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At least 487 records · Page 27Linked to original sources
PubMed2025

Implementing customized genomic sequencing reports to empower providers in safety-net neonatal intensive care units.

PURPOSE: Through our implementation study providing rapid genomic sequencing (rGS) in safety-net neonatal intensive care units (NICUs), we investigated the feasibility and perceived usefulness of customized "clinical interpretive reports" (CIRs) to help neonatal providers with interpreting, disclosing, and managing care based on rGS results. METHODS: Enrolled infants received rGS through a clinically accredited vendor. We developed 5 CIR types to provide customized interpretation of rGS results and link results to clinical management considerations, research opportunities, and resources. We developed workflows to triage, create, and deliver CIRs within 3 business days. Providers received the vendor reports and CIRs, disclosed results, and completed post-disclosure surveys. We analyzed summary statistics for the first 100 cases. RESULTS: We delivered 97 of 100 CIRs (97%) within our goal time frame (average 1.3 days) and provided clinical management recommendations in 40 of 100 (40%). Neonatal providers completed the post-disclosure surveys for 86 of 100 disclosures (86%). Most reported using the CIR before disclosure (80/86, 93%) and found it helpful at providing useful information beyond the vendor report (79/80, 99%). CONCLUSION: It is feasible and useful to develop customized rGS reports to assist non-genetics providers in safety-net NICU settings. Similar approaches may hold promise for equitably advancing genomic care in non-NICU settings.

Humans↗
PubMed2005

Governing genomics in the 21st century: between risk and uncertainty.

The paper reconstructs the governance of genomics by sketching the main features, modes of operation and tactics of the emerging genomics apparatus. Genomic governance in the 20th century is characterized by the simultaneous operation of a process of the stabilization of knowledge regimes, in particular via patenting. Furthermore, we observe a heterogenization and globalization of the actors and knowledge creating systems in genomics governance. A variety of different mechanisms and strategies of governance are mobilized simultaneously. The transition of governing via risk to governance by uncertainty is another important feature of contemporary genomics governance. The implications of these trends for the regulation of genomics are considerable and might lead to the emergence of new patterns and spaces of conflict and controversy. The governance of genomics in the 21st century could become a more complex challenge than currently anticipated by many policy makers and the scientific community.

Europe↗
PubMed1996

Salmonella typhi contains identical intervening sequences in all seven rrl genes.

Salmonella typhi Ty2 rrl genes contain intervening sequences (IVSs) in helix-25 but not in helix-45 on the basis of observed 23S rRNA fragmentation caused by IVS excision. We have confirmed this and shown all seven IVSs to be identical by isolating genomic DNA fragments containing each of the seven rrl genes from S. typhi Ty2 by use of pulsed-field gel electrophoresis; each rrl gene was amplified by PCR in the helix-25 and helix-45 regions and cycle sequenced. Thirty independent wild-type S. typhi strains, tested by genomic PCR and DraI restriction, also have seven rrl genes with helix-25 IVSs and no helix-45 IVSs. We propose that IVS homogeneity in S. typhi occurs because gene conversion drives IVS sequence maintenance and because adaptation to human hosts results in limited clonal diversity.

Base Sequence↗
PubMed1999

Genomics: the implications for ethics and education.

Over the past 10 years, the identification of disease genes has been expanding rapidly. Those identified in the earlier part of the decade were largely achieved through positional cloning and the majority are for relatively rare disorders which involve single genes. As the Human Genome Mapping Project has progressed, the rate of gene discovery has increased substantially through the development of new DNA sequencing techniques and in silico approaches. The human genome will have been largely sequenced by ther Spring 2000. We can expect the identification of large numbers of susceptibility genes for common multifactorial polygenic diseases as well as genes which are associated with human behavioural traits. Some of these advances hold out the prospects of real progress in the diagnosis and treatment of a wide range of disorders. However, for many individuals, increased knowledge about their genes will present ethical dilemmas which are difficult to resolve. There are also wider ethical issues which concern the use of genetic information by insurers and employers and yet others which concern ownership and access. In this chapter, the main ethical issues raised by the impact of genomics on healthcare are discussed. The role of education in enabling individuals and health professionals to meet these challenges is also considered.

Ethics, Medical↗
PubMed2005

[DNA and RNA random amplification polymorphism in 5-flurocytosine-resistant strains of Candida albicans from revul-vaginal candidasis].

