Norman Davidson

Kinetics of renaturation of DNA

The rate of renaturation of fully denatured DNA is kinetically a second-order reaction. The reaction rate increases as the temperature decreases below Tm†, reaching a broad flat maximum from 15 to 30 °C below Tm and then decreases with a further decrease in temperature. Let N be the complexity of the DNA or the number of base-pairs in non-repeating sequences per virus or cell for the given DNA, and L the average number of nucleotides per single strand of the denatured DNA preparation. Then, the second-order renaturation rate constants for all DNAs are given approximately by k2 = 3 × 105 L0.5N 1. mole−1 sec−1 at (Tm − 25) °C and at [Na+] = 1.0 mole 1.−1 in aqueous solution. The reaction rate increases slightly with the GC content of the DNA. The reaction rate at the temperature maximum (Tm − 25) °C is inversely proportional to solvent viscosity, when the viscosity is changed by the addition of components which either have a small (sucrose, glycerol, ethylene glycol) or a large (NaClO4) effect on Tm. It is proposed that the mechanism of the reaction involves the joining of short, homologous sites on the two strands followed by a fast, reversible zippering reaction with forward rate constant kt. A computer analysis for this model explains the temperature and the GC dependence. To explain the viscosity dependence it is proposed that kf is inversely proportional to viscosity; that is, the zippering reaction is hydrodynamically limited. Any simple theory predicts k2 ~ LN; the observed L0.5 length dependence is attributed to an excluded volume or steric hindrance effect, that is, to restricted interpenetration of the two complementary denatured DNA coils.

Methylmercury as a reversible denaturing agent for agarose gel electrophoresis

A method for agarose gel electrophoresis under denaturing conditions, with methylmercuric hydroxide as the denaturing agent, is described. Methylmercuric hydroxide is a strong and reversible denaturing agent. It appears that complete denaturation of any base-paired secondary structural feature of a nucleic aicd can be achieved at practical concentrations of CH3HgOH. The method has been tested by showing that singly nicked circular duplex PM2 DNA is dissociated into more rapidly migrating linear and circular single-strand forms by a CH3HgOH concentration within the 2.5–5.0 m m range. The mobilities of single-strand RNA molecules are decreased in the presence of sufficient CH3HgOH because their secondary structure is disrupted. For HeLa 28S rRNA, the melting transition occurs mainly in 2–3 m m range of CH3HgOH. For a series of RNAs, at 5 m m CH3HgOH, corresponding to complete denaturation, there is a linear dependence of log (molecular weight) on electrophoretic mobility and of log (mobility) on agarose concentration, just as in other gel electrophoresis systems.

The inward rectifier potassium channel family.

Recent cloning of a family of genes encoding inwardly rectifying K+ channels has provided the opportunity to explain some venerable problems in membrane biology. An expanding number of novel inwardly rectifying K+ channel clones has revealed multiple channel subfamilies that have specialized roles in cell function. The molecular determinants of inward rectification have been largely elucidated with the discovery of endogenous polyamines that act as voltage-dependent intracellular channel blockers, and with the identification of a critical site in the channel that mediates high-affinity block by both polyamines and Mg2+.

Retinal ganglion cells do not extend axons by default: promotion by neurotrophic signaling and electrical activity.

We investigate the signaling mechanisms that induce retinal ganglion cell (RGC) axon elongation by asking whether surviving neurons extend axons by default. We show that bcl-2 overexpression is sufficient to keep purified RGCs alive in the absence of any glial or trophic support. The bcl-2-expressing RGCs do not extend axons or dendrites unless signaled to do so by single peptide trophic factors. Axon growth stimulated by peptide trophic factors is remarkably slow but is profoundly potentiated by physiological levels of electrical activity spontaneously generated within embryonic explants or mimicked on a multielectrode silicon chip. These findings demonstrate that these surviving neurons do not constitutively extend axons and provide insight into the signals that may be necessary to promote CNS regeneration.

Selective dissociation of histones from calf thymus nucleoprotein

Abstract The extent of the dissociation of histones from nucleohistone by increasing concentrations of sodium chloride and sodium perchlorate has been measured. Dissociated histones were separated from nucleohistone in a given salt medium by differential centrifugation. The histone fractions were identified by gel electrophoresis, or, in some preliminary experiments, by column chromatography. Histone fraction I is dissociated from nucleohistone by NaCl in the range of 0.4 to 0.5 m . Histone II is dissociated by 0.8 to 1.2 m -NaCl. Histone III–IV consists of two components B1 and A, by gel electrophoresis. The B1 component is dissociated from native nucleohistone in the range 0.8 to 1.2 m -NaCl, the A component in the range 0.9 to 1.6 m . The NaClO4 studies are not as detailed, but the general result is that the concentration of NaClO4 needed to dissociate a certain histone fraction is about half the concentration of NaCl needed. The dissociation of a histone fraction is somewhat co-operative and takes place over a rather small salt concentration range. These results imply that both electrostatic and non-electrostatic interactions contribute to the strength of the binding between histones and DNA. The results of the sedimentation separations were confirmed by electrophoretic separations in different salt concentrations. Furthermore, and as expected, the more histone dissociated from a nucleohistone, the greater the electrophoretic velocity of the latter. The optical melting profiles of partially dissociated nucleohistones in a medium of low salt concentration are intermediate between those of fully covered nucleohistone and DNA. The melting curves for the partially covered materials are broader than those for fully covered nucleohistone or for DNA, but not clearly biphasic. The results therefore suggest that the histones are somewhat heterogeneously distributed along the DNA chain. Fully covered native nucleohistone is insoluble at sodium chloride concentrations in the range 0.15 to 0.30 m . Nucleohistone, from which histone I has been removed by extraction with 0.6 m -NaCl, is soluble in 0.15 to 0.30 m -NaCl.

