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Biomedical subjects

B Wittig

Publications and source records attributed to B Wittig.

At least 91 records · Page 5Linked to original sources
PubMed1990

Direct determination of chloramphenicol acetyltransferase (CAT) activity in homogenates of transfected mammalian cells by high performance liquid chromatography.

The conversion of chloramphenicol to its monoacetylated form by homogenates of pRSV CAT-transfected mammalian cells was assayed by HPLC. The method differed from conventional procedures, in that the extraction of chloramphenicol and its acetylated forms into an organic solvent was replaced by treatment of the cell homogenates with acetonitrile. This allows a rapid, direct analysis by HPLC with few experimental steps.

Acetonitriles↗
PubMed1990

A strategy for the construction of double-stranded DNA microcircles with circumferences less than 50 bp.

A procedure for the construction of double-stranded DNA microcircles is described that overcomes the natural limits of established circularization procedures. The assembly strategy needs only three components, i.e. two synthetic oligonucleotides containing any sequence of interest and T4 DNA ligase. All components can be provided in large quantities and only two enzymatic reaction steps are required. Such microcircles can serve as a model for topologically fixed and highly bent DNA molecules and the amounts needed for X-ray crystallography, n.m.r. spectroscopy or drug binding studies can readily be produced. The construction of a 42 base pair double-stranded DNA microcircle is presented, by far the smallest double-stranded DNA circle yet described either in vivo or in vitro.

Alkaline Phosphatase↗
PubMed1989

Construction of a 42 base pair double stranded DNA microcircle.

A procedure for the construction of double stranded DNA microcircles is described that overcomes the natural limits of established circularization procedures. Starting with two synthetic oligonucleotides which are able to form dumbbell shaped structures, two subsequent ligation reactions yield a microcircle of double stranded DNA of 42 base pairs. This is by far the smallest circle of double stranded DNA yet described. These microcircles can be constructed in quantities required for high resolution structural analyses such as X-ray crystallography and NMR spectroscopy.

Base Composition↗
PubMed1989

The level of Z-DNA in metabolically active, permeabilized mammalian cell nuclei is regulated by torsional strain.

Permeabilized nuclei from mammalian cells encapsulated within agarose microbeads in an isotonic buffer are active in transcription and replication (Jackson, D. A., and P. R. Cook. 1985. EMBO (Eur. Mol. Biol. Organ.) J. 4:913-918). Their DNA is intact and the nuclei are accessible to macromolecules. Myeloma nuclei prepared in this way were used to probe the extent of DNA negative supercoiling and the effects of altering torsional strain by binding radioactively labeled monoclonal antibodies to Z-DNA. Control experiments used monoclonal antibodies against a nonhistone chromosomal protein, HMG-17. On increasing the amount of anti-HMG-17 added, a binding plateau was reached encompassing a 200-fold range of antibody concentration. On binding anti-Z-DNA antibody, a similar broad plateau of constant binding was found encompassing a 100-fold range of antibody concentration. The latter result was taken as a measure of preexisting Z-DNA in the nuclei. Additional anti-Z-DNA antibody binding can be "induced" in the presence of much higher concentration of antibody, apparently by perturbing the B-DNA/Z-DNA equilibrium. On inhibiting topoisomerase I with camptothecin, an elevated antibody binding plateau was found, suggesting that elastic torsional strain in the DNA is responsible for stabilizing the preexisting Z-DNA. This interpretation is supported by the fact that addition of small, nicking amounts of DNase I leads to a complete loss of antibody binding in the Z-DNA plateau region but not in the region of "induced" Z-DNA.

Animals↗
PubMed1987

Transcription termination and processing of transcripts from tRNA-related Xenopus satellite DNA sequences.

Nucleotide sequence homologies have been found between the transcriptional unit of a Xenopus laevis satellite DNA containing 1037-bp monomeric repeats and tRNA-coding genes. Since the tRNA-homologous region forms only part of the transcriptional unit, we have analyzed the size of transcripts being made after injection of cloned DNA into Xenopus oocyte nuclei. Incubation for 6 h mainly results in three RNA species comprising 130, 180 and 196 nucleotides, which originate at the same initiation site but terminate at different T stretches. Longer incubation reveals a complex pattern of smaller RNAs most likely resulting from 3'-end processing of primary transcripts. All these RNAs are related to tRNA, since they contain the tRNA-homologous region which is close to the 5' end of the transcription unit.