OBJECTIVE: To investigate the DNA genome and RNA expression in 5-flurocytosine-resistant strains of Candida albicans from vaginal candidasis. METHODS: Sixteen strains of Candida albicans were selected from clinically diagnosed revul-vaginal candidasis. Eight 5-flurocytosine-sensitive isolates and 8 resistant isolates were examined by France Media FUNGUS sensitive test. DNA genome was detected with random amplification polymorph DNA. RNA expression was detected with random amplification polymorph RNA method. RESULTS: There were no distinct differences between 5-flurocytosine-sensitive and resistant Candida albicans in DNA genome, while RNA expression showed significant differences between 5-flurocytosine-resistant and sensitive strains. CONCLUSION: Clinical 5-flurocytosine-resistant strains of Candida albicans from revul-vaginal candidasis may be related to phenotype changes.

Adolescent↗
PubMed1988

Recombination-dependent replication of plasmids during bacteriophage T4 infection.

The replication of plasmids containing fragments of the T4 genome, but no phage replication origins, was analyzed as a possible model for phage secondary (recombination-dependent) replication initiation. The replication of such plasmids after T4 infection was reduced or eliminated by mutations in several phage genes (uvsY, uvsX, 46, 59, 39, and 52) that have previously been shown to be involved in secondary initiation. A series of plasmids that collectively contain about 60 kilobase pairs of the T4 genome were tested for replication after T4 infection. With the exception of those known to contain tertiary origins, every plasmid replicated in a uvsY-dependent fashion. Thus, there is no apparent requirement for an extensive nucleotide sequence in the uvsY-dependent plasmid replication. However, homology with the phage genome is required since the plasmid vector alone did not replicate after phage infection. The products of plasmid replication included long concatemeric molecules with as many as 35 tandem copies of plasmid sequence. The production of concatemers indicates that plasmid replication is an active process and not simply the result of passive replication after the integration of plasmids into the phage genome. We conclude that plasmids with homology to the T4 genome utilize the secondary initiation mechanism of the phage. This simple model system should be useful in elucidating the molecular mechanism of recombination-dependent DNA synthesis in phage T4.

DNA Replication↗
PubMed2000

Detection of chromosomal aberrations by a whole-genome microsatellite screen.

Chromosomal aberrations are a common cause of multiple anomaly syndromes that include developmental and growth retardation. Current microscopic techniques are useful for the detection of such aberrations but have a limit of resolution that is above the threshold for phenotypic effect. We hypothesized that a genomewide microsatellite screen could detect chromosomal aberrations that were not detected by standard cytogenetic techniques in a portion of these individuals. To test this hypothesis, we performed a genomewide microsatellite screen of patients, by use of a currently available genetic-marker panel that was originally designed for meiotic mapping of Mendelian traits. We genotyped approximately 400 markers on 17 pairs of parents and their children who had normal karyotypes. By using this approach, we detected and confirmed two cases of segmental aneusomy among 11 children with multiple congenital anomalies. These data demonstrate that a genomewide microsatellite scan can be used to detect chromosomal aberrations that are not detected by microscopic techniques.

Abnormalities, Multiple↗
PubMed2004

Development of a species-specific probe for Pythium insidiosum and the diagnosis of pythiosis.

Pythium insidiosum, the only species in the genus that infects mammals, is the etiological agent of pythiosis, a granulomatous disease characterized by cutaneous and subcutaneous lesions and vascular diseases. Accurate diagnosis of pythiosis and identification of its causal agent are often inconsistent with current immunological diagnostic methods. A species-specific DNA probe was constructed by using a 530-bp HinfI fragment from the ribosomal DNA intergenic spacer of P. insidiosum. When the probe was incubated with dot blots of genomic DNA from 104 Pythium species, it hybridized only to the DNA of P. insidiosum and P. destruens-two species that have been considered conspecific. The probe also hybridized to DNA from 22 P. insidiosum isolates in this study, regardless of their geographic origin or animal host. When tested against genomic DNA from other pathogenic organisms (Aspergillus fumigatus, Basidiobolus ranarum, Conidiobolus coronatus, Lagenidium giganteum, Paracoccidioides brasiliensis, and Prototheca wickerhamii), no cross-hybridization of the probe was detected. The specificity of the probe to hybridize to genomic DNA from all isolates of P. insidiosum and not cross-react with DNA from other Pythium species or pathogens that cause symptoms similar to pythiosis in their hosts makes it a powerful tool for the accurate diagnosis of pythiosis. In addition, the probe has the potential for pathological and environmental diagnostic applications.

DNA Probes↗
PubMed1994

Minimal sequence requirements of a functional human immunodeficiency virus type 1 primer binding site.

