Mechanically stretched chromosomes as targets for high-resolution FISH mapping.

Abstract

When used with metaphase chromosomes, fluorescence in situ hybridization (FISH} makes it possible to localize probes to individual chromosome bands and to establish the order of probes separated by ~2-3 Mb in dual-color hybridizations. We evaluated the use of mechanically stretched chromosomes as hybridization targets for increased mapping resolution. Mapping resolution was tested by pair-wise hybridizations with probes from the Ip32-p33 region, spanning distances from 20 to -1500 kb. Probes separated by ~170 kb could be ordered relative to one another and to the centromere-telomere axis of the chromosome. The advantages of the technique are the simple procedure for preparing the slides, the straightforward interpretation of the results, and the ability to score the predominant order from <10 stretched chromosomes. However, because of the variability of stretching from one sample to another, the calculation of actual physical distances between probes is not possible. To illustrate the utility of this method, we showed that the gene for receptor tyrosine kinase TIE lies centromeric to COLgA2, ~F, and L-b'iYC genes at Ip32. The use of mechanically stretched chromosomes provides ~lO-fold increased mapping resolution as compared with conventional metaphase FISH. Thus, the technique effectively bridges the gap between metaphase mapping and ultra-high-resolution mapping [I-300 kb} techniques, such as the DNA fiber FISH.High-resolution physical mapping, ordering of probes, and construction of contigs are essential steps in positional cloning projects. Rapid developments in fluorescence in situ hybridization (FISH) techniques have paved the way for the application of new visual physical mapping methods. The use of FISH with metaphase spreads and prometaphase preparations allows the ordering of probes with 2-3-and 1-Mb resolutions, respectively. Interphase nuclei in which the chromatin is -20-fold less condensed than chromatin in prometaphase and paometaphase nuclei have been used as hybridization targets for ordering the probes within the range 50-1000 kb (Trask 1991). However, interphase FISH mapping is more demanding than the metaphase approach. It requires nuclei that are arrested perfectly in the GI cell cycle phase, and the ordering of probes can be accomplished only after a statistical evaluation of the predominant order in -100 nuclei.The recently developed DNA fiber FISH techniques provide straightforward high-resolution mapping in the range 1-300 kb (Heng et al. 1992; Wiegant et al. 1992;Parra and Windle 1993;Haaf and Ward 1994a; Heiskanen et al. 1994;Senger et al. 1994). However, when using the DNA fiber FISH techniques, the mapping of probes separated by >300 kb becomes increasingly problematic and the direct establishment of the te!omeric-centromeric orientation is often difficult. Overall, a robust and accurate mapping technique is required in the range 300-3000 kb. This technique would bridge the gap between (pro)metaphase FISH and DNA fiber FISH techniques.Mechanically stretched chromosomes, prepared by ...

Chromosome Mapping of Dragline Silk Genes in the Genomes of Widow Spiders (Araneae, Theridiidae)

“…Based on the resolution of FISH with metaphase chromosomes, we deduce that this gene cluster is within a 2–3 Mb region [40]. Neighboring of multicopy genes has also been observed in other organisms, such as DAZ fertility factor genes on the human Y chromosome, patatin genes in potato, and histone genes in grasshopper [41] [42]–[43].…”

Chromosome Mapping of Dragline Silk Genes in the Genomes of Widow Spiders (Araneae, Theridiidae)

“…Based on the resolution of FISH with metaphase chromosomes, we deduce that this gene cluster is within a 2–3 Mb region [40]. Neighboring of multicopy genes has also been observed in other organisms, such as DAZ fertility factor genes on the human Y chromosome, patatin genes in potato, and histone genes in grasshopper [41] [42]–[43].…”

The mapping and visual ordering of the human syndecan-1 and N- myc genes near the telomeric region of chromosome 2p

“…1). This was further verified using mechanically stretched chromosomes (Laan et al 1995). In every (10/10) stretched chromosome analyzed, the signals could be seen to be clearly separated (Fig.…”

“…Although there seemed to be a few more signals in the order centromere-syndecan-N-myc, it was not feasible to draw any conclusions from the data. According to our previous experience, these results suggested that the genes were within 1500 kb of each other (Trask et al 1991;Laan et al 1995). With prometaphase spreads as targets for FISH, 20 out of 30 examined prometaphases showed distinct red and green signals within the chromosome (Fig.…”

“…Based on nearly overlapping signals on metaphases, separate signals on most of the prometaphases but distinct separation of the signals on all mechanically extended chromosomes, an estimation of a distance of 700 kb-1 Mb is likely (Laan et al 1995). Due to the variation in chromosomal stretching during cytocentrifugation, the actual physical distance could not be determined more accurately.…”

A dual-color FISH gene map of the proximal region of rat Chromosome 4 and comparative analysis in human and mouse

