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Ultrastructural analysis of an active gene
12
Cell Biology International
s29
THE EXPRESSION OF MICROTUBULE GENES DURING NEURITE OUTGROWTH Irith Ghuburg, Department of Neurobiology, The Weixmann Institute of Science,Rehovot 76100, Israel. The diversity of microtubule (MT) functions and the temporal employment of MI’s through cell division, growth, and differentiation, suggestthat the expression of MT genes may be tightly regulated. MTs are particularly important in the nervous system, where they are involved in neurite outgrowth and axonal transport. Brain MTs are composed of tubulin and MT-associated proteins, which copolymerixe with tubulin, promote MT assembly in vitro, and colocalii with MTs in cells. Two major MAP gtoups have been identified in brahi extracts. These include the tau proteins of 50-70 kDa, and the high molecular weight proteins MAEl-5 of 250-350 kDa. A variety of data implicate MAPS and, in particular, tau in regulating the stability of Ml’s in neuronal cells. The NGF-msponsive EC12 cell line. has been used as a model system to study the expression of cytoskeletal genes, during net&e outgrowth and development. One of the initial events in neuronal differentiation is the cell adhesion to the substratum. Neuronal interactions with the extracellular matrix (ECM) components stimulate neurite extension, and is crucial for axon growth and guidance during development and nerve regeneration. The effect of various ECM components on the expression of MT genesdtaing FClZinduced differentiation was, therefore, folhnvrxL It was observed that using tubulin specific cDNA probes, dte expression of pTa26 mRNA mcmasesconUnuousIydming the differentiation process, whik the expression of PTal mRNA is down-regulated and returns to its basal level after 48 h of induction. The use of specific antisenseoligodeoxynucl, which block the expmssion of specific transcripts, was another approach employed to study the involvement of individual gene transcripts during EC12 differentiation. Specific inhibition of neurite outgrowth was observed in cells treated with antisense oliiodwxynucleotides coneqmnding to tubulin and tau sequences. The results contribute to our understanding of the involvement of MT gene products in neuronal differentiation and their temporal interaction with other cell compcoents. Supportedby the Israel National Council for Research and Development, the European Economic Council. the Minerva Foundation. and the Forschheimer Centerfor Molecular Biology.
s30
s31
S32
A NOVEL DNA STRUCURE AT THE TELOMERlC TIPS OF CHROMOSOMES Aaron Klug, Wesley A Sundquist. MRC Laboratory of Molecular Biology, Cambridge, England. Oligonuclwtides encoding Tetrohymena and Oxyticha telomeres dimerize to form stable complexes in solution, the dimerization being mediated entirely by the single-stranded 3’terminal overhang. These overhangs form intermolecular hairpin loops which dime&e to form antiparallel quaduplexes containing cyclic guanine base tetrads. These novel hairpin dimers may be important in telomere association and recombination and could also provide a general mechanism for pairing two double helices in other recombinational processes.
Reports, Vol. 14, Abstracts Supplement
1990
CGNTROL OF ORGANELLE DJSTRTBUTfON ANDSE4PEBYCYTOPLASMIC~ John Heuser, Department of Cell Biology & Physi&ogy, Washington Unlversitv Med. School. St. Louis. MO 63110 The question of how cells establish the characteristic shape and spatial distribution of their organelles has begun to be answered by many recent advances in our understanding of the biochemistry and in vitro behavior of cytoplasmic motor proteins. Still lacking, however, is any clear understanding of how different competing motors produce the arderIy patterns of organelks seen b a. Our recent Ending that cytoplasmic pH controls the balance between the anterograde and retrograde forces that move endosomes and lysosomts around in <ured ceils (J. Cell Biol. 10s: 855-864) introduced one means to aria&e such Jo &otor-c&rol mechanisms. In that study we observed that lowering cytoplasmic pH (to ca. 6.5) inhibited and even reversed the normal retrograde flow of endosomal memhrapes. This suggested that low pH might inhibit cytoplasmic dynein and/or stimulate I&K.& motors. Here, we will describe additional findings that support this conclusion: most importantly, the tJnding that exogenously applied vandate (2 mM) directly inhibits retrograde organelle movement in macrophageswithout altering their pH or otherwise damaging them This allows us to demonstrate the direct induction of “reversed” or anterograde endosome movements upon lowering cytoplasmic pH, even after dynein activity has been suppressed. Thus, the macrophage has clearly separate motors for anterograde and retrograde movements of this class of organelles. A further manifestation of this fact is that when endosomes and lysosomesare made to fuse together by loading them with weak bases, a motor “competition” ensues which stretches these organelles out into thin tubules that closely parallel cytoplasmic microtubules. Supported by U.S.P.H.S. Grant #GM29645 and the M.D.AA.
