Intracellular sites of the synthesis of sweet potato mitochondrial F1ATPase subunits

Summary Five subunits (-, -, -, - and -subunits) of the six -and -subunits) in the F₁ portion (F₁ATPase) of sweet potato (Ipomoea batatas) mitochondrial adenosine triphosphatase were isolated by an electrophoretic method. The - and -subunits were not distinguishable immunologically but showed completely different tryptic peptide maps, indicating that they were different molecular species. In vitro protein synthesis with isolated sweet potato root mitochondria produced only the -subunit when analyzed with anti-sweet potato F₁ATPase antibody reacting with all the subunits except the -subunit. Sweet potato root poly(A)⁺RNA directed the synthesis of six polypeptides which were immunoprecipitated by the antibody: two of them immunologically related to the -subunit and the others to the - and -subunits. We conclude that the -subunit of the F₁ATPase is synthesized only in the mitochondria and the -, - and -subunits are in the cytoplasm.     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The purified isopropylmalate synthase of Alcaligenes eutrophus H 16 reacted with the following -keto acids and acyl-coenzyme A derivatives (in the sequence of decreasing affinities): -ketoisovalerate, -keto-n-valerate, -ketobutyrate and pyruvate; acetyl-CoA, propionyl-CoA, butyryl-CoA. malonyl-CoA, valeryl-CoA, and crotonyl-CoA. -Ketoisocaproate, however, is a strong inhibitor of the enzyme. All reactions catalyzed by isopropylmalate synthase were inhibited to the same extent by the endproduct l-leucine. the substrate saturation curves of -ketoisovalerate or other -keto acids and of acetyl-coenzyme A or other acyl-CoA derivatives had intermediary plateau regions; the Hill coefficient alternated between n _H -values higher and lower than 1.0, indicating changes from positive to negative and from negative to positive cooperativity for the substrates. The products, isopropylmalate and free coenzyme A, showed competitive inhibition patterns against both substrates (-ketoisovalerate and acetyl-CoA). Free coenzyme A (1 M) inactivated the enzyme irreversibly. The 3-phosphate of coenzyme A and the free carboxyl group of -ketoisovalerate were involved in optimal binding of these substrates, but 3-dephospho-acetyl-coenzyme A and the methylester of -ketoisovalerate were also converted by this enzyme. A CH₃–CH₂-grouping of the -keto acids seemed to be necessary for binding this substrate.Abbreviations Used CoA Coenzyme A - Tris Tris(hydroxymethyl)aminomethane hydrochloride - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - IPM -Isopropylmalate - KIV -Ketoisovalerate Prepared from doctoral thesis of the University of Göttingen 1973     

Sialidase of swine influenza A viruses: variation of the recognition specificities for sialyl linkages and for the molecular species of sialic acid with the year of isolation

The sialidase of swine influenza A viruses of N1 and N2 subtypes, isolated from 1930 to 1992, was studied for substrate specificity with ganglio-series, lacto-series type II and GM3 gangliosides containing Neu5Ac2-3Gal, Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. All viral sialidases tested showed that the activity for hydrolysing substrates with Neu5Ac2-3Gal was higher than the activities with Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. When GM1b, GM3 and sialylparagloboside were used as substrates, the earliest strain (A/Wisconsin/15/30 H1N1, isolated in 1930) showed the activity ratio of Neu5Ac2-6Gal to Neu5Ac2-3Gal to be 0.13:0.2, and the ratio Neu5Gc2-3Gal/Neu5Ac2-3Gal to be 0.19:0.37, while those strains isolated from 1978 to 1992 exhibited ratios of 0.29:0.58 for Neu5Ac2-6Gal/Neu5Ac2-3Gal and 0.51:0.76 for Neu5Gc2-3Gal/Neu5Ac2-3Gal. The above results indicate that the substrate specificities of sialidases from swine influenza A viruses towards sialyl linkages and the molecular species of sialic acid are related to the year of isolation, i.e. strains isolated after 1978 exhibited higher activity towards Neu5Ac2-6Gal and Neu5Gc2-3Gal linkages when compared with strains isolated in an earlier year, 1930.Abbreviation Neu5Ac 5-N-acetylneuraminic acid - Neu5Gc 5-N-glycolyneuraminic acid - Gal d-galactose - Glc d-glucose - Cer Ceramide - II³(Neu5Ac)Lac Neu5Ac2-3Gal1-4Glc - GM3(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-4Glc1-Cer - GM3(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-4Glc1-Cer - GM1b(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-3GalNac1-4Gal1-4Glc1-Cer - GMlb(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-3GalNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Ac)nLc4Cer Neu5Ac2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Gc)nLc4Cer Neu5Gc2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV⁶(Neu5Ac)nLc4Cer Neu5Ac2-6Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - TDC taurodeoxycholate.     

