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Dictyostelium nuclear plasmid DdP1: Investigation of plasmid encoded functions
Cell Biology
DICTYOSTELZUM NUCLEAR PLASMID P437 INVESTIGATIONOFPLASMIDENCODEDFUNCTIONS
International
DDP1:
Christine B.Gurniak and Angelika A. Noegel Max-Planck-Instimt fiir Bicchemie, 8033 Martinsried/FRG Various strains of Dictyostelium carry nuclearplasmids. They differ in size from 1.3kb to 27kb and do not show homology amongst each other, to chromosomal, ribosomal -dr mitochondxial DNA. Ddpl (13Skb) codes for 9 partially overlapping transcripts. A sequence of S.lkb of Ddpl encompassing four open reading frames (ORFs) has been determined and the corresponding transcripts have been identified. One of the transcripts is affected in its accumulation pattern by CAMP, an importantsignalduringDictyostelium development. None of the ORF% shows significant homologies to known proteins. The plasmid-soe&ied products are investigated lollowing two ities: a) expression of ORFs in E.coli and production of antibodies, b) expression in Dictyostelium-after introductionof a suitablkepitbpeinto the gene under investigation. The determined sequence also contains the oriti of reolication. The seouence reuuired for replication extends over !LOkb. A rranscxiit is loca& within this region which could code for a urotein of 34kD and mav be involv& in replication of Ddpl.*Although a transformation vector generated on the basis of Ddpl sequences remains extrachromosomal, though at a reduced copy number, this plasmid cannot be maintained extrachromosomal once it carries additional Dictyostelium sequences.The 2.Okb fragment was investigated in South-Western blot analyses for specific binding to nuclear proteins. In nuclear extracts of plasmid containing cells a protein of 32kD was detected whoese binding could be competed with excess of cold fragment and which was not present in nuclear extracts of a plasmid free Dictyostelium strain.
P439 TNg DRCXCIPHILA POLP(A) BINDING PROTEIN: CLONING AND HIPBBSSION TNROUGOIJTDgVNLOPNgNT. Valirie Lefr&re, Alain Vincent, Francois Amalric. CRBGC du CNRS, 118 route de Narbonne 31062 Toulouse cedex, FRANCE Two classes of cDNAs encoding the Drosophila poly(A) binding protein (PABP), which differ by the position of their respective 3' end separated by about 1 kb, were isolated by screening an embryonic cDNA librairy. These cDNAs hybridize to a single chromosomal site at position 55B on the right arm of the second chromosome. A unique 3.8 kb mRNA was detected, suggesting that "long" cDNAs correspond to full length cDNAs and that the 3' untranslated region of the mRNA is close to 1.5 kb long. The PABP transcript accumulates in oocytes, is maternally inherited by the embryo and present at every other developmental stage tested. The PABP cDNAs contain a 1722 nt long open reading frame encoding a 64 kDa protein with high affinity for poly(A). This protein contains four RNA binding domains which show limited primary sequence evolution, contrasting to the carboxy-terminal third of the protein; The strikingly long 3' untranslated region of the Drosophila PABP mRNA is shown to exist also in other eukaryotes, including vertebrate species. It suggests that important regulatory sequences intrinsic to the PABP mNNA are present in its 3' untranslated region. Data on the expression of the protein throughout development will be presented.
Reports,
P438
Vol. 74, Abstracts
HEAT-INDUCED LAMPBRUSH ENVIRONMENTAL
Supplement
CHANGES CHROMOSOMES CONDITIONS.
