DNA microarrays are powerful tools for the analysis of gene expression on a genomic scale. The im... more DNA microarrays are powerful tools for the analysis of gene expression on a genomic scale. The importance of individual regulatory events for the process under study can however not be deduced unequivocally without additional experiments. We devised a strategy to identify central regulators of cancer drug responses by combining the results of microarray experiments with efficient methods for phenotypic testing of candidate genes. We exposed murine FL5.12 pro-B cells to cisplatin, camptothecin, methotrexate or paclitaxel, respectively and analysed the patterns of gene expression with cDNA microarrays. Drug-specific regulatory events as well as intersections between different apoptotic pathways, including previously studied responses to staurosporine and interleukin-3 (IL-3) deprivation, were identified. Genes shared by at least three pathways were chosen for further analysis. Ectopic expression of three such genes, TEAP, GP49B, and Lipin1 was found to have an anti-proliferative effect on pro-B cells. Interestingly, we identified hemoglobin alpha as a strong pro-apoptotic regulator. While hemoglobin-expressing cells were growing normally in the presence of IL-3, they displayed accelerated apoptosis with similar kinetics as Bax overexpressing cells upon IL-3 removal. The proapoptotic effect of hemoglobin was suppressed by Bcl-2 and was characterized by enhanced stimulation of caspase activity.
Proceedings of the National Academy of Sciences of the United States of America, Jul 9, 1996
The core oligosaccharide Glc3MangGlcNAc2 is assembled at the membrane of the endoplasmic reticulu... more The core oligosaccharide Glc3MangGlcNAc2 is assembled at the membrane of the endoplasmic reticulum on the lipid carrier dolichyl pyrophosphate and transferred to selected asparagine residues of nascent polypeptide chains. This transfer is catalyzed by the oligosaccharyl transferase complex. Based on the synthetic phenotype of the oligosaccharyl transferase mutation wbpl in combination with a deficiency in the assembly pathway of the oligosaccharide in Saccharomyces cerevisiae, we have identified the novel ALG9 gene. We conclude that this locus encodes a putative mannosyl transferase because deletion of the gene led to accumulation of lipid-linked Man6GlcNAc2 in vivo and to hypoglycosylation of secreted proteins. Using an approach combining genetic and biochemical techniques, we show that the assembly of the lipid-linked core oligosaccharide in the lumen of the endoplasmic reticulum occurs in a stepwise fashion. N-linked glycosylation is an essential modification of proteins and follows a highly conserved pathway in eukaryotic cells (1-3). The core oligosaccharide Glc3Man9GlcNAc2 is assembled on the lipid carrier dolichyl pyrophosphate and transferred to selected asparagine residues of nascent polypeptide chains. This transfer is catalyzed by the enzyme oligosaccharyl transferase (4, 5). Abbreviations: CPY, carboxypeptidase Y; Dol, dolichol; Endo H, endo-13-N-acetylglucosaminidase H; ER, endoplasmic reticulum. Data deposition: The sequence reported in this paper has been deposited in the GeneBank data base (accession no. X96417).
Background: This short communication focuses the on articular cartilage and the subchondral bone,... more Background: This short communication focuses the on articular cartilage and the subchondral bone, both of which play important roles in the development of osteoarthritis (OA). There are indications that estrogendeficiency, as the post-menopausal state, accelerate the development of OA. Findings: We investigated, which extracellular matrix (ECM) protein, proteases and different pro-inflammatory factors was up-or down-regulated in the knee joint tissue in response to estrogen-deficiency in rats induced by ovariectomy. These data support previous findings that several metalloproteinases (MMPs) and cysteine proteases are co-regulated with numerous collagens and proteoglycans that are important for cartilage integrity. Furthermore quite a few pro-inflammatory cytokines were regulated by estrogen deprivation. Conclusion: We found multiple genes where regulated in the joint by estrogen-deficiency, many of which correspond well with our current knowledge of the pathogenesis of OA. It supports that estrogen-deficiency (e.g. OVX) may accelerate joint deterioration. However, there are also data that draw attention the need for better understanding of the synergy between proteases and tissue turnover.
