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The c-Myb protein is a sequence-specific DNA binding protein that activates transcription in hematopoietic cells. Three imperfect repeats (R1, R2, and R3) that contain regularly spaced tryptophan residues form the DNA binding domain of c-Myb. A fragment of c-Myb that contained the R2 and R3 regions bound specifically to a DNA sequence recognized by c-Myb plus ten additional base pairs at the 3' end of the element. The R2R3 fragment was predicted to contain two consecutive helix-turn-helix (HTH) motifs with unconventional turns. Mutagenesis of amino acids in R2R3 at positions that correspond to DNA-contacting amino acids in other HTH-containing proteins abolished specific DNA binding without affecting nonspecific DNA interactions.

A new method for tritiating indole derivatives was studied. The procedure consisted first of the formation of an o-nitrophenylsulfenyl tryptophanyl derivative by reacting the compound with o-nitrophenylsulfenyl chloride; then the sulfenylated product was submitted to catalytic hydro-genolysis in the presence of tritium gas. This latter reaction led to the desulfenylation of the compound and permitted the replacement of the thioether function by a tritium atom. By this procedure l-tryptophan, N-acetyl-l-tryptophanamide and a gastrin-like pentapeptide derivative, Boc-Gly-Trp-Met-Asp-Phe(NH2), have been tritiated; 15, 16 and 7 Ci/mmol have been obtained respectively. The labelled compounds have retained the properties of the native materials.

An improved high yield, racemization free synthesis of 85% 13C(U)-L-Asparagine by β- carboxamide formation on 13C(U) enriched L-Aspartic acid is reported.

The four disulfide bonds of nine homologous short curare-like polypeptides are cleaved by reduced dithiothreitol. Air oxidation renaturations of the reduced compounds are followed by far-ultraviolet circular dichroism analysis, and the kinetics of refolding thus determined are compared. They indicate that three toxins refold 4--10 times more slowly than the six others. It is shown that a significant difference between the refolding kinetics still subsists when renaturations are made in the presence of various concentrations of thiol-disulfide exchange reagents or at various pH values. From an examination of the toxin sequences, it is proposed that a single additional amino acid insertion is responsible for the difference in the observed kinetics. This proposal is supported by temperature studies of renaturation kinetics.

The luminescence properties (fluorescence and phosphorescence) of luliberin have been investigated at liquid-nitrogen temperature (77 K) in 50% ethylene glycolaqueous buffer at various pH's. Calculation of the energy-transfer efficiency between Trp and Tyr residues leads to an evaluation of the distance separating these residues. In alkaline medium, when tyrosine is ionized, a 100% transfer efficiency occurs from Trp to Tyr- at the singlet level and also from Tyr- to Trp at the triplet level indicating that the TRP-Tyr distance is less than about 5A. When luliberin is at pH 7.8 (or 4.5), transfer efficiency from Tyr to Trp at the singlet level is 85-90% which should correspond to a distance between Trp and Tyr of about 10-12 A. These results are discussed with respect to the role played by aromatic amino acids both in the hormone conformation and in the hormone biological potency. Moreover, comparison with the data obtained from fluorescence or circular dichroism studies at room temperature allows us to deduce some characteristics of luliberin conformation. Structure-activity relationships in the aromatic region of luliberin are also discussed.

The subunits of yeast RNA polymerases A(I) and B(II) were characterized using several techniques. The present studies demonstrate that the A and B enzymes possess three subunits, which are indistinguishable on the basis of molecular weight, isoelectric point, and fingerprint pattern. The three common subunits belong to the small molecular weight components of the enzymes. By polyacrylamide gel electrophoresis with sodium dodecyl sulfate they migrate with apparent molecular weights of 27,000, 23,000, and 14,500, respectively. A two-dimensional subunit mapping technique on polyacrylamide gel was used to separate the subunits according to isoelectric point and molecular weight. The common polypeptides co-migrated on three spots corresponding to isoelectric points of 9.2 (27,000), 4.5 (23,000), and 4.6 (14,500). The fingerprints of the 35S-labeled tryptic peptides of the presumptive common subunits were found to be essentially identical. Finally, the presence of common subunits was supported by the fact that antibodies against pure RNA polymerase A cross-react with and inhibit RNA polymerase B. Except for the common subunits, it is likely that RNA polymerases A and B are primarily made of distinct gene products for the following reasons. A total of 13 polypeptide chains are present in enzyme A, whereas 10 polypeptides are found in enzyme B. The molecular weight, isoelectric point, and sulfur content of the majority of these polypeptide chains are different in the two enzymes. No similarity was found in the 35S-peptide fingerprint from a number of A and B subunits of slightly different molecular weight. Finally, antibodies against the largest subunit from RNA polymerase A do not cross-react with or inhibit RNA polymerase B. The data are discussed in terms of structural organization of eukaryotic RNA polymerases.

