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Introduction: Patients with high-consequence infectious diseases (HCID) are rare in Western Europe. However, high-level isolation units (HLIU) must always be prepared for patient admission. Case fatality rates of HCID can be reduced by providing optimal intensive care management. We here describe a single centre's preparation, its embedding in the national context and the challenges we faced during the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic., Methods: Ten team leaders organize monthly whole day trainings for a team of doctors and nurses from the HLIU focusing on intensive care medicine. Impact and relevance of training are assessed by a questionnaire and a perception survey, respectively. Furthermore, yearly exercises with several partner institutions are performed to cover different real-life scenarios. Exercises are evaluated by internal and external observers. Both training sessions and exercises are accompanied by intense feedback., Results: From May 2017 monthly training sessions were held with a two-month and a seven-month break due to the first and second wave of the SARS-CoV-2 pandemic, respectively. Agreement with the statements of the questionnaire was higher after training compared to before training indicating a positive effect of training sessions on competence. Participants rated joint trainings for nurses and doctors at regular intervals as important. Numerous issues with potential for improvement were identified during post processing of exercises. Action plans for their improvement were drafted and as of now mostly implemented. The network of the permanent working group of competence and treatment centres for HCID (Ständiger Arbeitskreis der Kompetenz- und Behandlungszentren für Krankheiten durch hochpathogene Erreger (STAKOB)) at the Robert Koch-Institute (RKI) was strengthened throughout the SARS-CoV-2 pandemic., Discussion: Adequate preparation for the admission of patients with HCID is challenging. We show that joint regular trainings of doctors and nurses are appreciated and that training sessions may improve perceived skills. We also show that real-life scenario exercises may reveal additional deficits, which cannot be easily disclosed in training sessions. Although the SARS-CoV-2 pandemic interfered with our activities the enhanced cooperation among German HLIU during the pandemic ensured constant readiness for the admission of HCID patients to our or to collaborating HLIU. This is a single centre's experience, which may not be generalized to other centres. However, we believe that our work may address aspects that should be considered when preparing a unit for the admission of patients with HCID. These may then be adapted to the local situations., Competing Interests: The authors have declared that no competing interests exist.

Introduction: The complete inactivation of infectious tissues of large animal carcasses is one of the most challenging tasks in high-containment facilities. Steam sterilization is a method frequently in use to achieve biological inactivation of liquid and solid waste. Objective: This study aims to highlight parameters most effective in creating reproducible cycles for steam sterilization of pig and calf carcasses. Methods: Two pigs or 1 calf were sterilized by running a liquid cycle (n = 3) at 121°C for at least 120 minutes in a pass-through autoclave. To assess the physical and biological parameters, temperature data loggers and biological indicators (BIs) with spores of Geobacillus stearothermophilus (ATCC 7953) were placed at defined positions within animal carcasses. After completion of each cycle, data loggers were analyzed and BIs were incubated for 7 days at 60°C. Results: Initial testing with an undissected pig carcass resulted in suboptimal temperatures at the tissue level with growth on 1 BI. After modifications of the used stainless-steel boxes and by placing the reference probe of the autoclave in the animal carcass, reproducible cycles could be created. A complete inactivation of BIs and a temperature profile of >121°C for at least 20 minutes could be achieved in almost all probed tissues. Conclusion: Only minor modifications in carcass preparation and the used sterilization equipment resulted in effective and reproducible cycles to inactivate large animal carcasses by using a steam autoclave., (Copyright 2021, ABSA International 2021.)

