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Biomedical subjects

J Griffith

Publications and source records attributed to J Griffith.

At least 199 records · Page 11Linked to original sources
PubMed1982

Electron microscopic visualization of recA-DNA filaments: evidence for a cyclic extension of duplex DNA.

As visualized by electron microscopy, RecA protein binds in a highly cooperative manner to single-stranded fd DNA in solutions of 0.01 M Tris (pH 7.5). The resulting nucleoprotein filament loops are 1.25 micrometers in length, have a fiber diameter of 12 nm and show an indication of a 4.5 nm repeat along the axis of the compact fibers. RecA binds to linear duplex fd DNA in solutions of 0.01 M Tris (pH 7.5) to yield chains of beads which, in the presence of Mg2+ and ATP, coalesce into smooth filaments with a length of 1.9 micrometers (the length of protein-free fd duplex DNA) and have a fiber diameter of 12 nm. In solutions containing Mg2+ and ATP-gamma-S, however, RecA binds to duplex DNA in a highly cooperative manner to yield rigid filaments 3.0 micrometers in length. These filaments are 12 nm in diameter and show a very clear 7.5 nm axial repeat. This extension of DNA to 150% of its usual length in the apparent absence of any single-stranded components suggests that the DNA helix must also be highly unwound and provides new insights into the mode of RecA action.

Adenosine Triphosphate↗
PubMed1982

Escherichia coli single-strand binding protein organizes single-stranded DNA in nucleosome-like units.

Electron microscopy shows that complexes of the single-strand DNA binding protein (SSB) of Escherichia coli and phage fd DNA appear as beaded fiber loops containing an average of 38 beads, 1 per 170 bases of DNA. Extensive digestion of native unfixed SSB-fd DNA complexes with micrococcal nuclease reveals a protected DNA fragment of 145 bases, while shorter digestion periods result in a sequence of fragments in multiples of 160 +/- 25 bases. Digestion of these complexes with DNase I produces a repeating pattern of bands, multiples of approximately 15 bases with strong bands at 60, 105, 118, 130, 145, 150, and 210 bases. Isopycnic banding in CsCl solution yields densities of 1.272 and 1.700 g/ml, respectively, for SSB alone and for fd DNA and, after fixation, of 1.388 g/ml for fd DNA-SSB beaded fibers and 1.373 g/ml for the individual protein-DNA beads. Based on these data and the molecular weights of SSB and fd DNA, we suggest that the nucleoprotein chain consists of eight molecules of SSB bound to 145 bases of DNA, with these units linked by roughly 30 bases of protein-free DNA. The excellent concord between results obtained by enzyme digestion of unfixed native samples and, after fixation, by electron microscopy and density banding supports the conclusion that SSB organizes single-stranded DNA in a manner similar to the organization of duplex DNA by histones.

Carrier Proteins↗
PubMed1981

Filamentous bacteriophage contract into hollow spherical particles upon exposure to a chloroform-water interface.

The bacteriophage M13 is a 1 micrometer long filament consisting of a circular single-stranded DNA loop firmly held within a tubular protein and capsid. We report here that exposure to a chloroform-water interface initiates a 20 fold contraction of each filament into a hollow protein sphere. In these 0.04 micrometer diameter particles, termed M13 "spheroids," two thirds of the DNA is apparently extruded through a hole in the wall of the spheroid; the portion of DNA remaining inside the shell centers about the origins of M13 DNA replication. These results suggest that the filament, upon exposure to a membrane environment, undergoes an ordered change whereby the DNA is released into the cell and the coat protein is changed to a form more easily solubilized by the membrane lipids.

Binding Sites↗
PubMed1981

Mechanism of coliphage M13 contraction: intermediate structures trapped at low temperatures.

The filamentous coliphage M13 can be transformed into a spherical particle (termed spheroid) by exposure to an interface of water and slightly polar but hydrophobic solvent such as chloroform-water at 24 degrees C. We report here that exposure of M13 filaments to a chloroform-water interface at 2 degrees C trapped the phage particles in forms morphologically intermediate to filaments and spheroids. These structures were rods 250 nm long and 15 nm wide, and each had a closed, slightly pointed end, an open flaired end, and a hollow central channel. The final contraction of these intermediates (termed I-forms) into spheroids was dependent upon both temperature and the presence of the solvent-water interface but was apparently independent of both the minor phage coat proteins and the virion DNA. Although stable in an aqueous environment, I-forms, in contrast to filaments, were readily disrupted by detergents, suggesting that the phage structure had been altered to a form more easily solubilized by membrane lipids. These solvent-induced changes might be related to the initial steps of phage penetration in vivo.

Chloroform↗
PubMed1977

Association of a protein structure of probable membrane derivation with HeLa cell mitochondrial DNA near its origin of replication.