ULTRASTRUCTURAL
ANALYSIS OF AN ACTIVE GENE
Bertil Daneholt, Christer Ericsson, Birgitta Bjorkroth, and Ulrich Grossbach.. Denartment of Molecular Genetics. Rarolinska Institutet. Stockholm. Sweden; *3td Dqartment of Zcology-Developmental Biology; University of G(lttingen, GtXtingen,Federal Republic of Germany The native chmmatin template of transcriptionsEy active geneshas been visualixed by electron microscopic methods. The Balbiani ring genes in the salivary glandsof ChiroMrmcs rearanshave been anaIyxed specifiially: these genes are exceptionally long (37 kb) and very active in transcription. The most abundant configuration of the template is an extended fiber, approximately 5 MI in diameter. When the distance between adjacent RNA polymerases is unusuaEy long, the template is packed into a 10 nm fiber. OccasionaEy, the fiber can further fold into a loose coil forming a more or less distinct 30 nm fiber. It is concluded that a large part of the chromatin axis is in a fully extended form during transcription of the Balbiani ring genes. However, if a given segment of the template is not continuously occupied by RNA polymerases it can be packed into a single nucleosome, into a string of nucleosomes(the thin f&r) or even into a supercoiled string of nucleosomes (the thick fiber). Apparently the transcribing gene is a dynamic structure that most commonly is extended, but that packs into nucleosomecontahdng fibers in the absetuze. of RNA polymeram. The localization of histone II1 on the Balbiani ring genes was investigated using immtmo &ctmn microscopy. Histone H 1 was found not only on genetically inactive, compact chmmatin but also on the highly active Balbii ring geneseven when they are in their maximally extended confommtion. This suggeststhat the hismnea,including histone Hl, remain bound to the template and that the nucleosomes are unfolded at the RNA
pal-.
Recently it has also been feasible to obtain a clearer view of the upstream andQwnstrernn~ofaBalbianiringgene.Thcgcncstartsandendsin psckedc~.TbeSandthe3’regioasaresepsratefromeachotherand evidently a cIosed loop is not formed. The -nonuanscribedmgion is found as a 200 MI long nucleoiRament fiber loosely coiled into a 30 nm fiber. The promoter region consists of a 0.5 kb long nucleosome-free, extended fiber. This structure would be compatible with the concept that only a few tramcriptioafactors interactwith the RNA polymerase at the initiation site and that some such factors might act, at least in part, by preventing nucleostnne3to be formed on the promoter region.
Cell Biology International
s29
THE EXPRESSION OF MICROTUBULE GENES DURING NEURITE OUTGROWTH Irith Ghuburg, Department of Neurobiology, The Weixmann Institute of Science,Rehovot 76100, Israel. The diversity of microtubule (MT) functions and the temporal employment of MI’s through cell division, growth, and differentiation, suggestthat the expression of MT genes may be tightly regulated. MTs are particularly important in the nervous system, where they are involved in neurite outgrowth and axonal transport. Brain MTs are composed of tubulin and MT-associated proteins, which copolymerixe with tubulin, promote MT assembly in vitro, and colocalii with MTs in cells. Two major MAP gtoups have been identified in brahi extracts. These include the tau proteins of 50-70 kDa, and the high molecular weight proteins MAEl-5 of 250-350 kDa. A variety of data implicate MAPS and, in particular, tau in regulating the stability of Ml’s in neuronal cells. The NGF-msponsive EC12 cell line. has been used as a model system to study the expression of cytoskeletal genes, during net&e outgrowth and development. One of the initial events in neuronal differentiation is the cell adhesion to the substratum. Neuronal interactions with the extracellular matrix (ECM) components stimulate neurite extension, and is crucial for axon growth and guidance during development and nerve regeneration. The effect of various ECM components on the expression of MT genesdtaing FClZinduced differentiation was, therefore, folhnvrxL It was observed that using tubulin specific cDNA probes, dte expression of pTa26 mRNA mcmasesconUnuousIydming the differentiation process, whik the expression of PTal mRNA is down-regulated and returns to its basal level after 48 h of induction. The use of specific antisenseoligodeoxynucl, which block the expmssion of specific transcripts, was another approach employed to study the involvement of individual gene transcripts during EC12 differentiation. Specific inhibition of neurite outgrowth was observed in cells treated with antisense oliiodwxynucleotides coneqmnding to tubulin and tau sequences. The results contribute to our understanding of the involvement of MT gene products in neuronal differentiation and their temporal interaction with other cell compcoents. Supportedby the Israel National Council for Research and Development, the European Economic Council. the Minerva Foundation. and the Forschheimer Centerfor Molecular Biology.
s30
s31
S32
A NOVEL DNA STRUCURE AT THE TELOMERlC TIPS OF CHROMOSOMES Aaron Klug, Wesley A Sundquist. MRC Laboratory of Molecular Biology, Cambridge, England. Oligonuclwtides encoding Tetrohymena and Oxyticha telomeres dimerize to form stable complexes in solution, the dimerization being mediated entirely by the single-stranded 3’terminal overhang. These overhangs form intermolecular hairpin loops which dime&e to form antiparallel quaduplexes containing cyclic guanine base tetrads. These novel hairpin dimers may be important in telomere association and recombination and could also provide a general mechanism for pairing two double helices in other recombinational processes.