The proton pumping mechanism of cytochrome c oxidase on a molecular level is highly disputed. Recently theoretical calculations and real time electron transfer measurements indicated the involvement of residues in the vicinity of the ring A propionate of heme a3, including Asp399 and the CuB ligands His 325, 326. In this study we probed the interaction of Asp399 with the binuclear center and characterize the protonation state of its side chain. Redox induced FTIR difference spectra of mutations at the site in direct comparison to wild type, indicate that below pH 5 Asp 399 displays signals typical for the deprotonation of the acidic residue with reduction of the enzyme. Interestingly at a pH higher than 5, no contributions from Asp 399 are evident. In order to probe the interaction of the site with the binuclear center we followed the rebinding of CO by infrared spectroscopy for mutations on residue Asp399 to Glu, Asn and Leu. Previously different CO conformers have been identified for bacterial cytochrome c oxidases, and its pH dependent behaviour discussed to be relevant for catalysis. Interestingly we observe the lack of this pH dependency and a strong influence on the observable conformers for all mutants studied here, clearly suggesting a communication of the site with the heme-copper center and the nearby histidine residues.

The aa(3)-type cytochrome c oxidases from mitochondria and bacteria contain a cation-binding site located in subunit I near heme a. In the oxidases from Paracoccus denitrificans or Rhodobacter sphaeroides, the site is occupied by tightly bound calcium, whereas the mitochondrial oxidase binds reversibly calcium or sodium that compete with each other. The functional role of the site has not yet been established. D477A mutation in subunit I of P. denitrificans oxidase converts the cation-binding site to a mitochondrial-type form that binds reversibly calcium and sodium ions [Pfitzner, U., Kirichenko, A., et al. (1999) FEBS Lett. 456, 365-369]. We have studied reversible cation binding with P. denitrificans D477A oxidase and compared it with that in bovine enzyme. In bovine oxidase, one Ca(2+) competes with two Na(+) for the binding, indicating the presence of two Na(+)-binding sites in the enzyme, Na(+)((1)) and Na(+)((2)). In contrast, the D477A mutant of COX from P. denitrificans reveals competition of Ca(2+) (K(d) = 1 microM) with only one sodium ion (K(d) = 4 mM). The second binding site for Na(+) in bovine oxidase is proposed to involve D442, homologous to D477 in P. denitrificans oxidase. A putative place for Na(+)((2)) in subunit I of bovine oxidase has been found with the aid of structure modeling located 7.4 A from the bound Na(+)((1)) . Na(+)((2)) interacts with a cluster of residues forming an exit part of the so-called H-proton channel, including D51 and S441.

A combined electrochemical and FTIR spectroscopic approach was used to identify the vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans which change upon electron transfer and coupled proton transfer. Electrochemically induced FTIR difference spectra of the Tyr-D4-labeled cytochrome c oxidase reveal that only small contributions arise from the tyrosines. Contributions between 1600 and 1560 cm(-1) are attributed to nu8a/8b(CC) ring modes. The nu19(CC) ring mode for the protonated form of tyrosines is proposed to absorb with an uncommonly small signal at 1525-1518 cm(-1) and for the deprotonated form at 1496-1486 cm(-1), accompanied by the increase of the nu19(CC) ring mode of the Tyr-D(4)-labeled oxidase at approximately 1434 cm(-1). A signal at 1270 cm(-1) can be tentatively attributed to the nu7'a(CO) and delta(COH) mode of a protonated tyrosine. Uncommon absorptions, like the mode at 1524 cm(-1), indicate the involvement of Tyr280 in the spectra. Tyr280 is a crucial residue close to the binuclear center and is covalently bonded to His276. The possible changes of the spectral properties are discussed together with the absorbance spectra of tyrosine bound to histidine. The vibrational modes of Tyr280 are further analyzed in combination with the mutation to histidine, which is assumed to abolish the covalent bonding. The electrochemically induced FTIR difference spectra of the Tyr280His mutant point to a change in protonation state in the environment of the binuclear center. Together with an observed decrease of a signal at 1736 cm(-1), previously assigned to Glu278, a possible functional coupling is reflected. In direct comparison to the FTIR difference spectra of the D4-labeled compound and comparing the spectra at pH 7 and 4.8, the protonation state of Tyr280 is discussed. Furthermore, a detailed analysis of the mutant is presented, the FTIR spectra of the CO adduct revealing a partial loss of Cu(B). Electrochemical redox titrations reflect a downshift of the heme a3 midpoint potential by 95 +/- 10 mV. Another tyrosine identified to show redox dependent changes upon electron transfer is Tyr35, a residue in the proposed D-pathway of the cytochrome c oxidase.

