Interconvertible hairpin structure found in the replication origin of phage G4 single-stranded DNA

We found a synthetic GCGAAAGC fragment with a mobility greater than that of other oligodeoxyribonucleotides in gel electrophoresis to take on a stable hairpin structure possessing two terminal G-C base pairs. The GCGAAAGC sequence is also found in the replication origin of phage G4 single-stranded DNA, but the hairpin structure originally proposed differs from that of the GCGAAAGC fragment we have studied. Possibility of rearrangement of the secondary structure in the replication origin of phage G4 was examined in relation to its replication initiation mechanism.     

Synthetic oligodeoxyribonucleotides showing abnormal mobilities on polyacrylamide gel electrophoresis

We found that synthetic DNA fragments containing a GCGAAAGC sequence showed higher mobilities than oligonucleotides without the sequence on denaturing polyacrylamide gel electrophoresis. For example, the fragment, GCGAAAGCT (9mer), showed higher mobility than the corresponding 8mer (CGAAAGCT). In addition, on Maxam-Gilbert sequencing, a 21mer containing the GCGAAAGC sequence showed an abnormal pattern, which were similar to those due to compression observed on sequencing of DNAs with high GC contents, as recently reported. It was suggested that this compression was due to the increased mobilities of the specific fragments with the GCGAAAGC sequence and that these fragments took on abnormal conformations.     

Extraordinarily stable mini-hairpins: electrophoretical and thermal properties of the various sequence variants of d(GCGAAAGC) and their effect on DNA sequencing.

A small DNA fragment having a characteristic sequence d(GCGAAAGC) has been shown to form an extraordinarily stable mini-hairpin structure and to have an unusually rapid mobility in polyacrylamide gel electrophoresis, even when containing 7M urea. Here, we have studied the stability of the various sequence variants of d(GCGAAAGC) and the corresponding RNA fragments. Many such sequence variants form stable mini-hairpins in a similar manner to the d(GCGAAAGC) sequence. The RNA fragment, r(GCGAAAGC) also forms a mini-hairpin structure with less stability. The DNA mini-hairpins with GAAA or GAA loop are much more stable than DNA and RNA mini-hairpins with other loop sequence so far as has been examined. The stability difference between DNA and RNA mini-hairpins may be deduced to the stem structures formed by DNA (B form) and RNA (A form). The stable hairpins consisting of the GCGAAAGC sequence cause strong band compression on the sequencing gel. This phenomenon should be carefully considered in DNA sequencing.     

Unique hairpin structures occurring at the replication origin of phage G4 DNA

Recently we reported that a DNA fragment, GCCAAAGC, forms an extraordinarily stable hairpin structure with two G-C pairs at the terminus and A-A-A stacked structure. The sequence is present at the replication origin of bacteriophage G4 ssDNA, and so on. Several kinds of possible hairpin structures, corresponding to the replication origin of phage G4, were synthesized and their secondary structures were examined. It was found that the fragments are able to form interconvertible hairpin structures depending on the length of the base-paired regions. The hairpin structure consisting of GCGAAAGC was not digested by the exonuclease activity of T4 DNA polymerase and it was stable enough to be only minimally bound by a single-stranded DNA binding protein.     

Single-stranded DNA fragments holding unusual conformation

In the chemical synthesis of DNA, we found that the single-stranded DNA (ssDNA) fragments containing the sequence GCGAAAGC showed higher mobilities than the fragments without this sequence on a denaturing polyacrylamide gel electrophoresis. Physical structure of these DNA fragments was studied by enzyme digestion and optical analysis. The abnormal mobilities on electrophoresis seem to depend on an unusual conformation.     

Stability difference between DNA and RNA mini-hairpins containing two G-C pairs.

The deoxyribonucleotide fragments, d(GCGAAAGC) and d(GCGAAGC) form extraordinarily stable mini-hairpin structures containing only two G-C pairs. In contrast, the corresponding ribonucleotide fragments, r(GCGAAAGC) and r(GCGAAGC) are not so stable, although they also form mini-hairpin structures. The stability difference between these DNA and RNA mini-hairpins was examined by NMR studies and the molecular mechanics calculations, and by comparing with the stability of several sequence variants. Their stability was deduced to vary delicately depending on helical patterns formed by DNA (B form) and RNA (A form) structures.     

