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E A Ostrander

Publications and source records attributed to E A Ostrander.

84 records · Page 5Linked to original sources
PubMed1993

Local domains of supercoiling activate a eukaryotic promoter in vivo.

Experiments correlating template topology with transcriptional activity suggest that DNA topology plays a role in eukaryotic gene expression. Linear templates transfected into cultured cells produce far fewer transcripts than do circular transcription templates, and no transcripts can be detected from linear templates injected into Xenopus oocytes. Further, when transcriptionally active circular templates in Xenopus oocytes are linearized by injection of a restriction enzyme, transcription dramatically decreases. Here we show that transcription by phage T7 RNA polymerase from a divergent promoter can partially replace the requirement for circular Xenopus ribosomal RNA transcription templates in Xenopus oocytes. Supercoiled domains can apparently be generated on short pieces of DNA having no known sequences that result in association with the nuclear architecture, suggesting that localized, transient domains of supercoiling fulfil the minimum topological needs for Xenopus rRNA transcription in vivo.

Animals↗
PubMed1993

Identification and characterization of dinucleotide repeat (CA)n markers for genetic mapping in dog.

A large block of simple sequence repeat (SSR) polymorphisms for the dog genome has been isolated and characterized. Screening of primary libraries by conventional hybridization methods as well as by screening of enriched marker-selected libraries led to the isolation of a large number of genomic clones that contained (CA)n repeats. The sequences of 101 clones showed that the size and complexity of (CA)n repeats in the dog genome were similar to those reported for these markers in the human genome. Detailed analysis of a representative subset of these markers revealed that most markers were moderately to highly polymorphic, with PIC values exceeding 0.70 for 33% of the markers tested. An association between higher PIC values and markers containing longer (CA)n repeats was observed in these studies, as previously noted for similar markers in the human genome. A list of primer sequences that tag each characterized marker is provided, and a comprehensive system of nomenclature for the dog genome is suggested.

Alleles↗
PubMed1992

Construction of small-insert genomic DNA libraries highly enriched for microsatellite repeat sequences.

We describe an efficient method for the construction of small-insert genomic libraries enriched for highly polymorphic, simple sequence repeats. With this approach, libraries in which 40-50% of the members contain (CA)n repeats are produced, representing an approximately 50-fold enrichment over conventional small-insert genomic DNA libraries. Briefly, a genomic library with an average insert size of less than 500 base pairs was constructed in a phagemid vector. Amplification of this library in a dut ung strain of Escherichia coli allowed the recovery of the library as closed circular single-stranded DNA with uracil frequently incorporated in place of thymine. This DNA was used as a template for second-strand DNA synthesis, primed with (CA)n or (TG)n oligonucleotides, at elevated temperatures by a thermostable DNA polymerase. Transformation of this mixture into wild-type E. coli strains resulted in the recovery of primer-extended products as a consequence of the strong genetic selection against single-stranded uracil-containing DNA molecules. In this manner, a library highly enriched for the targeted microsatellite-containing clones was recovered. This approach is widely applicable and can be used to generate marker-selected libraries bearing any simple sequence repeat from cDNAs, whole genomes, single chromosomes, or more restricted chromosomal regions of interest.

Animals↗
PubMed1990

Template supercoiling by a chimera of yeast GAL4 protein and phage T7 RNA polymerase.

Fusion of the DNA-binding domain of yeast GAL4 protein to the amino terminus of bacteriophage T7 RNA polymerase yields a chimera that retains the characteristics of its components. The presence of the GAL4 peptide allows the chimeric enzyme to anchor itself on the DNA template, and this anchoring in turn drives the formation of a supercoiled DNA loop, in linear or circular templates, when RNA synthesis at the polymerase site forces a translocation of the DNA relative to the site. Nonspecific interaction between the chimeric enzyme and DNA appears to be sufficient to effect supercoiling during transcription. Transcription by the chimeric polymerase is strictly dependent on the presence of a T7 promoter; thus it provides a tool in vitro and in vivo for specifically supercoiling DNA segments containing T7 promoter sequences.

Amino Acid Sequence↗
PubMed1988

High resolution psoralen mapping reveals an altered DNA helical structure in the SV40 regulatory region.

Preferential psoralen photobinding sites have been mapped in vitro on restriction fragments spanning the SV40 origin region and surrounding sequences by a new fine structure analysis technique. Purified DNA fragments were photoreacted with 3H-5-methylisopsoralen (3H-5-MIP), a psoralen derivative which forms only monoadducts. Fragments were then end-labeled and digested with lambda exonuclease, a 5' processive enzyme which we have determined pauses at 5-MIP monoadducts. When photobinding sites were mapped on denaturing sequencing gels, it was observed that 5-MIP binds preferentially to 5'-TA sites, and to a lesser degree to 5'-AT sites. Utilizing this approach, we have identified a psoralen hypersensitive region in which the binding sites were much stronger than those in the surrounding sequences. This region extends from 150 base pairs (bp) to the late side of the enhancers to the early enhancer/promoter boundary. We suggest that this region contains a sequence directed structural alteration of the DNA helix which can be detected by the psoralen mapping approach described.

Base Sequence↗
PubMed1988

DNA structural alterations in the SV40 enhancer region are retained in vivo.