A method was devised for directing RNA polymerase on a single promoter site on T7 DNA. Initiation complexes were formed on each of the three main promoter sites using one dinucleotide plus one nucleoside triphosphate. The ternary initiation complexes are resistant to rifampicin action, to inhibition by (rI)n at 0 degrees C and are stable at high salt concentrations. A minimum of a trinucleotide is required to form a stable ternary complex. To determine which promoter site was selected by RNA polymerase during initiation, the (rI)n-resistant RNA was digested by RNAse III to generate three characteristic initiator RNA fragments, resolved by gel electrophoresis. The three major promoter sites could be selected individually by using different primer and substrate combinations ApC plus ATP selected promoter A3, CpG plus CTP selected A2 and CpC plus ATP specified preferentially A1. A number of primer-substrate combinations specified each site at low salt concentration but the substrate requirement became very stringent at high salt concentration, suggesting that the postulated local opening of the promoter site could be more or less extensive, depending on the ionic strength. The minimum opening observed at high salt concentration corresponded to the insertion of a leader trinucleotide sequence. The promoter region melted by RNA polymerase at low salt concentration was (G plus C)-rich and corresponded to about 9 to 11 base pairs. Sequences of the melting recognition regions were tentatively inferred from the results.

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.

The diastereoisomeric pairs of cyclodipeptides cis- and trans-cyclo(Ala-Ala), cyclo(Ala-Phe), cyclo(Val-Val) and cyclo(Leu-Leu) containing 85% 13C enriched amino-acid residues were synthesized and their 13C-13C coupling constants were measured. The combination of 13C-13C and 1H-1H coupling constants enabled to estimate unequivocally the side chain conformation of the valine and leucine residues.

A new RNA-dependent ATPase has been isolated from yeast chromatin extracts and partially characterized. The protein has a sedimentation coefficient of about 7 S. The enzyme hydrolyzes specifically ATP (or dATP) to ADP (or dADP) and Pi in the presence of Mg2+ or Mn2+ ions and requires a single-stranded polynucleotide as cofactor. The order of efficiency of synthetic polymers is poly(rU) > poly(rI) greater than or equal to poly(dU) > poly(rA) greater than or equal to poly(rC). Among natural polymers, single-stranded DNA and poly(rA)-containing mRNA from yeast are also active but less so than poly(rU). The enzyme exhibits a pH optimum of 8 and is fully inhibited by 0.25 M NaCl. The Km for ATP is0.2 mM. The resemblance between this ATPase and DNA-dependent ATPases from other sources, as well as the termination factor rho, is discussed.

The pattern of restriction DNA fragments transferred from agarose gel to nitrocellulose membranes (Southern's technique) can be affected by the salt concentration of the transfer solvent.

GH3 cells which had been exposed for 30 min to [3H]-TRH and had performed their maximum biological response, i.e. increase of prolactin (PRL) release, retained intracellular radioactivity which consisted of chemically unmodified TRH (>93%). Such preloaded GH3 cells were found able to spontaneously release into the culture medium a radioactive material [3H]-RM) which was analyzed. The kinetics of binding of [3H]-RM to intact GH3 cells and competition with unlabeled TRH were indistinguishable from [3H]-TRH. [3H]-RM was able to stimulate PRL release from GH3 cells. In addition thin layer electrophoresis (TLE) revealed that 90% of [3H]-RM migrated like TRH reference. All these features strongly suggest that [3H]-RM is identical to [3H]-TRH.