The transcriptional response of mouse pro-B cells to two dierent apoptotic stimuli was investigat... more The transcriptional response of mouse pro-B cells to two dierent apoptotic stimuli was investigated. First, interleukin-3 (IL-3) deprivation was used to trigger programmed cell death in IL-3 dependent FL5.12 cells. Alternatively, cells were treated with the protein kinase C (PKC) inhibitor staurosporine. The temporal pattern of gene expression was followed with cDNA microarrays, covering over 8700 dierent mouse cDNA sequences corresponding to approximately 7900 unique genes. Messenger RNA levels of 315 genes were found to be regulated by more than twofold upon IL-3 removal, while 125 genes reacted to staurosporine treatment. Cross-comparison revealed an intersection of 34 genes similarly regulated in both pathways and thus representing candidates for common apoptosis regulators. For many expressed sequence tags (ESTs) our data suggest for the ®rst time functions in the control of apoptosis, stress response or the cell cycle. IL-3 removal led to the repression of genes required for proliferation and to the induction of genes, linked to apoptotic and signaling pathways. Staurosporine caused predominantly activation of genes, some of which had previously been described to be involved in in¯ammation. Our ®ndings indicate that cellular responses to both apoptotic stimuli in¯uence various physiological pathways which had not previously been known to be linked. Oncogene (2000) 19, 5073 ± 5082.
Saccharomyces cerevisiae cnm67Δ cells lack the spindle pole body (SPB) outer plaque, the main att... more Saccharomyces cerevisiae cnm67Δ cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein–labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the γ-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Δ cells Spc72–γ-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Δ cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs.
, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (S... more , a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion of CNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi-and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67⌬1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half-bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. Although CNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy of cnm67⌬1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.
Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules ... more Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules emanating from the spindle pole bodies (SPBs). Herein, we show by in vivo fluorescence microscopy that cells lacking Spc72, the SPB receptor of the cytoplasmic ␥-tubulin complex, can only generate very short (Ͻ1 m) and unstable astral microtubules. Consequently, nuclear migration to the bud neck and orientation of the anaphase spindle along the mother-bud axis are absent in these cells. However, SPC72 deletion is not lethal because elongated but misaligned spindles can frequently reorient in mother cells, permitting delayed but otherwise correct nuclear segregation. High-resolution time-lapse sequences revealed that this spindle reorientation was most likely accomplished by cortex interactions of the very short astral microtubules. In addition, a set of double mutants suggested that reorientation was dependent on the SPB outer plaque and the astral microtubule motor function of Kar3 but not Kip2/Kip3/Dhc1, or the cortex components Kar9/Num1. Our observations suggest that Spc72 is required for astral microtubule formation at the SPB half-bridge and for stabilization of astral microtubules at the SPB outer plaque. In addition, our data exclude involvement of Spc72 in spindle formation and elongation functions.
As part of EUROFAN (European Functional Analysis Network), we investigated 21 novel yeast open re... more As part of EUROFAN (European Functional Analysis Network), we investigated 21 novel yeast open reading frames (ORFs) by growth and sporulation tests of deletion mutants. Two genes (YNL026w and YNL075w) are essential for mitotic growth and three deletion strains (ynl080c, ynl081c and ynl225c) grew with reduced rates. Two genes (YNL223w and YNL225c) were identified to be required for sporulation. In addition we also performed green fluorescent protein (GFP) tagging for localization studies. GFP labelling indicated the spindle pole body (Ynl225c-GFP) and the nucleus (Ynl075w-GFP) as the sites of action of two proteins. Ynl080c-GFP and Ynl081c-GFP fluorescence was visible in dot-shaped and elongated structures, whereas the Ynl022c-GFP signal was always found as one spot per cell, usually in the vicinity of nuclear DNA. The remaining C-terminal GFP fusions did not produce a clearly identifiable fluorescence signal. For 10 ORFs we constructed 5'-GFP fusions that were expressed from the regulatable GAL1 promoter. In all cases we observed GFP fluorescence upon induction but the localization of the fusion proteins remained difficult to determine. GFP-Ynl020c and GFP-Ynl034w strains grew only poorly on galactose, indicating a toxic effect of the overexpressed fusion proteins. In summary, we obtained a discernible GFP localization pattern in five of 20 strains investigated (25%). A deletion phenotype was observed in seven of 21 (33%) and an overexpression phenotype in two of 10 (20%) cases.
