A selective insecticidal protein from Pseudomonas mosselii for corn rootworm control

The coleopteran insect western corn rootworm (WCR, Diabrotica virgifera virgifera) is an economically important pest in North America and Europe. Transgenic corn plants producing Bacillus thuringiensis (Bt) insecticidal proteins have been useful against this devastating pest, but evolution of resistance has reduced their efficacy. Here, we report the discovery of a novel insecticidal protein, PIP‐47Aa, from an isolate of Pseudomonas mosselii. PIP‐47Aa sequence shows no shared motifs, domains or signatures with other known proteins. Recombinant PIP‐47Aa kills WCR, two other corn rootworm pests (Diabrotica barberi and Diabrotica undecimpunctata howardi) and two other beetle species (Diabrotica speciosa and Phyllotreta cruciferae), but it was not toxic to the spotted lady beetle (Coleomegilla maculata) or seven species of Lepidoptera and Hemiptera. Transgenic corn plants expressing PIP‐47Aa show significant protection from root damage by WCR. PIP‐47Aa kills a WCR strain resistant to mCry3A and does not share rootworm midgut binding sites with mCry3A or AfIP‐1A/1B from Alcaligenes that acts like Cry34Ab1/Cry35Ab1. Our results indicate that PIP‐47Aa is a novel insecticidal protein for controlling the corn rootworm pests.     

The alloantibody response in the allogeneically pregnant rat. VI. Detection in maternal serum of specific antigen-antibody complexes

We have examined prepartum and postpartum sera from allogeneically pregnant rats for the presence of paternal antigen using a mAb sandwich assay. Paternal class I antigen, RT1Aa, was frequently detected in postpartum and prepartum rat sera. Postpartum sera from high responders also include anti-RT1Aa alloantibodies, some of which were complexed with the RT1Aa antigen. Although the concentration of antigen is low, the high uterine blood flow causes the amount of class I antigen entering the pregnant female to be in the low microgram per day range. Such an amount could have immunoregulatory significance.     

Location of the Bombyx mori 175kDa cadherin-like protein-binding site on Bacillus thuringiensis Cry1Aa toxin

To identify and gain a better understanding of the cadherin-like receptor-binding site on Bacillus thuringiensis Cry toxins, it is advantageous to use Cry1Aa toxin, because its 3D structure is known. Therefore, Cry1Aa toxin was used to examine the locations of cadherin-like protein-binding sites. Initial experiments examining the binding compatibility for Cry1Aa toxin of partial fragments of recombinant proteins of a 175kDa cadherin-like protein from Bombyx mori (BtR175) and another putative receptor for Cry1Aa toxin, amino peptidaseN1, from Bo.mori (BmAPN1), suggested that their binding sites are close to each other. Of the seven mAbs against Cry1Aa toxin, two mAbs were selected that block the binding site for BtR175 on Cry1Aa toxin: 2A11 and 2F9. Immunoblotting and alignment analyses of four Cry toxins revealed amino acids that included the epitope of mAb 2A11, and suggested that the area on Cry1Aa toxin blocked by the binding of mAb 2A11 is located in the region consisting of loops2 and 3. Two Cry1Aa toxin mutants were constructed by substituting a Cys on the area blocked by the binding of mAb 2A11, and the small blocking molecule, N-(9-acridinyl)maleimide, was introduced at each Cys substitution to determine the BtR175-binding site. Substitution of Tyr445 for Cys had a crippling effect on binding of Cry1Aa toxin to BtR175, suggesting that Tyr445 may be in or close to the BtR175-binding site. Monoclonal antibodies that blocked the binding site for BtR175 on Cry1Aa toxin inhibited the toxicity of Cry1Aa toxin against Bo.mori, indicating that binding of Cry1Aa toxin to BtR175 is essential for the action of Cry1Aa toxin on the insect.     

Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin

We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.     

Importance of Cry1 delta-endotoxin domain II loops for binding specificity in Heliothis virescens (L.)

We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens. Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with (125)I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H. virescens. Cry1Ac competed with high affinity (competition constant [K(com)] = 1.1 nM) for (125)I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for (125)I-Cry1Ab binding sites, though the K(com) values ranged from 179 to 304 nM. Cry1Ab competed for (125)I-Cry1Ac binding sites (K(com) = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the (125)I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H. virescens. Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H. virescens. The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H. virescens.     

