Biomedical subjects

Hisakazu Yamane

Publications and source records attributed to Hisakazu Yamane.

At least 19 recordsLinked to original sources

PubMed2006

The Sphingomonas plasmid pCAR3 is involved in complete mineralization of carbazole.

We determined the complete 254,797-bp nucleotide sequence of the plasmid pCAR3, a carbazole-degradative plasmid from Sphingomonas sp. strain KA1. A region of about 65 kb involved in replication and conjugative transfer showed similarity to a region of plasmid pNL1 isolated from the aromatic-degrading Novosphingobium aromaticivorans strain F199. The presence of many insertion sequences, transposons, repeat sequences, and their remnants suggest plasticity of this plasmid in genetic structure. Although pCAR3 is thought to carry clustered genes for conjugative transfer, a filter-mating assay between KA1 and a pCAR3-cured strain (KA1W) was unsuccessful, indicating that pCAR3 might be deficient in conjugative transfer. Several degradative genes were found on pCAR3, including two kinds of carbazole-degradative gene clusters (car-I and car-II), and genes for electron transfer components of initial oxygenase for carbazole (fdxI, fdrI, and fdrII). Putative genes were identified for the degradation of anthranilate (and), catechol (cat), 2-hydroxypenta-2,4-dienoate (carDFE), dibenzofuran/fluorene (dbf/fln), protocatechuate (lig), and phthalate (oph). It appears that pCAR3 may carry clustered genes (car-I, car-II, fdxI, fdrI, fdrII, and, and cat) for the degradation of carbazole into tricarboxylic acid cycle intermediates; KA1W completely lost the ability to grow on carbazole, and the carbazole-degradative genes listed above were all expressed when KA1 was grown on carbazole. Reverse transcription-PCR analysis also revealed that the transcription of car-I, car-II, and cat genes was induced by carbazole or its metabolic intermediate. Southern hybridization analyses with probes prepared from car-I, car-II, repA, parA, traI, and traD genes indicated that several Sphingomonas carbazole degraders have DNA regions similar to parts of pCAR3.

Base Sequence↗

PubMed2006

Crystallization and preliminary X-ray diffraction studies of the terminal oxygenase component of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177.

Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular-position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. CARDO consists of a terminal oxygenase component and two electron-transfer components: ferredoxin and ferredoxin reductase. The terminal oxygenase component (43.9 kDa) of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177 was crystallized at 293 K using the hanging-drop vapour-diffusion method with PEG 8000 as the precipitant. The crystals diffract to 2.3 A resolution and belong to space group C2.

Bacterial Proteins↗

PubMed2006

Bacterial lipopolysaccharides induce defense responses associated with programmed cell death in rice cells.

PAMP (pathogen-associated molecular pattern) recognition plays an important role during the innate immune response in both plants and animals. Lipopolysaccharides (LPS) derived from Gram-negative bacteria are representative of typical PAMP molecules and have been reported to induce defense-related responses, including the suppression of the hypersensitive response, the expression of defense genes and systemic resistance in plants. However, the details regarding the precise molecular mechanisms underlying these cellular responses, such as the molecular machinery involved in the perception and transduction of LPS molecules, remain largely unknown. Furthermore, the biological activities of LPS on plants have so far been reported only in dicots and no information is thus available regarding their functions in monocots. In our current study, we report that LPS preparations for various becteria, including plant pathogens and non-pathogens, can induce defense responses in rice cells, including reactive oxygen generation and defense gene expression. In addition, global analysis of gene expression induced by two PAMPs, LPS and chitin oligosaccharide, also reveals a close correlation between the gene responses induced by these factors. This indicates that there is a convergence of signaling cascades downstream of their corresponding receptors. Furthermore, we show that the defense responses induced by LPS in the rice cells are associated with programmed cell death (PCD), which is a finding that has not been previously reported for the functional role of these molecules in plant cells. Interestingly, PCD induction by the LPS was not detected in cultured Arabidopsis thaliana cells.

Adsorption↗

PubMed2006

Differentiation of carbazole catabolic operons by replacement of the regulated promoter via transposition of an insertion sequence.

