Biochemical and genetic factors in the heat inactivation of murine β-glucuronidase

The enzymes coded for by two alleles at the glucuronidase structural locus (Gus) were compared in their response to pH, buffering anion, buffer molarity, ionic strength, and temperature. The heat-labile Gus^h gene product responded in a qualitatively similar but quantitatively reduced manner compared to the relatively heat-stable Gus ^b gene product. In all buffers tested, the enzyme was most heat stable at pH 5.0. Ranking of the various buffer anions tested, according to increasing heat stabilization, was water acetate phosphate < citrate. Varying the molarity of the buffers from 0.01 to 0.6 m at pH 5.0 revealed further differences among the buffers. Increasing ionic strength exerted a destabilizing force on the protein. The half-life of the enzyme decreased by as much as a hundredfold between 71 and 75 C. The Gus^h/Gus^h genotype also results in decreased activity levels in all tissues, reportedly because of decreased synthesis. The heat inactivation curves of Gus^b/Gus^h heterozygotes were incompatible with any theoretical curve based on the assumption that the Gus^b and Gus^h chromosomes in the heterozygote behave in a manner similar to that seen in the homozygotes.This research was supported by a Basil O'Connor Starter Research Grant from the National Foundation—March of Dimes (R. J. M.) and by a grant from The Jane Coffin Childs Memorial Fund for Medical Research (K. H.).Fellow of The Jane Coffin Childs Memorial Fund for Medical Research.     

Action of light and gibberellic acid on the growth of excised embryos from Phacelia tanacetifolia seeds

Summary Germination of Phacelia tanacetifolia seeds is inhibited by light. Embryos freed of endosperm grow irrespective of light. However, if they are held in 0.3 M mannitol plus 2% sucrose, light sensitivity is reinstated: growth (i. e., germination) occurs in darkness but not in light. Gibberellic acid (GA₃) releases the inhibition due to light. These results suggest that (a) the photoreceptor site of the seed is in the embryo; (b) GA₃ acts directly on the embryo; and (c) darkimbibed embryos appear to have a higher water-uptake potential.This work was supported in part by the Jane Coffin Childs Memorial Fund for Medical Research, and by the U.S. Atomic Energy Commission under Contract No. AT(11-1)-1338.     

The cyclization of mouse satellite DNA

Sheared fragments of mouse satellite DNA can form rings and other circular structures by several techniques. Folded rings are formed if the sheared fragments are simply annealed, indicating that shearing produces single-chain terminals, and that the repetitious sequence is shorter than the exposed ends. The occurrence of folded rings can be sharply reduced by prior treatment with single-chain specific endonuclease, and significantly increased if the fragments are treated with exonuclease III. Denaturation of satellite DNA followed by reassociation of the single chains results in the formation of slipped rings. These characteristics of the DNA lead to the conclusion that the sequences of the mouse satellite DNA are arranged in a tandemly repetitious manner.-About 20% of the DNA fragments from the main band cyclize after partial exonuclease III degradation, but not before this treatment. This indicates that a large fraction of the main band DNA is tandemly repetitious, but that the length of the repetitious sequence is on the average longer than the single-chain terminals produced by shearing.Reed Pyeritz is the recipient of a NSF predoctoral fellowship. C. S. Lee is a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. This investigation has been supported by grants from the National Institutes of Health (AI08186), the National Science Foundation (GB-8611), and the Jane Coffin Childs Memorial Fund for Medical Research.     

The new lead citrate method for the ultracytochemical demonstration of activity of non-specific alkaline phosphatase (orthophosphoric monoester phosphohydrolase)

Summary The new method, utilizing lead citrate as capture reagent, for the ultracytochemical demonstration of non-specific alkaline phosphatase activity at high alkaline ranges of pH was introduced. Results obtained by the new lead citrate method in various tissues of the rat were presented and discussed in connection with observations made by other methods. The method can be used at the light microscopic level also.Supported by grants from the U.S. Public Health Service MH 12269-01, the Jane Coffin Childs Memorial Fund for Medical Research (Project No. 196), the Anna Fuller Fund and the Department of Education, the Japanese Government (Sogokenkyu UDC 611.015.3:611-018:576.311).     

