Primary structure of hemoglobin β-chain fromColumba livia (gray wild pigeon)

Primary structure of -chain of pigeon is presented. It was determined by amino acid sequence analysis of intact -chain and its peptides obtained by the enzymatic and chemical cleavage. Comparison of amino acid sequence of the chain with other available data shows 14 Ile, 61 Lys, and 113 Ile as residues specific to pigeon. One important replacement at ₁₁ contact is 55 MetSer.     

Initial Stages of Trisporic Acid Synthesis in Blakeslea trispora

Biotransformation of -carotene with enzyme preparations isolated from the mycelium of Blakeslea trispora resulted in the formation of its hydroxylated metabolite and apocarotenals, products of oxidative degradation of this compound. Based on its spectral, chromatographic, and chemical properties, the -carotene derivative was identified as 4-hydroxy--carotene (isocryptoxanthine). One of the products of oxidative degradation of -carotene, -apo-13-carotenone, was modified in the presence of enzyme preparations from Blakeslea trispora to form trisporic acid precursors. -Apo-13-carotenone transformation proceeded more rapidly than -carotene oxidation at the carbon atom at position 4. The data suggest that, under oxidative stress, oxidative degradation of -carotene into -apo-13-carotenone leads to the formation of considerable amounts of trisporic acids.     

Biotransformation of codeine to morphine in freely suspended cells and immobilized cultures of Spirulina platensis

Both freely suspended cells and immobilized cultures of Spirulina platensis, a blue-green alga, biotransformed exogenously fed codeine, an opium alkaloid, to morphine. The external addition of codeine to the culture medium did not affect the growth of S. platensis. Immobilization of Spirulina in a calcium alginate gel matrix was optimized by using 2% (w/v) sodium alginate and reducing the concentration of nutrients of Zarrouk's medium, which caused destabilization of the calcium alginate gel. The accumulation of morphine increased gradually and reached maxima of 330g 100ml^–1 culture at 105h in freely suspended and 351g 100ml^–1 at 96h in immobilized Spirulina cultures. Accumulation of morphine was detected only in the medium, whereas cells did not show accumulation. The immobilized Spirulina cultures showed marginally higher conversion of codeine to morphine over freely suspended cultures.     

Kinetics of Δψ-Dependent Sucrose Transport in Plasma Membrane Vesicles from Higher Plant Cells

The inside-out vesicles of plasma membranes were isolated from pumpkin stem cells, and the kinetics of sucrose efflux induced by the K⁺-diffusion potential (_D) was studied by measuring light transmission. Two phases differing in their rates and duration were identified in _D-dependent changes of light transmission. The increase in _Delevated the rate and magnitude of the fast phase in light transmission changes but did not markedly affect the rate of the slow phase. These two phases also differed in their sensitivity to inhibitors and to changes in sucrose concentration in the external medium. Measurements of _Dduring sucrose transport by means of the fluorescence probe dis-C₃-(5) revealed differences in the magnitude of _Dand its stability in vesicles loaded with sucrose and mannitol, as well as under the action of inhibitors. The two-phase dependence of sucrose efflux from vesicles on the applied diffusion potential is discussed in the context of modern concepts on the functioning of sucrose carriers in the membranes.     

Characterization of chromosomes with hybrid genes for Hb Lepore-Washington,Hb Lepore-Baltimore,Hb P-Nilotic,and Hb Kenya

Summary This study concerns the characterization of chromosomes with hybrid genes for Hb Lepore-Washington (44 chromosomes), for Hb Lepore-Baltimore (5 chromosomes), for Hb P-Nilotic (8 chromosomes), and for Hb Kenya (7 chromosomes) by determining a relatively large number of restriction enzyme polymorphism. Two, and possibly three, different Hb Lepore-Washington chromosomes were identified by specific haplotypes, while the haplotype of the Hb Lepore-Baltimore chromosome had its own characteristic pattern. A likely conclusion is that the crossovers leading to the formation of these chromosomes have occurred as independent events within the populations. Chromosomes with the -Lepore-Washington hybrid gene maintained specific characteristies (such as increased Hb F levels in heterozygotes, and high or low ^G values in this Hb F) which have been observed in normal individuals with chromosomes having comparable haplotypes. Only one haplotype was observed for each of the chromosomes carrying either the -P-Nilotic hybrid gene or the ^A hybrid gene of Hb Kenya.     

The molecular basis of HbH disease in Taiwan

Summary We have determined the molecular characteristics of -thalassemia in 12 HbH subjects from Taiwan by restriction endonuclease mapping with -and -specific probes. We have found four types of defects in the -thalassemia-2 genetic determinant: ^3.7 type I; ^4.2; ^CS; and ^T. All HbH subjects carried the ——^SEA genotype in the -thalassemia-1 determinant. At least two different subtypes of ——^SEA genotype were observed in this study.     

