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Magnetic isolation of poly a mRNA from eucaryotic cells
Cell Biology
International
Reports,
Vol. 14, Abstracts
Supplement
ANALYSIS OF IN VZVO RED-OX STATUS OF THIOLS IN HUMAN PLASMA Mohammad A. Mansoor, Asbjem M. Svardal and Per M. Ueland.Department of Pharmacology and Toxicology, University of Bergen, Armauer
P554
P553
Hansens Hus, N-5021 Bergen, Norway. Sulfur exists within a variety of both organic and inorganic molecules in biological systems. Many of these sulfur containing biomolecules have been demonstrated to have pivotal roles in the functionality of many cellular biochemical mechanisms. The facile oxidation of organic sulfhydryl (SH) compounds leads to the existence withii biological systems of a variety of disulfide forms. In plasma these include lmwt compounds such as cystine, cysteinylglycine disulfide, glutathione disulfide and homocystine, mixed adducts between lmwt thiols and protein thiols. Disulfides within proteins themselves also exist. A method for quantitative analysis of the reduced, oxidized and protein-bound forms of cysteine, cysteinylglycine, homocvsteine and elutathione in human ulasma has been develo&. Blood wa’i collected into evacuated NbeS containing monobromobimane (mBrB) and N-etbylmaleimide respectively.
Fromtheformerthereducedfornx werequantitatedandfrom
the latter the oxidized forms. The oxidized and protein-bound fo&ns were reduced with NaBIQ prior to deriv‘atization with n&B. The thiol-bimane adducts were separated and quantitated by HPLC and fluorescence detection. This assay is the fast of its kind allowing determination of the red-ox thiol status in
Lars
1990
MAGNETIC ISOLATION OF POLY A mRNAIFROMBUCARYOT$C CELLS. Korsnes
Erik
Homes
.
1 Dynal A.S,'Apbthekernes Laboratorium A.S, Postboks 158, Sk&en, N-0212 Oslo 2, Norway. Purification involves
of messenger several extraction,
precipitation steps. In all enzymatic
RNA normally
and/or chromatographic methods physical and
degradation
of mRNA is
a
potential problem. We have developed a method for rapid purification of high quality polyA mRNAusing superparamagnetic monodisperse polymer particles (Dynabeads). The time required from the start of cell lysis until polyA mRNA is isolatet is five minutes. The mRNAis at this point in E$ 0 or
any
low
salt
buffer
ready
for
further use such as Northern analysis, cDNA synthesis or storage. This method
can also be employed on crude materials, i.e. containing high amounts of proteins, DNA or unsoluble substances. Our results show that more than 90% of cytoplasmic polyA mRNA is
obtained using the magnetic purification
method.
humanplasma.
P555 . .
m CNR,
EXPRESSION ANTIBODY
via Marx
OF INTRACELLULAR FV AND FAS FRAGMENTS IN MAMMALIAN CELLS
and Antonino 15, Roma.
Cattaneo.
lstituto
di Neurobiologia,
The possibility of expressing and targeting functional antibodies in the cytoplasm or in the nucleus of mammalian cells by replacing the secretory leader of the light and the heavy chain with cytoplasmic or nuclear signals hal; been recently reported (Biocca et al. EMBO J.&, 101-l 08,199O). By cloning monoclonal antibodies of desired specificity in vectors modified for intracellular expression we have suggested that we can in viva interfere with or inactivate a cytosolic or nuclear protein. In order to improve the efficiency of this method we have modified, by site-directed mutagenesis, the available shuttle vectors for the forced cloning of any variable region. Stop codons were introduced at the end of the light and heavy variable region (Fv fragments) and at the end of the first exon of the heavy chain constant region (Fab fragment). The mutant immunoglobulin domainswere equipped with N-terminus sequences found in cytoplasmic proteins with long half-life (lactic dehydrogenase and phosphoglicerokinase).The activity of the constructs was assayed after transfection of the plasmid DNA in cuftered cells. The results obtained will allow to compare the half-life of these antibody domains as well as their capacity to reconstitute antigen binding activity in different intracellular compartments and, therefore, to produce the suitable modified vectors for intracellular expression of any specific antibody or antibody domain.
