Biochemical and genetic studies on rabbit hemoglobin. II. Electrophoretic polymorphism of theα-chain

A genetic polymorphism of rabbit (Oryctolagus cuniculus) hemoglobin chain is demonstrated by means of acid starch gel electrophoresis. The polymorphism is not detected by isoelectric focusing and may be based upon neutral for neutral amino acid substitutions in accordance with previous findings by means of amino acid sequencing. Segregation analysis was performed on 15 matings with 49 offspring and confirmed the initial genetic hypothesis of three common codominant alleles at an autosomal locus. The calculated gene frequencies in a random sample of 86 unrelated individuals areHBA*1=0.73,HBA*2=0.22, andHBA*3=0.05.     

Inheritance and biochemical analysis of four electrophoretic variants of β-conglycinin from soybean

Summary Three genes which code for variant -conglycinin subunits were identified. Alleles Cgy ₁ ^S and Cgy ₂ ^S were codominant with Cgy ₁ and Cgy ₂ and produced and subunits, respectively, with reduced electrophoretic mobility. Allele Cgy ₃ ^D increased the mobility of at least one polypeptide in the subunit family and exhibited incomplete dominance. Gene loci Cgy ₂/Cgy ₂ ^S and Cgy ₃ ^D /cgy ₃ ^D were linked, whereas Cgy ₁/Cgy ₁ ^S / cgy ₁ segregated independently of the others. Techniques developed for purification of normal -conglycinin subunits were effective in purifying the altered subunits. Deglycosylated variant proteins from seeds containing the alleles Cgy ₁ ^S , Cgy ₂ ^S , or Cgy ₃ ^D also has altered mobility relative to deglycosylated normal proteins. Therefore, the altered subunits contained changes in their amino acid sequences rather than in their carbohydrate moieties. This interpretation is consistent with the observed codominant or incompletely dominant mode of inheritance for these alleles and suggests that each contains an altered nucleotide sequence in the structural gene. A fourth variant, which exhibited doublet and a electrophoretic bands, was inherited in a recessive fashion. Deglycosylated subunit proteins from this variant were identical in electrophoretic mobility to those of the deglycosylated normal protein. This suggests that the doublet phenotype resulted from an alteration in the carbohydrate moiety of these subunits. The gene or genes which condition this variant presumably are required for normal post-translational modification of the subunit carbohydrates and as such may be useful for investigating these events.Cooperative research of USDA-ARS and the Indiana Agric. Exp. Stn., Purdue Univ., West Lafayette, IN 47907, USA. Indiana Agric. Exp. Stn. Journal Article 10,323. Financial support from the American Soybean Research Foundation is gratefully acknowledged     

HMW and LMW glutenin alleles among putative tetraploid and hexaploid European spelt wheat (<Emphasis Type="Italic">Triticum spelta</Emphasis> L.) progenitors

The allelic compositions of high- and low-molecular-weight subunits of glutenins (HMW-GS and LMW-GS) among European spelt (Triticum spelta L.) and related hexaploid and tetraploid Triticum species were investigated by one- and two-dimensional polyacrylamide-gel electrophoresis (PAGE) and capillary electrophoresis (CE). A total of seven novel glutenin alleles (designated A1a*, B1d*, B1g*, B1f*, B1j*, D1a* at Glu-1 and A3h at the Glu-3 loci, respectively) in European spelt wheat were detected by SDS-PAGE, which were confirmed further by employing A-PAGE and CE methods. Particularly, two HMW-GS alleles, Glu-B1d* coding the subunits 6.1 and 22.1, and Glu-B1f* coding the subunits 13 and 22*, were found to occur in European spelt with frequencies of 32.34% and 5.11%, respectively. These two alleles were present in cultivated emmer (Triticum dicoccum), but they were not observed in bread wheat (Triticum aestivum L.). The allele Glu-B1g* coding for 13* and 19* subunits found in spelt wheat was also detected in club wheat (Triticum compactum L.). Additionally, two alleles coding for LMW-GS, Glu-A3h and Glu-B3d, occurred with high frequencies in spelt, club and cultivated emmer wheat, whereas these were not found or present with very low frequencies in bread wheat. Our results strongly support the secondary origin hypothesis, namely European spelt wheat originated from hybridization between cultivated emmer and club wheat. This is also confirmed experimentally by the artificial synthesis of spelt through crossing between old European emmer wheat, T. dicoccum and club wheat, T. compactum.Communicated by H.F. Linskens     

