Leonard H. Cohen

The fate of the small micromeres in sea urchin development

We show that in sea urchin embryos, the daughter cells of the small micromeres become part of the coelomic sacs, in contrast to the long-held view that these sacs are purely of macromere origin. In addition, after prolonged mitotic quiescence, and following their incorporation into the coelomic sacs, these cells resume dividing, contrary to the previous view that they do not divide. Since coelomic sac cells give rise to much of the adult urchin, our results indicate that the small micromeres are founders of cell lineages involved in the formation of adult tissues. The setting aside of these cells in a nondividing state may be analogous to a phenomenon in Drosophila development, in which primordial imaginal and germ cells divide approximately once after the blastoderm stage and do not resume dividing until the larval stage.

Stage-specific mRNAs coding for subtypes of H2A and H2B histones in the sea urchin embryo

Abstract Multiple electrophoretic forms of histones 1, 2A and 2B, stage-specific with respect to the timing of their synthesis in embryogenesis, have previously been detected in embryos of Stongylocentrotus purpuratus. We now present evidence that most of these can be synthesized in vitro by using mRNAs extracted from sea urchin polysomes as templates for a heterologous cell-free translation system (wheat germ). The in vitro products were identified by electrophoresis in two fundamentally different gel systems; the effects of oxidation of methionyl residues on affinities of the proteins for Triton X-100; the relative extents of incorporation of different amino acids into the various proteins; and correspondence of in vitro products and in vivo histones with respect to developmental stage specificity. Since a single translation system synthesized different sets of proteins in response to polysomal mRNAs from different stages (blastula, mesenchyme blastula and gastrula), the proteins most probably differ from each other in primary structure rather than in post-translational modifications. We therefore refer to them as subtypes of their respective classes. Some of the previously described multiplicity of the histone genes is needed to code for such subtypes. The data also show that functional mRNAs encoding the late subtypes (β, γ and δ) are not detectable in the total RNA of the embryo until the stage at which late subtypes begin to be synthesized in vivo. This indicates that control mechanisms preceding translation are involved in the onset of synthesis of those subtypes.

Isolation, properties and cellular distribution of Dl, a chromosomal protein of Drosophila

The protein D1 was obtained from nuclei of Drosophila melanogaster embryos and purified by perchloric acid fractionation and preparative gel electrophoresis. In nuclei its amount is approximately 1% of the amount of DNA by weight. D1 is soluble in 5% perchloric acid and extractable from nuclei by solutions of moderate ionic strength (0.35 M NaCl). Amino acid analysis shows that it is rich in both basic (20%) and acidic (27%) aminoacids. In all these properties D1 resembles HMG proteins (high mobility group; Johns et al., 1975) of vertebrates; however, its apparent molecular weight (∼50,000) is much higher. The distribution of D1 in salivary gland polytene chromosomes was investigated by immunofluorescence. Two levels of fluorescence intensity were observed: 1) Very bright fluorescence at chromosomal positions 81F, 83E, 101F, 102C and 102F; these sites are shown, by double labeling techniques, to coincide with quinacrine bright sites. 2) Medium to low fluorescence at many sites widely distributed throughout all chromosomes. In order to interpret these results and to relate them to the in vivo distribution of D1, we have investigated the pattern of immunofluorescence staining as a function of the methods of chromosome preparation and salivary gland fixation. The immunological specificity of the anti-D1 serum was studied by comparing its reactivity with D. melanogaster and D. virilis chromosome spreads and whole salivary glands, and by using reagents that minimize non-specific antibody interactions. We conclude that Dl is widely distributed throughout cytoplasm and nucleus, present in many chromomeres but most abundant in chromosomal sites that contain the AT-rich satellite DNA of density 1.672. This distribution, together with available evidence about the nucleotide sequences present in this satellite, suggests that D1 binds preferentially to chromatin containing sequences AATAT and/or AATATAT.

The alterations in H1 histone complement during mouse spermatogenesis and their significance for H1 subtype function

The principal histone H1 subtypes of mouse prepachytene spermatocytes were identified as H1a and H1c, proteins that are metabolically unstable and have been postulated to promote extended or flexible chromatin conformations. We suggest that the unusual H1 subtype composition in these cells favors chromatin conformations compatible with the precise chromosomal pairing necessary for genetic recombination and also that it may expedite histone replacement during spermiogenesis.

[25] Analysis of histone subtypes and their modified forms by polyacrylamide gel electrophoresis

Publisher Summary The electrophoretic techniques used in this laboratory to study histones are variations and combinations of three methods. The first is electrophoresis in acetic acid-urea gels, which separates molecules, to a large extent, on the basis of charge. This method allows the separation of modified and unmodified forms of the histones and also the partial separation of the various subtypes of H 1 and their phosphorylated forms. The second technique is electrophoresis in Triton-acetic acid-urea (TAU) gels to separate the various subtypes of H2A, H2B, and H3 and their modified forms. The third is electrophoresis in gels containing sodium dodecyl sulfate (SDS), which separates principally on the basis of mass and, in one or two instances, also separates certain phosphorylated forms of H 1 from other phosphorylated forms and from the parent molecule presumably because these phosphorylations affect the conformation of the protein. Although electrophoresis by any of these methods alone is often used for particular purposes, resolution of all the forms of histories found in cells or tissues requires electrophoresis in two dimensions, the first in acid-urea gels, with or without Triton ×-100, and the second in SDS gels.

