Hans Bünemann

Genes and transcripts for the P700 chlorophylla apoprotein and subunit 2 of the photosystem I reaction center complex from spinach thylakoid membranes

A photosystem I reaction center complex has been purified to homogeneity by a procedure involving partial solubilization of spinach thylakoid membranes, ion exchange chromatography and centrifugation in sucrose gradients. The complex contains 7 polypeptides: the P700 chlorophylla apoprotein with an apparent molecular weight of 67 kd, which at high resolution splits into two bands, and smaller polypeptides of 22 (subunit 2), 18.5, 18, 16, 12 and 10 kd.Stable transcripts for the P700 chlorophylla apojprotein and subunit 2 were found in plastid and cytosolic RNA, respectively. The apoprotein product obtained by translation in a mRNA-dependent cell-free rabbit reticulocyte lysate and also by DNA-programmed transcription-translation of cloned plastid DNA fragments inE. coli lysates was indistinguishable immunologically and electrophoretically from the authentic protein. However, the product immunologically related to subunit 2 was 4 kd larger than the mature compound indicating that this protein is encoded in the nucleus and synthesized as a precursor.The gene for the P700 chlorophylla apoprotein has been physically mapped on the spinach plastid chromosome by hybrid selection mapping and DNA-programmed cell-free transcription-translation using cloned restriction fragments of plastid DNA. There is one gene copy per chromosome and it is located centrally in the large single-copy region of the circular DNA molecule. This gene is uninterrupted and is transcribed in the same direction as that of the large subunit of ribulose bisphosphate carboxylase/oxygenase. Its transcript is approximately 4 kb longer than the 2 kbp structural gene.

The isolation of DNA from agarose gels by electrophoretic elution onto malachite green-polyacrylamide columns.

Electrophoretic elution of DNA coupled with direct adsorption onto malachite green-polyacrylamide columns was used to isolate double- and single-stranded DNA from agarose gels. Subsequently, DNA was eluted with a high salt buffer and filtered through Sephadex which permitted recovery of the DNA in a low salt buffer at concentrations suitable for heteroduplex analysis by electron microscopy. This method was tested by examining heteroduplexes formed from the isolated complementary single strands of T7 wild type DNA and a T7 deletion mutant. More than 80% of the reannealed molecules were intact heteroduplexes showing the deletion loop. Irradiation of single-stranded DNA with 254 nm light resulted in distorted, convoluted heteroduplexes while 366 nm light did not show this effect.

On the binding and specificity of 3,6-bis-(acetatomercurimethyl)-dioxane to DNAs of different base composition

Abstract From ultraviolet absorption spectra it has been observed that DNAs from bacterial (Micrococcus luteus, Escherichia coli, Clostridium perfringens) and mammalian (calf thymus) sources react reversibly with 3,6-bis-(acetatomercurimethyl)-dioxane (BAMD). The reversibility could be obtained by adding excess of NaCl or EDTA. The spectral characteristics are comparable to those observed in the case of Hg2 +-DNA interaction. The values of r b (moles of mercury compound bound per mole of nucleotide base pair) for 3,6-bis-(acetatomercurimethyl)-dioxane and Hg2 + were determined by equilibrium dialysis. Dithizone was used for the estimation of mercurials. When different DNAs are at equilibrium with the same concentration of 3,6-bis-(acetatomercurimethyl)-dioxane or Hg2 +, r b (when it is less than 0.5) generally increases in the order: M. luteus, E. coli, calf thymus, Cl. perfringens, poly[d(A-T)]. The relative difference in r b for different DNAs (at the same concentration) varies with the free 3,6-bis-(acetatomercurimethyl)-dioxane or Hg2 + concentration at equilibrium. At higher free 3,6-bis-(acetatomercurimethyl)-dioxane concentration ( > 9.0 · 10 −6 M ) r b values corresponding to different DNAs are altered in an irregular manner. At the same equilibrium concentration of free mercurials r b for denatured DNA is higher than the corresponding native DNA. Both Hg2 + and 3,6-bis-(acetatomercurimethyl)-dioxane lower the viscosity and enhance sedimentation coefficient of a DNA solution to a comparable extent.

