Fig. 4

Observed aberrations of an extended neochromosome (nChr 2) in wild-type cells (a) The shorter (p) arm of nChr 1 was extended with non-coding (nc)DNA and a second ARS, added by cloning components into precursor v1 to create v2. (b) Agarose gel showing I-SceI digestion products for eDA146 (control) and eDA155. (c) Validation of nChr 2 by PCR before K. phaffii transformation (for oligonucleotides sets see Additional file 2: Table 1). (d) De novo assembly analysis of the WGS for the resultant strains (yDA174 and yDA175) revealed a preponderance of fused chromosomes indicating instability (see Fig. 5 and Additional file 1: Figs. S7 and S8). Strain yDA174 carries a triple-fusion between a nChr 2 q arm (i.e. lacking its p arm), another nChr 2 q arm; and a copy of nChr 2 lacking the p arm telomere; the junction formed between q arms involves the inverted-repeat regions (IRs, 99% sequence identity) of the centromeres but it was not possible to ascertain the nature of this fusion event. In strain yDA175 an additional copy of nChr 2, lacking its p arm telomere, appears fused to the triple-fusion structure seen in yDA174.