PubMed Health⌕ Search

Biomedical subjects

J D Hoheisel

Publications and source records attributed to J D Hoheisel.

At least 55 records · Page 3Linked to original sources
PubMed1995

High-resolution cosmid mapping of the left arm of Saccharomyces cerevisiae chromosome XII; a first step towards an ordered sequencing approach.

For the sequencing of the left arm of chromosome XII of Saccharomyces cerevisiae, we fine-mapped the entire 450 kb fragment between the ribosomal DNA (rDNA) and the left telomere. Total yeast DNA in agarose blocks was digested with I-PpoI, which exclusively cuts once in each repeat unit of the rDNA. The resulting fragment was isolated from pulsed-field gels, together with the equally sized chromosome IX. A cosmid library of some 30-fold chromosome coverage was generated from this material, with the cloning efficiency being around 20,000 clones per microgram genomic DNA. The chromosome XII and IX specific clones were identified by complementary hybridizations with the respective chromosomes. For the left arm of chromosome XII, a contiguous cosmid array (contig) with an average map resolution better than 9 kb was generated by clone hybridization procedures. The ordered library serves as a tool for the physical mapping of genetic markers. Also, a minimal set of 15 clones was selected that covers the entire fragment. This subset forms the basis for the generation of a template map of much higher resolution for a directed sequencing of the left arm of chromosome XII.

Chromosome Mapping↗
PubMed1995

Genome analysis.

Explore the source record for details and available documents.

Chromosome Mapping↗
PubMed1995

The human homologue of the Drosophila melanogaster flightless-I gene (flil) maps within the Smith-Magenis microdeletion critical region in 17p11.2.

The Smith-Magenis syndrome (SMS) appears to be a contiguous-gene-deletion syndrome associated with a proximal deletion of the short arm of chromosome 17 in band p11.2. The spectrum of clinical findings includes short stature, brachydactyly, developmental delay, dysmorphic features, sleep disturbances, and behavioral problems. The complex phenotypic features suggest deletion of several contiguous genes. However, to date, no protein-encoding gene has been mapped to the SMS critical region. Recently, the Drosophila melanogaster flightless-I gene, fliI, and the homologous human cDNA have been isolated. Mutations in fliI result in loss of flight ability and, when severe, cause lethality due to incomplete cellularization with subsequent abnormal gastrulation. Here, we demonstrate that the human homologue (FLI) maps within the SMS critical region. Genomic cosmids were used as probes for FISH, which localized this gene to the 17p11.2 region. Somatic-cell hybrid-panel mapping further localized this gene to the SMS critical region. Southern blot analysis of somatic-cell hybrids and/or FISH analysis of lymphoblastoid cell lines from 12 SMS patients demonstrates the deletion of one copy of FLI in all SMS patients analyzed.

Animals↗
PubMed1994

Relational genome analysis using reference libraries and hybridisation fingerprinting.

The genomes of eukaryotic organisms are studied by an integrated approach based on hybridisation techniques. For this purpose, a reference library system has been set up, with a wide range of clone libraries made accessible to probe hybridisation as high density filter grids. Many different library types made from a variety of organisms can thus be analysed in a highly parallel process; hence, the amount of work per individual clone is minimised. In addition, information produced on one analysis level instantly assists in the characterisation process on another level. Genetic, physical and transcriptional mapping information and partial sequencing data are obtained for the individual library clones and are cross-referenced toward a comprehensive molecular understanding of genome structure and organisation, of encoded functions and their regulation. The order of genomic clones is established by hybridisation fingerprinting procedures. On these physical maps, the location of transcripts is determined. Complementary, partial sequence information is produced from corresponding cDNAs by hybridising short oligonucleotides, which will lead to the identification of regions of sequence conservation and the constitution of a gene inventory. The hybridisation analysis of the cDNA clones, and the genomic clones as well, could potentially be expanded toward a determination of (nearly) the complete sequence. The accumulated data set will provide the means to direct large-scale sequencing of the DNA, or might even make the sequence analysis of large genomic regions a redundant undertaking due to the already collected information.

Animals↗
PubMed1994

Application of hybridization techniques to genome mapping and sequencing.

Establishment of a variety of hybridization techniques for the analysis of large genomic areas has paved the way for a parallel examination of genomes on many levels within the framework of the various genome projects. Here, I discuss some recent achievements in the application of DNA hybridization techniques, in particular oligomer hybridization.