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA(3Lys) primer bound near the 5' end of the genomic RNA at a position termed the primer binding site (PBS). The PBS is an 18-nucleotide sequence of the HIV-1 genome which is complementary to the 3'-terminal 18 nucleotides of the tRNA(3Lys). To investigate the sequence specificity of the interaction between tRNA(3Lys) and the PBS, we have constructed proviral genomes containing mutations in the PBS region. A mutant PBS was constructed in which the 18 nucleotides complementary to tRNA(3Lys) were substituted with 18 nucleotides predicted to be complementary to the 3'-terminal bases of a tRNA(Phe) molecule [pHXB2PBS(phe)]. A second proviral genome was constructed in which the PBS complementary to tRNA(Phe) was changed such that the first six nucleotides correspond to the wild-type PBS [pHXB2PBS(pheC)]. In all models of reverse transcription, the complementarity between the minus- and plus-strand PBS DNA facilitates the template switch and elongation of plus-strand DNA, resulting in a complete proviral genome. To test this model, we have inserted a five-nucleotide sequence 6 bp 3' of the mutant PBSs, which corresponds to the last five nucleotides of the wild-type PBSs [pHXB2PBS(phe+5) and pHXB2PBS(pheC+5)]. Transfection of plasmids containing the wild-type or mutant proviral genomes into COS-1 cells resulted in similar levels of intracellular expression of HIV-1 gag and env gene products as determined by immunoprecipitation with sera from AIDS patients and release of virus as determined by p24 assay. Transfection of pHXB2PBS(phe) or pHXB2PBS(phe+5) did not result in the production of infectious virus, while replication-competent viruses from cells transfected with pHXB2PBS(pheC) were detected very infrequently. Transfection of pHXB2PBS(pheC+5), however, consistently resulted in the production of infectious virus, although the appearance of the virus was delayed compared with those from cells transfected with pHXB2(wild type). Reinfection of SupT1 cells with equal amounts of p24 antigen resulted in similar kinetics of replication. PCR was used to amplify the PBS, and individual DNA products were subcloned into M13mp18. Sequence analysis of the PBS region of integrated proviruses derived from transfection of pHXB2PBS(pheC+5) revealed that the 18-nucleotide PBS complementary to tRNA(3Lys) was regenerated with a deletion of 6 bp 3' to the PBS region in all phage clones examined.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

The genomic termini of wild-type and vaccine strains of measles virus.

The genomic termini from 18 strains of measles virus (MV) including wild-type MVs from the pre-vaccine period, recent wild-type isolates and various vaccine strains were sequenced. The first 25 nucleotides of the 3' terminus and last 52 nucleotides of the 5' terminus were conserved in all of the viruses examined. Nucleotides 26 and 42 of the 3' leader were A and G, respectively, in all genotype A viruses except Edmonston wild-type (Ed-WT). All non-genotype A viruses and Ed-WT had U in both positions. No consistent substitution pattern was found in the 5' trailer region of the genome. The nucleotide substitutions at positions 26 and 42 in the 3' leader region were introduced into a MV-CAT mini-genome to test for their effect on the production of reporter protein in both a vaccinia T7-driven, plasmid-based replication assay as well as in a helper virus system. Regardless of the source of the polymerase proteins or the natural leader sequence of the helper viruses, the mini-genome 26A42G produced more CAT protein than 26U42U. The nucleotide substitution at 26 had the greatest effect on CAT production. These results indicated that naturally occurring nucleotide variations in the 3' leader region can affect the levels of reporter protein synthesis, and presumably affected the level of replication of the virus.

3' Untranslated Regions↗
PubMed2003

A genome-wide screen for linkage disequilibrium in Australian HLA-DRB1*1501 positive multiple sclerosis patients.

The association of multiple sclerosis with alleles/haplotypes from the HLA region on chromosome 6p21 is well established although the remainder of the genome remains relatively unexplored. We have completed a genome-wide screen for linkage disequilibrium in a cohort of Australian multiple sclerosis patients positive for HLA-DRB1*1501. A total of 4346 microsatellite markers provided through the "Genetic Analysis of Multiple sclerosis in EuropeanS" (GAMES) collaborative were analysed in DNA separately pooled from cases (n=217) and controls (n=187). Associations were found in four genomic regions (12q15, 16p13, 18p11 and 19q13) previously identified in linkage genome screens. Three additional regions of novel association were also identified (11q12, 11q23 and 14q21). Further analysis of these regions is required to establish whether the associations observed are due to epistatic interaction with the HLA locus.

Adult↗
PubMed2001

Trans-NIH neuroscience initiatives on mouse phenotyping and mutagenesis.