Enhanced killing capacity of human Kupffer cells after activation with human granulocyte/macrophage-colony-stimulating factor and interferon γ

In this study we investigated the effect of the cytokines human granulocyte/macrophage-colony-stimulating Factor (hGM-CSF) and interferon (IFN) on human Kupffer-cell-mediated cytotoxicity against the SW948 coloncarcinoma cell line. Kupffer cells were isolated from small liver wedge biopsies, taken from 14 patient who had had abdominal surgery for colon carcinoma or partial hepatectomy. The cells were incubated with hGM-CSF (100 ng/ml), or with IFN (100 U/ml) or with their combination and the perecentage cytotoxicity was determined using a recently described modified assay. Additional experiments were performed with tumour-necrosis-factor-(TNF)-sensitive U937 cells as target. The TNF secretion of Kupffer cells was measured and we evaluated the effect of TNF on colon tumour targets. We performed human-Kupffer cell-mediated cytotoxicity blocking experiments with anti-TNF and used paraformaldehydefixed Kupffer cells to demonstrate lysis of TNF-sensitive WEHI-164 cells and of SW948 cells. The overall cytotoxicity against SW948 caused by unactivated Kupffer cells (n=14), and by Kupffer cells activated with hGM-CSF (n=14), IFN (n=6) or their combination (n=6) was respectively: 19.5±2.6%, 25.3±2.9% 41±9.4% and 45.6±8% at E/T=1 and 28.2±2.9%, 35.6±3.2%, 55.6±9.7% and 62.8% at E/T=5. All differences were statistically significant (P<0.05). No growth-promoting activity by hGM-CSF on the SW948 tumour cells was observed. U937 cells were highly susceptible to Kupffer-cell-mediated cytotoxicity. The TNF secretion by human Kupffer cells increased in parallel to their cytotoxicity after incubation with these cytokines. Soluble TNF had only a slight anti-proliferative effect on SW948 cells, while specific anti-TNF blocked Kupffer cell cytotoxicity by up to 80%. Finally, paraformaldehyde-fixed Kupffer cells were able to lyse WEHI-164 and SW948 cells. This indicates that expression of cell-associated TNF is the main cytolytic mechanism of human-Kupffer-cell-mediated cytotoxicity. The implications for the use of hGM-CSF and IFN in vivo are discussed.     

Efficient determination of angles subtended by Cα-Hα and N-HN vectors in proteins via dipole-dipole cross-correlation§

The angle CH,NHN subtended by the internuclear vectors 13C-H and 15N-HN in doubly-labeled proteins can be determined by observing the effect of cross-correlation between the dipolar interactions on zero- and double-quantum coherences involving 13C and 15N. Two complementary 2D experiments with the appearance of 15N-HN correlation spectra yield signal intensities that depend on the rate of interconversion through cross-correlated relaxation of in-phase and doubly antiphase zero- and double-quantum coherences. The ratio of the signal intensities in the two experiments bears a simple relationship to the cross-correlation rate, and hence to the angle CH,NHN. Assuming planarity of the peptide bond, the dihedral angle (between C and C) can be determined from the knowledge of CH,NHN. The experiments are very time-effective and provide good sensitivity and excellent spectral resolution.     

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.     