Animals↗
PubMed1987

Chromatin from transcribed genes contains HMG17 only downstream from the starting point of transcription.

Monoclonal antibodies specific for the non-histone chromosomal protein HMG17 were used to isolate oligonucleosomes from the transcriptionally active chromatin of chicken liver and oviduct. The distribution of HMG17 with respect to the coding region of three genes was analyzed in these oligonucleosomes by employing two independent experimental approaches. In the vitellogenin II gene (active in liver) and the lysozyme and ovalbumin genes (active in oviduct) HMG17 was found only downstream from the respective starting points of transcription. The transition from HMG17-free to HMG17-containing chromatin is located at the transcription start. This directly demonstrates that the distribution of an abundant nuclear protein correlates with the observation of moderate DNase I-sensitivity in upstream regions and of high sensitivity in the coding regions of active genes.

Animals↗
PubMed1987

Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene.

Inhibition of herpes simplex virus (HSV) thymidine kinase (TK) gene transcription (pHSV-106, pML-BPV-TK4) by DNA methylation is an indirect effect, which occurs with a latency period of approximately equal to 8 hr after microinjection of the DNA into TK- rat 2 and mouse LTK- cells. We have strong evidence that chromatin formation is critical for the transition of the injected DNA from methylation insensitivity to methylation sensitivity. Chromatin was reconstituted in vitro by using methylated and mock-methylated HSV TK DNA and purified chicken histone octamers. After microinjection, the methylated chromatin was always biologically inactive, as tested by autoradiography of the cells after incubation with [3H]thymidine and by RNA dot blot analysis. However, in transformed cell lines, reactivation of the methylated chromatin occurred after treatment with 5-azacytidine. Furthermore, integration of the TK chromatin into the host genome is not required to block expression of the methylated TK gene. Mouse cells that contained the pML-BPV-TK4 chromatin permanently in an episomal state also did not support TK gene expression as long as the TK DNA remained methylated.

Animals↗
PubMed1986

Isolation of oligonucleosomes from active chromatin using HMG17-specific monoclonal antibodies.

We report the preparation of HMG17-containing oligonucleosomes from chicken embryos and from liver and oviduct of laying hens. Monoclonal antibodies against HMG17 were used for their isolation. An unusual size distribution with respect to their repeat number was observed. The oligonucleosomes of repeat number up to N6 were highly enriched for DNA of the vitellogenin II gene (liver) and for DNA of the ovalbumin and lysozyme genes (oviduct).

Animals↗
PubMed1986

Gluconeogenic-glycolytic capacities and metabolic zonation in liver of rats with streptozotocin, non-ketotic as compared to alloxan, ketotic diabetes.

Activities (mumol X min-1 X g liver) and zonal distributions of key enzymes of carbohydrate metabolism were studied in livers of streptozotocin-diabetic rats and compared to the values in alloxan-diabetes. Streptozotocin led to a non-ketotic diabetes with blood glucose being increased by more than fivefold but ketone bodies being in the normal range, while alloxan produced a ketotic diabetes with blood glucose, acetoacetate and beta-hydroxybutyrate being elevated by more than fivefold. Portal insulin was decreased to about 20% in streptozotocin- and more drastically to about 7% in alloxan-diabetes. Conversely, portal glucagon was increased in the two states to about 250% and 180%, respectively. The glucogenic key enzyme phosphoenolpyruvate carboxykinase (PEPCK) was enhanced in streptozotocin- and alloxan-diabetes to over 300%, while the glycolytic pyruvate kinase L (PKL) was lowered to 65% and 80%, respectively. The normal periportal to perivenous gradient of PEPCK of about 3:1, as measured in microdissected tissue samples, was maintained with elevated activities in the two zones. The normal periportal to perivenous gradient of PKL of 1:1.7 was diminished with lowered activities in the two zones. The glucogenic glucose-6-phosphatase (G6Pase) was increased in streptozotocin- and alloxan-diabetes to 130% and 140%, respectively, while the glucose utilizing glucokinase (GK) was decreased to 60% and 50%, respectively. The normal periportal to perivenous gradient of G6Pase, demonstrated histochemically, remained unaffected. Carnitine palmitoyltransferase (CPT) was increased to over 190% and acetyl-CoA carboxylase (ACC) was decreased to 60% in streptozotocin, non-ketotic diabetes, while the two enzymes were altered more drastically to 400% and 50%, respectively, in alloxan, ketotic diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Alloxan↗
PubMed1985

Metabolic zonation in liver of diabetic rats. Zonal distribution of phosphoenolpyruvate carboxykinase, pyruvate kinase, glucose-6-phosphatase and succinate dehydrogenase.