The carbazole catabolic car operons from Pseudomonas resinovorans CA10 and Janthinobacterium sp. J3 have nearly identical nucleotide sequences in their structural and intergenic regions but not in their flanking regions. Transposition of ISPre1 from the anthranilate catabolic ant operon located an inducible promoter Pant upstream of the carCA10 operon, which is regulated by the AraC/XylS family activator AntR in response to anthranilate. The transposed Pant drives transcription of the carCA10 operon, which is composed of the car-AaAaBaBbCAcAdDFECA10 structural genes. Transcriptional fusion truncating Pant upstream of carAaCA10 resulted in constitutive luciferase expression. Primer extension analysis identified a transcription start point of the constitutive mRNA of the carCA10 operon at 385 nucleotides upstream of the carAaCA10 translation start point, and the PcarAa promoter was found. On the other hand, a GntR family regulatory gene carRJ3 is divergently located upstream of the carJ3 operon. The Pu13 promoter, required for inducible transcription of the carJ3 operon in the presence of carbazole, was identified in the region upstream of carAaJ3, which had been replaced with the Pant promoter in the carCA10 operon. Deletion of carRJ3 from a transcriptional fusion resulted in high level constitutive expression from Pu13. Purified CarRJ3 protein bound at two operator sequences OI and OII, showing that CarRJ3 directly represses Pu13 in the absence of its inducer, which was identified as 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoate, an intermediate of the carbazole degradation pathway.

Base Sequence↗

PubMed2006

Electron transfer complex formation between oxygenase and ferredoxin components in Rieske nonheme iron oxygenase system.

Carbazole 1,9a-dioxygenase (CARDO), a member of the Rieske nonheme iron oxygenase system (ROS), consists of a terminal oxygenase (CARDO-O) and electron transfer components (ferredoxin [CARDO-F] and ferredoxin reductase [CARDO-R]). We determined the crystal structures of the nonreduced, reduced, and substrate-bound binary complexes of CARDO-O with its electron donor, CARDO-F, at 1.9, 1.8, and 2.0 A resolutions, respectively. These structures provide the first structure-based interpretation of intercomponent electron transfer between two Rieske [2Fe-2S] clusters of ferredoxin and oxygenase in ROS. Three molecules of CARDO-F bind to the subunit boundary of one CARDO-O trimeric molecule, and specific binding created by electrostatic and hydrophobic interactions with conformational changes suitably aligns the two Rieske clusters for electron transfer. Additionally, conformational changes upon binding carbazole resulted in the closure of a lid over the substrate-binding pocket, thereby seemingly trapping carbazole at the substrate-binding site.

Bacterial Proteins↗

PubMed2006

Functional and transcriptional analyses of the initial oxygenase genes for acenaphthene degradation from Sphingomonas sp. strain A4.

Sphingomonas sp. strain A4 is capable of utilizing acenaphthene as its sole carbon and energy source. To isolate the genes responsible for acenaphthene degradation, transposon mutagenesis was performed on strain A4 and four mini-Tn5-inserted mutants lacking the ability to utilize acenaphthene were isolated. In three of the four mini-Tn5 inserted mutants, the mini-Tn5s were inserted into the same locus (within about 16 kb) as the arhA1A2 genes, which had previously been identified as the genes encoding the terminal oxygenase components for the initial oxygenation of acenaphthene. The nucleotide sequence analysis of the corresponding 16.4 kb DNA fragment revealed the existence of 16 ORFs and a partial ORF. From these ORFs, the genes encoding the ferredoxin (ArhA3) and ferredoxin reductase (ArhA4) complementary to ArhA1A2 were identified. RT-PCR analysis suggested that a 13.5 kb gene cluster, consisting of 13 ORFs and including all the arhA genes, forms an operon, although it includes several ORFs that are apparently unnecessary for acenaphthene degradation. Furthermore, using gene disruption and quantitative RT-PCR analyses, the LysR-type activator, ArhR, required for expression of the 13.5 kb gene cluster was also identified. Transcription of the gene cluster by ArhR was induced in the presence of acenaphthene (or its metabolite), and a putative binding site (T-N11-A motif) for ArhR was found upstream from the transcription start point of arhA3.

