Long range structural communication between sequences in supercoiled DNA. Sequence dependence of contextual influence on cruciform extrusion mechanism

Sequence context may profoundly alter the character of structural transitions in supercoiled DNA (Sullivan, K. M., and Lilley, D. M. J. (1986) Cell 47, 817-827). The A + T-rich sequences of ColE1, which flank the inverted repeat, are responsible for cruciform extrusion following a mechanistic pathway which proceeds via a relatively large denatured region. This C-type mechanism results in kinetic properties which are very different from those of the S-type pathway, the normal mechanism of cruciform extrusion in the absence of the ColE1 flanking sequences. We have analyzed the sequence requirements for the induction of the C-type pathway. The 100-base pair left side sequence of ColE1 (colL) was subjected to systematic deletion using Bal31 exonucleolysis, showing that removal of 30 base pairs from its right end abolished extrusion by the C-type process. A cloned oligonucleotide of the same 30-base pair sequence was sufficient to confer C-type cruciform extrusion on an adjacent inverted repeat. An A + T-rich sequence from Drosophila was found to act like the ColE1 sequences. We have studied the effects of introducing sequences between the A + T-rich colL, and the inverted repeat on which it acts. A range of such fragments was found, from those which augment the effect of colL to those which block it completely. In general, it appears that the ability of a sequence to block the effect of colL depends on both the length and G + C content of the fragment. The sequences which are responsible for the extrusion by the C-type pathway are termed C-type inducing sequences, while sequences which are interposed between the inducing sequence and the inverted repeat, and which may either augment or attenuate the effect, but which cannot function as inducing sequences in isolation, are termed transmitting sequences. The results of these studies are most readily consistent with long range destabilization of DNA structure via telestability effects.     

Hemoglobin-membrane interaction at physiological ionic strength and temperature

The spin-label electron paramagnetic resonance (EPR) technique has been used to study the interaction between human hemoglobin and erythrocyte membranes as a function of temperature and ionic strength. We show, for the first time, experimental evidence for the existence of the interaction at physiological pH, ionic strength and temperature. In addition to the pH dependence that we have previously reported, the interactions are also temperature and ionic strength dependent. Using a simple two-state equilibrium model to analyze the EPR data, we obtain an equilibrium dissociation constant of about 8.1 +/- 5.6 X 10(-5) M for hemoglobin-membrane systems in 5 mM phosphate with 150 mM NaCl at pH 7.4 and 37 degrees C.     

The Flexible Feminine in Vietnam: ‘Ordinary’ Goddesses and Extraordinary Ethnography

Book reviewed in this article: Philip Taylor . Goddess on the Rise: Pilgrimage and Popular Religion in Vietnam     

Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction.

We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions. A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding of recombination events in DNA. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The conclusions are based upon a comparison between a series of many identical DNA molecules which have been labeled on different positions, rather than a determination of a few absolute distances. Details of the FRET analysis are presented; features of the analysis with particular relevance to DNA structures are emphasized. Three methods were employed to determine the efficiency of FRET: (1) enhancement of the acceptor fluorescence, (2) decrease of the donor quantum yield, and (3) shortening of the donor fluorescence lifetime. The FRET results indicate that the arms of the four-way junction are arranged in an antiparallel stacked X-structure when salt is added to the solution. The ion-related conformational change upon addition of salt to a solution originally at low ionic strength progresses in a continuous noncooperative manner as the ionic strength of the solution increases. The mode of ion interaction at the strand exchange site of the junction is discussed.     

Immobilization of Penicillium chrysogenum: spore growth on Celite

Summary Penicillium chrysogenum spores have been immobilized by adsorption on two grades of wet or dry diatomaceous earth particles, Chromosorb-W and Celite R-633. Almost 90% of the spores were adsorbed within 2 h and those remaining in suspension were removed by washing to minimise the growth of free mycelia. After germination the immobilized biomass was almost independent of the spore loading on the particles and whether or not the spore suspension was added to wet or dry particles. The free biomass obtained was less than 5% of the immobilized biomass.     

