Showing papers in "Nucleic Acids Research in 1981"
TL;DR: In this article, a dynamic programming algorithm was proposed to fold an RNA molecule that finds a conformation of minimum free energy using published values of stacking and destabilizing energies, based on applied mathematics.
Abstract: This paper presents a new computer method for folding an RNA molecule that finds a conformation of minimum free energy using published values of stacking and destabilizing energies. It is based on a dynamic programming algorithm from applied mathematics, and is much more efficient, faster, and can fold larger molecules than procedures which have appeared up to now in the biological literature. Its power is demonstrated in the folding of a 459 nucleotide immunoglobulin gamma 1 heavy chain messenger RNA fragment. We go beyond the basic method to show how to incorporate additional information into the algorithm. This includes data on chemical reactivity and enzyme susceptibility. We illustrate this with the folding of two large fragments from the 16S ribosomal RNA of Escherichia coli.
3,202 citations
TL;DR: Gel electrophoresis in studies of equilibrium binding, site distribution, and kinetics of protein-DNA interactions found that binding to the so-called third operator site (03) is 15-18 fold weaker than operator binding, and that the binding reactions with the first and third operators are uncoupled, implying that there is no communication between the sites.
Abstract: We describe the use of gel electrophoresis in studies of equilibrium binding, site distribution, and kinetics of protein-DNA interactions. The method, which we call protein distribution analysis, is simple, sensitive and yields thermodynamically rigorous results. It is particularly well suited to studies of simultaneous binding of several proteins to a single nucleic acid. In studies of the lac repressor-operator interaction, we found that binding to the so-called third operator site (03) is 15-18 fold weaker than operator binding, and that the binding reactions with the first and third operators are uncoupled, implying that there is no communication between the sites. Pseudo-first order dissociation kinetics of the repressor-203 bp operator complex were found to be temperature sensitive, with delta E of 80 kcal mol-1 above 29 degrees C and 26 kcal mol-1 below. The half life of the complex (5 min at 21 degrees C) is shorter than that reported for very high molecular weight operator-containing DNAs, but longer than values reported for much shorter fragments. The binding of lac repressor core to DNA could not be detected by this technique: the maximum binding constant consistent with this finding is 10(5) M-1.
2,394 citations
TL;DR: A multipurpose cloning site has been introduced into the gene for beta-galactosidase on the single-stranded DNA phage M13mp2 with the use of synthetic DNA and two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations.
Abstract: A multipurpose cloning site has been introduced into the gene for beta-galactosidase (beta-D-galactosidegalactohydrolase, EC 3.21.23) on the single-stranded DNA phage M13mp2 (Gronenborn, B. and Messing, J., (1978) Nature 272, 375-377) with the use of synthetic DNA. The site contributes 14 additional codons and does not affect the ability of the lac gene product to undergo intracistronic complementation. Two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations. Using the new phage M13mp7, DNA fragments generated by cleavage with a variety of different restriction endonucleases can be cloned directly. The nucleotide sequences of the cloned DNAs can be determined rapidly by DNA synthesis using chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceeding the new array of restriction sites.
2,360 citations
TL;DR: The method eliminates the need to size the insert DNA fragments and prevents formation of clones containing short or multiple inserts and a rapid and efficient method for preparing plasmid and cosmid DNA is described.
Abstract: We present a procedure for cosmid cloning that allows rapid and efficient cloning of individual DNA fragments of between 32kb and 45kb. By appropriate treatment of the cloning vector, pJb8, we make left-hand and right-hand vector ends that are incapable of self-ligation but which accept dephosporylated insert DNA fragments. The inserted fragments are generated by partial digestion with MboI or Sau3A and are dephosphorylated to prevent ligation and insertion of non-contiguous fragments. The method eliminates the need to size the insert DNA fragments and prevents formation of clones containing short or multiple inserts. 1 microgram of target Drosophila DNA gives about 5 x 10(5) clones, with an average insert size of 38kb. We also describe a rapid and efficient method for preparing plasmid and cosmid DNA.
1,778 citations
TL;DR: It is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed, and this gel method is applied to the study of the E. coli lactose operon regulatory system.
Abstract: The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified. This gel method is applied to the study of the E. coli lactose operon regulatory system. At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild type lac promotor, but not with a CAP-insensitive mutant. Formation of a stable "open" or "melted-in" complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP. Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed.
