Hl histones from mammalian testes : Hlt is associated with spermatogenesis in humans

Hlt is a testis-specific Hl histone variant associated with meiosis and post-meiotic stages of male germ cell development. We have now made tentative identification of Hlt in humans by a variety of criteria including electrophoretic and extractive properties. While Hlt was readily identified in extracts from normal testes, it was not detectable in extracts from aspermatogenic testes or from placenta. Identification of Hlt in humans confirms the widespread association of this unusual Hl variant with spermatogenesis among mammals ranging from rodents to primates.     

Changes in endogenous cell surface galactosyltransferase activity during in vitro limb bud chondrogenesis

When the subridge mesoderm of the embryonic chick limb bud is cultured in the absence of the apical ectodermal ridge and adjacent ectoderm, the cells rapidly progress through the various stages of chondrogenesis. During the first day of culture, the cells initiate condensation, and during subsequent days, deposit a cartilage matrix. In the present study, we show that early in the first day there is a progressive 2-fold increase in cell surface galactosyltransferase activity towards endogenous acceptors. Later in the first day, although the cells are still in condensation, endogenous galactosyltransferase activity has decreased, suggesting in situ galactosylation of surface acceptors. During subsequent development, when cartilage matrix is being deposited, surface galactosyltransferase activity remains low. All controls have been performed to insure cell surface localization of enzyme activity. Two other surface glycosyltransferases show very low levels of activity, which do not change significantly during culture. We suggest that during cellular condensation, an interaction between surface galactosyltransferases and acceptors on adjacent cells occurs, and this interaction may be causally related to subsequent chondrogenic differentiation.     

Gene expression in normal and virally transformed mouse 3T3B and hamster BHK21 cells

Cell lines 3T3B (mouse), 3T3B-SV40, BHK21 (hamster) and BHK21 polyoma virus (PyY) were labelled with [³⁵S]methionine under conditions in which 500–600 cpm were incorporated per cell during a 20 h incubation period. Two-dimensional gel electrophoresis analysis of the total [³⁵S]methionine-labelled polypeptides from 200–300 cells followed by fluorography revealed about 500 acidic (isoelectric focusing, IEF) and 150 basic polypeptides (non-equilibrium pH gradient electrophoresis, NEPHGE) whose position could be reproducibly assessed. Counting of 33 abundant acidic polypeptides present in both 3T3B and 3T3B-SV40 revealed significant changes in the relative proportion of ten of them. Seven, including the subunit of the 100 Å filaments ‘fibroblast type’ (55K) (1.1% in 3T3B; 0.6% in 3T3B-SV40), three cytoarchitectural proteins and three soluble proteins, corresponded to a decrease of 40% or more in the radioactivity of the spots in transformed cells, and only in three cases was there a significant increase in radioactivity of polypeptides in 3T3B-SV40 cells. Among the polypeptides that show less than 40% variation we have identified total actin (42K) (13% of total label in 3T3B; 10% in 3T3B-SV40), α- and β-tubulin (55K) (1.6% of total label in 3T3B; 2% in 3T3B-SV40), eleven polypeptides present in Triton skeletons, and nine soluble proteins. We have also observed 25 obvious changes in polypeptide intensities (16 acidic and 9 basic) but these were not quantitated. Only three polypeptides were found in transformed cells that were not detected in normal cells. One of these corresponded to the large T antigen and the other two to Triton-soluble proteins of a molecular weight in the range of 52–54K. Similar quantitative studies on the hamster BHK21/BHK21PyY pair confirmed at least the major observations made in 3T3B and 3T3B-SV40.     

The co-induction of sister chromatid exchanges and virus synthesis in mammalian cells

The induction of virus synthesis and sister chromatid exchange (SCE) formation was investigated in several mammalian cell lines. Ultraviolet light co-induced the production of virus and SCEs in Simian virus 40 (SV40) transformed hamster cells. Post-irradiation treatment with caffeine enhanced virus induction, though it caused a smaller, less consistent elevation of SCE formation. Co-induction of oncovirus synthesis and SCEs was also observed in three murine cell lines exposed to increasing concentrations of 5-bromodeoxyuridine. These and previous data demonstrate a correlation between the induction of virus synthesis and SCE formation in rodent cells exposed to several agents, although inter-agent variation in the correlation may reflect differences between the two processes.     