Resonance Raman and Fourier transform infrared spectroscopies have been used to study the aa(3)-type cytochrome c oxidase and the Y280H mutant from Paracoccus denitrificans. The stability of the binuclear center in the absence of the Tyr(280)-His(276) cross-link is not compromised since heme a(3) retains the same proximal environment, spin, and coordination state as in the wild type enzyme in both the oxidized and reduced states. We observe two C-O modes in the Y280H mutant at 1966 and 1975 cm(-1). The 1975 cm(-1) mode is assigned to a gamma-form and represents a structure of the active site in which Cu(B) exerts a steric effect on the heme a(3)-bound CO. Therefore, the role of the cross-link is to fix Cu(B) in a certain configuration and distance from heme a(3), and not to allow histidine ligands to coordinate to Cu(B) rather than to heme a(3), rendering the enzyme inactive, as proposed recently (Das, T. K., Pecoraro, C., Tomson, F. L., Gennis, R. B., and Rousseau, D. L. (1998) Biochemistry 37, 14471-14476). The results provide solid evidence that in the Y280H mutant the catalytic site retains its active configuration that allows O(2) binding to heme a(3). Oxygenated intermediates are formed by mixing oxygen with the CO-bound mixed-valence wild type and Y280H enzymes with similar Soret maxima at 438 nm.

NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR) in Arabidopsis. Using the yeast two-hybrid system, we have identified three novel genes, NIMIN-1, NIMIN-2 and NIMIN-3 (NIMIN for NIM1-interacting) that encode structurally related proteins interacting physically with NPR1/NIM1. NIMIN-1 and NIMIN-2 both bind strongly to NPR1/NIM1 via a common binding motif interacting with the C-terminal moiety of NPR1/NIM1, whereas NIMIN-3 interacts with NPR1/NIM1 via the N-terminal part of NPR1/NIM1. In addition, NIMIN-1, NIMIN-2, and NIMIN-3 are able to interact via NPR1/NIM1 with basic leucine zipper transcription factors of the TGA family in a yeast tri-hybrid system. A mutant protein of NPR1/NIM1, npr1-2, which has been shown to be severely impaired in induction of SAR gene expression, failed to bind the NIMIN proteins. The NIMIN genes are expressed in Arabidopsis plants in response to SAR-inducing treatments, and the NIMIN proteins, like NPR1/NIM1, carry functional nuclear localization signals as revealed by expression of fusion proteins in yeast and in transgenic plants. Taken together, these data indicate that the NIMIN proteins, via physical interaction with NPR1/NIM1, are part of the signal transduction pathway leading to SAR gene expression in Arabidopsis.

Heme-copper terminal oxidases use the free energy of oxygen reduction to establish a transmembrane proton gradient. While the molecular mechanism of coupling electron transfer to proton pumping is still under debate, recent structure determinations and mutagenesis studies have provided evidence for two pathways for protons within subunit I of this class of enzymes. Here, we probe the D-pathway by mutagenesis of the cytochrome c oxidase of the bacterium Paracoccus denitrificans; amino acid replacements were selected with the rationale of interfering with the hydrophilic lining of the pathway, in particular its assumed chain of water molecules. Proton pumping was assayed in the reconstituted vesicle system by a stopped-flow spectroscopic approach, allowing a reliable assessment of proton translocation efficiency even at low turnover rates. Several mutations at positions above the cytoplasmic pathway entrance (Asn 131, Asn 199) and at the periplasmic exit region (Asp 399) led to complete inhibition of proton pumping; one of these mutants, N131D, exhibited an ideal decoupled phenotype, with a turnover comparable to that of the wild-type enzyme. Since sets of mutations in other positions along the presumed course of the pathway showed normal proton translocation stoichiometries, we conclude that the D-pathway is too wide in most areas above positions 131/199 to be disturbed by single amino acid replacements.