1,759 citations
TL;DR: The observed asymmetry suggests that purines in positions -3 and +4 might facilitate recognition of the AUG condon during formation of initiation complexes and a modified version of the scanning mechanism is proposed.
Abstract: Sequences flanking the initiator codon in eukaryotic mRNAs are not random. Out of 153 messages examined, 151 have either a purine in position -3, or a G in position +4, or both. Thus, [A/G]XXAUGG emerges as the favored sequence for eukaryotic initiation sites. Nucleotides flanking nonfunctional AUG triplets, which occur in the 5'-noncoding region of a few eukaryotic messages, are different from those found at most functional sites. Whereas most authentic initiator codons are preceded by a purine (usually A) in position -3, most nonfunctional AUGs have a pyrimidine in that position. The observed asymmetry suggests that purines in positions -3 and +4 might facilitate recognition of the AUG condon during formation of initiation complexes. To test this idea, in vitro binding studies were carried out with 32P-labeled oligonucleotides. Binding of AUG-containing oligonucleotides to wheat germ ribosomes was significantly enhanced by placing a purine in position -3 or +4. The scanning model, which postulates that 40S ribosomal subunits attach at the 5'-end of a message and migrate down to the AUG codon, is discussed in light of these new observations. A modified version of the scanning mechanism is proposed.
1,137 citations
TL;DR: The nucleic acid sequence bank now contains 161 mRNAs, 43 new genes are added, and internal regulation of mRNA expression by different third base choices between quartet and duet codons is proposed for bacterial genes.
Abstract: The nucleic acid sequence bank now contains 161 mRNAs, 43 new genes are added. One sequence, that of B. mori fibroin, is dropped due to uncertainty on the starting point for translation. Frequencies of all codons are given for each gene added and for each genome type in the total bank. A new series of correspondence analyses on codon use is presented, substantiating the genome hypothesis. Internal regulation of mRNA expression by different third base choices between quartet and duet codons is proposed for bacterial genes.
910 citations
TL;DR: The possibility that the 72 bp repeat region in SV40 may act as a bi-directional entry site for RNA polymerase B such that promoter sequences linked to the repeat are more efficiently utilised is discussed.
Abstract: By introduction of recombinant plasmids into monkey CV1 cells, we have unambiguously demonstrated that sequences entirely within the 72 bp repeat, which is located upstream of the SV40 early region, are crucial for T-antigen expression in vivo. We have also shown that a DNA fragment containing the 72 bp repeat, inserted directly before chicken conalbumin or adenovirus-2 major late promoter sequences in chimeric plasmids where these promoters replace that of the SV40 early genes, caused a dramatic increase in the expression of T-antigen in vivo. This effect was independent of the orientation of the 72 bp repeat, but was sensitive to its location within the plasmid, when the 72 bp repeat was separated from the promoter sequences, T-antigen expression was reduced. Insertion of the 72 bp repeat into equivalent plasmids containing no known eukaryotic promoter sequences (plasmids which were not detectably expressed in vivo) gave rise to a measurable, but smaller level of expression. The stimulation of expression by the 72 bp repeat is cis-acting : it required covalent linkage to the recombinant. We discuss the possibility that the 72 bp repeat region in SV40 may act as a bi-directional entry site for RNA polymerase B such that promoter sequences linked to the repeat are more efficiently utilised.
609 citations
TL;DR: Comparison of sequences near the 5' end of the hGH mRNA with a similar region of the alpha subunit of the human glycoprotein hormones reveals an unexpected region of homology between these otherwise unrelated peptide hormones.
Abstract: We have determined the complete sequence of the human growth hormone (hGH) gene and the position of the mature 5' end of the hGH mRNA within the sequence. Comparison of this sequence with that of a cloned hGH cDNA shows that the gene is interrupted by four intervening sequences. S1 mapping shows that one of these intervening sequences has two different 3' splice sites. These alternate splicing pathways generate hGH peptides of different sizes which are found in normal pituitaries. Comparison of sequences near the 5' end of the hGH mRNA with a similar region of the alpha subunit of the human glycoprotein hormones reveals an unexpected region of homology between these otherwise unrelated peptide hormones.
468 citations
TL;DR: R beta G13Mix was found to hybridize specifically to globin DNA under conditions where oligonucleotides forming single base pair mismatches do not, and was shown to hybridized specifically to colonies containing a plasmid with a globinDNA insert.
