Genetic analysis of two wheat cultivars, ‘Sonalika’ and ‘WL 711’ for reaction to leaf rust (Puccinia recondita)

Summary Genetic analysis for leaf rust reaction of two widely adapted cultivars, Sonalika and WL 711, has been done using 21 near isogenic Lr lines and rust culture IL004 — avirulent on the two cultivars and all the Lr lines used. The segregation pattern in the F₂ generation indicated the presence of a recessive gene in Sonalika and of a dominant gene in WL 711. These genes in cultivars Sonalika and WL 711 have been identified as Lr 11 and Lr 13, respectively. Gene Lr 13 is no longer effective in WL 711 but it continues to give field resistance in the backgrounds of Chris, Prelude and Thatcher. There has been no significant change in the virulence spectrum of the leaf rust pathogen in India with the release of WL 711. High susceptibility of WL 711 seems to be due to the evolution of more aggressive forms of the pathogen to this cultivar. The gene Lr 11, which behaves as a recessive in Sonalika, was effective against leaf rust when this cultivar was released. The high susceptibility of Sonalika is probably due to an increase in the frequency of race 77 virulent on Lr 11. Lr 11 has shown a dominance reversal in the background of Sonalika. Present results suggest that interaction of resistance genes with the background genotype must be studied for their effective use in breeding programme.     

DoesAgapetus fuscipes cultivate algae in its case?

The presence of algae within the cases ofAgapetus fuscipes was investigated. Cases recognised as dirty or clean with the naked eye had more and less algal growth, respectively. Larvae in the former survived significantly longer when starved in the laboratory. It is suggested that the presence of algae within the cases would be of ecological advantage during periods of flood.     

Intrinsic fluorescence studies on saccharide binding toArtocarpus integrifolia lectin

The combining region ofArtocarpus integrifolia lectin has been studied by using the ligand-induced changes in the fluorescence of the lectin. The saccharide binding properties of the lectin show that C-l, C-2, C-4, and C-6 hydroxyl groups of D-galactose are important loci for sugar binding. The -anorner of galactose binds more strongly than its -counterpart. Inversion in the configuration at C-4 as in glucose results in a loss of binding to the lectin. The C-6 hydroxyl group is also presumably involved in binding as D-fucose does not bind to the lectin.The lectin binds to the Thomsen-Friedenreich antigen (Gal(13)GalNAc) more strongly than the other disaccharides studied, viz. Gal/ (14) Gal and Gal (13) GlcNAc, which are topographically similar to T-antigen. This observation suggests that the combining region ofArtocarpus lectin is complementary to that of T-antigen.Solvent accessibility of the protein fluorophores have been probed by the quenching of protein fluorescence by Iodide ion in the absence and presence of sugar. In the presence of sugar a slight inaccessibility of the fluorophores to the solvent has been observed.Abbreviations MeGal 1-O-methyl--glucopyranoside - MeGal 1-O-methyl--glucopyranoside - GalNAc 2-acetamido-2-deoxy-galactose - Gal Galactose     

Genetic analysis of brown planthopper resistance in twenty varieties of rice,Oryza saliva L.

Summary The inheritance of resistance to brown planthopper, Nilaparvata lugens (Stol.), of 20 rice cultivars was studied. Single dominant genes that are allelic to Bph 3 condition the resistance in cultivars Ptb 19, Gangala (Acc. 7733), Gangala (Acc. 15207), Horana Mawee, Kuruhondarwala, Mudu Kiriyal and Muthumanikam. Single recessive genes that are allelic to bph 4 govern the resistance in cultivars Gambada Samba, Heenhoranamawee, Hotel Samba, Kahata Samba, Kalukuruwee, Lekam Samba, Senawee, Sulai, Thirissa and Vellai Illankali. The resistance in Ptb 33, Sudu Hondarwala, and Sinna Sivappu is governed by one dominant and one recessive gene which segregate independently of each other. The dominant resistance genes in these cultivars appear allelic to either Bph 1 or Bph 3. Similarly, the recessive genes in these cultivars seem allelic to either bph 2 or bph 4. Further investigations are needed to conclusively determine the allelic relationships of resistance genes in Ptb 33, Sudu Hondarwala and Sinna Sivappu.     