In higher plants, as-1-like cis elements mediate auxin- and salicylic acid-inducible transcription. Originally found in viral and T-DNA promoters, they are also functional elements of plant promoters activated during the defence response against pathogens. Tobacco bZIP transcription factor TGA1a was the first recombinant protein shown to bind to as-1. cDNAs for two novel tobacco as-1-binding bZIP proteins (TGA2.1 and TGA2.2) were isolated. Revealing a high degree of amino acid identity in the bZIP domain (89%) and the C-terminus (79%), the two TGA2 factors differ remarkably with respect to the length of the N-terminus (170 amino acids in TGA2.1 versus 43 amino acids in TGA2.2). TGA2.1 and TGA2.2, but not TGA1a, interacted with ankyrin repeat protein NPR1, a central activator of the plant defence response. In contrast, TGA2.1 and TGA1a, but not TGA2.2, functioned as transcriptional activators in yeast. Apart from conferring transcriptional activation, the N-terminal domain of TGA2.1 led to reduced in vitro as-1-binding activity and almost completely abolished binding to one half site of this bifunctional element. When being part of a heterodimer with TGA2.2, TGA2.1 was efficiently recruited to a single half site, though double occupancy of the element was still preferred. In contrast, TGA1a preferred to bind to only one palindrome, a feature that was also maintained in heterodimers between TGA1a and TGA2.1 or TGA2.2.

Recent structure determinations suggested a new binding site for a non-redox active metal ion in subunit I of cytochrome c oxidase both of mitochondrial and of bacterial origin. We analyzed the relevant metal composition of the bovine and the Paracoccus denitrificans enzyme and of bacterial site-directed mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca2+ was found, the bacterial wild-type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer. Mutants in Asp-477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme a visible absorption upon Ca addition, which was reversed by Na ions. This spectral behavior, largely comparable to that of the mitochondrial enzyme, was not observed for the bacterial WT oxidase. Further structure refinement revealed a tightly bound water molecule as an additional Ca2+ ligand.

Pathogenesis-related proteins of group 1 (PR-1) are strongly induced in plants by pathogen attack, exposure of the plants to (acetyl)salicylic acid (ASA, SA), and by developmental cues. Functional analysis of the PR-1a promoter identified a region of 139 bp (from -691 to -553) mediating expression of the GUS reporter gene in response to ASA. Inspection of this region revealed two TGACG elements reminiscent of activation sequence-1 (as-1). Recently, as-1 has been reported to be responsive to SA in the context of the CaMV 35S RNA promoter. To address the question of whether the as-1-like sequence may be of functional significance for the expression of the PR-1a gene, gel shift assays were performed with TGA1a, a protein been shown to interact with as-1 in vitro. TGA1a was found to bind to the PR-1a as-1-like sequence with similar specificity and affinity as to as-1. Furthermore, mutations were introduced in the as-1-like sequence in the context of the inducible 906 bp PR-1a promoter which are impaired in binding TGA1a in vitro. Significantly reduced levels of GUS reporter gene activity were obtained with the mutant promoter regions as compared to the wild-type PR-1a promoter in response to all stimuli in transgenic tobacco plants. Yet, mutation of the as-1-like sequence did not abolish induction of reporter gene expression. Taken together, these results suggest that the level of expression of the tobacco PR-1a gene is controlled by an as-1-like sequence motif in the PR-1a upstream region, possibly interacting with a factor related to TGA1a.