The 13C chemical shifts of several 85% 13C-enriched amino acids and small peptides were studied as a function of pH. The results show that the chemical shifts of carbon atoms of ionizable groups vary significantly within the zone of their pK. Generally with the pH going from 7 to 1 all the δC are shifted more or less upfield with the exception of the carbonyl group of the second last residue which is shifted slightly downfield. This suggests the formation of an hydrogen bond at acid pH involving in a seven-membered ring the C=O in question and the COOH terminal. The percentage of cis and trans conformers of glycyl-l-proline and glycyl-l-prolylglycine were studied as a function of pH. The trans form is always preponderant whatever the pH. The accessibility of the carbonyl group to protonation of the proline residue strongly influences the cis-trans equilibrium. Thus, with the pH varying from 7 to 1, the trans isomer changes from 61 to 85% for glycyl-l-proline and only from 77 to 80% for glycyl-l-prolylglycine. The proton NMR studies underline the important differences existing between the two molecular forms of glycyl-l-proline. The cis conformation is characterized with regard to the trans form by the non-equivalence of the α-protons of the glycine residue, by a lower pK1 and by a larger ΔδHα of the proline residue as a function of pH. These results could suggest an end-to-end interaction in the cis form of the glycyl-l-proline molecule. The 13C-13C coupling constants were also studied as a function of pH. The results show that JCo-Cα of a C-terminal residue, varying from 5 to 6 Hz and reflecting the pK of the carboxylate group, is a linear function of δCo and δCα as in the case of the amino acids. The total variation of the electron density of those two carbons in an amino acid is approximately 40% weaker than in a C-terminal residue. The charge distribution along the Cα−Co bond, however, is practically the same in both cases. Finally the ratios of the conversion rate constants of the two isomers cis-trans of glycyl-proline were calculated at different pH values; the relations between the isomer percentages and δCo, δCα on the one hand and the JCo-Cα on the other were established.

Iodinated peptides or proteins (125I, 131I) are used as tracers for radioimmono assays and for detection in biological systems. The labelling takes place on the aromatic side chains. However, when disulfide bridges are present, iodination must be carried out under conditions preventing their degradation. The rate of iodination of L-tyrosine, L-histidine and L-histidine containing peptides by iodine monochloride (ICI) are studied by spectroscopy. Free hitidine increases the hydrolysis rate of aqueous solution of ICI. The interactions between ICI and some simple disulfide are investigated by circular dichroism measurements. These reactions are presented as models for degradation of disulfide bridges in peptide or protein during the iodination process.

Yeast RNA polymerase A (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) can be converted to a new form of enzyme, called RNA polymerase A*, which is lacking two polypeptide chains of 48,000 and 37,000 daltons. Apart from these two missing polypeptides the subunit structures of RNA polymerases A and A* are indistinguishable. RNA polymerase A* differs from the complete enzyme in its electrophoretic and chromatographic behavior, template requirements, and alpha-amanitin sensitivity. RNA polymerase A* transcribes the alternated copolymer d(A-T)n with the same efficiency as RNA polymerase A but its specific activity is greatly reduced with native calf thymus DNA as template. The transcription of a variety of synthetic templates is also altered by removal of the two polypeptide chains. RNA polymerase A* is inhibited by high concentrations of alpha-amanitin (500 mug/ml), whereas RNA polymerase A is comparatively less sensitive to the toxic peptide. The data are discussed in terms of possible roles of the two dissociable polypeptides.

Cytoplasmic elongation factor 1 alpha (EF-1 alpha) was purified to homogeneity from the yeast Saccharomyces cerevisiae using a large-scale procedure. The three steps of purification used were batch adsorption on phosphocellulose, phosphocellulose chromatography and, as the last step, GDP-Sepharose or Biorex column chromatography. The protein is very basic (pI = 9.2) and has an apparent molecular mass of 49 kDa, as determined by polyacrylamide gel electrophoresis using denaturing conditions. It is one of the most abundant proteins in yeast (about 5% of total soluble protein), as shown by two-dimensional gel electrophoresis and by immunological titration. A strong immunological and structural homology was found between yeast EF-1 alpha and elongation factors from other sources. Common immunological features were found between yeast and wheat germ EF-1 alpha. Tryptic hydrolysis of yeast EF-1 alpha in the presence of 25% glycerol generated a large trypsin-resistant polypeptide (Mr = 43,000) which had the same NH2-terminal sequence as the proteolyzed product from rabbit reticulocyte, Artemia salina EF-1 alpha and Escherichia coli EF-Tu. Completed DNA sequence determination of one structural gene for yeast EF-1 alpha confirmed a remarkable conservation of several protein sequence domains in yeast and animal EF-1 alpha (Cottrelle, P., Thiele, D., Price, V., Memet, S., Micouin, J.Y., Marck, C., Buhler, J.M. Sentenac, A., and Fromageot, P. (1985) J. Biol. Chem. 260, 3090-3096).

