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R L Margolis

Publications and source records attributed to R L Margolis.

At least 91 records · Page 5Linked to original sources
PubMed1995

Cloning and expression of the rat atrophin-I (DRPLA disease gene) homologue.

Dentatorubral pallidoluysian atrophy (DRPLA) is a rare, progressive, fatal neuropsychiatric disorder similar to Huntington's disease, caused by an expansion of a CAG trinucleotide repeat encoding glutamine. We have cloned the cDNA of the rat homologue of this gene. The cDNA contains a 3549 base pair open reading frame that is 88.2% identical to the human cDNA, with a predicted amino acid sequence that is 93.6% identical to the human sequence. The consecutive glutamine repeat is only five residues in length (normal range in human: 7-35 glutamines) and is followed by a polymorphic region of alternating glutamine and proline residues (QQQQQPQPQPQPQQ). The sequence also includes a polymorphic proline repeat, a serine repeat, and a region of alternating acidic and basic residues. Northern analysis and in situ hybridization indicate that the gene is widely expressed as a 4.5 kb mRNA, with a neuronal distribution in the brain. The widespread expression of this gene is consistent with the possibility that DRPLA, like other glutamine repeat diseases, is a result of an abnormality at the protein level.

Animals↗
PubMed1995

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells.

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase alpha as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase alpha are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.

Base Sequence↗
PubMed1995

Characterization of cDNA clones containing CCA trinucleotide repeats derived from human brain.

Expansion mutation is the cause of eight neuropsychiatric disorders. Thus far each disease is the result of expansion of a C-G rich trinucleotide repeat that is polymorphic for length in the general population. We now report the identification of seven novel cDNA clones with CCA or equivalent trinucleotide repeats obtained by screening a human frontal cortex cDNA library. The repeat lengths of two clones, CCA11 (linked to D20S101, expressed in human brain as a 3.2 kb message) and CCA38 (linked to D5S404), are highly polymorphic in a normal human population. CCA54, mapped to chromosome 19, appears to correspond to a portion of the human gene encoding the alpha 1 subunit of a P-type calcium channel. Expansion mutations at these loci should be considered as possible candidates in evaluating the genetic etiologies of diseases linked to chromosomes 5, 19, and 20.

Base Sequence↗
PubMed1995

Microsatellite evolution--evidence for directionality and variation in rate between species.

Microsatellite DNA sequences are rapidly becoming the dominant source of nuclear genetic markers for a wide range of applications, from genome mapping to forensic testing to population studies. If misinterpretation is to be avoided, it is vital that we understand fully the way in which microsatellite sequences evolve. We have therefore compared allele length distributions for 42 microsatellites in humans with their homologues in a range of related primates. We find a highly significant trend for the loci to be longer in humans, showing that microsatellites can evolve directionally and at different rates in closely related species.

Alleles↗
PubMed1995

Delay of HeLa cell cleavage into interphase using dihydrocytochalasin B: retention of a postmitotic spindle and telophase disc correlates with synchronous cleavage recovery.

The molecular signals that determine the position and timing of the cleavage furrow during mammalian cell cytokinesis are presently unknown. We have studied in detail the effect of dihydrocytochalasin B (DCB), a drug that interferes with actin assembly, on specific late mitotic events in synchronous HeLa cells. When cleavage furrow formation is blocked at 10 microM DCB, cells return to interphase by the criteria of reformation of nuclei with lamin borders, degradation of the cyclin B component of p34cdc2 kinase, and loss of mitosis specific MPM-2 antigens. However, the machinery for cell cleavage is retained for up to one hour into G1 when cleavage cannot proceed. The components retained consist prominently of a "postmitotic" spindle and a telophase disc, a structure templated by the mitotic spindle in anaphase that may determine the position and timing of the cleavage furrow. Upon release from DCB block, G1 cells proceed through a rapid and synchronous cleavage. We conclude that the mitotic spindle is not inevitably destroyed at the end of mitosis, but persists as an integral structure with the telophase disc in the absence of cleavage. We also conclude that cell cleavage can occur in G1, and is therefore an event metabolically independent of mitosis. The retained telophase disc may indeed signal the position of furrow formation, as G1 cleavage occurs only in the position where the retained disc underlies the cell cortex. The protocol we describe should now enable development of a model system for the study of mammalian cell cleavage as a synchronous event independent of mitosis.

Actins↗
PubMed1995

Activation-induced T-cell death is cell cycle dependent and regulated by cyclin B.

