Showing papers by "Cold Spring Harbor Laboratory published in 1975"

Mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments.

Abstract: Cytoplasmic RNA, isolated from cells late after infection by adenovirus type 2 and fractionated by hybridization to specific fragments of adenovirus DNA produced by cleavage with the endonuclease R-EcoRI, was used as template for protein synthesis in cell-free mammalian extracts. Each of the R-EcoRI fragments of DNA selects RNA that encodes specific subsets of the viral polypeptides. From the known order of the R-EcoRI fragments, the following partial map is deduced: (III, IIIa, IVa2, V, P-VII, IX), (II, P-VI), 100K, IV-where the relative order of the components enclosed in parentheses has not yet been determined.

Association of actin and myosin with secretory granule membranes.

Abstract: ACTIN and myosin have been found in many non-muscle cells1. They are presumed to function in the generation of the force required in the various expressions of cytoplasmic or cell motility. Although both have been found in association with the plasma membrane2–5, nothing is known about the nature or consequences of this association. Equally uncertain is the mechanism of movement of secretory granules : synaptic vesicles, for example, are transported along neurones at high rates (up to 20 cm d−1)6, probably in association with microtubules7. Roles for actomyosin in this process8 and in exocytosis9 have been proposed. We have used a new approach to the problem of actomyosin-membrane interaction, and report here the binding of actin and myosin in vitro to a purified cytoplasmic membrane component.

Slab gel electrophoresis apparatus

Abstract: Apparatus for vertical slab gel electrophoresis has upper and lower tanks having open tops. The tanks are supported in stepped relation to each other by a vertical support plate which forms a front wall of one tank and a rear wall of the other. At least part of the wall of the upper tank formed by the support plate has a top edge which is lower than the other top edges of the tank. A substantially open-ended chamber is formed by clamping a plastic plate to a glass plate with strip members between side edges of the plates. The plastic plate is held against the spport plate for reinforcing the rigidity of the plastic plate with the support plate. A portion of the top edge of the plastic plate is lower than the top edge of the glass plate and intermediate the lower edge portion of the wall of the upper tank formed by the support plate and the other top edges of the upper tank. Partitions extend across the chamber from the plastic plate to the glass plate and down into the chamber from the top, lower edge of the plastic plate at least to a slab of gel which fills the chamber to form wells over the gel for receiving samples. An electrolytic buffer solution is placed in both tanks, the solution in the upper tank flowing over the lower edges of the support plate and plastic plate to the glass plate to cover the samples in the wells. Opposite electric potentials of the appropriate polarity are then applied to electrodes in both tanks to cause the samples to migrate electrophoretically into the gel, the position of the samples in the gel then being an indication of the composition of the samples.

Isolation and characterization of revertant cell lines. VII. DNA synthesis and mitotic rate of serum-sensitive revertants in non-permissive growth conditions

Abstract: The ability to synthesize DNA and enter mitosis was studied in Balb/c and Swiss 3T3 cells, SV40 and MSV-transformed 3T3 cells and revertants of these transformed cells in cultures of different serum concentrations and cell densities. Three ways were found by which cells were able to maintain a constant cell number in non-permissive growth conditions: cessation of DNA synthesis, synthesis of DNA coupled with failure to enter mitosis, and the slow traverse of the cell cycle coupled with cell shedding. Growth control of the revertant of an MSV-transformed Balb/3T3 cell most closely resembled that of Balb or Swiss 3T3. This line did not grow in 1% serum and did not synthesize DNA in either non-permissive condition. Serum-sensitive revertants of SV40-transformed 3T3 cells are also unable to grow in 1% serum and also do not grow beyond confluence in 10% serum, but these cells differ from 3T3 in the manner in which this growth arrest is accomplished. In 1% serum, revertants synthesize DNA but do not enter mitosis. At confluence in 10% serum, they slowly traverse the cell cycle, with dividing cells replacing cells that are shed into the medium.

Mutation blocking the specific degradation of reinitiation polypeptides in E. coli.

Abstract: A NONSENSE mutation in a bacterial gene leads to the synthesis of two kinds of incomplete polypeptides1–4. These are the termination, or T, fragments extending from the normal N terminus to the site of mutation, and the reinitiation, or R, fragments initiating distal to the end of the T fragment and continuing to the normal C terminus. Both the T and the R fragments produced by nonsense mutations in the β-galactosidase gene of Escherichia coli (z gene) are rapidly and selectively degraded in vivo5–7. This contrasts strongly with the wild-type β-galactosidase, which is stable5,7. The degradation of mutant polypeptides implies the existence of proteolytic systems which can distinguish these unstable proteins from the stable ones.

The synthetase gene of the RNA phages R17, MS2 and f2 has a single UAG terminator codon

Abstract: Translation of the RNA from the wild-type bacteriophages R17, MS2, and f2 in bacterial cell-free extracts containing an amber suppressor yields 30-40% of the synthetase with an approximate molecular weight of 63 500, slightly larger than the major synthetase product (63 000 daltons). The occurrence of the 63 500 dalton in vitro product is dependent on the presence of an amber suppressor, and we predict that it is due to read-through of a UAG termination codon at the end of the synthetase gene. Previous results of Capecchi and Klein (Nature, 226, 1029-1033, 1070) showed that antibodies to both release factors RF1 and RF2 are required to block release of synthetase, suggesting that synthetase is released at a UAA codon. If the interpretations of both experiments are correct, the termination and release may not be synonomous and may be spatially separated. In addition there is the unexplained fact that 7% of the synthetase made in vitro in both su+ and su- extracts with either R17, MS2 or f2 as template has an apparent molecular weight of 66 000.

Transcription of SV40 in Lytically Infected and Transformed Cells

Abstract: As long as we take reasonable care, we can grow stocks of Simian Virus that are remarkably homogeneous. Each infectious particle contains a single molecule of DNA weighing 3.4 x 106 daltons—enough to code for perhaps 5–8 polypeptides of average size. The manner in which this virus-coded information is expressed is highly cell-dependent. For example, after infection with SV40, monkey cells undergo a productive or lytic response, during which there is an ordered appearance of virus-specific functions, with some virus products present at all times and others detectable only in the late stages of the viral growth cycle. The infection progresses through a well-defined series of episodes, which culminate in cell death and the concomitant liberation of a new crop of virus particles. The events are multiplicity-independent in that they occur in the same order, albeit at different rates, in cells infected either with one infectious particle or with a hundred. By contrast, the outcome of exposing mouse cells to SV40 is very different.

The Cell Nucleus

Abstract: The ultimate book on the nucleus would require about a million pages just to present the complete base sequence of DNA in a single human nucleus, alphabetically. The three volumes of The Cell Nucleus, of course, make no pretense of offering that kind of information, but they provide a wide range of up-to-date information on the structure and function of the nucleus.Behind the deceptively bland, lightmicroscopic image of the cell nucleus lies the complexity necessary to selectively transcribe portions of the million pages of information; to transmit the transcribed messages to the protein synthesizing mechanism in the cytoplasm; and, on occasion, to replicate the entire store of information errorlessly. The devices employed to perform these nuclear functions are under analysis, with an impressive array of techniques detailed in these volumes. Three of the more spectacular recent approaches can be mentioned here. Gurdon tells the story of the utilization of