In the post-genomic era, the laboratory mouse will excel as a premier mammalian system to study normal and disordered biological processes, in part because of low cost, but largely because of the rich opportunities that exist for exploiting genetic tools and technologies in the mouse to systematically determine mammalian gene function. Many robust models of human disease may therefore be developed, and these in turn will provide critical clues to understanding gene function. The full potential of the mouse for understanding many of the neural and behavioral phenotypes of relevance to neuroscientists has yet to be realized. With the full anatomy of the mouse genome at hand, researchers for the first time will be able to move beyond traditional gene-by-gene approaches and take a global view of gene expression patterns crucial for neurobiological processes. In response to an action plan for mouse genomics developed on the basis of recommendations from the scientific community, seven institutes of the National Institutes of Health (NIH) initiated in 1999 a mouse genetics research program that specifically focused on neurobiology and complex behavior. The specific goals of these neuroscience initiatives are to develop high-throughput phenotyping assays and to initiate genome-wide mutagenesis projects to identify hundreds of mutant strains with heritable abnormalities of high relevance to neuroscientists. Assays and mutants generated in these efforts will be made widely available to the scientific community, and such resources will provide neuroscientists unprecedented opportunities to elucidate the molecular mechanisms of neural function and complex behavior. Such research tools ultimately will permit the manipulation and analysis of the mouse genome, as a means of gaining insight into the genetic bases of the mammalian nervous system and its complex disorders.

Animals↗
PubMed2004

Translation termination in Arabidopsis thaliana: characterisation of three versions of release factor 1.

Translation termination is mediated in all eukaryotes by the two release factors eRF1 and eRF3. Most organisms have a single eRF1 gene, however, three isogenes of eRF1 are found in Arabidopsis thaliana. They have no introns in the coding region which may indicate that some are pseudogenes. However, each was expressed and able to rescue a temperature sensitive eRF1-mutant of Saccharomyces cerevisiae indicating functional redundancy in A. thaliana. While normally a highly accurate process, translation termination can be directed to fail by sequence elements within an messenger RNA (mRNA). Interestingly, a well-characterised readthrough element follows the stop codon in one of these three isogenes (designated eRF1-1). This element was shown to be capable of inducing readthrough in an in vitro assay using a dual luciferase reporter, but surprisingly readthrough could not be detected using the complete gene context. The results highlight the diversity and duplication of genes within plant genomes, but also emphasize the conservation of the translation process across kingdoms.

Amino Acid Motifs↗
PubMed2005

High-throughput screen for genes predominantly expressed in the ICM of mouse blastocysts by whole mount in situ hybridization.

Mammalian preimplantation embryos provide an excellent opportunity to study temporal and spatial gene expression in whole mount in situ hybridization (WISH). However, large-scale studies are made difficult by the size of the embryos ( approximately 60mum diameter) and their fragility. We have developed a chamber system that allows parallel processing of embryos without the aid of a microscope. We first selected 91 candidate genes that were transcription factors highly expressed in blastocysts, and more highly expressed in embryonic (ES) than in trophoblast (TS) stem cells. We then used the WISH to identify 48 genes expressed predominantly in the inner cell mass (ICM) and to follow several of these genes in all seven preimplantation stages. The ICM-predominant expressions of these genes suggest their involvement in the pluripotency of embryonic cells. This system provides a useful tool to a systematic genome-scale analysis of preimplantation embryos.

Animals↗
PubMed2004

Characterization of the brassinosteroid insensitive 1 genes of cotton.

Suppression of brassinosteroid (BR) biosynthesis in cotton ovules by treatment with brassinazole inhibits fiber formation, indicating that BR plays an important role in cotton fiber development. Plant responses to brassinosteroids (BR) are mediated through a plasma membrane-bound leucine-rich repeat (LRR) receptor-like protein kinase known as BRI1. Mutations in the BRI1 genes of several species result in dwarfed plants with reduced sensitivity to BR. A single expressed sequence tag (EST) from cotton with strong sequence similarity to Arabidopsis BRI1 ( AtBRI1 ) was identified in a search of publicly available databases. With this EST as a starting point, full-length cDNAs and genomic coding sequences from upland cotton ( Gossypium hirsutum ) BRI1 ( GhBRI1 ) were obtained and characterized. Ectopic expression of this coding sequence in BR-insensitive Arabidopsis plants resulted in recovery of normal growth indicating that GhBRI1 is a functional homologue of AtBRI1. G. hirsutum is an allotetraploid (AADD) derived from diploid ancestors. Analysis of several GhBRI1 cDNAs showed two distinct sequences indicating the presence of two GhBRI1 genes, denoted GhBRI1-1 and GhBRI1-2. Sequence comparisons between these GhBRI1 coding sequences and those from related A and D genome diploid Gossypium species ( G. arboreum and G. thurberi ) indicated that GhBRI1-1 is likely to the A sub-genome orthologue while GhBRI1-2 is from the D sub-genome.

