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PubMed1975

Formaldehyde as a probe of DNA structure. I. Reaction with exocyclic amino groups of DNA bases.

A comprehensive description is given of both the equilibrium and the kinetic aspects of the reaction of formaldehyde with the exocyclic amino groups of derivatives of adenine, cytosine, and guanine; the results extend previous data in the literature to the point where formaldehyde can now be used as a quantitative probe of DNA structure and dynamic behavior. The main results are: (i) the reaction product is proven (by isolation followed by nuclear magnetic resonance (NMR) spectroscopy) to be a hydroxymethyl group; (ii) a dihydroxymethyl adduct is shown to exist at high formaldehyde concentrations; (iii) equilibrium constants at 25 degrees for forming the monoadduct with adenine and cytosine compounds are about 12 (M-1), while those for forming the dihydroxymethyl adduct are about 0.4 (M-1); (iv) the standard enthalpies for forming the monoadducts with adenine and cytosine compounds are about minus 4 to minus 6 kcal/mol; (v) indirect evidence is presented suggesting that a monohydroxymethyl group on adenine or cytosine derivatives exists preferentially as that rotational isomer which blocks Watson-Crick hydrogen bonding; (vi) in derivatives of guanine, it is shown that the N-1 endocyclic imino group can react with formaldehyde, as well as the amino group, the overall equilibrium constant being about 6 (M-1); (vii) all rate constants are reported, as well as their response to temperature, pH, and various solvent additives known to perturb DNA structure; (viii) using a series of substituted anilines, a linear free energy relation is obtained between the logarithm of both the forward and the reverse rate constant for the formaldehyde reaction and the amine pK, over a range of 10-8 change in amie basicity; (ix) using this relation, the pK's for protonating the nucleoside amino groups are estimated to lie in the range of minus 2 to minus 4; (x) a reaction mechanism is proposed; and (xi) some implications of these results forpolynucleotide studies are discussed.

Adenine↗
PubMed1975

Formaldehyde as a probe of DNA structure. II. Reaction with endocyclic imino groups of DNA bases.

We describe the equilibrium and kinetic aspects of the formaldehyde reaction with the endocyclic imino groups of derivatives of thymine, uracil, and a series of halogenated uracils, as well as poly(uridylic acid) and poly(inosinic acid). The main results are: (i) the equilibrium constants for forming a hydroxymethyl adduct remain quite constant at about 2-2.5 (M-1) for all the compounds studied, independent of their pK; (ii) both forward and reverse rate constants with 5'-TMP are specific base catalyzed in the pH range of about 4-9; (iii) the response of the rate constants to temperature and to several solvent additives are measured; (iv) at neutral pH, for the series of pyrimidine compounds, a linear free energy relation is observed between the logarithm of both the forward and the reverse rate constant and the pK for deprotonation; (v) the unstructured polynucleotides, poly(U) and poly(I) react very similarly to their constituent monomers; (vi) a reaction mechanism is proposed; and (vii) some implications for polynucleotide studies are discussed. In an appendix, a method of spectral analysis is derived to obtain accurate estimates of the quite small equilibrium constants; this should be applicable to all similar two-component systems in which the final product is unobtainable, either by isolation or by saturation. Together with the results of the previous paper on the formaldehyde reaction with exocyclic amino groups (J. D. McGhee and P. H. von Hippel, preceding paper), these results form a reasonably comprehensive account of the basic chemical controls required to use formaldehyde as a quantitative probe of DNA structure.

Chemical Phenomena↗
PubMed1977

Association of e antigen with Dane particle DNA in sera from asymptomatic carriers of hepatitis B surface antigen.

