Biomedical subjects
Publications and source records attributed to J Huet.
Personal experience with the use of musculocutaneous flaps from pectoralis major confirmed classical documented data. These flaps are reliable in use, they allow wide tumoral excision to be performed, and are therefore perfectly adapted for cervicofacial cancer surgery; however, carcinologic results have to be weighed against the operative procedures necessary.
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Specific antibodies directed against each polypeptide component of yeast RNA polymerases A or B were prepared and their affinity spectrum determined by protein blot immunodetection. The majority of enzyme A or B subunits were specifically recognized by their respective antiserum. A direct correspondence was established between the polypeptides immunologically related in the three forms of RNA polymerases A, B, and C by reacting the different antibodies with enzymes subunits transferred to a nitrocellulose membrane. Subunit-specific antibodies and antibodies to native enzymes A and B were used to probe the activity of RNA polymerases A, B, and C. Based on DNA protection experiments, the largest subunit of enzymes A and B as well as the common subunit ABC23 appear to be involved in DNA binding.
The archaebacterial DNA-dependent RNA polymerases have a complex structure containing eight or more components. Immunochemical analysis shows an extensive homology between the components of the enzymes of nine different species. Two enzyme subtypes can be distinguished: that of the thermoacidophilic and/or sulfur-metabolizing archaebacteria with the composition BACDEFGHIJ and that of the methanogenic plus halophilic archaebacteria with the composition ABB'C(D).... Components B and B' of the latter subtype probably evolved by the division of the large component B of the BACD... type enzyme. The existence of the two subtypes corroborates the division of the archaebacteria into two phylogenetic main branches.
DNA-dependent RNA polymerases of archaebacteria not only resemble the nuclear RNA polymerases of eukaryotes rather than the eubacterial enzymes in their complex component patterns but also show striking immunochemical, i.e., structural, homology with the eukaryotic polymerases at the level of single components. Thus, eukaryotic and archaebacterial RNA polymerases are indeed of the same type, distinct from the eubacterial enzymes, which, however, are also derived from a common ancestral structure.
Incubation with 9-hydroxyellipticine (9-OH-E) decreases the survival of X-irradiated CHO cells. The survival decreases in the case of exponentially growing and plateau-phase cells, although cells in exponential phase of growth are more sensitive to the drug alone. Radiosensitivity increases with the drug concentration and whether the cells are incubated with the drug for 1 hr prior to or immediately after irradiation. 9-OH-E inhibits the repair of potentially lethal damage, but recovery from sublethal radiation damage is suppressed only by high drug concentrations. The interaction between 9-OH-E and X-ray damage and repair has been examined. 9-OH-E itself induces DNA single-strand breaks or alkali-labile sites which are repaired by the cells. When drug-incubated cells are X irradiated, a repair-inhibiting action of the drug is observed.
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Monoclonal antibodies were raised in mouse against native RNA polymerase A from Saccharomyces cerevisiae. After screening with the spot-immunodetection technique, 14 hybridomas were selected and the antibodies produced in mice. Their specificity, analyzed by blot-immunodetection, was found to be markedly biased towards a few RNA polymerase subunits: A135 , A49 , A43 , and A14.5. A different monoclonal antibody directed against the largest subunit, A190 , was obtained by immunizing a mouse with RNA polymerase A dissociated into its subunits with SDS. Two antibodies, which probably recognized the same antigenic determinant on subunit A135 , inhibited in vitro RNA synthesis. Inhibition was prevented by preincubation of the enzyme with DNA, suggesting a role for the A135 subunit in template binding. The antibody directed against A14.5 interacted with the A14.5 kd subunit present in all three forms of the yeast nuclear RNA polymerases but did not interfere with RNA polymerase activity. These antibody probes will be useful to study subunit function in reconstituted transcription systems.
Antisera were raised against native RNA polymerases A or B, as well as against each individual subunit of RNA polymerase A from the yeast Saccharmoyces cerevisiae. The affinity spectrum of antibodies was evaluated by reacting electrophoretically separated enzyme subunits, transferred to a membrane, with 125I-labeled immunoglobulins. Alternatively, the subunit . immunoglobulin complex was revealed by 125I-labeled Protein A. Antibodies directed against native RNA polymerase A recognized the majority of the polypeptides forming the enzyme. When challenged with RNA polymerases B or C, this antibody preparation demonstrated the presence of polypeptides common to the three enzymes. A small cross-reaction was also found at the level of the large subunits of Enzyme B as well as some additional polypeptides of Enzyme C. Similar experiments with antibodies directed against native RNA polymerase B confirmed the presence of common subunits and also showed that the large polypeptides of the three enzymes share a few immunological determinants. Common subunits are AC40, ABC27, ABC23, AC19, and ABC14.5. Immunologically related sites were conserved in the large subunits of RNA polymerase A from remote yeast species. Similarly, yeast and wheat germ RNA polymerase B share immunological determinants on the large subunit as well as on a small peptide. On the other hand, there was no significant cross-reaction between yeast and mammalian Enzyme B or Escherichia coli RNA polymerase. Antibodies raised against the different polypeptide components of RNA polymerase A reacted specifically with the corresponding subunits. Inhibition studies with these subunit-specific antibodies showed that the common subunits are not always similarly exposed to antibody attack within the three enzymes. The data are discussed in terms of the structural similarity, organization and evolution of eukaryotic RNA polymerases.