Summary 3H-gastrin I, 45 Ci/mmole, has been obtained by a procedure including the preparation of intermediary iodinated gastrin I. 3H-gastrin I and mono-iodo gastrin I exhibited the same activity towards stimulation of gastric HCl secretion and the same potency in radioimmunological assays as the parent hormone. By contrast, di-iodo gastrin I showed a 2/3 reduction in its effect on HCl secretion, whereas its immunological characteristics were not altered.

Two ribonucleases H, which specifically degrade RNA in RNA-DNA hybrid structures, have been extensively purified from extracts of Saccharomyces corevisiae. Ribonuclease H1 can be obtained in high yield by a rapid purification procedure mainly involving ammonium sulfate fractionation and phosphocellulose chromatography. The enzyme preparation was shown to be homogeneous by acrylamide gel electrophoresis with sodium dodecylsulfate. Ribonuclease H2 was also obtained in an essentially homogeneous form by phosphocellulose and Amberlite chromatography. About 60 mg of ribonuclease H1 and 2 mg of ribonuclease H2 were obtained from 100 g of wet cells. Using T7 DNA · RNA, hybrids, the specific activity of ribonuclease H2 was approximately 10-fold higher than that of ribonuclease H1.

Resume Nous avons construit un plasmide qui renferme une sequence de 186 nucleotides codant pour une neurotoxine d'origine animale: l'erabutoxine a du venin du serpent marin Laticauda semifasciata. Outre un codon d'initiation, la partie 5′ de cette sequence comporte une region nucleotidique codant pour un fragment de 20 acides amines hydrophobes. Ce fragment est probablement implique dans le processus de biosynthese de la neurotoxine et plus particulierement dans celui de son excretion. La region 3′ de la sequence qui code pour la toxine s'acheve par un codon d'arret lequel est immediatement suivi par une sequence non traduite de 240 nucleotides environ a l'exclusion du fragment poly(A).

The template specificity of yeast RNA polymerase B (or II) has been investigated using single-stranded homopolymers, helical homopolymers, and alternated copolymers. The enzyme exhibited a remarkable base sequence specificity for single-stranded polynucleotides; only those containing pyrimdine nucleotides were transcribed with (dC)n g (dT)n. Homopolymer pairs were transcribed asymmetrically, the pyrimidine-containing strand acting as template. The best synthetic template was the alternated d(I-C)n copolymer. The results signal a preference by the enzyme for initiating in pyrimidine-rich and (dC)-rich regions of the DNA. The structure of the RNA product was studied with ribonucleases of different specificity, RNases A and Ti, RNase H, and RNase III. It appeared that the dissociation of the RNA product from the template strand was determined by the relative stabilities of the helical structures involved. Net synthesis of RNA was favored by the release of the RNA chain from the template. Among polyribonucleotides, (rC)n was a very efficient template, turnip yellow mosaic virus RNA had some activity, but no other ribopolymer tested was found to have a significant activity. Competition experiments with polyribonucleotides indicated a different specificity with respect to the binding reaction, since (rG)n and (rU)n, which had no template activity, are much better inhibitors of transcription than (rC)n.

A conformational analysis of palladium (II)-thioether (side chains of free amino-acids and of residues) complexes was carried out by circular dichroism and magnetic circular dichroism. Two types of thioethers were studied : those in linear side chains as in L-methionine and those in cyclic side chains as in L-thiazolidine-4-carboxylic acid). In the complexes the carboxylic group of amino-acids Is free of Interaction with palladium at acid pH, as shown by C.D. titration. For the diketoplperazine of the cyclic thioether e.g. cycloglycyl-L-thiazolidine 4-carboxylic acid complexed with Pd (II) a possible correlation appears to exist between the variation of the Cotton effect at 230 nm and a change of conformation (Sy — exo to Sy — endo) of the thiazolidine ring, this occurring by increasing pH from acid to neutral. Magnetic circular dichroism revealed charge transfer (CT) bands at 230 nm having varied Intensities. For the complexes of Pd (II) with amino-acids the smaller the chelate ring the larger is the Faraday Effect. A peptide containing two methionyl residues showed about double the Faraday Effect than a peptide having a single methionine residue.On the other hand, Ion-pair reversed phase chromatography was performed to separate the palladium (II) complexes. By using cetyltrimethylammonium as a counter ion it has been possible to resolve the complexes according to a dynamic ion exchange mechanism.Finally, the optical and chromatographic properties of palladium (II)-thloether peptides complexes seem to be correlated : the larger the magnetic ellipticity, the longer is the retention time.

