Biomedical subjects

E Prigmore

Publications and source records attributed to E Prigmore.

6 recordsLinked to original sources

PubMed2006

Ultra-high resolution array painting facilitates breakpoint sequencing.

OBJECTIVE: To describe a considerably advanced method of array painting, which allows the rapid, ultra-high resolution mapping of translocation breakpoints such that rearrangement junction fragments can be amplified directly and sequenced. METHOD: Ultra-high resolution array painting involves the hybridisation of probes generated by the amplification of small numbers of flow-sorted derivative chromosomes to oligonucleotide arrays designed to tile breakpoint regions at extremely high resolution. RESULTS AND DISCUSSION: How ultra-high resolution array painting of four balanced translocation cases rapidly and efficiently maps breakpoints to a point where junction fragments can be amplified easily and sequenced is demonstrated. With this new development, breakpoints can be mapped using just two array experiments: the first using whole-genome array painting to tiling resolution large insert clone arrays, the second using ultra-high-resolution oligonucleotide arrays targeted to the breakpoint regions. In this way, breakpoints can be mapped and then sequenced in a few weeks.

Adult↗

PubMed2005

The complex nature of constitutional de novo apparently balanced translocations in patients presenting with abnormal phenotypes.

OBJECTIVE: To describe the systematic analysis of constitutional de novo apparently balanced translocations in patients presenting with abnormal phenotypes, characterise the structural chromosome rearrangements, map the translocation breakpoints, and report detectable genomic imbalances. METHODS: DNA microarrays were used with a resolution of 1 Mb for the detailed genome-wide analysis of the patients. Array CGH was used to screen for genomic imbalance and array painting to map chromosome breakpoints rapidly. These two methods facilitate rapid analysis of translocation breakpoints and screening for cryptic chromosome imbalance. Breakpoints of rearrangements were further refined (to the level of spanning clones) using fluorescence in situ hybridisation where appropriate. RESULTS: Unexpected additional complexity or genome imbalance was found in six of 10 patients studied. The patients could be grouped according to the general nature of the karyotype rearrangement as follows: (A) three cases with complex multiple rearrangements including deletions, inversions, and insertions at or near one or both breakpoints; (B) three cases in which, while the translocations appeared to be balanced, microarray analysis identified previously unrecognised imbalance on chromosomes unrelated to the translocation; (C) four cases in which the translocation breakpoints appeared simple and balanced at the resolution used. CONCLUSIONS: This high level of unexpected rearrangement complexity, if generally confirmed in the study of further patients, will have an impact on current diagnostic investigations of this type and provides an argument for the more widespread adoption of microarray analysis or other high resolution genome-wide screens for chromosome imbalance and rearrangement.

Cell Line↗

PubMed2004

Applications of combined DNA microarray and chromosome sorting technologies.

The sequencing of the human genome has led to the availability of an extensive mapped clone resource that is ideal for the construction of DNA microarrays. These genomic clone microarrays have largely been used for comparative genomic hybridisation studies of tumours to enable accurate measurement of copy number changes (array-CGH) at increased resolution. We have utilised these microarrays as the target for chromosome painting and reverse chromosome painting to provide a similar improvement in analysis resolution for these studies in a process we have termed array painting. In array painting, chromosomes are flow sorted, fluorescently labelled and hybridised to the microarray. The complete composition and the breakpoints of aberrant chromosomes can be analysed at high resolution in this way with a considerable reduction in time, effort and cytogenetic expertise required for conventional analysis using fluorescence in situ hybridisation. In a similar way, the resolution of cross-species chromosome painting can be improved and we present preliminary observations of the organisation of homologous DNA blocks between the white cheeked gibbon chromosome 14 and human chromosomes 2 and 17.

Cell Line↗

PubMed2003

Array painting: a method for the rapid analysis of aberrant chromosomes using DNA microarrays.

OBJECTIVE: The authors describe a method, termed array painting, which allows the rapid, high resolution analysis of the content and breakpoints of aberrant chromosomes. METHODS: Array painting is similar in concept to reverse chromosome painting and involves the hybridisation of probes generated by PCR of small numbers of flow sorted chromosomes on large insert genomic clone DNA microarrays. RESULTS: and CONCLUSIONS: By analysing patients with cytogenetically balanced chromosome rearrangements, the authors show the effectiveness of array painting as a method to map breakpoints prior to cloning and sequencing chromosome rearrangements.

Adult↗

PubMed1998

Cryptic Rac-binding and p21(Cdc42Hs/Rac)-activated kinase phosphorylation sites of NADPH oxidase component p67(phox).

Rac1 is a member of the Rho family of small molecular mass GTPases that act as molecular switches to control actin-based cell morphology as well as cell growth and differentiation. Rac1 and Rac2 are specifically required for superoxide formation by components of the NADPH oxidase. In binding assays, Rac1 interacts directly with p67(phox), but not with the other oxidase components: cytochrome b, p40(phox), or p47(phox) (Prigmore, E., Ahmed, S., Best, A., Kozma, R. , Manser, E., Segal, A. W., and Lim, L. (1995) J. Biol. Chem. 270, 10717-10722). Here, the Rac1/2 interaction with p67(phox) has been characterized further. Rac1 and Rac2 can bind to p67(phox) amino acid residues 170-199, and the N terminus (amino acids 1-192) of p67(phox) can be used as a specific inhibitor of Rac signaling. Deletion of p67(phox) C-terminal sequences (amino acids 193-526), the C-terminal SH3 domain (amino acids 470-526), or the polyproline-rich motif (amino acids 226-236) stimulates Rac1 binding by approximately 8-fold. p21(Cdc42Hs/Rac)-activated kinase (PAK) phosphorylates p67(phox) amino acid residues adjacent to the Rac1/2-binding site, and this phosphorylation is stimulated by deletion of the C-terminal SH3 domain or the polyproline-rich motif. These data suggest a role for cryptic Rac-binding and PAK phosphorylation sites of p67(phox) in control of the NADPH oxidase.

3T3 Cells↗

PubMed1995

A 68-kDa kinase and NADPH oxidase component p67phox are targets for Cdc42Hs and Rac1 in neutrophils.

Cdc42Hs and Rac1 are members of the Ras superfamily of small molecular weight (p21) GTP binding proteins. Cdc42Hs induces filopodia formation in Swiss 3T3 fibroblasts while Rac1 induces membrane ruffling. Rac1 also activates superoxide production by the components (cytochrome b, p40phox, p67phox, and p47phox) of the neutrophil oxidase. To isolate target proteins involved in these signaling pathways, we have probed proteins from neutrophil cytosol immobilized on nitrocellulose with Cdc42Hs labeled with [gamma-32P]GTP. Cdc42Hs probe detected binding protein(s) of 66-68 kDa in neutrophil cytosol. Rac1 probe also detected the 66-68-kDa proteins, suggesting the possibility that p67phox may be a binding protein for both of these p21 proteins. Indeed, Cdc42Hs and Rac1 were found to bind specifically to purified recombinant p67phox but not the other oxidase components. A 68-kDa Cdc42Hs binding protein was purified from neutrophil cytosol and found to be related to the recently described p65pak kinase from brain. These results suggest that the p68 kinase and p67phox are targets for Cdc42Hs and Rac1 in neutrophils.

Brain↗