Effect of liquid culture on the growth and development of miniature rose (Rosa chinensis Jacq. ‘Minima’)

The growth of miniature rose (Rosa chinensis Jacq. Minima) shoots cultured on liquid medium was greater relative to those cultured on two-phase (solid + liquid) medium or solid medium alone. Shoot multiplication ratio (number of multiple shoots per explant per subculture) on liquid medium was higher with 17.8–26.6 M 6-benzyladenine at compared to that at 0–8.9 M. Shoots grown on 30 ml or more of liquid medium had a higher multiplication ratio than those grown on 10 or 20 ml. The growth and multiplication ratio increased when the culture period was extended from 3 to 6 weeks, although plantlets began to exhibit some chlorosis by the 6^th week. These conditions were maintained over four subcultures for cultivars Baby Katie, Lavender Jewel, Red Sunblaze and Royal Sunblaze, with no significant change in multiplication ratio over time.Abbreviations BA 6-benzyladenine - NAA 1-naphthaleneacetic acid     

RFLP markers to identify the alleles on the Mla locus conferring powdery mildew resistance in barley

Summary To identify the mildew resistance locus Mla in barley with molecular markers, closely linked genomic RFLP clones were selected with the help of near-isogenic lines having the Pallas and Siri background. Out of 22 polymorphic clones 3 were located around the Mla locus on chromosome 5 with a distance of 5.1 + 2.9 cM (MWG 1H068), 4.2±1.7 cM (MWG 1H060) and 0.7 ± 0.7 cM (MWG 1H036), respectively. The polymorphic clone MWG 1H036 displayed the same RFLP pattern in both Pallas and Siri near-isogenic lines and in different varieties digested with six restriction enzymes possessing the same mildew resistance gene. The alleles of the Mla locus were grouped in 11 classes according to their specific RFLP patterns; 3 of these groups contain the majority of Mla alleles already used in barley breeding programs in Europe.     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: a consequence of the formation of "native-like conformation".

The influence of n-propanol on the overall -helical conformation of -globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of -helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of -helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of -globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The -helical conformation induced into - and -globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the -helical conformation of both - and -chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced -helical conformation of globins and the -helical conformation of - and -chains. A cross-correlation of the n-propanol induced increase in the fluorescence of -globin with the corresponding increase in the -helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the -helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a native-like structure. The induction of -helical conformation into these proteins in the presence of n-propanol and the consequent generation of native-like conformation is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an -helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume -helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high -helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of -helical proteins. Consistent with this concept, the induction of -helical conformation into shorter polypeptide fragments of 30 residues, (e.g., _1-30, which exists in an -helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

Specific in vitro phosphorylation of plant eIF2α by eukaryotic eIF2α kinases

Phosphorylation of the subunit of eukaryotic initiation factor 2 (eIF2) is known to be an important translational control mechanism in all eukaryotes with the major exception of plants. Regulation of mammalian and yeast eIF2 activity is directly governed by specific phosphorylation on Ser-51. We now demonstrate that recombinant wheat wild-type (51S) but not mutant 51-Ala (51A) protein is phosphorylated by human PKR and yeast GCN2, which are defined eIF2 kinases. Further, only wheat wild-type eIF2 is a substrate for plant-encoded, double-stranded RNA-dependent kinase (pPKR) activity. Plant PKR and GCN2 phosphorylate recombinant yeast eIF2 51S but not the 51A mutant demonstrating that pPKR has recognition site capability similar to established eIF2 kinases. A truncated version of wild-type wheat eIF2 containing 51S but not the KGYID motif is not phosphorylated by either hPKR or pPKR suggesting that this putative eIF2 kinase docking domain is essential for phosphorylation. Taken together, these results demonstrate the homology among eukaryotic eIF2 species and eIF2 kinases and support the presence of a plant eIF2 phosphorylation pathway.     

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

A simple brassinolide analogue 2α, 3α-dihydroxy-17β-(3-methyl-butyryloxy)-7-oxa-B-homo-5α-androstan-6-one which induces bean second internode splitting

