Lack of specific correlation of the deoxycytidine triphosphate pool level with rate of DNA synthesis.

The levels of the four deoxyribonucleoside triphosphate pools and the distribution of cells in the various phases of the cell cycle have been examined in Chinese hamster cells as thymidine, present as a regular constituent in the growth medium, was removed in stages. The results indicate that: 1. Duration of the DNA synthetic phase was lengthened when thymidine was removed from the growth medium. 2. Temporally correlated with lengthening of the DNA synthetic phase upon thymidine removal was a 7-fold increase in level of the dCTP pool, reduction in the dGTP pools, and little or no change in dATP pool. 3. Radioactive labeling procedures indicated that expansion of the dCTP pool could be completely accounted for by increased ribonucleotide reductase activity and that the dTTP pool switched from a largely exogenous thymidine source to endogenous dTTP synthesis as the extracellular thymidine concentration was reduced. 4. Deoxyuridine and thymidine were apparently transported by the same system in Chinese hamster cells, while deoxycytidine was transported by a different system. Although deoxycytidine transport was unaffected by thymidine, phosphorylation of intracellular deoxycytidine compounds to the triphosphate level was stimulated by thymidine. Cytidine transport was not significantly affected by thymidine.

Terminal deoxynucleotidyl transferase in the diagnosis of leukemia and malignant lymphoma.

Neoplastic cells from 253 patients with leukemia and 46 patients with malignant lymphoma were studied for the presence of terminal deoxynucleotidyl transferase (TdT) by biochemical and fluorescent antibody technics. TdT was detected in circulating blast cells from 73 of 77 patients with acute lymphoblastic leukemia, 24 of 72 patients with chronic myelogenous leukemia examined during the blastic phase of the disorder and in cell suspensions of lymph nodes from nine of nine patients with diffuse lymphoblastic lymphoma. Blast cells from six of 10 patients with acute undifferentiated leukemia were TdT positive, but the enzyme was found in only two of 55 patients with acute myeloblastic leukemia. TdT was not detected in other lymphocytic or granulocytic leukemias or in other types of malignant lymphomas. The fluorescent antibody assay for TdT permits rapid and specific identification of the enzyme in single cells. The TdT assay is clinically useful in confirming the diagnosis of acute lymphoblastic leukemia, evaluating patients with blastic chronic myelogenous leukemia, and distinguishing patients with lymphoblastic lymphoma, whose natural history includes rapid extranodal dissemination, from patients with other poorly differentiated malignant lymphomas.

Enzymatic elongation of microsatellite oligomers for use in direct-label chemiluminescent hybridizations.

Short, synthetic oligonucleotide sequences representing microsatellites and other short tandem repeats can be elongated (concatamerized) using a simple method in which complementary strands are annealed, phosphorylated, primer extended and ligated. When used in direct-label chemiluminescent hybridizations, the elongated microsatellite sequences provide an approximately 30-fold increase in signal strength compared with microsatellite oligomers that have not been concatamerized. Concatamerization of simple repeat oligomers further enables the use of relatively short oligonucleotide sequences in direct-label chemiluminescent hybridization experiments, thereby reducing the overall need for radioisotopes in certain commonly performed laboratory procedures such as DNA fingerprinting and selection of clones containing microsatellite sequences.

Sequence-dependent conformational variations in the B-DNA double-helix of poly d(AATT).poly d(AATT).

X-ray diffraction data from well oriented and polycrystalline fibers of the lithium salt of poly d(AATT).poly d(AATT) are isomorphous with those from B-DNA. The double-helix consists of conformationally identical antiparallel strands and the molecular symmetry is 2 5(2); the asymmetric unit is a tetranucleotide, AATT, and 5 tetranucleotides span two turns per strand. Two double helices pass through a monoclinic unit cell of dimensions a = 31.05, b = 22.62, c = 33.85 A (fiber axis) and gamma = 90 degrees. In each repeating motif, the four nucleotides have distinct conformations, TpA displays an axial P-O bond and there is shortening of minor groove in the central region.

High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules.

We are developing a laser based technique for the rapid sequencing of large fragments (approximately 40 kb) of DNA based upon the detection of single, fluorescently tagged nucleotides cleaved from a single DNA fragment. We have demonstrated significant progress on several of the important steps of this technique. The projected rate of sequencing is several hundred bases per second which is orders of magnitude faster than existing methods. Once developed, this technology could be utilized by investigators for rapid sequencing of genetic material from virtually any source.

Reactions induced in vitro between model DNA and benzo[a]pyrene by near-ultraviolet radiation.