The development of miniaturized arrays, displaying thousands of genes on a surface smaller than a... more The development of miniaturized arrays, displaying thousands of genes on a surface smaller than a thumb-nail, can be regarded as a technological breakthrough of similar importance as the introduction of the PCR reaction. For the development of novel cancer treatment regimens, microarrays will in the future be indispensable in the elucidation of tumor development and progression, in the detection and monitoring of tumor heterogeneity, and the identification of putative risk factors. Microarrays will furthermore help in the identification of novel, more specific molecular targets crucial in the development of primary tumors as well as in the process of metastasis. In addition, multiparallel analysis of thousands of genes in tissues treated with putative anticancer drugs, will lead to a better, genome-wide understanding of the drugs efficacy, specificity and potentially harmful side-effects. The hope is to come up in the future with more potent and more specific anticancer agents at increased speed and chemical diversity. Die Entwicklung miniaturisierter angeordneter Muster von beispielsweise Nukleinsauren („microarrays”), mit denen sich tausende von Genen auf einer Flache kleiner als ein Daumennagel reprasentieren lassen, kann als ein technischer Durchbruch angesehen werden, der sich mit der Einfuhrung der PCR vergleichen lasst. Fur die Entwicklung neuer Tumortherapien werden Microarrays in Zukunft unverzichtbar sein fur die Analyse von Tumorentstehung und -progression, fur die Bestimmung der Heterogenitat im Tumor und bei der Identifizierung moglicher Risikofaktoren. Weiterhin werden Microarrays hilfreich sein bei der Definition neuer, spezifischerer Zielmolekule, die sowohl bei der Enstehung von Primartumoren als auch Metastasen eine Rolle spielen. Daruber hinaus wird die multiparallele Analyse tausender von Genen in Tumorgewebe unter Behandlung mit moglichen antineoplastischen Substanzen zu einem besseren, genomweiten Verstandnis der Effizienz, Spezifitat und moglicher unerwunschter Nebenwirkungen dieser Substanzen fuhren. Es besteht Hoffnung zu der Annahme, dass in Zukunft wirksamere und spezifischere Krebsmedikamente in kurzerer Zeit und mit hoherer chemischer Vielfalt entwickelt werden konnen.
We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and... more We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosaccharomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii. Both chimeric modules and the cognate S. kluyveri HIS3 gene were tested in transformations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1•4 kb chimeric Sz. pombe module (HIS3MX6) performed best. With less than 5% incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX. The rare false-positive His + transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA-plasmids with HIS3MX6 and kanMX markers. The 0•9 kb GFP reporters consist of wild-type GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator. PCR-synthesized 2•4 kb-long double modules flanked by 40-45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S. cerevisiae genes. We could estimate that only about 10% of the transformants carried inactivating mutations in the GFP reporter. 1997 John Wiley & Sons, Ltd.
DNA microarrays are powerful tools for the analysis of gene expression on a genomic scale. The im... more DNA microarrays are powerful tools for the analysis of gene expression on a genomic scale. The importance of individual regulatory events for the process under study can however not be deduced unequivocally without additional experiments. We devised a strategy to identify central regulators of cancer drug responses by combining the results of microarray experiments with efficient methods for phenotypic testing of candidate genes. We exposed murine FL5.12 pro-B cells to cisplatin, camptothecin, methotrexate or paclitaxel, respectively and analysed the patterns of gene expression with cDNA microarrays. Drug-specific regulatory events as well as intersections between different apoptotic pathways, including previously studied responses to staurosporine and interleukin-3 (IL-3) deprivation, were identified. Genes shared by at least three pathways were chosen for further analysis. Ectopic expression of three such genes, TEAP, GP49B, and Lipin1 was found to have an anti-proliferative effect on pro-B cells. Interestingly, we identified hemoglobin alpha as a strong pro-apoptotic regulator. While hemoglobin-expressing cells were growing normally in the presence of IL-3, they displayed accelerated apoptosis with similar kinetics as Bax overexpressing cells upon IL-3 removal. The proapoptotic effect of hemoglobin was suppressed by Bcl-2 and was characterized by enhanced stimulation of caspase activity.
Proceedings of the National Academy of Sciences of the United States of America, Jul 9, 1996
The core oligosaccharide Glc3MangGlcNAc2 is assembled at the membrane of the endoplasmic reticulu... more The core oligosaccharide Glc3MangGlcNAc2 is assembled at the membrane of the endoplasmic reticulum on the lipid carrier dolichyl pyrophosphate and transferred to selected asparagine residues of nascent polypeptide chains. This transfer is catalyzed by the oligosaccharyl transferase complex. Based on the synthetic phenotype of the oligosaccharyl transferase mutation wbpl in combination with a deficiency in the assembly pathway of the oligosaccharide in Saccharomyces cerevisiae, we have identified the novel ALG9 gene. We conclude that this locus encodes a putative mannosyl transferase because deletion of the gene led to accumulation of lipid-linked Man6GlcNAc2 in vivo and to hypoglycosylation of secreted proteins. Using an approach combining genetic and biochemical techniques, we show that the assembly of the lipid-linked core oligosaccharide in the lumen of the endoplasmic reticulum occurs in a stepwise fashion. N-linked glycosylation is an essential modification of proteins and follows a highly conserved pathway in eukaryotic cells (1-3). The core oligosaccharide Glc3Man9GlcNAc2 is assembled on the lipid carrier dolichyl pyrophosphate and transferred to selected asparagine residues of nascent polypeptide chains. This transfer is catalyzed by the enzyme oligosaccharyl transferase (4, 5). Abbreviations: CPY, carboxypeptidase Y; Dol, dolichol; Endo H, endo-13-N-acetylglucosaminidase H; ER, endoplasmic reticulum. Data deposition: The sequence reported in this paper has been deposited in the GeneBank data base (accession no. X96417).