A new synthetic brassinolide analogue, 2,3-dihydroxy-17-(3-methylbutyryloxy)-7-oxa-B-homo-5-androstan-6-one (11), has been shown to exhibit typical brassinolide activity characterised by elongation, swelling, twisting and splitting of the bean second internode. It was prepared from the known lactone 2,3,17-trihydroxy-7-oxa-B-homo-5-androstan-6-one (4) which was transformed to an isopropylidenedioxy derivative. After protection of the 2- and 3-hydroxy groups it yielded the 2,3-isopropylidenedioxy-17-(3-methyl-butyryloxy)-7-oxa-B-homo-5-androstan-6-one (7) on treating with 3-methylbutyryl chloride in pyridine. The analogue with a 2-methylbutyric moiety (10, 2,3-dihydroxy-17-(2-methyl-butyryloxy)-7-oxa-B-homo-5-androstan-6-one) in position 17 stimulated only elongation and swelling of the bean second internode. However, in this bioassay 100 times more 10 or 11 compared to 24-epibrassinolide is required to obtain the same effects. Analogues with -oriented hydroxyl groups at C-2 and C-3 (14,15), a 6-ketone (17,18) or 6-oxa-7-oxo-lactone system (12,13) in ring B lack the typical brassinolide activity. In addition, the active brassinosteroids applied to the second internode stimulated a similar, but 30% lower elongation of the first internode. From data presented here we conclude that the presence of two hydroxy groups in the positions 22 and 23 of the brassinolide side chain, which are considered as a key structural requirement, is not absolutely necessary for a compound to exhibit typical brassinosteroid activity. Nevertheless, these compounds have generally 2–10 times lower activity than that having 22,23-vicinal diol in the side chain.     

Comparisons of a new triazole retardant PP 333 with ancymidol and other compounds on pot-grown tulips

PP 333 was compared with ancymidol, CGA 65993, dikegulac-sodium (as Atrinal) and maleic hydrazide for its ability to restrict stem extension in tulip cvs Paul Richter, Apeldoorn and Trance. Single, 300 ml compost drenches were applied one day after housing fully cooled bulbs grown in a sphagnum peat/sand compost (3:1 v/v).Experiments in 1979/1980 and 1980/1981 with mid- and late-season crops showed that PP 333, like ancymidol, could reduce stem extension without deleterious responses. However, higher amounts of PP 333 (0.8–33.3 mg a.i./pot) were required than of ancymidol (0.625–2.5 mg a.i./pot). Cv. Paul Richter was much less responsive to PP 333 and ancymidol than Apeldoorn, particularly when grown as a late-season crop. Dikegulac was the most effective chemical in the latter situation, especially as it restricted post-flowering extension growth. PP 333 and ancymidol were better able to control such growth in the mid-season crops. Other than for the above purpose, dikegulac proved unsuitable because it increased flower bud blasting and gave rise to abnormally coloured perianth segments. Similarly, marked reductions in stem length of Apeldoorn and Paul Richter with CGA 65993 were associated with unacceptable side-effects, namely, smaller flowers (both cvs) and more bud blasting in Apeldoorn. Maleic hydrazide (5–500 mg a.i./pot) had little influence on stem length in any of the three cultivars.The trials indicated the need to test each cultivar/retardant combination, as well as to take into account the time of forcing because, whereas Paul Richter and Apeldoorn were adequately dwarfed by PP 333 and ancymidol without adverse effects, both compounds caused about 50% of Trance flowers to blast. No treatment influenced flowering date in cv. Paul Richter but PP 333 delayed flowering by two days in Apeldoorn and Trance, as did the higher doses of ancymidol in Apeldoorn.     

Adrenergic regulation of ion transport by primary cultures of canine tracheal epithelium: Cellular electrophysiology

Summary We examined the effect of adrenergic agents on the cellular electrical properties of primary cultures of canine tracheal epithelium. Both isoproterenol and epinephrine stimulated Cl secretion, as evidenced by an increase in transepithelial voltage and a fall in transepithelial resistance. Moreover, both agents appear to increase the conductance of apical and basolateral membranes. However, the pattern of response was different. Isoproterenol initially depolarized apical voltage _a and decreased the fractional resistance of the apical membranef _R. These changes are consistent with an initial increase in apical Cl conductance. In contrast, epinephrine acutely hyperpolarized _a and increasedf _R, changes consistent with an initial increase in basolateral K conductance. Following the acute effect of epinephrine, _a depolarized andf _R decreased to values not significantly different from those observed with isoproterenol. The acute increase in basolateral K conductance produced by epinephrine appeared to result from stimulation of adrenergic receptors because it was reproduced by addition of the agonist phenylephrine, and blocked by the antagonist phentolamine. The ability of prazosin but not yohimbine to block the acute epinephrine-induced increase in K permeability indicates the presence of ₁ adrenergic receptors. The acute adrenergic-induced increase in basolateral K conductance may be mediated by an increase in cell Ca because the response was mimicked by addition of the Ca ionophore A23187. In contrast, the response to isoproterenol was similar to that observed with addition of 8-bromo-cAMP and theophylline. These results indicate that both and adrenergic agents mediate the ion transport processes in canine tracheal epithelium. adrenergic agents have their primary effect on the apical Cl conductance, probably via an increase in cAMP. adrenergic agents exert their primary effect on the basolateral K conductance, possibly via an increase in cell Ca.     