Adult human retinal cells in culture. Identification of cell types and expression of differentiated properties

A method for culturing adult mammalian retinal neurons in serum-free N2 medium supplemented with nerve growth factor (NGF) is described. Identification of neurons in cultures of dispersed human retina was based upon morphology, immunocytochemical localization of bound tetanus toxin, and autoradiographic localization of 3H-neurotransmitter candidates (gamma-aminobutyric acid, glycine, dopamine) accumulated by high-affinity uptake mechanisms. Neurons would not attach to glass or plastic substrates, consequently the present studies were performed using neurons plated upon a feeder layer. Serum was required for the initial phase of attachment. The feeder layer was derived from retinal cells that had been plated on glass or plastic in the presence of serum and had later been passaged. Since these cells exhibited glial fibrillary acidic protein (GFAP) immunoreactivity, they were tentatively identified as being glial in origin. Under these conditions, neuron- and glia-specific properties were retained up to 28 days. The presence of interstitial retinol-binding protein (IRBP) in medium of cultures of neuronal cells on feeder layers was demonstrated by an immunoblot technique using rabbit antibovine IRBP antibodies. No IRBP was detected in medium in which the feeder layers alone had been cultured. IRBP biosynthesis was demonstrated by incubation of the cultures with [35S]methionine. Immunoprecipitable [35S]IRBP was detected only in medium from cultures containing neurons; cells of the feeder layer did not synthesize and secrete this glycoprotein. These findings are consistent with the hypothesis that IRBP, a 135K constituent of the interphotoreceptor matrix, is synthesized in vivo by a neuronal cell, specifically, the photoreceptors.     

Genetic complementation in hybrid cells derived from two metabolic co-operative defective mammalian cell lines

A series of hybrid clones have been isolated following the somatic cell fusion of two mammalian cell lines, each defective in junctional transfer of metabolites. One of these parental lines is a variant isolated by selection from the metabolic co-operation competent embryonal carcinoma line PC13TG8. The other parent is LMTK^? in which inability to transfer was found to be a pre-existing property. Hybrids between these two cell lines are restored in their ability to co-operate, indicating the existence of at least two genetically distinct lesions affecting metabolic co-operation, each of which is recessive. This is the first demonstration that more than one locus is involved in junctional communication.     

Immunocytochemical localization of chromatin regions UV-microirradiated in S phase or anaphase. Evidence for a territorial organization of chromosomes during cell cycle of cultured Chinese hamster cells

Chinese hamster cells (M3-1 line) in S phase were laser-UV-microirradiated (lambda, 257 nm) at a small site of the nucleus. Cells were fixed either immediately thereafter or in subsequent stages of the cell cycle, including prophase and metaphase. The microirradiated chromatin was visualized by indirect immunofluorescence microscopy using antibodies specific for UV-irradiated DNA. During the whole post-incubation period (4-15 h) immunofluorescent labelling was restricted to a small part of the nucleus (means, 4.5% of the total nuclear area). In mitotic cells segments of a few chromosomes only were labelled. Following microirradiation of chromosome segments in anaphase, immunofluorescent labelling was observed over a small part of the resulting interphase nucleus. A territorial organization of interphase chromosomes, i.e. interphase chromosomes occupying distinct domains, has previously been demonstrated by our group for the nucleus of Chinese hamster cells in G1. Our present findings provide evidence that this organization pattern is maintained during the entire cell cycle.     

Extent of histone modifications and H1 content during cell cycle progression in the presence of butyrate

The effects of butyrate upon the extents of phosphorylation of histones H1 and H1(0) during cell-cycle progression have been investigated. Chinese hamster (line CHO) cells were synchronized in early S phase and released into medium containing 0 or 15 mM butyrate to resume cell-cycle traverse into G1 of the next cell cycle. Cells were also mechanically selected from monolayer cultures grown in the presence of colcemid and 0 or 15 mM butyrate to obtain greater than 98% pure populations of metaphase cells. Although cell cycle progression is altered by butyrate, electrophoretic patterns of histones H1, H1(0), H3, and H4 indicate that butyrate has little, if any, effect on the extents of H1 and H1(0) phosphorylation during the cell cycle or the mitotic-specific phosphorylation of histone H3. Butyrate does, however, inhibit removal of extraordinary levels of histone H4 acetylation (hyperacetylation) during metaphase, and it appears to cause an increase in the content of H1(0) in chromatin during the S or G2 phases of the cell cycle.     

