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A Poustka

Publications and source records attributed to A Poustka.

139 records · Page 8Linked to original sources
PubMed1990

Physical maps of 4p16.3, the area expected to contain the Huntington disease mutation.

The gene for Huntington disease, a neurodegenerative disorder with autosomal dominant inheritance, has been localized to the terminal portion of the short arm of human chromosome 4 (4p16.3) by linkage analysis. Since eventual isolation of the gene requires the application of high-resolution genetic analysis coupled with long-range DNA mapping and cloning techniques, we have constructed a physical map of the chromosomal region 4p16.3 using more than 20 independently derived probes. We have grouped these markers into three clusters which have been ordered and oriented by genetic and somatic cell genetic mapping information. The mapped region extends from D4S10 (G8) toward the telomere and covers minimally 5 Mb.

Chromosome Mapping↗
PubMed1990

The murine GABAA receptor delta-subunit gene: structure and assignment to human chromosome 1.

The murine chromosomal gene for the GABAA receptor delta subunit was isolated and characterized by high-resolution mapping and DNA sequencing. Spanning 13 kb, it comprises nine exons and displays an intron pattern comparable, but not identical, to that seen in members of the nicotinic acetylcholine receptor family. Notably, the second transmembrane domain thought to line the ion channel and conserved among different GABAA receptor subunits, is interrupted by an intron. The 5'-flanking region of the delta gene displays features characteristic of a CpG island and lacks canonical promoter elements such as TATA and CCAAT consensus sequences in proximity to the transcriptional initiation site. The human delta subunit gene was localized on the short arm of chromosome 1.

Amino Acid Sequence↗
PubMed1990

The (6;9) chromosome translocation, associated with a specific subtype of acute nonlymphocytic leukemia, leads to aberrant transcription of a target gene on 9q34.

The specific (6;9)(p23;q34) chromosomal translocation is associated with a defined subtype of acute nonlymphocytic leukemia (ANLL). The 9q34 breakpoint is located at the telomeric side of the c-abl gene. Through a combination of chromosome jumping, long-range mapping, and chromosome walking, the chromosome 9 breakpoints of several t(6;9) ANLL patients were localized within a defined region of 8 kilobases (kb), 360 kb telomeric of c-abl. Subsequent cDNA cloning revealed that this region represented an intron in the middle of a gene, called Cain (can), encoding a 7.5-kb transcript. Disruption of the can gene by the translocation resulted in the expression of a new 5.5-kb can mRNA from the 6p- chromosome. Isolation of chromosome 6 sequences showed that breakpoints on 6p23 also clustered within a limited stretch of DNA. These data strongly suggest a direct involvement of the translocation in the leukemic process of t(6;9) ANLL.

Blotting, Southern↗
PubMed1990

A yeast artificial chromosome telomere clone spanning a possible location of the Huntington disease gene.

The Huntington disease (HD) gene has been mapped to the most distal subband of chromosome 4p. Analysis of recombination events has not provided an unequivocal location of the HD gene, but it indicates a position very close to the telomere as one possibility. We have constructed a yeast artificial chromosome (YAC) vector (containing a rare-cutter polylinker) for the cloning of mammalian telomeres, used it to prepare a BssHII-telomere library with DNA from an individual homozygous for HD, and have identified a 115-kb clone containing the telomere of 4p. One probable recombinant would confine the telomeric candidate location for the gene to the region covered by the YAC, which makes it possible that the clone described here contains the HD locus in its mutant form.

Chromosomes, Fungal↗
PubMed1989

Clustering of multiallele DNA markers near the Huntington's disease gene.

Five highly informative multiallele restriction fragment length polymorphisms (RFLPs) of value for preclinical diagnosis of Huntington's disease (HD) have been genetically characterized. One RFLP was uncovered by expansion of the D4S43 locus while three others are at D4S111 and D4S115, loci defined by NotI-linking clones. The final marker, D4S125, represents a recently discovered VNTR locus. All four loci map closer to the HD gene and to the telomere than D4S10, the original linked marker for HD. In combination with two multiallele RFLPs previously identified for D4S43 and another linked locus, D4S95, these five new multiallele markers will dramatically improve the speed and accuracy of predictive testing in HD, and increase its applicability by maximizing the chances of an informative test for anyone with appropriate family structure.

Alleles↗
PubMed1988

Construction of a NotI linking library and isolation of new markers close to the Huntington's disease gene.

