Showing papers on "Base pair published in 1979"

Rapid evolution of animal mitochondrial DNA.

Abstract: Mitochondrial DNA was purified from four species of higher primates (Guinea baboon, rhesus macaque, guenon, and human) and digested with 11 restriction endonucleases. A cleavage map was constructed for the mitochondrial DNA of each species. Comparison of the maps, aligned with respect to the origin and direction of DNA replication, revealed that the species differ from one another at most of the cleavage sites. The degree of divergence in nucleotide sequence at these sites was calculated from the fraction of cleavage sites shared by each pair of species. By plotting the degree of divergence in mitochondrial DNA against time of divergence, the rate of base substitution could be calculated from the initial slope of the curve. The value obtained, 0.02 substitutions per base pair per million years, was compared with the value for single-copy nuclear DNA. The rate of evolution of the mitochondrial genome appears to exceed that of the single-copy fraction of the nuclear genome by a factor of about 10. This high rate may be due, in part, to an elevated rate of mutation in mitochondrial DNA. Because of the high rate of evolution, mitochondrial DNA is likely to be an extremely useful molecule to employ for high-resolution analysis of the evolutionary process.

Molecular structure of a left-handed double helical DNA fragment at atomic resolution

Abstract: The DNA fragment d(CpGpCpGpCpG) crystallises as a left-handed double helical molecule with Watson-Crick base pairs and an antiparallel organisation of the sugar phosphate chains. The helix has two nucleotides in the asymmetric unit and contains twelve base pairs per turn. It differs significantly from right-handed B-DNA.

Electron microscopic evidence for the circular form of RNA in the cytoplasm of eukaryotic cells.

Abstract: LINEAR single-stranded RNA genomes extracted from several RNA viruses were found to exist in a circular conformation when examined in the electron microscope in partially denaturing conditions1–4. The circular conformation of viral RNA presumably arises from base pairing between RNA sequences at the two ends of the RNA molecules. The biological significance of the circular structure of single-stranded RNA is still unknown. Here we report the observation of the circular form of RNA extracted from the cytoplasm of several eukaryotic cells.

Helical repeat of DNA in solution.

Abstract: The helical repeat of DNA in solution has been measured directly by analyzing the gel electrophoretic patterns of pairs of covalently closed DNAs with length differences between 1 and 58 base pairs, out of a total length of about 4350 base pairs per DNA molecule. The method is based on the observation that for a covalently closed DNA of a fixed size of n base pairs (n of the order of several thousand), under appropriate conditions, two topological isomers (topoisomers) differing by 1 in their linking numbers are well resolved by gel electrophoresis. If the size of the DNA is increased to n + x base pairs, unless x is an integral multiple of the helical repeat h, the bands of the topoisomers with n + x base pairs per molecule are all shifted relative to the bands of the topoisomers with n base pairs per molecule. The magnitude of the shift is directly related to the nonintegral residual of x/n. Analysis of the set with x ranging from 1 to 58 gives the DNA helix repeat in solution as 10.4 base pairs per turn under physiological conditions, with an estimated probable error of +/- 0.1. This result strongly supports the double helix structure of DNA and rejects the side-by-side model of Rodley et al. [Rodley, G.A., Scobie, R.S., Bates, R.H. T & Lewitt, R.M. (1976) Proc. Natl. Acad. Sci. USA 73, 2959-2963]. The helical repeat of DNA measured in solution is significantly different from the value 10.0 base pairs per turn for the B form fiber structure.

Organisation and sequences at the 5′ end of a cloned complete ovalbumin gene

Abstract: A clone which contains the complete chicken ovalbumin gene, including its leader coding sequences, has been isolated. From electron microscopic analysis of this DNA we conclude that the minimal size of the transcriptional unit for ovalbumin is 7.7 kilobases. The DNA sequence of the region surrounding the 5' end of the ovalbumin gene is presented. Comparison of this sequence with those of other eukaryotic genes reveals striking similarities, possibly related to a promoter region, approximately 30 base pairs upstream from the site coding for the 5' end of the mRNA.

Hypercycles and the origin of life.

Abstract: Perhaps the most difficult step to explain in the origin of life is that from the replication of molecules (RNA for example) in the absence of specific proteins, to the appearance of polymerases and other proteins involved in the replication of RNA and themselves coded for by that RNA. Suppose we start with a population of replicating RNA molecules. Without specific enzymes the accuracy of replication is low and hence the length of RNA which could be precisely replicated small. Before replication can be reasonably accurate, there must as a minimum be a specific polymerase, as well as synthetases and tRNAs, which in turn implies an RNA genome of considerable length. Thus, even if one supposes an initially very limited set of codons, one cannot have accurate replication without a length of RNA, say, 2000 or more base pairs, and one cannot have that much RNA without accurate replication. This is the central problem discussed in a series of papers by Manfred Eigen and Peter Schuster proposing the ‘hypercycle’ as a necessary intermediate stage.