Cation effects on the denaturation of DNA

The midpoint ( T m ) of the thermal denaturation curve of a DNA sample increases linearly with the logarithm of the ionic strength for 0·0003 M μ M . This ionic strength dependence does not depend on the base ratio of the DNA sample. The transition breadth increases with decreasing ionic strength. The broadening effect is interpreted as indicating that the transition is less cooperative at low μ . At μ = 3 × 10 −4 M , the divalent ions Mg 2+ and Co 2+ are bound almost stoicheiometrically by DNA and T m increases by 35 to 45°C. The transition breadth is greater for 1/2 equivalent of divalent ion per mole of DNA phosphate than for zero or one equivalent; this is attributed to stronger ion binding by native than by denatured DNA. Silver ion at a ratio of 0·2 Ag + per base increases T m by about 40°C and broadens the transition. The pH values for denaturation by acid and base for several DNAs are reported.

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli: I. Structure of F-prime factors

Abstract The sequence relations between some bacterial F-prime factors in Escherichia coli have been determined by observing, in the electron microscope, the pattern of duplex and single-stranded regions in heteroduplexes consisting of one strand from one episome and the complementary strand from another. All of the F-primes studied have a short piece of F missing. They appear to have been formed by excision from their respective Hfrs by a type I process (Scaife, 1967), leaving a piece of F remaining in the chromosome. The piece of F missing is the same in F lac and in the close relatives F450 and F 1s , although the former F-prime was derived from Hfr2 and the latter two from Hfr3. Thus, there is evidence for hot spots on F for insertion at different places in the Escherichia coli K12 chromosome to give Hfrs and hot spots for excision to give F-primes. However, a different sequence of F is missing in the episome F 8 . The physical structure of a number of F 8 episomes has been studied. In particular, we have studied the physical and genetic structures of a set of deleted F 8 episomes, prepared by P1 kc transduction of the original F 8 , and mostly deleted in some of the transfer genes. Thus, the various genetic markers have been physically mapped. A general model for the structure of F is proposed.

Transcripts of the six Drosophila actin genes accumulate in a stage- and tissue-specific manner

We have surveyed expression of the six Drosophila actin genes during ontogeny. Unique portions of cloned actin genes were used to monitor levels of respective mRNAs in developmentally staged whole organisms and dissected body parts. We find that each gene is transcribed to form functional mRNA, which accumulates with a distinct pattern. Two of the genes, act5C and act42A, are expressed in undifferentiated cells and probably encode cytoplasmic actins. Act57A and act87E are expressed predominantly in larval, pupal, and adult intersegmental muscles; act88F in muscles of the adult thorax; and act79B in the thorax and leg muscles. These composite data define three main patterns of actin gene expression which are correlated with changing Drosophila morphology, particularly muscle differentiation and reorganization.

A rat brain na+ channel α subunit with novel gating properties

Abstract We have constructed a full-length rat brain Na + channel α subunit cDNA that differs from the previously reported a subunit of Noda et al. at 6 amino acid positions. Transcription of the cDNA in vitro and injection into Xenopus oocytes resulted in the synthesis of functional Na + channels. Although the single-channel conductance of the channels resulting from cloned cDNA was the same as that of channels resulting from injection of rat brain RNA, we observed two significant differences in the gating properties of the channels. The Na + currents from cloned cDNA displayed much slower macroscopic inactivation compared with those from rat brain mRNA. In addition, the current-voltage relationship for currents from cloned cDNA was shifted 20–25 mV in the depolarizing direction compared with currents from rat brain RNA. Coinjection of low MW rat brain RNA restored normal inactivation of the channels indicating the presence of a component, either a structural subunit of the channel complex or a modifying enzyme, necessary for normal gating of the channel.

A Role for Endothelial NO Synthase in LTP Revealed by Adenovirus-Mediated Inhibition and Rescue

Pharmacological studies support the idea that nitric oxide (NO) serves as a retrograde messenger during long-term potentiation (LTP) in area CA1 of the hippocampus. Mice with a defective form of the gene for neuronal NO synthase (nNOS), however, exhibit normal LTP. The myristoyl protein endothelial NOS (eNOS) is present in the dendrites of CA1 neurons. Recombinant adenovirus vectors containing either a truncated eNOS (a putative dominant negative) or an eNOS fused to a transmembrane protein were used to demonstrate that membrane-targeted eNOS is required for LTP. The membrane localization of eNOS may optimally position the enzyme both to respond to Ca2+ influx and to release NO into the extracellular space during LTP induction.