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D J Forbes

Publications and source records attributed to D J Forbes.

At least 37 records · Page 2Linked to original sources
PubMed1993

Mitotic repression of transcription in vitro.

A normal consequence of mitosis in eukaryotes is the repression of transcription. Using Xenopus egg extracts shifted to a mitotic state by the addition of purified cyclin, we have for the first time been able to reproduce a mitotic repression of transcription in vitro. Active RNA polymerase III transcription is observed in interphase extracts, but strongly repressed in extracts converted to mitosis. With the topoisomerase II inhibitor VM-26, we demonstrate that this mitotic repression of RNA polymerase III transcription does not require normal chromatin condensation. Similarly; in vitro mitotic repression of transcription does not require the presence of nucleosome structure or involve a general repressive chromatin-binding protein, as inhibition of chromatin formation with saturating amounts of non-specific DNA has no effect on repression. Instead, the mitotic repression of transcription appears to be due to phosphorylation of a component of the transcription machinery by a mitotic protein kinase, either cdc2 kinase and/or a kinase activated by it. Mitotic repression of RNA polymerase III transcription is observed both in complete mitotic cytosol and when a kinase-enriched mitotic fraction is added to a highly simplified 5S RNA transcription reaction. We present evidence that, upon depletion of cdc2 kinase, a secondary protein kinase activity remains and can mediate this in vitro mitotic repression of transcription.

Animals↗
PubMed1991

A complex of nuclear pore proteins required for pore function.

A family of proteins bearing novel N-acetylglucosamine residues has previously been found to be required to form functional nuclear pores. To begin to determine which of the proteins in this family are essential for pore function, antisera were raised to each of three members of the family, p62, p58, and p54. With these antisera, it was possible to deplete nuclear reconstitution extracts of the proteins and to test the depleted nuclei for nuclear transport. In the course of the experiments, it was found that the three proteins exist as a complex; antisera to any one, while specific on a protein blot, coimmunoprecipitated all three proteins. This complex of pore proteins is stable to 2 M salt, 2 M urea, and the detergent Mega 10, indicating the presence of specific and tight protein-protein interactions. By gel filtration, the complex has a molecular mass of 550-600 kD. Nuclei containing pores depleted of the complex are found to be defective for nuclear transport; moreover, we observe a strict linear correlation between the amount of complex present in nuclei and the amount of nuclear transport of which those nuclei are capable. Thus, the p62-p58-p54 complex defines a group of proteins with strong protein-protein interactions that form a unit of pore structure essential for pore function.

Animals↗
PubMed1990

Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored.

Biochemically altered nuclear pores specifically lacking the N-acetylglucosamine-bearing pore proteins were constructed in a nuclear assembly extract in order to assign function to these proteins. The depleted pores do not bind nuclear signal sequences or actively import nuclear proteins, but they are functional for diffusion. These defects can be fully repaired by assembly with readded Xenopus pore glycoproteins. Strikingly, isolated rat pore glycoproteins also restore transport. Electron microscopy reveals that depleted pores have largely normal morphology. Thus, the pore glycoproteins are not required for assembly of the nuclear envelope, the major structures of the pore, or a pore diffusional channel. Instead, they are essential for active protein import and, unexpectedly, for construction of the part of the pore necessary for signal sequence recognition.

Acetylglucosamine↗
PubMed1990

An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore.

We described previously an assay for authentic nuclear protein import in vitro. In this assay, exogenous nuclei are placed in an extract of Xenopus eggs; a rhodamine-labeled protein possessing a nuclear localization signal is added, and fluorescence microscopy is used to measure nuclear uptake. The requirement in this system for a cytosolic extract suggests that nuclear import is dependent on at least one cytosolic factor. We now confirm this hypothesis. Treatment of the cytosol with N-ethylmaleimide (NEM) abolishes nuclear protein import; readdition of a cytosolic fraction to the NEM-inactivated extract rescues transport. Thus, at least one NEM-sensitive factor required for transport is supplied by the cytosol. This activity, called nuclear import factor-1, or NIF-1, is ammonium-sulfate-precipitable, protease-sensitive, and heat-labile; it is therefore at least partly proteinaceous. NIF-1 stimulates, in a concentration-dependent manner, the rate at which individual nuclei accumulate protein. The effect of NIF-1 is enhanced by a second cytosolic NEM-sensitive factor, NIF-2. Earlier we identified two steps in the nuclear import reaction: (a) ATP-independent binding of a signal-sequence-bearing protein to the nuclear pore; and (b) ATP-dependent translocation of that protein through the pore. We now show that NEM inhibits signal-mediated binding, and that readdition of NIF-1 restores binding. Thus, NIF-1 is required for at least the binding step and does not require ATP for its activity. NIF-1 may act as a cytoplasmic signal receptor that escorts signal-bearing proteins to the pore, or may instead promote signal-mediated binding to the pore in another manner, as discussed.