RNA-dependent DNA polymerase: Presence in normal human cells☆

Abstract RNA-dependent DNA polymerase has been reported in oncogenic RNA viruses, as well as in human leukemia cells, suggesting a close relationship between this activity and malignancy. However, we have detected an RNA-dependent DNA polymerase activity in normal human lymphocytes stimulated with phytohemagglutinin, indicating either that this enzyme is not unique to RNA-viruses, or that a viral genome is present in non-malignant human cells. Therefore, the use of this activity as an indication of malignancy or a target for the control of cancer should be approached with caution.

Multiple forms of fumarase of pig heart

Abstract Crystalline pig heart fumarase was found to be highly heterogeneous by gel electrophoresis and ion-exchange chromatography. The enzyme was resolved into five active components by chromatography. These components in turn appear heterogeneous; one yielded four electrophoretic bands. Each chromatographic component has the same molecular weight as the original sample; contains four subunits of molwt 48,500; retains its particular chromatographic behavior on rechromatography; can be reconverted to active enzyme after urea dissociation of subunits. Each chromatographic component yielded three or four chromatographic peaks after dissociation and reassociation, but no two yielded the same pattern, showing that there are differences in primary structure between the subunits. The data are consistent with the presence of at least three different subunit types.

Isolation of sea urchin embryo histone H2Aα1 and immunological identification of other stage-specific H2A proteins

Abstract H2Aα1, the principal H2A histone synthesized prior to the blastula stage of the sea urchin, was isolated free of other putative H2A subtypes and other histones. Its amino acid composition provides confirmation that H2Aα1 is the H2A protein encoded in the histone gene cluster carried by pCO2. An antibody prepared against this protein cross-reacts strongly with CS2A (a putative H2A synthesized only during the cleavage stage) as well as with H2Aβ, H2Aγ, and H2Aδ (putative H2As synthesized principally after the blastula stage) but not with non-H2A core histones or other nuclear proteins. The data support the view that CS2A, H2Aα1, H2Aβ, H2Aγ, and H2Aδ are all H2A proteins.

Distribution of histone H1α among cells of the sea urchin embryo

Abstract We have used immunofluorescent staining of sea urchin embryos to study how histone H1α is distributed among progeny cells formed after the cessation of its synthesis. Our results are consistent with H1α being distributed to both daughter cells at mitosis, resulting in it being most concentrated in cells that stop dividing shortly after H1α synthesis ends, while cells that continue to divide dilute their H1α content in proportion to the number of cell divisions. This rules out our earlier suggestion that H1α becomes segregated in dividing cells. In addition, our results show that most dividing cells of the 3-day embryo contain predominantly H1β and H1γ. Since these subtypes are known not to undergo phosphorylation, this finding has implications regarding the roles of H1 phosphorylation in the cell cycle.

Multiple DNA polymerases displayed by isoelectric focusing.

We have been using the high resolving power of isoelectric focusing to analyze DNA polymerase from animal tissues. Systems were studied in which changes occur in both the rate of DNA synthesis and in the genetic expression of the cells, namely human lymphocytes that have been stimulated to divide, and embryonic tissues in which differentiation and cell determination is occurring. It seemed plausible that, in such systems more than one type of DNA polymerase might function and that one might be able to relate their properties to the physiological changes of the cells. In addition, we wanted to know whether normal or neoplastic tissues contain DNA polymerases that can use RNA as template or primer. T o minimize the problem of enzyme instability, we routinely established the pH gradient in the ampholyte column before loading the sample, as we have previously reported in studies of the isoenzymes d fumarase.1 The sample containing the amount of sucrose or glycerol needed to locate it at the desired region of the gradient is then inserted into the gradient through Teflon tubing. The benefits of this procedure are threefold: First, trace metal contaminants of the reagents that might inactivate the enzyme migrate out of the gradient before the sample is added; second, the time required for electrofocusing is considerably reduced; and, most importantly, the proteins are not exposed to extreme conditions of pH. We found that this method resulted in improved recovery of enzyme activity and could be used to electrofocus crude extracts of tissues to study multiple enzyme forms.1 For example, FIGURE 1 shows the profiles of fumarase obtained with a crude tissue extract (upper figure), and with the crystalline enzyme (lower ligure). At least 20 peaks can be detected in the fluorescent tracing of the protein in the crystalline enzyme and most of these are detected also by measurement of enzyme activity. These are not artifacts-these peaks run true as single peaks when isolated and reelectrofocused. Interestingly, the best yield of activity is obtained at 23” C, and the enzyme was found to be cold labile.1 This enzyme is a tetramer.2 By isoelectric focusing the urea-dissociated enzyme, we have detected at least six subunit types and separated the four major ones. Each of the subunit types isolated in this wiy formed active tetramers when renatured and also formed active hybrids with each of the other types, thus accounting for the high degree of multiplicity of enzyme forms.1 One can see by comparing the isoelectric points of the crude and crystalline enzyme that the patterns are virtually identical. We know that fumarase is a “sticky” enzyme, in that it adsorbs to cell particles of all types at low ionic strength.8 The fact that this property did not interfere with isoelectric focusing of the crude extract encouraged us to use the method for separating DNA polymerases in cell extracts. Human lymphocytes are cells that do not divide and virtually do not synthesize