Acenaphthenes↗

PubMed2006

Plasmid pCAR3 contains multiple gene sets involved in the conversion of carbazole to anthranilate.

The carbazole degradative car-I gene cluster (carAaIBaIBbICIAcI) of Sphingomonas sp. strain KA1 is located on the 254-kb circular plasmid pCAR3. Carbazole conversion to anthranilate is catalyzed by carbazole 1,9a-dioxygenase (CARDO; CarAaIAcI), meta-cleavage enzyme (CarBaIBbI), and hydrolase (CarCI). CARDO is a three-component dioxygenase, and CarAaI and CarAcI are its terminal oxygenase and ferredoxin components. The car-I gene cluster lacks the gene encoding the ferredoxin reductase component of CARDO. In the present study, based on the draft sequence of pCAR3, we found multiple carbazole degradation genes dispersed in four loci on pCAR3, including a second copy of the car gene cluster (carAaIIBaIIBbIICIIAcII) and the ferredoxin/reductase genes fdxI-fdrI and fdrII. Biotransformation experiments showed that FdrI (or FdrII) could drive the electron transfer chain from NAD(P)H to CarAaI (or CarAaII) with the aid of ferredoxin (CarAcI, CarAcII, or FdxI). Because this electron transfer chain showed phylogenetic relatedness to that consisting of putidaredoxin and putidaredoxin reductase of the P450cam monooxygenase system of Pseudomonas putida, CARDO systems of KA1 can be classified in the class IIA Rieske non-heme iron oxygenase system. Reverse transcription-PCR (RT-PCR) and quantitative RT-PCR analyses revealed that two car gene clusters constituted operons, and their expression was induced when KA1 was exposed to carbazole, although the fdxI-fdrI and fdrII genes were expressed constitutively. Both terminal oxygenases of KA1 showed roughly the same substrate specificity as that from the well-characterized carbazole degrader Pseudomonas resinovorans CA10, although slight differences were observed.

Bacterial Proteins↗

PubMed2006

Characterization of the replication, maintenance, and transfer features of the IncP-7 plasmid pCAR1, which carries genes involved in carbazole and dioxin degradation.

Isolated from Pseudomonas resinovorans CA10, pCAR1 is a 199-kb plasmid that carries genes involved in the degradation of carbazole and dioxin. The nucleotide sequence of pCAR1 has been determined previously. In this study, we characterized pCAR1 in terms of its replication, maintenance, and conjugation. By constructing miniplasmids of pCAR1 and testing their establishment in Pseudomonas putida DS1, we show that pCAR1 replication is due to the repA gene and its upstream DNA region. The repA gene and putative oriV region could be separated in P. putida DS1, and the oriV region was determined to be located within the 345-bp region between the repA and parW genes. Incompatibility testing using the minireplicon of pCAR1 and IncP plasmids indicated that pCAR1 belongs to the IncP-7 group. Monitoring of the maintenance properties of serial miniplasmids in nonselective medium, and mutation and complementation analyses of the parWABC genes, showed that the stability of pCAR1 is attributable to the products of the parWAB genes. In mating assays, the transfer of pCAR1 from CA10 was detected in a CA10 derivative that was cured of pCAR1 (CA10dm4) and in P. putida KT2440 at frequencies of 3 x 10(-1) and 3 x 10(-3) per donor strain, respectively. This is the first report of the characterization of this completely sequenced IncP-7 plasmid.

Bacterial Proteins↗

PubMed2006

Characterization of novel carbazole catabolism genes from gram-positive carbazole degrader Nocardioides aromaticivorans IC177.