Side reactions in the synthesis of phosphotyrosine-containing peptides

Summary A series of phosphopeptides Tyr(PO₃H₂)-Val-Pro-Xxx-Leu (Xxx=Met, Met(O), Nle, Dab or Cys), derived from the native platelet-derived growth factor- receptor (PDGF-) sequence, has been prepared to study their interaction with the src-homology 2 (SH2) domains of the p85 subunit of PI3 kinase. The phosphopeptides were synthesized using Fmoc methodology incorporating N-Boc dibenzyl-protected phosphotyrosine (Boc-Tyr[PO₃(Bzl)₂]) as the N-terminal amino acid, since the benzyl groups can be removed during resin cleavage with TFA. Only peptides containing methionine were found to exist partially as S-benzyl sulfonium salts after TFA cleavage from the resin. The desired peptide could be obtained from the S-benzyl sulfonium salt by hydrogenolysis.     

Expression of Streptomyces genes encoding extracellular enzymes in Brevibacterium lactofermentum: secretion proceeds by removal of the same leader peptide as in Streptomyces lividans

The -amylase gene (amy) from Streptomyces griseus IMRU 3570 and the -galactosidase gene (lac) from S. lividans were subcloned into Brevibacterium lactofermentum or B. lactofermentum/Escherichia coli shuttle vectors. The amy gene was not expressed in B. lactofermentum from its own promoter but was efficiently expressed when the promoter of the kanamycin resistance gene (kan) was inserted upstream of the promoterless amylase gene. The lac gene from S. lividans was subcloned without its native promoter and was expressed when placed downstream of pBL1 promoters P₂ or P₃. The -amylase was secreted extracellularly by removal of the same 28-amino acid leader peptide as in S. lividans. The amy and lac genes provide useful markers for selection of transformants and will facilitate the study of protein secretion in B. lactofermentum. Correspondence to: J. F. Martín     

Sequence variability and gene structure at the self-incompatibility locus of Solanum tuberosum

Summary Allelic complexity is a key feature of self-incompatibility (S) loci in gametophytic plants. We describe in this report the allelic diversity and gene structure of the S locus in Solanum tuberosum revealed by the isolation and characterization of genomic and cDNA clones encoding S-associated major pistil proteins from three alleles (S ₁, S _r1, S ₂). Genomic clones encoding the S₁ and S₂ proteins provide evidence for a simple gene structure: Two exons are separated by a small intron of 113 (S ₁) and 117 by (S ₂). Protein sequences deduced from cDNA clones encoding S₁ and S_r1 proteins show 95% homology. 15 of the 25 residues that differ between these S ₁and S _r1alleles are clustered in a short hypervariable protein segment (amino acid positions 44–68), which corresponds in the genomic clones to DNA sequences flanking the single intron. In contrast, these alleles are only 66% homologous to the S ₂allele, with the residues that differ between the alleles being scattered throughout the sequence. DNA crosshybridization experiments identify a minimum of three classes of potato S alleles: one class contains the alleles S ₁, S _r1and S ₃, the second class S ₂and an allele of the cultivar Roxy, and the third class contains at present only S ₄. It is proposed that these classes reflect the origin of the S alleles from a few ancestral S sequence types.     