Abstract: Two oligonucleotides 14-bases long were synthesized, one complementary to rabbit beta-globin DNA (R beta G14A) and the other with the same sequence except for a single base change (T for C) (R beta G14B). Hybridization conditions were established such that R beta G14A would hybridize to globin DNA while R beta G14B would not. We also synthesized a mixture of 13-base long oligonucleotides (R beta G13Mix), representing eight of the possible coding sequences for amino acids 15-19 of rabbit beta-globin. One of the eight is complementary to globin DNA. R beta G13Mix was found to hybridize specifically to globin DNA under conditions where oligonucleotides forming single base pair mismatches do not. Furthermore, R beta G13Mix was shown to hybridize specifically to colonies containing a plasmid with a globin DNA insert. These results are discussed with respect to a general procedure for screening recombinant clones for those containing DNA coding for a protein of known amino acid sequence.
465 citations
TL;DR: The complete primary structure (8031 base pairs) of an infectious clone of cauliflower mosaic virus strain CM1841 is determined using the strategy of cloning shotgun restriction fragments in the sequencing vector M13mp7.
Abstract: We have determined the complete primary structure (8031 base pairs) of an infectious clone of cauliflower mosaic virus strain CM1841. The sequence was obtained using the strategy of cloning shotgun restriction fragments in the sequencing vector M13mp7. Comparison of the CM1841 sequence with that published for another caMV strain (Strasbourg) reveals 4.4% changes, mostly nucleotide substitutions with a few small insertions and deletions. The six open reading frames in the sequence of the Strasbourg isolate are also present in CM1841.
TL;DR: The dideoxynucleotide chain termination method was used to sequence completely a 4257 bp EcoRI fragment of bovine mitochondrial DNA, and completion of the entire sequence required the construction of only a single library.
Abstract: A method for DNA sequencing has been developed that utilises libraries of cloned randomly-fragmented DNA. The DNA to be sequenced is first subjected to limit attach by a non-specific endonuclease (DNase I in the presence of Mn++), fractionated by size and cloned in a single-stranded phage vector. Clones are then picked at random and used to provide a template for sequencing by the dideoxynucleotide chain termination method. This technique was used to sequence completely a 4257 bp EcoRI fragment of bovine mitochondrial DNA. The cloned fragments were evenly distributed with respect to the EcoRI fragment, and completion of the entire sequence required the construction of only a single library. In general, once a clone library has been prepared, the speed of this approach (greater than 1000 nucleotides of randomly selected sequence per day) is limited mainly by the rate at which the data can be processed. Because the clones are selected randomly, however, the average amount of new sequence information per clone is substantially diminished as the sequence near completion.
TL;DR: A cloned fragment of spinach chloroplast DNA carrying the gene for the large subunit of ribulose bisphosphate (RuBP) carboxylase has been analysed by electron microscopy of R-loops, by hybridization to Northern blots of chloropleft RNA, by S1 nuclease mapping and by DNA sequencing.
Abstract: A cloned fragment of spinach chloroplast DNA carrying the gene for the large subunit of ribulose bisphosphate (RuBP) carboxylase has been analysed by electron microscopy of R-loops, by hybridization to Northern blots of chloroplast RNA, by S1 nuclease mapping and by DNA sequencing. The transcribed region of the gene is 1690 +/- 3 nucleotides long and co-linear with its mRNA. It comprises a 178-179 bp 5' untranslated sequence, a 1425 bp coding region and an 85-88 bp 3' untranslated region. The deduced sequence of the 475 amino acids of the spinach large subunit protein shows 10% divergence from that of the maize large subunit protein (1). The nucleotide sequence divergence between spinach and maize over the same coding region is 16% but in the transcribed flanking regions it is 35%. Features of the spinach chloroplast gene which resemble those of bacterial genes include a 5-base Shine-Dalgarno sequence complementary to a sequence near the 3' end of chloroplast and bacterial 16S rRNA, a promoter region partially homologous to a consensus sequence of bacterial promoters, and a transcription termination region capable of forming a typical stem and loop structure.
TL;DR: The sequences of the displacement-loop (D-loop) regions of mitochondrial DNA from mouse L cells and human KB cells have been determined and provide physical maps to aid in the identification of sequences involved in the regulation of replication and expression of mammalian mtDNA.