Ring-like nucleoid does not play a key role in radioresistance of Deinococcus radiodurans

The conclusion based on transmission electron microscopy, "the tightly packed ring-like nucleoid of the Deinococcus radiodurans R1 is a key to radioresistance", has instigated lots of debates. In this study, according to the previous research of PprI’s crucial role in radioresistance of D. radiodurans, we have attempted to examine and compare the nucleoid morphology differences among wild-type D. ra-diodurans R1 strain, pprI function-deficient mutant (YR1), and pprI function-complementary strains (YR1001, YR1002, and YR1004) before and after exposure to ionizing irradiation. Fluorescence mi-croscopy images indicate: (1) the majority of nucleoid structures in radioresistant strain R1 cells ex-hibit the tightly packed ring-like morphology, while the pprI function-deficient mutant YR1 cells carrying predominate ring-like structure represent high sensitivity to irradiation; (2) as an extreme radioresistant strain similar to wild-type R1, pprI completely function-complementary strain YR1001 almost displays the loose and irregular nucleoid morphologies. On the other hand, another radioresistant pprI partly function-complementary strain YR1002’s nucleiods exhibit about 60% ring-like structure; (3) a PprI C-terminal deletion strain YR1004 consisting of approximately 60% of ring-like nucleoid is very sensi-tive to radiation. Therefore, our present experiments do not support the conclusion that the ring-like nucleoid of D. radiodurans does play a key role in radioresistance.     

An ADAM metalloprotease is a Cry3Aa Bacillus thuringiensis toxin receptor

Bacillus thuringiensis insecticidal proteins toxic action relies on the interaction with receptor molecules on insect midgut target cells. Here, we describe an ADAM metalloprotease as a novel type of B. thuringiensis toxin receptor on the basis of the following data: (i) by ligand blot and N-terminal analysis, we detected a Colorado potato beetle Cry3Aa toxin binding molecule that shares homology with an ADAM10 metalloprotease; (ii) Colorado potato beetle brush border membrane vesicles display ADAM activity since it cleaves an ADAM fluorogenic substrate; (iii) Cry3Aa acts as a competitor of the cleavage of the ADAM fluorogenic substrate; (iv) Cry3Aa sequence contains the recognition motif R(345)FQPGYYGND(354) present in ADAM10 substrates. Accordingly, a peptide representative of the recognition motif localized within loop 1 of Cry3Aa domain II (Ac-F(341)HTRFQPGYYGNDSFN(358)-NH(2)) effectively prevented Cry3Aa proteolytic processing and nearly abolished pore formation, evidencing the functional significance of the Cry3Aa-ADAM interaction in relation to this toxin mode of action.     

Location of the Bombyx mori Aminopeptidase N Type 1 Binding Site on Bacillus thuringiensis Cry1Aa Toxin

We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of ⁵⁰⁸STLRVN⁵¹³ and ⁵⁸²VFTLSAHV⁵⁸⁹. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.     

Down Regulation of Genes Involved in T Cell Polarity and Motility during the Induction of Heart Allograft Tolerance by Allochimeric MHC I

Background

The allochimeric MHC class I molecule [α1h1/u]-RT1.Aa that contains donor-type (Wistar Furth, WF; RT1u) epitopes displayed on recipient-type (ACI, RT1a) administered in conjunction with sub-therapeutic dose of cyclosporine (CsA) induces indefinite survival of heterotopic cardiac allografts in rat model. In vascularized transplantation models, the spleen contributes to graft rejection by generating alloantigen reactive T cells. The immune response in allograft rejection involves a cascade of molecular events leading to the formation of immunological synapses between T cells and the antigen-presenting cells.

Methodology/Principal Findings

To elucidate the molecular pathways involved in the immunosuppressive function of allochimeric molecule we performed microarray and quantitative RTPCR analyses of gene expression profile of splenic T cells from untreated, CsA treated, and allochimeric molecule + subtherapeutic dose of CsA treated animals at day 1, 3 and 7 of post transplantation. Allochimeric molecule treatment caused down regulation of genes involved in actin filament polymerization (RhoA and Rac1), cell adhesion (Catna1, Vcam and CD9), vacuolar transport (RhoB, Cln8 and ATP6v1b2), and MAPK pathway (Spred1 and Dusp6) involved in tubulin cytoskeleton reorganization and interaction between actin and microtubule cytoskeleton. All these genes are involved in T cell polarity and motility, i.e., their ability to move, scan and to form functional immunological synapse with antigen presenting cells (APCs).