Sera containing hepatitis B surface antigen from 30 asymptomatic blood donors were assayed for e antigen (HBeAg) and antibody to e antigen (anti-HBe) by rheophoresis,. Fourteen samples (47%) had detectable HBeAg, ten (33%) had anti-HBe, and six (20%) had neither. DNA was extracted from 26 of these sera and assayed for its ability to anneal to a [32P]-DNA probe that is a copy of Dane particle DNA. All 10 HBeAg-positive samples tested contained DNA that formed specific hybrids with the DNA probe, as did one of 10 anti HBe-positive samples. Hybridization was not detected in nine sera containing anti-HBe and six sera without HBeAg or anti-HBe. Because the Dane particle is thought to be the hepatitis B virus, this association between HBeAg positivity and Dane particle DNA strongly supports the hypothesis that e antigen is a marker of the presence of the virus and, consequently, potential infectivity.

Antibodies, Viral↗
PubMed1977

Virus-specific RNA in selected cell lines derived from RSV-induced tumour.

The presence of virus-specific RNA in mammalian tumour cell lines of RSV aetiology was studied using molecular hybridization between the PR-RSV 3H-DNA probe synthetized by the endogenous RNA-directed DNA polymerase reaction and cellular RNA. It was found that both virogenic XC cells harbouring the full PR-RSV genome and cryptovirogenic RVP3 cells harbouring an incomplete PR-RSV genome contain virus-specific RNA. The degree of homology between such RNA and PR-RSV 3H-DNA probe reached 10--12% in XC cells and 28--30% in RVP3 cells. It was found that RVP3 cells contain larger amounts of virus-specific RNA than XC cells and that it is localized in the gradient zone corresponding to 20--25S.

Animals↗
PubMed1975

Terbium as a fluorescent probe for DNA and chromatin.

Terbium reacted with DNA and chromatin to form a complex in which terbium acted as a sensitive fluorescent probe. By measuring the narrow-line emission of Tb-3+ when DNA is selectively excited, the relative amount of Tb-3+ bound to the DNA can be calculated. Terbium was bound to DNA until one Tb-3+ was present for each phosphate group. After this point no more terbium was bound. TbCl3 was bound to chromatin in a linear manner until approximately 0.48 TbCl3 was added for each phosphate group in the chromatin-DNA solution. From these data it appears that 52% of the phosphate groups in chromatin were unavailable for binding. The binding of Tb-3+ to DNA can be reversed by prolonged dialysis against 0.5 M NaCl and chelating agents. The terbium ion is ideal in that it binds DNA tight enough so that completion of the reaction can be assumed but loose enough so that it can be removed by gentle means. Low concentrations of salt (up to 2 mM NaCl) enhance the quantum efficiency. Below pH 3 and above pH 7 the DNA-terbium complex will not form. Between pH 3 and pH 7 the quantum efficiency of the DNA terbium complex increases from either pH to a maximum at pH 5.5 to 5.6. Several biochemical uses for Tb-3+ ion are suggested.

Animals↗
PubMed1978

Amplification of DNA sequences during chicken cartilage and neural retina differentiation.

[3H]BrdUrd-substituted DNA probes were prepared from organ cultures of differentiating chicken neural retina and cell cultures of stage 24 chicken limb buds. Reassociation reactions using the neural retina probe demonstrated amplification of DNA sequences during differentiation of neural retina. This probe also contained sequences present in greater numbers in heart DNA than in DNA from undifferentiated neural retina. Reassociation reactions of both differentiated cartilage and differentiated neural retina DNA with both the neural retina probe and the cartilage probe demonstrated that at least part of the amplified sequences are tissue specific.

Animals↗
PubMed1976

Circular dichroism as a probe of DNA structure inside reconstituted nucleohistones.

Reconstituted nucleohistones were obtained by mixing in given conditions acid extracted histones and eukaryotic DNA. The histone/DNA ratio (w/w) was in the range 0.35 - 0.95. With the four histones (H2A2B) we have been able to obtain subunits (nucleosomes or upsilon-bodies). The variation of cirsular dichroism signal with temperature at 280 nm was measured to follow structural changes of the DNA inside the complex. The true change of ellipticity (see article) of histone-bound DNA regions, is similar for reconstituted nucleohistone and H1-depleted chromatin, and is therefore a physical probe of the presence of nucleosomes.

