Physical mapping of chromosomes VII and XV ofSaccharomyces cerevisiae at 3·5 kb average resolution to allow their complete sequencing

Abstract

The high resolution complete physical maps of chromosomes VII and XV were constructed to form the basis for sequencing these chromosomes as part of the European systematic sequencing programme of the yeast genome, using a unique cosmid library from strain FY1679, and an original top‐down mapping strategy involving I‐Sce I chromosome fragmentation. A total of 138 and 196 cosmid clones were used to construct the maps for VII and XV, respectively, forming two unique contigs that cover the entirety of chromosomes (1091 kb each), except the telomeric repeats. Colinearity of the cosmid inserts with yeast DNA was verified, and the physical maps were eventually compared with the independently generated genetic maps. © 1998 John Wiley & Sons, Ltd.

Eucaryotic genome evolution through the spontaneous duplication of large chromosomal segments

“…We used DNA combing to map precisely the intrachromosomal segmental duplications within chromosome XV (Bensimon et al , 1994; Michalet et al , 1997). DNA from five recombinant cosmid clones (Tettelin et al , 1998), whose inserts are localized in the duplicated regions, were labeled either with biotin or digoxigenin and detected by red or green fluorochromes, respectively. Joint hybridization of these five probes onto stretched DNA from the WT strain led to an alternation of red and green signals with a defined interval length between them (Figure 2).…”

“…Direct tandem organization of an intrachromosomal duplicated segment visualized by molecular combing. Molecular combing of the genomic DNA of the parental strain (top) and of YKF1022 (bottom) performed with fluorescent probes derived from recombinant cosmids 130, 1338, 488, 323 and 183 of chromosome XV (Tettelin et al , 1998). Red and green colors correspond to biotin‐ and digoxigenin‐labeled probes, respectively.…”

“…In situ hybridization of fixed DNA was performed using cosmids from the right arm of chromosome XV as probes. For both strains, cosmids 130, 1338, 488, 323 and 183 (Tettelin et al , 1998) were labeled either with biotin‐dUTP (1338, 488 and 183) or dig‐dUTP (130 and 323). Signals were amplified by several consecutive layers of fluorophore‐conjugated antibodies and finally detected with Texas red (for biotin probes) or FITC (for digoxigenin probes).…”

“…html). Among 191 deletant strains expected to be slow growing, 134 presented no growth defect on a rich medium (Tettelin et al, 1998). Red and green colors correspond to biotin-and digoxigenin-labeled probes, respectively.…”

Histone H2A.Z has a conserved function that is distinct from that of the major H2A sequence variants

“…To clone the yHTZ1 gene, a 2.3 kb yHTZ1 fragment was amplified with primers yH2A.Z-1 (CGGAATTCTTTCTCCC-CTTCTCTTAC) and yH2A.Z-2 (CCCGGAATTCCTTGCT-TTTAGTCCTCTT), digested with NsiI and SpeI and the resulting 1.2 kb fragment was ligated to the PstI and SpeI sites of pBSIISKto make pJJ1. A longer 1.6 kb yHTZ1 fragment was amplified from cosmid pEOA215 (13), which contains a 29 kb fragment from chromosome XV that includes yHTZ1, using oligonucleotides yH2A.Z-1 and yH2A.Z-20 (GGTAT-GCTCGAGTTGCAACGCACAAAGCTT). This PCR product was blunt end ligated to the SmaI sites of both pBSISK + and pRS313 ( 14), a HIS3 CEN6 ARSH4 plasmid, to make pJJ42 and pJJ43, respectively.…”