Amino Acid Sequence↗
PubMed1998

Screening blood donations for viral genomes: multicenter study of real-time simulation using pooled samples on the model of hepatitis C virus RNA detection.

BACKGROUND: The systematic screening for several blood-borne viral genomes in blood donations is a complementary safety measure planned or discussed by the national authorities of several countries. STUDY DESIGN AND METHODS: To appreciate the feasibility of such screening using pooled samples, a multicenter study of real-time simulation has been performed on the model of hepatitis C virus (HCV) infection. Four blood transfusion center laboratories and two research and diagnosis laboratories simultaneously screened, by several HCV RNA polymerase chain reaction assays, a panel of plasma sample pools of different sizes (500, 100, and 10 samples), collected from HCV-infected or HCV-uninfected blood donors. One viremic plasma was introduced in each pool. HCV RNA was detected by in-house polymerase chain reaction procedures or by standardized manual or semi-automated polymerase chain reaction assays. RESULTS: The results indicate the feasibility of sample pooling, which renders the screening for viral genomes by molecular biology techniques applicable in the short term and the importance of the experience of laboratory personnel in the use of molecular biology tools. CONCLUSION: The improvement of standardized assays needs to be continued, and training of laboratory staff members appears to be a crucial step before systematic screening of blood donations for viral genomes by molecular biology techniques can occur.

Blood Donors↗
PubMed1991

Genotyping and haplotyping of polymorphisms directly from genomic DNA via coupled amplification and sequencing (CAS).

Coupled amplification and sequencing (CAS) allows a segment of DNA to be sequenced directly from genomic DNA. An initial PCR amplification stage selects and amplifies the target. During a subsequent stage both strands of the target segment are sequenced simultaneously and amplified further. We show that CAS can readily identify variant base pairs. Genotyping of a population for known sequence variation can be achieved simply and directly from genomic DNA of each organism by performing CAS only for the variant bases. The procedure supercedes development and optimization of alternative typing assays based on oligonucleotide hybridization or ligation. In addition, we show that competitive oligonucleotide priming with allelic primers can be readily performed in concert with the second stage of CAS. The combination of techniques allows sequencing of a single chromosome from a heterozygous genomic sample and direct haplotyping of the polymorphism at the priming site with any others encompassed within the amplified segment.

Animals↗
PubMed2006

Construction and characterization of a highly redundant Pseudomonas aeruginosa genomic library prepared from 12 clinical isolates: application to studies of gene distribution among populations.

OBJECTIVE: To create, array, and characterize a pooled, high-coverage, genomic library composed of multiple biofilm-forming clinical strains of the opportunistic pathogen, Pseudomonas aeruginosa (PA). Twelve strains were obtained from patients with otorrhea, otitis media, and cystic fibrosis as a resource for investigating: difference in the transcriptomes of planktonic and biofilm envirovars; the size of the PA supragenome and determining the number of virulence genes available at the population level; and the distributed genome hypothesis. METHODS: High molecular weight genomic DNAs from 12 clinical PA strains were individually hydrodynamically sheared to produce mean fragment sizes of approximately 1.5 kb. Equimolar amounts of the 12 sheared genomic DNAs were then pooled and used in the construction of a genomic library with approximately 250,000 clones that was arrayed and subjected to quality control analyses. RESULTS: Restriction endonuclease and sequence analyses of 686 clones picked at random from the library demonstrated that >75% of the clones contained inserts larger than 0.5 kb with the desired mean insert size of 1.4 kb. Thus, this library provides better than 4.5x coverage for each of the genomes from the 12 components clinical PA isolates. Our sequencing effort ( approximately 1 million nucleotides to date) reveals that 13% of the clones present in this library are not represented in the genome of the reference P. aeruginosa strain PA01. CONCLUSIONS: Our data suggests that reliance on a single laboratory strain, such as PA01, as being representative of a pathogenic bacterial species will fail to identify many important genes, and that to obtain a complete picture of complex phenomena, including bacterial pathogenesis and the genetics of biofilm development will require characterization of the P. aeruginosa population-based supra-genome.

Child, Preschool↗