Identification and characterization of the functional alpha origin of DNA replication of the R6K plasmid and its relatedness to the R6K beta and gamma origins

Summary The functional R6K origin is composed of two DNA elements, one of 580 bp carrying the origin sequences and the other of 277 bp containing the seven 22 bp direct repeats previosly identified as also required for and origin activity. These two genetic elements are separated by approximately 3,000 bp of R6K sequences which are dispensable for origin activity. The function of the origin depends on the presence in cis of the 580 bp and the 277 bp fragments and requires that they be oriented as in the intact R6K. Activation of the origin depends on the R6K replication initiation protein .Within the 580 bp of the origin, there is a sequence of 98 bp which appears as an inverted repeat of 96 bp in the replicon. Deletion of the 96 bp or 98 bp results in inactivation of the and the origins respectively. These long repeats are palindromic and it is suggested that these may serve as the recognition signals for initiation of DNA replication in the and the origins of R6K. DNA homology analysis performed on , and origin sequences, also reveals 10–23 bp sequences in the and the origins that are related to the family of 22 bp direct repeats in the origin which were shown previously to be binding sites for the protein.     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

Genetics of resistance to Puccinia graminis tritici in ‘Chris’ and ‘W3746’ wheats

Summary Chris wheat possessed genes Sr5, Sr7a, Sr8a, Sr9g and Sr12. W3746, derived from the cross Chris/Baart, possessed Sr7a and Sr12. The response conferred by Sr7a was influenced by the genetic background. Although Sr7a or Sr12 alone conferred no observable resistance upon adult plants, the adult resistances of Chris and W3746 to predominant pathotypes appeared to be associated with the interaction of Sr7a and Sr12, or genes at closely linked loci.     

Structural diversity of O-polysaccharides and serological classification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">garcae</Emphasis> and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I:3)--L Rha (12)-- L Rha (12)--L-Rha-(13)--L Rha (1;II: 2)--L-Rha-(13) -L-Rha-(12)--L-Rha-(13)--L Rha (1.The branched O-polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588^T have the same L-rhamnan backbone with repeating units I and II and a lateral chain of 14)- or 13)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral -D-Fuc3NAc residues were characterized.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 777–789.Original Russian Text Copyright © 2004 by Ovod, Zdorovenko, Shashkov, Kocharova, Knirel.     

Spontaneous chlorophyll mutants of Pennisetum americanum: Genetics and chlorophyll quantities

Summary Thirteen spontaneously occurring chlorophyll deficient phenotypes have been described and their genetic basis was established. Ten of these — white, white tipped green, patchy white, white virescent, white striping 1, white striping 2, white striping 4, fine striping, chlorina and yellow virescent showed monogenic recessive inheritance and the remaining three — yellow striping, yellow green and light green seedling phenotypes showed digenic recessive inheritance. The genes for (i) white tipped green (wr) and yellow virescent (yv) and (ii) patchy white (pw) and white striping 1 (wst 1) showed independent assortment. Further, the genes for white (w), white tipped green (wr) and yellow virescent (yv) were inherited independently of the gene for hairy leaf margin (Hm).In the mutants — white tipped green, patchy white, white striping 1, white striping 2, fine striping, chlorina, yellow virescent, yellow striping, yellow green and light green phenotypes total quantity of chlorophyll was significantly less than that in the corresponding controls, while in white virescent there was no reduction in the mature stage. For nine of the mutants the quantity of chlorophyll was also estimated in F₁'s (mutant x control green). In F₁'s of six of the mutants — white tip, patchy white, chlorina, yellow virescent, fine striping and yellow striping the quantity of chlorophyll was almost equal to the wild type. In the F₁'s of three of the mutants — white striping 1, white striping 2 and light green an intermediate value between the mutant and wild types was observed. In yellow virescent retarded synthesis of chlorophyll, particularly chlorophyll a was observed in the juvenile stage. Reduced quantity of chlorophyll was associated with defective chloroplasts. In the mutants — white tipped green, white virescent, fine striping, chlorina, yellow striping, yellow green and light green defective plastids were also observed. In patchy white secondary destruction of chlorophylls and the presence of defective plastids were found to be associated with reduced chlorophyll quantity at maturity.Paper chromatographic studies of leaf flavonoids revealed some variation between the inbreds, but there were three common spots, 7, 8 and 9, except for PDP in which the spot 8 was absent. Chlorophyll deficient mutants differed from their respective controls in the absence of one or more of the spots present in the controls and in the presence of new spots in some of the mutants.Most of the chlorophyll mutants showed higher survival rate in the Kharif season than in Rabi season which was attributed to the higher mean day temperature and longer day light period in the Kharif season than in Rabi season.     

