David M. Prescott

Enucleation of mammalian cells with cytochalasin B

Abstract L cells and chinese hamster cells growing on coverslips can be enucleated by centrifugation at 3 000 g (glass coverslips) or 5 800 g (plastic coverslips) for 40 min at 35 ° to 37 °C in medium containing 10 μg/ml of cytochalasin B. The enucleated cells incorporate 3H-leucine, support replication of vaccinia virus DNA, and are viable for at least 3 days.

Genome gymnastics: unique modes of dna evolution and processing in ciliates

In some ciliates, the DNA sequences of the germline genomes have been profoundly modified during evolution, providing unprecedented examples of germline DNA malleability. Although the significance of the modifications and malleability is unclear, they may reflect the evolution of mechanisms that facilitate evolution. Because of the modifications, these ciliates must perform remarkable feats of cutting, splicing, rearrangement and elimination of DNA sequences to convert the chromosomal DNA in the germline genome (micronuclear genome) into gene-sized DNA molecules in the somatic genome (macronuclear genome). How these manipulations of DNA are guided and carried out is largely unknown. However, the organization and manipulation of ciliate DNA sequences are new phenomena that expand a general appreciation for the flexibility of DNA in evolution and development.

An evaluation of the double thymidine block for synchronizing mammalian cells at the G1-S border☆

The effect of a double 2 mM thymidine block on L cells and CHOP cells has been investigated. The S phase following release from the second 2 mM thymidine block is about 5 h which represents a shortening by about one-third. Unlabeled mitoses are observed in the first hours after release of the block and labeled mitoses are observed 5 h after the block, 32P-orthophosphate is incorporated in the DNA during the thymidine block at about one-third the rate for non-blocked, control cells. The results indicate that cells continue to synthesize DNA during the thymidine block at about one-third the normal rate and do not accumulate at the G 1-S border.

DNA of ciliated protozoa: DNA sequence diminution during macronuclear development of oxytricha

We have measured the reassociation kinetics of DNA from the micronucleus and from the macronucleus of the hypotrichous ciliate Oxytricha. The micronuclear DNA reassociates with at least a two-component reaction, indicating the presence of both repeated and non-repeated sequences. The kinetic complexity of micronuclear non-repeated DNA is in the range of 2 to 15 X 10(11) daltons; the haploid DNA content of the micronucleus is 4 X 10(11) daltons (0.66 pg), measured microspectrophotometrically. The DNA of the macronucleus reassociates as a single second-order reaction, with a kinetic complexity of 3.6 X 10(10) daltons. A comparison of the kinetic complexities of micronuclear and macronuclear DNAs suggest a 5 to 30 fold reduction in DNA sequence complexity during the formation of a macronucleus from a micronucleus. Macronuclear DNA is in pieces with an average molecular weight of 2.1 X 10(6) daltons. Since the kinetic complexity of macronuclear DNA is 3.6 X 10(10) daltons, the macronucleus must contain about 17,000 different kinds of DNA pieces. Each macronucleus contains 3.5 X 10(13) daltons (58 pg) of DNA, indicating that each sequence must be present about 1000 times per macronucleus or 2000 times per cell.

The Cell Cycle and the Control of Cellular Reproduction

Publisher Summary This chapter summarizes about the cell life cycle. An effort has been made to integrate the information into a general scheme of operation for the cell cycle with comments about mechanisms that may provide for cycle continuity and for interruption of the cycle in the regulation of cell reproduction. Cell cycle continuity is considered to be based on the sequential transcription and translation of the set of cell cycle genes that are held in temporal order by the activation of one cell cycle gene, by the function of an antecedently activated gene. The outlines of the cell cycle have been established, and this has led to clearer definition of the problems of cell growth and reproduction. The major tasks are to determine the basic causal sequence of molecular events that underlies the procession of G,-S-G2-D, to explain how the many, various growth activities participate in or are coordinated with this procession, and to discover how the cycle is arrested in G, to provide regulation of cell reproduction.