The molecular processes concomitant with the redox reactions of wild-type and mutant cytochrome c oxidase from Paracoccus denitrificans were analyzed by a combination of protein electrochemistry and Fourier transform infrared (FTIR) difference spectroscopy. Oxidized-minus-reduced FTIR difference spectra in the mid-infrared (4000-1000 cm-1) reflecting full or stepwise oxidation and reduction of the respective cofactor(s) were obtained. In the 1800-1000 cm-1 range, these FTIR difference spectra reflect changes of the polypeptide backbone geometry in the amide I (ca. 1620-1680 cm-1) and amide II (ca. 1560-1540 cm-1) region in response to the redox transition of the cofactor(s). In addition, several modes in the 1600-1200 cm-1 range can be tentatively attributed to heme modes. A peak at 1746 cm-1 associated with the oxidized form and a peak at 1734 cm-1 associated with the reduced form were previously discussed by us as proton transfer between Asp or Glu side chain modes in the course of the redox reaction of the enzyme [Hellwig, P., Rost, B., Kaiser, U., Ostermeier, C., Michel, H., and Mäntele, W. (1996) FEBS Lett. 385, 53-57]. These signals were resolved into several components associated with the oxidation of different cofactors. For a stepwise potential titration from the fully reduced state (-0.5 V) to the fully oxidized state (+0.5 V), a small component at 1738 cm-1 develops in the potential range of approximately +0.15 V and disappears at more positive potentials while the main component at 1746 cm-1 appears in the range of approximately +0.20 V (all potentials quoted vs Ag/AgCl/3 M KCl). This observation clearly indicates two different ionizable residues involved in redox-induced proton transfer. The major component at 1746 cm-1 is completely lost in the FTIR difference spectra of the Glu 278 Gln mutant enzyme. In the spectrum of the subunit I Glu 278 Asp mutant enzyme, the major component of the discussed difference band is lost. In contrast, the complete difference signal of the wild-type enzyme is preserved in the Asp 124 Asn, Asp 124 Ser, and Asp 399 Asn variants, which are critical residues in the discussed proton pump channel as suggested from structure and mutagenesis experiments. On the basis of these difference spectra of mutants, we present further evidence that glutamic acid 278 in subunit I is a crucial residue for the redox reaction. Potential titrations performed simultaneously for the IR and for the UV/VIS indicate that the signal related to Glu 278 is coupled to the electron transfer to/from heme a; however, additional involvement of CuB electron transfer cannot be excluded.

One of the challenging features of energy-transducing terminal oxidases, like the aa3 cytochrome c oxidase of Paracoccus denitrificans, is the translocation of protons across the cytoplasmic membrane, which is coupled to the transfer of electrons to oxygen. As a prerequisite for a more advanced examination of the enzymatic properties, several amino acid residues, selected on the basis of recent three-dimensional structure determinations, were exchanged in subunit I of the Paracoccus enzyme by site-directed mutagenesis. The properties of the mutated oxidases were analyzed by different methods to elucidate whether they are involved in the coupled and coordinated transfer of protons via two different pathways either to the site of oxygen reduction or through the enzyme from the cytoplasm to the periplasmic side.

Crystals of UDP-N-acetylglucosamine O-acyltransferase (lpxA) from Escherichia coli have been obtained from solutions of sodium/potassium phosphate and dimethylsulfoxide. These crystals belong to the cubic space group P2(1)3 (a = 99.0 A), diffract X-rays to approximately 2.5 A resolution and contain one subunit of the enzyme in the asymmetric unit.

Pathogenesis-related proteins (PR proteins) are a heterogeneous group of proteins which are induced in plants by diverse stimuli, e.g. PR proteins are elicited by pathogen attack in the course of the hypersensitive defense reaction of the plant. To examine the regulation of these genes, the 5'-flanking region of the tobacco (Nicotiana tabacum cv. Wisconsin 38) PR-1a gene up to position -1533 was isolated from genomic DNA by the polymerase chain reaction. Two chimeric gene constructs containing 1533 bp and 906 bp, respectively, of the PR-1a upstream region fused to the GUS reporter gene were stably integrated into the tobacco genome. All primary transformants exhibited induced expression of the reporter gene after infection of the plants with tobacco mosaic virus or treatment with acetylsalicylic acid. In addition, similar expression of the reporter gene was observed in leaves of adult transgenic plants without any prior inductive treatments. To study this phenomenon in more detail, the F1 progeny of independent transgenic lines were monitored during the ontogeny of the plants. In normally developing tobacco plants, strong GUS activities were typically detected approximately 12 weeks after germination in the lowest leaves of vegetative plants. When successive leaves of individual plants were tested during the following weeks, a clear gradient of reporter gene activity had developed in the green leaves including the sepals from the bottom to the top of the plants. In all cases analyzed, this gradient of reporter gene expression was strictly parallelled by the expression of the endogenous PR-1 proteins. These results suggest that the acidic PR-1 proteins from tobacco fulfill a role during the later stages of plant development and that the PR-1a upstream region -906 to -335 contains positive regulatory elements for both environmental and developmental signals.