Background: This short communication focuses the on articular cartilage and the subchondral bone,... more Background: This short communication focuses the on articular cartilage and the subchondral bone, both of which play important roles in the development of osteoarthritis (OA). There are indications that estrogendeficiency, as the post-menopausal state, accelerate the development of OA. Findings: We investigated, which extracellular matrix (ECM) protein, proteases and different pro-inflammatory factors was up-or down-regulated in the knee joint tissue in response to estrogen-deficiency in rats induced by ovariectomy. These data support previous findings that several metalloproteinases (MMPs) and cysteine proteases are co-regulated with numerous collagens and proteoglycans that are important for cartilage integrity. Furthermore quite a few pro-inflammatory cytokines were regulated by estrogen deprivation. Conclusion: We found multiple genes where regulated in the joint by estrogen-deficiency, many of which correspond well with our current knowledge of the pathogenesis of OA. It supports that estrogen-deficiency (e.g. OVX) may accelerate joint deterioration. However, there are also data that draw attention the need for better understanding of the synergy between proteases and tissue turnover.
The transcriptional response of mouse pro-B cells to two dierent apoptotic stimuli was investigat... more The transcriptional response of mouse pro-B cells to two dierent apoptotic stimuli was investigated. First, interleukin-3 (IL-3) deprivation was used to trigger programmed cell death in IL-3 dependent FL5.12 cells. Alternatively, cells were treated with the protein kinase C (PKC) inhibitor staurosporine. The temporal pattern of gene expression was followed with cDNA microarrays, covering over 8700 dierent mouse cDNA sequences corresponding to approximately 7900 unique genes. Messenger RNA levels of 315 genes were found to be regulated by more than twofold upon IL-3 removal, while 125 genes reacted to staurosporine treatment. Cross-comparison revealed an intersection of 34 genes similarly regulated in both pathways and thus representing candidates for common apoptosis regulators. For many expressed sequence tags (ESTs) our data suggest for the ®rst time functions in the control of apoptosis, stress response or the cell cycle. IL-3 removal led to the repression of genes required for proliferation and to the induction of genes, linked to apoptotic and signaling pathways. Staurosporine caused predominantly activation of genes, some of which had previously been described to be involved in in¯ammation. Our ®ndings indicate that cellular responses to both apoptotic stimuli in¯uence various physiological pathways which had not previously been known to be linked. Oncogene (2000) 19, 5073 ± 5082.
Saccharomyces cerevisiae cnm67Δ cells lack the spindle pole body (SPB) outer plaque, the main att... more Saccharomyces cerevisiae cnm67Δ cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein–labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the γ-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Δ cells Spc72–γ-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Δ cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs.
, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (S... more , a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion of CNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi-and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67⌬1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half-bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. Although CNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy of cnm67⌬1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.
Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules ... more Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules emanating from the spindle pole bodies (SPBs). Herein, we show by in vivo fluorescence microscopy that cells lacking Spc72, the SPB receptor of the cytoplasmic ␥-tubulin complex, can only generate very short (Ͻ1 m) and unstable astral microtubules. Consequently, nuclear migration to the bud neck and orientation of the anaphase spindle along the mother-bud axis are absent in these cells. However, SPC72 deletion is not lethal because elongated but misaligned spindles can frequently reorient in mother cells, permitting delayed but otherwise correct nuclear segregation. High-resolution time-lapse sequences revealed that this spindle reorientation was most likely accomplished by cortex interactions of the very short astral microtubules. In addition, a set of double mutants suggested that reorientation was dependent on the SPB outer plaque and the astral microtubule motor function of Kar3 but not Kip2/Kip3/Dhc1, or the cortex components Kar9/Num1. Our observations suggest that Spc72 is required for astral microtubule formation at the SPB half-bridge and for stabilization of astral microtubules at the SPB outer plaque. In addition, our data exclude involvement of Spc72 in spindle formation and elongation functions.