Nocardioides aromaticivorans IC177 is a gram-positive carbazole degrader. The genes encoding carbazole degradation (car genes) were cloned into a cosmid clone and sequenced partially to reveal 19 open reading frames. The car genes were clustered into the carAaCBaBbAcAd and carDFE gene clusters, encoding the enzymes responsible for the degradation of carbazole to anthranilate and 2-hydroxypenta-2,4-dienoate and of 2-hydroxypenta-2,4-dienoate to pyruvic acid and acetyl coenzyme A, respectively. The conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and mononuclear iron, the Rieske-type [2Fe-2S] cluster, and flavin adenine dinucleotide were found in the deduced amino acid sequences of carAa, carAc, and carAd, respectively, which showed similarities with CarAa from Sphingomonas sp. strain KA1 (49% identity), CarAc from Pseudomonas resinovorans CA10 (31% identity), and AhdA4 from Sphingomonas sp. strain P2 (37% identity), respectively. Escherichia coli cells expressing CarAaAcAd exhibited major carbazole 1,9a-dioxygenase (CARDO) activity. These data showed that the IC177 CARDO is classified into class IIB, while gram-negative CARDOs are classified into class III or IIA, indicating that the respective CARDOs have diverse types of electron transfer components and high similarities of the terminal oxygenase. Reverse transcription-PCR (RT-PCR) experiments showed that the carAaCBaBbAcAd and carDFE gene clusters are operonic. The results of quantitative RT-PCR experiments indicated that transcription of both operons is induced by carbazole or its metabolite, whereas anthranilate is not an inducer. Biotransformation analysis showed that the IC177 CARDO exhibits significant activities for naphthalene, carbazole, and dibenzo-p-dioxin but less activity for dibenzofuran and biphenyl.

Acetyl Coenzyme A↗

PubMed2006

Induction of resistance against rice blast fungus in rice plants treated with a potent elicitor, N-acetylchitooligosaccharide.

The mode of action of a potent elicitor, N-acetylchitooligosaccharide, in rice plants was examined. In intact seedlings, no significant uptake of the elicitor via the roots was observed within 3 h, whereas rapid uptake was observed in excised leaves. Rapid and transient expression of an elicitor-responsive gene, EL2, was induced in the leaves of intact seedlings sprayed with the elicitor or in the roots and leaves of intact seedlings by immersing roots in the elicitor solution. Histochemical analysis indicated that EL2 was expressed in cells exposed to the elicitor of root and leaves. In seedlings treated with the elicitor for 1 d or longer, hyphal growth of rice blast fungus was significantly delayed, and an accumulation of auto-fluorescence around the infection site was observed. Two defense-related genes, PR-1 and PR-10 (PBZ1), were induced in a systemic and local manner by elicitor treatment, in correlation with the induction of resistance against rice blast fungus. N-Acetylchitoheptaose did not inhibit the hyphal growth of the fungi. These results indicate the occurrence of systemic signal transmission from N-acetylchitooligosaccharide in rice plants.

Gene Expression Regulation, Plant↗

PubMed2006

Characterization of a rice gene family encoding type-A diterpene cyclases.

We have previously isolated and characterized the rice (Oryza sativa) cDNAs, OsCyc1/OsCPS4, OsCyc2/OsCPS2, OsKS4, OsDTC1/OsKS7, OsDTC2/OsKS8 and OsKS10, which encode cyclases that are responsible for diterpene phytoalexin biosynthesis. Among the other members of this gene family, OsCPS1 and OsKS1 have been suggested as being responsible for gibberellin biosynthesis, OsKSL11 has recently been shown to encode stemodene synthase, and the functions of the three other diterpene cyclase genes in the rice genome, OsKS3, OsKS5 and OsKS6, have not yet been determined. In this study, we show that recombinant OsKS5 and OsKS6 expressed in E. coli converted ent-copalyl diphosphate into ent-pimara-8(14),15-diene and ent-kaur-15-ene, respectively. Neither product is a hydrocarbon precursor required in the biosynthesis of either gibberellins or phytoalexins. OsKS3 may be a pseudogene from which the translated product is a truncated enzyme. These results suggest that the diterpene cyclase genes responsible for gibberellin and phytoalexin biosynthesis are not functionally redundant.

Amino Acid Sequence↗

PubMed2005

Crystallization and preliminary X-ray diffraction analysis of the electron-transfer complex between the terminal oxygenase component and ferredoxin in the Rieske non-haem iron oxygenase system carbazole 1,9a-dioxygenase.