Blood-gas,acid-base and catecholamine responses to lactic acid infusion in larval and adult Ambystoma tigrinum

Larval and adult Ambystoma tigrinum were subjected to acidosis by infusing lactic acid (2 M·g^-1) into the peritoneal cavity. Blood was sampled at intervals to establish the time-course of the ensuing acidosis. Both larvae and adults became significantly acidotic after 1 h. The larval acidosis was more pronounced (-4 pH units versus-2 pH units) than adults due to greater extracellular buffering capacity (higher [HCO₃ ^-]) in adults. Both forms recovered in about 8 h. Larvae showed a typical increase in circulating norepinephrine during the initial stages of the acidosis; adults did not, having significantly lower norepinephrine titer than larvae during the acidosis. Both larvae and adults showed transient increases in PO₂ during the acidosis. The ₁ and ₂ antagonists, timolol and butoxamine respectively, (0.2 g·g^-1) were administered to separate groups of larvae. Butoxamine (₂) delayed the recovery from the acidosis by prolonging the increase in arterial PCO₂ and reversing the recovery of [HCO₃ ^-]. Timolol (₁) did not delay recovery. We conclude that ₂ receptors are involved in the catecholamine responses to acidosis in larvae. Catecholamines appear not to play the same role in adult acid-base disturbances as they seem to in larvae.Abbreviations RBC red blood cell     

Recombinant Thymosin α1

An artificial gene encoding thymosin ₁ was obtained by chemoenzymatic synthesis and cloned into Escherichia coli. An expressing recombinant plasmid containing the hybrid protein gene, which encodes amino acid sequences of thymosin ₁ and the Saccharomyces cerevisiae intein Sce VMA, was constructed. The expression of the hybrid protein from the resulting hybrid gene in E. coli, the properties of the resulting hybrid protein, and the conditions for its nonenzymatic cleavage to thymosin ₁ were studied.     

Relative mobility and activity of leaf malate dehydrogenase in flax(Linum usitatissimum) genotrophs and genotypes

Malate dehydrogenase (MDH) band relative mobility (R _m) and activity were examined in leaf extracts of Durrant's flax genotrophs, L and S, and flax genotypes, R and M. MDH activity in leaves from just below the inflorescence was higher in the two smaller, sparsely branched plant types, S and M, than in the larger, more branched plant types, L and R. The MDH electrophoretic banding pattern in flax leaf extracts consisted of three major anionic bands, MDH-1, MDH-2, and MDH-3. NoR _m differences were detected between corresponding isozymes of genotypes R and M. For the genotrophs, however, all three bands of S migrated faster than the corresponding bands of L. Codominance was absent in F₁ hybrids; SR _m was dominant for MDH-2 and MDH-3 and LR _m was dominant for MDH-1. The observations suggest that MDHR _m in L and S may be controlled by a modifier locus (or loci). Previous studies indicate that a modifier locus may also control heritable genotrophic differences in peroxidase (PER) and acid phosphates (AP)R _m. The three enzyme systems are compared.The financial assistance of the Natural Sciences and Engineering Research Council of Canada is acknowledged with thanks.     

Biochemical characteristics of guanine nucleotide binding protein alpha-subunit recombinant protein and three mutants: investigation of a domain motion involved in GDP-GTP exchange

Our previous studies using molecular dynamics have shown a hinge bending motion between the helical and the GTPase domains of G_T (Mello et al., 1998). The hypothesis that this motion is allowed by residues Gly56 and Gly179 and that this motion may affect the ligand exchange was tested in this work. Mutations of Gly 56 were carried out and the mutant proteins were expressed in Sf9 cells using the Baculovirus expression system. The recombinant proteins were purified using Ni-NTA affinity chromatography. The results for the (GDP/GTP) exchange assays showed that G56S and double mutants (D55G/G56S) proteins differ significantly from the wild type and D55G mutant forms. The K _d values for GTPS binding of those mutants have decreased by approximately 10-fold. No difference in the GTPase activity was detected for the mutants. Thus, the biochemical results obtained support the conclusions of the computational studies.     

