Long Pyrimidine Tracts of L-Cell DNA: Localization to Repeated DNA

Abstract

Like HeLa DNA, L-cell DNA contains a significant number of unexpectedly long pyrimidine tracts. 1.3% of the thymidine residues of L-cell DNA are found in these long polypyrimidine tracts. Analysis of L-cell DNA fractions with different rates of reassociation indicates that polypyrimidine tracts are associated with "repeated" DNA. Longer pyrimidine tracts appear to have a higher repetition frequency than.shorter tracts, and some may be repeated as many as 5 X 104 times in L-cell DNA.Limited treatment of DNA with acid results in the release of purine bases and the breakage of the phosphodiester backbone. Since pyrimidine nucleotides are quite stable to this treatment they appear as nucleotides and a series of pyrimidine-containing oligonucleotides (1). When HeLa (human) DNA was degraded in this way, unexpectedly long pyrimidine tracts were found, in addition to the usual products of digestion (2). Both the size and number of these tracts differed greatly from that predicted assuming a random distribution of pyrimidines. Their unusual nature led us to examine DNA from a variety of eukaryotes, and similar polydeoxynucleotides were detected in several samples (Birnboim & Straus, manuscript in preparation). In particular, mouse L-cell DNA was found to contain more of these sequences than did other DNA's, and it was therefore chosen for further study. In the present report, we have utilized hydioxyapatite chromatography to fractionate L-cell DNA into "unique" and "repeated" sequences and have shown that long pyrimidine tracts are primarily localized in "repeated" DNA. min. This mixture was centrifuged at 3000 X g for 10 min and the aqueous phase was added to a column (2.54-cm diameter) containing 2 g of hydroxyapatite (HTP, Bio-Rad Laboratories). The hydroxyapatite was washed with the 8 M urea-PB solution until the 3H cpm/ml eluting from the column dropped to 1000 cpm/mi, and then with 0.014 M PB until the index of refraction of the effluent equaled that of the eluant.[8H]DNA was eluted with 0.

Enzymic determination of nonrandom incorporation of 5-bromodeoxyuridine in rat DNA

“…The apparent lability of middle repetitive rat DNA to single-strand specific nucleases following mild depurination is unclear. This property may be accounted for by a high A + T content, as was recently described for mouse repeated DNA sequences (Straus and Birnboim, 1974). Long eukaryotic-specific pyrimidine runs in repetitive DNA sequences of sea urchin embryos were recently reported as well (Case and Baker, 1975b).…”

Characterization of polypyrimidines in Drosophila and L-cell DNA

“…Selective Loss of Long Pyrimidine Tracts Following Formic Acid-Diphenylamine Hydrolysis. Pyrimidine tract analyses in this and earlier experiments (Birnboim et al, 1973;Straus and Birnboim, 1974) were carried out using the Burton (1967) procedure for hydrolyzing DNA. Short pyrimidine tracts represent the bulk of the nucleotide material in such hydrolysates, and they may readily be freed of formic acid and diphenylamine by ether extraction (Burton, 1967; Table I).…”

“…The procedures for isolating L-cell [3H]thymidine-labeled and unlabeled DNA have been described elsewhere (Straus and Birnboim, 1974). The specific activity of the labeled DNA was 250,000 dpm/^g.…”

“…The frequency distribution of pyrimidine isostichs in this size range has been examined for several DNA samples and small differences from random' have been reported (Bellett et al, 1972;Sneider, 1971;Tate and Peterson, 1974). We have recently analyzed HeLa cell DNA and L-cell DNA to see if longer pyrimidine tracts are present (Birnboim et al, 1973;Straus and Birnboim, 1974). Since fragments of this size might be lost on an ion-exchange column, polyacrylamide gel electrophoresis was used for size separation.…”

Electron microscopy of DNA crosslinked with trimethylpsoralen: a probe for chromatin structure

“…Portions of the mouse genome that contain substantial polypyrimidine stretches are expected to form larger uncross-linked regions than predicted by the random theory. However, long pyrimidine tracts and their complementary purine tracts comprise only 1-1.5% of the mouse genome (Straus and Birnboim, 1974), not enough to noticeably shift the Me3psoralen cross-linking distributions.…”