Repetitive DNA sequences in the rat genome and their expression in normal and transformed rat cells
(1) The properties of RNA populations from different Rat-1 cells were examined by neutral agarose gel electrophoresis, denaturing sucrose gradients, poly(U) Sepharose chromatography and hydroxyapatite chromatography. No difference in the size, poly(A) content, or elution profiles was found between the hnRNA populations of normal and transformed Rat-1 cells. (2) Examination of the restriction enzyme pattern of rat liver nuclear DNA revealed the presence of a number of repetitive families. Two major Eco R1 bands, 2360 and 1240 b.p. in length were examined in finer detail by hybridization and restriction enzyme analysis. These two major Eco R1 bands were shown to be related, but not composed of identical sequences. The Eco R1 bands are not complementary to rat satellite -1 DNA or to rDNA sequences. However a relationship to other cryptic satellites of rat liver nuclear DNA was not excluded. (3) Examination of the hybridization patterns of Rat-l cell hnRNA to Rat-1 cell DNA, led to identification of a number of restriction fragments. The detection of the restriction fragments was due to the hybridization of repetitive DNA transcripts or abundant RNA sequences. However, it was not possible to distinguish between repetitive DNA transcripts or abundant RNA sequences. Whilst most of the sequences detected were common to both Rat-1 normal and Rat-1 GE 11 hnRNA populations, one restriction fragment was detected only with Rat-1 normal hnRNA hybridization probe, and one with only Rat-1 GE 11 hnRNA hybridization probe. The Rat-1 normal hnRNA specific fragment was shown to be complementary to the 1240 b.p. Eco R1 fragment of rat liver nuclear DNA. (4) Examination of the hybridization pattern of Rat-1 normal mRNA to Rat-1 cell DNA led to the detection of a number of repetitive DNA transcripts (or abundant RNA sequences). Some of the restriction fragments detected were complementary to those detected by Rat-1 normal and Rat-1 GE 11 hnRNA populations. No restriction fragments were observed in the hybridization pattern of Rat-1 GE 11 mRNA to Rat-1 cell DNA. (5) Hybridization of Rat-1 normal mRNA to rat liver nuclear DNA restriction digests led to the detection of a prominent Hae III fragment, 560 b.p. in length. This prominent Hae III fragment was shown to be relatively homogeneous with respect to base composition and was possibly complementary to the 1240 b.p. Eco R1 fragment from rat liver nuclear DNA. A number of restriction fragments were detected by the hybridization of Rat-1 normal and GE 11 hnRNA populations to restriction digests of rat liver nuclear DNA. Sequences showing complementarity to the Eco R1 fragments 2360 and 1240 b.p. in length were also detected. (6) Construction of recombinant plasmids containing the two Eco R1 bands 2360 and 1240 b.p. in length was attempted; however, this was unsuccessful. A very small bank of recombinant plasmids containing rat liver nuclear DNA sequences was constructed by the use of the DNA Vector, pBR322 and the restriction enzymes, Bam H1 and Ava 1. Some of the clones examined briefly : pBAR 2, 32, 33, 51 and 52 were found to be complementary to Rat-1 normal hnRNA sequences. Sequences showing complementarity to pBAR 2, 32, 33, 35 and 51 were not detected in the Rat-1 GE 11 hnRNA populations. However, sequences complementary to pBAR7 and 52 were detected in Rat-1 GE 11 populations by the use of Grunstein and Hogness filters. pBAR 33, 35 and 32 were shown to contain interspersed repetitive elements from rat liver nuclear DNA. pBAR32, whilst containing an interspersed repetitive element, was complementary to a repetitive element seemingly organised as a tandem repeating array in the rat liver genome. (7) The methylation pattern of Rat-1 cell DNA was examined by the use of Msp 1 and Hpa II restriction enzymes. The DNA from Rat-1 GE 11 was shown to be more heavily methylated at the internal cytosine of the Hpa II site than that from Rat-1 normal DNA. Sequences complementary to pBAR33 were shown to be more heavily methylated in Rat-1 GE 11 and Rat-1 La24 35° DNA compared to that from Rat-1 normal and Rat-1 La24 40° DNA. This may be coupled with the differential expression of pBAR33 in Rat-1 cells.