Tobacco plants containing the N' resistance gene exhibit a hypersensitive defense reaction when infected with tomato mosaic virus (ToMV); infection results in necrotic lesions at the primary infection sites. In an attempt to investigate the molecular mechanism(s) underlying this plant-pathogen interaction, the ToMV coat protein gene was joined by a transcriptional fusion to the strong constitutive 35S RNA promoter from cauliflower mosaic virus. This chimeric gene was introduced via Agrobacterium-mediated transformation into isogenic tobacco cultivars differing only with respect to the N' gene. Strong necrotic reactions were observed on most emerging calli of the N' genotype, but never on calli lacking the N' resistance gene. These data indicate that the coat protein of ToMV is, on its own, sufficient to induce a hypersensitive reaction in tobacco. Thus, recognition of a single viral gene product may be the only prerequisite for the induction of a specific defense reaction in higher plants.

PR-1 genes are induced by various environmental stimuli such as pathogen attack or exposure of the plants to certain chemicals. To examine the regulation of these genes, the 5' flanking regions of the PR-la gene and of two PR-1 pseudogenes were joined by a transcriptional fusion to the Escherichia coli beta-glucuronidase (GUS) gene. These constructs were stably integrated into the tobacco genome and independent primary transformants were monitored for the expression of the reporter gene. Unexpectedly, out of 55 transformants analysed, four plants exhibited considerable GUS activities without any inductive treatment of the plants. Expression of the endogenous PR-1 genes, however, could not be detected in these plants. Primer extension analyses revealed correct initiation of the PR1/GUS hybrid transcripts from the PR-1a TATA box. When the plants were analysed at the cellular level, clear differences regarding the tissue specificity of expression of the reporter gene were observed. These results strongly suggest that the PR1/GUS hybrid promoter expression cassettes may be activated when integrated in the vicinity of heterologous enhancer elements dispersed in the tobacco genome. In order to support this hypothesis, domain B of the enhancer of the 35S RNA promoter from cauliflower mosaic virus (CaMV) was fused to various PR1/GUS hybrid genes upstream as well as downstream from the RNA start site. These constructs were stably introduced into the tobacco genome. In any primary transformant analysed, strong GUS activities were observed with the PR1/GUS hybrid RNAs originating from the normal transcription start site of the PR-1a gene. The tissue specificity of gene expression was identical to that described previously for the CaMV 35S domain B enhancer element. Thus, modulations of the transcriptional activity of the PR-1 promoter can be achieved by heterologous enhancers in transgenic plants and may be encountered upon random integration of PR-1 promoter constructs into the tobacco genome.

Pathogenesis-related (PR) proteins are a heterogeneous group of host encoded, low-molecular-mass proteins that are induced in plants by various external stimuli, such as pathogen attack or exposure of the plants to certain chemicals. To examine the regulation of these genes, the 5'-flanking region of the tobacco PR-1a gene [Pfitzner U.M., Pfitzner, A.J.P. & Goodman, H.M. (1988) Mol. Gen. Genet. 211, 290-295] was joined by a transcriptional fusion to the Escherichia coli beta-glucuronidase (GUS) gene. Expression of the reporter gene was monitored in transient expression assays as well as in stable transformants. The PR-1a 5'-flanking sequences from -335, -149 or -71 to +28 are functional promoter elements in tobacco and carrot protoplasts, as determined by transient expression. These constructs direct correct initiation at the normal transcription-start site of the PR-1a gene. The level of gene expression was about twofold less than that obtained with the cauliflower mosaic virus 35S RNA promoter. Regulation of gene expression by acetylsalicylic acid, however, could not be detected in the transient assays. When the same constructs were stably integrated into the tobacco genome, neither constitutive nor induced beta-glucuronidase activity was observed. A comparison of the results from the transient and the stable transfection experiments suggests that expression of the reporter gene may be due to a constitutive transcriptional activity of the PR-1a 5'-flanking regions under the conditions of the transient assays and that the PR-1a promoter may contain at least two functional domains.