IN
AMPHIBIAN DEPEND
1990
ON
Maria-LuzRodriguez-Martin,NicoleMoreau. C%annsineHerbcrts, Nicole Angelier. UPR 3101, Centre de Biologic Cellulaire du CNRS,94205My-sur-Seine Cedex, France. At ll~nnal breedingtemperahlre(20°c)amphibianlampb~sh chromosomes art characterized by the presence oflaterslloops wbicharedirectlytelatcdtothe transcriptional processes.Inthe oocytes of the newt PZeurod&swalrl, heat treatment induces striking modifications in rhe structure of lampbrush loops but the nature and tbniw of these modifications depend on hyperthermic stressconditions. Weed. our data demonstrate thatat the same high temperature
(WC),
lampbrush
chmnosomes
modifications induced
by in viva heat treatment are different from those induced in in vine experimental conditions. When heat treatment (34°C for 45 min to 4 days) is directly applied to females ( in vivo treatment) lampbrush chromosomes undergo a gradual disorganization of loop matrices. TMs was clearly visualized for dense matrix loops which successivelyexhibit globular, granular and finally normal aspects. However, such adisorganization was only visualized in IV-V stage oocytes.InVIstageoocytes,hcat treatmentresultcdin avariable loop condensation. For heat shock applied in virro , i.e. when isolated oocytes were directly submitted to the same high temperaaue,aprogiessivelsteialloop condensationoccursevenfor shon periods of stress(5 to 20 min) whatever the oocyte stage. In order to relate modifications that occur in lampbrush loop morphology to possible changes in RNA syntheses, autoradiogmphies of control and suessed lampbrush chromosomes were performed after incorporation of labeled RNA precursors. Differences between data obtained in the two experimental conditions suggest the intervention of maternal control in the oocyte stress response. Our results emphasize the necessity for studying gene expression both in isolated oocytes and in oocytes maintained undertheamtmlofmatemalorganism. This work was supported
by ARC (no 6309)
HJLIAR CELL WLTRAOTRUCTWRIZ IN WTANT OF CUCUKIS SATIWS 9 CrYeiun Cmnatantin Cerneanu C GabrielI, Cr&ciun Vereniea2. (1) Craieva University: Genetics Lab.. 1100-Craiova: (2) C1u.j University, die&y Bept.,340&luj, Rei&ia. ke a result ef the aetien ef a thcnaic sheck (-15.C) on Cucwaio sativuls WV Farbio seeas, there were ebtained the dwarf type mutant plants with other multiple mutatincr affecting the shape and dietributien rf the leaves, the chleraphyllmuo pigment aP440
YWARF
meunt, Rrewth ultreatructura
type was
e.8.e.
The
established
folier
cells
en the ma-
ture led fragaentcs (prefixetion: % glutaraldehyde fsr 3 h; fixetien: 1% Yillenir fixater -for 1 1/2-h; centreating: uranyi and leeil citrate). Normal plants acetate present in paliaede parcnchyma ~11s chloroplaets periphericelly diBpese1. Laeuaar parenchyma is lax, with large intereelluler speaes end relatively few eyt*plecam and chloroplests. The chlerepleste are evalc, with a big number l f atareh granulee. The dwarf mutant with opposite leeves withetmt petiele, down imbriceted, present a dense parcrekyma cells with many chlereplaete compared te the normal plants. The chleroplaat ultrastructure (Crane, interrrana) is mermally. favorable ior whet@Keep&i the normal plaatb at e bynthcsia. lower light intelnelty rer 24 h.determinate the cempiete Pieapp&arenee l ? the starch granules frem ehloreplaete. The nucleus euchrwaatine with blecka l f present fine the heterechrematinc et its periphery.
DICTYOSTELZUM NUCLEAR PLASMID P437 INVESTIGATIONOFPLASMIDENCODEDFUNCTIONS
International
DDP1:
Christine B.Gurniak and Angelika A. Noegel Max-Planck-Instimt fiir Bicchemie, 8033 Martinsried/FRG Various strains of Dictyostelium carry nuclearplasmids. They differ in size from 1.3kb to 27kb and do not show homology amongst each other, to chromosomal, ribosomal -dr mitochondxial DNA. Ddpl (13Skb) codes for 9 partially overlapping transcripts. A sequence of S.lkb of Ddpl encompassing four open reading frames (ORFs) has been determined and the corresponding transcripts have been identified. One of the transcripts is affected in its accumulation pattern by CAMP, an importantsignalduringDictyostelium development. None of the ORF% shows significant homologies to known proteins. The plasmid-soe&ied products are investigated lollowing two ities: a) expression of ORFs in E.coli and production of antibodies, b) expression in Dictyostelium-after introductionof a suitablkepitbpeinto the gene under investigation. The determined sequence also contains the oriti of reolication. The seouence reuuired for replication extends over !LOkb. A rranscxiit is loca& within this region which could code for a urotein of 34kD and mav be involv& in replication of Ddpl.*Although a transformation vector generated on the basis of Ddpl sequences remains extrachromosomal, though at a reduced copy number, this plasmid cannot be maintained extrachromosomal once it carries additional Dictyostelium sequences.The 2.Okb fragment was investigated in South-Western blot analyses for specific binding to nuclear proteins. In nuclear extracts of plasmid containing cells a protein of 32kD was detected whoese binding could be competed with excess of cold fragment and which was not present in nuclear extracts of a plasmid free Dictyostelium strain.