The activities and zonal distribution of key enzymes of carbohydrate metabolism were studied in livers of diabetic rats. 48 h after alloxan treatment the following alterations were observed, intermediate values being reached after 24 h: Blood glucose, acetoacetate and beta-hydroxybutyrate were increased to more than 500%; liver glycogen was reduced to about 10%. Portal vein insulin was reduced to below 10%, portal glucagon was increased to almost 200%. The glucogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were enhanced to 320% and 150%, respectively. The glycolytic enzymes glucokinase and pyruvate kinase L (differentiated from the M2 isoenzyme with a specific anti-L-antibody) were lowered to 50% and 75%, respectively. The citrate cycle enzyme succinate dehydrogenase remained unchanged. The normal periportal to perivenous gradient of phosphoenolpyruvate carboxykinase of about 3:1, as measured in microdissected tissue samples, was enhanced to about 4:1 with activities elevated to 230% and 190%, respectively, in the two zones. The normal periportal to perivenous gradient of pyruvate kinase L of about 1:1.7, as determined with the microdissection technique, was reduced to about 1:1.4 with levels lowered to 55% and 45%, respectively, in the two zones. The even zonal distribution of pyruvate kinase M2 remained unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Glucostat capacity and metabolic zonation in rat liver after portocaval anastomosis.

The activities and zonal distribution of key enzymes of carbohydrate metabolism were studied in livers of rats after end-to-side portocaval anastomosis. Sham-operated control animals with the same periods of interruption of hepatic blood supply as the shunted animals were pair-fed. The following alterations were observed: Food uptake was reduced to about 20% at the first postoperational day; it was then increased continuously to about 70% at day 8. Body weight, after a small 10% postoperational decrease, remained unaltered, but liver weight was lowered to 55% at day 8 and then stayed constant. The total glycogen reserves of the liver (g X 100 g body weight-1) were reduced, after a transient fall to about 10% at day 1-4, to about 25%. The total activity of the glucogenic phosphoenolpyruvate carboxykinase (mumol . min-1 X 100 g body weight-1) was diminished, after a transient increase to 190% and 150% at day 1 and 2 respectively, to about 55% from day 8 onwards. The total activity of the glucogenic glucose-6-phosphatase was lowered without a transient rise to about 30%. The total activities of the glycolytic pyruvate kinase isoenzyme L and glucokinase were decreased continuously to about 40% at day 8; that of the citrate cycle enzyme succinate dehydrogenase was lowered parallel with liver weight to 55%. The transient decrease of the glycogen reserves and the intermediate increase of the phosphoenolpyruvate carboxykinase capacity were due to the operational stress, since they were observed also in the sham-operated control animals. All other alterations, the decrease of liver weight and of the capacities of both gluconeogenic and glycolytic key enzymes, were specific for the portocaval anastomosis. The normal periportal to perivenous gradient of phosphoenolpyruvate carboxykinase of about 3.5:1, as measured in microdissected tissue samples, remained the same with specific activities reduced to about 80% each in the two zones. The normal periportal to perivenous gradient of pyruvate kinase L of about 1:1.7 was equalized with levels lowered to 35% and 23%, respectively, in the two zones. The normal periportal to perivenous gradients of glucose-6-phosphatase and succinate dehydrogenase, demonstrated histochemically, were essentially maintained with perivenous bridging occurring transiently at day 4 and 8.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1982

Site directed in-vitro assembly of nucleosomes.

An in-vitro system is described allowing for the assembly of nucleosomes on preselected sites of a cloned tRNA gene. The system consists of a soluble nucleoprotein fraction, a histone source, and circular DNA containing a single stranded stretch (sssDNA). Nucleosomes assemble on the sssDNA, if the three components are incubated as a highly concentrated solution in the presence of the four deoxyribonucleotidetriphosphates and of ATP. The single stranded stretch is rendered double stranded during incubation. The middle axis of one assembled nucleosome always coincides roughly with the midpoint of the original single stranded DNA stretch.

Chromatin↗