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At least 235 records · Page 13Linked to original sources
PubMed1995

Rapid identification of polymorphic CA-repeats in YAC clones.

Positional cloning of rare disease genes depends on the availability of highly polymorphic markers near the disease loci. The most abundant class of polymorphic markers in the human genome is CA-repeats. We have developed a strategy for the rapid isolation of highly polymorphic CA-repeats from YAC clones. Total DNA of yeast clones containing partly overlapping YACs is digested with frequent cutter restriction enzymes, blotted and hybridized with a poly(CA/GT) probe under high stringency conditions that enable preferential detection of long CA-repeats. The repeats detected in this way are isolated by PCR using vectorette linkers, sequenced, and appropriate flanking markers are constructed for genotyping. All of the CA-repeats identified using this approach were highly polymorphic. This simple and rapid approach should allow the development of highly polymorphic markers at any genomic region cloned in YACs.

Blotting, Southern↗
PubMed1999

Isolation of CpG islands from large genomic clones.

Positional cloning is a powerful method for the identification of genes. Using genetic and physical mapping methods the genomic region within which a particular gene is located can relatively easily be narrowed down to a comparatively small area contained within cosmid, PAC or BAC clones. It is then a matter of identifying genes within these clones. Here we describe the appli-cation of a technique, which has been successfully used for the bulk purification of CpG islands from whole genomes, to the isolation of CpG island sequences from such clones. As CpG islands overlap transcription units they can be used to isolate full-length cDNAs for associated genes, either by probing cDNA libraries or by searching databases. CpG islands are linked with approximately 60% of human genes and because their isolation is independent of the expression profile of these genes this approach would complement other expression-based methods of gene identification. By applying this technique to a cosmid clone known to contain the PAX6 gene we successfully isolated the CpG island for this gene along with other CpG island-like sequences. Closer examination revealed that an extensive genomic region around the 5'-end of PAX6 is unusual with regard to methylation and GC content. CpG island sequences were also successfully isolated from a PAC clone carrying the MBD1 gene. These included the complete CpG island containing the first exon and regulatory sequences from MBD1.

Base Sequence↗
PubMed1999

Cloning genes for non-syndromal hearing impairment.

Over 45 genes that cause autosomal non-syndromic hearing impairment (NSHI) have been localized and many more are predicted to exist. To clone these genes, a number of different strategies can be used. This paper focuses on four general approaches: functional cloning, positional cloning, position-dependent candidate gene cloning, and position-independent candidate gene cloning.

Cloning, Organism↗
PubMed1999

Screening of transcriptionally regulated genes following iron chelation in human astrocytoma cells.

Deferoxamine is an effective iron chelator and a potential therapeutic agent for use in minimizing free radical-mediated injury following trauma. Iron chelation may also be an effective means of limiting tumor growth by decreasing bioavailable iron. Deferoxamine can modulate gene expression through manipulation of intracellular iron levels; specifically at the posttranscription level by changing the activity of iron regulatory proteins (IRPs). The effect of iron chelation on the transcription of genes is still unclear, but iron-binding sites on DNA have been reported. Here we investigate the influence of deferoxamine on gene transcription. Two-directional (forward and backward) suppression subtraction hybridization (SSH) was performed on human astrocytoma cells (SW1088) cultured in either standard media or treated for 48 hours with deferoxamine. To restrict the number of false-positive clones, reverse Northern blotting was used to further verify the differentially expressed cDNA clones. Positive clones were sequenced and the mRNAs were re-examined on Northern blots for changes in expression over time of deferoxamine exposure. The results of these analyses have identified both novel and known genes whose expression is influenced by iron chelation. The known genes include a group related to energy production and a group related to protease function. These results provide examples of genes not previously known to be directly influenced by iron availability, and as such may be potential targets for iron chelation therapy.

Astrocytoma↗