Reports, Vol. 14, Abstracts Supplement
1990
CGNTROL OF ORGANELLE DJSTRTBUTfON ANDSE4PEBYCYTOPLASMIC~ John Heuser, Department of Cell Biology & Physi&ogy, Washington Unlversitv Med. School. St. Louis. MO 63110 The question of how cells establish the characteristic shape and spatial distribution of their organelles has begun to be answered by many recent advances in our understanding of the biochemistry and in vitro behavior of cytoplasmic motor proteins. Still lacking, however, is any clear understanding of how different competing motors produce the arderIy patterns of organelks seen b a. Our recent Ending that cytoplasmic pH controls the balance between the anterograde and retrograde forces that move endosomes and lysosomts around in <ured ceils (J. Cell Biol. 10s: 855-864) introduced one means to aria&e such Jo &otor-c&rol mechanisms. In that study we observed that lowering cytoplasmic pH (to ca. 6.5) inhibited and even reversed the normal retrograde flow of endosomal memhrapes. This suggested that low pH might inhibit cytoplasmic dynein and/or stimulate I&K.& motors. Here, we will describe additional findings that support this conclusion: most importantly, the tJnding that exogenously applied vandate (2 mM) directly inhibits retrograde organelle movement in macrophageswithout altering their pH or otherwise damaging them This allows us to demonstrate the direct induction of “reversed” or anterograde endosome movements upon lowering cytoplasmic pH, even after dynein activity has been suppressed. Thus, the macrophage has clearly separate motors for anterograde and retrograde movements of this class of organelles. A further manifestation of this fact is that when endosomes and lysosomesare made to fuse together by loading them with weak bases, a motor “competition” ensues which stretches these organelles out into thin tubules that closely parallel cytoplasmic microtubules. Supported by U.S.P.H.S. Grant #GM29645 and the M.D.AA.
ULTRASTRUCTURAL
ANALYSIS OF AN ACTIVE GENE
Bertil Daneholt, Christer Ericsson, Birgitta Bjorkroth, and Ulrich Grossbach.. Denartment of Molecular Genetics. Rarolinska Institutet. Stockholm. Sweden; *3td Dqartment of Zcology-Developmental Biology; University of G(lttingen, GtXtingen,Federal Republic of Germany The native chmmatin template of transcriptionsEy active geneshas been visualixed by electron microscopic methods. The Balbiani ring genes in the salivary glandsof ChiroMrmcs rearanshave been anaIyxed specifiially: these genes are exceptionally long (37 kb) and very active in transcription. The most abundant configuration of the template is an extended fiber, approximately 5 MI in diameter. When the distance between adjacent RNA polymerases is unusuaEy long, the template is packed into a 10 nm fiber. OccasionaEy, the fiber can further fold into a loose coil forming a more or less distinct 30 nm fiber. It is concluded that a large part of the chromatin axis is in a fully extended form during transcription of the Balbiani ring genes. However, if a given segment of the template is not continuously occupied by RNA polymerases it can be packed into a single nucleosome, into a string of nucleosomes(the thin f&r) or even into a supercoiled string of nucleosomes (the thick fiber). Apparently the transcribing gene is a dynamic structure that most commonly is extended, but that packs into nucleosomecontahdng fibers in the absetuze. of RNA polymeram. The localization of histone II1 on the Balbiani ring genes was investigated using immtmo &ctmn microscopy. Histone H 1 was found not only on genetically inactive, compact chmmatin but also on the highly active Balbii ring geneseven when they are in their maximally extended confommtion. This suggeststhat the hismnea,including histone Hl, remain bound to the template and that the nucleosomes are unfolded at the RNA
pal-.
Recently it has also been feasible to obtain a clearer view of the upstream andQwnstrernn~ofaBalbianiringgene.Thcgcncstartsandendsin psckedc~.TbeSandthe3’regioasaresepsratefromeachotherand evidently a cIosed loop is not formed. The -nonuanscribedmgion is found as a 200 MI long nucleoiRament fiber loosely coiled into a 30 nm fiber. The promoter region consists of a 0.5 kb long nucleosome-free, extended fiber. This structure would be compatible with the concept that only a few tramcriptioafactors interactwith the RNA polymerase at the initiation site and that some such factors might act, at least in part, by preventing nucleostnne3to be formed on the promoter region.