DNA variants within the 5′-flanking region of milk-protein-encoding genes II. The β-lactoglobulin-encoding gene

For the detection of polymorphisms within the 5-flanking region of the -lactoglobulin (-LG) -encoding gene a nucleotide sequence containing 795 bp of the promoter and 59 bp of exon I was cloned and sequenced. After comparing the sequence from the DNA of 11 diverse cows (different breeds and milk-protein yields), 14 singlebp substitutions were identified within the 5-flanking region and two in the 5-untranslated region (5-UTR) of exon I. Some of the variants are located in potential binding sites for trans-acting factors or in the 5-UTR. A PCR-based RFLP analysis was performed, and the genotypes of an additional 60 cows were identified at five variable 5-flanking sites. The results reveal three frequent combinations between the A and B alleles of the protein-coding region and the novel 5-flanking DNA variants. This finding may explain the differences of the protein-variant-dependent -LG synthesis (A>B) observed in vivo. A sequence comparison of the bovine and ovine promoters reveals an homology of 92.8% and shows a higher degree of conservation between positions -600 and -300.     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.     

Enzymatic regioselective synthesis of sucrose acrylate esters

Using a protease (at 100 g l^–1) from Bacillus licheniformis, enzymatic acryloylation of sucrose (1 M) with vinyl acrylate (4 M) was carried out in anhydrous pyridine and yielded sucrose acrylate esters with more than 90% of sucrose converted in 24 h. After 5 days of reaction, the ester products consisted of 70% sucrose monoacrylate and 30% sucrose diacrylate. The monoester product was a sucrose 1-acrylate and the diester products consisted of sucrose 6,1-diacrylate and sucrose 6,1-diacrylate in the ratio of 3:2.     

TROSY experiment for refinement of backbone psi and phi by simultaneous measurements of cross-correlated relaxation rates and 3,4J(H alpha HN) coupling constants

The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential H-C/H^N-N dipole/dipole and H-C/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the ^3,4 J _{H HN} coupling constants measured in an E.COSY manner. The conformational restraints for the and angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants. Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle. The application of the [¹⁵N,¹³C]-DQ/ZQ-[¹⁵N,¹H]-TROSY-E.COSY experiment to the 16 kDa apo-form of the E. coli Heme Chaperon protein CcmE is described. Overall good agreement is achieved between and angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformers.     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

Does an animal peptide:N-glycanase have the dual role as an enzyme and a carbohydrate-binding protein?

Recently, we have reported purification and characterization of a de-N-glycosylating enzyme, peptide:N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase [Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994)J Biol Chem 269, 17611–18]. The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g.K _m=114 µm for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain. In this study, we report the new findings of the mannan-binding property of L-929 PNGase; the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Man1 3(Man1 6)Man, but not by mannose and -methyl-d-mannoside. Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L-929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition proteinin vivo. Such dual properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases — PNGase A and PNGase F, respectively.Abbreviations GLC gas liquid chromatography - GlcNAc-Asn 2-acetamido-1--(l-aspartamido)-1,2-dideoxy-d-glucose - BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Man d-mannose; triomannose, Man1 3(Man1 6)Man; - MES 2-(N-morphorino)ethanesulfonic acid - NeuAc N-acetyl-neuraminic acid - PNGase peptide:N ⁴-(N-accetyl-glucosaminyl)asparagine amidase (peptide:N-glycanase,EC 3.5.1.52) - PNP p-nitrophenyl     

Structure-function relationships of β- D-glucan endo- and exohydrolases from higher plants

(13),(14)--d-Glucans represent an important component of cell walls in the Poaceae family of higher plants. A number of glycoside endo- and exohydrolases is required for the depolymerization of (13),(14)--d-glucans in germinated grain or for the partial hydrolysis of the polysaccharide in elongating vegetative tissues. The enzymes include (13),(14)--d-glucan endohydrolases (EC 3.2.1.73), which are classified as family 17 glycoside hydrolases, (14)--d-glucan glucohydrolases (family 1) and -d-glucan exohydrolases (family 3). Kinetic analyses of hydrolytic reactions enable the definition of action patterns, the thermodynamics of substrate binding, and the construction of subsite maps. Mechanism-based inhibitors and substrate analogues have been used to study the spatial orientation of the substrate in the active sites of the enzymes, at the atomic level. The inhibitors and substrate analogues also allow us to define the catalytic mechanisms of the enzymes and to identify catalytic amino acid residues. Three-dimensional structures of (13),(14)--d-glucan endohydrolases, (14)--d-glucan glucohydrolases and -d-glucan exohydrolases are available or can be reliably modelled from the crystal structures of related enzymes. Substrate analogues have been diffused into crystals for solving of the three-dimensional structures of enzyme-substrate complexes. This information provides valuable insights into potential biological roles of the enzymes in the degradation of the barley (13),(14)--d-glucans during endosperm mobilization and in cell elongation.     