Linking clones contain sequences flanking recognition sites for enzymes cutting rarely in mammalian DNA. They can be used to obtain and correlate both physical and genetic mapping information over subregions of mammalian chromosomes. We have constructed and used a NotI linking clone library representing unmethylated NotI sites from HHW693 DNA, a hamster hybrid cell line containing 4p15-4pter and a fragment of 5p as its only human chromosome contribution. Human clones were identified by hybridisation with a cloned human repeat sequence, and localised further to subregions of human chromosome 4p15-4pter using a panel of additional hybrids. Clones from the region distal to the DNA probes (D4S10, D4S43, D4S95) linked to the Huntington's disease mutation, were further analysed. Four markers close to the HD gene: D4S111, D4S113, D4S114 and clone 417 are described here. In addition to serving as markers in physical and genetic mapping experiments, these linking clones provide probes next to cleavable NotI sites, and can therefore be used to screen NotI based chromosome jumping libraries. They also provide indications for potential gene sequences, identifiable as evolutionarily conserved sequences.

Chromosome Mapping↗
PubMed1987

A family of cosmid vectors with the multi-copy R6K replication origin.

A family of cosmid vectors was constructed which contain replication origins (ori) derived from the multicopy plasmid R6K, a kanamycin resistance gene and two cos sites, permitting efficient library construction. Additional features of later constructs are (i) the presence of NotI sites flanking the site of insertion to allow intact excision of inserts, (ii) the facility for selective cloning of the ends of inserts for rapid chromosome walking, and (iii) the use of a mutated R6K ori leading to an increased copy number.

Bacteriophage lambda↗
PubMed1985

Analysis of cosmids using linearization by phage lambda terminase.

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage lambda terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage lambda (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.

Animals↗
PubMed1984

Selective isolation of cosmid clones by homologous recombination in Escherichia coli.

A procedure for selection of specific cosmid clones by homologous recombination between cosmid clones from a library and sequences cloned into a plasmid has been developed. Cosmid libraries constructed in a rec- host strain are packaged in vivo into lambda particles. Appropriate aliquots are then introduced into a rec+ host containing the sequence used for selection cloned into a plasmid vector without sequence homology to the cosmid vector. After a short time for recombination, the cosmids are packaged in vivo. Cosmids that have taken up the plasmid by homologous recombination are isolated by plating under conditions selecting for the antibiotic resistance markers carried by both vectors. The recombined cosmids can lose the inserted sequence by another homologous recombination event and, after packaging in vivo, these revertants can be identified on appropriate indicator plates.

Animals↗
PubMed1983

Lambda replacement vectors carrying polylinker sequences.

To simplify the construction and screening of genomic libraries, we have made a new family of lambda replacement vectors (EMBL1, EMBL2, EMBL3, EMBL4) and derivatives containing amber mutations (EMBL3 Sam, EMBL3 AamBam, EMBL3 AamSam). These vectors have a large capacity and polylinker sequences flanking the middle fragment. The polylinkers allow a choice of cloning enzymes and, especially useful in the case of cloning of Sau3A partial digests, the excision of the entire insert by flanking SalI (EMBL3) or EcoRI (EMBL4) sites. Phages with inserts can be selected either biochemically (particularly EMBL3) or genetically by their Spi- phenotype. Amber derivatives of the EMBL3 vector allow the application of genetic screening procedures based on selection for the products of homologous recombination events, and for the selective cloning of DNA sequences linked to supF genes.

Animals↗
PubMedDate not supplied

Construction and use of human chromosome jumping libraries from NotI-digested DNA.

A basic difficulty in the molecular analysis of genes identified by mutations in the mammalian genome is the need to cover genetic distances corresponding to several hundred kilobases or more by molecular techniques like chromosome walking. In chromosome jumping, this limitation is overcome by the deletion of all but the extreme ends of large DNA molecules before cloning. We describe here the construction and characterization of a NotI 'jumping library' from human DNA. To characterize this library, random clones were analysed by restriction mapping. Clones carrying unique end fragments were characterized further by hybridization to Southern blots of NotI-cleaved human DNA separated on pulsed field gradient (PFG) gels. As a first step in a directional walk, the library was screened with a clone containing a NotI site cleaved in genomic DNA ('NotI linking clone') localized to the distal third of the short arm of human chromosome 4 (A.-M.F. & T.P., unpublished data). Starting and end points of two identified clones were positioned within a restriction map covering 850 kilobases.

Base Sequence↗