Inhibition of dihydrofolate reductase gene expression following serum withdrawal or db-cAMP addition in methotrexate-resistant mouse fibroblasts

We have used a methotrexate (MTX)-resistant mouse 3T6 cell line (M50L3), that overproduces dihydrofolate reductase (DHFR) and its mRNA by a factor of 300, to study the mechanism for turning off DHFR gene expression following withdrawal of serum factors or elevation of the intracellular level of cAMP. When resting (G0) M50L3 cells are serum-stimulated to reenter the cell cycle, the level of DHFR activity begins to increase at about the same time the cells begin synthesizing DNA. The increase in enzyme activity is preceded by increases in the synthesis rate of the enzyme, and the content and production rate of DHFR mRNA. These increases, as well as entry into S phase, are blocked when the cells are serum-stimulated in the presence of dibutyryl cyclic AMP (db-cAMP) and theophylline. In this study, we found that when these drugs were added, or the serum stimulus was withdrawn during S phase (20 h following stimulation), the subsequent increase in DHFR level was blocked. Immunoprecipitation of DHFR from pulse-labelled cells showed that both treatments led to a rapid decrease in synthesis rate of the enzyme. The effect on total protein synthesis was much less than on DHFR synthesis. In DNA-excess filter hybridization experiments, we found that the content of cytoplasmic DHFR mRNA decreased in parallel with the synthesis rate of the enzyme. This was due in part to a decrease in the production rate of DHFR mRNA relative to total mRNA. In addition, drug addition or serum withdrawal led to a significant destabilization of DHFR (as well as total) mRNA. About 85% of poly(A)(+) DHFR mRNA was associated with polysomes in resting, growing, or cAMP-treated cells, suggesting that DHFR gene expression was not controlled at the translational level under these conditions.     

Synchronous exocytosis in Paramecium cells. I. A novel approach

From a total number of approximately 1100-1300 secretory organelles ("trichocysts") in a Paramecium tetraurelia cell, approximately 90% are docked to the cell membrane. Approximately 90% of this subpopulation can be discharged from the cells within seconds, when exposed to the novel trigger agent aminoethyldextran (AED) at a concentration of 10(-6) M. No deleterious side effects were recognized with this trigger agent even over long time periods. By application of AED close to cells with the use of a micropipette we found that triggering of trichocyst release by AED involves a local, non-propagated effect and that all regions of the cell body are equally reactive. It requires exogenous Ca2+. It is independent of ciliary Ca2+ channels, since deciliated cells or ciliary mutations with "Ca2+-tight" cilia respond to AED with normal exocytosis performance. The massive and rapid occurrence of trichocyst release in response to AED allowed for a freeze-fracture analysis of intramembraneous changes (see Olbricht et al., Exp cell res 151 (1984) 14 [23]) which also shows the involvement of exocytosis) as well as for a long-term study of the re-attachment of trichocysts (see Haacke & Plattner, Exp cell res 151 (1984) 21 [10]) under synchronous conditions.     

Double minutes replicate once during S phase of the cell cycle

Double minutes (dm) characteristically exhibit greater numerical heterogeneity among tumor cells than do chromosomes. The biological basis of this heterogeneity was studied in human carcinoma cell line S 18. Pulse labeling of asynchronous cells with [³H]dThd, continuous labeling of synchronized cells with BrdUrd and prematurely condensed chromosome (PCC) studies of G1 and G2 phase S 18 cells indicate that dm-DNA replicates only once during S phase of the cell cycle. No evidence was found for replication of dm-DNA at G1 phase, G2 phase or mitotis. Cells observed at anaphase show imprecise distribution of dm to daughter cells. These studies suggest numerical heterogeneity of dm results from anomalous mitotic segregation rather than anomalous replication of dmDNA.     

Size changes of protamine 1 mRNA provide a molecular marker to monitor spermatogenesis in wild-type and mutant mice

We utilized a cDNA encoding the cysteine-rich, tyrosine-containing mouse protamine, mouse protamine 1 (MP1), to detect the presence of several classes of differentiating germ cells in testicular extracts from wild-type and male sterile mutant mice. This assay is based on the changes in the poly (A) length of MP1-mRNA during spermatogenesis. Testicular extracts of sexually mature CD-1 mice contain a heterogeneous population of protamine-1 mRNA ranging in length from 450 to 580 nucleotides. When the protamine-1 probe was hybridized to testicular RNA preparations from 16- to 20-day-old animals, no MP1-mRNA was detected. Twenty-four-day-old mice contain only the 580-nucleotide form of MP1-mRNA. This size class of protamine mRNA is also present in purified populations of round spermatids, whereas elongating spermatids and residual bodies contain mRNAs ranging from 450 to 580 nucleotides in length, which are identical in size to those present in the testes of sexually mature animals. When the protamine cDNA probe was used to examine the progression of spermiogenesis in three male sterile mouse mutants, blind sterile (bs), quaking (qk) and testicular feminization (Tfm), the results demonstrated that each mutant is pathologically distinct. Analysis of the bs mutant revealed a diminution in the amount of both size classes of MP1-mRNA, in agreement with the cytological reports of reduced numbers of haploid spermatogenic cells in these animals. The presence of both size classes of protamine mRNA in the qk mutant indicates that germ-cell differentiation has proceeded at least to the step-12 spermatid in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship of adhesiveness of cells in culture with specific enzyme activity.