Animals↗
PubMed1989

Nuclear transport in vitro.

In this paper, progress towards the goal of understanding communication between the nucleus and cytoplasm using an in vitro system is reviewed. To probe the mechanism of nuclear targeting, we developed an in vitro transport system and have begun to dissect the highly selective process of nuclear transport. The basic parameters of transport were defined using an easily isolated nuclear protein, nucleoplasmin. To study the interaction of nuclear targeting signals with the pore, an artificial nuclear transport substrate was constructed, which consists of human serum albumin coupled to the signal sequence of the SV40 T-antigen. A similar peptide-protein conjugate was made using a mutant signal sequence. These conjugates were fluorescently labeled and/or tagged with gold and tested for transport in the in vitro system. High levels of nuclear transport of the wild-type signal sequence-containing protein were observed, while no transport of the mutant signal sequence-containing protein was seen. Thus, the in vitro system correctly recognizes the single amino acid change between the wild-type and mutant signal sequences. We found that the observed nuclear transport was completely dependent on the presence of ATP. Using the in vitro system we identified a specific inhibitor of nuclear transport, the lectin wheat germ agglutinin (WGA), which we find binds directly to the nuclear pore. Probing blots of nuclear proteins with 125I-WGA identified a family of nuclear pore glycoproteins, including one major glycoprotein of 62K (K = 10(3)Mr) molecular weight. With the inhibitor and the in vitro assay, it has been possible to experimentally separate nuclear transport into two steps: (1) a step in which the signal sequence-bearing protein binds to the pore, followed by (2) a step in which the protein translocates through the pore. It is this second step which is the ATP-dependent step of transport, since pore binding but not translocation was seen to occur in the absence of ATP.

Acetylglucosamine↗
PubMed1988

Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation.

Large nuclear proteins must possess a signal sequence to pass through the nuclear pores. Using an in vitro system, we have been able experimentally to dissect nuclear protein transport into two distinct steps: binding and translocation. In the absence of ATP, we observe a binding of nuclear proteins to the pore that is signal sequence-dependent. Translocation through the pore, on the other hand, strictly requires ATP. These steps, visualized in the fluorescence and electron microscopes, were observed both with a natural nuclear protein, nucleoplasmin, and a synthetic nuclear protein, composed of the signal sequence of SV40 T antigen coupled to HSA. When a mutant signal sequence was coupled to HSA, neither transport nor binding were observed, indicating that both result from the presence of a functional signal sequence. An inhibitor of transport, the lectin WGA, also arrested nuclear proteins in a bound state at the cytoplasmic face of the pore. Therefore, only the translocation step is sensitive to the inhibitor WGA, which is known to bind specifically to proteins of the nuclear pore.

Adenosine Triphosphate↗
PubMed1987

Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores.

Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. With this system, we screened for inhibitors of transport. We found that the lectin, wheat germ agglutinin (WGA), completely inhibits the nuclear transport of fluorescently labeled nucleoplasmin. No other lectin tested affected nuclear transport. The inhibition by WGA was not seen when N-acetylglucosamine was present and was reversible by subsequent addition of sugar. When rat liver nuclei that had been incubated with ferritin-labeled WGA were examined by electron microscopy, multiple molecules of WGA were found bound to the cytoplasmic face of each nuclear pore. Gel electrophoresis and nitrocellulose transfer identified one major and several minor nuclear protein bands as binding 125I-labeled WGA. The most abundant protein of these, a 63-65-kD glycoprotein, is a candidate for the inhibitory site of action of WGA on nuclear protein transport. WGA is the first identified inhibitor of nuclear protein transport and interacts directly with the nuclear pore.

Acetylglucosamine↗
PubMed1987

Co-culture of rat trigeminal ganglion neurons and corneal epithelium.

Corneal epithelium and the trigeminal ganglion neurons which normally innervate the epithelium have been grown in adjacent chambers of a 35 mm tissue culture plate. Dissociated nerve cells from late embryonic rats were plated inside an 8 mm cloning cylinder attached to the center of the culture plate by silicone grease. In 7-10 days neurites extended out of this inner chamber by growing through the grease seal and along parallel scratches in the collagen coating of the tissue culture plate. Once this occurred, pure corneal epithelial explants were isolated from young adult rats and plated in the area surrounding the cloning cylinder, i.e. in the outer chamber. Cultures were monitored regularly with phase microscopy and, at various times, were fixed for ultrastructural examination. Within 24-48 hours of the epithelial plating, there were both individual neurites and bundles of neurites in contact with the epithelium. This interaction increased substantially over the next few days. Growth cones of the neurites could be seen to approach the microvilli-covered surface of the epithelium, travel over the surface and penetrate between the epithelial cells. This tissue culture model of the innervated ocular surface may prove valuable in the study of a variety of ocular conditions or diseases, as well as provide a means to study functional relationships and mechanisms of cellular interaction between neurons and their target cells.

