Secretory proteins in the reproductive tract of the snapping turtle, Chelhydra serpentina.

SDS-polyacrylamide gel electrophoresis was used to separate the secretory proteins produced by the epithelial and endometrial glands of the uterine tube and uterus in the snapping turtle Chelydra serpentina. The proteins were analyzed throughout the phases of the reproductive cycle from May to August, including preovulatory, ovulatory, postovulatory or luteal, and vitellogenic phases. The pattern of secretory proteins is quite uniform along the length of the uterine tube, and the same is true of the uterus, but the patterns for uterine tube and uterus are clearly different. We identify 13 major proteins in C. serpentina egg albumen. Bands co-migrating with 11 of these are found in the uterine tube, but at most 4 are found in the uterus, suggesting that the majority of the albumen proteins are most likely secreted in the uterine tube, not in the uterus. Although some of the egg albumen proteins are present in the uterine tube only at the time of ovulation, most of the bands corresponding to albumen proteins are present throughout the breeding season even though the snapping turtle is a monoclutch species. These results suggest that the glandular secretory phase in the uterine tube is active and quite homogeneous in function regardless of location or phase of the reproductive cycle.

On the structure of the scaffolding core of bacteriophage T4.

The scaffolding core in bacteriophages is a temporary structure that plays a major role in determining the shape of the protein shell that encapsulates the viral DNA. In the currently accepted structure for the scaffolding core in bacteriophage T4, there is a symmetry mismatch between the protein shell, which has fivefold symmetry, and the scaffolding core, which is believed to consist of six helical chains. The analysis of T4 giant prohead data that was used to determine this structure made an implicit assumption about the manner in which giant proheads flatten during preparation for electron microscopy. Namely, it was assumed that techniques for analysis of Fourier transforms of flattened single-layer cylinders could be applied independently to the shell and the core. This analysis makes the implicit assumption that connections between the core and the shell do not affect the flattening process, and thus are stretched or broken during the flattening process. Reexamination of the experimental data shows that this assumption is likely to be incorrect. A reanalysis shows that the data could be consistent with six, eight, or 10 helical chains, and is a better match for eight or 10 helical chains. Ten helical chains would match the fivefold symmetry of the shell. The 10-helix core model is particularly attractive because it suggests a Vernier mechanism, which is able to explain the process of length determination in giant head mutants of T4. It is possible that the same assumption has been made for structural analysis of other biological systems. If this is the case, any results obtained should also be reexamined.

Evidence that the endogenous histone H1 phosphatase in HeLa mitotic chromosomes is protein phosphatase 1, not protein phosphatase 2A.

Histone H1 is highly phosphorylated in mitotic HeLa cells, but is quickly dephosphorylated in vivo at the end of mitosis and in vitro following cell lysis. We show here that okadaic acid and microcystin-LR block the in vitro dephosphorylation of H1 and that they do so directly by inhibiting the histone H1 phosphatase rather than by some indirect mechanism. The concentrations of microcystin and okadaic acid required for inhibition strongly suggest that the histone H1 phosphatase is either PP1 or an unknown protein phosphatase with okadaic acid-sensitivity similar to PP1. The histone H1 phosphatase is predominantly located in chromosomes with at most one copy for every 86 nucleosomes. This tends to support its identification as PP1, since localization in mitotic chromosomes is a characteristic of PP1 but not of the other known okadaic acid-sensitive protein phosphatases. We also show that treatment of metaphase-arrested HeLa cells with staurosporine and olomoucine, inhibitors of p34cdc2 and other protein kinases, rapidly induces reassembly of interphase nuclei and dephosphorylation of histone H1 without chromosome segregation. This result indicates that protein kinase activity must remain elevated to maintain a mitotic block. Using this as a model system for the M- to G1-phase transition, we present evidence from inhibitor studies suggesting that the in vivo histone H1 phosphatase may be either PP1 or another phosphatase with similar okadaic acid-sensitivity, but not PP2A.

Okadaic acid induces dephosphorylation of histone H1 in metaphase-arrested HeLa cells.

It is shown here that treatment of metaphase-arrested HeLa cells with okadaic acid (0.15-2.5 microM) leads to dephosphorylation of histone H1. This effect is presumably due to the specific ability of okadaic acid to inhibit protein phosphatases 1 and/or 2A, because okadaic acid tetraacetate, which is not a phosphatase inhibitor, has no effect. Dephosphorylation of H1 does not occur if okadaic acid-treated cells are simultaneously treated with 20 nM calyculin A, or if the okadaic acid concentration is 5.0 microM or greater. The mechanism behind this phenomenon is not known. However, the results suggest that the chain of events leading to histone dephosphorylation may be negatively controlled by a protein phosphatase 2A, while the phosphatase which actually dephosphorylates H1 could be a protein phosphatase 1. It remains to be determined whether the phosphatase involved here is the same enzyme as that which dephosphorylates H1 at the end of normal mitosis.