Base Sequence↗
PubMed1993

Use of reference libraries and hybridisation fingerprinting for relational genome analysis.

The concept of relational genome analysis by hybridisation has been developed into a working system. Various genomic and cDNA libraries have been generated and are distributed via a reference system. Analysis procedures have been tested successfully in the mapping of the entire Schizosaccharomyces pombe genome. In another test-case for their refinement, analyses on the Drosophila genome are well under way. Human and mouse libraries are being studied on all levels, from generating YAC maps to partially sequencing representative cDNA libraries. The automation of the involved processes and the development of improved image detection and analysis are well advanced.

Animals↗
PubMed1993

High resolution cosmid and P1 maps spanning the 14 Mb genome of the fission yeast S. pombe.

Gridded on high density filters, a P1 genomic library of 17-fold coverage and a cosmid library of 8 genome equivalents, both made from S. pombe strain 972h-, were ordered by hybridizing genetic markers and individual clones from the two libraries. Yeast artificial chromosome (YAC) clones covering the entire genome were used to subdivide the libraries, and hybridization of short oligonucleotides and DNA pools made from randomly selected cosmids provided further mapping information. Restriction digests were generated as an independent confirmation of the clone order. The high resolution clone map was aligned to the genetic map and the physical Notl and YAC maps. The usefulness of the various mapping techniques and cloning procedures could be assessed upon the different data sets.

Bacteriophage P1↗
PubMed1993

On the activities of Escherichia coli exonuclease III.

Exonuclease III (Exo III) of Escherichia coli is a DNA-modifying enzyme very frequently used in molecular biology. The experiments described here were carried out with the aims of reliably controlling exonuclease activity and of learning more about the enzyme's specificities. The dependence of Exo III activity on factors such as temperature (including heat inactivation), the concentrations of Exo III and NaCl, and the concentration and shape of 3' termini was investigated. Double stranded DNA was found to be a competitive inhibitor of the enzyme activity. Some four nucleotide 3' protrusions were shown to be sensitive to Exo III digestion. The synchronism of deletion was also examined. Implications for the proposed mechanism of activity are discussed.

Base Sequence↗
PubMed1992

Hybridization fingerprinting of high-density cDNA-library arrays with cDNA pools derived from whole tissues.

As part of an integrated mapping and sequencing analysis of genomes, we have developed an approach allowing the characterization of large numbers of cDNA library clones with a minimal number of experiments. Three basic elements used in the analysis of cDNA libraries are responsible for the high efficiency of this new approach: (1) high-density library arrays allowing thousands of clones to be screened simultaneously; (2) hybridization fingerprinting techniques to identify clones abundantly expressed in specific tissues (by hybridizations with labeled tissue cDNA pools) and to avoid the repeated selection of identical clones and of clones containing noncoding inserts; and (3) a computerized system for the evaluation of hybridization data. To demonstrate the feasibility of this approach, we hybridized high-density cDNA library arrays of human fetal brain and embryonal Drosophila with radiolabeled cDNA pools derived from whole mouse tissues. Fingerprints of the library arrays were generated, localizing clones containing cDNA sequences from mRNAs expressed at middle to high abundance (> 0.1-0.15%) in the respective tissue. Partial sequencing data from a number of clones abundantly expressed in several tissues were generated to demonstrate the value of the approach, especially for the selection of cDNA clones for the analyses of genomes based on expressed sequence tagged sites. Data obtained by the technique described will ultimately be correlated with additional transcriptional and sequence information for the same library clones and with genomic mapping information in a relational database.

Animals↗
PubMed1992

Complete coverage of the Schizosaccharomyces pombe genome in yeast artificial chromosomes.

The genome of the fission yeast, Schizosaccharomyces pombe, consists of some 14 million base pairs of DNA contained in three chromosomes. On account of its excellent genetics we used it as a test system for a strategy designed to map mammalian chromosomes and genomes. Data obtained from hybridization fingerprinting established an ordered library of 1,248 yeast artificial chromosome clones with an average size of 535 kilobases. The clones fall into three contigs completely representing the three chromosomes of the organism. This work provides a high resolution physical and clone map of the genome, which has been related to available genetic and physical map information.