Influence of factors affecting germination on respiration of Phacelia tanacetifolia seeds

Summary Germination of the seeds of Phacelia tanacetifolia is inhibited by light. Removal of that part of the covering structures of the seeds which directly covers the radicle allows full germination in light. The rate of O₂ uptake in the seeds increases following imbibition, and reaches the same steady rate in light and in darkness after 3 hr. From the 14 th hour on, dark-imbibed seeds show a linear increase in the rate of respiration. This increase is not observed in dormant seeds incubated in light. In normal dark germination, protrusion of the radicle begins at 12 th hour following soaking, and by the end of 18 th hour approximately 60% of the seeds have germinated. The seeds which have been scarified at the radicle end and germinate readily in light show a steady increase in Q _{O 2}. If scarified seeds are allowed to imbibe 0.3 M mannitol and are then incubated in light, the embryo does not grow and the pattern of O₂ uptake becomes identical with that of intact seeds in light. Mannitol, however, does not inhibit respiration by itself. These observations indicate that the increased O₂ uptake is the result rather than the cause of seed germination, and that light does not cause dormancy by inhibiting O₂ uptake. Measures effective in releasing dormancy (dark incubation, mechanical scarification, gibberellin treatment) do not induce germination by facilitating oxygen entry.This work was supported in part by the Jane Coffin Childs Memorial Fund for Medical Resarch, and by the U. S. Atomic Energy Commission under Contract No. AT (11-1)-1338.     

Differentiation of epithelial cells in the mouse thymus

Summary The foetal and post-natal development of the mouse thymus was studied with the electron microscope paying particular attention to the differentiation of the epithelial cells. At about 13 days' gestation, the thymus was composed principally of undifferentiated epithelial cells and some lymphoblasts. The latter accumulated rapidly but did not show much evidence of mitotic activity until after the development of differentiated cortical epithelial cells which appeared during the 15th day of gestation. Further differentiation of epithelial cells did not occur until near term when medullary cystic epithelial cells appeared, and post-natally when small Hassall's corpuscles were developed. Undifferentiated and dividing epithelial cells were seen in the medulla and were present in all postnatal animals examined.This is publication number 1400 from the Walter & Eliza Hall Institute of Medical Research.The author is grateful to Prof. G. J. V. Nossal, Dr. J. F. A. P. Miller and Dr. P. J. Russell for their interest and assistance with various aspects of this study. Special thanks are due to Miss Mary Bravington for her skilled technical assistance. This investigation was supported by grants from the Jane Coffin Childs Memorial Fund for Medical Research and the National Health and Medical Research Council of Australia. The Electron Microscope Laboratory was equipped and supported by grants from the Australian Research Grants Committee, J. B. Were and Sons and the Potter Foundation.     

A rapid and specific enrichment procedure forHyphomicrobium spp.

An enrichment procedure for the isolation of stalked bacteria of the genusHyphomicrobium is described. The method is based on the use of an organic C₁ compound as carbon and energy source for growth together with anaerobic incubation in the presence of nitrate as an electron acceptor. Optimal conditions for the growth of a number ofHyphomicrobium isolates have been investigated. Applying these conditions,Hyphomicrobium spp. have been enriched from a wide range of natural habitats within 1–2 weeks. This work was supported by a Grant from the Medical Research Fund, University of Sheffield, England, to M.M.A.     