Elution of DNA from agarose gels after electrophoresis.

Abstract: Publisher Summary This chapter discusses the elution of deoxyribonucleic acid (DNA) from agarose gels after electrophoresis. The studies of genome structure and function rely heavily on the isolation and analysis of the defined DNA fragments. Gel electrophoresis is a simple, high-resolution method of separating specific DNA fragments on the basis of size. Agarose gels at concentrations of 0.1–2.5% resolve DNA from 150–880,000 base pairs, whereas acrylamide gels, ranging from 3–20%, afford a good resolution of fragments in the size range of 10–2,000 base pairs. The chapter describes three new or revised methods for the recovery of DNA from agarose gels. The evaluation is based on the simplicity, speed, yield, and amenability of the purified DNA. The first method involves the electroelution of DNA into slots. The second method involves the electroelution of DNA onto dialysis membranes. The third method describes the process of dissolving gel slices in perchlorate solution.

Comparison of total sequence of a cloned rabbit beta-globin gene and its flanking regions with a homologous mouse sequence.

Abstract: The nucleotide sequence of a cloned rabbit chromosomal DNA segment of 1620 nucleotides length which contains a beta-globin gene is presented. The coding regions are separated into three blocks by two intervening sequences of 126 and 573 base pairs, respectively. The rabbit sequence was compared with a homologous mouse sequence. The segments flanking the rabbit gene, as well as the coding regions, the 5' noncoding and part of the 3' noncoding messenger RNA sequences are similar to those of the mouse gene; the homologous introns, despite identical location, are distinctly dissimilar except for the junction regions. Homologous introns may be derived from common ancestral introns by large insertions and deletions rather than be multiple point mutations.

Inverted repeats in chloroplast DNA from higher plants

Abstract: The circular chloroplast DNAs from spinach, lettuce, and corn plants have been examined by electron microscopy and shown to contain a large sequence repeated one time in reverse polarity. The inverted sequence in spinach and lettuce chloroplast DNA has been found to be 24,400 base pairs long. The inverted sequence in the corn chloroplast DNA is 22,500 base pairs long. Denaturation mapping studies have shown that the structure of the inverted sequence is highly conserved in these three plants. Pea chloroplast DNA does not contain an inverted repeat. All of the circular dimers of pea chloroplast DNA are found to be in a head-to-tail confirmation. Circular dimers of spinach and lettuce were also found to have head-to-tail conformation. However, approximately 70-80% of the circular dimers in preparations of lettuce and spinach chloroplast DNA were found to be in a head-to-head conformation. We propose that the head-to-head circular dimers are formed by a recombination event between two circular monomers in the inverted sequence.

Circular dichroism and DNA secondary structure

Abstract: The change in average rotation of the DNA helix has been determined for the transfer from 0.05 M NaCl to 3.0 M CsCl, 6.2 M LiCl and 5.4 M NH4Cl. This work, combined with data at lower salt from other laboratories, allows us to relate the intensity of the CD of DNA at 275 nm directly to the change in the number of base pairs per turn. The change in secondary structure for the transfer of DNA from 0.05 M NaCl (where it is presumably in the B-form) to high salt (where the characteristic CD has been interpreted as corresponding to C-form geometry) is found to be -0.22 (+/- 0.02) base pairs per turn. In the case of mononucleosomes, where the CD indicates the "C-form", the change in secondary structure (including temperature effects) would add -0.31 (+/- 0.03) turns about the histone core to the -1.25 turns estimated from work on SV40 chromatin. Accurate winding angles and molar extinction coefficients were determined for ethidium.

Anisotropic flexibility of DNA and the nucleosomal structure

Abstract: Potential energy calculations of the DNA duplex dimeric subunit show that the double helix may be bent in the direction of minor and major grooves much more easily than in other directions. It is found that the total winding angle of DNA decreases upon such bending. A new model for DNA folding in the nucleosome is proposed on the basis of these findings according to which the DNA molecule is kinked each fifth base pair to the side of the minor and major grooves alternatively. The model explains the known contradiction between a C-like circular dichroism for the nucleosomal DNA and the nuclease digestion data, which testify to the B-form of DNA.

Nucleotide sequence of small ColE1 derivatives: structure of the regions essential for autonomous replication and colicin E1 immunity.

Abstract: A small ColE1 derivative, pAO2, which replicates like the original ColE1 and confers immunity to colicin E1 on its host cell has been constructed from a quarter region of ColE1 DNA (Oka, 1978). The entire nucleotide sequence of pAO2 (1,613 base pairs) was determined based on its fine cleavage map. The sequence of a similar plasmid, pAO3, carrying additional 70 base pairs was also deduced.