Carbazole 1,9a-dioxygenase, which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. The electron-transport complex between CARDO-O and CARDO-F crystallizes at 293 K using hanging-drop vapour diffusion with the precipitant PEG MME 2000 (type I crystals) or PEG 3350 (type II). Blossom-shaped crystals form from a pile of triangular plate-shaped crystals. The type I crystal diffracts to a maximum resolution of 1.90 A and belongs to space group P2(1), with unit-cell parameters a = 97.1, b = 89.8, c = 104.9 A, alpha = gamma = 90, beta = 103.8 degrees. Diffraction data for the type I crystal gave an overall Rmerge of 8.0% and a completeness of 100%. Its VM value is 2.63 A3 Da(-1), indicating a solvent content of 53.2%.

Bacterial Proteins↗

PubMed2005

Characterization of fructose-bisphosphate aldolase regulated by gibberellin in roots of rice seedling.

Fructose-bisphosphate aldolase is a glycolytic enzyme whose activity increases in rice roots treated with gibberellin (GA). To investigate the relationship between aldolase and root growth, GA-induced root aldolase was characterized. GA3 promoted an increase in aldolase accumulation when 0.1 microM GA3 was added exogenously to rice roots. Aldolase accumulated abundantly in roots, especially in the apical region. To examine the effect of aldolase function on root growth, transgenic rice plants expressing antisense aldolase were constructed. Root growth of aldolase-antisense transgenic rice was repressed compared with that of the vector control transgenic rice. Although aldolase activity increased by 25% in vector control rice roots treated with 0.1 microM GA3, FBPA activity increased very little by 0.1 microM GA3 treatment in the root of aldolase-antisense transgenic rice. Furthermore, aldolase co-immunoprecipitated with antibodies against vacuolar H+ -ATPase in rice roots. In the root of OsCDPK13-antisense transgenic rice, aldolase did not accumulate even after treatment with GA3. These results suggest that the activation of glycolytic pathway function accelerates root growth and that GA3-induced root aldolase may be modulated through OsCDPK13. Aldolase physically associates with vacuolar H-ATPase in roots and may regulate the vacuolar H-ATPase mediated control of cell elongation that determines root length.

Blotting, Western↗

PubMed2005

Diversity of carbazole-degrading bacteria having the car gene cluster: isolation of a novel gram-positive carbazole-degrading bacterium.

Twenty-seven carbazole-utilizing bacterial strains were isolated from environmental samples, and were classified into 14 groups by amplified ribosomal DNA restriction analysis. Southern hybridization analyses showed that 3 and 17 isolates possessed the car gene homologs of Pseudomonas resinovorans CA10 and Sphingomonas sp. strain KA1, respectively. Of the 17 isolates, 2 isolates also have the homolog of the carAa gene of Sphingomonas sp. strain CB3. While the genome of one isolate, a Gram-positive Nocardioides sp. strain IC177, showed no hybridization to any car gene probes, PCR and sequence analyses indicated that strain IC177 had tandemly linked carAa and carC gene homologs whose deduced amino acid sequences showed 51% and 36% identities with those of strain KA1.

Actinomycetales↗

PubMed2005

Leader peptide-mediated transcriptional attenuation of lysine biosynthetic gene cluster in Thermus thermophilus.

The molecular mechanism for regulation of the genes involved in the biosynthesis of amino acids is poorly identified in Thermus thermophilus. In this study, we analyzed the transcriptional control of the major lysine biosynthetic gene cluster in T. thermophilus. S1 nuclease mapping revealed that the transcription, which is repressed by lysine, starts at 111 bp, upstream of the translational start codon, ATG, for the homocitrate synthase (hcs) gene. The 5'-leader region of 111 bp carries a sequence that can encode a short peptide of 14 amino acids with tandem-arranged lysine residues in its sequence. The nucleotide sequence of the region suggests that the transcript can form complicated secondary structures. Deletion of most of the 5'-leader region or mutation of the tandem lysine codons suppressed the transcriptional repression by lysine. Mutation of the tandem codons from lysine to glutamine resulted in glutamine-dependent repression of the gene connected downstream, indicating that the leader peptide mediated the transcriptional attenuation of the gene expression. This is the first report demonstrating the transcriptional regulation of amino acid biosynthesis in T. thermophilus.

Bacillus subtilis↗

PubMed2005

Crystal structure of the ferredoxin component of carbazole 1,9a-dioxygenase of Pseudomonas resinovorans strain CA10, a novel Rieske non-heme iron oxygenase system.