Correlation of structure and RNA synthesis in the nucleolus-organizing polytene chromosomes of Phaseolus vulgaris

The relationship between RNA synthesis and morphology of the nucleolus-organizing polytene chromosomes in the highly endopolyploid suspensor cells of Phaseolus vulgaris has been studied by actinomycin D treatment, temperature lowering, and H³-uridine autoradiography. Actinomycin D and low temperature induce a condensation of the giant chromosomes, particularly of the nuoleolus organizers and of the intranucleolar regions of the chromosomes. RNA synthesis occurs in the extended state of the chromosomes, but it ceases in the highly condensed state caused by the treatment of the cells either with actinomycin D or with low temperature.     

Isolation, fractionation and template activity of the continuously-condensed chromatin of Euglena gracilis

A technique for isolating whole chromatin from nuclei of the lower eukaryote Euglena gracilis is presented. This chromatin, which appears under the electron microscope as uniformly condensed fibers, can, nevertheless, be subfractionated into distinct heterochromatic and euchromatic fractions. The euchromatin, comprising about 14% of the total DNA of the nucleus, contains over 80 % of the total endogenous RNA polymerase activity measured. The K_m for this enzyme is higher than that found for prokaryotes, but falls in the range found for other eukaryotes. Stability constants, calculated from cation-chromatin binding data, suggest that internal carboxyl groups of chromosomal proteins, at least, are involved in the condensation of Euglena chromatin. The relationship between Euglena chromatin and that of higher eukaryotes is discussed.     

Structure analysis of three T7 late mRNA ribosome binding sites

The 5′-terminal regions of the three T7 late RNA species IIIb, IV and V have been characterized. These regions contain the protein synthesis initiation sites for the T7 genes 17, 9 and 10, respectively. Each of these is located between 60 and 90 nucleotides from the 5′ terminus of an in vitro synthesized RNA species. The sequence 5′ A-C-U-U-U-A-A-G-Pu-A-G-Pu, which is common to these ribosome binding regions, contains an impressive stretch of complementarity to the sequence 5′ A-C-C-U-C-C-U-U-A, at the 3′ terminus of 16 S ribosomal RNA. The nuclease mapping technique of Wurst et al. (1978) has been used to probe intramolecular structural interactions involving these initiation regions in the RNA. My results indicate that all three initiation codons, together with other portions of the ribosome binding regions are protected, under non-denaturing conditions, against the actions of both the single-strand-specific nuclease S₁ and RNAase T₁.     

Some studies of erythroid differentiationin vitro

Summary When rat marrow cells are pre-incubated for periods of up to 20 days they retain the capacity to respond to exogenous erythropoietin. This is true whether the response is measured in terms of increased rates of RNA synthesis, protein synthesis, iron-uptake, or hemoglobin synthesis. The patterns of response to erythropoietin by pre-incubated cells are complex and involve secondary waves of increased rates of synthesis that are not yet understood. Life Insurance Medical Research Fund Fellow. Operated by the University of Chicago for the United States Atomic Energy Commission.     

Related base sequences in the DNA of simple and complex organisms. IV. Evolutionary divergence of base sequence in mouse L-cell cytoplasmic and nucleus-restricted RNA

Nuclear and cytoplasmic RNAs of mouse L-cells were compared for base sequence relatedness between and within them. The relationship of each of these RNAs to the DNA of other rodents and the rabbit was determined. RNA restricted to the mouse L-cell nucleus is unrelated in base sequence to cytoplasmic RNA. There is less diversity of base sequence within the gene families transcribing nucleus-restricted RNA than within those transcribing cytoplasmic RNA, suggesting more recent evolutionary origin. The gene families which code for mouse L-cell unique nuclear RNA are far more divergent from those of other rodents than are cytoplasmic RNA gene families. They are unrelated in base sequence to any gene families in rabbit DNA, suggesting that these gene families evolved after the separation of the ancestors of the orders Rodentia and Lagomorpha. The known characteristics of the RNA restricted to the nucleus in eukaryotic cells are summarized.This investigation was supported by U.S. Public Health Service Training Grant GM 00182 and Research Grant GM 12449 from the Institute of General Medical Sciences.     