Resonance assignment of methionine methyl groups and χ3angular information from long-range proton—carbon and carbon—carbon J correlation in a calmodulin—peptide complex

Summary Several simple 3D experiments are used to provide J correlations between methionine C methyl carbons and either the CH₂ protons or C and C. The intensity of the J correlations provides information on the size of the three-bond J couplings and thereby on the ³ torsion angle. In addition, a simple 3D version of the HMBC experiment provides a sensitive link between the CH₃ methyl protons and C. The methods are demonstrated for a 20 kDa complex between calmodulin and a 26-residue peptide fragment of skeletal muscle myosin light chain kinase.     

The general occurrence of “plastid initials” and their development into plastids in cortical cells of woody plants in winter

Summary Electron microscopic studies revealed that plastid initials, presumed precursors of plastids, occur in cortical cells of the following plants studied in February and March: Betula ermanii Cham.; Prunus sargentii Rhed.; Pyrus communis L.; Ribes sinanense F. Maekawa; Salix matsudana Koidz. forma tortuosa Rhed.; and Sambucus sieboldiana var. miquelii Hara. Since plastid initials were found previously in Malus pumila Mill., Morus bombycis Koidz. and Populus euramericana cv. gelrica (Sagisaka 1991), plastid initials have been found in all woody plants examined to date. In P. euramericana cv. gelrica, at later stages of the development of the initials in March, the conglomerates of plastid initials became heterogeneous in terms of size, extent of thylakoid formation and ability to form starch granules. The formation of prolamellar structures was frequently observed in cells of Magnolia kobus var. borealis Sarg., which was sampled on April 19. These observations suggest the course of events in the development of the plastid initial and the continuity of the life of amyloplasts over a year in the life of woody plants.     

Gamma carbonic anhydrase like complex interact with plant mitochondrial complex I

We report the identification by two hybrid screens of two novel similar proteins, called Arabidopsis thaliana gamma carbonic anhydrase like1 and 2 (AtCAL1 and AtCAL2), that interact specifically with putative Arabidopsis thaliana gamma Carbonic Anhydrase (AtCA) proteins in plant mitochondria. The interaction region that was located in the N-terminal 150 amino acids of mature AtCA and AtCA like proteins represents a new interaction domain. In vitro experiments indicate that these proteins are imported into mitochondria and are associated with mitochondrial complex I as AtCAs. All plant species analyzed contain both AtCA and AtCAL sequences indicating that these genes were conserved throughout plant evolution. Structural modeling of AtCAL sequences show a deviation of functionally important active site residues with respect to CAs but could form active interfaces in the interaction with AtCAs. We postulate a CA complex tightly associated to plant mitochondrial complex.     

Nucleotide sequence of the maize chloroplast rpo B/C1/C2 operon: Comparison between the derived protein primary structures from various organisms with respect to functional domains

Summary The genes (rpo B/C₁/C₂) coding for the , , subnits of maize (Zea mays) chloroplast RNA polymerase have been located on the plastome and their nucleotide sequences established. The operon is part of a large inversion with respect to the tobacco and spinach chloroplast genomes and is flanked by the genes trnC and rps2. Notable features of the nucleotide sequence are the loss of an intron in rpoC₁, and an insertion of approximately 450 by in rpOC₂ compared to the dicotyledons tobacco, spinach and liver-wort. The derived amino acid sequence of this additional monocotyledon specific sequence is characterized by acidic heptameric repeat units containing stretches of glutamic acid, tyrosines and leucines with regular spacing. Other structural motifs, such as a nucleotide binding domain in the subunit and a zinc finger in the subunit, are compared at the amino acid level throughout the RNA polymerase subunits with the enzymes from other organisms in order to identify functionally important conserved regions.The sequence data presented in this paper will appear in the EMBL/Gen Bank/DDBJ Nucleotide Databases under the accession number X17318     

Patterns and conformations of commonly occurring supersecondary structures (basic motifs) in protein data bank

Short peptides connecting-helices and-strands have been analyzed in 240 proteins refined at resolutions of 0.25 nm or better. Connecting peptides of lengths between one and five residues have been classified as part of supersecondary motifs of four types:, , , and. Careful consideration has been given to the definition of secondary structures on the basis of hydrogen bonds and main-chain conformational angles. Using five classes of residue conformation—a, b, e, l, t—in the nonregular structure regions of, space, 34 classes of supersecondary motifs occurring at least five times have been identified. Among these 34 classes, 11 classes that occur more than 25 times are commonly occurring supersecondary structure motifs. The patterns and conformations of the 11 commonly occurring supersecondary structure motifs have been characterized, demonstrating that patterns and conformations adopted by supersecondary structure motifs are limited. The results have relevance to structure prediction, comparative modeling, and protein folding.     