“…PACs giving rise to overlapping signals on the metaphase and prometaphase levels of resolution were again subjected to dual-color FISH, this time together with a PAC representing a third gene, on interphase chromatin in resting cell nuclei. Thus, three levels of resolution were obtained in the dual-color FISH analysis, ranging from approximately 1-3 Mb for metaphase chromosomes (Trask et al 1991;Laan et al 1995), 0.5-1 Mb for prometaphase chromosomes (Lichter et al 1990;Haaf and Ward 1994), and 50 kb-2 Mb for interphase chromatin (Yokota et al 1995;Heiskanen et al 1996). Using this procedure, we could unambiguously determine the relative order of most loci, as exemplified in Fig.…”

Chromosome Mapping of Dragline Silk Genes in the Genomes of Widow Spiders (Araneae, Theridiidae)

“…Based on the resolution of FISH with metaphase chromosomes, we deduce that this gene cluster is within a 2–3 Mb region [40]. Neighboring of multicopy genes has also been observed in other organisms, such as DAZ fertility factor genes on the human Y chromosome, patatin genes in potato, and histone genes in grasshopper [41] [42]–[43].…”

The mapping and visual ordering of the human syndecan-1 and N- myc genes near the telomeric region of chromosome 2p

“…1). This was further verified using mechanically stretched chromosomes (Laan et al 1995). In every (10/10) stretched chromosome analyzed, the signals could be seen to be clearly separated (Fig.…”

“…Although there seemed to be a few more signals in the order centromere-syndecan-N-myc, it was not feasible to draw any conclusions from the data. According to our previous experience, these results suggested that the genes were within 1500 kb of each other (Trask et al 1991;Laan et al 1995). With prometaphase spreads as targets for FISH, 20 out of 30 examined prometaphases showed distinct red and green signals within the chromosome (Fig.…”

“…Based on nearly overlapping signals on metaphases, separate signals on most of the prometaphases but distinct separation of the signals on all mechanically extended chromosomes, an estimation of a distance of 700 kb-1 Mb is likely (Laan et al 1995). Due to the variation in chromosomal stretching during cytocentrifugation, the actual physical distance could not be determined more accurately.…”

A dual-color FISH gene map of the proximal region of rat Chromosome 4 and comparative analysis in human and mouse

“…PACs giving rise to overlapping signals on the metaphase and prometaphase levels of resolution were again subjected to dual-color FISH, this time together with a PAC representing a third gene, on interphase chromatin in resting cell nuclei. Thus, three levels of resolution were obtained in the dual-color FISH analysis, ranging from approximately 1-3 Mb for metaphase chromosomes (Trask et al 1991;Laan et al 1995), 0.5-1 Mb for prometaphase chromosomes (Lichter et al 1990;Haaf and Ward 1994), and 50 kb-2 Mb for interphase chromatin (Yokota et al 1995;Heiskanen et al 1996). Using this procedure, we could unambiguously determine the relative order of most loci, as exemplified in Fig.…”

Chromosome Mapping of Dragline Silk Genes in the Genomes of Widow Spiders (Araneae, Theridiidae)

“…Based on the resolution of FISH with metaphase chromosomes, we deduce that this gene cluster is within a 2–3 Mb region [40]. Neighboring of multicopy genes has also been observed in other organisms, such as DAZ fertility factor genes on the human Y chromosome, patatin genes in potato, and histone genes in grasshopper [41] [42]–[43].…”

The mapping and visual ordering of the human syndecan-1 and N- myc genes near the telomeric region of chromosome 2p

“…1). This was further verified using mechanically stretched chromosomes (Laan et al 1995). In every (10/10) stretched chromosome analyzed, the signals could be seen to be clearly separated (Fig.…”

“…Although there seemed to be a few more signals in the order centromere-syndecan-N-myc, it was not feasible to draw any conclusions from the data. According to our previous experience, these results suggested that the genes were within 1500 kb of each other (Trask et al 1991;Laan et al 1995). With prometaphase spreads as targets for FISH, 20 out of 30 examined prometaphases showed distinct red and green signals within the chromosome (Fig.…”

“…Based on nearly overlapping signals on metaphases, separate signals on most of the prometaphases but distinct separation of the signals on all mechanically extended chromosomes, an estimation of a distance of 700 kb-1 Mb is likely (Laan et al 1995). Due to the variation in chromosomal stretching during cytocentrifugation, the actual physical distance could not be determined more accurately.…”

A dual-color FISH gene map of the proximal region of rat Chromosome 4 and comparative analysis in human and mouse

“…PACs giving rise to overlapping signals on the metaphase and prometaphase levels of resolution were again subjected to dual-color FISH, this time together with a PAC representing a third gene, on interphase chromatin in resting cell nuclei. Thus, three levels of resolution were obtained in the dual-color FISH analysis, ranging from approximately 1-3 Mb for metaphase chromosomes (Trask et al 1991;Laan et al 1995), 0.5-1 Mb for prometaphase chromosomes (Lichter et al 1990;Haaf and Ward 1994), and 50 kb-2 Mb for interphase chromatin (Yokota et al 1995;Heiskanen et al 1996). Using this procedure, we could unambiguously determine the relative order of most loci, as exemplified in Fig.…”