Morphological changes of sperm nuclei during spermatogenesis in the brown alga Cystoseira hakodatensis (Fucales, Phaeophyceae)

Morphological changes and chromatin condensation of sperm nuclei were observed during spermatogenesis in the fucalean brown alga Cystoseira hakodatensis (Yendo) Fensholt. Ultrastructural studies have shown that the mature spermatozoid has an elongated and concave nucleus with condensed chromatin. The morphological changes and the chromatin condensation process during spermatogenesis was observed. Nuclear size decreased in two stages during spermatogenesis. During the first stage, spherical nuclei decreased in size as they were undergoing meiotic divisions and the subsequent mitoses within the antheridium. During the second stage, the morphological transformation from a spherical into an elongated nucleus occurred. Afterwards, chromatin condensed at the periphery in each nucleus, and chromatin‐free regions were observed in the center of the nucleus. These chromatin‐free regions in the center of nucleus were compressed by the peripheral chromatin‐condensed region. As the result, the elongated and concave nucleus of the mature sperm consisted of uniformly well‐condensed chromatin.     

Spontaneous cell shedding by tumor cells in monolayer culture

Summary Sarcoma 180 monolayers spontaneously shed single cells and small multicellular aggregates into the surrounding medium to produce a dual population of floating and substratum-attached cells. Shedding was a motility-associated event that occurred when cells attempted to migrate over one another. It resulted from a combination of cell shape change and active motility, which increased sensitivity to fluid shear dislodgement by reducing a cell's surface area of adhesive contact and increasing strain tension at its adhesive contact points. Shedding occurred at all phases of the cell cycle. Extracellular matrix but not conditioned medium enhanced the floating subpopulation by slowing the kinetics of rattachment to plastic and cellular substrata. Although sarcoma 180 cells are anchorage independent in the sense that they grow readily in single cell suspension, they nevertheless exhibited anchorage modulation of their cell cycle. Short periods in suspension produced a mild G₁ accumulation, whereas longer periods of anchorage deprivation led to a mild G₂ accumulation which appeared to result from an interference with cytokinesis. This work was supported by grants from the Medical Research Council of Canada, The National Cancer Institute of Canada, the Alberta Heritage Savings and Trust Fund for Applied Cancer Research, and the Alberta Heritage Fund for Medical Research.     

Cellular organization in the synganglion of the mite Macrocheles muscaedomesticae (Acarina: Macrochelidae)

Summary A large DNA containing body is found in oocytes of the house cricket, Acheta domesticus. Little or no RNA synthesis is associated with the DNA body during the leptotene, zygotene, and pachytene stages of meiotic prophase I. During the early diplotene stage of development, large masses of nucleolar material begin to accumulate at the periphery of the DNA body. The onset of RNA synthesis correlates with a change in the histochemically detectable histone proteins associated with the DNA body. In ovaries of animals injected with uridine-H³, most of the label accumulates in ribosomal RNA. Autoradiographic studies show that the cytoplasm of late diplotene stage cells accumulates uridine label to a greater extent than does the cytoplasm of early diplotene stage cells. Increased transport of nucleolar material through the nuclear envelope of late diplotene stage cells accounts for the increased cytoplasmic labeling.This investigation was supported by PHS Research Grant No. GM 16440 from the Institute of General Medical Sciences, and by Grants No. L-16 and J-1 from the Health Research and Services Foundation.The authors gratefully acknowledge the technical assistance of Mrs. Marcia Andrews and Miss Celeste Malinoski.     

Enhancement of rat ACT-1 tumor clonogenicity by xenogeneic mouse macrophages

Summary In vitro growth of rat atriocaval epithelial tumor cells (ACT-1) was enhanced by the inclusion of xenogeneic mouse adherent peritoneal exudate cells (PECs) in a two-layer soft agar system. A linear relationship was found between the number of cells plated and the number of colonies when ACT-1 tumor cells were plated at plating densities of between 1 and 5×10⁵ cell/60 mm plate (r=0.9,P<0.001). Inclusion of irradiated PECs in the bioassay for tumor stem cells resulted in a two and a half-fold increase in colony formation in three separate experiments (P<0.001). This work was supported by grants from the Cancer Research Trust, the University of Otago Cancer Research Fund and by the Medical Research Committee (Golden Kiwi).     

