Juergen Bode | Hannover Medical School (original) (raw)
Papers by Juergen Bode
ÄESTRACT v) P B ltlthln nouse cl27 cells, plasold BPv-Bvl, carrylag the hunan Lotefferoo-B gene a... more ÄESTRACT v) P B ltlthln nouse cl27 cells, plasold BPv-Bvl, carrylag the hunan Lotefferoo-B gene and lts regulatory sequeoces' ls uostable. A stable clone, n4, has elloloated all plokeryqtlc (pBRd) and sone hunan sequeoces from the shuttle vector. A chrouatitr{applng-stuäy shows that a htgh perceatege of the nultlcopy vecto! hes lts lnterferon getres packaged luto nuclgosoreä, äv"n alter an lnductlon protocol, suggestlng that only a subpopulatl-on becomes actlve. ifrascä nucleosooes are alio detected la an upstrea eleoeat, derlved fton fhe large lntron of the hunan $-globla gene. Thls eleuent, whlch beJ.ongs to the stablllzlng sequences of the vector, ls unlque ln havlng afflnlty to the nuclear scaffold.
ÄESTRACT v) P B ltlthln nouse cl27 cells, plasold BPv-Bvl, carrylag the hunan Lotefferoo-B gene a... more ÄESTRACT v) P B ltlthln nouse cl27 cells, plasold BPv-Bvl, carrylag the hunan Lotefferoo-B gene and lts regulatory sequeoces' ls uostable. A stable clone, n4, has elloloated all plokeryqtlc (pBRd) and sone hunan sequeoces from the shuttle vector. A chrouatitr{applng-stuäy shows that a htgh perceatege of the nultlcopy vecto! hes lts lnterferon getres packaged luto nuclgosoreä, äv"n alter an lnductlon protocol, suggestlng that only a subpopulatl-on becomes actlve. ifrascä nucleosooes are alio detected la an upstrea eleoeat, derlved fton fhe large lntron of the hunan $-globla gene. Thls eleuent, whlch beJ.ongs to the stablllzlng sequences of the vector, ls unlque ln havlng afflnlty to the nuclear scaffold.
Sequencing of complete genomes (Genomics), the detection of all gene products (Proteomics), and t... more Sequencing of complete genomes (Genomics), the detection of all gene products (Proteomics), and the characterization of specific molecular functions have enabled the development of cell-based expression principles (Cytomics). In the past, we have pioneered three fundamentally new strategies to generate cell lines with predictable expression properties, which can be combined with advantage:
Biochemistry Usa, May 1, 1994
Scaffold-attached regions (SAR elements) increase transcriptional rates for integrated but not ep... more Scaffold-attached regions (SAR elements) increase transcriptional rates for integrated but not episomal templates, and this effect can be potentiated by using an epigenetically active reagent, butyrate.
Nature Cell Biology, 2000
ABSTRACT DNA replication occurs in tight association with the nuclear matrix, where binding of th... more ABSTRACT DNA replication occurs in tight association with the nuclear matrix, where binding of the replication origin to the nuclear matrix must precede the onset of S phase1–4. We have shown previously that the origin of replication of the simian virus 40 (SV40) genome linked to a human scaffold/matrix-attached region (S/MAR) allows sustained episomal replication (where an episome is autonomous, self-replicating DNA) that is independent of the expression of the virally encoded large T-antigen5. A vector with this combination of SV40 origin and potential for matrix association is maintained in cultured cells for at least 100 cell generations, in the absence of selection5. Here we show, by in situ hybridization and nuclear-fractionation procedures, that there is a specific interaction of this vector with the nuclear matrix and the chromosome scaffold, presumably through proteins that both structures have in common. This interaction correlates with replication of the vector as an episome. These observations allow a mechanistic explanation for the episomal replication and mitotic stability of this new type of vector.