Genetic polymorphism at the κ chain locus in mice: Comparisons of restriction enzyme hybridization fragments of variable and constant region genes

Variable (V) and constant (C) region genes of the mouse kappa light chain have been compared in inbred strains and in geographically isolated or genetically separated populations of mice by Southern blot analysis of endonuclease-restricted germline DNA. In most cases, the C gene is found on a single restriction fragment while the V genes of the V19 and V21 groups are each found on several (6–18) fragments. The restriction fragment (RF) patterns of V19 and V21 groups are both polymorphic when compared among inbred mouse strains. Southern blot patterns of V21 and V19 of inbred strains are also found among some geographically isolated populations of mice, suggesting that inbred strains acquired kappa loci from different subspecies. Some populations of geographical isolates show V21, V19, and C contexts similar to inbred mice while more distantly related species within the genus Mus and laboratory rats show no apparent similarity in context to inbred strains. Variable region genes determining the RF patterns of V19 and V21 appear to be linked to each other and to the C and Lyt-3 loci.     

The Use of the [13C]/[12C] Ratio for the Assay of the Microbial Oxidation of Hydrocarbons

The study deals with a comparative analysis of the relative abundances of the carbon isotopes ¹²C and ¹³C in the metabolites and biomass of the Burkholderia sp. BS3702 and Pseudomonas putida BS202-p strains capable of utilizing aliphatic (n-hexadecane) and aromatic (naphthalene) hydrocarbons as sources of carbon and energy. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of Burkholderia sp. BS3702 on n-hexadecane (¹³C = –44.6 ± 0.2) were characterized by the values of ¹³C_{CO 2} = –50.2 ± 0.4, ¹³C_biom = –46.6 ± 0.4, and ¹³C_exo = –41.5 ± 0.4, respectively. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of the same bacterial strain on naphthalene (¹³C = –21 ± 0.4) were characterized by the isotope effects ¹³C_{CO 2} = –24.1 ± 0.4, ¹³C_biom = –19.2 ± 0.4, and ¹³C_exo = –19.1 ± 0.4, respectively. The possibility of using the isotope composition of metabolic carbon dioxide for the rapid monitoring of the microbial degradation of petroleum hydrocarbons in the environment is discussed.     

Disulfide linked αoLH-gelonin conjugate failed to recombine with βoLH subunit to generate bioeffective hormonotoxin

Since, linking of ovine luteinizing hormone (oLH) to ribosome inactivating protein gelonin (in oLH-gelonin conjugate) occur via the alpha-subunit, oLH, an attempt has been made to develop a universal hormonotoxin for selective targeting to specific cells in the gonads. Four different molar ratios of oLH and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) were used to activate the epsilon amino (-NH₂) groups of oLH. The oLH-SPDP derivatives recombine to native beta subunit of oLH (oLH) and the purified recombinants retained substantial receptor binding, steroidogenic activity and immunoreactivity to native oLH. The disulfide linked oLH-S-S-gelonin conjugates prepared by SPDP method were purified by gel filtration chromatography and analysed by reverse-phase high performance liquid chromatography (RP-HPLC). In order to obtain specificity and bioeffectivity, the oLH-S-S-gelonin conjugates were allowed to recombine to native oLH and the recombination mixture was further purified by gel-filtration chromatography. The RP-HPLC analysis of these recombinants indicated that oLH-S-S-gelonin did not recombine to oLH. The failure of recombination may be due to the reasons. (i) The site of -NH₂ activation by SPDP may be different in the oLH than the native oLH. (ii) The activation site may be in close proximity to the annealing site which facilitates the recombination of -subunit but failured to reassociate to oLH-S-S-gelonin conjugate. (iii) The introduction of gelonin (30 kDa basic protein) might have induced some steric hinderence for oLH to recombine to the oLH site which might have been masked in oLH-S-S-gelonin conjugates. (Mol Cell Biochem120: 95–102, 1993)Abbreviations oLH ovine Luteinizing Hormone - oLH alpha subunit of oLH - oLH beta subunit of oLH - BSA Bovine Serum Albumin - DTT Dithiothreitol - RP-HPLC Reverse Phase High Performance Liquid Chromatography - TSH Thyroid Stimulating Hormone - FSH Follicle Stimulating Hormone - LH Luteinizing Hormone - eCG equine Chorionic Gonadotropin - DMEM Dulbecco's Modified Eagles Medium - HEPES 4-(2-Hydroxyethyl)-1 Piperazine Ethane Sulfonic acid - PAP Pokeweed Antiviral Protein - RIA Radioimmunoassay - hCG human Chorionic Gonadotropin - TRH Thyrotropin Releasing Hormone - CRF Corticotropin Releasing Factor - hPL human Placental Lactogen - TFA Trifluroacetic Acid - oLH-SPDP SPDP activated derivative of oLH