Neurospora glucamylase and a mutant affected in its regulation

Neurospora glucamylase is a glucose-repressible extracellular enzyme. The enzyme was purified to homogeneity and found to have a molecular weight of 82,000 and to release glucose from either maltose or amylose. The rate of glucamylase synthesis increases more than 100-fold when cells are transferred from a glucose-containing medium to a glucose-free medium. Increased production of glucamylase begins within 30 min of the transfer. Glucamylase is rapidly secreted into the medium. A mutant affecting the ability of glucose to repress the synthesis of the glucose-repressible extracellular enzymes glucamylase and invertase has been isolated and studied. The mutant constitutively synthesizes and secretes a glucamylase which is indistinguishable from the wild-type enzyme.Funds for this research were provided by Grant PCM-8011772 from the National Science Foundation and by a grant from the Research Development Fund of The Research Foundation of the State University of New York.     

Localization of repetitive DNA in the chromosomes of Microtus agrestis by means of in situ hybridization

Heterochromatin in the European field vole, Microtus agrestis, was studied using a special staining technique and DNA/RNA in situ hybridization. The heterochromatin composed the proximal 1/4 of the short arm and the entire long arm of the X chromosome, practically the entire Y chromosome and the centromeric areas of the autosomes. By using the DNA/RNA in situ hybridization technique, repeated nucleotide sequences are shown to be in the heterochromatin of the sex chromosomes.Supported in part by Research Grants DRG-1061 and 269 from the Damon Runyon Memorial Fund for Cancer Research, G-373 and G-267 from the Robert A. Welch Foundation.     

Radioautographic,ultrastructural and biochemical studies on the effect of fluorouracil on the RNA synthesis in the liver of rats

Summary The effects of fluorouracil and uracil on the ultrastructure and RNA synthesis in the liver of rats have been studied. High doses of uracil, contrary to fluorouracil, had no effects on nucleolar ultrastructure. Fluorouracil, even in low doses, depressed the RNA synthesis as studied after cytidine-³H administration. In the radioautographs the depression was especially seen to affect the cytoplasmic labelling. The biochemical analysis showed signs of a depression of ribosomal RNA synthesis and a block or delay in the maturation of ribosomal RNA. Repeated, high doses of uracil also depressed the RNA synthesis but there did not seem to be any delay in the maturation of ribosomal RNA. The results suggest that fluorouracil may affect the RNA metabolism in the nucleoli.The investigation was supported by grants from the Swedish Medical Research Council (Project K 67–12 X-623-03), the Swedish Cancer Society (Project 67:31) and the Medical Faculty of Uppsala.     

The structure and behavior of the nucleolus organizers in mammalian cells

The regularly occurring secondary constrictions on metaphase chromosomes of mammalian cells prove to be nucleolus organizers as expected. The expression of nucleolus organizers as secondary constrictions, however, varies from cell to cell and from tissue to tissue, including cultivation in vitro. Electron micrographs of the organizer region show that the nucleolus organizer at metaphase is not a constriction. The width of the organizer area is the same as the condensed chromosomal arms; but the filaments, which are the major components of this region, show a diameter of 50–70 Å. The condensed chromosome arms consist of filaments 150–200 Å in diameter. In some mammalian species, structures similar to the nucleolus organizer are located at the end of chromosomes. These may be terminal nucleolus organizers.Supported in part by Research Grants DRG-269 from Damon Runyon Memorial Fund for Cancer Research, E-286 from American Cancer Society, and HD-2590 from National Institutes of Health.