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Two ribonuclease H activities have been found in yeast RNA polymerase A. The nuclease activities comigrated with subunits A49 (Mr = 49,000) and A40 (Mr = 40,000), after electrophoresis in a sodium dodecyl sulfate polyacrylamide gel containing [32P](rG)n . (dC)n as substrate. Both activities were also found, among other nucleases, in a high salt chromatin extract. Several lines of evidence suggest that the chromatin RNase H of 49,000 daltons (RNase H49) is the same protein as subunit A49. They co-migrate on sodium dodecyl sulfate-gel electrophoresis, have the same chromatographic properties, and dissociate simultaneously from RNA polymerase A. Fractions containing RNase H49 stimulate RNA synthesis by RNA polymerase A* lacking A49 and A34.5 subunits. Finally, limited proteolysis of the protein band having RNase H49 activity yields the characteristic fingerprint of the A49 subunit. This subunit, therefore, exists in two states: bound to chromatin and associated with RNA polymerase A. On the other hand, it is not yet clear whether the RNase H activity of 40,000 daltons, associated with RNA polymerase A, is due to the A40 subunit or whether it represents a trace contamination by a very active nuclease tightly bound to the enzyme.
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Two forms of yeast RNA polymerase A are resolved by phosphocellulose chromatography. One of these, called RNA polymerase A, is lacking two polypeptide chains of 48,000 and 37,000 daltons. The properties of the two enzymes are compared in the present paper. RNA polymerase A transcribes d(A-T)n with a similar efficiency as the complete enzyme, but it is comparatively much less active with native DNA. The two enzymes can also be differentiated on the basis of their ionic strength and divalent cation requirements. RNA polymerase A has a particularly low activity at high salt and low Mg2+ concentrations. Thermal inactivation curves of the two enzymes are different when residual activity is assayed with native DNA. In contrast with d(A-T)n as template the apparent inactivation curves of the two enzymes are identical. The data suggest that the two dissociable polypeptide chains play an important role in transcription. The template specificity of yeast RNA polymerase B was further investigated using SV40 DNA-FI as template. RNA polymerase B is able to retain [3H]SV40 DNA-FI on nitrocellulose filters but the enzyme-DNA complex is very unstable. The observation that RNA polymerase B can transcribe to some extent a supercoiled DNA but not a linear double stranded template supports the hypothesis that the enzyme needs some unpaired DNA structure to initiate transcription.
1. Mitochondria from Candida utilis CBS 1516 and Sacchromyces cerevisiae JB 65 possess an ATPase-inhibitor activity. The inhibitor activity depends on the growth conditions of the yeast cells. It is markedly decreased when the cells are grown in the presence of a high concentration of glucose, which suggests that glucose represses the synthesis of the ATPase inhibitor or of a protein required for the insertion of the inhibitor into the inner mitochondrial membrane. 2. The ATPase inhibitor has been isolated from D. utilis mitochondria and purified to homogeneity. The minimal molecular weight calculated from amino acid composition is close to 7500. Dtermination of the molecular weight by sokium dodecylsulfate-polyacrylamide gel electrophoresis gives a value close to 6000. 3. The ATPas inhibitor of C. utilis mitochondria differs from the beef heart ATPase inhibitor by a number of properties. It has a lower molecular weight (6000-7500 vs 10500), a different amino acid composition, and a more acidic isoelectric point 5, 6 vs 7, 6). In spite of these differences, the C. utilis inhibitor cross-reacts with the ATPase of beef heart submitochondrial inhibitor-depleted particles. 4. The interaction of the C. utilis inhibitor with the ATPase of inhibitor-depleted particles requires the addition of Mg-2+-ATP or ATP in the incubation medium. 5. 14-C labelling of the C.utilis inhibitor has been achieved by growing C. utilis in a medium supplemented with [14-C]leucine. It has been found by titration experiments that the C. utilis 14-C-labelled inhibitor binds to the homologous submitochondrial inhibitor-depleted particles with a KD of about 10- minus 7 M. The number of binding sites is of the order of 0.1 nmol/mg protein.