Developing thymocytes and some T-cell hybridomas undergo activation-dependent programmed cell death. Although recent studies have identified some critical regulators in programmed cell death, the role of cell cycle regulation in activation-induced cell death in T cells has not been addressed. We demonstrate that synchronized T-cell hybridomas, irrespective of the point in the cell cycle at which they are activated, stop cycling shortly after they reach G2/M. These cells exhibit the diagnostic characteristics of apoptotic cell death. Although p34cdc2 levels are not perturbed after activation of synchronously cycling T cells, cyclin B- and p34cdc2-associated histone H1 kinase activity is persistently elevated. This activation-dependent induction of H1 kinase activity in T cells is associated with a decrease in the phosphotyrosine content of p34cdc2. We also demonstrate that transient inappropriate coexpression of cyclin B with p34cdc2 induces DNA fragmentation in a heterologous cell type. Finally, in T cells, cyclin B-specific antisense oligonucleotides suppress activation-induced cell death but not cell death induced by exposure to dexamethasone. We therefore conclude that a persistent elevation of the level of cyclin B kinase is required for activation-induced programmed T-cell death.

Amino Acid Sequence↗
PubMed1994

Programmed cell death: implications for neuropsychiatric disorders.

Programmed cell death, sometimes referred to as apoptosis, occurs through an active process requiring new gene transcription, in contrast to the passive cell death produced by metabolic toxins. Programmed cell death is an essential part of normal development, particularly in the nervous system. Spatial, temporal, or quantitative errors in the stimuli that initiate programmed cell death, or errors within the programmed cell death pathway itself, can result in an abnormal number of neurons and pathological neural development. Excesses and deficits in neuronal numbers have now been observed not only in typical neurodegenerative disorders such as Alzheimer's and Huntington's diseases, but also in several neurodevelopmental disorders, including schizophrenia and autism. Recent investigations into the mechanisms of cell death during C. elegans neurodevelopment thymocyte negative selection, and withdrawal of sympathetic ganglion cells trophic support provides intriguing clues to the etiology and pathophysiology of these neuropsychiatric disorders.

Apoptosis↗
PubMed1994

Growth factor-induced binding of dynamin to signal transduction proteins involves sorting to distinct and separate proline-rich dynamin sequences.

Dynamin, a 100 kDa GTPase, is critical for endocytosis, synaptic transmission and neurogenesis. Endocytosis accompanies receptor processing and plays an essential role in attenuating receptor tyrosine kinase signal transduction. Dynamin has been demonstrated to be involved in the endocytic processing at the cell surface and may play a general role in coupling receptor activation to endocytosis. Src homology (SH) domain dependent protein-protein interactions are important to tyrosine kinase receptor signal transduction. The C-terminus of dynamin contains two clusters of SH3 domain binding proline motifs; these motifs may interact with known SH3 domain proteins during tyrosine kinase receptor activation. We demonstrate here that SH3 domain-containing signal transduction proteins, such as phospholipase C gamma-1 (PLC gamma-1), do indeed bind to dynamin in a growth factor inducible manner. The induction of PLC gamma-1 binding to dynamin occurs within minutes of the addition of platelet derived growth factor (PDGF) to cells. Binding of these signal transduction proteins to dynamin involves specific sorting to individual proline motif clusters and appears to be responsible for co-immunoprecipitation of tyrosine phosphorylated PDGF receptors with dynamin following PDGF stimulation of mammalian cells. The binding of dynamin to SH3 domain-containing proteins may therefore be important for formation of the protein complex required for the endocytic processing of activated tyrosine kinase receptors.

3T3 Cells↗
PubMed1994

Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells.

The protein kinase inhibitor 2-aminopurine induces checkpoint override and mitotic exit in BHK cells which have been arrested in mitosis by inhibitors of microtubule function (Andreassen, P. R., and R. L. Margolis. 1991. J. Cell Sci. 100:299-310). Mitotic exit is monitored by loss of MPM-2 antigen, by the reformation of nuclei, and by the extinction of p34cdc2-dependent H1 kinase activity. 2-AP-induced inactivation of p34cdc2 and mitotic exit depend on the assembly state of microtubules. During mitotic arrest generated by the microtubule assembly inhibitor nocodazole, the rate of mitotic exit induced by 2-AP decreases proportionally with increasing nocodazole concentrations. At nocodazole concentrations of 0.12 microgram/ml or greater, 2-AP induces no apparent exit through 75 min of treatment. In contrast, 2-AP brings about a rapid exit (t1/2 = 20 min) from mitotic arrest by taxol, a drug which causes inappropriate overassembly of microtubules. In control mitotic cells, p34cdc2 localizes to kinetochores, centrosomes, and spindle microtubules. We find that efficient exit from mitosis occurs under conditions where p34cdc2 remains associated with centrosomal microtubules, suggesting it must be present on these microtubules in order to be inactivated. Mitotic slippage, the natural reentry of cells into G1 during prolonged mitotic block, is also microtubule dependent. At high nocodazole concentrations slippage is prevented and mitotic arrest approaches 100%. We conclude that essential components of the machinery for exit from mitosis are present on the mitotic spindle, and that normal mitotic exit thereby may be regulated by the microtubule assembly state.

