Base Pair Distance Research Articles

In Vivo Thermodynamic Analysis of Repression with and without Looping in lac Constructs: Estimates of Free and Local lac Repressor Concentrations and of Physical Properties of a Region of Supercoiled Plasmid DNA in Vivo

A strong-binding primary (O1) lac operator located 100 to 200 base-pairs (bp) upstream from a lac promoter control region reduces expression from a lac promoter controlled by a weaker-binding (Oc) lac operator between 3 and 20-fold on a multicopy plasmid in E. coli. We attribute this effect to loop formation in which a thermodynamically stable complex is formed between bidentate lac repressor tetramers and the O1 and Oc operators. A thermodynamic model for repression is developed to interpret these data in terms of the composite effects of free lac repressor concentration and of local repressor concentration (from looping) at the Oc site. The local repressor concentration is found to vary periodically with the distance in base-pairs between the O1 and the Oc operators, ranging from 2 to 20-fold larger than the free concentration (i.e. the bulk thermodynamic activity) of repressor in this F′Iq overproducing strain (estimated to be ≲0·5 μM). The amplitude of the periodic variation in expression and in local concentration appears to decrease with increasing interoperator distance in the range examined. Quantitative analysis of the looping data provides estimates of the physical properties of the intervening DNA region in vivo. For distances in the range 127 to 197 bp, the periodicity of modulation is uniformly 11·28(±0·04) bp, which we interpret as the helical repeat of this region of supercoiled plasmid DNA in vivo. Possible origins of this altered helical repeat include the global linking deficit of the supercoiled DNA and any local linking deficit induced by divergent transcription from promoters bracketing the interoperator region. DNA cyclization analysis yields an apparent in vivo persistence length of this interoperator region of 64(±26) Å (which is ∼15% of the in vivo result) and an in vivo torsional rigidity constant of 1·1(±0·1) × 10-19 erg cm, which is at the lower end of the range of values found in vitro.

Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae.

The Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The molecular mechanism of the induction was investigated using a fusion of the amyB promoter and the Escherichia coli uidA gene encoding beta-glucuronidase (GUS). To identify the region responsible for high-level expression and regulation within the amyB promoter, a series of deletion promoters was constructed and introduced into the A. oryzae met locus by homologous recombination. Deletion of the region between -377 to -290 (the number indicates the distance in base pairs from the translation initiation point (+1) to the deletion end point) significantly reduced of the GUS activity, but slight reduction of the GUS activity was observed in deletions up to -377. Northern blot analysis showed that reduction of the GUS activity depended upon the expression level of the GUS gene. The region between -377 to -290 is suggested to include the sequence required directly for high-level expression and regulation of the amyB gene.

An intragenic region downstream from the dihydrofolate reductase promoter is required for replication-dependent expression.

The gene encoding dihydrofolate reductase (DHFR) is down-regulated as myoblasts withdraw from the cell cycle and commit to terminal differentiation. To localize cis-acting elements involved in regulating DHFR gene expression, the DHFR promoter and upstream region, together with differing amounts of contiguous intragenic sequence, were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The resulting fusion genes were stably transformed into muscle cells, and CAT mRNA levels were measured in proliferative myoblasts and committed myocytes. A gene consisting of the -850/+465 region of DHFR (numbers refer to distance in base pairs from transcription initiation site) fused to CAT was efficiently expressed in proliferating myoblasts and was appropriately down-regulated during commitment. A gene consisting of the -850/+60 region of DHFR fused to CAT was poorly expressed in proliferating myoblasts and was not down-regulated during commitment. When inserted between the Rous sarcoma virus promoter and CAT sequence of RSVpCAT, the +61/+465 region of the DHFR gene augmented CAT mRNA expression in muscle cell transformants but did not confer a regulated pattern of expression. Our data indicate that DHFR sequences between +60 and +465 are required but are not sufficient for replication-dependent expression. The DHFR sequences may be operating at either a transcriptional or posttranscriptional level.

Molecular weight determination of the two segments of double-stranded RNA of infectious bursal disease virus, a member of the birnavirus group.

The molecular weights (mol. wts.) of the two double-stranded (ds) RNA segments of infectious bursal disease virus (IBDV) were determined using previously sequenced reovirus genes M3 and S2 as internal ds RNA reference molecules. Electrophoresis under fully denaturing conditions revealed mol. wts. of 2.26 X 10(6) daltons and 1.98 X 10(6) daltons. By direct length measurements under the electron microscope, using two different spreading conditions, the two segments were calculated to be composed of 3274 +/- 79 base pairs (bp) and 2821 +/- 59 bp or 3299 +/- 68 bp and 2830 +/- 73 bp, resulting in mol. wts. of 2.24-2.26 X 10(6) daltons and 1.93-1.94 X 10(6) daltons, respectively. Base pair distances of 2.67 +/- 0.08 A and 2.71 +/- 0.11 A in ds RNA were close to those of the A-RNA form; in ds DNA included as a control, the rise per base pair was 3.18 A, which is consistent with published results. Mol. wts. obtained for IBDV indicate that the RNAs of the other birnaviruses are also smaller than reported.

Use of polylysine for adsorption of nuclei acids and enzymes to electron microscope specimen films.

Enzymes and nucleic acids, both free and as bound in binary complexes, adsorb to electron microscope specimen films in well-distributed fashion if a dilute solution of polylysine is previously applied to the films. Electron micrographs are exhibited that demonstrate the usefulness of the technique in visualizing double- and single-stranded DNA, Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) in negative stain, and polymerase complexed to poly(dA-dT) and to an 1100 base-pair restriction fragment of bacteriophage T7 DNA containing the early promoters. The base-pair spacing of DNA prepared for electron microscopy by the polylysine method was found to be 0.326 nm. Four promoter sites on the T7 fragment were located at 215, 440, 560, and 670 base-pair distances from the left terminus. When poly(dA-dT) was incubated with a 20-to-1 weight ratio of polymerase the bound enzyme particles were found to be about two-thirds as closely packed as is sterically permissible.