Enzyme-catalyzed DNA unwinding: studies on Escherichia coli rep protein.

Abstract: Replication in vitro of the replicative form (RF) I DNA of bacteriophage ϕX174 requires the phage-induced cistron A (cisA) protein, the host rep protein, DNA-binding protein, ATP, and DNA polymerase III plus replication factors. The rep protein is a single-stranded DNA-dependent ATPase. In this paper we show that ϕX174 RF I DNA cut by the cisA protein acts as a duplex DNA cofactor for the rep protein ATPase activity, provided that DNA-binding protein is present. In this latter reaction the duplex DNA is unwound by the rep protein with concomitant hydrolysis of ATP. The extents of ATP hydrolysis, DNA unwinding, and, where appropriate, DNA synthesis are proportional to the amounts of DNA-binding protein present. Two ATP molecules are hydrolyzed per base pair unwound. We propose that the obligatory requirement for the cisA protein in the unwinding of ϕX174 RF I DNA is not simply due to its endonuclease activity but rather is due to its provision of a site for the binding of the rep protein. The rep protein in the presence of DNA-binding protein, but in the absence of cisA protein, unwinds duplex DNA when one strand extends to generate a single-stranded leader region preceding the duplex. We show that rep protein translocates along the leader single strand in a 5′-to-3′ direction only and then invades the duplex DNA. The rep protein shows a directional specificity for translocation and unwinding. A model is presented to explain the mechanism of DNA unwinding catalyzed by the rep protein.

Precise location of DNase I cutting sites in the nucleosome core determined by high resolution gel electrophoresis

Abstract: The precise locations of the DNase I cutting sites in the nucleosome core have been determined by analysis of the DNA products of a DNase I digestion of 32P end-labelled mucleosome cores on a high resolution gel electrophoresis system. This system is capable of resolving fragments of mixed sequence DNA differing by one base into the region of 160 bases in length. The DNase I cutting sites in the core are found to be spaced at multiples of about 10.4 (i.e. clearly different from 10.0) bases along the DNA, but show significant variations about this value. In addition to the location of the sites, the stagger between individual sites on opposite strands has been determined and is found to be inconsistent with at least one proposed mechanism for nuclease cleavage of chromatin DNA. Finally, a calculated distribution of fragment lengths in a DNase I digest of nuclei has been determined from the data obtained from the nucleosome core and found to be in reasonable agreement with the observed distribution. The periodicity of 10.4 is discussed with respect to the number of base pairs per turn of chromatin DNA and the number of superhelical turns of DNA per nucleosome.

Sites of inhibition of in vitro DNA synthesis in carcinogen- and UV-treated phi X174 DNA.

Abstract: THE availability of DNA molecules of known sequence and rapid methods for the determination of the sequence of particular DNA fragments1–3 make it possible to investigate the molecular action of mutagens and carcinogens more closely4,5. For example, there is considerable evidence that some types of lesion in DNA such as UV light-induced pyrimidine dimers and various chemical adducts are blocks to DNA synthesis both in vivo6–9 and in vitro10,11. The available data support the hypothesis that the block occurs at the level of the lesion on the template strand10,11. We have used DNA containing such lesions on a template of known sequence for in vitro DNA synthesis by DNA polymerase 1 to investigate the site of the block at the level of the nucleotide sequence. This method allows us to determine both the site at which lesions capable of blocking synthesis by DNA polymerase occur and exactly where synthesis stops in relation to the lesion.

Nucleotide sequence of Escherichia coli K-12 replication origin.

Abstract: From subfragments of an EcoRI fragment (9 kilobase pairs) that contained the replication origin of the Escherichia coli chromosome and had been cloned as a recombinant with a nonreplicating DNA fragment coding for ampicillin resistance, small derivative plasmids were constructed. The smallest of these, pTSO151, contained a segment of 463 base pairs as the chromosomal component. Another plasmid, pSY134, constructed from BamHI digests of the EcoRI fragment and mini-F(pMF21), contained a region of 422 base pairs identical with a corresponding region in pTSO151. We conclude that the replication origin of E. coli chromosome is located within this 422-base-pair segment. The nucleotide sequence of this segment is presented.