Animals↗
PubMed1986

In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins.

An in vitro system was developed that provides a quick microscopic assay for nuclear transport. The assay uses an extract of Xenopus eggs, normal or synthetic nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. This in vitro system accurately mimics in vivo nuclear transport, both in exclusivity and in the amount of accumulation observed (up to 17-fold). Selective accumulation of fluorescent nucleoplasmin is observed microscopically within 30 min with rat liver nuclei, Xenopus embryonic nuclei, regrown Xenopus sperm nuclei, or nuclei reconstituted in vitro from bacteriophage lambda DNA. This transport requires the signal domain of nucleoplasmin. Furthermore, the ability of nuclei to accumulate nucleoplasmin directly correlates with their ability to exclude the fluorescent non-nuclear proteins, FITC-immunoglobulin and phycoerythrin. An active transport model would predict that nuclear transport be temperature- and energy-dependent and that inhibition of transport by either low temperature or energy depletion would be reversible. Both predictions were confirmed in our system. Nucleoplasmin accumulation increases with temperature, while the protein is completely excluded at 0 degrees C. The effects of low temperature are reversible. As found for 125I-labeled nucleoplasmin (Newmeyer, D. D., J. M. Lucocq, T. R. Bürglin, and E. M. De Robertis, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:501-510), transport of fluorescent nucleoplasmin is inhibited by ATP depletion. This effect is reversed by later ATP addition. Under ATP-depleted conditions non-nuclear proteins continue to be excluded. These results argue for a direct role of ATP in transport rather than for a simple role in preserving envelope integrity. In a first step towards defining the minimum requirements for a transport medium, egg extracts were depleted of membrane vesicles. Membrane-depleted extracts neither support transport nor maintain the integrity of the nuclear envelope.

Animals↗
PubMed1984

Differential expression of multiple U1 small nuclear RNAs in oocytes and embryos of Xenopus laevis.

The small nuclear RNA, U1, is a highly conserved, 165 nucleotide long RNA which has been implicated in the processing of mRNA precursors. We present evidence that in the amphibian X. laevis there exist at least seven species of U1 RNA, which differ in sequence but not in length. Strikingly, these RNAs are not coordinately expressed. Two of the U1 RNAs are the predominant U1 species transcribed in the late blastula-early gastrula stages of Xenopus embryogenesis. These two RNAs, designated xU1a and xU1b, are not synthesized in significant amounts in stage 6 oocytes; a different set of U1 RNAs are expressed during late oogenesis. In a Xenopus cultured cell line, all of the U1 RNA species are expressed. Possible functions and developmental significance of these multiple U1 RNA species are discussed.

Animals↗
PubMed1984

The two embryonic U1 small nuclear RNAs of Xenopus laevis are encoded by a major family of tandemly repeated genes.

We have identified a large family of U1 RNA genes in Xenopus laevis that encodes two distinct species of U1 RNA. These genes are expressed primarily at the onset of transcription in the 4,000-cell embryo (D. J. Forbes, M. W. Kirschner, D. Caput, J. E. Dahlberg, and E. Lund, Cell 38:681-689, 1984). The two types of embryonic U1 RNA genes are interspersed and are organized in large tandem arrays. The basic 1.9-kilobase repeating unit contains a single copy of each of the embryonic genes and is reiterated ca. 500-fold per haploid genome. This repetitive U1 DNA accounts for more than 90% of all U1 DNA in X. laevis. In addition to this major family, there exist several minor families of dispersed U1 RNA genes, which presumably encode the oocyte and somatic species of X. laevis U1 RNA. Although the embryonic genes are normally inactive in stage VI oocytes, they are expressed when cloned copies are injected into oocyte nuclei.

Animals↗
PubMed1984

Characterization of rat corneal epithelium maintained in tissue culture.

Using Dispase-treated rat corneas, primary cultures of corneal epithelium have been established which are free of contaminating cell types. Cultures were maintained for periods up to 36 days and were monitored with morphological and electrophysiological methods. Phase and scanning electron microscopy revealed a mosaic of polygonal epithelial cells which migrated over the culture plate. Actively migrating cells at the periphery of the culture presented a complex border of ruffles and filopodia. Surface specializations of the epithelial cells, i.e. microvilli, reflected those seen in vivo. Transmission electron microscopy revealed many cytological features common to the intact cornea: bundles of tonofilaments, desmosomes between adjacent cells and glycocalyx-covered microvilli on all free surfaces. These features developed over time in culture. Membrane potentials of the cultured epithelial cells could be recorded intracellularly. Therefore, it appears that pure cultures of rat corneal epithelium which maintain the morphological characteristics of their in vivo counterparts can be grown for considerable periods of time and that their electrophysiological properties can be monitored. This system offers the possibility of studying, under the controlled conditions of tissue culture, a wide variety of factors which might influence the integrity of the corneal epithelium or alter its susceptibility to disease.