Yeast transcription factor τ interacts with the A and B blocks of the intragenic promoter of tRNA genes. The structure of τ was investigated by identifying the polypeptide chains specifically complexed to the tRNA3Glu gene. Highly purified factor, obtained by an improved purification procedure, contained several polypeptide chains, four of which (Mr = 145,000, 135,000, 100,000 and 65,000) comigrated with τ-DNA complex by polyacrylamide gel electrophoresis. Antibodies raised against the 145- and 100-kDa components altered the migration of τ-DNA complexes in band shift assays and inhibited tRNA synthesis in a reconstituted transcription system. These components are immunologically unrelated proteins. By UV cross-linking to 32P-body-labeled tDNA followed by extensive DNase treatment, two polypeptides of the same size (145 and 100 kDa) were found to be radioactively labeled. Factor τ, therefore, appears to be a multisubunit DNA-binding protein with two distinct polypeptides contributing to DNA recognition. Limited proteolysis of τ generated a protease-resistant τB (τB) domain that binds solely to the B block. τB-tDNA complexes were recognized by anti-145 IgG and contained a 120-kDa polypeptide that could originate from the 145-kDa component by proteolysis. These results strongly suggest that the 145-kDa polypeptide belongs to τB and is responsible for B block binding.

One gene coding for yeast cytoplasmic elongation factor 1 alpha (EF-1 alpha) was isolated by colony hybridization using a cDNA probe prepared from purified EF-1 alpha mRNA. A recombinant plasmid, pLB1, with a 6-kilobase yeast DNA insert, was found by hybrid selection and translation experiments to carry the entire gene. The nucleotide sequence of the gene with its 5'- and 3'-flanking regions was determined. The 5' and 3' ends of EF-1 alpha mRNA were localized by the S1 nuclease mapping technique. The cloned gene, called TEF1, encodes a protein of 458 amino acids (Mr = 50,071) in a single, uninterrupted reading frame. The amino acid sequence shows a strong homology with several domains of Artemia salina EF-1 alpha cytoplasmic factor, as evidenced by diagonal dot matrix analysis. Protein sequence homology is comparatively much lower with the yeast mitochondrial elongation factor. S1 nuclease mapping of the mRNA, hybridization analysis of chromosomal DNA using intragenic or extragenic DNA probes, and gene disruption experiments demonstrated the existence of two genes coding for the cytoplasmic elongation factor EF-1 alpha/haploid genome. The presence of an intact chromosomal TEF1 gene is not essential for growth of haploid yeast cells.

L’etude comparative en absorption electronique et en dichroisme circulaire magnetique de quinones (naphtoquinone-1,4, juglone, anthraquinone-9,10, chrysophanol et quinizarine) a permis de relier l’existence et le signe des bandes DCM a la presence et a la direction de polarisation de certaines transitions electroniques determinees par d’autres methodes. Ceci nous a conduit a attribuer les transitions electroniques de deux analogues naturels d’interet biologique, la Luteo-skyrine et la Rugulosine. L’etude en DCM des complexes (pigment — ion divalent — acide nucleique) a revele une association forte de Mg++ (ou de Mn2+ ) sur le pigment et une symetrie differente des deux types de complexes.

Resume L'analogue enkephalinergique FK 33-824 (Tyr- d -Ala-Gly-MePhe-Met(O)ol) a ete marque au tritium au niveau de son residu tyrosine. Il possede une haute radioactivite specifique (41 Ci/mmole) et se lie a des sites specifiques de haute affinite. Ces sites correspondent a ceux qui sont reconnus par les opiaces puisque la liaison est inhibee specifiquement par des faibles concentrations de morphine, de levallorphane et de β-endorphine. Les resultats obtenus indiquent que le FK 33-824 [3H] lie preferentiellement les sites de type μ. Les experiences de liaison a l'equilibre et les mesures des cinetiques d'association et de dissociation montrent que le FK 33-824 [3H] s'associe a deux classes de sites dont les affinites sont distinctes a 0° (K d = 1.3 et 5.8 nM) et tres proches l'une de l'autre a 37° (K d = 1.9 nM).

A direct fluorination of the imidazole side chain of thyroliberin using a photochemical method is described. Preparation of 5-fluoro-imidazole-thyroliberin and 2-fluoro-imidazole-thyroliberin was achieved by successive diazocoupling, amination and fluorination. Specifically substituted derivatives were purified and identified at each step. 5-fluoro-imidazole-thyroliberin was found as active as TRF on short-term prolactin release in GH3 cell lines and more active (4.5 times) than the parental peptide on long-term experiments. 2-fluoro-imidazole-thyroliberin biological activity test shall be delayed since its chemical stability is not known precisely.