The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 catalyzes the dioxygenation of carbazole; the 9aC carbon bonds to a nitrogen atom and its adjacent 1C carbon as the initial reaction in the mineralization pathway. The CARDO system is composed of ferredoxin reductase (CarAd), ferredoxin (CarAc), and terminal oxygenase (CarAa). CarAc acts as a mediator in the electron transfer from CarAd to CarAa. To understand the structural basis of the protein-protein interactions during electron transport in the CARDO system, the crystal structure of CarAc was determined at 1.9 A resolution by molecular replacement using the structure of BphF, the biphenyl 2,3-dioxygenase ferredoxin from Burkholderia cepacia strain LB400 as a search model. CarAc is composed of three beta-sheets, and the structure can be divided into two domains, a cluster-binding domain and a basal domain. The Rieske [2Fe-2S] cluster is located at the tip of the cluster-binding domain, where it is exposed to solvent. While the overall folding of CarAc and BphF is strongly conserved, the properties of their surfaces are very different from each other. The structure of the cluster-binding domain of CarAc is more compact and protruding than that of BphF, and the distribution of electric charge on its molecular surface is very different. Such differences are thought to explain why these ferredoxins can act as electron mediators in respective electron transport chains composed of different-featured components.

Bacterial Proteins↗

PubMed2005

Characterization of [3Fe-4S] ferredoxin DbfA3, which functions in the angular dioxygenase system of Terrabacter sp. strain DBF63.

Dibenzofuran 4,4a-dioxygenase (DFDO) from Terrabacter sp. strain DBF63 is comprised of three components, i.e., terminal oxygenase (DbfA1, DbfA2), putative [3Fe-4S] ferredoxin (ORF16b product), and unidentified ferredoxin reductase. We produced DbfA1 and DbfA2 using recombinant Escherichia coli BL21(DE3) cells as a native form and purified the complex to apparent homogeneity. We also produced and purified a putative [3Fe-4S] ferredoxin encoded by ORF16b, which is located 2.5 kb downstream of the dbfA1A2 genes, with E. coli as a histidine (His)-tagged form. The reconstructed DFDO system with three purified components, i.e., DbfA1A2, His-tagged ORF16b product, and His-tagged PhtA4 (which is a tentative reductase derived from the phthalate dioxygenase system of strain DBF63) could convert fluorene to 9-fluorenol (specific activity: 14.4 nmol min(-1) mg(-1)) and convert dibenzofuran to 2,2',3-trihydroxybiphenyl. This indicates that the ORF16b product can transport electrons to the DbfA1A2 complex; and therefore it was designated DbfA3. Based on spectroscopic UV-visible absorption characteristics and electron paramagnetic resonance spectra, DbfA3 was elucidated to contain a [3Fe-4S] cluster. Ferredoxin interchangeability analysis using several types of ferredoxins suggested that the redox partner of the DbfA1A2 complex may be rather specific to DbfA3.

Actinomycetales↗

PubMed2005

Analysis of PU.1/ICSBP (IRF-8) complex formation with various PU.1 mutants: molecular cloning of rat Icsbp (Irf-8) cDNA.

We isolated cDNA encoding a full-length Interferon (IFN) consensus sequence binding protein [Icsbp; also called IFN regulatory factor-8 (Irf-8)] from rat and analyzed interaction between ICSBP and PU.1, an Ets-family transcription factor regulating hematopoietic cell-specific promoters. Electrophoretic mobility shift assay indicated that PU.1 with deletion of the PEST domain could not bind to ICSBP, although loss of the PEST domain had no effect on DNA-binding ability of PU.1 itself. An amino-acid replacement of Ser147 by Ala of the PEST domain of PU.1 did not affect DNA-binding ability of PU.1 to form a binary complex, PU.1/DNA, but clearly decreased the ternary complex formation of PU.1/ICSBP/DNA. Phosphatase treatment of the ternary complex markedly decreased PU.1/ICSBP/DNA-complex formation. These results indicated that Ser147 of PU.1 is definitively required for forming a complex with ICSBP and phosphorylation of PU.1 and/or ICSBP is critical for formation of the ternary complex.

Animals↗