SEARCH · PubMed Health
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
DNA from dormant and germinated embryos exhibit differences in the kinetics of reassociation of thermal denatured nucleic acid. Within 24 h of germination a reduction of the renaturation, expressed as Cot1/2 of the redundant fraction, is detectable.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Calf thymus chromatin was partially deproteinized by 0.6-1 M NaCl extraction. From the shape of the temperature derivative plot of its melting curve, the DNA in each chromatin was resolved into regions of exposed DNA, and DNA still complexed with proteins. The partially deproteinized chromatin was capable of directing in vitro RNA synthesis in an amount proportional to the fraction of exposed DNA. The in vitro RNA transcript was hybridized to denatured calf DNA under hybridization conditions where only RNA's transcribed from repetitive DNA sequences were able to form hybrids. From the results it is deduced that the in vitro RNA transcripts of 0.6 M NaC1-extracted chromatin after the removal of HI histones plus one-quater of the non histones may be transcribed from repetitive DNA sequences and those of 1 M NaCl-extracted chromatin contain more transcripts from the unique DNA sequences.
Explore the source record for details and available documents.
In situ hybridisation of certain AT rich and GC rich satellite DNA complementary RNAs (cRNAs) to their homologous chromosomes at their respective optimal rate temperatures (TOPTS) after denaturation with various reagents (0.2 N HCl, 0.07 N NaOH, 90% formamide and heat) led to the following conclusions. -- Heat denaturation of chromosomal DNA in 0.1 X SSC at 100 degrees C gives significantly higher grain counts regardless of DNA base composition, HCl denaturation discriminates markedly against GC rich DNA. Chromosome morphology is best preserved after HCl and heat denaturation.
Explore the source record for details and available documents.
A simple and general model of reversible conformational changes in biopolymers that lends itself to accounting for cooperativity without resort to a detailed description of the elementary steps is presented. It is suggested that the model permits the description of transitions in specific instances in which long-range effects are present and no simplifying feature allows for a more detailed theory in a straightforward way. The proposed phenomenological approach is based on the concept of molecular field which led to the first theory of ferromagnetism. Equations are given for the temperature dependence of optical properties and of the specific heat, from which the cooperativity parameter introduced by the theory can be obtained when the reaction enthalpy of the elementary step or the number of concerted elements is known. In the limit of a strong molecular field, heterogeneity in composition of a melting sequence does not affect the sharpness of the corresponding transition. Accounting for long-range effects allows for all-or-none transitions that are sharper than those derived from the two-state model. The feasibility of applying the molecular field concept is illustrated by comparing the results for poly(A).2 poly(U) triple helices (which exhibit hysteresis) and those for poly(A).poly(U) double helices (which separate reversibly).Tertiary structure is considered, among the sources of cooperativity that possibly may be represented in terms of a molecular field. On the basis of recent results for tRNA(1) (val), it is suggested that the proposed approach may be applicable, in particular, to transfer ribonucleic acids.
1. Changes in circular dichroism (CD) spectra and thermal melting profiles of guinea pigliver DNA reassociated with histones and/or nonhistone proteins from the cerebral of liver chromatin are described. 2. In the DNA-histone complex, positive ellipiticity in the CD spectrum at 260-300 nm is progressively lod by a red-shift of the crossover point at around 260 nm. DNA in this complex is thermally stabilised to a considerable extent, but not to such a full extent as is shown with DNA in native chromatin. 3. DNA-nonhistone complex in 0.14 M NaCl is, in contrast to DNA-histone complex, not precipitable by centrifugation at 20 000 X g. DNA in this complex shows only a slight reduction in ellipticity at 260-300 nm, and a very weak thermal stabilisation. 4. Characteristics in the CD spectrum of the native chromatin are most satisfactorily reproduced in the DNA-histone-nonhistone complex. These include a large decrease in ellipticity at 260-300 nm, a red-shift of the crossover point at around 260 nm, and a slight negative band at around 305 nm. Also, DNA in this complex is thermally stabilised to the extent comparable with DNA in the native chromatin. 5. Addition of nonhistone proteins to the preformed DNA-histone complex in 3 M urea renders a half of the complex, named DNA-histone(-nonhistone), unprecipitable upon centrifugation at 20 000 X g in 0.14 M NaCl. CD spectrum and thermal melting profile of the precipitable DNA-histone(-nonhistone) complex are similar to those of the DNA-histone-nonhistone complex, while in the unprecipitable DNA-histone(-nonhistone) comples, the ellipticity at 260-300 nm is significantly elevated and the highest melting transition (at 80 degrees C) is lacking. 6. The CD spectrum of native cerebral chromatin closely resembles that of unprecipitable DNA-histone(-nonhistone) complex, while in liver chromatin, the spec.trum is an intermediate between those of the unprecipitable and pn of chromatin by nonhistone proteins. Cerebral nonhistone proteins bind to DNA and to the DNA-histone complex more extensively than liver nonhistone proteins. 7. It is concluded that, although the basic conformation of DNA in native chromatin is determined largely by histones, nonhistone proteins also play an individual role. There is also an indication that nonhistone proteins exert an organ-specific modification of chromatin superstructure.
Here we describe the 500-fold purification of an mRNA encoding an immunoglobulin lambda light chain derived from the mouse myeloma tumor, RPC-20. Purification involves the isolation of membrane-bound polysomes, oligo(dT)-cellulose chromatography, and sucrose gradient centrifugation under conditions favoring denaturation of polynucleotide complexes. The mRNA purified in this way directs the cell-free synthesis of a polypeptide which is five or six amino acids longer than the mature form of RPC-20 light chain. In addition to directing the synthesis of a precursor-like polypeptide, the mRNA migrates on electrophoresis as a band containing approximately 1150 nucleotides, about 500 more than required to encode the mature form of the light chain.