Two independent PR-1 lambda genomic clones (W38/1 and W38/3) were isolated and characterized from a tobacco (Nicotiana tabacum cv. Wisconsin 38) library. Neither clone is identical to the previously described PR-1 cDNA clones, and both clones carry mutations within the highly conserved PR-1 protein coding region. For example, clone W38/1 has a GAA Glu codon instead of the translation stop codon thus harbouring an open reading frame extended by 16 additional amino acids. Furthermore, both clones display considerable variations in the genomic flanking sequences when compared to the PR-1a gene. In order to test whether the encoded genes are active, their upstream sequences were fused to the E. coli beta-glucuronidase (GUS) reporter gene. While significant GUS activities as compared to the 35S RNA promoter from cauliflower mosaic virus (CaMV) were obtained with the W38/1 and W38/3 sequences in transient gene expression assays, no transcriptional activities could be observed upon stable transformation of the same constructs. In addition, the protein coding region of W38/1 was joined to the CaMV 35S RNA promoter and transgenic tobacco plants were generated. However, neither transcripts nor a protein could be detected deriving from the W38/1 structural gene with this chimaeric construct in the transformants. Taken together, these data indicate that the genes contained in lambda clones W38/1 and W38/3 are not active in planta.

Four separable isoforms of Strictosidine synthase, which catalyze condensation of tryptamine with secologanin to form Strictosidine, were purified to homogeneity from cultured cells of CATHARANTHUS ROSEUS and from leaves of C. ROSEUS plants. These enzymes are distinguished by their isoelectric points as well as by their catalytic properties. The specific activity of the main form (isoform III) is 104 nkat/mg, K (cat) = 3.2 s (-1). The relative molecular mass is 31 kDa as estimated by gel filtration, and 41.5 kDa as estimated by SDS gel electrophoresis. The pH-optimum is observed at pH 6.7. The apparent K (M)-value for tryptamine is 1.9 mM (isoform III). Secologanin shows a positive cooperativity with an n (H) of 2.2 (isoform III). Polyclonal antibodies raised against isoform III show cross reactivity against all four isoforms. Furthermore, these antibodies also react with Strictosidine synthases from cell cultures of other species of the family Apocynaceae but not with those of the family Rubiaceae.

Strictosidine synthase was partially purified from Catharanthus roseus cell suspension cultures and immobilized on CNBr-activated Sepharose. The immobilized enzyme exhibits a thermostability increased 300 fold over that of the soluble enzyme and catalyses exclusively the formation of the 3alpha(S)-isomer, strictosidine. Gram quantities of this biologically active glucoalkaloid, which hitherto had been difficult to synthesize and purify, were prepared.

Infection of the tobacco cultivar Samsun NN by tobacco mosaic virus (TMV) results in a hypersensitive response. During this defense reaction several host encoded proteins, known as pathogenesis-related proteins (PR-proteins), are induced. Poly(A)+ RNA from TMV infected tobacco plants was used to construct a cDNA library. Thirty two cDNA clones were isolated and after digestion with different restriction endonucleases, twenty clones were found to code for PR-1a, six clones for PR-1b, and four clones for PR-1c. Two independent cDNA clones of each class were further characterized by DNA sequence analysis. All clones analyzed contained the 138 amino acid coding regions of their respective mature proteins, but only partial sequences of the signal peptides. Minor differences between the nucleotide sequences for clones belonging to the same class were detected. Comparison of the amino acid sequence for PR-1a deduced from its nucleotide sequence with published data obtained by Edman degradation of the protein showed four differences. Analysis of the 3' ends of the cDNA clones indicates that various alternate poly(dA) addition sites are used. Southern blot analysis using these cDNAs as probes suggests the presence of multiple PR-protein genes in the genomes of tobacco and tomato plants.