P439 TNg DRCXCIPHILA POLP(A) BINDING PROTEIN: CLONING AND HIPBBSSION TNROUGOIJTDgVNLOPNgNT. Valirie Lefr&re, Alain Vincent, Francois Amalric. CRBGC du CNRS, 118 route de Narbonne 31062 Toulouse cedex, FRANCE Two classes of cDNAs encoding the Drosophila poly(A) binding protein (PABP), which differ by the position of their respective 3' end separated by about 1 kb, were isolated by screening an embryonic cDNA librairy. These cDNAs hybridize to a single chromosomal site at position 55B on the right arm of the second chromosome. A unique 3.8 kb mRNA was detected, suggesting that "long" cDNAs correspond to full length cDNAs and that the 3' untranslated region of the mRNA is close to 1.5 kb long. The PABP transcript accumulates in oocytes, is maternally inherited by the embryo and present at every other developmental stage tested. The PABP cDNAs contain a 1722 nt long open reading frame encoding a 64 kDa protein with high affinity for poly(A). This protein contains four RNA binding domains which show limited primary sequence evolution, contrasting to the carboxy-terminal third of the protein; The strikingly long 3' untranslated region of the Drosophila PABP mRNA is shown to exist also in other eukaryotes, including vertebrate species. It suggests that important regulatory sequences intrinsic to the PABP mNNA are present in its 3' untranslated region. Data on the expression of the protein throughout development will be presented.
Reports,
P438
Vol. 74, Abstracts
HEAT-INDUCED LAMPBRUSH ENVIRONMENTAL
Supplement
CHANGES CHROMOSOMES CONDITIONS.
IN
AMPHIBIAN DEPEND
1990
ON
Maria-LuzRodriguez-Martin,NicoleMoreau. C%annsineHerbcrts, Nicole Angelier. UPR 3101, Centre de Biologic Cellulaire du CNRS,94205My-sur-Seine Cedex, France. At ll~nnal breedingtemperahlre(20°c)amphibianlampb~sh chromosomes art characterized by the presence oflaterslloops wbicharedirectlytelatcdtothe transcriptional processes.Inthe oocytes of the newt PZeurod&swalrl, heat treatment induces striking modifications in rhe structure of lampbrush loops but the nature and tbniw of these modifications depend on hyperthermic stressconditions. Weed. our data demonstrate thatat the same high temperature
(WC),
lampbrush
chmnosomes
modifications induced
by in viva heat treatment are different from those induced in in vine experimental conditions. When heat treatment (34°C for 45 min to 4 days) is directly applied to females ( in vivo treatment) lampbrush chromosomes undergo a gradual disorganization of loop matrices. TMs was clearly visualized for dense matrix loops which successivelyexhibit globular, granular and finally normal aspects. However, such adisorganization was only visualized in IV-V stage oocytes.InVIstageoocytes,hcat treatmentresultcdin avariable loop condensation. For heat shock applied in virro , i.e. when isolated oocytes were directly submitted to the same high temperaaue,aprogiessivelsteialloop condensationoccursevenfor shon periods of stress(5 to 20 min) whatever the oocyte stage. In order to relate modifications that occur in lampbrush loop morphology to possible changes in RNA syntheses, autoradiogmphies of control and suessed lampbrush chromosomes were performed after incorporation of labeled RNA precursors. Differences between data obtained in the two experimental conditions suggest the intervention of maternal control in the oocyte stress response. Our results emphasize the necessity for studying gene expression both in isolated oocytes and in oocytes maintained undertheamtmlofmatemalorganism. This work was supported
by ARC (no 6309)
HJLIAR CELL WLTRAOTRUCTWRIZ IN WTANT OF CUCUKIS SATIWS 9 CrYeiun Cmnatantin Cerneanu C GabrielI, Cr&ciun Vereniea2. (1) Craieva University: Genetics Lab.. 1100-Craiova: (2) C1u.j University, die&y Bept.,340&luj, Rei&ia. ke a result ef the aetien ef a thcnaic sheck (-15.C) on Cucwaio sativuls WV Farbio seeas, there were ebtained the dwarf type mutant plants with other multiple mutatincr affecting the shape and dietributien rf the leaves, the chleraphyllmuo pigment aP440
YWARF
meunt, Rrewth ultreatructura
type was
e.8.e.
The
established
folier
cells
en the ma-
ture led fragaentcs (prefixetion: % glutaraldehyde fsr 3 h; fixetien: 1% Yillenir fixater -for 1 1/2-h; centreating: uranyi and leeil citrate). Normal plants acetate present in paliaede parcnchyma ~11s chloroplaets periphericelly diBpese1. Laeuaar parenchyma is lax, with large intereelluler speaes end relatively few eyt*plecam and chloroplests. The chlerepleste are evalc, with a big number l f atareh granulee. The dwarf mutant with opposite leeves withetmt petiele, down imbriceted, present a dense parcrekyma cells with many chlereplaete compared te the normal plants. The chleroplaat ultrastructure (Crane, interrrana) is mermally. favorable ior whet@Keep&i the normal plaatb at e bynthcsia. lower light intelnelty rer 24 h.determinate the cempiete Pieapp&arenee l ? the starch granules frem ehloreplaete. The nucleus euchrwaatine with blecka l f present fine the heterechrematinc et its periphery.