Die klassischen Techniken des Gentransfers sind wenig effizient und zuverlässig: das Ziel einer I... more Die klassischen Techniken des Gentransfers sind wenig effizient und zuverlässig: das Ziel einer Integration des "Transgens" in einen vorteilhaften chromosomalen Ort wird so selten erreicht, dass stets umfangreiche Selektionsarbeiten vorgenommen werden müssen. Wenn Integration erfolgt, dann zumeist in hoher Kopienzahl und in einer Anordnung, gegen die die Wirtszelle Abwehrmechanismen entwickelt hat ("Cosuppression"). Unerwünschte Mutationen und genomische Instabilität können die Folge sein. Das erstrebte Ziel einer vorhersagbaren, langfristig stabilen und bei gentherapeutischen Ansätzen sicheren Modifikation wird so nur zufällig erreicht.
While there is significant progress in the modification by episomal DNA of slowly-dividing tissue... more While there is significant progress in the modification by episomal DNA of slowly-dividing tissues like liver, muscle and brain, maintenance problems have so far limited the use of nonviral episomes for dividing cells. For liver, the most advanced vehicles appear to be "minicircles", small circular vectors that are exclusively composed from eukaryotic sequences. In contrast to linear DNA, minicircles are less prone to integration and, owing to their superhelical status, they are better transcriptional templates. The essential components of a minicircle derivative that replicates once per cell cycle have been defined, i.e. an active transcription unit and a S/MAR -so far the upstream bordering element from the huIFN-β chromatin domain and its derivatives. Present work explores the requirements to be met by the S/MAR in association with regulatory elements and it investigates alternative transfer routes to optimize gene expression
Arch Biochem Biophys, 1975
A) Multiple sets of heterospecific FRTs (half arrows marked in red or blue) enable simultaneous R... more A) Multiple sets of heterospecific FRTs (half arrows marked in red or blue) enable simultaneous RMCE reactions at two (or more) pre-defined genomic loci. Since each of the FRTs differs from the others in four spacer positions, unwanted cross-interactions are safely precluded.
Hoppe-Seyler´s Zeitschrift für physiologische Chemie, 1977
DNA was titrated with protamine under equilibrium conditions while binding was simultaneously fol... more DNA was titrated with protamine under equilibrium conditions while binding was simultaneously followed by electron microscopy and the fluorescamine technique. Positive cooperativity concluded from the binding assays was shown to be correlated with an aligned association of DNA fibres. Subsequent structural modi-fications, dependent upon the protamine/DNA ratio, suggest a mechanism for DNA compaction by protamines. In the framework of this model, protamine phosphorylation appears to promote formation of DNA interstrand links yielding a very pronounced filigree structure. Die Anontomie der kooperativen Bindung zwischen Protaminen und DNA Zusammenfassung: DNA wurde mit steigenden Mengen eines Protamins äquilibriert. Der jeweils
Scaffold-attached regions (SAR elements) have been identified for a number of eukaryotic genes wh... more Scaffold-attached regions (SAR elements) have been identified for a number of eukaryotic genes where they occur in association with established enhancers or as the borders of a chromatin domain. SAR elements themselves share some properties with both the enhancers (transcriptional stimulation independent of distance and orientation) and the locus-control regions (LCRs mediate a copy-number dependent expression and become active only after the integration of a gene construct into the genome). We have investigated the structural features of SAR elements responsible for transcriptional augmentation. For this purpose we have amplified, by oligomerization, the action of a certain core motif common to several SARs. The resulting elements enable an efficient separation of DNA strands under super helical tension . By this approach artificial elements of a minimum length (175 bp) are created which share both the affinity for the nuclear scaffold and the enhancing properties which are otherwise typical of a SAR comprising 2-3 kb of DNA. The expression of certain genes is stimulated by millimolar concentrations of butyrate. We have shown that any gene flanked by SAR elements is subject to this effect and, conversely, that all SAR-specific actions are amplified in the presence of millimolar concentrations of the fatty acid [3]. Among the many activities of butyrate, the hyperacetylation of the nuclear core histones has gained particular attention. Support for this level of action comes from the fact that comparable results have also be obtained with nanomolar amounts of (R)-Trichostatin A, a highly specific inhibitor of histone deacetylases . Based on their properties, SAR elements have been considered as domain openers. We demonstrate, by inverse PCR techniques, that they create a major target site for the integration of retroviral genomes. In the majority of these cases, the SAR character is located 3' to the retroviral DNA but there is no documented case, where a SAR region is hit centrally. Therefore the DNAse I hypersensitive site, which is commonly located at the border(s) of a SAR, is considered the primary signal for integration [5]. [1] Mielke C, Kohwi Y, Kohwi-Shigematsu T & Bode J (1990) Hierarchical binding of DNA fragments derived from scaffold-attached regions: correlation of properties in vitro and function in vivo. Biochemistry 29, 7475-7485. http://dx.doi.org/10.1021/bi00484a017 [3] Klehr D, Schlake T, Maass K & Bode J (1992), Scaffold-attached regions (SAR elements) mediate transcriptional effects due to butyrate. Biochemistry 31:3222-3229. http://dx.