Periodicity and fragment size of DNA from mouse TLT hepatoma chromatin and chromatin fractions using endogenous and exogenous nucleases

Summary The action of micrococcal nuclease, DNase I and DNase II on mouse TLT hepatoma chromatin revealing the periodicity of its structure as visualized by denaturing and nondenaturing gel electrophoresis, was consistent with the action of these enzymes on other chromatins. Micrococcal nuclease showed a complex subnucleosome fragment pattern based on multiples of 10 base pairs with a prominant couplet at 140/160 base pairs and the absence of the 80 base pair fragment. This couplet of the core and minimal nucleosome fragments was conspicuously present in the mononucleosomes found in the 11S fractions of a glycerol gradient centrifugation. DNase I and II produced a fairly even distribution of a 10 base pair increasing series of fragments to about 180 base pairs, a pattern also repeated in the DNA of nucleosome glycerol-gradient fractions. In limited digestions by these nucleases multinucleosomic DNA fragments are pronounced. These fragment lengths are multiples of an estimated average repeat length of nucleosome DNA of 180 base pairs. The action of the endogenous Mg/Ca-stimulated endonuclease produced only limited cuts in the hepatoma chromatin resulting primarily in multi-nucleosommc DNA fragment lengths and only upon lengthy digestion limited subnucleosomic, 10-base-pair multiple fragments are produced. The putative euchromatin-enriched fractions (50–75S) of the glycerol gradient centrifugation of autodigested chromatin, similarly, contained primarily the multinucleosomic DNA fragment lengths. These results are consistent with our previous electron microscopic demonstration that autodigested chromatin as well as the putative euchromatin-enriched fractions were composed of multinucleosomic chromatin segments containing a full complement of histones.     

短DNA片段缩合结构的比较研究

本文利用透射式电镜对四种短DNA片段(500、1100、1500、2700 bP)的缩合结构进行了比较研究得出很有意义的结果。定量研究证实短至500 bP的DNA分子仍可形成复曲面,且分子量相差5倍多的DNA片段缩合形成的复曲面尺度大小一致。复曲面外径为400A左右。从而进一步证实作者与Arscott及Bloomfield关于复曲面尺度独立于DNA分子量,及短DNA片段的缩合是多分子缩合的结论。此外,观测到缩合中间结构的尺度依DNA分子量大小不同而变化,同时分子量愈小的DNA片段产生另一种缩合结构—棒体的几率愈大。     

Separation of large DNA restriction fragments on a size-exclusion column by a nonideal mechanism

Double-strand DNA (dsDNA) restriction fragments were chromatographed on the DuPont Bioseries GF-250 column. Two anomolous chromatographic properties were observed. (1) A triphasic dependence of retention on dsDNA chain length was observed. Small DNA fragments (less than 500 base pairs) displayed typical size exclusion, intermediate size DNA (800-5000 base pairs) eluted in the void volume, and larger DNA fragments were increasingly retained. (2) The void volume for nucleic acids was less than that for large polypeptides. The retention of moderately large DNA fragments increased linearly as the square root of the chain length over the range 5.5 to 50 kilobase pairs (ca. 3-30 X 10(6) Mr). A number of eluant manipulations were carried out in order to examine the mechanism by which the larger DNA fragments were being retained and separated. Evidence was not obtained to support either ion exchange or reverse phase as the retention mechanism. The usefulness of such a column for molecular biological manipulations is illustrated by the rapid isolation of homogeneous viral DNA fragments resected from their cloning vectors with restriction endonucleases.     

Map of restriction sites on bacteriophage T4 cytosine-containing DNA for endonucleases bamHI, BglII, KpnI, PvuI, SalI, and XbaI.

