The seven rRNA operons (genes) of E. coli were physically mapped by restriction endonucleases, us... more The seven rRNA operons (genes) of E. coli were physically mapped by restriction endonucleases, using the Southern blotting technique. Two complete rRNA operons (rrnB and rrnD) were isolated by the recombinant DNA method, using the pBR322 plasmid cloning vehicle. The rRNA operon carried by the transducing phage λdaroE152 was found to be a hybrid of the rrnD and rrnE operons. The DNA sequence of the promoter region for rrnB was determined. The RNA-polymerase binding sites on the cloned rRNA operons were localized by filter-binding and electron-microscopic techniques. rRNA transcription was investigated by electron microscopy and hybridization analysis of the in vitro transcripts. All these findings were correlated with the DNA sequence, and a tentative model was proposed to explain the unusual properties of rRNA promoters.
The DNA of the specialized transducing phage lambdarifd18, which carries a bacterial rRNA transcr... more The DNA of the specialized transducing phage lambdarifd18, which carries a bacterial rRNA transcription unit, was digested with restriction enzymes EcoRI and/or BamHI. Attempts were made to clone fragments containing the presumed rRNA promoter region or the entire rRNA gene in RSF2124 or pBR313 plasmid vectors with the following results: (1) We failed to clone an EcoRI fragment with the rRNA promoter region in plasmid RSF2124. (2) A smaller EcoRI-BamHI fragment with the rRNA promoter was also unclonable by itself, but one recombinant was found containing this fragment together with another large (7 Mdaltons) fragment, derived from phage lambda. The presence of this large fragment proved to be essential. The identity of these DNA fragments in the recombinant clone was confirmed by redigestion with several restriction enzymes, hybridization with rRNA, and in vitro transcription experiments, which showed preferential rRNA transcription. (3) A BamHI fragment encompassing the entire rRNA gene was easily cloned. Such stable clones carried a doubled number of rRNA genes. In vitro transcription using the recombinant plasmid resulted in 70% rRNA transcription. These recombinant clones allow the easy purification of the relevant DNA fragments for further investigation including sequencing.
The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to t... more The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to the 1821st nucleotide upstream from the beginning of the sequence coding for mature 16 S rRNA. In vitro transcription experiments indicated the presence of two new promoters in this region, located more than 1 kb upstream from the known P1 and P2 promoters of rrnB. Previous electron microscopic studies demonstrated that these sites bind RNA-polymerase very strongly. In vitro transcription, starting at these sites reads through the entire region into the rrnB gene without termination. A similar uninterrupted transcription into rrnB in vivo can be demonstrated by S1-mapping, and by fusing the DNA containing the new promoters (but not P1 and P2) to the lacZ gene. Thus it seems likely that these promoters (P3 and P4) belong functionally to the rrnB gene and play some role in its regulation of expression.
Cartilage matrix protein (CMP), a major noncollagenous component of certain types of hyaline cart... more Cartilage matrix protein (CMP), a major noncollagenous component of certain types of hyaline cartilage, is synthesized by chondrocytes in a developmentally regulated manner. In this study, we monitored the accumulation of CMP in the developing chicken limb and sternum by immunostaining. In older embryos, the specific extracellular staining was restricted to the resting/proliferative zone of metaphyseal cartilage and to the immediately adjacent hypertrophic cartilage. A lack of staining was observed in the peripheral layers of articular cartilage. Data were compared with the accumulation of CMP mRNA measured by Northern analysis relative to other cartilage-specific messages in cell cultures representing different stages of chondrocyte differentiation, as well as with the steady state mRNA levels in tissue samples. We found a correlation between the gene expression pattern of the in vitro cultures and the one observed in certain in vivo differentiation stages. The high-density mesenchyme culture was utilized as a model for studying the events at early stage I (stage Ia) of chondrogenesis. This culture was characterized by relatively low steady state mRNA levels for cartilage proteins, including the later activation of the CMP gene as compared to type II collagen or link protein genes, and relatively high steady state mRNA levels for type VI collagen and beta-actin. Chicken embryo chondrocyte cultures obtained from sterna of 14-day-old embryos, however, consisted predominantly of stage Ib chondrocytes, and showed high steady state levels for cartilage proteins, but relatively lower levels for type VI collagen and beta-actin mRNAs. In accordance with the in vivo data, a relatively high steady state level was detected for CMP mRNA in cultures of hypertrophic (stage II) chondrocytes. We also performed transient expression assays in the various culture systems to study the role of the promoter upstream and intronic control regions in the tissue- and developmental stage-specific regulation of the CMP gene. We showed that the enhancer worked in a lineage-specific manner, by further stimulating the minimal promoter activity independent of the developmental stage of chondrocytes, while it did not in other tissues. The promoter upstream control regions, however, seemed to play a role in restricting the promoter activity to a certain chondrocyte developmental stage.