DETECTION OF ONWDENE ACTIYATION IN PARAFFIN-EMBEDDED TISSUE WIND THE POLYtlERASE CHAIN REACTION @CR). A.lolm*, A.Lania*.E.Miraglia &I Giudics*, S.Perrotta*, M.Badiali’, G.saglio” and S.Cutillo*; from*Dpt. of Pedlatrlcs University of Naples; University of ‘Bologna and ” Torino. Molecular analyses of malignancies have increasingly become relevsnt to clintcal practice In onc~lcgy. The standard sources of nucleic acids are perlphsral venousblood, bone marrow aspirates or tumor specimens.However obtaining large numbers of fresh samples of human tumor tissue, even from relatively commontumors is time consuming end difficultIn order to examine the Possibility that onccgenesplay a causativerole in a rare cancer, it could take tlzades before a significant number of c%sescould be studled. With accessto archival materials of pathologists, however, all the cases collected over the last 20 or 30 years could be immediately examinsd.For this reason we studied onccgene activation using a axnbination of techniques including specific in vitro gene amplification by PCR and detection of single base mutations by specific sequence illigonucleotides (.%I) in thin tissue sectionsIn this stu& it is shcwn that DNA&an beextracted from tissues prepared for routine histopatholcgical examination,proved to be suitable for analysis by PCR/SSDprocedure.We studied N-Ras activation at codon 12,13 and 6 1 in 32 msdulloblastomas and in IO pancreas carcinomas. DNAwas isolated from S- IO pm section of form&in-fixed paraffin-embs&d tissuesIn none of the pancreas samples was found a mutation in the relW8nt 12,13 or 61 codon. In 3 out of the examined msdulloblastomas we showed the involvsment of c&n 6 1 : C-A at positlon I (leading to a substitution of a glutamine residue for a lysine) in two casesand a mutation A-T at Position 3 ( tfys) The rapid and sensitive analysis of oncogeneactivation (by smgle basemutation) in human m8llgnancies using this technique may contribute significantly to identifying the role of onwgsne as a risk factor in human cancer.
P556
International
Reports,
Vol. 14, Abstracts
Supplement
ANALYSIS OF IN VZVO RED-OX STATUS OF THIOLS IN HUMAN PLASMA Mohammad A. Mansoor, Asbjem M. Svardal and Per M. Ueland.Department of Pharmacology and Toxicology, University of Bergen, Armauer
P554
P553
Hansens Hus, N-5021 Bergen, Norway. Sulfur exists within a variety of both organic and inorganic molecules in biological systems. Many of these sulfur containing biomolecules have been demonstrated to have pivotal roles in the functionality of many cellular biochemical mechanisms. The facile oxidation of organic sulfhydryl (SH) compounds leads to the existence withii biological systems of a variety of disulfide forms. In plasma these include lmwt compounds such as cystine, cysteinylglycine disulfide, glutathione disulfide and homocystine, mixed adducts between lmwt thiols and protein thiols. Disulfides within proteins themselves also exist. A method for quantitative analysis of the reduced, oxidized and protein-bound forms of cysteine, cysteinylglycine, homocvsteine and elutathione in human ulasma has been develo&. Blood wa’i collected into evacuated NbeS containing monobromobimane (mBrB) and N-etbylmaleimide respectively.
Fromtheformerthereducedfornx werequantitatedandfrom
the latter the oxidized forms. The oxidized and protein-bound fo&ns were reduced with NaBIQ prior to deriv‘atization with n&B. The thiol-bimane adducts were separated and quantitated by HPLC and fluorescence detection. This assay is the fast of its kind allowing determination of the red-ox thiol status in
Lars
1990
MAGNETIC ISOLATION OF POLY A mRNAIFROMBUCARYOT$C CELLS. Korsnes
Erik
Homes
.