A complete map of the cleavage sites of restriction endonucleases BamHI, BglII, KpnI, PvuI, SalI, and XbaI was determined for the cytosine-containing DNA of a bacteriophage T4 alc mutant. The 56 sequence-specific sites were assigned map coordinates based on a least-squares analysis of measured fragment lengths. Altogether, the lengths of 118 fragments from single and double enzyme digestions were measured by electrophoresis of the fragments in agarose gels. DNA fragments of known sequence or DNA fragments calibrated with fragments of known sequence were used as standards. The greatest deviation between an experimentally measured fragment length and its computed map coordinates was 3.0%; the average deviation was 0.8%. The total length of the wild-type T4 genome was calculated to be 166,200 base pairs.     

S4-alpha mRNA translation regulation complex. II. Secondary structures of the RNA regulatory site in the presence and absence of S4

The secondary structure of the Escherichia coli alpha mRNA leader sequence has been determined using nucleases specific for single- or double-stranded RNA. Three different length alpha RNA fragments were studied at 0 degrees C and 37 degrees C. A very stable eight base-pair helix forms upstream from the ribosome initiation site, defining a 29 base loop. There is evidence for base-pairing between nucleotides within this loop and for a "pseudo-knot" interaction of some loop bases with nucleotides just 3' to the initiation codon, forming a region of complex structure. A weak helix also pairs sequences near the 5' terminus of the alpha mRNA with bases near the Shine-Dalgarno sequence. Affinity constants for the translational repressor S4 binding different length alpha mRNA fragments indicate that most of the S4 recognition features must be contained within the main helix and hairpin regions. Binding of S4 to the alpha mRNA alters the structure of the 29 base hairpin region, and probably melts the weak pairing between the 5' and 3' termini of the leader. The pseudo-knot structure and the conformational changes associated with it provide a link between the structures of the S4 binding site and the ribosome binding site. The alpha mRNA may therefore play an active role in mediating translational repression.     

Optimization of fragmentation conditions for microarray analysis of viral RNA

An important consideration in microarray analysis of nucleic acids is the efficiency with which the target molecule is captured by, or hybridized to, surface-immobilized oligos. For RNA, secondary and tertiary structure of the target strand can significantly decrease capture efficiency. To overcome this limitation, RNA is often fragmented to reduce structural effects. In this study, the metal ion-catalyzed base hydrolysis fragmentation conditions for viral RNA extracted from influenza viruses were evaluated and the hybridization efficiency of the resulting fragments was determined as a function of fragment length. The amount of RNA captured was evaluated qualitatively by fluorescence intensity normalized to an internal standard. Optimized conditions for influenza RNA were determined to include a fragmentation time of 20-30 min at 75 degrees C. These conditions resulted in a maximum concentration of fragments between 38 and 150 nt in length and a maximum in the capture and label efficiency.     

A new packing for separation of DNA restriction fragments by high performance liquid chromatography

TSK-GEL SW was found to be useful as a packing in high performance liquid chromatography for the separation of double-stranded DNA restriction fragments. DNA fragments smaller than 300 base pairs were separated as discrete peaks depending solely upon difference in chain length. The recovery of DNA fragments was higher than 90%.     

Analysis of highly repeated DNA sequences of rat with EcoR1 endonuclease

Cleavage of rat liver nuclear DNA with EcolR1 restriction endonuclease yields 14 discrete fragments ranging from 2300 to 93 base pairs in length, representing approx. 10.5% of the rat genome. Fragments of 1500, 180, and 93 base pairs are reiterated over 100 000 times; fragments of 2300, 880, 290, and 200 base pairs are reiterated over 20 000 times; the remaining fragments are present in over 1000 copies per genome. When compared to whole rate DNA, 11 were 1-5% richer in A . T base pairs and five were 1.5-2.5 times more methylated. From the criteria of the banding patterns in complete and incomplete digests, base composition and extent of methylation, none of these fragments appeared to be generated as oligomers of a basic shorter repeat. The reassociation of EcoR1 fragments was monitored on hydroxyapatite and by S1 nuclease treatment in order to assess band reiteration frequency and the possibility of interpersion or short internal repeats. The renaturation of the four smallest EcoR1 fragments gave no indication of short internal repeats from hyperpolymer formation nor interpersion with lower frequency sequences by size reduction after S1 nuclease treatment. Anomalous renaturation of several large fragments was observed, possibly due to internal repeats.     