Animals↗
PubMed1983

Spontaneous formation of nucleus-like structures around bacteriophage DNA microinjected into Xenopus eggs.

We have found that injection of bacteriophage lambda DNA into unfertilized Xenopus eggs causes the assembly around the DNA of structures resembling typical eucaryotic cell nuclei. These spherical structures begin to form 60-90 min after injection. They contain lambda DNA and are bounded by a phase-dense envelope. Immunofluorescent staining of the lambda-DNA-containing structures with anti-lamin antibody reveals the presence of the lamin nuclear proteins at the periphery of the structure, a pattern identical to that of embryonic nuclei. Electron microscopy reveals that the injected DNA is surrounded by a double bilayer nuclear membrane containing nuclear pore complexes. The "nuclei" containing lambda DNA respond to modulators of the Xenopus cell cycle in a manner that mimics the response of embryonic nuclei to these modulators during mitosis. These results suggest that nuclear reassembly and breakdown occur independently of specific DNA sequence information.

Animals↗
PubMed1983

Small nuclear RNA transcription and ribonucleoprotein assembly in early Xenopus development.

The Xenopus egg and embryo, throughout the transcriptionally inactive early cleavage period, were found to contain a store of approximately 8 X 10(8) molecules of the small nuclear RNA (snRNA) U1, sufficient for 4,000-8,000 nuclei. In addition, when transcription is activated at the twelfth cleavage (4,000 cell-stage), the snRNAs U1, U2, U4, U5, and U6 are major RNA polymerase II products. From the twelfth cleavage to gastrulation, U1 RNA increases sevenfold in 4 h, paralleling a similar increase in nuclear number. This level of snRNA transcription is much greater than that typical of somatic cells, implying a higher rate of U1 transcription or a greater number of U1 genes active in the embryo. The Xenopus egg also contains snRNP proteins, since it has the capacity to package exogenously added snRNA into immunoprecipitable snRNP particles, which resemble endogenous particles in both sedimentation coefficient and T1 RNase digestibility. SnRNP proteins may recognize conserved secondary structure of U1 snRNA since efficient packaging of both mouse and Drosophila U1 RNAs, differing 30% in sequence, occurs. The Xenopus egg and embryo can be used to pose a number of interesting questions about the transcription, assembly, and function of snRNA.

Animals↗
PubMed1981

Neurogenesis in the trigeminal ganglion of the albino rat: a quantitative autoradiographic study.

The time of neuron origin in the trigeminal ganglion was examined in autoradiograms of 60-day-old rats that were exposed to a single pulse of 3H-thymidine on day 11, 12, 13, 14, or 15 of gestation. Heavily labeled neurons, representing cells in or near their last mitotic division at the time of the pulse label, were present in animals injected between embryonic days 11 and 13 with a peak on day 12. Within this time period, larger neurons were generated prior to smaller neurons with a peak for larger cells on day 12 and for smaller cells on day 13. Thus, the majority of trigeminal ganglion neurons are generated over a three-day period just after the midpoint of gestation. Neuron number, size, type, and cytoarchitectural organization were also examined in the ganglion. The mean neuron count per ganglion was 52,372. The size distribution of these cells ranged continuously from 7-61 microns (mean diameter) with no evidence for clearly defined subpopulations. The staining intensity and distribution patterns of the Nissl substance varied greatly from cell to cell precluding the classification of cells as light or dark. Little correspondence between these Nissl features and cell size was found. Among the clusters and rows of neurons in the ganglion, we did not see consistent cytoarchitectonic patterns which might reflect specific sensory receptive fields.

Animals↗
PubMed1979

Computer-assisted mapping with the light microscope.

With the system described specific features in a histological section such as neuronal degeneration, specific cell types, or the silver grains of autoradiography can be accurately localized. It is simple to operate, and allows for data storage and retrieval. The system consists of a Zeiss Universal microscope equipped with stepping motor drives on the X and Y axes and interfaced to a PDP-12 computer equipped with a printer/plotter. The user controls the system through a joystick control box and teletype. In spite of magnification changes all points in the field remain constant relative to a point of origin selected at the beginning of the mapping sequence. Therefore, the outline and major features of a section are entered at low magnification while the detail of neuronal degeneration, for example, is added in its precise location at a higher magnification. The plotting of points can be done in either random fashion or in a systemized series of horizontal or vertical scans. A graphic display is available throughout the procedure and all maps are both printed on a Versatec printer/plotter and stored on a disk for future recall and analysis. Use of the system in plotting neuronal degeneration is described.

Animals↗