Observations on the fine structure of the cercaria of Notocotylus attenuatus and formation of the cyst wall of the metacercaria

Summary The dorsal tegument of the mature cercaria of Notocotylus attenuatus is a syncytial, cytoplasmic layer, containing two types of secretory granule which are identifiable ultrastructurally. The type 1 secretory bodies are electron lucid, whereas most type 2 granules have a banded appearance. The ventral tegument contains granules which are secreted from the type 3 cells; the type 3 granules are membrane bound, electron dense, and consist of both an amorphous and a finely striated zone. The type 4 cells mainly contain cigar-shaped granules consisting of an amorphous core surrounded by concentric striations. The granules exhibit structural variability in shape and content. The type 4 cells undergo a cellular migration to the tegument during encystment. The structure of the posterior-lateral glands and mode of secretion of the granules are described. Possible functions of microtubules are discussed for each cell type. Details of some secretory processes involved in the formation of the hemispherical cyst wall are described. The layers of the cyst wall may be related to the granular contents of the various parenchymal cells of the cercaria. The tegument of the metacercaria originates primarily from the cytoplasm of the type 1, type 2, type 3 and type 4 cells.     

Secondary structural changes in the intact and the disulfide bridges cleaved beta-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

The relative proportions of -helix, -sheet, and unordered form in -lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of -lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% -helix and 41% -sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased -sheet up to 48% but did not affect the -helical proportion. The -helical proportions of nonreduced -lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the -helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The -sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.     

Further cytogenetic analyses of the Croatian triploid shallot “Ljutika” (Allium cepa var.viviparum, Alliaceae) and its comparison with the Indian triploid “Pran”

Feulgen and silver-stained karyotypes and meiosis of two triploid viviparous onion forms (Allium cepa var.viviparum), the Croatian Ljutika and the Indian Pran, were comparatively analyzed. The results of chromosome measurements show that Ljutika and Pran are karyologically not identical, although significant similarities were found in the morphology of their chromosomes. Five geographically distant clones of Ljutika showed good agreement in the number and gross morphology of the chromosomes and in the number and position of NORs and interphase nucleoli. Heterotrivalents were predominant in meiosis of Ljutika but a relatively high frequency of higher multivalents together with univalents and bivalents were also observed. The relationship between Ljutika and Pran and their possible origin are discussed.     

An NTP-binding motif is the most conserved sequence in a highly diverged monophyletic group of proteins involved in positive strand RNA viral replication

Summary NTP-motif, a consensus sequence previously shown to be characteristic of numerous NTP-utilizing enzymes, was identified in nonstructural proteins of several groups of positive-strand RNA viruses. These groups include picorna-, alpha-, and coronaviruses infecting animals and como-, poty-, tobamo-, tricorna-, hordei-, and furoviruses of plants, totalling 21 viruses. It has been demonstrated that the viral NTP-motif-containing proteins constitute three distinct families, the sequences within each family being similar to each other at a statistically highly significant level. A lower, but still valid similarity has also been revealed between the families. An overall alignment has been generated, which includes several highly conserved sequence stretches. The two most prominent of the latter contain the socalled A and B sites of the NTP-motif, with four of the five invariant amino acid residues observed within these sequences. These observations, taken together with the results of comparative analysis of the positions occupied by respective proteins (domains) in viral multidomain proteins, suggest that all the NTP-motif-containing proteins of positive-strand RNA viruses are homologous, constituting a highly diverged monophyletic group. In this group the A and B sites of the NTP-motif are the most conserved sequences and, by inference, should play the principal role in the functioning of the proteins. A hypothesis is proposed that all these proteins posses NTP-binding capacity and possibly NTPase activity, performing some NTP-dependent function in viral RNA replication. The importance of phylogenetic analysis for the assessment of the significance of the occurrence of the NTP-motif (and of sequence motifs of this sort in general) in proteins is emphasized.     