Abstract: The sequences of the displacement-loop (D-loop) regions of mitochondrial DNA (mtDNA) from mouse L cells and human KB cells have been determined and provide physical maps to aid in the identification of sequences involved in the regulation of replication and expression of mammalian mtDNA Both D-loop regions are bounded by the genes for tRNAPhe and tRNAPro This region contains the most highly divergent sequences in mtDNA with the exceptions of three small conserved sequence blocks near the 5' ends of D-loop strands, a 225 nucleotide conserved sequence block in the center of the D-loop strand template region, and a short sequence associated with the 3' ends of D-loop strands A sequence similar to that associated with the 3' termini of D-loop strands overlaps one of the conserved sequence blocks near the 5' ends of D-loop strands The large, central conserved sequence probably does not code for a protein since no open reading frames are discretely conserved Numerous symmetric sequences and potential secondary structures exist in these sequences, but none appear to be clearly conserved between species
TL;DR: A secondary structure model for 23S ribosomal RNA has been constructed on the basis of comparative sequence data, including the complete sequences from E. coli, Bacillus stearothermophilis, human and mouse mitochondria and several partial sequences.
Abstract: A secondary structure model for 23S ribosomal RNA has been constructed on the basis of comparative sequence data, including the complete sequences from E. coli. Bacillus stearothermophilis, human and mouse mitochondria and several partial sequences. The model has been tested extensively with single strand-specific chemical and enzymatic probes. Long range base-paired interactions organize the molecule into six major structural domains containing over 100 individual helices in all. Regions containing the sites of interaction with several ribosomal proteins and 5S RNA have been located. Segments of the 23S RNA structure corresponding to eucaryotic 5.8S and 25 RNA have been identified, and base paired interactions in the model suggest how they are attached to 28S RNA. Functionally important regions, including possible sites of contact with 30S ribosomal subunits, the peptidyl transferase center and locations of intervening sequences in various organisms are discussed. Models for molecular 'switching' of RNA molecules based on coaxial stacking of helices are presented, including a scheme for tRNA-23S RNA interaction.
TL;DR: The complete nucleotide sequence for the five structural genes of the trp operon of E. coli together with the internal and flanking regions of regulatory information are presented.
Abstract: The tryptophan (trp) operon of Escherichia coli has become the basic reference structure for studies on tryptophan metabolism. Within the past five years the application of recombinant DNA and sequencing methodologies has permitted the characterization of the structural and functional elements in this gene cluster at the molecular level. In this summary report we present the complete nucleotide sequence for the five structural genes of the trp operon of E. coli together with the internal and flanking regions of regulatory information.
TL;DR: In this article, a method for cloning mRNAs has been used, which results in a high yield of recombinants containing complete 5'-terminal mRNA sequences, and the data strongly support the conclusion that the 5'-untranslated region of the lysozyme mRNA is heterogeneous in length.
Abstract: A method for cloning mRNAs has been used which results in a high yield of recombinants containing complete 5'-terminal mRNA sequences. It is not dependent on self-priming to generate double-stranded DNA and therefore the S1 nuclease digestion step is not required. Instead, the cDNA is dCMP-tailed at its 3'-end with terminal deoxynucleotidyl transferase (TdT). The synthesis of the second strand is primed by oligo(dG) hybridized to the 3'-tail. Double-stranded cDNA is subsequently tailed with dCTP and annealed to dGMP-tailed vector DNA. This approach overcomes the loss of the 5'-terminal mRNA sequences and the problem of artifacts which may be introduced into cloned cDNA sequences. Chicken lysozyme cDNA was cloned into pBR322 by this procedure with a transformation efficiency of 5 x 10(3) recombinant clones per ng of ds-cDNA. Sequence analysis revealed that at least nine out of nineteen randomly isolated plasmids contained the entire 5'-untranslated mRNA sequence. The data strongly support the conclusion that the 5'-untranslated region of the lysozyme mRNA is heterogeneous in length.
TL;DR: The substrate specificity of micrococcal nuclease has been studied and Digestion of end-labelled linear DNA molecules of known sequence revealed that only a limited number of sites are cut, generating a highly specific pattern of fragments.