l-Glutamine is required by mouse teratoma cells and other mouse ascites tumor cells in the synthesis of complex carbohydrates involved in intercellular adhesion. Since l-glutamine is synthesized by the enzyme glutamine synthetase (GS) (EC 6.3.1.2), these studies were undertaken to determine if a relationship exists between cellular adhesiveness and GS specific activity. Two types of experiment were performed to examine this relationship. Actinomycin D enhanced both teratoma cell GS specific activity and cellular adhesiveness over controls in batch cultures at confluency. Also, the relationship between cell adhesiveness and GS specific activity during the cell cycle was studied using cell populations synchronized with thymidine plus Colcemid. In these synchronized cultures, cellular adhesiveness displayed an oscillatory pattern with peaks of GS specific activity occurring just prior to peaks of adhesiveness. The levels of GS specific activity and intercellular adhesiveness were enhanced by the addition of hydrocortisone, a steroid known to induce GS specific activity in mouse teratoma cells. These results demonstrate a correlation between GS specific activity and cellular adhesiveness. Based upon previous work which implicates l-glutamine in intercellular adhesion, it is not unreasonable to speculate that GS specific activity and cellular adhesiveness may be causally related.     

The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. I. Characterization of the chondrocyte-specific phenotypes

We have maintained chick embryo chondrocytes in culture for more than 2 months, passaging the floating cells in the absence of ascorbic acid. Throughout the culture period some of the cells attached to the dish, assuming an epithelial-like morphology and subsequently giving rise to new floating cells. The interconversion of the two cell populations was highest in primaries and decreased with the aging of the culture. Cartilage cells synthesized pro-alpha 1 (II) collagen and sulphated proteoglycans in vitro; compared with floaters, the epithelial-like cells secreted relatively large amounts of fibronectin. When ascorbic acid was added to the medium, all cells attached, maintaining their rounded shape; in this condition the pro-alpha, (II) collagen was matured and collagen fibres were detectable outside the cells. Other specific proteins synthesized by the chondrocytes in culture were also identified. One of these, a 64 K collagenase-sensitive protein, was not related to the type II collagen and may represent a new collagen type.     

Synchronous exocytosis in Paramecium cells. II. Intramembranous changes analysed by freeze-fracturing

Since Paramecium tetraurelia cells were found to discharge synchronously most of their secretory organelles ('trichocysts') when exposed to 10(-6) M aminoethyldextran (AED) [17], this was now used for a freeze-fracture and -etching analysis of intramembranous changes during exocytosis performance, in conjunction with a rapid freezing method. In controls the potential exocytosis sites of the cell membrane revealed a 'rosette' of approximately 8 membrane-intercalated particles (MIPs) within a 300 nm large double 'ring' of MIPs (see [18]). During exocytosis we found the following changes: (a) Membrane fusion starts as a focal event, the smallest recognizable openings measuring 20-30 nm in diameter. (b) The exocytotic opening always forms in the center of the rosette. (c) Rosette MIPs may stay very close to the exocytotic opening, or they may partly be dispersed as the exocytotic opening is formed. (d) No diaphragm is formed during exocytotic membrane fusion. (e) The exocytotic opening is increasing to a size where it fills the total fusogenic zone contained within a ring, but not any further. (f) Rosette MIPs become further dispersed through the rings. (g) Resealing involves the transformation of rings into a collapsed form ('parenthesis'). (h) A resealed exocytosis site contains no conspicuous MIP aggregates, such as rosettes or 'annulus' structures from the trichocyst membrane, indicating a clear separation of both components.     