2-Aminopurine↗
PubMed1993

Effect of cocaine, lidocaine kindling and carbamazepine on batrachotoxin-induced phosphoinositide hydrolysis in rat brain slices.

Repeated administration of a subconvulsant dose of a local anesthetic will eventually induce seizures, a phenomenon similar to electrical kindling. We have investigated the effect of repeated lidocaine and cocaine administration on the phosphoinositide (PI) hydrolysis induced by batrachotoxin (BTX), a specific Na channel activator. Rats were injected with cocaine or saline daily for 6 days and PI hydrolysis was assayed in sliced frontal cortex. Cocaine treatment had no effect on BTX-induced PI hydrolysis while in vitro cocaine blocked the BTX effect. In a second experiment, rats received daily injections of lidocaine or saline. After a rat developed at least two seizures, it was sacrificed together with a rat receiving lidocaine injections which had never seized and a rat receiving saline injections. Basal, BTX and ibotenic acid (IBO; a glutamate receptor agonist)-stimulated PI hydrolysis did not differ among the three groups in slices of either hippocampus (HC) or piriform cortex (PC) though IBO-stimulated PI hydrolysis was much greater in the HC than in the PC. Neither in vitro nor in vivo carbamazepine altered the effect of cocaine on BTX-induced PI hydrolysis. These results demonstrate that local anesthetic kindling does not alter PI hydrolysis coupled to Na channel or IBO activation.

Animals↗
PubMed1993

The telophase disc: its possible role in mammalian cell cleavage.

The molecular signals that determine the position and timing of the furrow that forms during mammalian cell cytokinesis are presently unknown. It is apparent, however, that these signals are generated by the mitotic spindle after the onset of anaphase. Recently we have described a structure that bisects the cell during telophase at the position of the cytokinetic furrow. This structure, the telophase disc, appears to be templated by the mitotic spindle during anaphase, and precedes the formation of the cytokinetic furrow. The relationship of the telophase disc to the myosin and actin based furrowing mechanism is discussed here. We propose that the telophase disc may determine the position and timing of cleavage by recruitment and alignment of myosin.

Actins↗
PubMed1993

Novel triplet repeat containing genes in human brain: cloning, expression, and length polymorphisms.

Human genes containing triplet repeats may markedly expand in length and cause neuropsychiatric disease, explaining the phenomenon of anticipation (increasing severity or earlier age of onset in successive generations in a pedigree). To identify novel genes with triplet repeats, we screened a human brain cDNA library with oligonucleotide probes containing CTG or CCG triplet repeats. Fourteen of 40 clones encoded novel human genes, and 8 of these inserts have been sequenced on both strands. All contain repeats, and 5 of the 8 have 9 or more consecutive perfect repeats. All are expressed in brain. Chromosomal assignments reveal a distribution of these genes on multiple autosomes and the X-chromosome. Further, the repeat length in two of the genes is highly polymorphic, making them valuable index linkage markers. We predict that many triplet repeat-containing genes exist; screening with the CTG probe suggests approximately 50-100 genes containing this type of repeat are expressed in the human brain. Since additional disorders, such as Huntington's disease, bipolar affective disorder, and possibly others, show features of anticipation, we suggest that these novel human genes with triplet repeats are candidates for causing neuropsychiatric diseases.

Base Sequence↗
PubMed1993

Genes with triplet repeats: candidate mediators of neuropsychiatric disorders.