Abstract: The substrate specificity of micrococcal nuclease (EC 3.1.4.7.) has been studied. The enzyme recognises features of nucleotide composition, nucleotide sequence and tertiary structure of DNA. Kinetic analysis indicates that the rate of cleavage is 30 times greater at the 5' side of A or T than at G or C. Digestion of end-labelled linear DNA molecules of known sequence revealed that only a limited number of sites are cut, generating a highly specific pattern of fragments. The frequency of cleavage at each site has been determined and it may reflect the poor base overlap in the 5' T-A 3' stack as well as the length of contiguous A and T residues. The same sequence preferences are found when DNA is assembled into nucleosomes. Deoxyribonuclease 1 (EC 3.1.4.5.) recognises many of the same sequence features. Micrococcal nuclease also mimics nuclease S1 selectively cleaving an inverted repeat in supercoiled pBR322. The value of micrococcal nuclease as a "non-specific" enzymatic probe for studying nucleosome phasing is questioned.
TL;DR: These results, which were obtained with mouse satellite DNA and two fragments from the plasmid pBR22, do not support the previous contention that it is the regions of high At-content which are initially cleaved by micrococcal nuclease.
Abstract: Micrococcal nuclease is shown to cleave DNA under conditions of partial digestion in a specific manner. Sequences of the type 5'CATA and 5'CTA are attacked preferentially, followed by exonucleolytic degradation at the newly generated DNA termini. GC-rich flanking sequences further increase the probability of initial attack. Unexpectedly, long stretches containing only A and T are spared by the nuclease. These results, which were obtained with spared by the nuclease. These results, which were obtained with mouse satellite DNA and two fragments from the plasmid pBR22, do not support the previous contention that it is the regions of high At-content which are initially cleaved by micrococcal nuclease. This specificity of micrococcal nuclease complicates its use in experiments intended to monitor the nucleoprotein structure of a DNA sequence in chromatin.
TL;DR: The sequence of the 26S rRNA of the yeast Saccharomyces carlsbergensis is presented as inferred from the gene sequence; the molecule is 3393 nucleotides long and consists of 48% G+C; 30 of the 43 methyl groups can be located in the sequence.
Abstract: We present the sequence of the 26S rRNA of the yeast Saccharomyces carlsbergensis as inferred from the gene sequence. The molecule is 3393 nucleotides long and consists of 48% G+C; 30 of the 43 methyl groups can be located in the sequence. Starting from the recently proposed structure of E. coli 23S rRNA (see ref. 25) we constructed a secondary structure model for yeast 26S rRNA. This structure is composed of 7 domains closed by long-range base pairings as n the bacterial counterpart. Most domains show considerable conservation of the overall structure; unpaired regions show extended sequence homology and the base-paired regions contain many compensating base pair changes. The extra length of the yeast molecule is due to a number of insertions in most of the domains, particularly in domain II. Domain VI, which is extremely conserved, is probably part of the ribosomal A site. alpha-Sarcin, which apparently inhibits the EF-1 dependent binding of aminoacyl-tRNA, causes a cleavage between position 3025 and 3026 in a conserved loop structure, just outside domain VI. Nearly all of the located methyl groups, like in E. coli, are present in domain II, V and VI and clustered to a certain extent mainly in regions with a strongly conserved primary structure. The only three methyl groups of 26S rRNA which are introduced relatively late during the processing are found in single stranded loops in domain VI very close to positions which have been shown in E. coli 23S rRNA to be at the interface of the ribosome.
TL;DR: The hemoglobin beta-1 gene of the rabbit was linked to a 244 bp DNA fragment from the beginning of the polyoma virus late region, not including the viral origin of replication, which will be particularly useful as a component of mammalian expression vectors since it almost exclusively yielded high levels of correct beta-globin gene transcripts.
Abstract: The hemoglobin beta-1 gene of the rabbit was linked to a 244 bp DNA fragment from the beginning of the polyoma virus late region, not including the viral origin of replication. After transfection of such recombinant DNAs into mouse 3T6 and human HeLa cells, the polyoma sequences were found to strongly enhance the level of correct beta-globin gene transcripts over a distance of at least 1400 bp. These findings are similar to those obtained with a segment of DNA from the corresponding region of the SV40 genome (J. Banerji, S. Rusconi and W. Schaffner, 1981, Cell, in press) which, however, shows very limited sequence homology to the polyoma 244 bp segment. Using the same assay, a complete copy of polyoma virus DNA was found to interfere with the enhancement of globin gene expression in a cell type-specific manner which may be due to incorrect transcription. In contrast to the complete polyoma virus genome, the 244 bp DNA fragment will be particularly useful as a component of mammalian expression vectors since it almost exclusively yielded high levels of correct beta-globin gene transcripts.