As part of EUROFAN (European Functional Analysis Network), we investigated 21 novel yeast open re... more As part of EUROFAN (European Functional Analysis Network), we investigated 21 novel yeast open reading frames (ORFs) by growth and sporulation tests of deletion mutants. Two genes (YNL026w and YNL075w) are essential for mitotic growth and three deletion strains (ynl080c, ynl081c and ynl225c) grew with reduced rates. Two genes (YNL223w and YNL225c) were identified to be required for sporulation. In addition we also performed green fluorescent protein (GFP) tagging for localization studies. GFP labelling indicated the spindle pole body (Ynl225c-GFP) and the nucleus (Ynl075w-GFP) as the sites of action of two proteins. Ynl080c-GFP and Ynl081c-GFP fluorescence was visible in dot-shaped and elongated structures, whereas the Ynl022c-GFP signal was always found as one spot per cell, usually in the vicinity of nuclear DNA. The remaining C-terminal GFP fusions did not produce a clearly identifiable fluorescence signal. For 10 ORFs we constructed 5'-GFP fusions that were expressed from the regulatable GAL1 promoter. In all cases we observed GFP fluorescence upon induction but the localization of the fusion proteins remained difficult to determine. GFP-Ynl020c and GFP-Ynl034w strains grew only poorly on galactose, indicating a toxic effect of the overexpressed fusion proteins. In summary, we obtained a discernible GFP localization pattern in five of 20 strains investigated (25%). A deletion phenotype was observed in seven of 21 (33%) and an overexpression phenotype in two of 10 (20%) cases.
The development of miniaturized arrays, displaying thousands of genes on a surface smaller than a... more The development of miniaturized arrays, displaying thousands of genes on a surface smaller than a thumb-nail, can be regarded as a technological breakthrough of similar importance as the introduction of the PCR reaction. For the development of novel cancer treatment regimens, microarrays will in the future be indispensable in the elucidation of tumor development and progression, in the detection and monitoring of tumor heterogeneity, and the identification of putative risk factors. Microarrays will furthermore help in the identification of novel, more specific molecular targets crucial in the development of primary tumors as well as in the process of metastasis. In addition, multiparallel analysis of thousands of genes in tissues treated with putative anticancer drugs, will lead to a better, genome-wide understanding of the drugs efficacy, specificity and potentially harmful side-effects. The hope is to come up in the future with more potent and more specific anticancer agents at increased speed and chemical diversity. Die Entwicklung miniaturisierter angeordneter Muster von beispielsweise Nukleinsauren („microarrays”), mit denen sich tausende von Genen auf einer Flache kleiner als ein Daumennagel reprasentieren lassen, kann als ein technischer Durchbruch angesehen werden, der sich mit der Einfuhrung der PCR vergleichen lasst. Fur die Entwicklung neuer Tumortherapien werden Microarrays in Zukunft unverzichtbar sein fur die Analyse von Tumorentstehung und -progression, fur die Bestimmung der Heterogenitat im Tumor und bei der Identifizierung moglicher Risikofaktoren. Weiterhin werden Microarrays hilfreich sein bei der Definition neuer, spezifischerer Zielmolekule, die sowohl bei der Enstehung von Primartumoren als auch Metastasen eine Rolle spielen. Daruber hinaus wird die multiparallele Analyse tausender von Genen in Tumorgewebe unter Behandlung mit moglichen antineoplastischen Substanzen zu einem besseren, genomweiten Verstandnis der Effizienz, Spezifitat und moglicher unerwunschter Nebenwirkungen dieser Substanzen fuhren. Es besteht Hoffnung zu der Annahme, dass in Zukunft wirksamere und spezifischere Krebsmedikamente in kurzerer Zeit und mit hoherer chemischer Vielfalt entwickelt werden konnen.
We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and... more We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosaccharomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii. Both chimeric modules and the cognate S. kluyveri HIS3 gene were tested in transformations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1•4 kb chimeric Sz. pombe module (HIS3MX6) performed best. With less than 5% incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX. The rare false-positive His + transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA-plasmids with HIS3MX6 and kanMX markers. The 0•9 kb GFP reporters consist of wild-type GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator. PCR-synthesized 2•4 kb-long double modules flanked by 40-45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S. cerevisiae genes. We could estimate that only about 10% of the transformants carried inactivating mutations in the GFP reporter. 1997 John Wiley & Sons, Ltd.
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