Yeast RNA polymerase C purified by a simple large scale method was resolved into multiple components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific antibodies directed against each polypeptide chain were prepared in rabbits and used as structural and functional probes. With minor exceptions, each antibody recognized specifically the corresponding polypeptide by blot-immunodetection. Cross-reactions with purified RNA polymerases A and B confirmed our previous description of the subunits shared by the three nuclear RNA polymerases. Immunoadsorption of RNA polymerase C at different stages of purification using antibodies to subunits C160 and C128 yielded the same collection of polypeptides as found in the purified enzyme: C160, C128, C82, C53, C40, C37, C34, C31, C27, C25, C23, C19, C14.5, C12.5, and C10. Subunit-specific antibodies were used to probe the activity of RNA polymerase C in a specific, reconstituted transcription system as well as on a nonspecific template. Transcription of the tRNAGlu3 gene in vitro was inhibited when RNA polymerase C was preincubated with antibodies directed to C128, C82, C53, C34, C23, or C19. Antibodies to C82, C53, and C34 were much less inhibitory in the nonspecific assay. Inhibition by anti-C128 or anti-C23 was relieved by preincubation of enzyme C with plasmid DNA prior to antibody addition. These results are discussed in terms of the participation of these polypeptides to the active enzyme molecule, and of their possible role in DNA binding or transcription factor recognition.

A lambda gt11 yeast genomic library was screened with antibodies directed against yeast RNA polymerases A, B, and C. Thirty-five individual recombinant phages that expressed proteins in Escherichia coli that were antigenically related to RNA polymerases A, B, or C were isolated by using 22 distinct antisera. Thus, all 22 genes for the RNA polymerase subunits were potentially cloned. In three cases (lambda A-43, lambda A-40, and lambda A-34.5), an antigenic protein was expressed in E. coli with the same molecular weight as the corresponding subunit. When lambda A-40 DNA was used to hybrid-select yeast mRNA, the protein translated in vitro was the expected size for the A-40 subunit, further supporting our isolation of the A-40 gene. However, mRNA hybrid selected by lambda A-27 DNA did not code for a protein of the correct size. The lengths of the mRNA that hybridized to phage lambda A-190 or lambda C-160 DNA on RNA blots were in agreement with the predicted sizes of the coding regions of the corresponding genes. As predicted by our previous immunological results, yeast DNA inserts of the lambda A-190 and lambda C-160 clones cross-hybridized to the B-220 subunit gene. The cloned genes for the RNA polymerase subunits will prove to be valuable tools for the study of the function, regulation, and genetics of the yeast RNA polymerases.

Snake curaremimetic toxins are known to bind to the nicotinic acetylcholine receptor (AcChoR) [Changeux et al. (1970) Proc. Natl Acad. Sci. USA, 67, 1241-1247], thus blocking neuromuscular transmission, and producing respiratory failure in mammals. In the present paper we show that the toxic effects of Naja nigricollis toxin alpha to mammals can be efficiently reversed by toxin-alpha-specific antibodies. In vivo we observed that return to normal breathing in toxin-alpha-intoxicated and ventilated rats was 12 times faster after injection of specific antiserum or monoclonal antibody (M-alpha 1) as compared with control animals. Ex vivo we observed that return to normal contraction of a toxin-alpha-blocked phrenic nerve-hemidiaphragm preparation was 14 times more rapid after treatment with specific antiserum than after washings. In vitro we observed that antibodies accelerated the reversal of binding of [3H]toxin alpha to AcChoR prepared from rat diaphragm. The observation made in vitro furthermore indicates that antibodies are capable of destabilizing the [3H]toxin-AcChoR complex. A similar destabilization phenomenon occurs also in vivo, as inferred from measurements of receptor occupancy by [3H]toxin alpha in diaphragm of anaesthetized rats in the presence or absence of antibodies. The property of antibodies to reverse neurotoxin binding to AcChoR may be considered as a critical test for evaluation of the quality of a neurotoxin-specific antisera.