Rapid analysis of mitotic histone H1 phosphorylation by cationic disc electrophoresis at neutral pH in minigels.

A new method is described for analysis of histone H1 and other basic proteins by cationic disc electrophoresis in polyacrylamide gels at neutral pH. The multiphasic buffer (disc) system uses Na+ as leading ion, L-histidine as trailing ion, and Hepes as buffering counterion. These "Hepes/histidine gels" have three advantages over conventional acid-urea gels for studies of H1 phosphorylation and dephosphorylation: speed, convenience, and the need for only small amounts of cells or chromatin. Core histones and their acetylated forms can also be separated in gels containing 0.4% Triton X-100. The difference in electrophoretic mobility between mitotic (superphosphorylated) and interphase H1 from HeLa cells is approximately twice as great at neutral pH as at pH 4.5, making it possible to separate these two H1 forms rapidly and easily in Hepes/histidine "minigels" only 5-cm long. Total histones can be rapidly prepared by simply neutralizing 0.2 N HCl extracts, and the entire analysis, from harvesting cells to destaining gels, can be carried out in 1 day. The stacking effect of the disc system produces sharp bands and high resolution even with relatively dilute samples.

Scaffold morphology in histone-depleted HeLa metaphase chromosomes.

In a study of HeLa metaphase chromosomes depleted of histones with 2M NaCl and spread with cytochrome c, two new types of images of chromosome scaffolds have been observed in the electron microscope. In the first type, scaffolds are very large and fibrous but still display the shape typical of metaphase chromosomes. The regularity and lack of distortion in these scaffolds, despite their openness and seeming fragility, support the notion that the underlying scaffold structure is an interconnected network of fibers. In the second type, fibrous regions and dense regions are juxtaposed in the same chromosome scaffold. These micrographs suggest that the dense appearance of some previously observed scaffolds may be the result of incomplete adherence to the cytochrome c monolayer, leading to collapse and aggregation during dehydration.

Low angle x-ray diffraction studies of HeLa metaphase chromosomes: effects of histone phosphorylation and chromosome isolation procedure.

To test whether gross changes in chromatin structure occur during the cell cycle, we compared HeLa mitotic metaphase chromosomes and interphase nuclei by low angle x-ray diffraction. Interphase nuclei and metaphase chromosomes differ only in the 30-40-nm packing reflection, but not in the higher angle part of the x-ray diffraction pattern. Our interpretation of these results is that the transition to metaphase affects only the packing of chromatin fibers and not, to the resolution of our method, the internal structure of nucleosomes or the pattern of nucleosome packing within chromatin fibers. In particular, phosphorylation of histones H1 and H3 at mitosis does not affect chromatin fiber structure, since the same x-ray results are obtained whether or not histone dephosphorylation is prevented by isolating metaphase chromosomes in the presence of 5,5'-dithiobis(2-nitrobenzoate) or low concentrations of p-chloromercuriphenylsulfonate (ClHgPhSO3). We also compared metaphase chromosomes isolated by several different published procedures, and found that the isolation procedure can significantly affect the x-ray diffraction pattern. High concentrations of ClHgPhSO3 can also profoundly affect the pattern.

Low angle x-ray diffraction studies of chromatin structure in vivo and in isolated nuclei and metaphase chromosomes.

Diffraction of x-rays from living cells, isolated nuclei, and metaphase chromosomes gives rise to several major low angle reflections characteristic of a highly conserved pattern of nucleosome packing within the chromatin fibers. We answer three questions about the x-ray data: Which reflections are characteristic of chromosomes in vivo? How can these reflections be preserved in vitro? What chromosome structures give rise to the reflections? Our consistent observation of diffraction peaks at 11.0, 6.0, 3.8, 2.7 and 2.1 nm from a variety of living cells, isolated nuclei, and metaphase chromosomes establishes these periodicities as characteristic of eukaryotic chromosomes in vivo. In addition, a 30-40- nm peak is observed from all somatic cells that have substantial amounts of condensed chromatin, and a weak 18-nm reflection is observed from nucleated erythrocytes. These observations provide a standard for judging the structural integrity of isolated nuclei, chromosomes, and chromatin, and thus resolve long standing controversy about the "tru" nature of chromosome diffraction. All of the reflection seen in vivo can be preserved in vitro provided that the proper ionic conditions are maintained. Our results show clearly that the 30-40-nm maximum is a packing reflection. The packing we observe in vivo is directly correlated to the side-by-side arrangement of 20- 30-nm fibers observed in thin sections of fixed and dehydrated cells and isolated chromosomes. This confirms that such packing is present in living cells and is not merely an artifact of electron microscopy. As expected, the packing reflection is shifted to longer spacings when the fibers are spread apart by reducing the concentration of divalent cations in vitro. Because the 18-, 11.0-, 6.0-, 3.8-, 2.7-, and 2.1-nm reflections are not affected by the decondensation caused by removal of divalent cations, these periodicities must reflect the internal structure of the chromaticn fibers.