Conclusions

These results indicate that the immunosuppressive function of allochimeric molecule may depend on the impairment of T cells'' movement and scanning ability, and possibly also the formation of immunological synapse. We believe that these novel findings may have important clinical implications for organ transplantation.     

Bacillus thuringiensis insecticidal Cry1Aa toxin binds to a highly conserved region of aminopeptidase N in the host insect leading to its evolutionary success.

Bacillus thuringiensis insecticidal protein, Cry1Aa toxin, binds to a specific receptor in insect midguts and has insecticidal activity. Therefore, the structure of the receptor molecule is probably a key factor in determining the binding affinity of the toxin and insect susceptibility. The cDNA fragment (PX frg1) encoding the Cry1Aa toxin-binding region of an aminopeptidase N (APN) or an APN family protein from diamondback moth, Plutella xylostella midgut was cloned and sequenced. A comparison between the deduced amino acid sequence of PX frg1 and other insect APN sequences shows that Cry1Aa toxin binds to a highly conserved region of APN family protein. In this paper, we propose a model to explain the mechanism that causes B. thuringiensis evolutionary success and differing insect susceptibility to Cry1Aa toxin.     

Receptor subtype-specific docking of Asp6.59 with C-terminal arginine residues in Y receptor ligands

Y receptors (YRs) are G protein-coupled receptors whose Y(1)R, Y(2)R, and Y(5)R subtypes preferentially bind neuropeptide Y (NPY) and peptide YY, whereas mammalian Y(4)Rs show a higher affinity for pancreatic polypeptide (PP). Comparison of YR orthologs and paralogs revealed Asp(6.59) to be fully conserved throughout all of the YRs reported so far. By replacing this conserved aspartic acid residue with alanine, asparagine, glutamate, and arginine, we now show that this residue plays a crucial role in binding and signal transduction of NPY/PP at all YRs. Sensitivity to distinct replacements is, however, receptor subtype-specific. Next, we performed a complementary mutagenesis approach to identify the contact site of the ligand. Surprisingly, this conserved residue interacts with two different ligand arginine residues by ionic interactions; although in Y(2)R and Y(5)R, Arg(33) is the binding partner of Asp(6.59), in Y(1)R and Y(4)R, Arg(35) of human PP and NPY interacts with Asp(6.59). Furthermore, Arg(25) of PP and NPY is involved in ligand binding only at Y(2)R and Y(5)R. This suggests significant differences in the docking of YR ligands between Y(1/4)R and Y(2/5)R and provides new insights into the molecular binding mode of peptide agonists at GPCRs. Furthermore, the proposed model of a subtype-specific binding mode is in agreement with the evolution of YRs.     

Cry11Aa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loop alpha-8 of domain II

Bacillus thuringiensis subs israelensis produces Cry toxins active against mosquitoes. Receptor binding is a key determinant for specificity of Cry toxins composed of three domains. We found that exposed loop alpha-8 of Cry11Aa toxin, located in domain II, is an important epitope involved in receptor interaction. Synthetic peptides corresponding to exposed regions in domain II (loop alpha-8, beta-4 and loop 3) competed binding of Cry11Aa to membrane vesicles from Aedes aegypti midgut microvilli. The role of loop alpha-8 of Cry11A in receptor interaction was demonstrated by phage display and site-directed mutagenesis. We isolated a peptide-displaying phage (P5.tox), that recognizes loop alpha-8 in Cry11Aa, interferes interaction with the midgut receptor and attenuates toxicity in bioassay. Loop alpha-8 mutants affected in toxicity and receptor binding were characterized.     

