Fluorescence in situ hybridization analysis of keratinocyte growth factor gene amplification and dispersion in evolution of great apes and humans

Abstract

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family. Portions of the gene encoding KGF were amplified during primate evolution and are present in multiple nonprocessed copies in the human genome. Nucleotide analysis of a representative sampling of these KGF-like sequences indicated that they were at least 95% identical to corresponding regions of the KGF gene. To localize these sequences to specific chromosomal sites in human and higher primates, we used fluorescence in situ hybridization. In human, using a cosmid probe encoding KGF exon 1, we assigned the location of the KGF gene to chromosome 15q15-21.1. In addition, copies of KGF-like sequences hybridizing only with a cosmid probe encoding exons 2 and 3 were localized to dispersed sites on chromosome 2q21, 9p11, 9q12-13, 18p11, 18q11, 21q11, and 21q21.1. The distribution of KGF-like sequences suggests a role for alphoid DNA in their amplification and dispersion. In chimpanzee, KGF-like sequences were observed at five chromosomal sites, which were each homologous to sites in human, while in gorilla, a subset of four of these homologous sites was identified; in orangutan two sites were identified, while gibbon exhibited only a single site. The chromosomal localization of KGF sequences in human and great ape genomes indicates that amplification and dispersion occurred in multiple discrete steps, with initial KGF gene duplication and dispersion taking place in gibbon and involving loci corresponding to human chromosomes 15 and 21. These findings support the concept of a closer evolutionary relationship of human and chimpanzee and a possible selective pressure for such dispersion during the evolution of higher primates.

Figure 1

Normal human chromosomes cohybridized with a digoxigenin-labeled cosmid probe for the exon 1 of KGF gene (red signal) and a biotinylated centromeric probe for chromosome 15 (yellow) counterstained with 4′,6-diaminido-2-phenylindole. Red rhodamine signal for KGF gene probe was observed on both chromosomes 15.

Figure 2

(a) Digital images of normal human chromosomes hybridized with a biotinylated p7–6 cosmid probe comprising exons 2 and 3 counterstained with propidium iodide. Chromosomes 2, 9, 15, 18, and 21 exhibit double symmetrical fluorescent signals. Chromosomes 9 and 18 have signals on both short and long arms and chromosomes 21 have two pairs of signals on the long arm. (b) The same metaphase as in a showing G-like banding pattern generated by contrast enhancement and LUT (look-up table) inversion of the image of 4′,6-diamidino-2-phenylindole-restained chromosomes. The resolution of the chromosome bands permits an accurate individual chromosome identification as well as the regional localization of the fluorescent signal. (c) Orangutan chromosomes hybridized with a biotinylated p7–6 cosmid probe. Two chromosome pairs exhibit hybridization signal on the long arm. (d) The same metaphase as in c rehybridized with human chromosomes 15 (biotin-labeled, yellow) and 21 (digoxigenin-labeled, red) probes for chromosome identification. These probes painted the orangutan chromosomes homologous to human 15 and 21. The long arm of chromosomes 15 and the distal segment of the long arm of chromosome 22 where KGF sequences are located are painted. (e) Gibbon chromosomes hybridized with a biotinylated p7–6 probe (yellow signal) and a digoxigenin-labeled chromosome 21 probe. Chromosome 15 in gibbon has homology with human chromosomes 15 and 21. Two chromosomes 15 exhibit fluorescent signal on the distal part of the long arm corresponding to human chromosome 21 as shown by painting (red).

Figure 3

Comparable sized digital images of human (HSA), chimpanzee (PTR), gorilla (GGO), and orangutan (PPY) single chromosomes showing hybridization fluorescent signals with corresponding idiograms on their left side. The position of KGF hybridization signal on the idiograms is indicated by red arrows.