Structure and organization of the rDNA intergenic spacer in lake trout (Salvelinus namaycush)

A total-genomic cosmid library was created to isolate complete copies of th e rDNA cistron of lake trout (Salvelinus namaycush) in order to study the s tructure and organization of the intergenic spacer (IGS) in this species. A total of 60 rDNA-positive clones (average inserts > 25 kb) was recovered b y screening the library with a rDNA-specific probe. Positive clones were as sayed for the presence of the two internal rDNA spacers (ITS-1 and ITS-2) a nd the entire IGS fragment was successfully amplified from 42 clones by PCR . Length of the IGS fragments ranged from 9.4 to 17.8 kb. Comparative restr iction mapping of the IGS-PCR products of several clones indicated two regi ons of extensive length variation surrounding a central region with sequenc e conservation. DNA sequence analysis was used to investigate the molecular basis of the IGS length variation and focused on identifying the region re sponsible for this variation. Over 9 kb of DNA sequence was obtained for on e clone (A1) with a total IGS length of approximately 12.4 kb. Sequence of a conserved central region contained two open reading frames and a number o f short direct repeats. Length variation in the IGS was determined by RFLP to result from differences in the number of copies of repetitive DNA sequen ces. These included an 89-bp tandem repeat (alpha repeats), an 82-bp elemen t (beta repeats), a 168-177-bp element (S repeats), and a 179-201-bp elemen t (delta repeats). Overall nucleotide composition of the IGS was biased tow ards A and T (%GC = 47.4). Maintenance of discrete rDNA-length variants in lake trout suggests that the rate of gene conversion is insufficient to pro duce homogeneous copies across the genome.