Changes in the mRNA population of chick myoblasts during myogenesis in vitro

We have analyzed the sequence complexity, frequency distribution and coding capacity of the mRNA populations of primary chick embryo muscle cultures at different stages of myogenesis. Prefusion cultures, fused myofibrillar cultures and cultures blocked for both fusion and myogenesis all contain about 17,000 different mRNA sequences, arranged in three of four abundance classes. The myofibril (96 hr) cultures, however, contain about 2500 sequences in higher concentration and six sequences in exceptionally high concentration, each present in about 15,000 copies per nucleus. These sequences are shown to be 10 times less common in premyogenic (26 hr) cultures and 40 times less common in cultures that have been blocked by BUdR against both fusion and myogenesis. The concentration of these sequences in cultures developing toward myofibril formation correlates well with the capacity of the mRNA to stimulate the cell-free synthesis of muscle-specific proteins. A more direct approach to the identity of the abundant class of myofibril mRNA indicates that it contains the templates for the synthesis of seven polypeptides that are synthesized in particularly large amounts in myogenic cultures, including myosin, actin and tropomyosin. Between 20 and 30% of the abundant mRNA is transcribed from moderately repetitive DNA sequences. The remainder of the abundant, and all of the less-abundant, mRNA is transcribed from single-copy DNA.     

Increased uptake of thymidine in the activation of sea urchin eggs. II. Cooperativity with phosphorylation, involvement of the cortex, and partial localization of the kinases

Uptake and phosphorylation of exogenously supplied thymidine are stimulated in Strongylocentrotus purpuratus eggs after fertilization. Before fertilization, the rate of uptake is low and less than 10% of the thymidine entering the egg is phosphorylated. After fertilization, the rate of uptake increases over 50-fold and greater than 90% of the thymidine is immediately phosphorylated. These results imply that there is close cooperativity between fertilization-induced uptake and phosphorylation of thymidine. To gain insight into the structural basis of this apparent cooperativity and to provide a partial localization of the kinases, uptake and phosphorylation were measured in centrifuged eggs, and in centrifuged nucleate and anucleate merogons. Electron micrographs show that in these cells, the inner cytoplasmic contents are stratified according to density and displaced within the egg, whereas the outer cortical region of the cytoplasm remains intact. Uptake and phosphorylation of thymidine are fully stimulated in these eggs and merogons after fertilization, suggesting that both processes are mediated by an intact egg cortex. In support of this suggestion, we report that controlled disruption of the egg cortex prior to fertilization by treatment with cytochalasin B (CB) significantly reduces the rates of uptake and phosphorylation after fertilization. The full stimulation of phosphorylation in nucleate and anucleate merogons eliminates any localization of the catalyzing enzymes (thymidine kinase and thymidylate kinase) in the maternal nucleus and other inner cytoplasmic contents differentially segregated by centrifugation.     

Tetrahymena calmodulin. Characterization of an anti-tetrahymena calmodulin and the immunofluorescent localization in Tetrahymena

A rabbit antiserum specific for Tetrahymena calmodulin was prepared and characterized: In Ouchterlony's immunodiffusion test, the antiserum gave rise to a single precipitin line only with calmodulin in the reaction with crude Tetrahymena extract and the antiserum cross-reacted with a calmodulin fraction from Paramecium, but not with several calmodulin fractions, from higher organisms. Calmodulins from the ciliates appear to share some antigenic determinants which are absent in calmodulins from higher organisms. The intracellular localization of calmodulin was investigated by indirect immunofluorescent method using anti-Tetrahymena calmodulin antibody purified on an antigen-Sepharose affinity column. Immunofluorescence was localized in the oral apparatus, cilia, basal bodies, the anterior end of the cell, and the contractile vacuole pores. The localization suggested involvement of calmodulin in food vacuole formation (nutrient uptake), excretion of contractile vacuole contents (regulation of osmotic pressure), and in ciliary movement (reversal). The suggestion was supported by the observation that trifluoperazine markedly suppressed food vacuole formation and excretion of contractile vacuole contents and affected the ciliary motion.     

A gene function required for cell cycle progression during the G1 portion of the cell cycle and for maintenance of macronuclear DNA synthesis in Paramecium tetraurelia

The ccl mutation in Paramecium tetraurelia reversibly and rapidly blocks cell cycle progression and DNA synthesis at the restrictive temperature. Progression through the cell cycle is blocked during both the G1 and S portions of the cell cycle, while at the restrictive temperature there is neither residual cell cycle progression nor induction of excess delay of subsequent cell cycle events. DNA synthesis activity is reduced to 50% of the normal level in about 5 min and is completely blocked at 30 min after a shift to restrictive temperature. On return to permissive conditions, DNA synthesis is reactivated with similar kinetics.