Topographical analysis of yeast ribosomal DNA by cleavage with restriction endonucleases. II. The physical map of EcoRI fragments

Abstract

Yeast ribosomal DNA (rDNA) was digested with the restriction endonuclease EcoRI. Eight distinct fragments were obtained with a molecular weight of 4.35 (1), 1.75 (2), 1.45 (3), 1.07 (4), 0.42 (5), 0.37 (6), 0.26 (7) and 0.22 x 10(6) (8) daltons, respectively. Except for fragment 1 with a molecular weight of 4.35 x 10(6) daltons, all fragments are derived from the multiple ribosomal transcription units. The 'spacer' sequences, on the other hand, gave rise to digestion products which are very heterogeneous in size. By analysis of the partial digestion products which are very heterogeneous in size. By analysis of the partial digestion products, together with the data obtained by digestion with a combination of two restriction enzymes (EcoRI and Hind II or Hind III) and redigestion of the Hind II-and Hind III-fragments with EcoRI, the physical map of the EcoRI cleavage sites in the ribosomal transcription unit could be established.

Genetic organization of the ribosomal transcription units of the yeast Saccharomyces carisbergensis

“…Although the occurrence of whiskers on either or both members of a pair of R-loops may influence the value of b for that particular pair, the absence of polarity in whisker formation (data not shown) ensures that there is no effect on the mean value of b. The value of b calculated in Table 1 corresponds to a molecular weight of double-stranded DNA of 6.23 ± 0.23x10 D, which is very close to the molecular weight of the RTU determined from renaturation studies (6.2 x 10 D), digestion of rDNA with restriction enzymes (5.9 x 106 D), or the size of the primary transcript (6.2 x 10 D) (1)(2)(3)12). Given the value of b (Table 1) and taking the molecular weight of the RTU as 6.1 x 106 D, two neighbouring RTUs can be separated by at most 0.4 x 10 D of nontranscribed spacer DNA which is equal to about 6% of the size of the RTU.…”

“…Indeed, digestion of rDNA with various restriction enzymes produced a set of distinct fragments, adding up to the length of the RTU, superimposed upon a continuous background of DNA fragments. No partial digestion products exceeding the length of the RTU could be positively identified (2,3). Moreover, the amount of heterogeneous DNA agreed well with the percentage of spacer DNA calculated from the melting studies (1) .…”

Structure and function of the nontranscribed spacer regions of yeast rDNA

“…Each rDNA unit comprises about 9000 bp and encodes two primary transcripts, one for 5S rRNA and one for 37S pre-rRNA (3), separated by two nontranscribed spacers both of which are roughly 1000 bp long (NTS1 and NTS2). Restriction analysis of yeast genomic DNA indicates that the transcription units as well as the spacers are homogeneous in length (4,5). This is in contrast with the length heterogeneity observed for the NTS of rDNA in Vertebrata and Arthropoda (6), which is caused by their internally repetitious nature.…”