An autonomous transcription unit, delimited by scaffold/matrix attachment regions (S/MARs), is ex... more An autonomous transcription unit, delimited by scaffold/matrix attachment regions (S/MARs), is expressed largely independent of the genomic integration site. Such a minidomain resists epigenetic silencing and mediates/maintains histone hyperacetylation. Once a suitable genomic target has been established, the site can be re-used by the Flp-RMCE (recombinasemediated cassette exchange) technology. In the extreme case a minidomain can be reduced to a circular entity with just a single S/MAR. These so-called minicircles represent episomes with an unusual replication potential. They represent a novel nonviral mammalian vector system with an exceptional potential for transgenesis.
Minicircles are episomes with unusual replication potental, representing a novel class of nonvira... more Minicircles are episomes with unusual replication potental, representing a novel class of nonviral mammalian vectors. A major goal of our efforts is the implementation of the system for gene therapeutic purposes, since it enables uses for both the transient and/or the stable expression of the relevant factors. Recent refinements concern optimization oft the intrinsic S/MAR element, and reduction of the over-all size to ~ 4kb, One of our central aims was the side-by-side establishment of different episomes for the regulated expression of, for instance, antibody H- and L-chains, which we show to be feasible..
ÄESTRACT v) P B ltlthln nouse cl27 cells, plasold BPv-Bvl, carrylag the hunan Lotefferoo-B gene a... more ÄESTRACT v) P B ltlthln nouse cl27 cells, plasold BPv-Bvl, carrylag the hunan Lotefferoo-B gene and lts regulatory sequeoces' ls uostable. A stable clone, n4, has elloloated all plokeryqtlc (pBRd) and sone hunan sequeoces from the shuttle vector. A chrouatitr{applng-stuäy shows that a htgh perceatege of the nultlcopy vecto! hes lts lnterferon getres packaged luto nuclgosoreä, äv"n alter an lnductlon protocol, suggestlng that only a subpopulatl-on becomes actlve. ifrascä nucleosooes are alio detected la an upstrea eleoeat, derlved fton fhe large lntron of the hunan $-globla gene. Thls eleuent, whlch beJ.ongs to the stablllzlng sequences of the vector, ls unlque ln havlng afflnlty to the nuclear scaffold.
ÄESTRACT v) P B ltlthln nouse cl27 cells, plasold BPv-Bvl, carrylag the hunan Lotefferoo-B gene a... more ÄESTRACT v) P B ltlthln nouse cl27 cells, plasold BPv-Bvl, carrylag the hunan Lotefferoo-B gene and lts regulatory sequeoces' ls uostable. A stable clone, n4, has elloloated all plokeryqtlc (pBRd) and sone hunan sequeoces from the shuttle vector. A chrouatitr{applng-stuäy shows that a htgh perceatege of the nultlcopy vecto! hes lts lnterferon getres packaged luto nuclgosoreä, äv"n alter an lnductlon protocol, suggestlng that only a subpopulatl-on becomes actlve. ifrascä nucleosooes are alio detected la an upstrea eleoeat, derlved fton fhe large lntron of the hunan $-globla gene. Thls eleuent, whlch beJ.ongs to the stablllzlng sequences of the vector, ls unlque ln havlng afflnlty to the nuclear scaffold.
