Elton Stubblefield

A new method for the rapid isolation of chromosomes, mitotic apparatus, or nuclei from mammalian fibroblasts at near neutral pH☆

Abstract A rapid isolation procedure for chromosomes, mitotic apparatus, or nuclei from mammalian fibroblasts has been developed. It is accomplished at a near neutral pH in a simple buffer solution. The morphology of each of the components at light microscopy and electron microscopy levels is comparable to previously published isolation procedures. The DNA, RNA, and protein content of chromosomes and nuclei are similar to values obtained by other methods. Active nuclear enzymes are present in the nuclei, and cytoplasmic enzymes are excluded. It is possible to obtain as the desired end product either metaphase chromosomes, intact mitotic apparatus, or interphase nuclei by slight modifications of the isolation procedure.

The effects of Colcemid inhibition and reversal on the fine structure of the mitotic apparatus of Chinese hamster cells in vitro

Electron microscope studies of Chinese hamster cells treated with Colcemid at a concentration sufficient to prevent mitosis (0.06 μg/ml) demonstrated that this alkaloid does not completely prevent the formation of certain elements of the mitotic apparatus. Instead, an aberrant apparatus formed whose organization appeared to be dictated by the position of the centrioles. The latter components assumed a position near the cell center, and the remaining structures (microtubules, chromosomes, and other organelles) became radially distributed around them. When Colcemid was removed from the culture medium, the entire cell population completed mitosis synchronously within 30–45 minutes. However, a complete mitotic apparatus formed only after the centrioles had migrated to opposite ends of the cells. The present study describes the ultrastructure of cells during Colcemid inhibition and at various stages during reversal. Normal cell division and the equidistribution of genetic material into two daughter cells appeared to be greatly dependent upon centriole behavior during the initial stages of mitosis. It is proposed that Colcemid inhibits mitosis by preventing the formation of certain elements of the mitotic apparatus (perhaps the assembly of continuous spindle filaments) necessary for centriole movement.

The fine structure of the kinetochore of a mammalian cell in vitro.

The chromosomes of Chinese hamster cells were examined with the electron microscope and the following observations were made concerning the structure and organization of the kinetochore. — The kinetochore consists of a dense core 200–300 Å in diameter surrounded hy a less dense zone 200–600 Å wide. The dense core consists of a pair of axial fibrils 50–80 Å in diameter which may be coiled together in a cohelical manner. The less dense zone about the axial elements is composed of numerous microfibrils which loop out at right angles to the axial fibrils. Together the structures comprise a lampbrush-like filament which extends along the surface of each chromatid. Some sections suggested that two such filaments may be present on each chromatid. The fine structure of kinetochores associated with spindle filaments was essentially the same as those free of filaments. The structure and organization of the kinetochore of these mammalian cells was compared to that of lampbrush chromosomes of certain amphibian oöcytes, dipteran polytene chromosome puffs, and the synaptinemal complex seen during meiotic prophase.

Architecture of the Chinese hamster metaphase chromosome

The development of procedures for the isolation of unfixed metaphase chromosomes has made feasible a direct analysis of their morphology. Wholemount stereo electron microscopy was used to examine intact and partially disrupted chromosomes produced by physical shearing and extraction with salt and urea solutions. A model of chromosome architecture was developed to accommodate evidence from studies using both light and electron microscopy. In the proposed model the chromatid (anaphase chromosome) consists of two half-chromatids; each half-chromatid contains two deoxyribonucleoprotein ribbons wound into a single fiber (termed the core), with many loops of chromatin (termed epichromatin) attached along its length. The core ribbons are each about 50 Å thick by 4000 Å wide and are composed of many parallel deoxyribonucleoprotein strands. The epichromatin loops appear to be 250 Å supercoiled fibers containing about 75 per cent of the chromosomal DNA. The epichromatin can be selectively removed from the core fibers by extraction with 2.0 M NaCl or 6.0 M urea solutions.

The vertebrate homolog of the putative transforming gene of avian myelocytomatosis virus: Characteristics of the DNA locus and its RNA transcript

Abstract Vertebrate genomes contain nucleotide sequences homologous to the putative transforming genes of at least several avian retroviruses. Each of these homologs is highly conserved throughout vertebrates; each may function during the course of normal cellular growth and development; and each may have served as a progenitor for a viral oncogene. The present communication substantiates and enlarges upon these conclusions by providing further characterization of proto-onc MCV , the cellular homolog of the putative transforming gene of avian myelocytomatosis virus (MC29V). We used three experimental approaches to characterize proto-onc MCV . First, we analyzed proto-onc MCV in DNA from each of 18 chickens by using restriction endonucleases. Our results suggest that proto-onc MCV is situated at a single constant locus in the chicken genome. By contrast, the number and location of loci harboring endogenous retrovirus genes varies from one chicken to another (Hughes et al. , 1979a). Second, we fractionated chicken chromosomes by rate-zonal centrifugation and found that proto-onc MCV is not linked to proto-src , the cellular homolog of the transforming gene of avian sarcoma virus. Third, we demonstrated that the principal transcript from proto-onc MCV is a 2.8-kb polyadenylated RNA located primarily in the cytoplasm of chick fibroblasts. Embryonic RNA from other avian species also contained a 2.8-kb RNA that shared homology with proto-onc MCV . We conclude that proto-onc MCV , like proto-src , is a normal locus in the vertebrate genome whose expression may be essential to cellular growth and/or development.