Structure of the functional form of the mosquito larvicidal Cry4Aa toxin from Bacillus thuringiensis at a 2.8-angstrom resolution

The Cry4Aa delta-endotoxin from Bacillus thuringiensis is toxic to larvae of Culex, Anopheles, and Aedes mosquitoes, which are vectors of important human tropical diseases. With the objective of designing modified toxins with improved potency that could be used as biopesticides, we determined the structure of this toxin in its functional form at a resolution of 2.8 angstroms. Like other Cry delta-endotoxins, the activated Cry4Aa toxin consists of three globular domains, a seven-alpha-helix bundle responsible for pore formation (domain I) and the following two other domains having structural similarities with carbohydrate binding proteins: a beta-prism (domain II) and a plant lectin-like beta-sandwich (domain III). We also studied the effect on toxicity of amino acid substitutions and deletions in three loops located at the surface of the putative receptor binding domain II of Cry4Aa. Our results indicate that one loop is an important determinant of toxicity, presumably through attachment of Cry4Aa to the surface of mosquito cells. The availability of the Cry4Aa structure should guide further investigations aimed at the molecular basis of the target specificity and membrane insertion of Cry endotoxins.     

Shared Binding Sites for the Bacillus thuringiensis Proteins Cry3Bb,Cry3Ca,and Cry7Aa in the African Sweet Potato Pest Cylas puncticollis (Brentidae)

Bacillus thuringiensis Cry3Bb, Cry3Ca, and Cry7Aa have been reported to be toxic against larvae of the genus Cylas, which are important pests of sweet potato worldwide and particularly in sub-Saharan Africa. However, relatively little is known about the processing and binding interactions of these coleopteran-specific Cry proteins. The aim of the present study was to determine whether Cry3Bb, Cry3Ca, and Cry7Aa proteins have shared binding sites in Cylas puncticollis to orient the pest resistance strategy by genetic transformation. Interestingly, processing of the 129-kDa Cry7Aa protoxin using commercial trypsin or chymotrypsin rendered two fragments of about 70 kDa and 65 kDa. N-terminal sequencing of the trypsin-activated Cry7Aa fragments revealed that processing occurs at Glu⁴⁷ for the 70-kDa form or Ile⁸⁸ for the 65-kDa form. Homologous binding assays showed specific binding of the two Cry3 proteins and the 65-kDa Cry7Aa fragment to brush border membrane vesicles (BBMV) from C. puncticollis larvae. The 70-kDa fragment did not bind to BBMV. Heterologous-competition assays showed that Cry3Bb, Cry3Ca, and Cry7Aa (65-kDa fragment) competed for the same binding sites. Hence, our results suggest that pest resistance mediated by the alteration of a shared Cry receptor binding site might render all three Cry toxins ineffective.     

Ring-like nucleoid does not play a key role in radioresistance of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

The conclusion based on transmission electron microscopy, “the tightly packed ring-like nucleoid of the Deinococcus radiodurans R1 is a key to radioresistance”, has instigated lots of debates. In this study, according to the previous research of Pprl’s crucial role in radioresistance of D. radiodurans, we have attempted to examine and compare the nucleoid morphology differences among wild-type D. radiodurans R1 strain, pprf function-deficient mutant (YR1), and pprl function-complementary strains (YR1001, YR1002, and YR1004) before and after exposure to ionizing irradiation. Fluorescence microscopy images indicate: (1) the majority of nucleoid structures in radioresistant strain R1 cells exhibit the tightly packed ring-like morphology, while the pprl function-deficient mutant YR1 cells carrying predominate ring-like structure represent high sensitivity to irradiation; (2) as an extreme radioresistant strain similar to wild-type R1, pprl completely function-complementary strain YR1001 almost displays the loose and irregular nucleoid morphologies. On the other hand, another radioresistant pprl partly function-complementary strain YR1002’s nucleiods exhibit about 60% ring-like structure; (3) a Pprl C-terminal deletion strain YR1004 consisting of approximately 60% of ring-like nucleoid is very sensitive to radiation. Therefore, our present experiments do not support the conclusion that the ring-like nucleoid of D. radiodurans does play a key role in radioresistance.     

