Biomedical subjects
Publications and source records attributed to P Hossenlopp.
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DNA-dependent RNA polymerase class C (or III) has been solubilized from either uninfected or adenovirus-2-infected HeLa cells and purified by chromatography on phosphocellulose, DNA-cellulose, CM-Sephadex and DEAE-Sephadex. The last column separated the enzyme into three forms CI, CII and CIII, which were completely free of RNA polymerases class A and B and of DNase and RNase. The total and the relative amount of these different enzyme C forms did not vary whether purified from uninfected or infected cells. Irrespective of the stage of purification, the three enzyme forms transcribed deproteinized adenovirus-2DNA very efficiently. This transcription was highly sensitive to elevated ionic strength (especially in the presence of Mg2+) and was accompanied by continuous reinitiation as shown by adding poly(rI), a potent inhibitor of initiation. In addition heparin-resistant initiation complexes could be formed at elevated temperature. The RNA synthesized in vitro on deproteinized intact adenovirus-2 DNA by the different forms of RNA polymerase class C, has been characterized. Analysis of the transcripts by gel electrophoresis, RNA self-annealing, hybridization to separated adenovirus-2 DNA strands and to restriction endonuclease (BamHI, HindIII), adenovirus-2 DNA fragments have demonstrated that restriction endonuclease (BamHI, HindIII), adenovirus-2 DNA fragments have demonstrated that the various regions of the adenovirus-2 genome were randomly transcribed. In addition, hybridization of RNA transcripts labelled at their 5' end by either [gamma32P]ATP or [gamma-32P]GTP indicated that not only elongation but also initiation occurred randomly through the entire adenovirus-2 genome, irrespective of the form of the enzyme and of the origin of the cells (normal or infected). The results are discussed in terms of the components which are possibly involved in specific transcription.
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DNA-dependent RNA polymerase was solubilized from normal and adenovirus-2 infected HeLa cells. Multiple peaks of enzyme activity were separated by DEAE-Sephadex chromatography. In addition to class A and B enzyme activities (respectively insensitive and sensitive to inhibition by 10 nM alpha-amanitin), three peaks of class C enzyme activity were found which are sensitive to inhibition by alpha-amanitin only at much higher concentrations (0.1 mM). Rechromatography of these class C peaks indicates that they are not chromatographic artifacts. Class C enzymes differ from class A and B enzymes by several criteria including inhibition by alpha-amanitin, immunological properties, and the ability to transcribe native calf thymus DNA at high ionic strength. However, the ionic strength optimum and the divalent cation requirements of class C enzymes are not invariant characteristics of the enzymes and are markedly dependent on the nature and the amount of template in the reaction. No differences, either qualitative or quantitative, were found between the multiple enzymes isolated from normal or adenovirus-2 infected cells. All of the partially purified HeLa cell RNA polymerases were able to transcribe an intact double-stranded adenovirus-2 DNA under conditions where no transcription occurred with purified calf thymus AI and B RNA polymerases. Since the multiple enzymes were devoid of endonuclease and exonuclease activities, the ability of the partially purified enzymes to transcribe adenovirus-2 DNA cannot be ascribed to initiation of RNA synthesis at nicks of single-stranded regions of the DNA. No differences in transcriptional ability between corresponding enzyme classes from normal or infected cells, but a comparison of the ability of the various enzyme classes to transcribe intact viral DNA revealed large differences. Although partially purified HeLa class A and B enzymes were able to initiate on the intact viral DNA to a limited extent only, it appears that the class C enzymes transcribe intact duplex DNA much more efficiently than any other class of eukaryotic polymerase yet reported.
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Rat liver explants in culture release into the culture medium IGF and their carrier which form a complex with an apparent molecular weight of similar to or approximately 40,000. With gel filtration in acetic acid the IGF are separated from their carrier and each can then be measured by radioligand assay. In terms of their hormonal control "in vitro", GH, insulin and cortisol have an independent action on each. Pituitary and various brain tissues in organ culture also produce IGF and their carrier. The "in vitro" regulation of the IGF and their carrier has been investigated through assays of their levels in human serum under normal and pathological conditions, such as hypopituitarism, acromegaly, coeliac disease, Cushing's syndrome and idiopathic short or tall stature.