Cyclic AMP levels in synchronized mammalian cells

Abstract Cyclic adenosine 3′5′ monophosphate (cAMP) levels were measured in Chinese hamster ovary cells synchronized by mitotic shake-off. Comparing the various stages with the S period, we observed mitotic cells to have a depressed cAMP level, early G1 cells to have an elevated cAMP level, and the late G1 period to possess a cAMP level similar to cells in S phase. Isoleucine deficiency, which blocks cells in the G1 period, led to a depressed level of cAMP, possibly due to the inhibited protein synthesis which accompanies isoleucune deficiency.

Molecular operations for DNA processing in hypotrichous ciliates

Genes in the germline nucleus (micronucleus) of ciliates are interrupted by multiple, short, noncoding segments called internal eliminated segments, or IESs. During development of a somatic nucleus (macronucleus) from a micronucleus, IESs are spliced out of genes, and the gene segments separated by IESs, called macronuclear destined segments, or MDSs, are ligated to form transcriptionally competent genes. In some genes of hypotrichous ciliates the MDSs have been rearranged into a scrambled disorder. During macronuclear development scrambled MDSs are ligated in the orthodox order. Here we present a set of molecular operations that explain and account for IES excision, unscrambling of MDSs, and MDS ligation to accomplish gene assembly during macronuclear development. In the appendix we present a reasoning system that formalizes the gene assembly process based on these operations. The system presented here is applied to all known cases of gene assembly in hypotrichous ciliates.

Cellular Sites of RNA Synthesis

Publisher Summary The chapter discusses the more recent experimental work on the sites of RNA synthesis and the intracellular transport or redistribution of the RNA molecules. A primary question is whether all the cellular classes of RNA are synthesized under the immediate control of DNA templates or whether the cells contain mechanisms for self-replication of one or more cellular RNA types. A decisive answer is provided, by an abundance of new experimental work of a type representing, to a large degree, a cross between the biochemical and cytological approaches. A large body of evidence shows that the synthesis of all ribosomal ribonucleic acid (rRNA), molecular ribonucleic acid (mRNA), and small ribonucleic acid (sRNA) is immediately DNA-dependent and restricted to the cell nucleus. RNA synthesis occurs independently on both the chromosomes and in the nucleoli. Some evidence suggests that nucleoli may accumulate RNA from other parts of the nucleus, but there is no satisfactory evidence that nucleolar RNA passes to the chromosomes. There is no evidence that RNA of these classes can undergo self-replication, within the cells, and all cytoplasmic RNA must be derived from the nucleus.

Gene-sized DNA molecules of the macronuclei in three species of hypotrichs: Size distributions and absence of nicks

Macronuclear DNAs from three related hypotrichous ciliated protozoans were compared by agarose gel electrophoresis. Each was shown to be composed of DNA duplexes that yielded a unique pattern of bands overlying a continuous distribution of DNA sizes ranging from ∼400 bp to ∼20,000 bp. By EM, the number average molecular sizes for doublestranded DNA were 2,200 bp for Oxytricha sp., 2,514 bp for Stylonychia pustulata and 1,836 bp for Euplotes aediculatus. Contrary to previous reports we present evidence that the macronuclear DNAs in each of these three organisms lack single-stranded interruptions.

Induction of sister chromatid exchanges in chromosomes of rat kangaroo cells by tritium incorporated into DNA

Abstract The frequency of sister chromatid exchanges increases with increasing amounts of incorporation of 3H-thymidine into the chromosomes of a line of rat kangaroo cells. The relation only holds over a range of very low levels of incorporated radioactivity. Increases in the incorporated radioactivity above the amount necessary to produce about 0.4 grains/day along the length of the chromosome in autoradiographs does not result in any further increase in the frequency of exchanges. Sister chromatid exchanges therefore appear to be induced by the tritium that must be incorporated in order to detect the exchanges. The data suggest that all exchanges are radiation-induced, and that no exchanges occur spontaneously.