Ring-like nucleoid does not play a key role in radioresistance of Deinococcus radiodurans

The conclusion based on transmission electron microscopy, “the tightly packed ring-like nucleoid of the Deinococcus radiodurans R1 is a key to radioresistance”, has instigated lots of debates. In this study, according to the previous research of Pprl’s crucial role in radioresistance of D. radiodurans, we have attempted to examine and compare the nucleoid morphology differences among wild-type D. radiodurans R1 strain, pprf function-deficient mutant (YR1), and pprl function-complementary strains (YR1001, YR1002, and YR1004) before and after exposure to ionizing irradiation. Fluorescence microscopy images indicate: (1) the majority of nucleoid structures in radioresistant strain R1 cells exhibit the tightly packed ring-like morphology, while the pprl function-deficient mutant YR1 cells carrying predominate ring-like structure represent high sensitivity to irradiation; (2) as an extreme radioresistant strain similar to wild-type R1, pprl completely function-complementary strain YR1001 almost displays the loose and irregular nucleoid morphologies. On the other hand, another radioresistant pprl partly function-complementary strain YR1002’s nucleiods exhibit about 60% ring-like structure; (3) a Pprl C-terminal deletion strain YR1004 consisting of approximately 60% of ring-like nucleoid is very sensitive to radiation. Therefore, our present experiments do not support the conclusion that the ring-like nucleoid of D. radiodurans does play a key role in radioresistance. Supported by the National Basic Research Program of China (Grant No. 2004CB19604), the National Natural Science Foundation of China (Grant No. 30330020), and the National Science fund for Distinguished Young Scholars (Grant No. 30425038)     

The LEW.BN(2R) strain: a recombinant in the rat MHC

Cell-mediated cytolytic (CMC) responses resulting from immunizations between rat strains considered to be RT1 (Ag-B) identical (LEW.B3:BN) are capable of detecting a membrane determinant(s) controlled by a locus linked to RT1, which has been designated Ag-L. The Ag-L gene region has been isolated in a recombinant line, tentatively designated as LEW.BN(2R), and has been assigned the RT1r5 haplotype. The data presented demonstrate that the genes responsible for MLR stimulation in the 2R strain are of LEW origin. In addition, LEW.B3 anti-BN CTL appear to recognize multiple specificities, only one of which is in the 2R strain. Some of the remaining specificities in BN may be the result of interactions between undetected genes that have been separated in the LEW.B3 and 2R strains.     

Testing of Arg-8-gonadotropin-releasing hormone-directed antisera by immunological and immunocytochemical methods for use in comparative studies

Three polyclonal antisera raised in rabbits against the mammalian molecular form of gonadotropin-releasing hormone (GnRH) were tested in enzyme-linked immunosorbent assays for crossreactivity with naturally occurring GnRHs and with GnRH analogues. Antisera were then tested immunocytochemically in order (i) to identify amino acids essential for the binding of each antiserum, and (ii) to evaluate the specificity of the immunocytochemical reaction in brain sections from various species of cyclostomes, amphibians, reptiles, and birds. Antiserum GnRH 80/1, recognizing mainly a discontinuous determinant including the NH2- and COOH-termini, crossreacts with GnRHs the molecular bending of which enables the spatial approach of both terminal amino acid residues. Antiserum GnRH 80/2, by requiring the COOH-terminus for binding and not tolerating substitutions by aromatic amino acids in the middle region of the molecule, recognizes chicken I GnRH, however, not the salmon form. The use of this antiserum is appropriate in species synthesizing the mammalian and/or the chicken I form of GnRH. GnRH antiserum 81/1 is specific mostly for mammalian GnRH. The results obtained by ELISAs are confirmed by immunocytochemical studies. A comparison between the results obtained in ELISA and in immunocytochemistry involving mammalian-, chicken I-, chicken II-, salmon-, and lamprey-directed GnRH antisera resulted in the following conclusions: (1) An antiserum recognizing the discontinuous antigen determinant including both NH2- and COOH-termini may be reactive in most vertebrate brain sections thus being appropriate for phylogenetically directed immunocytochemical studies. (2) Moreover, this discontinuous determinant seems to be immunocytochemically reactive in all parts of the neurons in the GnRH system, whereas, in some species, determinants located in the middle region of the molecule(s) tend to become reactive only during the axonal transport. (3) A crossreaction between tissue-bound antigen and antibodies recognizing the above cited discontinuous determinant indicates an appropriate bending of the molecule even in case of severe molecular differences, e.g., in lamprey form of GnRH. (4) It follows that in phylogenetic studies, an immunologically well characterized antiserum can be substituted for a species-directed antiserum.