Phosphorylation of histones 1 and 3 and nonhistone high mobility group 14 by an endogenous kinase in HeLa metaphase chromosomes.

We have developed a simple in vitro system for studying phosphorylation in isolated HeLa metaphase chromosomes which utilizes the endogenous protein kinase and phosphoprotein phosphatase activities in the chromosomes. Because the isolated chromosomes retain the specificity of phosphorylation seen in vivo, this system offers unique possibilities for studying the properties and regulation of the kinase and phosphatase by adding exogenous substances and observing their effects. It should also be useful for studying the sites of phosphorylation, since proteins can be more easily labeled to high specific activity with 32P in this system than in vivo. The pattern of proteins phosphorylated in isolated metaphase chromosomes appears to be nearly identical with the pattern found in vivo. Among the histones (H) only H1 and H3 are phosphorylated, but several nonhistone proteins, including high mobility group (HMG) 14, are also phosphorylated. Since HMG 14 has been implicated as a structural protein of actively transcribing chromatin, our results suggest that phosphorylation of chromatin proteins may be involved in the shutoff of transcription during mitosis. Tryptic peptide maps and analysis of the phosphorylated amino acids indicate that H1A, H1B, HMG 14, and H3 are phosphorylated at the same sites in vitro in metaphase chromosomes as in mitotic cells in vivo. The major site of phosphorylation of histone H3, both in vivo and in vitro, has been identified as serine 10. HMG 14 is phosphorylated both at serine and threonine residues.

Isolation of chromosome clusters from metaphase-arrested HeLa cells.

We have developed a simplified approach for the isolation of metaphase chromosomes from HeLa cells. In this method, all the chromosome from a cell remain together in a bundle which we call a "metaphase chromosome cluster". Cells are arrested to 90-95% in metaphase, collected by centrifugation, extracted with non-ionic detergent in a low ionic strength buffer at neutral pH, and homogenised to strip away the cytoskeleton. The chromosome clusters which are released can then be isolated in a crude state by pelleting or they can be purified away from nearly all the interphase nuclei and cytoplasmic debris by banding in a PercollTM density gradient. -- This procedure has the advantages that it is quick and easy, metaphase chromatin is recovered in high yield, and Ca++ is not needed to stabilise the chromosomes. Although the method does not yield individual chromosomes, it is nevertheless very useful for both structural and biochemical studies of mitotic chromatin. The chromosome clusters also make possible biochemical and structural studies of what holds the different chromosomes together. Such information could be useful in improving chromosome isolation procedures and for understanding suprachromosomal organisation of the nucleus.

Sulfhydryl reagents prevent dephosphorylation and proteolysis of histones in isolated HeLa metaphase chromosomes.

It has been shown by D'Anna et al. [Nucleic Acids Res. 5, 3195-3207 (1978)] that histones H1 and H3, which are highly phosphorylated during mitosis in mammalian cells, become rapidly dephosphorylated during conventional metaphase chromosome isolation procedures. We show here that this dephosphorylation can be completely prevented by including sulfhydryl reagents, such as p-chloromercuriphenyl sulfonate or 5,5'-dithiobis(2-nitrobenzoate), in the chromosome isolation buffers. These reagents also efficiently inhibit the endogenous proteases present in isolated HeLa chromosomes and nuclei.

Isolation of a protein scaffold from mitotic HeLa cell chromosomes.

We have recently shown that, after the histones and most of the nonhistone proteins are gently removed from HeLa metaphase chromosomes, the chromosomal DNA is still highly organized and relatively compact. The structure of these histone-depleted chromosomes is due to the presence of a number of nonhistone proteins that form a central scaffold that retains the approximate size and shape of intact chromosomes and to which the DNA is attached, predominantly forming loops. We now demonstrate that the protein scaffold may be isolated independently of the DNA by treating HeLa chromosomes with micrococcal nuclease before removing the histones.The chromosomal scaffolds may be isolated by sucrose density gradient centrifugation as a well-defined peak that is stable in 2 M sodium chloride, but is dissociated by treatment with proteases, 4 M urea, or 0.1% sodium dodecyl sulfate. Polyacrylamide gel electrophoresis reveals that the protein content of scaffold preparations is identical to that of histone-depleted chromosomes. Fluorescence microscopy of purified scaffolds in isolation buffer shows that the particles still possess the familiar chromosome morphology. When the scaffolds are examined in the electron microscope, a fibrous structure with the approximate size and shape of intact, paired chromatids is seen. Less than 0.1% of the chromosomal DNA and virtually no histones are associated with the purified scaffold structures.