorphisms and genomic organization of repetitive DNA from centromeric regions of Arabidopsis chromosomes.

A highly abundant repetitive DNA sequence family of Arabidopsis, AtCon, is composed of 178-bp tandemly repeated units and is located at the centromeres of all five chromosome pairs. Analysis of multiple copies of AtCon showed 95% conservation of nucleotides, with some alternative bases, and revealed two boxes, 30 and 24 bp long, that are 99% conserved. Sequences at the 3' end of these boxes showed similarity to yeast CDEI and human CENP-B DNA-protein binding motifs. When oligonucleotides from less conserved regions of AtCon were hybridized in situ and visualized by using primer extension, they were detected on specific chromosomes. When used for polymerase chain reaction with genomic DNA, single primers or primer pairs oriented in the same direction showed negligible amplification, indicating a head-to-tail repeat unit organization. Most primer pairs facing in opposite directions gave several strong bands corresponding to their positions within AtCon. However, consistent with the primer extension results, some primer pairs showed no amplification, indicating that there are chromosome-specific variants of AtCon. The results are significant because they elucidate the organization, mode of amplification, dispersion, and evolution of one of the major repeated sequence families of Arabidopsis. The evidence presented here suggests that AtCon, like human alpha satellites, plays a role in Arabidopsis centromere organization and function.

Arabidopsis↗
PubMed2001

Possible dosage effect of maternally expressed genes on visual recognition memory in Prader-Willi syndrome.

Seventeen patients with Prader-Willi syndrome (7 with paternal deletion of chromosome 15q11-q13 and 10 with maternal uniparental disomy [UPD]), and 9 controls performed a computerized visual recognition task. A series of color digital photographs were presented; most were presented twice, but the remainder appeared only once. Photographs presented twice were separated in their presentation by either 0, 10, 30, 50 or 100 intervening photographs. Subjects indicated whether each photograph had been presented previously. This procedure was implemented twice, once using photographs of foods, and once using photographs of nonfood objects. As the number of intervening photographs between the first and second presentation increased, subjects were less likely to remember having seen the photograph before. Performance by UPD subjects was less affected by increasing the number of intervening photographs relative to the other two groups, suggesting they had superior visual recognition memory. This raises the possibility of a beneficial effect of having two copies maternally expressed genes on chromosome 15. UBE3A is suggested as a possible candidate for this effect.

Adolescent↗