Low molecular weight peptides from calf thymus cause a strong dose-dependent stabilization of the DNA. The strength od DNA-peptide interaction is pH-dependent and decreases repidly above pH 6.5. Moreover the complete kinetics of DNA denaturation and renaturation demonstrates that the peptide fraction increases significantly the DNA renaturation mostly at low temperature, showing that the interaction DNA-thymic effector helps the recombination of complementary DNA segments. The DNA stabilization rate by the peptide fraction is comparable to that obtained by means of high concentration of histones or synthetic polycationic peptides. However, the lack of basic amino acids in the peptide structure is not in favor of strong electrostatic interactions and implies a specific binding of peptide to DNA. The possible correlation of the specific thymic peptides-DNA interaction with the stereochemical kinking scheme of DNA is discussed.
Electron microscopic visualization of molecular hybrids formed in situ is feasible at the present time. It can be accomplished by two alternative approaches. In one, the in situ hybridization is carried out on ultrathin sections of target embedded in glycol methacrylate. In the other, whole cells are used for hybridization and they are subsequently prepared for electron microscopy. The choice of the method to be adopted depends on the type of target tissue. When there is a choice, the second approach seems preferable. Some of the important technical steps in the hybridization procedure, such as DNA denaturation in ultrathin sections, have been discussed and attention has been drawn to practical problems that may arise during the preparatory steps. Our light microscope experiments demonstrate that preparations made after glutaraldehyde fixation have a lower hybridization efficiency than those fixed with 3 : 1 methanol-acetic acid. Attempts are therefore being made to explore the possibility of using methanol-acetic acid for electron microscope in situ hybridization. First results of straight-forward fixation show that the preservation of nuclear structure may be fairly satisfactory for the purpose. However, the cumultative effects of subsequent treatments in the procedure still remain to be examined. For electron microscope autoradiograph (EM ARG) of hybridized preparations, the most suitable emulsion at present appears to be Ilford L4. Various factors conductive to optimum resolution consistent with maximum efficiency in this emulsion have been pointed out. Practical problems that may arise in autoradiographs of hybridized preparations such as background and variation of grain density in adjacent sections have also been considered.
Specific complementary DNAs (cDNAs) for the messenger RNAs coding for sheep alpha-, betaA-, betaB-, betaC-, and gamma-globins were prepared by thermal denaturation of heterologous hybrids (e.g. alphabetaB-cDNA-alphagamma-mRNA) followed by hydroxylapatite chromatography. Each cDNA represented a nearly full-length copy of its globin mRNA complement as determined by electrophoretic analysis in polyacrylamide gels containing 98% formamide. The purity of each cDNA fraction was estimated by hybridization analysis and thermal denaturation. The beta- and gamma-cDNAs contained 5 to 20% contaminating alpha-cDNA while the alpha-cDNA was 25 to 30% contaminated with non-alpha-cDNA. The melting temperatures (Tm) of homologous duplexes between each non-alpha chain cDNA and its mRNA complement ranged from 69.5-71.5 degrees in 50% formamide while alpha-alpha duplexes melted with a Tm of 75-76 degrees. The Tm values of heterologous duplexes formed between each non-alpha-cDNA and the various globin mRNAs (e.g. betaB-cDNA-Hb C mRNA) ranged between 64.5 degrees and 68 degrees and thus were only 1.5-5.0degrees below that of homologous duplexes. These results suggest that the nucleotide sequence divergence among the various non-alpha-mRNAs (or cDNAs) is not greatly different from the minimum predicted from the amino acid sequence differences of the corresponding globins. When annealing reactions were performed above the Tm of the heterologous hybrids (68 degrees), each non-alpha-cDNA hybridized only to its own complementary mRNA. Thus the purified cDNAs provide molecular probes for the quantitation of alpha-, beta-, and gamma-globin-specific nucleotide sequences.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
RNA-driven complementary DNA (cDNA) hybridization experiments have been carried out in order to detect complementary sequences in RNA prepared from a mouse T cell lymphoma line (EL4). In conditions where efficient hybridization of L-chain cDNA with homologous P3 myeloma mRNA was observed, poor hybridization was observed with EL4 mRNA unless a low criterion of hybrid formation was employed (i.e. hydroxyapatite fractionation). The hybrid formed between EL4 mRNA and L-chain cDNA was found to melt about 5 degrees C below the homologous hybrid indicating that the sequence detected in EL4 mRNA is similar but not identical with the P3 mRNA sequence. However, a similar sequence was detectable in a Clambda-producing myeloma cell line but in this line the concentration of the sequence was found to be an order of magnitude lower than in EL4 cells.
Increasing concentrations of dimethylsulfoxide (DMSO) strip tobacco mosaic virus (TMV) stepwise from the 3'end. The RNA tail increases in length up to 2,000 nucleotides (nu) reaching a region of very strong protein-RNA affinity. Thereafter, uncoating occurs from the other end and produces a second RNA tail 500 nu long. Further stripping of TMV proceeds from both ends, the long tail increasing in length up to 4,000 nu and the short one increasing more moderately and remaining below 2,000 nu. The region of strongest protein-RNA affinity is located between 4,000 and 5,000 nu away from the 3' end. Using the same conditions as for in vitro TMV reassembly, it is possible to recoat the RNA tails with viral protein preferentially in the 5' direction. The advantages of DMSO in studies of TMV protein-RNA interactions are discussed.