Almost all (about 95%) of the mitochondrial DNA molecules released by Triton X-100 lysis of HeLa cell mitochondria in the presence of 0.15 M salt are associated with a single protein-containing structure varying in appearance between a 10-20 nm knob and a 100-500 nm membrane-like patch. Analysis by high resolution electron microscopy and by polyacrylamide gel electrophoresis after cleavage of mitochondrial DNA with the endonucleases EcoRI, HindIII, and Hpa II has shown that the protein structure is attached to the DNA in the region of the D-loop, and probably near the origin of mitochondrial DNA replication. The data strongly suggest that HeLa cell mitochondrial DNA is attached in vivo to the inner mitochondrial membrane at or near the origin of replication, and that a membrane fragment of variable size remains associated with the DNA during the isolation. After sodium dodecyl sulfate extraction of mitochondrial DNA, a small 5-10 nm protein is found at the same site on a fraction of the mitochondrial DNA molecules.

DNA Replication↗
PubMed1977

phiX174 cistron A protein is a multifunctional enzyme in DNA replication.

The cistron A protein induced by phage varphiX174 nicks (produces a single-strand break in) the viral strand of the superhelical varphiX duplex DNA, thereby forming a complex with the DNA. The protein, seen bound to the DNA in the electron microscope, was located in the restriction endonuclease fragment between nucleotides 4290 and 4330 on the varphiX map [Sanger, F., Air, G. M., Barrel, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison, C. A., III, Slocomb, P. M. Y. & Smith, M. (1977) Nature 265, 687-695]. Replication also was initiated at this point, thus identifying the site of cistron A protein nicking and binding as the origin of replication. The cisA-DNA complex (separated from free cistron A protein), upon the addition of Escherichia coli rep protein, ATP, and DNA binding protein, is unwound to generate a single-stranded linear [presumably the nicked (+) strand] and a circular [presumably the (-) strand] molecule. The cisA-DNA complex, upon the further addition of DNA polymerase III holoenzyme and deoxynucleoside triphosphates, supports replication to generate viral, single-stranded circles, as many as 15 circles per cisA-DNA complex. The replicating intermediates seen in the electron microscope are a novel form of "rolling circle" [Gilbert, W. & Dressler, D. H. (1969) Cold Spring Harbor Symp. Quant. Biol. 33, 473-485]. The 5' end (presumably with the cistron A protein bound to it) is locked in the replication fork and loops back to accompany the strand-separation and replication fork around the template [(-) strand] circle. Thus, the multiple functions of cistron A protein include: (i) nicking the viral strand at the origin of replication to initiate a round of replication, (ii) participating in a complex which supports fork movement in strand separation and replication, (iii) nicking again at the regenerated origin to produce a unit-length DNA, and (iv) ligating the newly generated 3'-OH end to the 5'-phosphate-complexed end to form a circular viral molecule.

Coliphages↗
PubMed1977

Salt and divalent cations affect the flexible nature of the natural beaded chromatin structure.

A natural chromatin containing simian virus 40 (SV40) DNA and histone has been used to examine changes in chromatin structure caused by various physical and chemical treatments. We find that histone H1 depleted chromatin is more compact in solutions of 0.15M NaCl or 2 mM MgCl2 than in 0.01 M NaCl or 0.6M NaCL, and is compact in 0.01 M NaCl solutions if histone H 1 is present. Even high concentrations of urea did not alter the fundamental beaded structure, consisting of 110A beads of 200 base pair content, each joined by thin DNA bridges of 50 base pairs. The physical bead observed by EM therefore contains more DNA than the 140 base pair "core particle". The natural variation in the bridge length is consistent with the broad bands observed after nuclease digestion of chromatin. Chromatin prepared for EM without fixation containing long 20A to 30A fibers possibly complexed with protein.

Cations, Divalent↗
PubMed1977

Characterization of components released by alkali disruption of simian virus 40.

Treatment of simian virus 40 (SV40) particles at pH 9.8 in the presence of 1 mM dithiothreitol for 5 min at 37 degrees C disrupted the virions into a 60S DNA-protein complex and DNA-free 7S protein particles. The DNA-protein complex contained approximately equal amounts of DNA and protein, and appeared by electron microscopy to be relaxed circular structures with an average of 21 beads joined by short, thin bridges. The major protein components in the complex were host cell histones, but SV40-specific proteins VP3 and VP2 were also present. The 7S protein particles were almost exclusively VP1 and, in negatively stained samples, resembled the capsomer structures of intact virions.

DNA, Circular↗
PubMed1975

Electron microscope localization of a protein bound near the origin of simian virus 40 DNA replication.

A salt-stable complex of protein and viral DNA obtained from Simian virus 40 (SV40)-infected monkey cells or mature SV40 virions has a novel structure. When viewed by high resolution electron microscopy, the circular SV40 DNA molecule has bound to it one to three globular protein "knobs". Using ecoRI and hpaII restriction endonucleases, each of which can cleave SV40 DNA once at a known location (10, 11, 12, 14), the bound protein can be localized at 0.7 plus or minis 0.05 on the SV40 DNA physical map (SV40 fractional length, clockwise from the ecoRI endonuclease-cleavage site).

Animals↗