Les neurotoxines courtes des venins des cobras sont des polypeptides chimiquement et structuralement homologues constitues de 60 a 62 acides amines et quatre liaisons disulfures. La rupture de ces dernieres, par un agent reducteur par exemple, provoque une desorganisation de l'architecture native des neurotoxines. La reaction inverse est spontanee et consiste en un reploiement de la chaine polypeptidique accompagne par une reoxidation des groupements thiols.Afin de savoir si les substitutions d'acides amines qui interviennent d'une toxine a l'autre modifient ou non le processus de reploiement de ces polypeptides homologues, nous avons etudie, par dichroisme circulaire, les cinetiques de renaturation de huit neurotoxines dans des conditions experimentales identiques. Les resultats auxquels nous sommes parvenus montrent que deux neurotoxines (l’erabutoxine b et la cobrotoxine), se renaturent environ huit fois plus lentement que les six autres. L'analyse des cinetiques en fonction de la temperature indique que les huit toxines etudiees se renaturent avec la meme enthalpie d'activation. L'examen des sequences des toxines suggere que la difference de comportement observee est due a l'insertion en position 19 d'un acide amine supplementaire au sein des sequences de l'erabutoxine b et de la cobrotoxine. Ce residu additionnel est localise au cœur d'une structure coudee qui semble importante pour le deroulement du processus de renaturation des neurotoxines.

The present paper describes the “toxic” site and one topologically distinct antigenic determinant of N. nigricollis toxin α.

Specific methods of tritium labelling onto C-2 and (or) C-5 positions of the imidazole ring were successfully applied to thyroliberin (TRF, L-Glu-L-His-L-Pro-NH2). Diazocoupling by diazotized sulphanilic acid and iodination by iodine monochloride of the histidyl residue were chosen to prepare precursors and to quantify the distribution of tritium atoms which were introduced. Catalytic hydrogenolysis in the presence of tritium gas of 5-iodohistidine2 - and 2(4-sulfophenylazo)histidine2 - derivatives allowed to label these positions with specific radioactivities of 30 Ci and 25 Ci/mmole respectively. For comparison thyroliberin tritiated by direct catalytic exchange is labelled essentially onto C-2 with a specific radioactivity of 30 Ci/mmole.

By using ion-pair reversed-phase chromatography with cetyltrimethylammonium or trioctylmethylammonium as the counter ion, two classes of Pd(II) complexes with completely different properties were distinguished, depending on whether the thioether group is inside a linear moiety (L-methionine and relted peptides, S-ethyl-L-cysteine) or intracyclic (L-thiazolidine-4-carboxylic acid and related peptides). The retention times clearly indicate that in the complexes these ligands interact differently with the metal through their sulphus atom. Fixation of the palladium(II)-S-ethyl-L-cystene complex on the stationary phase is useful for the characterization of D- and L-methionine. Magnetic circular dichroism studies indicated the existence of a charge-transfer band arising from the interaction of the metal with the ligand, the intensity of which varies as a function of the chemical structure of the ligand, either cyclic or linear, and its sequence. There is a good correlation between the intensity of the charge-transfer band and the retention time of the complexes

This paper describes a method for the isolation of RNA polymerase A (or I) from Saccharomyces cerevisiae which is rapid and achieves a 2,000-fold purification. The method involves mainly a batchwise adsorption of enzyme on phosphocellulose and on DEAE-cellulose and a sedimentation on glycerol gradient. The enzyme obtained can be differentiated from RNA polymerase B (or II) by a number of criteria including electrophoretic migration on polyacrylamide gel, sensitivity to α-amanitin, subunit structure, and optimal conditions for RNA synthesis. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate reveals that yeast RNA polymerase A (or I) is made up of two large subunits in equimolar amount of 190,000 and 135,000 daltons and several smaller polypeptide chains, 1 (48,000), 1 (41,000), 2 (29,000), and 2 (16,000). The RNA synthesized on native calf thymus or yeast DNA is constituted of many short chains and of a small percentage (about 10%) of very long chains which represent the bulk of the RNA. Termination factor rho from Escherichia coli inhibits 50% the transcription of native DNA.

Tyrosine derivatives were introduced onto the N-terminal deprotected Pentagastrin to yield precursors of labelling. The tritiated derivative Boc-3,5-3H2-Tyr-Pentagastrin of high specific radioactivity (45 Ci/mmol) was obtained by catalytic dehalogenation of the corresponding di-iodo-derivative in the presence of tritium gas. Iodination of Boc-Tyr-Pentagastrin by NaI and chloramine T led to the formation of several compounds separated by HPLC. Conditions to obtain the mono-iodinated non-oxidized peptide, the only biologically active derivative, were investigated. Analytical chromatographic behaviour as well as biological activity of the tritiated and of the various iodinated peptides were reported.