Sequencing of complete genomes (Genomics), the detection of all gene products (Proteomics), and t... more Sequencing of complete genomes (Genomics), the detection of all gene products (Proteomics), and the characterization of specific molecular functions have enabled the development of cell-based expression principles (Cytomics). In the past, we have pioneered three fundamentally new strategies to generate cell lines with predictable expression properties, which can be combined with advantage:
Biochemistry Usa, May 1, 1994
Scaffold-attached regions (SAR elements) increase transcriptional rates for integrated but not ep... more Scaffold-attached regions (SAR elements) increase transcriptional rates for integrated but not episomal templates, and this effect can be potentiated by using an epigenetically active reagent, butyrate.
Nature Cell Biology, 2000
ABSTRACT DNA replication occurs in tight association with the nuclear matrix, where binding of th... more ABSTRACT DNA replication occurs in tight association with the nuclear matrix, where binding of the replication origin to the nuclear matrix must precede the onset of S phase1–4. We have shown previously that the origin of replication of the simian virus 40 (SV40) genome linked to a human scaffold/matrix-attached region (S/MAR) allows sustained episomal replication (where an episome is autonomous, self-replicating DNA) that is independent of the expression of the virally encoded large T-antigen5. A vector with this combination of SV40 origin and potential for matrix association is maintained in cultured cells for at least 100 cell generations, in the absence of selection5. Here we show, by in situ hybridization and nuclear-fractionation procedures, that there is a specific interaction of this vector with the nuclear matrix and the chromosome scaffold, presumably through proteins that both structures have in common. This interaction correlates with replication of the vector as an episome. These observations allow a mechanistic explanation for the episomal replication and mitotic stability of this new type of vector.
Die klassischen Techniken des Gentransfers sind wenig effizient und zuverlässig: das Ziel einer I... more Die klassischen Techniken des Gentransfers sind wenig effizient und zuverlässig: das Ziel einer Integration des "Transgens" in einen vorteilhaften chromosomalen Ort wird so selten erreicht, dass stets umfangreiche Selektionsarbeiten vorgenommen werden müssen. Wenn Integration erfolgt, dann zumeist in hoher Kopienzahl und in einer Anordnung, gegen die die Wirtszelle Abwehrmechanismen entwickelt hat ("Cosuppression"). Unerwünschte Mutationen und genomische Instabilität können die Folge sein. Das erstrebte Ziel einer vorhersagbaren, langfristig stabilen und bei gentherapeutischen Ansätzen sicheren Modifikation wird so nur zufällig erreicht.
While there is significant progress in the modification by episomal DNA of slowly-dividing tissue... more While there is significant progress in the modification by episomal DNA of slowly-dividing tissues like liver, muscle and brain, maintenance problems have so far limited the use of nonviral episomes for dividing cells. For liver, the most advanced vehicles appear to be "minicircles", small circular vectors that are exclusively composed from eukaryotic sequences. In contrast to linear DNA, minicircles are less prone to integration and, owing to their superhelical status, they are better transcriptional templates. The essential components of a minicircle derivative that replicates once per cell cycle have been defined, i.e. an active transcription unit and a S/MAR -so far the upstream bordering element from the huIFN-β chromatin domain and its derivatives. Present work explores the requirements to be met by the S/MAR in association with regulatory elements and it investigates alternative transfer routes to optimize gene expression
Arch Biochem Biophys, 1975
A) Multiple sets of heterospecific FRTs (half arrows marked in red or blue) enable simultaneous R... more A) Multiple sets of heterospecific FRTs (half arrows marked in red or blue) enable simultaneous RMCE reactions at two (or more) pre-defined genomic loci. Since each of the FRTs differs from the others in four spacer positions, unwanted cross-interactions are safely precluded.