Characterization of a second carotenoid β-hydroxylase gene from Arabidopsis and its relationship to the LUT1 locus

Xanthophylls are oxygenated carotenoids that perform critical roles in plants. -carotene hydroxylases (-hydroxylases) add hydroxyl groups to the -rings of carotenes and have been cloned from several bacteria and plants, including Arabidopsis. The lut1 mutation of Arabidopsis disrupts -ring hydroxylation and has been suggested to identify a related carotene hydroxylase that functions specifically on -ring structures. We have used library screening and genomics-based approaches to isolate a second -hydroxylase genomic clone and its corresponding cDNA from Arabidopsis. The encoded protein is 70% identical to the previously reported Arabidopsis -hydroxylase 1. Phylogenetic analysis indicates a common origin for the two proteins, however, their different chromosomal locations, intron positions and intron sizes suggest their duplication is not recent. Although both hydroxylases are expressed in all Arabidopsis tissues analyzed, -hydroxylase 1 mRNA is always present at higher levels. Both cDNAs encode proteins that efficiently hydroxylate the C-3 position of -ring containing carotenes and are only weakly active towards -ring containing carotenes. Neither -hydroxylase cDNA maps to the LUT1 locus, and the genomic region encompassing the LUT1 locus does not contain a third related hydroxylase. These data indicate that the LUT1 locus encodes a protein necessary for -ring hydroxylation but unrelated to -hydroxylases at the level of amino acid sequence.     

Biosynthesis of RNA polymerase in Escherichia coli

Summary Effect of temperature-sensitive, assembly-defective mutations in Escherichia coli RNA polymerase (rpoB) or subunit gene (rpoC) was investigated on the expression of wild-type rpoB ⁺C⁺operon, which was introduced by infection of a lambda transducing phage drif ⁺ (rpoB ⁺)-6 after UV-irradiation of the mutant cells. In rpoB2·rpoB7 strain which accumulates assembly-intermediates, free , ₂ complex and premature core, the expression of rpoB ⁺C⁺operon measured by the rate of subunit synthesis was considerably inhibited whereas that of EF(translation elongation factor)-Tu, ribosomal proteins L1 and L7/L12, and some -coded proteins remained unaffected. On the other hand, the expression was enhanced specifically for only rpoB ⁺C⁺operon in either rpoC4 or rpoC1 mutants, which are defective in the association of ₂ complex and subunit or the activation of premature core enzyme, respectively. Upon preincubation of the mutant cells at 42° C prior to phage infection, during which assembly intermediates degraded rapidly, the rate of subunit synthesis relative to other phage-corded proteins increased remarkably in rpoB2·rpoB7 mutant as well as in rpoC4 and rpoC1 mutants. These observations strongly suggested the autogenous regulation for at least (rpoB ⁺C⁺) operon by some trans-active diffusible protein complexes built of RNA polymerase subunits. Nature of the regulatory molecules is discussed.Paper VI in this series is Saitoh and Ishihama (1977)     

Anti-u.v. activity of lignin biopolymers on Euglena gracilis

Sulphur-free lignin biopolymer and its oxidized and reduced derivatives have been prepared and their inhibitory activity against u.v.-induced mutagenesis in Euglena gracilis was evaluated. The structure- and dose-dependent anti-u.v. activity of lignins was observed at concentrations higher than 250gml^–1. The oxidized lignin showed the most antimutagenic activity, followed by the reduced lignin and the unmodified lignin had the least antimutagenic activity.     

Structure-function relationships in human α- and γ-thrombins

Summary Human pro-coagulant -thrombin may be proteolyzed under controlled conditions to the non-coagulant - and -thrombin forms. These derivative forms nonetheless retain esterase and amidase activities with small substrates as well as several other thrombin functions. Structurally, human -thrombin consists of three non-covalently associated fragments which retain structural integrity as measured by several spectroscopic criteria as well as enzymatic function. The protein folding characteristics of three-chain -thrombin indicate that each fragment (domain) contains sufficient information to result in a correct renaturation of protein conformation. Those subtle structural differences which distinguish - from -thrombin are most likely the obstructions to fibrinogen binding which account for the loss of clotting activity.     

RNA polymerase subunit biosynthesis in Bacillus subtilis

Summary The relative rates of RNA polymerase biosynthesis in Bacillus subtilis has been examined under steady-state growth conditions. The synthesis of RNA polymerase subunits (, , , ) has been followed by subunit fractionation of immunoprecipitated [³H]-labelled samples on SDS-polyacrylamide gels. The stoichiometries of ::: subunits have been determined from cultures pulse-labelled during steady-state growth. The results suggest that an unassembled pool of the -subunit exists from which the holoenzyme is formed.Upon shift-up from acetate to glycerol containing medium, a rapid rise in the differential rate of core enzyme synthesis was observed, while the rate of synthesis of the -subunit was not stimulated. During shift-down, a concomitant reduction in the rate of synthesis of all subunits occurred for the first 20 min after the shift; thereafter, a rate of synthesis characteristic of the new growth rate was established.As cultures enter sporulation, an immediate reduction in the rate of -subunit synthesis was demonstrated.