Base Composition↗
PubMed1991

Use of high coverage reference libraries of Drosophila melanogaster for relational data analysis. A step towards mapping and sequencing of the genome.

Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated. Also, a jumping library has been created by a new method that takes advantage of methylation differences between genomic DNA and vector. Thirdly, two cDNA libraries have been picked. All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones. As a reference system, such filters are distributed and identified clones are provided. Single-copy probes have identified on average 1.4 cosmids per genome equivalent. Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations. cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization.

Animals↗
PubMed1990

Quantitative measurements on the duplex stability of 2,6-diaminopurine and 5-chloro-uracil nucleotides using enzymatically synthesized oligomers.

2,6-Diaminopurine and 5-chloro-uracil 2'-deoxynucleoside 5'-triphosphates were synthesized from their 2'-deoxynucleosides. Using a method of creating oligonucleotides by enzymatic primer extension, dodecanucleotides representing an XbaI/SalI site and the complementary SalI/XbaI site were generated containing these base modifications. Their duplex stability was quantitatively compared by thin-layer chromatography to oligomers containing 2'-deoxyadenosine and 2'-deoxythymidine. The two unmodified oligomers already showed significant differences in dissociation temperature and binding equilibrium. Substitution with 5-chloro-2'-deoxyuridine did not affect the dissociation temperature of either oligomer, the 2,6-diaminopurine, however, led to an increase of 1.8 degrees C or 1.5 degrees C per modified base, respectively. While in the XbaI/SalI oligomer both base modifications changed the binding equilibrium, the 2,6-diaminopurine by a factor of 1.32, the 5-chloro-uracil by 0.65, no such effect was found with the complementary oligomer.

Base Sequence↗
PubMed1990

Effect of 5-bromo- and 5-methyldeoxycytosine on duplex stability and discrimination of the NotI octadeoxynucleotide. Quantitative measurements using thin-layer chromatography.

Octadeoxynucleotides based on the recognition sequence of the restriction endonuclease NotI were synthesized containing unmodified nucleotides and nucleotides with methyl and bromide additions at the C5 position of the pyrimidine ring of deoxycytosine. On annealing to single-stranded DNA bearing one NotI site, thin-layer chromatography (TLC) of the different oligonucleotides was used quantitatively to determine differences in dissociation temperature (Td) and binding equilibrium. Buffers used in filter hybridization experiments could be used in this TLC system. In addition, actual hybridizations were carried out to filter-bound DNA with and without a NotI site. The incorporation of 5-methyldeoxycytosine and 5-bromodeoxycytosine led to a significant increase in stability of homoduplex formation during hybridization, due to a shift in the binding equilibrium and an increase of the Td, thereby improving discrimination considerably. Some implications of the results for several techniques involving oligomer hybridization are discussed.

5-Methylcytosine↗
PubMed1990

Ordering of cosmid clones covering the herpes simplex virus type I (HSV-I) genome: a test case for fingerprinting by hybridisation.

To allow the efficient construction of ordered clone libraries, we have been investigating the use of 'oligonucleotide fingerprinting' as an approach to identify overlapping clones, and ultimately restore the linear order of the clone set. To test the effectiveness of the procedure, we have constructed a cosmid library from the genome of the human DNA virus HSV-I and used hybridisation to multiple oligonucleotides selected from the nucleotide sequence to reconstruct the order of clones and oligonucleotides on the genome.

Base Sequence↗
PubMed1989

Control of partial digestion combining the enzymes dam methylase and MboI.

A method is described which allows the preparation of reproducible partial digests without previous establishment of the incubation conditions. It is based on a combined application of dam methylase and the restriction endonuclease MboI, both recognizing the sequence 5'-GATC-3' but MboI unable to cut the methylated site. Due to their competition for the same substrate the DNA is partially digested, with the size of the resulting fragments strongly dependent on the ratio of enzymes. The Km of the dam methylase was determined to be 115 ng DNA/microliters indicating a variance in fragment sizes generated at low DNA-concentrations. This effect is minimized above 150 ng/microliters. Any influence of digestion time is avoided, because the reaction runs until complete modification of all sites. The dependence on enzyme concentration and presence of agarose was checked. Knowledge of these parameters allows an accurate prediction of fragment sizes generated at different conditions. The technique was successfully used to construct libraries from different sources, in particular chromosome-specific libraries from small amounts of flow-sorted material.

DNA↗