1 Dynal A.S,'Apbthekernes Laboratorium A.S, Postboks 158, Sk&en, N-0212 Oslo 2, Norway. Purification involves
of messenger several extraction,
precipitation steps. In all enzymatic
RNA normally
and/or chromatographic methods physical and
degradation
of mRNA is
a
potential problem. We have developed a method for rapid purification of high quality polyA mRNAusing superparamagnetic monodisperse polymer particles (Dynabeads). The time required from the start of cell lysis until polyA mRNA is isolatet is five minutes. The mRNAis at this point in E$ 0 or
any
low
salt
buffer
ready
for
further use such as Northern analysis, cDNA synthesis or storage. This method
can also be employed on crude materials, i.e. containing high amounts of proteins, DNA or unsoluble substances. Our results show that more than 90% of cytoplasmic polyA mRNA is
obtained using the magnetic purification
method.
humanplasma.
P555 . .
m CNR,
EXPRESSION ANTIBODY
via Marx
OF INTRACELLULAR FV AND FAS FRAGMENTS IN MAMMALIAN CELLS
and Antonino 15, Roma.
Cattaneo.
lstituto
di Neurobiologia,
The possibility of expressing and targeting functional antibodies in the cytoplasm or in the nucleus of mammalian cells by replacing the secretory leader of the light and the heavy chain with cytoplasmic or nuclear signals hal; been recently reported (Biocca et al. EMBO J.&, 101-l 08,199O). By cloning monoclonal antibodies of desired specificity in vectors modified for intracellular expression we have suggested that we can in viva interfere with or inactivate a cytosolic or nuclear protein. In order to improve the efficiency of this method we have modified, by site-directed mutagenesis, the available shuttle vectors for the forced cloning of any variable region. Stop codons were introduced at the end of the light and heavy variable region (Fv fragments) and at the end of the first exon of the heavy chain constant region (Fab fragment). The mutant immunoglobulin domainswere equipped with N-terminus sequences found in cytoplasmic proteins with long half-life (lactic dehydrogenase and phosphoglicerokinase).The activity of the constructs was assayed after transfection of the plasmid DNA in cuftered cells. The results obtained will allow to compare the half-life of these antibody domains as well as their capacity to reconstitute antigen binding activity in different intracellular compartments and, therefore, to produce the suitable modified vectors for intracellular expression of any specific antibody or antibody domain.
DETECTION OF ONWDENE ACTIYATION IN PARAFFIN-EMBEDDED TISSUE WIND THE POLYtlERASE CHAIN REACTION @CR). A.lolm*, A.Lania*.E.Miraglia &I Giudics*, S.Perrotta*, M.Badiali’, G.saglio” and S.Cutillo*; from*Dpt. of Pedlatrlcs University of Naples; University of ‘Bologna and ” Torino. Molecular analyses of malignancies have increasingly become relevsnt to clintcal practice In onc~lcgy. The standard sources of nucleic acids are perlphsral venousblood, bone marrow aspirates or tumor specimens.However obtaining large numbers of fresh samples of human tumor tissue, even from relatively commontumors is time consuming end difficultIn order to examine the Possibility that onccgenesplay a causativerole in a rare cancer, it could take tlzades before a significant number of c%sescould be studled. With accessto archival materials of pathologists, however, all the cases collected over the last 20 or 30 years could be immediately examinsd.For this reason we studied onccgene activation using a axnbination of techniques including specific in vitro gene amplification by PCR and detection of single base mutations by specific sequence illigonucleotides (.%I) in thin tissue sectionsIn this stu& it is shcwn that DNA&an beextracted from tissues prepared for routine histopatholcgical examination,proved to be suitable for analysis by PCR/SSDprocedure.We studied N-Ras activation at codon 12,13 and 6 1 in 32 msdulloblastomas and in IO pancreas carcinomas. DNAwas isolated from S- IO pm section of form&in-fixed paraffin-embs&d tissuesIn none of the pancreas samples was found a mutation in the relW8nt 12,13 or 61 codon. In 3 out of the examined msdulloblastomas we showed the involvsment of c&n 6 1 : C-A at positlon I (leading to a substitution of a glutamine residue for a lysine) in two casesand a mutation A-T at Position 3 ( tfys) The rapid and sensitive analysis of oncogeneactivation (by smgle basemutation) in human m8llgnancies using this technique may contribute significantly to identifying the role of onwgsne as a risk factor in human cancer.
P556