Activities of growth inhibitors isolated from light-grown dwarf pea shoots

The growth inhibitory activities of 6 endogenous growth inhibitors isolated from light-grown dwarf peas (Pisum sativum cv. Progress No. 9) were examined in the epicotyl of dark-grown seedlings of the same cultivar in the dark in order to examine the possible contribution of these compounds to the growth inhibition brought about by red light. The activities of these natural inhibitors, including two A-2 and A-2 of as yet undetermined structure, were compared with those of synthetic growth retardants and benzyladenine. Samples were applied directly into the epicotyls via a glass capillary tube. In 24-h tests doses for a 25% inhibition (I₂₅) were: A-2, 4.3 × 10^-2: cis-xanthoxin, 1.2 × 10^-1 ; A-2, 1.6 × 10^-1; trans-xanthoxin, 1.2; R,S-dihydromaleimide, 3.5 × 10² and pisatin, 4.0 × 10² nmol plant^-1 . In 72-h tests, I₂₅'s were: benzyladenine, 1.5; AMO-1618 (ammonium-(5-hydroxycarvacryl)-trimethylchloride piperidine carboxylate), 2.4; R,S-dihydromaleimide, 4.0 × 10² and CCC (chlorocholine chloride), 1.1 × 10³ nmol plant^-1. -D-Glucosyl-R-dihydromaleimide had no activity at all. Benzyladenine caused the thickening as well as elongation inhibition of the epicotyls of intact plants. The possible involvement of A-2 and in the red light growth inhibition of dwarf peas is discussed.Abbreviations AMO-1618 ammonium-(5-hydroxycarvacryl)-trimethylchloride piperidine carboxylate - CCC chlorocholine chloride - G-DHMD -D-glucosyl-R-dihydromaleimide - I₂₅ dose required for a 25% growth inhibition - R red light author for correspondence     

Studies on metabolic role of 5′-methylthioadenosine in Ochromonas malhamensis and other microorganisms

Several compounds containing a thiomethyl group were found to replace vitamin B₁₂ in a protozoan, Ochromonas malhamensis. The order of the effectiveness was as follows: 5-methylthioadenosine > S-adenosylmethionine > 5-methylthioribose > L-methionine. A similar order was obtained with respect to the permeability of these compounds into the protozoan cells, except for S-adenosylmethionine. 5-Methylthioadenosine and 5-methylthioribose as well as l-methionine markedly increased the intracellular content of l-methionine. The level of S-adenosylmethionine was also increased by them, but to a lesser degree. The thiomethyl group of the compounds was established to be incorporated into S-adenosylmethionine. The metabolic fate of the thiomethyl group of 5-methylthioadenosine cannot be distinguished from that of l-methionine. A high activity of 5-methylthioadenosine nucleosidase was detected in the cell-free extracts of the protozoan. These results strongly suggest that 5-methylthioadenosine would be metabolized to l-methionine via 5-methylthioribose and then the l-methionine would be converted to S-adenosylmethionine. Like l-methionine and vitamin B₁₂, 5-methylthioadenosine and 5-methylthioribose may play an important role in maintenance of the C-1 pool in Ochromonas malhamensis.Neither 5-methylthioadenosine nor 5-methylthioribose replaced vitamin B₁₂ in some vitamin B₁₂-requiring bacteria. This result is consistent with the fact that neither compounds was significantly taken up by these bacteria.Abbreviations MTA 5-methylthioadenosine - AdoMet S-adenosylmethionine - MTR 5-methylthioribose - TCA trichloroacetic acid Paper II in the series. The first paper of the series has been published (Sugimoto and Fukui, 1974)     

HLA class II alleles and measles virus-specific cytokine immune response following two doses of measles vaccine

Measles virus-specific T cells and the production of cytokines play a critical role in the immune response following measles immunization. To understand the genetic factors that influence variation in IFN- and IL-4 responses following measles immunization and to provide insight into the factors influencing both cellular and humoral immunity to measles, we assessed associations between human leukocyte antigen (HLA) class II genes and measles-specific Th1 and Th2-type cytokine responses in peripheral blood lymphocytes from 339 children previously vaccinated with two doses of measles-mumps-rubella vaccine (MMR-II). Median values for measles-specific IFN- and IL-4 secretion levels were 40.73 and 9.71 pg/ml, respectively. The global tests suggested associations between measles-specific IFN- response and alleles of the DRB1 and DQB1 loci (P=0.07 and P=0.02, respectively). Specifically, DRB1*0301, *0901, and *1501 alleles were significantly associated with IFN- secretion. The alleles that suggested evidence of an HLA association with IL-4 secretion were DRB1*0103, *0701, and *1101. Th1 cytokine responses and DQB1 allele associations revealed that the alleles with the strongest association with IFN- secretion were DQB1*0201, *0303, *0402, and *0602. Specific alleles with a suggestive association with low measles-specific Th2 cytokine responses were DQB1*0202 and *0503. In addition, DPB1*0101, *0201, and *0601 alleles provided suggestive evidence of an HLA association with measles-induced IFN- response, while DPB1*0501 was associated with an IL-4 response. These data suggest that IFN- and IL-4 cytokine responses to measles may be genetically restricted in part by HLA class II genes, which in turn can restrict the cellular immune response to measles vaccine.     