Xylem and Phloem Import of Na+, K+, Rb+, Cs+ and Sb(SO4)2- in Tomato Fruits: Differential Contributions from Stem and Leaf

Wolterbeek, H. Th. and De Bruin, M. 1986. Xylem and phloem importof Na⁺, K⁺ , Rb⁺, Cs⁺ and in tomato fruits: differential contributions from stem and leaf.—J.exp. Bot. 37: 928–939. The transport of Na⁺, K⁺, Rb⁺, Cs⁺ and into developing fruits of tomato (an inbred lineof Lycopersicon esculentum Mill. cv. Tiny Tim) was measured.Element solutions were introduced into the transpiration streamthrough the cut stem bases of plant parts consisting of a stempart with single green fruit, both with and without attachedfully expanded leaf. Measurements were carried out of the accumulationin the fruit of the gamma-ray emitting radiotracers ²⁴Na⁺, ⁴²K⁺,⁸⁶Rb⁺, ¹³⁴Cs⁺ and The transport into the fruit was expressed by a single parameter taking intoaccount volume flows varying with time and experiments. Xylemto phloem transfer in the stem as a source of fruit elementsupply was shown to be inversely related with the velocity offlow of the stem xylem. The results also indicated that thetransfer system in the stem was more rapidly equilibrated thanit was in the leaf. Stem loading of the phloem is suggested as a possible mechanismregulating the solute influx in fruits under varying flow velocitiesof the stem xylem, while fruit influx of phloem solutes, whichwere loaded in the leaf, may play a major role in influx regulationunder conditions of varying solute concentrations. Key words: Alkali ions, tomato fruits, stem and leaf phloem loading     

Changes in Abscisic Acid Content in Axis and Cotyledons of Developing Phaseolus vulgaris Embryos and their Physiological Consequences

Prevost, I. and Le Page–Degivry, M. Th. 1985. Changesin absicisic acid content in axis and cotyledons of developingPhaseolus vulgaris embryos and their physiological consequences.—J.exp. Bot. 36: 1900–1905.Changes in abscisic acid (ABA)content with time were measured in embryonic axes and in cotyledonsof Phaseolus vulgaris embryos using a radio–immunoassay.During embryogenesis, a similar pattern was observed in bothtissues: ABA increased to a maximum 29 d after an thesis, followedby a decrease as the seed matured. The level of ABA in the cotyledonswas always much higher than that in the axes. In in vitro cultures,the duration of the lag phase before germination of isolatedembryonic axes increased with ABA content. The presence of cotyledonsalways lengthened the lag phase; longer lag phases were associatedwith greater concentrations of ABA in the cotyledons. Moreoverthe presence of cotyledons stimulated the growth of seedlings. Key words: ABA distribution, embryo maturation, axis and embryo germinability     

Investigation of the instability of plasmids directing the expression of Met-prochymosin in Escherichia coli

The causes of the instability of a multicopy plasmid, pCT70, which directs the expression of calf prochymosin in Escherichia coli, were investigated. Plasmid pAT153 and its derivative, pCT54, were stable for more than 90 generations in continuous culture with glucose limitation. The multicopy plasmid pCT66, which expressed very low levels of prochymosin due to poor translational efficiency, and low copy number plasmids which efficiently expressed the prochymosin gene, were also stable. These results indicated that high level translation of the recombinant gene was the cause of the instability of pCT70. The maximum specific growth rate of E. coli(pCT70) was reduced by 30% compared with E. coli(pCT66). To fulfil the requirements of a production system, a dual origin plasmid with controllable copy number was developed. Both this plasmid (pMG165) and a derivative which contained the prochymosin gene (pMG168) were stable when maintained at low copy number. When the copy number of plasmid pMG168 was increased by putting replication under the control of the lambda PR promoter and the cI857 temperature sensitive repressor, expression of prochymosin was achieved. This strategy enables large-scale production of prochymosin without the need for antibiotic selection or other methods of preventing plasmid loss.