A high performance liquid chromatographic (HPLC) method is described for separation of L-methionine and L- methionine dipeptides complexed with palladium (II). Under isocratic conditions, at room temperatures, with the appropriate selection of counter-ion (cetyltrimethylammonium or trioctylmethylammonium), it was possible by ion-pairing reversed phase chromatography to resolve the palladium (II) complexes studied. Stainless steel and polythylene columns were used. The chroma-tograms from both the two different materials made columns indicate about the same ratio of capacity factor of the palladium (II) complexes.

The Saccharomyces cerevisiae mutant rpo B1 produces a DNA-dependent RNA polymerase B defective in RNA synthesis in vitro. RNA polymerase B purified from the mutant is altered both structurally and functionally. The enzyme is defective in the RNA chain initiation and elongation reactions. Enzyme-DNA binding is comparatively much less affected. These enzymological defects in the mutant enzyme are enhanced at elevated ionic strengths. Purified rpo B1 RNA polymerase B is lacking B32 and B16.5 subunits. However, the low activity of the mutant enzyme cannot be accounted for only by the loss of these two polypeptides. Wild type enzyme devoid of B32 and B16.5 subunits can be obtained after a mild urea treatment. This enzyme variant, called RNA polymerase B, does not share the enzymological properties of the mutant RNA polymerase. Immunoprecipitation of the enzyme from crude extracts shows that, in the rpo B1 mutant, a normal amount of RNA polymerase B is synthesized which contains the full complement of subunits. The polypeptide chain altered by the rpo B1 mutation was identified by partial proteolysis with proteinase K in the presence of sodium dodecyl sulfate. The 35S-labeled peptide pattern generated from the B220 subunit of the mutant enzyme differs markedly from the peptide pattern of the wild type subunit. The rpo B1 mutation therefore alters the B220 subunit, suggesting a role for this subunit in RNA chain elongation and in the association of the B32 and B16.5 subunits to the RNA polymerase molecule.

To understand fully interactions between peptides and cellular receptors, peptide side chain conformation must be defined. In many cases the complexity of proton nuclear magnetic resonance (NMR) prevents this but the present work demonstrates this problem can be solved by using 13C enrichment. Selective 13C enrichment of a natural peptide hormone has been achieved by preparing [85% 13C-enriched proline]thyrotropin releasing factor which was examined by 13C NMR spectroscopy at various pH values. Because of the 13C enrichment, one-bonded and three-bonded (vicinal) 13C-13C coupling constants have been determined. The latter vary from 0 to 5 Hz and show bond angle dependence. These data indicate that in this hormone the pyrrolidine ring is not free but fixed in the Cgamma-endo puckered conformation. It has also been possible to assign chemical shift values for a second order 13C NMR spectrum.

Isoelectric focusing in a thin layer of polyacrylamide gel is shown to be a suitable method for the resolution of intermediates trapped during the refolding process of reduced cystine-containing proteins. The method has been applied to the well-characterized snake neurotoxin erabutoxin b. An explanation is offered for the relatively low rate of refolding of this polypeptide.

We have demonstrated that the dissociation of Naja nigricollis alpha-toxin from the two acetylcholine receptor sites [Weber and Changeux, Molec. Pharmac. 10, 1-14 (1974); Rousselet et al., Eur. J. Biochem. 140, 31-37 (1984)], is markedly accelerated by a monoclonal neurotoxin-specific antibody. The dissociation of the toxin occurs in a biphasic manner in the presence of a 900 molar excess of immunoglobulin (with respect to toxin concn). The progress curves are characterized by first-order kinetics. Under these conditions the maximal dissociation rate is achieved as further rate enhancement cannot be induced by exposure to an increased immunoglobulin level. In contrast when a toxin-immunoglobulin complex is incubated with a large excess of receptor, the dissociation kinetics of the complex are not enhanced. The data fit a kinetic model which implicates the existence of a transient ternary complex involving the receptor, the toxin and the antibody.

Tritiation of thyroliberin (L-(Glu-L-His-L-Pro-NH2) starting from the L-[Δ3-Pro3]-TRF, on prolyl and histidyl residues is being dealt with below. By catalytic tritiation of the double bond, followed by iodination of histidyl residue and catalytic deshalogenation in presence of tritium gas, TRF was labelled with a specific radioactivity close to the theoretical value. 3H-[2,5-His2-3,4-Pro3]-TRF retained all biological potencies.