Purification and characterization of β‐methylaspartase from Fusobacterium varium

Methylaspartase (EC 4.3.1.2) was purified 20fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAESepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg²⁺ (K_m = 0.27 ± 0.01 mM) and K⁺ (K_m = 3.3 ± 0.8 mM) for maximum activity. Methylaspartasecatalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)3methylaspartate (major product) and (2S,3R)3methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)3methylaspartate (K_m = 0.51 ± 0.04 mM) was observed at pH 9.7. The Nterminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.     

Characterization of the G protein coupling of SRIF and β-adrenergic receptors to the maxi KCa channel in insulin-secreting cells

Modulation of the Ca- and voltage-dependent K channel—K_Ca—by receptors coupled to the G proteins G _i /G _o and G _s has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches somatostatin (somatotropin-releasing inhibitory factor; SRIF) caused concentration-dependent inhibition of the K_Ca channel, an effect that was prevented by pertussis toxin (PTX). In inside-out patches, exogenous subunits of either G _i or G _o -type G proteins also inhibited the K_Ca channel (IC₅₀ 5.9 and 5.7 pM, respectively). These data indicate that SRIF suppresses K_Ca channel activity via a membrane-delimited pathway that involves the subunits of PTX-sensitive G proteins G _i and/or G _o . In outside-out patches, activation of G _s either by -agonists or with cholera toxin (CTX) increased K_Ca channel activity, consistent with a membrane-delimited stimulatory pathway linking the -adrenergic receptor to the K_Ca channel via G _s . In outside-out patches, channel inhibition by SRIF suppressed the stimulatory effect of -agonists but not that of CTX, while in inside-out patches CTX reversed channel inhibition induced by exogenous _i or _o . Taken together these data suggest that K_Ca channel activity is enhanced by activation of G _s and blocked by activated G _i and/or G _o . Further, K_Ca channel stimulation by activated G _s may be direct, while inhibition by G _i /G _o may involve deactivation of G _s . In inside-out patches K_Ca channel activity was reduced by an activator of protein kinase C (PKC) and enhanced by inhibitors of PKC, indicating that PKC also acts to inhibit the K_Ca channel via a membrane delimited pathway. In outside-out patches, chelerythrine, a membrane permeant inhibitor of PKC prevented the inhibitory effect of SRIF, and in inside-out patches PKC inhibitors prevented the inhibitory effect of exogenous _i or _o . These data indicate that PKC facilitates the inhibitory effect of the PTX-sensitive G proteins which are activated by coupling to SRIF receptors. To account for these results a mechanism is proposed whereby PKC may be involved in G _i /G _o -induced deactivation of G _s .The authors would like to thank Dr. S. Ciani for many helpful discussions, Dr. A.E. Boyd III for supplying the HIT cells, Drs. J. Codina and L. Birnbaumer for supplying the alpha subunits of the G proteins G _i and G _o , and Mrs. Satoko Hagiwara for preparing and maintaining the cell cultures.This work was supported by grant DCB-8919368 from the National Science Foundation and a research grant (W-P 880513) from the American Diabetes Association to B.R., and by grant RO1-DK39652 from the National Institutes of Health to G.T.E.     