The seven rRNA operons (genes) of E. coli were physically mapped by restriction endonucleases, us... more The seven rRNA operons (genes) of E. coli were physically mapped by restriction endonucleases, using the Southern blotting technique. Two complete rRNA operons (rrnB and rrnD) were isolated by the recombinant DNA method, using the pBR322 plasmid cloning vehicle. The rRNA operon carried by the transducing phage λdaroE152 was found to be a hybrid of the rrnD and rrnE operons. The DNA sequence of the promoter region for rrnB was determined. The RNA-polymerase binding sites on the cloned rRNA operons were localized by filter-binding and electron-microscopic techniques. rRNA transcription was investigated by electron microscopy and hybridization analysis of the in vitro transcripts. All these findings were correlated with the DNA sequence, and a tentative model was proposed to explain the unusual properties of rRNA promoters.
The DNA of the specialized transducing phage lambdarifd18, which carries a bacterial rRNA transcr... more The DNA of the specialized transducing phage lambdarifd18, which carries a bacterial rRNA transcription unit, was digested with restriction enzymes EcoRI and/or BamHI. Attempts were made to clone fragments containing the presumed rRNA promoter region or the entire rRNA gene in RSF2124 or pBR313 plasmid vectors with the following results: (1) We failed to clone an EcoRI fragment with the rRNA promoter region in plasmid RSF2124. (2) A smaller EcoRI-BamHI fragment with the rRNA promoter was also unclonable by itself, but one recombinant was found containing this fragment together with another large (7 Mdaltons) fragment, derived from phage lambda. The presence of this large fragment proved to be essential. The identity of these DNA fragments in the recombinant clone was confirmed by redigestion with several restriction enzymes, hybridization with rRNA, and in vitro transcription experiments, which showed preferential rRNA transcription. (3) A BamHI fragment encompassing the entire rRNA gene was easily cloned. Such stable clones carried a doubled number of rRNA genes. In vitro transcription using the recombinant plasmid resulted in 70% rRNA transcription. These recombinant clones allow the easy purification of the relevant DNA fragments for further investigation including sequencing.
The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to t... more The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to the 1821st nucleotide upstream from the beginning of the sequence coding for mature 16 S rRNA. In vitro transcription experiments indicated the presence of two new promoters in this region, located more than 1 kb upstream from the known P1 and P2 promoters of rrnB. Previous electron microscopic studies demonstrated that these sites bind RNA-polymerase very strongly. In vitro transcription, starting at these sites reads through the entire region into the rrnB gene without termination. A similar uninterrupted transcription into rrnB in vivo can be demonstrated by S1-mapping, and by fusing the DNA containing the new promoters (but not P1 and P2) to the lacZ gene. Thus it seems likely that these promoters (P3 and P4) belong functionally to the rrnB gene and play some role in its regulation of expression.
Cartilage matrix protein (CMP), a major noncollagenous component of certain types of hyaline cart... more Cartilage matrix protein (CMP), a major noncollagenous component of certain types of hyaline cartilage, is synthesized by chondrocytes in a developmentally regulated manner. In this study, we monitored the accumulation of CMP in the developing chicken limb and sternum by immunostaining. In older embryos, the specific extracellular staining was restricted to the resting/proliferative zone of metaphyseal cartilage and to the immediately adjacent hypertrophic cartilage. A lack of staining was observed in the peripheral layers of articular cartilage. Data were compared with the accumulation of CMP mRNA measured by Northern analysis relative to other cartilage-specific messages in cell cultures representing different stages of chondrocyte differentiation, as well as with the steady state mRNA levels in tissue samples. We found a correlation between the gene expression pattern of the in vitro cultures and the one observed in certain in vivo differentiation stages. The high-density mesenchyme culture was utilized as a model for studying the events at early stage I (stage Ia) of chondrogenesis. This culture was characterized by relatively low steady state mRNA levels for cartilage proteins, including the later activation of the CMP gene as compared to type II collagen or link protein genes, and relatively high steady state mRNA levels for type VI collagen and beta-actin. Chicken embryo chondrocyte cultures obtained from sterna of 14-day-old embryos, however, consisted predominantly of stage Ib chondrocytes, and showed high steady state levels for cartilage proteins, but relatively lower levels for type VI collagen and beta-actin mRNAs. In accordance with the in vivo data, a relatively high steady state level was detected for CMP mRNA in cultures of hypertrophic (stage II) chondrocytes. We also performed transient expression assays in the various culture systems to study the role of the promoter upstream and intronic control regions in the tissue- and developmental stage-specific regulation of the CMP gene. We showed that the enhancer worked in a lineage-specific manner, by further stimulating the minimal promoter activity independent of the developmental stage of chondrocytes, while it did not in other tissues. The promoter upstream control regions, however, seemed to play a role in restricting the promoter activity to a certain chondrocyte developmental stage.
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Papers by Ibolya Kiss