Analysis of Long Pyrimidine Polynucleotides in HeLa Cell Nuclear DNA: Absence of Polydeoxythymidylate

Abstract

HeLa cell nuclear DNA contains an unexpectedly large amount of long pyrimidine polynucleotides.These sequences were detected in DNA labeled with ['HI thymidine by treatment with formic acid-diphenylamine and subsequent analysis by polyacrylamide gel electrophoresis. About 0.5% of the total thymine residues were found in polynucleotides that migrated more slowly than 4S RNA. No polynucleotides of comparable size were detected in Escherichia coli DNA. The pyrimidine polynucleotides contained no pure poly(dT) sequences (less than 0.0015% of the total residues), as judged by thermal chromatography of a complex formed with poly(A).When high molecular weight DNA is treated with acid under relatively mild conditions (1, 2), purine bases are selectively removed, the phosphodiester backbone is broken, and pyrimidine clusters having the general formula, p(Pyp)5, are released. The largest pyrimidine oligonucleotides previously isolated from animal cell DNA contain 15-20 nucleotides (3)(4)(5). Small amounts of longer polynucleotides have been detected but not defined. Because relatively long polyadenylate segments (6-11) and uridylate-rich regions (12) have been found in cellular RNA, we have examined pyrimidine polynucleotides in HeLa cell nuclear DNA to see if corresponding sequences could be detected. Pyrimidine polynucleotides were separated according to chain length by polyacrylamide gel electrophoresis and, unexpectedly, a large amount of long polynucleotides was found. Since their size and amount differ very greatly from that predicted assuming a random distribution of pyrimidines, they may represent sequences of special significance in the genome. When annealed with commercial poly(A), almost no pure poly(dT) sequences could be detected. MATERIALS AND METHODSPreparation of ['HIDNA. HeLa cells used in these experiments were grown in spinner culture in Eagle's minimal essential medium with 5% calf serum without antibiotics. 400 ml of cell suspension (3 X 105/ml) were incubated with [methyl-'H ]thymidine at a concentration of 1 gCi/ml, 0.5 AM, for 24 hr. Nuclei were isolated as described by Penman (13); the nuclear pellet was suspended in 0.5 ml of hypotonic buffer (10 mM NaCl-10 mM Tris HCl-3 mM MgCl2) and rapidly mixed with 8 ml of lysing solution [1 M LiCl, 0.1 M EDTA, 0.1 M Tris HCl (pH 8.0), and 1% Na dodecyl sulfate]. The suspension was heated at 600 for 5 min, then cooled to room temperature and passed twice through a no. 26 needle to reduce its viscosity. The preparation was deproteinized by shaking at room temperature for 30 min with an equal 2189 volume of chloroform containing 0.1 ml n-octanol. The aqueous phase was recovered by centrifugation and the procedure was repeated three times more. DNA was precipitated with ethanol and dissolved in 2 ml of a solution containing 10 mM NaCl, 1 mM EDTA, 1 mM Tris -HCl (pH 7.4), and 0.2% Na dodecyl sulfate. NaOH was added to 0.15 N, and the DNA was heated at 600 for 10 min to hydrolyze RNA. The sample was neutralized with 3 N acetic acid, and the DNA was pre...

Ending the message: poly(A) signals then and now

“…RNase digestion (pancreatic RNase cuts at C and U residues, while T1 RNase cuts at G) of this mRNA preparation revealed a resistant fraction presumed to be poly(A) (Lim and Canellakis 1970;Edmonds et al 1971;Adesnik et al 1972;Mendecki et al 1972;Birnboim et al 1973). Since long poly(A) tracts were not thought to be DNA-templated (Birnboim et al 1973;Jelinek et al 1973), a poly(A) polymerase was sought and found that was subsequently shown to be responsible for poly(A) tail formation on mRNA (Winters and Edmonds 1973a,b). The function of mRNA poly(A) could only be guessed at in these initial studies.…”

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

“…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.…”

“…Ninety percent formic acid (1.5 ml) -2.7% diphenylamine was added and the mixture was incubated for 18-20 hr at 300 in a darkened, Teflon-stoppered tube. Formic acid and diphenylamine were removed by a slight modification of our previous procedure (2). One milliliter of solution containing 10 M urea, 1 M sodium acetate, and 100 lig of tRNA was added to the hydrolysis mixture and it was extracted three times with 5 ml of diethyl ether.…”

“…The precipitate was dissolved in 0.2 ml of the acetate-Tris-CDTA solution. After a further precipitation with ethanol, nucleotide material was analyzed by polyacrylamide gel electrophoresis as described previously (2).…”

Transcription of Polydeoxythymidylate Sequences in the Genome of the Cellular Slime Mold, Dictyostelium discoideum

“…There is evidence that these poly(A) sequences are not encoded in DNA, but are added post-transcriptionally: (a) cordycepin blocks synthesis of poly(A) and appearance of mRNA into the cytoplasm, but does not inhibit synthesis of HnRNA (5,7,8,11,12); (b) the number and size of poly(dT) sequences in mammalian nuclear DNA has been determined by isolation of polypyrimidine sequences in DNA after depurination; these poly(dT) sequences could account for only a very small fraction of the poly(A)200 sequences in mRNA (13); and (c) adenovirus type-2 mRNA contains poly(A) sequences of 100-200 bases, yet adenovirus DNA contains no poly(dT) sequences of equivalent size detectable by RNA DNA hybridization (14).…”