Animals↗
PubMed1977

Carbodiimide modification of superhelical PM2 DNA: considerations regarding reaction at unpaired bases and the unwinding of superhelical DNA with chemical probes.

Superhelical PM2 DNA I can be modified with N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethyl carbodiimide (CMC). The transition of the sedimentation coefficient uncorrected for buoyant density change (S20,*) vs. % reactivity in terms of base pairs shows the following characteristics. The S20,* increases by 4.5 S units upon 1% modification. There is a plateau in S20,* between 1 and 4% reactivity. The extent of reactivity was determined by buoyant density and 14C radioactive CMC binding measurements. Further reactivity was not explored since Pulleyblank and Morgan's (22) data of S20,* vs. % reactivity from 6 to 34% was previously published. The initial results obtained in this study are complementary to the cited results of the above authors. Consequently, both sets of data taken together represent a complete description of S20,* vs. % reactivity with CMC. It is shown that the model in which superhelical DNA is proposed to contain small intrastrand hairpin regions can be extended to account for the observed transitions in S20,* vs. reactivity.

Carbodiimides↗
PubMed1979

Precise localization of human beta-globin gene complex on chromosome 11.

Cloned DNA probes were used in combination with a panel of five hybrid cell clones containing a series of different terminal deletions in human chromosome 11 to map precisely the human hemoglobin beta and delta chain structural genes contained on this chromosome. The region of deletion in each clone of the panel has been defined by biochemical, immunologic, and cytogenetic markers. DNA from clones containing successively larger terminal deletions was tested with appropriate DNA probes to determine the point on the chromosome at which DNA for these two closely linked hemoglobin genes is deleted. These genes, and by inference the closely linked G gamma and A gamma globin genes as well, have been assigned to the intraband region 11p1205 leads to 11p1208 on the short arm of chromosome 11, an interval containing approximately 4500 kilobases of DNA. The approach appears to have potential for even greater resolution and reasonably wide applicability for gene mapping.

Animals↗
PubMed1978

Hormonal control of mRNA synthesis studied by nucleic acid hybridization.

The regulation of protein synthesis by steroids is thought to be due to hormonal effects primarily on mRNA concentration. Experimental evidence to support this conclusion has come largely from the use of DNA probes complementary (cDNA) to mRNA molecules or by translation of the mRNA in vitro. In this review the experimental procedures involved and the application to hormone action of cDNA hybridization will be reviewed. (1) mRNA concentrations can be assayed in tissue RNA samples by hybridization with radiolabelled complementary DNA probes (cDNA). From the rate of hybridization of an mRNA preparation to a cDNA probe it is possible to estimate specific mRNA concentrations and thereby study their hormonal regulation within tissues of subcellular fractions (2) Rates of synthesis of a specific RNA can be measured by hybridization of pulse-labelled RNA with excess cold cDNA as illustrated in studies of the glucocorticoid induction of MMTV RNA. (3) Hormore-induced alterations of mRNA populations as a whole can be investigated. From the kinetics of hybridization of mRNA with its complementary DNA it is possible to estimate the number of different RNA sequences in tissues and to approximate the number of copies of each sequence per cell. Consequently, by comparing mRNA samples isolated from tissues of different hormonal status it is possible to demonstrate specific hormone-inducible mRNA species and, in some cases, identify their translation products.

Androgens↗
PubMed1979

Distribution of RNA transcripts from structural and intervening sequences of the ovalbumin gene.