Near-ultraviolet (300--480 nm wavelength) irradiation of the single-strand polydeoxynucleotide poly[d(A,C,G,T)] and carbon-14 labeled benzo[alpha]-pyrene (B[alpha] P) in aqueous dimethylsulfoxide (DMSO) solution led to appreciable binding of labeled hydrocarbon to the polynucleotide. Nuclease digests of polydeoxynucleotide-B[alpha]P complexes were examined by chromatography on Sephadex LH-20; at high fluences of near-ultraviolet light deoxyguanosine (dG) residues of the polymer were largely destroyed when the hydrocarbon was present. Approximately 85% of the B[alpha] P of the digests were recovered as hydrophilic derivatives not adsorbed by Sephadex LH-20. Elution of the columns with an aqueous-methanol gradient indicated that substances similar to the covalent deoxynucleoside-B[alpha] P adducts formed between microsomally-oxidized B[alpha] P and DNA were likewise present in the digests. When the deoxyadenosine (dA), deoxycytidine (dC) or dG moieties of the polymer were tritium-labeled, substances doubly-labeled with tritium and carbon-14 were found; ratios of the two radioactivities indicated that equimolar amounts of deoxynucleoside and hydrocarbon were present.

Evidence of 2-aminopurine-cytosine base mispairs involving two hydrogen bonds.

It is shown that the mutagen base analogue 2-aminopurine is hydrogen-bonded at its 1-ring position when annealed with cytosine in DNA. The presence of stably hydrogen-bonded regions proximal to the 2-aminopurine-cytosine base mispair is a prerequisite for the occurrence of two hydrogen bonds coupling the bases at their 1-3- and 2-2-positions. We consider the possibility that, in the resulting heteroduplex base mispair, 2-aminopurine or cytosine may be present as a disfavored imino tautomer.

CD of six different conformational rearrangements of poly[d(A-G).d(C-T)] induced by low pH.

On the basis of circular dichroism (CD) data, we have now identified six different conformational states (other than the duplex) of poly[d(A-G).d(C-T)] at pH values between 8 and 2.5 (at 0.01M Na+; 20 degrees C). Three of these structural rearrangements were observed as the pH was lowered from 8 to 2.5, and three additional rearrangements were observed as the pH was raised from 2.5 back to neutral pH. The major components of the six conformational states were defined using appropriate combinations of the CD spectra of the duplex, triplex, and denatured forms of this polymer, as well as the CD spectra of the individual single strands and their respective acid-induced self-complexes. Our results show that the acid-induced rearrangements of poly[d(A-G).d(C-T)] include not only the poly[d(C+-T).d(A-G).d(C-T)] triplex, but also include the poly[d(C-T)] loop-out structure and a self-complexed form of the poly[d(A-G)] strand that is pH-dependent.

CD of homopolymer DNA-RNA hybrid duplexes and triplexes containing A-T or A-U base pairs.

CD spectra and difference-CD spectra of (a) two DNA X RNA hybrid duplexes (poly[r(A) X d(U)] and poly[r(A) X d(T)]) and (b) three hybrid triplexes (poly-[d(T) X r(A) X d(T)], poly[r(U) X d(A) X r(U)], and poly[r(T) X d(A) X r(T)]) were obtained and compared with CD spectra of six A X U- and A X T-containing duplex and triplex RNAs and DNAs. We found that the CD spectra of the homopolymer duplexes above 260 nm were correlated with the type of base pair present (A-U or A-T) and could be interpreted as the sum of the CD contributions of the single strands plus a contribution due to base pairing. The spectra of the duplexes below 235 nm were related to the polypurine strands present (poly-[r(A)] or poly[d(A)]). We interpret the CD intensity in the intermediate 255-235 nm region of these spectra to be mainly due to stacking of the constituent polypurine strands. Three of the five hybrids (poly[r(A) X d(U)], poly[r(A) X d(T)], and poly[d(T) X r(A) X d(T)]) were found to have heteronomous conformations, while poly[r(U) X d(A) X r(U)] was found to be the most A-like and poly[r(T) X d(A) X r(T)], the least A-like.

Circular dichroism spectra of DNA oligomers show that short interior stretches of C.C+ base pairs do not form in duplexes with A.T base pairs.

