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H Kleinert

Publications and source records attributed to H Kleinert.

At least 55 records · Page 3Linked to original sources
PubMed1991

A single base pair deletion from the inactive octamer-like motif of the 7S K distal sequence element brings full functionality in vivo.

Octamer sequence elements were analyzed for their capacity to induce the 7S K "core" promoter in vivo. The U6 distal sequence element (DSE) which contains a consensus sequence octamer, was able to support efficient 7S K expression in vivo. In contrast, no such function could be attributed to the octamer-like element alone, which is present within the 7S K DSE. However, conversion of this octamer-like element (ATTTaGCAT) to the octamer consensus sequence ATTTGCAT generated a potent DSE, even in the absence of the CACCC box, which constitutes the major functional element of the 7S K DSE. Both the consensus and the octamer-like sequences revealed no cooperativity with the CACCC box. Together, these results demonstrate that the octamer-like element of the wild-type 7S K DSE is definitely not functional in vivo. Furthermore, our experiments indicate that in contrast to the RNA polymerase II-transcribed small nuclear RNA genes, in intact cells a single functional DSE motif is necessary and sufficient for maximal transcription by RNA polymerase III of the 7S K RNA gene.

Base Composition↗
PubMed1990

Activating-transcription-factor (ATF) regulates human 7S L RNA transcription by RNA polymerase III in vivo and in vitro.

The gene-external part of the human 7S L promoter was analyzed by transcription in vitro and in vivo. Compared to the wild type promoter (-178), a -66 5'deletion mutant revealed full activity in vitro but was inefficiently transcribed in vivo. Further deletion to -37 reduced template activity to 50% in vitro and to basal level expression in vivo (below 5%). A DNase I footprint observed around position -50 protected an ATF-like binding site ('TGACGT'). With respect to 7S L transcription regulation, the functionality of this ATF-like binding site was confirmed in competition experiments and by mutation analysis. Furthermore, S100 extracts of cells pretreated with forskolin in vivo to induce the cAMP system, revealed significantly increased transcription of 7S L RNA in vitro, with no effect on a 7S K RNA gene, lacking such an ATF binding site. Thus, the 7S L RNA gene too is controlled by a regulatory element originally defined in class II promoters and represents another rare example where a specific type of transcription regulation in vivo can be mimicked with cell-free extracts in vitro.

Activating Transcription Factors↗
PubMed1990

Sequence and factor requirements for faithful in vitro transcription of human 7SL DNA.

We have analysed the transcription of a functional human 7SL gene by RNA polymerase III (RNAPIII) in S100 extracts in vitro. Accurate and efficient synthesis of 7S L RNA depends on the presence of (i) an upstream sequence and (ii) an internal promoter element located within the first 22 bp of the gene. These findings were substantiated by DNase I footprinting. Mutations of the internal promoter identified the doublet CG [nucleotide (nt) +15/+16] outside the A-box homologue (nt +5 to +14) as being essential for both proper promoter function in the in vitro transcription assay and competition in the template-exclusion assay. Fractionation of S100 extracts identified two fractions required in addition to RNAPIII for faithful transcription of the gene. Each of these two fractions gave rise to one of two footprints observed in DNase I protection experiments, indicating that at least two DNA-binding factors are involved.

Base Sequence↗
PubMed1990

Expression of a human 7S K RNA gene in vivo requires a novel pol III upstream element.

The 5'-flanking sequences required for expression of a human 7S K RNA gene have been defined by mutant analysis. A -111 upstream deletion mutant showed full activity when analysed by in vitro transcription with HeLa cell extracts. In contrast, upon transfection into intact cells, this mutant only revealed a basal level activity of approximately 6% as compared to the wild-type promoter up to position -252. The deleted upstream sequence element acts as a transcriptional activator in vivo, in a strictly position and orientation-dependent manner. Two octamer-like binding motifs observed within this upstream sequence were both dispensable for proper function of this RNA polymerase III promoter in vivo. Instead, a detailed analysis of this region identified a CACCC-box element, together with its surrounding base pairs, as the essential upstream element required for expression of this 7S K RNA gene in vivo. Furthermore, this CACCC-box is centered within a footprint obtained with HeLa cell nuclear proteins.

Base Sequence↗
PubMed1990

Glucose content and efficiency of glycolysis in protected ischemic kidneys of different species.

In ischemic canine kidneys protected by Bretschneider's HTK solution the glycolytic lactate production is limited by a low renal substrate content. However, for anaerobic energy supply ischemic organs depend on glycolysis. To evaluate the role of glycolysis in renal protection, the relationship between lactate production and anaerobic energy supply was examined in protected kidneys of dogs, sheep, and swine. Additionally, in canine kidneys an attempt was made to improve anaerobic energy provision by adding glucose to the protective solution. The results were as follows: (1) According to increasing lactate production from swine to dog to sheep, intraischemic ATP decay was delayed least in swine and most in sheep. (2) Glucose addition (10 mM) to the HTK solution roughly doubled the time for ATP to fall to 1 mumol/g dry wt (tATP) in dogs. (3) The greater the lactate production in all three species, the lower the decrease in SAN (ATP + ADP + AMP) from 5 to 120 min of ischemia. (4) A glucose additive in the protective solution led to a significant (p less than .005) increase of SAN in dogs at 120 min of ischemia. A sufficient substrate supply seems to be an essential component of a reliable renal protection.

Adenine Nucleotides↗
PubMed1989

Intraischemic metabolic effects of different disaccharides on protected canine kidneys.

The addition of the disaccharides maltose (10, 20, 30 mM) and sucrose (30, 60 mM) to Bretschneider's organ protective HTK solution was evaluated to improve renal protection by an enhanced glycolytic energy supply. Canine kidneys were perfused for 8 min with either HTK solution or HTK solution containing additional disaccharides. After nephrectomy the kidneys were incubated at 25 degrees C and metabolic parameters were determined at regular intervals. Maltose and sucrose are slowly cleaved during renal ischemia but maltose distinctly faster than sucrose. Maltose increases intraischemic ATP supply. However, 30 mM maltose was no better than 10 mM. 60 mM sucrose was about as effective for glycolysis as 10 mM maltose. However, possibly due to fructose release there was an accelerated decrease of adenine nucleotides with sucrose. Although fructose enters glycolysis it seems to have negative side-effects. Hence, probably neither sucrose nor fructose are appropriate for renal substrate supply during ischemia.

Adenosine Triphosphate↗
PubMed1988

Differential regulation of transcription of human 7 S K and 7 S L RNA genes.

Two functional human genes coding for 7 S RNA species K and L were analyzed for promoter requirements by in vitro transcription experiments with cytoplasmic S-100 extracts. Since accurate and efficient transcription of both genes is dependent on the presence of 5'-flanking sequences, hybrid genes representing crossover fusions between the 5' external control regions and the coding sequences of both genes were analyzed for their capacity to direct RNA synthesis in vitro. Differing results were obtained with both types of constructs. While the 5'-flanking L-7 S K gene fusion revealed no activity in the in vitro transcription assay, the 5'-flanking sequence of the 7 S K RNA gene did confer the ability for accurate in vitro transcription to the 7 S L coding sequence. However, a 5'-flanking L sequence element including the first 22 nucleotides of the 7 S L RNA coding sequence was active in promoting transcription of the 7 S K RNA gene. Together, these results demonstrated that the 7 S L promoter is located inside and outside the coding region, whereas the 7 S K RNA gene is exclusively controlled by an upstream promoter element.

Base Sequence↗