TL;DR: The laser-Raman spectra show a striking congruence, which indicates clearly that the high-salt form of the aqueous polymer has the left-handed Z-DNA structure of the crystalline oligomer.
Abstract: The laser-Raman spectra of crystalline d(CpGpCpGpCpG) and of aqueous poly(dG-dC).poly(dG-dC) in high salt (4M NaCl) and low salt (0.1M NaCl) solutions have been measured and compared. The spectra of the crystal and the high-salt solution show a striking congruence, which indicates clearly that the high-salt form of the aqueous polymer has the left-handed Z-DNA structure of the crystalline oligomer. These two spectra differ substantially from that of the low-salt form of the polymer, which has been found previously to have spectral characteristics of the B-form of DNA. The high salt spectrum shows a unique line due to guanine residues at 625 cm-1 which should be useful for qualitative and possibly quantitative assessment of the amount of Z-structure present in a sample of DNA.
TL;DR: Viable mutants within the Py DNA region between the replication origin and the beginning of late protein coding sequences have been constructed and analysed for viability, early gene expression and viral DNA replication.
Abstract: Deletion mutants within the Py DNA region between the replication origin and the beginning of late protein coding sequences have been constructed and analysed for viability, early gene expression and viral DNA replication. Assay of replicative competence was facilitated by the use of Py transformed mouse cells (COP lines) which express functional large T-protein but contain no free viral DNA. Viable mutants defined three new nonessential regions of the genome. Certain deletions spanning the PvuII site at nt 5130 (67.4 mu) were unable to express early genes and had a cis-acting defect in DNA replication. Other mutants had intermediate phenotypes. Relevance of these results to eucaryotic "enhancer" elements is discussed.
TL;DR: Not only is 2-5A present, but the 2- 5A-dependent nuclease also appears to be active, in interferon-treated, EMC-infected L-cells.
Abstract: Ribosomal RNA (rRNA) in intact ribosomes is cleaved into discrete products on incubation of reticulocyte lysates or L-cell extracts with ppp(A2'p)3A Cleavage of rRNA may, therefore, provide a useful assay for 2-5A (ppp)A2'p)nA; n = 2 to 4) or for the presence of a 2-5A-dependent nuclease The results with reticulocyte lysates differed from those obtained in the L-cell-free system in that (a) a different RNA cleavage pattern was produced (with added L-cell ribosomes) and (b) cleavage was fully activated by the analogue ppp(A2'p)3A3'pCp As might be expected from the relatively high levels of 2-5A present in interferon-treated, encephalomyocarditis virus (EMC)-infected L-cells, rRNA extracted from these cells was also cleaved The cleavage pattern observed overlapped with that obtained on incubation of an L-cell-free system with 2-5A Thus, not only is 2-5A present, but the 2-5A-dependent nuclease also appears to be active, in interferon-treated, EMC-infected L-cells
TL;DR: It is found that the intra- and intermolecular condensation of DNA induced by spermine is highly selective even in the presence of added protein or triphosphates.
Abstract: We have examined the selectivity of the precipitation of DNA by spermine. We have found that the intra- and intermolecular condensation of DNA induced by spermine is highly selective even in the presence of added protein or triphosphates. We have also investigated the influence of buffer components on the threshold concentration of spermine required for DNA precipitation. Representative applications exploiting the selectivity of the precipitation reaction are also described.
TL;DR: The resulting amino acid sequence agrees with previous information obtained about sigma including the amino acid composition, partial sequence data for the N-terminus, the highly acidic nature of the polypeptide, and the cleavage pattern at cysteines.
Abstract: We have determined the nucleotide sequence of the rpoD gene which codes for the sigma subunit of RNA polymerase from E coli K12 The gene, which we formerly cloned as a HindIII restriction fragment in the transducing phage, charon 25, was recloned into several plasmids We have determined a 2600 base pair DNA sequence which includes the entire structural gene for sigma The resulting amino acid sequence agrees with previous information obtained about sigma including the amino acid composition, partial sequence data for the N-terminus, the highly acidic nature of the polypeptide, and the cleavage pattern at cysteines The molecular weight of 70,263 daltons calculated for the 613 amino acid polypeptides is significantly lower than had been determined previously by SDS polyacrylamide gel analysis
TL;DR: A multipurpose plasmid, pUR222, was constructed that contains six unique cloning sites (PstI, SalI, AccI, HindII, BamHI and EcoRI) in a small region of its lac Z-gene part.