Five TGA “stop” codons occur within the translated sequence of the yeast mitochondrial gene for cytochrome c oxidase subunit II

Abstract: A mitochondrial mutation that genetically maps in the middle of the gene coding cytochrome c oxidase subunit II has been found to be a single-base-pair deletion. Three independently isolated spontaneous revertants of this mutant have different single-base-pair insertions within 15 nucleotides of the mutation. These findings clearly identify the location of the gene and suggest that the mutation causes a frame-shift. The sequence of about 900 base pairs surrounding the mutation has been determined and found to have several chain termination codons in every possible reading frame. The sequence can, however, be translated in one frame by assuming that the codon TGA does not cause chain termination in yeast mitochondira, as was recently suggested for the human organelle [Barrell, B. G., Bankier, A. T. & Drouin, J. (1979) Nature (London), in press]. If TGA codes for tryptophan residues, as is apparently the case in human mitochondria, a polypeptide can be read from the yeast mtDNA that is identical to bovine cytochrome oxidase subunit II at 37.8% of its residues. Furthermore, the DNA sequences of the frame-shift revertants discussed above predict relative isolectric point differences between the wild-type and various revertant forms of the polypeptide. The detection of these isolectric point differences by two-dimensional electrophoresis of subunit II from the various strains independently confirms the presumed reading frame of the gene. It is concluded that TGA is translated in yeast mitochondria, most probably as tryptophan.

RNA polymerase unwinds an 11-base pair segment of a phage T7 promoter.

Abstract: WHEN RNA polymerase binds to a promoter site, it must unwind part of the DNA double helix so as to expose the template bases. Wang et al.1, Melnikova et al.2 and Hsieh and Wang3 have used different experimental techniques to measure the degree of unwinding. Their estimates range from 7 (ref. 1) to 15 (ref. 2) base pairs unwound per bound Escherichia coli RNA polymerase (EC 2.7.7.6). These studies, however, do not establish which part of a promoter is melted out. A new technique described here directly proves unwinding and furthermore identifies the exact region that RNA polymerase opens in the A3 promoter of phage T7. This promoter is one of three strong E. coli RNA polymerase promoters used early in the life cycle of phage T7 (ref. 4).

Analysis of integrated avian RNA tumor virus DNA in transformed chicken, duck and quail fibroblasts.

Abstract: The state of integration of avian sarcoma virus DNA in the genomes of transformed chicken, duck, and quail fibroblasts was deduced by means of restriction enzyme digestion of total cell DNA, gel electrophoresis, and subsequent analysis by the procedure of Southern. The cells used in these studies were either mass-infected cultures or clones of infected cells selected by their ability to form colonies in agar. For both mass-infected cultures and clones of cells of all three species, we found that integration occurred at a specific site on the viral genome but appeared to occur at many sites on the cell genome. At least some of the integrated viral DNA existed as intact nonpermuted species flanked by direct terminal repeats of at least 0.134 megadalton (217 base pairs). For each of 12 transformed quail clones studied, it was possible to detect, after digestion with Kpn I, unique junctions between viral and cellular DNA. That is, at our level of analysis, the integration site on the cell genome for each clone was different. However, within each of the 17 chicken and 9 duck clones of transformed cells, a heterogeneity presumably occurred during the outgrowth of the cell clone population, in that we could not readily detect identifiable cell-virus junction fragments.

Nucleotide sequence of the transposable DNA-element IS2

Abstract: The complete sequence of the transposable DNA element IS2 in gal OP-308:: IS2 (I) has been determined. This element is 1.327 bp long. The integrated element is flanked by a five base pair long sequence duplication. The termini of IS2 are not perfect inverted repeats, but a close approximation.

Conformation and circular dichroism of DNA

Abstract: CD spectra of calf thymus, C. perfringens, E. coli, and M. luteus DNA have been measured in the vacuum-uv region to about 168 nm for the A-, B-, and C-forms. The positive band at about 187 nm and the negative band at about 170 nm found for each type and form of DNA are sensitive to the source of the DNA and the base–base interactions of the double-stranded helix. The A-form spectra confirm that these bands are indeed sensitive to secondary structure. In the near-uv, the CD of B-form DNA is well analyzed as a linear combination of 27% A-form and 78% C-form. However, an analysis of the extended spectrum demonstrates that the near-uv analysis is not correct. The extended analysis shows that the base–base interactions are similar for B- and C-forms in solution, which implies that these two forms have nearly the same number of base pairs per turn. Various types of CD difference spectra are also discussed.

Fidelity of replication of phage phi X174 DNA by DNA polymerase III holoenzyme: spontaneous mutation by misincorporation.

Abstract: DNA from phi X174 is replicated in vitro with a fidelity similar to that found genetically. A mutation of TAG leads to TGG may be induced, however, by varying the concentrations of deoxynucleoside triphosphates, with a frequency proportional to [dGTP]2/[dATP]. This complex concentration dependence is consistent with the active participation of a proofreading mechanism that hydrolytically excises mismatched base pairs as they are formed. A simple kinetic analysis predicts that the frequency of misincorporation depends on the ratio of incorrect to correct deoxynucleoside triphosphates times the concentration of the next triphosphate in the sequence to be added. This suggests that spontaneous mutation by misincorporation depends crucially on the composition of the deoxynucleoside triphosphate pool.