Hoppe-Seyler´s Zeitschrift für physiologische Chemie, 1977
DNA was titrated with protamine under equilibrium conditions while binding was simultaneously fol... more DNA was titrated with protamine under equilibrium conditions while binding was simultaneously followed by electron microscopy and the fluorescamine technique. Positive cooperativity concluded from the binding assays was shown to be correlated with an aligned association of DNA fibres. Subsequent structural modi-fications, dependent upon the protamine/DNA ratio, suggest a mechanism for DNA compaction by protamines. In the framework of this model, protamine phosphorylation appears to promote formation of DNA interstrand links yielding a very pronounced filigree structure. Die Anontomie der kooperativen Bindung zwischen Protaminen und DNA Zusammenfassung: DNA wurde mit steigenden Mengen eines Protamins äquilibriert. Der jeweils
Scaffold-attached regions (SAR elements) have been identified for a number of eukaryotic genes wh... more Scaffold-attached regions (SAR elements) have been identified for a number of eukaryotic genes where they occur in association with established enhancers or as the borders of a chromatin domain. SAR elements themselves share some properties with both the enhancers (transcriptional stimulation independent of distance and orientation) and the locus-control regions (LCRs mediate a copy-number dependent expression and become active only after the integration of a gene construct into the genome). We have investigated the structural features of SAR elements responsible for transcriptional augmentation. For this purpose we have amplified, by oligomerization, the action of a certain core motif common to several SARs. The resulting elements enable an efficient separation of DNA strands under super helical tension . By this approach artificial elements of a minimum length (175 bp) are created which share both the affinity for the nuclear scaffold and the enhancing properties which are otherwise typical of a SAR comprising 2-3 kb of DNA. The expression of certain genes is stimulated by millimolar concentrations of butyrate. We have shown that any gene flanked by SAR elements is subject to this effect and, conversely, that all SAR-specific actions are amplified in the presence of millimolar concentrations of the fatty acid [3]. Among the many activities of butyrate, the hyperacetylation of the nuclear core histones has gained particular attention. Support for this level of action comes from the fact that comparable results have also be obtained with nanomolar amounts of (R)-Trichostatin A, a highly specific inhibitor of histone deacetylases . Based on their properties, SAR elements have been considered as domain openers. We demonstrate, by inverse PCR techniques, that they create a major target site for the integration of retroviral genomes. In the majority of these cases, the SAR character is located 3' to the retroviral DNA but there is no documented case, where a SAR region is hit centrally. Therefore the DNAse I hypersensitive site, which is commonly located at the border(s) of a SAR, is considered the primary signal for integration [5]. [1] Mielke C, Kohwi Y, Kohwi-Shigematsu T & Bode J (1990) Hierarchical binding of DNA fragments derived from scaffold-attached regions: correlation of properties in vitro and function in vivo. Biochemistry 29, 7475-7485. http://dx.doi.org/10.1021/bi00484a017 [3] Klehr D, Schlake T, Maass K & Bode J (1992), Scaffold-attached regions (SAR elements) mediate transcriptional effects due to butyrate. Biochemistry 31:3222-3229. http://dx.
An autonomous transcription unit, delimited by scaffold/matrix attachment regions (S/MARs), is ex... more An autonomous transcription unit, delimited by scaffold/matrix attachment regions (S/MARs), is expressed largely independent of the genomic integration site. Such a minidomain resists epigenetic silencing and mediates/maintains histone hyperacetylation. Once a suitable genomic target has been established, the site can be re-used by the Flp-RMCE (recombinasemediated cassette exchange) technology. In the extreme case a minidomain can be reduced to a circular entity with just a single S/MAR. These so-called minicircles represent episomes with an unusual replication potential. They represent a novel nonviral mammalian vector system with an exceptional potential for transgenesis.
Minicircles are episomes with unusual replication potental, representing a novel class of nonvira... more Minicircles are episomes with unusual replication potental, representing a novel class of nonviral mammalian vectors. A major goal of our efforts is the implementation of the system for gene therapeutic purposes, since it enables uses for both the transient and/or the stable expression of the relevant factors. Recent refinements concern optimization oft the intrinsic S/MAR element, and reduction of the over-all size to ~ 4kb, One of our central aims was the side-by-side establishment of different episomes for the regulated expression of, for instance, antibody H- and L-chains, which we show to be feasible..