Abstract: A multipurpose plasmid, pUR222, was constructed. It contains six unique cloning sites (PstI, SalI, AccI, HindII, BamHI and EcoRI) in a small region of its lac Z-gene part. Insertion of foreign DNA into the plasmid can be easily detected. Bacteria harbouring recombinant plasmids generally give rise to white colonies, while those containing only vector DNA form blue colonies on indicator plates. Plasmid DNA purified by a rapid method (Birnboim, H.C. and Doly, J. (1979) Nucl. Acids. Res. 7, 1513-1523) can be used for chemical sequencing of the cloned insert DNA. Labeled fragments need not be isolated after cutting with the proper restriction enzymes and are treated directly according to the sequencing protocol of Maxam and Gilbert.
TL;DR: An improved procedure for detecting poly A tracts in situ by hybridization of 3H poly U achieves significantly higher retention of RNA and better morphologic preservation than does Carnoy's, and measurement of the increase in poly A concentration after fertilization by solution titration and by in situ hybridization are in excellent agreement.
Abstract: We present an improved procedure for detecting poly A tracts in situ by hybridization of 3H poly U. Glutaraldehyde fixation achieves significantly higher retention of RNA and better morphologic preservation than does Carnoy's. A dramatic increase in signal to noise is obtained by prehybridization treatment of glutaraldehyde-fixed sections with proteinase K and acetic anhydride. Measurement of the increase in poly A concentration after fertilization by solution titration and by in situ hybridization are in excellent agreement indicating that in situ measurements yield accurate relative estimates of local RNA concentrations in sections. Examination of the grain density distribution in section of sea urchin eggs and cleaving embryos reveals no major cytoplasmic localization of poly A+ RNA, although nuclei show much less labelling and micromeres of 16-cell embryos have a small, but significant, reduction in poly A concentration.
TL;DR: The knotted P4 head DNA can be used in a sensitive assay for the detection of a type II DNA topoisomerase even in the presence of excess type I DNATopoisomerases.
Abstract: DNA molecules isolated from bacteriophage P4 are mostly linear with cohesive ends capable of forming circular and concatemeric structures. In contrast, almost all DNA molecules isolated form P4 tailless capsids (heads) are monomeric DNA circles with their cohesive ends hydrogen-bonded. Different form simple DNA circles, such P4 head DNA circles contain topological knots. Gel electrophoretic and electronmicroscopic analyses of P4 head DNA indicate that the topological knots are highly complex and heterogeneous. Resolution of such complex knots has been studied with various DNA topoisomerases. The conversion of highly knotted P4 DNA to its simple circular form is demonstrated by type II DNA topoisomerases which catalyze the topological passing of two crossing double-stranded DNA segments [Liu, L. F., Liu, C. C. & Alberts, B. M. (1980) Cell, 19, 697-707]. The knotted P4 head DNA can be used in a sensitive assay for the detection of a type II DNA topoisomerase even in the presence of excess type I DNA topoisomerases.
TL;DR: It is determined, using pyrimidine tract analysis, that nucleosomes will not form over a sufficiently long segment of poly(dA).poly(dT) in a recombinant DNA molecule, which may be relevant to nucleosome positioning in chromatin.
Abstract: We have been unable to "force" double-stranded RNA to fold into nucleosome-like structures using several different histone-RNA "reconstitution" procedures. Even if the histones are first stabilized in octameric form by dimethylsuberimidate cross-linking they are still unable to form specific complexes with the RNA. Moreover double-stranded RNA is unable to induce histones to assemble into octamers although we confirm that the non-nucleic acid homopolymer, polyglutamic acid, has this ability. We have also determined, using pyrimidine tract analysis, that nucleosomes will not form over a sufficiently long segment of poly(dA).poly(dT) in a recombinant DNA molecule. Thus nucleosomes cannot fold DNA containing an 80 base pair poly(dA).poly(dT) segment but a 20 base pair segment can be accommodated in nucleosomes fairly well. Segments of intermediate length can be accommodated but are clearly selected against. Poly(dA).poly(dT) differs only slightly from natural DNA in helix structure. Therefore either this homopolymer resists folding, or nucleosomes are very exacting in the nucleic acid steroid parameters they will tolerate. Such constraints may be relevant to nucleosome positioning in chromatin.