1,316 research outputs found
My career in molecular biology
Norman Davidson's training as a physical chemist led him to make key early contributions to the chemistry of DNA. He described the details of DNA denaturation and renaturation, concepts that still form the basis for understanding hybridization. He also applied the single-molecule resolution of the electron microscope to describing the chemistry of circular DNA, mapping specific genes, and characterizing heteroduplexes. The latter became a dominant tool for the study of nucleic acids and contributed to our knowledge of transcription, polyadenylation, and retroviral structure. The advent of cDNA cloning and restriction enzymes enabled Davidson to describe the diversity of Drosophila actin genes and to isolate the gene encoding cAMP phosphodiesterase. Davidson then turned his attention to neuroscience and participated in cDNA cloning, oocyte expression, and structure-function studies of nicotinic acetylcholine receptors, voltage-gated sodium channels, a GABA transporter, a G protein-gated potassium channel, and calcium channels. His interests also extended to synaptic plasticity, and he helped to define the role of neuronal nitric oxide synthase and of trkB receptors. His final experiments concerned the role of protein kinase A in long-term potentiation. (The abstract was written posthumously by a colleague.
Secondary structures in polyoma DNA
Three reproducible secondary-structure features were observed on single strands of polyoma virus DNA mounted for electron microscopy by the T4 gene 32 protein technique: (i) a hairpin fold-back extending from 92.9 +/- 0.8 to 95.0 +/- 0.7 map units; (ii) a small loop extending from 63.2 +/- 3.1 to 68.5 +/- 2.8 map units; and (iii) a big loop extending from 51.9 +/- 2.3 to 68.9 +/- 2.1 map units. Both loops are bounded by inverted repeat stems of length 40 +/- 20 base pairs. The stem sequences around 68.5 and 68.9 of the large and small loops overlap, either partially or completely. Several lines of evidence indicate that the inverted repeat stems of the two secondary-structure loops lie in the regions of polyoma virus DNA flanking and probably very close to the sequences that are spliced out in the formation of the late 16S and 18S messages, whereas the hairpin fold-back appears to map at a splicing point of an early message. These structures may therefore be important for the processing of the primary transcripts to form the early and late messages
Evidence that neuronal G-protein-gated inwardly rectifying K+ channels are activated by Gβγ subunits and function as heteromultimers
Guanine nucleotide-binding proteins (G proteins) activate K+ conductances in cardiac atrial cells to slow heart rate and in neurons to decrease excitability. cDNAs encoding three isoforms of a G-protein-coupled, inwardly rectifying K+ channel (GIRK) have recently been cloned from cardiac (GIRK1/Kir 3.1) and brain cDNA libraries (GIRK2/Kir 3.2 and GIRK3/Kir 3.3). Here we report that GIRK2 but not GIRK3 can be activated by G protein subunits Gβ1 and G2 in Xenopus oocytes. Furthermore, when either GIRK3 or GIRK2 was coexpressed with GIRK1 and activated either by muscarinic receptors or by Gβ subunits, G-protein-mediated inward currents were increased by 5- to 40-fold. The single-channel conductance for GIRK1 plus GIRK2 coexpression was intermediate between those for GIRK1 alone and for GIRK2 alone, and voltage-jump kinetics for the coexpressed channels displayed new kinetic properties. On the other hand, coexpression of GIRK3 with GIRK2 suppressed the GIRK2 alone response. These studies suggest that formation of heteromultimers involving the several GIRKs is an important mechanism for generating diversity in expression level and function of neurotransmitter-coupled, inward rectifier K+ channels
Heteroduplex analysis of tra delta f' plasmids and the mechanism of their formation
Four tra delta FargG+ plasmids, derived from matings between Hfr AB312 and a recA recipient, have been shown to have deletions of at least 50% of the F genome, including the region in which the tra genes map. The mutant plasmids do contain the F genes required for plasmid maintenance. Correlations can be made between, on the one hand, the F genes present on the tradelta F' plasmids and the F genes transferred early by an Hfr donor, and, on the other hand, the F genes deleted from the tradelta F' plasmids and the F genes transferred late by an Hfr donor. A biased representation of proximally and distally transferred chromosomal markers among the tradelta F' elements was also demonstrated. Taken Taken together, the asymmetrical representation of Hfr genes and the cis dominance of the Tra phenotype of these mutants can best be explained by the hypothesis that the tradelta F' plasmids are formed by repliconation of the transferred exogenote in a recA recipient
Electron Microscope Studies of Heteroduplex DNA from a Deletion Mutant of Bacteriophage phi X-174
A population of double-stranded replicative form of DNA molecules from bacteriophage phi X-174 carrying a deletion of about 9% of the wild-type DNA has been discovered in a sample cultivated under conditions where the phage lysozyme gene is nonessential. The structures of deleted monomers, dimers, and trimers were studied by the electron microscope heteroduplex method. The dimers and trimers are head-to-tail repeats of the deleted monomers. Some interesting examples of the dynamical phenomenon of branch migration in vitro have been observed in heteroduplexes of deleted dimer and trimer strands with undeleted monomer viral strands from the wild-type phage
The isolation and properties of the enzyme uricase
A method has been evolved whereby the enzyme
uricase can be isolated from pig liver, and a preparation
500 - 700 times more active than the starting
material has been obtained. The purest preparatlon
has a specific activity of 85 - 90 μl. per mg . per
min . compared with 0.12 - 0 .15 μ1. per mg . per min.
for the dry liver powder used as starting material.The pure enzyme is a white protein, insoluble in
water , almost insoluble in phosphate buffer pH 7.4
but soluble in alkaline solutions such as borate buffer
pH 10 . Solutions of the enzyme are almost colourless .
The enzyme contains 0 .15 - 0.20% iron , a mere trace of
copper, no cobalt or manganese, and 14.4% of nitrogen .The activity of the enzyme is retained for several
weeks when it is preserved in t he form of a solution
in borate buffer at 0° but the free protein loses its
activity more rapidly . The enzyme cannot be dried
without great loss of activity.The velocity of the enzyme action is proportional
to the oxygen pressure being only 7% as great in a
mixture of 2% oxygen and 98% argon as in 100% oxygen.
When argon is replaced by carbon monoxide no inhibition
occurs .On the other hand the enzyme is completely inhibited
by cyanides in a concentration as low as
M/20,000.Inhibition by cyanide suggests that the enzyme is
a heavy metal compound and the possibility exists that
iron is the active group of the enzyme although thi s
has not been conclusively proved.The iron appears to be tightly bound to the protein
and all attempts to remove it have failed
alphabeta sequence of F is IS31
Previous studies have shown that there is a deoxyribonucleic acid (DNA) segment, of length 1.3 kb and denoted as the alphabeta sequence, which occurs twice on the F plasmid at corrdinates 93.2 to 94.5/OF kb and 13.7 to 15.0F kb. In the present investigation, heteroduplexes were prepared between a phage DNA carrying the insertion sequence IS3 and suitable F-prime DNAs. The hybrids formed show that IS3 is the same as alphabeta. This result plus previous studies support the view that: (i) the insertion sequence IS2 and IS3 occur on F and, in multiple copies, on the main bacterial chromosome of Escherichia coli K-12; and (ii)these IS sequences on the main bacterial chromosomes are hot spots for Hfr formation by reciprocal recombination with the corresponding sequences of F
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