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Biomedical subjects

R A Laskey

Publications and source records attributed to R A Laskey.

At least 91 records · Page 5Linked to original sources
PubMed1980

Nucleosome assembly.

Histones and DNA can spontaneously associate to form the nucleosome subunits of eukaryotic chromatin, but two proteins which occur in the eukaryotic nucleus can facilitate nucleosome assembly and greatly extend the conditions which permit assembly to occur. Recently several laboratories have reported new experimental systems which help to analyse, reconsruct and exploit the cellular mechanisms of chromatin assembly.

Animals↗
PubMed1980

Regulated replication of DNA microinjected into eggs of Xenopus laevis.

Purified circular DNA of SV40 or polyoma virus has been injected into unfertilized eggs of Xenopus laevis. Injected DNA initiates and completes multiple rounds of semiconservative replication while observing cellular regulatory signals. Thus replication initiation of double-stranded templates is induced after the oocyte is matured in vitro by progesterone. Only one round of replication of injected DNA is observed in a single cell cycle. When protein synthesis is inhibited unreplicated molecules continue to initiate replication at an undiminished rate, but reinitiation on previously replicated molecules is completely and selectively abolished. The DNA sequence requirements for the replication of injected DNA have been investigated. A variety of procaryotic DNA molecules and circularized fragments of SV40 or polyoma DNA replicate, regardless of whether they contain the viral origin of DNA replication. These results suggest that a specialized DNA sequence is not essential for the initiation of semiconservative DNA replication in the Xenopus embryo, nor is a specialized sequence essential for the mechanism which prevents reinitiation on a molecule which has already replicated within a cell cycle. The possibility is discussed that viral origins of replication are not valid models for the eucaryotic chromosome but are adaptations for uncoupling viral replication from the mechanism which prevents reinitiation within a cell cycle.

Animals↗
PubMed1980

Assembly of nucleosomes: the reaction involving X. laevis nucleoplasmin.

We analyze the nucleosome core assembly reaction which is mediated in vitro by a protein previously purified from Xenopus laevis eggs, now named nucleoplasmin in reference to its occurrence in the soluble phase of the nucleus of a wide range of vertebrate cell types. Nucleoplasmin is present in solution as a pentamer. We use nuclease digestion analysis to show that the protein assembles bona fide nucleosome cores in vitro from purified histones and DNA. Nucleoplasmin itself binds neither to DNA nor to the nucleoprotein particles which it assembles in vitro. However, it interacts with histones in vitro in such a way that histones no longer adhere to negatively charged surfaces. We have found no evidence for sterically specific interactions with particular histones. The initial rate of the nucleosome core assembly reaction mediated by purified nucleoplasmin in vitro is essentially identical with the rate of the nucleosome assembly reaction which occurs in the cell-free extracts of Xenopus eggs from which nucleoplasmin was purified. This rate is sufficient to account for the rate of nucleosome assembly required during the early development of Xenopus embryos.

Amino Acids↗
PubMed1977

Assembly of SV40 chromatin in a cell-free system from Xenopus eggs.

A cell-free system is described which assembles chromatin from purified DNA in 1 hr under physiological incubation conditions. It consists of a 145,000 x g (maximum) supernatant fraction from eggs of Xenopus laevis. It converts SV40 DNA to a nucleoprotein which co-sediments with naturally occurring SV40 chromatin and which can be cleaved by micrococcal nuclease to a highly ordered pattern of DNA fragments resembling those from digestion of liver chromatin. It inserts superhelical turns into relaxed, covalently closed DNA. The assembly process is not cooperative. Under limiting conditions, each DNA molecule becomes partially assembled. Assembly does not require replication of the DNA or protein synthesis, but occurs from a stored histone pool of at least 40 ng per egg. Under conditions of DNA excess, assembly becomes dependent upon the amount of exogenous histones added to the incubation. Apart from histones and a nicking-closing activity, chromatin assembly requires an additonal thermolabile factor which is present in the egg supernatant.

Animals↗
PubMed1977

Injected amphibian oocytes: a living test tube for the study of eukaryotic gene transcription?

