Construction and characterization of a DNA library from mouse chromosomes 19 purified by flow cytometry

Abstract

In spite of the constant development concerning physical mapping of eukaryotic genomes, the mouse chromosome 19 remains poorly characterized. In order to improve the possibilities for studying this chromosome, we have constructed a chromosome-specific EcoRI DNA fragment library from mouse chromosomes 19 sorted by flow cytometry. The resulting library contains about 3 X 10(4) recombinant clones. The identified inserts range in size from about 0.2-10 kb, with a 4 kb average size and with no observable redundancy. The purity of the library has been analyzed by flow-blot. For that purpose, chromosomes from 2 cell lines, 1 with a normal karyotype and 1 with translocated chromosome 19, were sorted on nylon filters and hybridized with 9 clones of the library. Results show that 5 clones out of the 9 clearly originate from sorted chromosomes 19 and 3 and are likely to be derived from its DNA, thus indicating that the library of chromosome 19 is of high purity.

Genetic analysis of MRL-lpr mice: relationship of the Fas apoptosis gene to disease manifestations and renal disease-modifying loci.

“…The clones used for spedfically cited chromosomal markers are listed in Table 1. The unpublished clones included: L15 for Lyw-57 (lymphocyte antigen workshop-57, kind gift of P. Cohen, University of North Carolina, Chapel Hill, NC); bMT008 for D12Sell (DNA segment, Chr 12, Seldin-1), a clone randomly selected from a mouse thymus cDNA library (Stratagene, La Jolla, CA); and MJ55 for D19SeI3 (DNA segment, Chr 19, Seldin-3), a genomic clone derived from a flow sorted mouse Chr 19 library (38). In addition, several clones were derived by PC1L amplification of Fas sequences from a mouse thymus cDNA library (Stratagene).…”

Chromosomes in the flow to simplify genome analysis

“…In a similar work, Krumlauf et al (1982) created libraries from autosomes 21 and 22, and ultimately two complete sets of small-insert DNA libraries for each of the 24 human chromosome types were created by the US National Laboratory Gene Library Project (Van Dilla et al 1986; van Dilla and Deaven 1990). Comparable libraries were constructed for various animals (Baron et al 1990; Shepel et al 1998) and in wheat (Wang et al 1992). Construction of short-insert libraries became easier after the introduction of methods for representative amplification of chromosomal DNA as only a few hundred or thousand sorted chromosomes (Miller et al 1992; Vooijs et al 1993; Macas et al 1996) or even a single chromosome (Van Devanter et al 1994) was sufficient as starting material.…”

Analysis and sorting of chromosomes by flow cytometry: New trends

“…Somatic hybrids in which unwanted chromosomes have already been eliminated may also be used [79,84]. In certain cases, such as the mouse, it is essential to use a cell line with translocated chromosomes in order to have access to a well-individualised chromosome, since chromosomes adjacent to the desired chromosome are shifted on the histogram following manipulation [3,5,6]. Another possibility is to use cells with Robertsonian fusions which move chromosomes of interest outside the flow karyotype area with overlapping peaks or spots [2,4].…”

“…These physical parameters are also valuable in establishing human and large mammal mapping strategies, such as cloning scheme design and rescaling of physical and composite maps [14,62,67]. The undeniable advantages of flow cytogenetics are that it can be used for isolation of pure chromosomal fractions, whether for specifying or completing the flow karyotype [10,26,56,60,68,73,88], rapid localisation of genes on a chromosome [24,39,50,70], characterisation of abnormal chromosomes [16,37,76], and above all, for construction of chromosome-specific genes librairies [2,3,5,6,17,21,23,25,32,46,61,84,85].…”