A study was made of the function of the intervening sequences in the ovalbumin gene, Radioactively labeled DNA probes for the intervening sequences were prepared and RNA's were isolated from whole cells, nuclei, and polysomes of estrogen-stimulated chick oviducts. The concentrations of messenger RNA (mRNA) transcripts from ovalbumin structural sequences (mRNAov) and transcripts corresponding to intervening sequences were then estimated by hybridization to cloned DNA probes. Oviduct tissue contains approximately 58,000 molecules of mRNAov sequences per tubular gland cell and most of these sequences are present in the cytoplasm. In contrast, there are 200 to 300 molecules of RNA per cell which are transcribed from the intervening sequences of the natural ovalbumin gene and almost all of these are found in the nucleus. The difference in distribution of structural and intervening sequence transcripts suggests that, unlike mature mRNA, the intervening sequences are not preferentially transported to cytoplasmic polysomes.

Animals↗
PubMed1978

Procollagen complementary DNA, a probe for messenger RNA purification and the number of type I collagen genes.

Type I procollagen mRNAs were separated from contaminating low-abundance messenger and nuclear RNAs by chromatography over Sepharose 4B in 0.65 M NaCl at room temperature. All of 27S rRNA and four-fifths of procollagen mRNAs bind to Sepharose under these conditions, while 18S rRNA and about three-fourths of other poly(A)-containing RNAs do not bind. AMV reverse transcriptase was used to prepare complementary DNA to procollagen mRNA at each purification step. Hybridization studies, in RNA excess, were carried out to establish the enrichment at each step both with respect to total RNA and to poly(A)-containing RNA. While "purified" procollagen mRNA preparations still consist of about 50% 27S rRNA, over 80% of cDNA prepared from it back hybridizes to its template at a log of cr0t1/2 of -1.9. This type I procollagen cDNA hybridizes in DNA excess to DNA isolated from chicken erythrocytes and from embryonic chick calvaria at a log c0t1/2 of 3.1, demonstrating that procollagen cDNA is complementary to unique gene sequences in both tissues and that procollagen genes are not reiterated.

Animals↗
PubMed1979

Tumor-specific DNA sequences in human gliomas.

Utilizing the technique of hydroxyapatite chromatography, normal cellular DNAs were used to recycle off the repeat or normal sequences found in [3H]DNA copied off 70S RNA from malignant astrocytomas. The recycled [3H]DNA were then used to hybridize against DNAs from normal human brain tissues and DNAs from malignant astrocytomas or Grade IV astrocytomas. The results indicated the presence of tumor-specific DNA sequences in malignant astrocytomas, absent in normal brain tissues. The percentages were 88% and 7%, respectively. When recycled medulloblastoma 70S[3H]DNA probes were utilized against DNA'S FROM Grade IV astrocytomas and from normal brain, similar results were obtained. The respective percentages of hybridization were 67% and 7%. Thus it would appear that malignant gliomas contain tumor-specific DNA sequences which are not found in normal brain tissues.

Astrocytoma↗
PubMed1978

Impairment of RNA synthesis and its recovery in angelicin photosensitized mammalian cells. A probe for DNA damage and repair.

The template activity of DNA for ribosomal RNA transcription has been investigated in monkey kidney CV-1 cells after angelicin photosensitization in order to monitor the induction of lesions in the DNA and their possible subsequent disappearance, i.e. repair. Separate confluent cultures were submitted to a single angelicin treatment at different time intervals before incubation with [3H]uridine. The labeled RNA prepared from whole cells was analysed by polyacrylamide-agarose gel electrophoresis. The results indicate that: Angelicin monoadditions on DNA constitute transcription-terminating lesions which depress overall RNA synthesis, give rise to shortened RNA chains and modify the expression of transcriptional linked genes. CV-1 cells are able to repair, at least partially, the induced transcription-terminating lesions and progressively recover RNA synthesis with a reversion of the initially observed modifications. The repair seems to be independent of semiconservative DNA synthesis since fluorodeoxyuridine does not affect the recovery of RNA transcription. The present work also confirms the arrangement of rRNA genes in tandem behind a common operator in the order 18--28 S as previously determined in the same cells by a radiological mapping technique and reinforces the potential applicability of transcription analysis to the study of repair processes operating on physically or chemically induced damage in DNA.

Cell Line↗