Thymidine kinase activity in synchronized HeLa cell cultures.

Abstract Thymidine kinase activity increases in HeLa cells which have been treated with amethopterin to produce a thymidineless state. The addition of thymidine after 16 hours results in a synchronous wave of DNA synthesis during which the enzyme activity continues to increase. After division of the cells this activity drops to the level of logarithmically growing, control cultures.

Chicken macrochromosomes contain an endogenous provirus and microchromosomes contain sequences related to the transforming gene of ASV.

Chicken chromosomes from a euploid Mareks lymphoma cell line have been partially fractionated according to size by rate zonal centrifugation in a zonal rotor. DNA-DNA hybridization tests, using unlabeled DNA extracted from gradient fractions and labeled single-stranded, virus-specific DNAs prepared in vitro, indicate that large macrochromosomes harbor the provirus for the endogenous RNA tumor virus of chickens (RAVO), whereas a cellular sequence related to the transforming gene of avian sarcoma virus (ASV) is located in microchromosomes. In support of the method, we have also shown that the single gene for ovalbumin can be assigned to macrochromosomes.

Synchronized mammalian cell cultures:III. Variation of ribonucleotide reductase activity during the replication cycle of Chinese hamster fibroblasts

Abstract Ribonucleotide reductase activity was studied during the replication cycle of Colcemid synchronized Chinese hamster fibroblasts in culture. Enzyme activity dropped to a minimum value during the G1 phase, returned to the level observed in the metaphase cells as the cells entered the S phase, and continued to increase during the S and G2 phases. Actinomycin D, cycloheximide, and hydroxyurea were added to the cell cultures at the time of lowest activity (1.5 h after synchronization). With over 90 % inhibition of RNA synthesis by actinomycin D, enzyme activity returned by 4.5 h to the level observed in the metaphase cells, but a further increase was not seen. With inhibition of over 90 % of protein synthesis by cycloheximide, enzyme activity did not rise from the low level observed in the G1 phase. The addition of hydroxyurea produced an elevated level of reductase activity which rose during the S-phase to more than twice that of the control. Experiments in which extracts from cultures at different time points were mixed and assayed indicated that an inhibitor was not reponsible for the low levels of activity during the G1 phase.

DNA SYNTHESIS AND CHROMOSOMAL MORPHOLOGY OF CHINESE HAMSTER CELLS CULTURED IN MEDIA CONTAINING N-DEACETYL-N-METHYLCOLCHICINE (COLCEMID) 1

Publisher Summary This chapter describes the DNA synthesis and chromosomal morphology of Chinese hamster cells cultured in media containing N-deacetyl-N-methylcolchicine (Colcemid). Colcemid and the parent compound colchicine have been used in cytogenetics for many years. Almost all chromosomal studies of mammalian tissue culture cells employ one of these drugs to arrest metaphase. However, in spite of widespread use, careful studies of the many effects of these drugs in mammalian tissue culture systems are sparse in the literature. The chapter presents a study in which a careful analysis of a time-lapse film showing the effects of Colcemid on the mitotic index of a culture was made. All the studies reported here were made with a male diploid cell line designated as strain “Don” grown in McCoys medium 5a supplemented with 20 per cent fetal calf serum. Stocks were maintained as monolayer cultures and were routinely sub-cultured by trypsinization every 2 or 3 days. Time-lapse motion pictures were taken using cultures grown in T-30 flasks modified to permit phase-contrast microscopy. In the observed field, 163 cells could be studied throughout the duration of the first metaphase. The number of mitotic cells increased rapidly for about 8 h and then more slowly until about 15 h, when about half of the cells were in metaphase. As the metaphase cells returned to interphase, the mitotic index dropped. By 45 h, all of the cells examined had succeeded in circumventing the mitotic inhibitor and had returned to the interphase state.

Quantitative tritium autoradiography of mammalian chromosomes

The rates of tritiated thymidine accumulation in each of the chromosome types in Chinese hamster line Don and strain Don-C have been assayed by quantitative tritium autoradiography. The late-replicating nature of the X and Y chromosomes is readily apparent. Many chromosomes exhibit three separate steps of synthesis, with reduced rates of thymidine incorporation between 3 and 4 hours and again between 5 and 6 hours. The same three-step pattern can be seen in scintillation data from FUdR synchronized cells, with 40% of the DNA made in each of the first two steps and 20% in the final step.