Recently a new form of human mutation-expansion of trinucleotide repeats-has been found to cause the diseases of fragile X syndrome, spinal and bulbar muscular atrophy, myotonic dystrophy and, most recently, Huntington's disease. We review the emerging data on the genetics and neurobiology of these disorders. Three are characterized by unusual patterns of inheritance, in particular, genetic 'anticipation', in which the severity of the disorder increases and the age of onset decreases in successive generations of a pedigree. Several idiopathic neuropsychiatric disorders have features of inheritance consistent with anticipation. In bipolar affective disorder, there is evidence for both earlier age of onset and more severe illness in the second generation of a subset of unilineal pedigrees. There is also the suggestion of anticipation in some forms of schizophrenia, spinocerebellar atrophy and autism. Triplet repeats are present in additional known genes, both in coding regions and untranslated regions. Furthermore, many novel genes with triplet repeats are expressed in the human brain, and these are candidates to cause some forms of these neuropsychiatric disorders.

Humans↗
PubMed1993

Huntington's disease gene (IT15) is widely expressed in human and rat tissues.

Huntington's Disease (HD) is notable for selective neuronal vulnerability in the basal ganglia and cerebral cortex. We have investigated in human and rodent tissues the expression of the gene (IT15) whose mutation causes HD. IT15 is widely expressed, with highest levels of expression in brain, but also in lung, testis, ovary, and other tissues. Within the brain, expression is widespread with a neuronal pattern and is not enriched in the basal ganglia. Expression of IT15 is not reduced in the brain of HD patients when corrected for actin (though it is slightly decreased in the striatum when uncorrected, consistent with neuronal loss). Thus, the widespread distribution of IT15 expression does not correspond with the restricted distribution of neuropathologic changes in HD. We suggest that pathophysiology may relate to abnormal cell type-specific protein interactions of the HD protein.

Animals↗
PubMed1992

Ca(2+)-calmodulin regulated effectors of microtubule stability in neuronal tissues.

In general, microtubules are labile structures which depolymerize at low temperature and are sensitive to Ca2+. However, in brain tissue, axonal microtubules are disassembly-resistant and can exist without attachment to a microtubule organizing center. Stable microtubules cannot be purified by usual recycling procedures and this has made the elucidation of the molecular mechanisms involved in their stabilization difficult. This paper summarizes previous work in our laboratories, aimed at the identification of brain microtubule stabilizing proteins. We present assay methods which allow the detection of microtubule stability effectors in complex extracts and in chromatographic column fractions. Applied to brain crude extracts, they result in the isolation of Ca(2+)-calmodulin binding and Ca(2+)-calmodulin regulated proteins. One, called STOP, appears to account for microtubule stabilization in neurons. A second protein with similar activity is myelin basic protein. Non-neuronal tissues also contain Ca(2+)-calmodulin-regulated effectors which appear to differ in structure from their neuronal counterparts. Thus, in all tissues examined, microtubule stability seems to be accounted for by unique Ca(2+)-calmodulin regulated proteins, showing tissue specificity.

Animals↗
PubMed1992

Ca(2+)-calmodulin regulated effectors of microtubule stability in bovine brain.

Stable microtubules (as defined by resistance to Ca2+, drug or cold temperature induced disassembly) form in abundance during tubulin assembly in brain crude extracts. We have previously shown that, in rat brain crude extracts, all microtubule stabilizing activity could be ascribed to a single Ca(2+)-calmodulin binding and Ca(2+)-calmodulin regulated protein, called "stable tubule only polypeptide", STOP145 [Pirollet, F., Rauch, C. T., Job, D., & Margolis, R. L. (1989) Biochemistry 28, 835-842]. We have now performed an exhaustive study of STOP-like effectors in bovine brain high-speed supernatants. All activity binds to cation exchangers and to Ca(2+)-calmodulin affinity columns. The activity can be resolved into two peaks on sizing columns. The first eluted peak contains a prominent 220-kDa protein. The second peak contains an apparently homogeneous 20-kDa polypeptide. A monoclonal antibody specific to rat brain STOP145 recognizes the 220-kDa protein, but not the 20-kDa species. The 220-kDa protein can be purified on a STOP antibody column and accounts for the bulk of stabilizing activity in the first peak. The 20-kDa protein does not bind to STOP antibody affinity columns. Sequence analysis of oligopeptide fragments of the 20-kDa protein shows 100% homology with bovine myelin basic protein (MBP). Anti-MBP antibodies recognize the 20-kDa, but not the 220-kDa species. We conclude that the 220-kDa protein is the bovine equivalent to rat brain STOP145 and that the 20-kDa species is MBP. Microtubule stabilization by MBP and STOP220 is abolished in the presence of Ca(2+)-calmodulin, and inhibition curves are similar for both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