Circular dichroism (CD) experiments were carried out on a series of DNA oligomers to determine if short internal stretches of protonated cytosine-cytosine (C.C+) base pairs could coexist with adenine-thymine (A.T) base pairs. (1) C.C+ base pairs did form in the absence of A.T base pairs in the individual oligomers d(AACC)5 and d(CCTT)5, as indicated by the appearance of a long-wavelength CD band centered at 282-284 nm, when the pH was lowered to 6 or 5 at 0.5 M Na+. A comparison of measured with calculated spectra showed that d(CCTT)5 at pH 5, 0.5 M Na+, 20 degrees C, likely adopted a structure with a central core of stacked C.C+ base pairs and looped-out thymines. Under the same conditions, it appeared that C.C+ base pairs also formed in d(AACC)5, but with the adenines remaining intrahelical. Each of these oligomers showed a cooperative transition for formation of C.C+ base pairs as the temperature was lowered, with C.C+ base pairs forming at a higher temperature in d(CCTT)5 than in d(AACC)5. A.T base formed in equimolar mixtures of d(AACC)5 plus d(CCTT)5 as monitored by an increase in the negative magnitude of the 250-nm CD band. However, a large increase did not appear at about 285 nm in CD spectra of the mixtures, showing that there were no stacked C.C+ base pairs in the d(AACC)5.d(CCTT)5 duplex even though they formed under the same conditions in the individual strands. Thus, in this duplex, A.T base pairs prevented the formation of neighboring internal C.C+ base pairs. (2) CD measurements were also made of d(A10C4T10).(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of the circular dichroism spectra of synthetic DNA sequences of the homopurine . homopyrimidine and mixed purine- pyrimidine types.

We have obtained the ultraviolet circular dichroism spectra of two repeating trinucleotide DNAs, poly [d(A-G-G).d(C-C-T)] and poly[d(A-A-G).d(C-T-T)], that have all purines on one strand and all pyrimidines on the other. These spectra, together with spectra of other synthetic polymers, can be combined to give 3 first-neighbor calculations of the spectrum of poly[d(A).d(T)] and 2 first-neighbor calculations of the spectrum of poly [d(G).d(C)]. The results show (1) that first-neighbor calculations utilizing only spectra of homopurine.homopyrimidine DNA sequences are no more accurate than are similar calculations that involve spectra of mixed purine-pyrimidine sequences, demonstrating that double-stranded homopurine.homopyrimidine sequences do not obviously belong to a special class of secondary conformations, and (2) that the wavelength region above 250 nm in the CD spectra of synthetic DNAs is least predictable from first-neighbor equations, probably because this region is especially sensitive to sequence-dependent conformational differences.

Circular dichroism measurements show that C.C+ base pairs can coexist with A.T base pairs between antiparallel strands of an oligodeoxynucleotide double-helix.

We have studied the coil-to-helix transition of the DNA oligomer d(C4A4T4C4), using circular dichroism measurements to monitor the formation of A.T base pairs within the central self-complementary A4T4 region and the formation of protonated C.C+ base pairs at the ends of the oligomer. We found that both A.T and C.C+ base pairs formed in a coordinated fashion as the temperature and pH were lowered. The CD data of the helix form of the oligomer were consistent with the presence of paired oligomers, but not with hairpin loops. The pKa for formation of C.C+ base pairs between the C4 ends of the oligomer was higher than the pKa for formation of C.C+ base pairs in d(C8), indicating that the formation of C.C+ base pairs in the oligomer was influenced by the presence of a paired A4T4 region. We conclude that A.T and C.C+ base pairs coexist in the self-complex of the oligomer and, therefore, that C.C+ base pairs can form between antiparallel DNA strands.

CD evidence that the alternating purine-pyrimidine sequence poly[d(A-C).d(G-T)], but not poly[d(A-T).d(A-T)], undergoes an acid-induced transition to a modified secondary conformation.

Circular dichroism and UV absorption data showed that poly[d(A-C).d(G-T)] (at 0.01M Na+ (phosphate), 20 degrees C) underwent two reversible conformational transitions upon lowering of the pH. The first transition was complete at about pH 3.9 and resulted in an acid form of the polymer that was most likely a modified, protonated duplex. The second transition occurred between pH 3.9 and 3.4 and consisted of the denaturation of this protonated duplex to the single strands. UV absorption and CD data also showed that the separated poly[d(A-C)] strand formed two acid-induced self-complexes with pKa values of 6.1 and 4.7 (at 0.01M Na+). However, neither one of these poly[d(A-C)] self-complexes was part of the acid-induced rearrangements of the duplex poly[d(A-C).d(G-T)]. Acid titration of the separated poly[d(G-T)] strand, under similar conditions, did not show the formation of any protonated poly[d(G-T)] self-complexes. In contrast to poly[d(A-C).d(G-T)], poly[d(A-T).d(A-T)] underwent only one acid-induced transition, which consisted of the denaturation of the duplex to the single strands, as the pH was lowered from 7 to 3.