[27] Hybrid selection of specific rnas using DNA covalently coupled to macroporous supports

Publisher Summary For immobilization of sonicated denatured DNA, macroporous supports of the Sepharose CL and Sephacryl S type can be used advantageously instead of fine-grained cellulose or Sephadex. Direct comparison of parameters important for sensitive hybridization reactions with sonicated DNAs or RNAs clearly proves the macroporous supports to be equivalent or superior to their solid counterparts. No substantial difference is found for the rate of hybridization reaction for DNAs immobilized to macroporous or solid supports as long as the hybridization is performed with sonicated DNA. In general, this heterogeneous type of hybridization reaction proceeds about one order of magnitude slower for all supports tested so far than for the comparable renaturation reaction in homogeneous solution. Based on these results, DPTE-Sephacryl S-500, DBM-Sephacryl S-500, and BrCN-Sephacryl S-500 are recommended as favorable supports for immobilization of DNA.

Interaction of 4,5-dibromo-2,7-di-(acetatomercuri)-fluorescein with DNAs of different base composition.

The changes in absorption spectra in the visible region observed on adding different naturally occurring and synthetic DNA duplexes to solutions of 4,5-dibromo-2,7-di-(acetatomercuri)-fluorescein indicate that the mercurial reacts with polynucleotides of this type. The reaction is reversible as proved by adding excess of KCN which restores the original spectra of the free dye. The interaction is characterised also by quenching of the fluorescence of the dye and the induction of optical activity in it. The extent of these spectral effects depends strongly on the (A+T) content of the complexed DNA and decreased in the order: poly [d(A-T)], Clostridium perfringens DAN, Escherichia coli DNA, Micrococcus luteus DNA and poly(dC). From equilibrium-dialysis experiments the same order in affinity is obtained when these poly-nucleotides are at equilibrium with the same concentration of 4,5-dibromo-2,7-di-(acetatomercuri)-fluorescein. From the changes produced by different mercurials in the ORD spectra and viscosity of a DNA solution it has been concluded that 4,5-dibromo-2,7-di(acetatomercuri)-fluorescein does not cause any drastic alteration of the secondary structure of DNA.

The interaction of calf thymus DNA with mercuric acetate and 3,6-bis-(acetatomercurimethyl)-dioxane. Small-angle X-ray scattering and viscosity studies.

The binding of Hg2+ and 3,6-bis-(acetatomercurimethyl)-dioxane (BAMD) to sonicated calf thymus DNA was studied by small-angle X-ray scattering and viscosity measurements. The scattering experiments with DNA complexed by different amounts of mercurials (for Hg2+ rb = 0-0.79, for BAMD rb = 0-0.86 mol of mercurial bound per mol of base pairs) established that the rod-like character of the DNA molecules is maintained up to high binding ratios. They revealed further a steady decrease of the cross-section radius of gyration Rc for the DNA · Hg2+ complex and a similar decrease of Rc for the DNA · BAMD complex up to rb = 0.35. This behaviour is certainly caused by the incorporation of both mercurials near the axis of the DNA helix. Binding of BAMD at rb > 0.35 led to an increase of Rc which behaviour obviously reflects the location of mercury atoms at large distances from the axis, possibly on the surface of the helix. The increase of the mass per unit length Mc upon binding of the mercurials was found to be much higher than expected. This finding established that the length of the DNA helix decreases by 0.10±0.01 nm per bound mercurial at low binding ratios (i.e. up to rb = 1/3 for BAMD, up to possibly rb = 0.5 for Hg2+). A similar conclusion was also drawn from the observed decrease of intrinsic viscosity [rj] with increasing rb. The analysis of Mc at high binding ratios suggests that every BAMD molecule bound beyond rb=1/3 decreases the length of the DNA by 0.21 ±0.05 nm whereas Hg2+ when bound beyond rb = 0.5 causes no change of the length.