Somatic-cell nuclei injected in Xenopus laevis oocytes remain transcriptionally active for up to 4 weeks. The rate of RNA synthesis increases as the somatic-cell nuclei enlarge. The message activity of the RNA synthesized by the injected nuclei can be demonstrated, as new proteins are made a few days after injection of HeLa-cell nuclei. Several lines of evidence demonstrate that these proteins are coded for by the HeLa-cell genome and that they result from RNA synthesized within the oocytes in the course of several days. Furthermore gene expression by the somatic-cell nuclei is selective, since only a few gene products can be detected. The oocyte cytoplasm is able to reprogramme the injected nuclei in such a way that only genes of the type normally active in oocytes are expressed. We describe experiments which indicate that genes previously inactive in the somatic cells can be switched on by the oocyte cytoplasm. These experiments have involved the use of the frog somatic-cell nuclei injected into newt oocytes. In addition, we have also found that even purified DNA is transcribed when injected into the nucleus of Xenopus oocytes. The identification of the molecules presumed to regulate chromatin transcription has been hampered by the lack of a satisfactory experimental system for assaying the biological activity of purified macromolecules. Living oocytes injected with somatic-cell nuclei (or cloned DNA) may provide such a system, since these regulatory factors could be introduced by a second injection.

Animals↗
PubMed1975

Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography.

Methods which use the scintillator PPO to record film images of 3H in chromatograms and polyacrylamide gels (fluorography) have been described elsewhere. This paper demonstrates that pre-exposure of the film to a brief flash of light greatly increases the sensitivity of fluorography. Pre-exposure also permits quantitative interpretation of the film image, because it corrects the non-linear relationship between radioactivity of the sample and absorbance of the film image. Therefore the distribution of radioactivity in the sample is accurately represented by microdensitometry of the image obtained on pre-exposed film. Using pre-exposed film 300 dis. 3H/min or 30 dis. 14C/min can be detected in a band in a gel in a 24-h exposure. The Appendix describes revisions and extensions of existing fluorographic procedures, including application to agarose gels and a rapid procedure for recovering PPO for re-use.

Carbon Radioisotopes↗
PubMed1975

The developmental capacity of nuclei transplanted from keratinized skin cells of adult frogs.

Nuclei from keratinized skin cells of adult Xenopus foot-webs have been transplanted to enucleated eggs of the same species. 2. The cells used to provide donor nuclei were obtained as a monolayer outgrowth from cultured foot-web explants. When explants were cultured without plasma for 3 days, over 99-9% of the outgrowth cells contained keratin as revealed by the binding of monospecific fluorescent antibody prepared against purified Xenopus keratin. Nuclei were transplanted from cells which had been cultured for 31/2 days. 3. None of the first transfer embryos developed as far as tadpoles. Eleven clones of embryos were prepared from the nuclei of partial first-transfer blastulae by use of serial nuclear transplantation. Eight of these clones contained swimming tadpoles with functional muscle and nerve cells, and six clones contained tadpoles with beating hearts, well differentiated eyes, and other organs. 4. To prove that the nuclei of nuclear-transplant tadpoles were derived from the transplanted skin cell nuclei and not from a failure of ultraviolet light to inactivate the recipient egg nucleus, 1-nu skin cell nuclei were transplanted to eggs laid by 2-nu frogs. Several advanced tadpoles from six clones were analysed for nucleolar and chromosome number and found to be 1-nu diploids. 5. The six clones of advanced tadpoles which were proved to carry the donor nuclear marker represent six first-transfer nuclei in a total sample of 129 skin cell nuclei originally transplanted. The probability that all six nuclei were derived from the 0-1% of the donor cell population not proved to contain keratin is less than one in 10(10). 6. We conclude that cell specialization does not involve any loss, irreversible inactivation or permanent change in chromosomal genes required for development.

Animals↗
PubMed1975

In vitro differentiation of a homogeneous cell population--the epidermis of Xenopus laevis.

A simple culture technique is described for obtaining homogeneous populations of differentiating epidermal cells from adult amphibian skin. This population of cells continues its normal differentiation, namely keratinization, in vitro. By the third day of culture more than 99-9% of the cells in the population contain immunoreactive keratin. During the next 5-7 days of culture these cells synthesize increasing amounts of a tissue-specific keratin-like protein until this protein constitutes more than 25% of the total extractable carboxymethylated proteins of these cells. Under the conditions described cell division does not occur during the course of differentiation. When vitamin A is added to the culture medium the rate of keratinization is decreased; when its antagonist citral is added, keratinization is accelerated. These factors influence only the rate but not the direction of differentiation.

Animals↗