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.     

The inheritance of host plant resistance and its effect on the relative infection efficiency of Magnaporthe grisea in rice cultivars

The inheritance of host plant resistance and its effect on the relative infection efficiency for leaf blast was studied in the crosses IR36/CO39 (partially resistant × highly susceptible) and IR36/IR64 (both partially resistant). On the natural scale, gene action appeared multiplicative. After log transformation, additive effects described most of the genetic variation in the cross IR36/CO39, while additive and dominance effects were about equal in magnitude in the cross IR36/IR64. Dominance was towards increased resistance. No transgressive segregation occurred in the cross IR36/CO39. The number of genes that reduce lesion number was estimated to be zero in CO39 and five or more in IR36. The cross IR36/IR64 showed transgressive segregation in both directions, and IR36 and IR64 each contain at least one gene that is not present in the other cultivar. The heritabilities (narrow sense) in the F₂ were low (range 0.06–0.16), while narrow sense heritabilities based on F₃ lines were much higher (range 0.41–0.68). Lesion numbers in F₃ lines were reasonably correlated with those in F₅ progenies derived from the same F₂ plant (r was±0.6 in both crosses). Partial resistance can be effectively improved by selecting the most resistant plants from the most resistant F₃ lines.     

Identification and mapping of the Gli-R3 locus on chromosome 1R of rye (Secale cereale L.)

Summary The progenies of two different rye test-crosses were analyzed for secalin proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using unreduced and reduced aqueous ethanol extracts. Segregation for two high-molecular-weight secalin bands (Glu-R1 or Sec3), one -secalin band (Gli-R1 or Sec-1), two 40K -secalin bands (Gli-R1 or Sec1) and two -type secalin bands (new locus) were studied. One recombinant between - and -secalins was found in one test-cross. The new locus, designated Gli-R3 or Sec-4, was mapped between Glu-R1 and Gli-R1, more displaced towards Gli-R1. In test-cross 1 recombination between Glu-R1 and Gli-R3 was 33.80±3.22%, and between Gli-R3 and Gli-R1, 12.04±2.21%. In the other test-cross the map distances were relatively similar but smaller, likely due to less recombination within two different species of Secale. Genes coding for 40K -secalins at Gli-R1 were likely proximal to the centromere with respect to genes coding for -secalins at the same complex locus.     

Efficient and precise measurement of H(alpha)-C(alpha), C(alpha)-C', C(alpha)-C(beta) and H(N)-N residual dipolar couplings from 2D H(N)-N correlation spectra

A suite of experiments are presented for the measurement of H–C, C–C, C–C and H^N–N couplings from uniformly ¹⁵N, ¹³C labeled proteins. Couplings are obtained from a series of intensity modulated two-dimensional H^N–N spectra equivalent to the common ¹H–¹⁵N–HSQC spectra, alleviating many overlap and assignment issues associated with other techniques. To illustrate the efficiency of this method, H–C, C–C, and H^N–N isotropic scalar couplings were determined for ubiquitin from data collected in less than 4.5 h, C–C data collection required 10 h. The resulting couplings were measured with an average error of ±0.06, ±0.05, ±0.04 and ±0.10 Hz, respectively. This study also shows H–C and C–C couplings, valuable because they provide orientation of bond vectors outside the peptide plane, can be measured in a uniform and precise way. Superior accuracy and precision to existing 3D measurements for C–C couplings and increased precision compared to IPAP measurements for H^N–N couplings are demonstrated. Minor modifications allow for acquisition of modulated H^N–C 2D spectra, which can yield additional well resolved peaks and significantly increase the number of measured RDCs for proteins with crowded ¹H–¹⁵N resonances.