Examination of chromosome number
In RCC individuals, 87.5% of the chromosomal metaphases had 100 chromosomes (Figure. 1a). In 3nG individuals, 90% of the chromosomal metaphases had 150 chromosomes (Figure. 1b). In 2nCC individuals, 89.5% of the chromosomal metaphases had 100 chromosomes (Figure. 1c). In 3nCC individuals, 94.5% of the chromosomal metaphases had 150 chromosomes (Figure. 1d).
5S rDNA sequences
Three 5S PCR fragments (approximately 200, 340, and 500 bp) were amplified from the genomes of RCC, 3nG, 2nCC and 3nCC (Figure. 2). Based on the BLASTn analysis, all fragments from RCC, 3nG, 2nCC and 3nCC were confirmed as 5S rDNA repeat units, comprising a complete 5S rDNA region and a whole NTS region (Figure. 3). In RCC, three monomeric 5S rDNA (designated class I, class II and class III) were characterized by distinct NTS types (designated NTS-I: 83-bp, NTS-II: 220-bp and NTS-III: 357-bp). 3nG had three monomeric 5S rDNAs inherited from RCC (Figure. 4a and Figure. 5a-c). The coding region similarities (class I, class II and class III) between RCC and 3nG were 98.3%, 100% and 99.1%, respectively. In 2nCC, three monomeric 5S rDNAs (designated class IV, class V and class VI) possessed three NTS types (designated NTS-IV: 83-bp, NTS-V: 220-bp and NTS-VI: 370-bp). 3nCC had three monomeric 5S rDNAs, which were completely from 2nCC (Figure. 4b) and Figure. 5d-f). The coding region similarities (class IV, class V and class VI) between 2nCC and 3nCC were 97.5%, 100% and 100%, respectively. The accession numbers of 5S rDNA sequences in GenBank are listed in Table 1.
Fluorescence in situ Hybridization
The 5S rDNA probe (class I: 203 bp) hybridized with the metaphase chromosomes of RCC and 3nG. The 5S rDNA probe (class IV: 203 bp) hybridized with the metaphase chromosomes of 2nCC and 3nCC. The FISH results showed that eight 5S rDNA gene loci were present in 2nCC and RCC (Figure. 6a, c, respectively; Table 2). Twelve 5S rDNA gene loci were present in 3nG and 3nCC (Figure. 6b, d, respectively; Table 2).
The 5S rDNA probe (class II: 340 bp) hybridized with the metaphase chromosomes of RCC and 3nG. The 5S rDNA probe (class V: 340 bp) hybridized with the metaphase chromosomes of 2nCC and 3nCC. The FISH results showed that four 5S rDNA loci were present in 2nCC and RCC (Figure. 7a, c, respectively; Table 2). Six 5S rDNA loci were found in 3nG and 3nCC (Figure. 7b, d, respectively; Table 2).
The 5S rDNA probe (class III: 477 bp) hybridized with the metaphase chromosomes of RCC and 3nG. The 5S rDNA probe (class VI: 490 bp) hybridized with the metaphase chromosomes of 2nCC and 3nCC. The FISH results showed that eight 5S rDNA gene loci were identified in 2nCC and RCC (Figure. 8a, c, respectively; Table 2). Twelve 5S rDNA gene loci were identified in 3nG and 3nCC (Figure. 8b, d, respectively; Table 2).