Role of fructose-1,6-bisphosphatase, fructose phosphotransferase, and phosphofructokinase in saccharide metabolism of four C3 grassland species under elevated CO2

We studied the effects of 15-months of elevated (700 μmol mol⁻¹) CO₂ concentration (EC) on the CO₂ assimilation rate, saccharide content, and the activity of key enzymes in the regulation of saccharide metabolism (glycolysis and gluconeogenesis) of four C₃ perennial temperate grassland species, the dicots Filipendula vulgaris and Salvia nemorosa and the monocots Festuca rupicola and Dactylis glomerata. The acclimation of photosynthesis to EC was downward in F. rupicola and D. glomerata whereas it was upward in F. vulgaris and S. nemorosa. At EC, F. rupicola and F. vulgaris leaves accumulated starch while soluble sugar contents were higher in F. vulgaris and D. glomerata. EC decreased pyrophosphate-D-fructose-6-phosphate l-phosphotransferase (PFP, EC 2.7.1.90) activity assayed with Fru-2,6-P₂ in F. vulgaris and D. glomerata and increased it in F. rupicola and S. nemorosa. Growth in EC decreased phosphofructokinase (PFK, EC 2.7.1.11) activity in all four species, the decrease being smallest in S. nemorosa and greatest in F. rupicola. With Fru-2,6-P2 in the assay medium, EC increased the PFP/PFK ratio, except in F. vulgaris. Cytosolic fructose-1,6-bisphosphatase (Fru-1,6-P₂ase, EC 3.1.3.11) was inhibited by EC, the effect being greatest in F. vulgaris and smallest in F. rupicola. Glucose-6-phosphate dehydrogenase (G6PDH EC 1.1.1.49) activity was decreased by growth EC in the four species. Activity ratios of Fru-1,6-P₂ase to PFP and PFK suggest that EC may shift sugar metabolism towards glycolysis in the dicots.     

The S11 and S13 self incompatibility alleles in Solanum chacoense Bitt. are remarkably similar

A genomic clone of the S11 allele from the self-incompatibility locus (S locus) in Solanum chacoense Bitt. has been isolated by cross-hybridization to the S. chacoense S13 allele and sequenced. The sequence of the S11 allele contains all the features expected for S genes of the Solanaceae, and S11 expression, as assessed by northern blots and RNA-PCR, was similar to that of other S. chacoense S alleles. The S11 protein sequence shares 95% identity with the phenotypically distinct S13 protein of S. chacoense and is the gametophytic S allele with the highest similarity to an existing allele so far discovered. Only 10 amino acid changes differentiate the mature proteins from these two alleles, which sets a new lower limit to the number of changes that can produce an altered S allele specificity. The amino acid substitutions are not clustered, suggesting that an accumulation of random point mutations can generate S allele diversity. The S11 intron is unusual in that it could be translated in frame with the coding sequence, thus suggesting an additional mechanism for the generation of new S alleles.     

Molecular analysis of two functional homologues of the S 3 allele of the Papaver rhoeas self-incompatibility gene isolated from different populations

The S ₃ allele of the S gene has been cloned from Papaver rhoeas cv. Shirley. The sequence predicts a hydrophilic protein of 14.0 kDa, showing 55.8% identity with the previously cloned S ₁ allele, preceded by an 18 amino acid signal sequence. Expression of the S ₃ coding region in Escherichia coli produced a form of the protein, denoted S₃e, which specifically inhibited S₃ pollen in an in vitro bioassay. The recombinant protein was ca. 0.8 kDa larger than the native stigmatic form, indicating post-translational modifications in planta, as was previously suggested for the S₁ protein. In contrast to other S proteins identified to date, S₃ protein does not appear to be glycosylated. Of particular significance is the finding that despite exhibiting a high degree of sequence polymorphism, secondary structure predictions indicate that the S₁ and S₃ proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the P. rhoeas S alleles share some limited homology with the SLG and SRK genes from Brassica oleracea. Previously, cross-classification of different populations of P. rhoeas had revealed a number of functionally identical alleles. Probing of western blots of stigma proteins from plants derived from a wild Spanish population which contained an allele functionally identical to the Shirley S ₃ allele with antiserum raised to S₃e, revealed a protein (S ₃ s) which was indistinguishable in pI and M _r from that in the Shirley population. A cDNA encoding S ₃ s was isolated, nucleotide sequencing revealing a coding region with 99.4% homology with the Shirley-derived clone at the DNA level, and 100% homology at the amino acid level.