Nucleosome structure in Aspergillus nidulans

The structure of chromatin from Aspergillus nidulans was studied using micrococcal nuclease and DNAase I. Limited digestion with micrococcal nuclease revealed a nucleosomal repeat of 154 base pairs for Aspergillus and 198 base pairs for rat liver. With more extensive digestion, both types of chromatin gave a similar quasi-limit product with a prominent fragment at 140 base pairs. The similarity of the two limit digests suggests that the structure of the 140 base pair nucleosome core is conserved. This implies that the difference in nucleosome repeat lengths between Aspergillus and rat liver is caused by a difference in the length of the DNA between two nucleosome cores. Digestion of Aspergillus chromatin with DNAase I produced a pattern of single-stranded fragments at intervals of 10 bases which was similar to that produced from rat liver chromatin.     

Transient electric birefringence study of the length and stiffness of short DNA restriction fragments

Transient electric birefringence measurements of the rotational diffusion constant of five short restriction fragments of the plasmid pBR322 show that the hydrodynamic length is independent of sodium ion concentration in the range of 0.2 to 2.5 mM. The fragments are too stiff to be modeled as wormlike molecules. The rotational relaxation times of the fragments, which range from 64 to 124 base pairs, have been used to calculate the rise per base pair using six different theoretical expressions for the length dependence of the rotational diffusion coefficient of straight cylinder. The best estimate for the rise per base pair of Na-DNA in solution is 3.3 ± 0.1 Å.     

A site of discontinuity in the interaction between DNA and histones in nucleosomes of sea urchin embryo chromatin.

Digestion of chromatin DNA in nuclei of sea urchin embryos with pancreatic nuclease and with micrococcal nuclease give additional details concerning the interaction between DNA and histones. A specific site of hydrolysis appears to be located on the nucleosome in such a position as to split the DNA unit length in two equivalent fragments of about 60–70 base pairs in length. The complete digestion of chromatin DNA appears to depend on the low stability of the nucleosome containing the split DNA fragments.     

Adenoassociated virus has a unique chromatin structure

The organization of intranuclear adenoassociated virus DNA (AAV) was examined following micrococcal nuclease digestion of nuclei prepared from cells coinfected with AAV type 2 (AAV-2) and adenovirus type 2 (Ad2). Blot-hybridization analysis of the DNA with AAV-2, Ad2, and cellular DNA probes revealed that AAV-2 chromatin has a unique structure, which upon nuclease digestion gives rise to a smear of oligomeric DNA fragments from 600-2200 base pairs in length with only a very faint band about 160 base pairs and no discrete multimers. This structure was similar to, but distinguishable from, Ad2 chromatin and completely unrelated to eukaryotic chromatin.     

Iodination as a probe for small regions of disrupted secondary structure in double-stranded DNAs.

Conditions were established where the thallium-catalyzed iodination of random coil DNA proceeded 100-200 times faster than for native DNA. This reaction was explored as a probe for localized regions of disrupted base pairs in duplex DNA. A heteroduplex was constructed between DNA fragments produced by Hind II + III cleavage of phi80 plac DNA and phi80 plac DNA containing the Ll deletion (73 nucleotides in length). This heteroduplex incorporated twelve times as much iodine as the parent homoduplex fragments. Hence the technique could reveal the presence of a few (two or more) nonpaired cytosines, if they existed within an otherwise helical DNA fragment 789 base pairs long. Iodination studies were performed on superhelical SV40 DNA and on linear lambdaplac DNA. Analysis of the relative amount of iodine in restriction endonuclease fragments of these DNA's revealed the absence of localized single-stranded regions.     

Probing the structure of bacterial deoxyribonucleoproteins with exogenous and endogenous nucleases

During digestion of deoxyribonucleoproteins (DNP) of gram-negative bacteria by micrococcal nuclease and Ca2+, Mg2+-dependent endonuclease in situ regular series fragments-and large nuclease-resistent fragments of DNP were revealed by electrophoresis. The DNP length of the smallest DNP-fragment was tentatively 120-140 base pairs. In investigated bacterial species DNP contained at least two basic proteins which had electrophoretic mobility similar to that of histone H4 of eucaryot. It is suggested that bacterial DNPs have common regular structure.