Psoralen accessibility in the SV40 regulatory region has been analyzed in vivo on viral minichromosomes and extracellular virus particles using a psoralen mapping technique which we have recently described. This approach takes advantage of the fact that lambda exonuclease digestion of double-stranded DNA is inhibited by the presence of 5-methylisopsoralen monoadducts. Previous analysis of purified restriction fragments from this region irradiated in vitro revealed a region of psoralen hypersensitivity which encompassed the repeated 72-base-pair (bp) enhancers and sequences 150 bp upstream toward the late region. The studies reported here demonstrate that changes in the structure of the DNA helix due to supercoiling do not affect the pattern of psoralen accessibility in this region. Additionally, no significant difference in the precise pattern of the psoralen hypersensitive domain is observed between late nucleoprotein complexes and extracellular virus. Finally, virus particles that have been through a freeze-thaw cycle, a process which we have shown eliminates the preferential photoaddition of the regulatory region in chromatin, also exhibit the same pattern of psoralen hypersensitivity.

DNA, Viral↗
PubMedDate not supplied

Hereditary canine spinal muscular atrophy is phenotypically similar but molecularly distinct from human spinal muscular atrophy.

Hereditary canine spinal muscular atrophy (HCSMA) is an autosomal dominant motor neuron disease that is similar in pathology and clinical presentation to various forms of human motor neuron disease. We have tested the hypothesis that the canine survival motor neuron (SMN) gene is responsible for HCSMA by genetic and molecular analysis of a colony of mixed breed dogs, all descended from a single affected individual. We cloned the canine SMN gene and determined the DNA sequence in an affected and an unaffected dog. We found no germline mutations in the SMN gene of the affected individual. Using conventional linkage analysis with canine-specific microsatellite repeat markers we screened the canine genome and identified a single linkage group likely to contain the HCSMA gene. Analysis with a panel of canine/rodent hybrid cell lines revealed that the SMN gene did not map to the same chromosome as the HCSMA linkage group. Collectively these results suggest that the molecular basis for HCSMA is distinct from that of phenotypically similar human disorders caused by inherited mutations in the SMN gene. This further suggests that additional studies on the molecular nature of HCSMA may reveal an unknown element of the molecular pathway leading to motor neuron disease.

Amino Acid Sequence↗
PubMedDate not supplied

Toward a framework linkage map of the canine genome.

Selective breeding to maintain specific physical and behavioral traits has made the modern dog one of the most physically diverse species on earth. One unfortunate consequence of the common breeding practices used to develop lines of dogs with the desired traits is amplification and propagation of genetic diseases within distinct breeds. To map disease loci we have constructed a first-generation framework map of the canine genome. We developed large numbers of highly polymorphic markers, constructed a panel of canine-rodent hybrid cell lines, and assigned those markers to chromosome groups using the hybrid cell lines. Finally, we determined the order and spacing of markers on individual canine chromosomes by linkage analysis using a reference panel of 17 outbred pedigrees. This article describes approaches and strategies to accomplish these goals.

Animals↗
PubMedDate not supplied

Genetic diversity and fitness in black-footed ferrets before and during a bottleneck.

The black-footed ferret (Mustela nigripes) is an endangered North American carnivore that underwent a well-documented population bottleneck in the mid-1980s. To better understand the effects of a bottleneck on a free-ranging carnivore population, we used 24 microsatellite loci to compare genetic diversity before versus during the bottleneck, and compare the last wild population to two historical populations. We also compared genetic diversity in black-footed ferrets to that of two sibling species, the steppe polecat (Mustela eversmanni) and the European polecat (Mustela putorius). Black-footed ferrets during the bottleneck had less genetic diversity than steppe polecats. The three black-footed ferret populations were well differentiated (F(ST) = 0.57 +/- 0.15; mean +/- SE). We attributed the decrease in genetic diversity in black-footed ferrets to localized extinction of these genetically distinct subpopulations and to the bottleneck in the surviving subpopulation. Although genetic diversity decreased, female fecundity and juvenile survival were not affected by the population bottleneck.

Animals↗
PubMedDate not supplied

Establishing appropriate genome-wide significance levels for canine linkage analyses.

A threshold of 3.3 for a genome-wide maximum LOD score (MAXLOD) has been demonstrated in human linkage studies as corresponding to a type I error rate of 5%. Generalization of this work to other species assumes the presence of an infinitely dense marker map. While this assumption is increasingly realistic for the human genome, it may be unrealistic for the dog genome. In this study we establish the analytic and empirical thresholds for MAXLOD in canine linkage studies corresponding to type I error rates of 5% and 1% for autosomal traits. Empirical thresholds are computed via simulation assuming a 10 cM map with no fine mapping performed. Pedigree structures for simulations were drawn from two canine disease studies. Five thousand replicates of genome-wide null genotype data were simulated and analyzed for each disease. We determined that MAXLOD thresholds of 3.2 and 2.7 correspond to analytic and empirical type I error rates of 5%, respectively. In all cases, the MAXLOD thresholds from simulations were always at least 0.5 LOD units below the corresponding analytic thresholds. We therefore recommend that a threshold of 3.2 be used for canine linkage studies when fine mapping is performed, and that researchers perform their own simulation studies to assess genome-wide empirical significance levels when no fine mapping is performed.

Animals↗
PubMedDate not supplied

Comparison of MultiMap and TSP/CONCORDE for constructing radiation hybrid maps.

Radiation hybrid (RH) map construction allows investigators to locate both type I and type II markers on a given genome map. The process is composed of two steps. The first consists of determining the pattern distribution of a set of markers within the different cell lines of an RH panel. This is mainly done by polymerase chain reaction (PCR) amplification and gel electrophoresis, and results in a series of numbers indicating the presence or the absence of each marker in each cell line. The second step consists of a comparison of these numbers, using various algorithms, to group and then order markers. Because different algorithms may provide (slightly) different orders, we have compared the merits of the MultiMap and TSP/CONCORDE packages using a data set of information currently under analysis for construction of the canine genome RH map.

Animals↗