Origin and evolution of the sulawesi macaques 2. complete amino acid sequences of seven β chains of three molecular types

Seven β chains were identified as the typical molecular types carried by the seven species of Sulawesi macaques based on isoelectric focusing and urea starch gel electrophoresis. These β chains include the β3 chains ofmaura, tonkeana, nigra, andbrunnescens, β1 chains ofhecki andochreata and β5 chain ofnigra. The results of chromatography on cation-exchange and reversed phase columns and the amino acid compositions of the tryptic peptides suggested substitutions at the 9th and 13th amino acids from the N-terminal. Sequence analyses of these seven β chains from the N-terminal to the 18th amino acid and those of purified tryptic peptides from βT3 to βT15 by Edman degradation revealed the following facts: (1) the amino acid sequences of the β3 chains carried by the four species coincided with each other and as did those of the β1 chains of the two named species; and (2) the 9th and 13th amino acids were Lys and Thr in β3, Asn and Asn in β1, and Asp and Thr in the β5 chain, respectively. These three β chains are related with each other by at least two-base changes. The evolution of the β chains of the Sulawesi macaques was inferred to be as follows. (1) The β3 chain might have been dominant β chain in the past among Sulawesi macaques, since peripheral species separately carried this chain; (2) the β1 and β5 chains might have derived from a “missing link” because of more than two-base substitutions between β3 and β1 and between β3 and β5; (3) eight other macaque species, including the lion-tailed macaque (M. silenus), bear Asn and Thr at these two positions, while the Barbary macaque (M. sylvanus) has Thr and Thr; and (4) thus, if the parsimonious rule is followed, the type with Asn-Thr is the most plausible “missing link,” since only the Asn-Thr type can combine these five β chains by minimum one-base change. Two genetic events are postulated in the evolutionary process of the Sulawesi β chains: the first Lys-Thr type (β3) was distributed over the whole island, and next Asn-Thr, the common type in other macaques, produced Asn-Asn (β1) and Asp-Thr (β5).     

The amino acid sequences of two large glycopeptides derived from the carbohydrate-carrying region of α1-acid glycoprotein

Specific fragmentation with cyanogen bromide and subsequent reduction and carboxymethylation of α₁-acid glycoprotein, a normal human plasma globulin, permitted isolation of a large fragment which was shown to represent the amino-terminal half and to contain the total carbohydrate moiety of this protein. The amino acid sequences of two large glycopeptides derived from this fragment were established. One glycopeptide was composed of 22 amino acid residues and one carbohydrate unit, and the other consisted of 65 amino acid residues and carried four carbohydrate units.     

The amino acid sequences of the α subunits of the lectins from the seeds of Lathyrus hirsutus and Lathyrus tingitanus

The amino acid sequences of the α₁ and α₂ subunits of the isolectins (LhL1 and LhL2) from seeds of Lathyrus hirsutus and the α subunit of the lectin from L. tingitanus were determined by analysis of peptides derived from the proteins by separate digestions with chymotrypsin and the protease from S. aureus V8. The α₁ subunit of the L. hirsutus LhL1 isolectin differed from the α₂ form in LhL2 only in having an extra lysine inserted near the C-terminus. The L. tingitanus α subunit differed from the L. hirsutus α₁ in three positions and from L. hirsutus α₂ in four.     

Influence of cation–π interactions to the structural stability of prokaryotic and eukaryotic translation elongation factors

We have investigated the role of cation–π interactions on translation elongation factors. In our investigation, an average of four significant cation–π interactions were found, that is, an average of one cation–π interaction per 44 residues in the ten elongation factors were observed. The analysis on the influence of short (<±4), medium (>±4 to <±20) and long (>20) range contacts showed that cation–π interactions are mainly formed by medium and long-range contacts. Arg-Tyr pair was found largest in number but energetic contribution of Arg-Trp pair was found most. Preferred secondary structural conformation analysis of the residues involved in cation–π interaction indicates that the cationic Arg prefers to be in helix and Lys having equal probability for helix and strand, whereas the aromatic Phe and Trp were found mostly in helix while Tyr in strand regions. The cation–π interaction residues involved in these proteins were found highly conserved with 48.86% residues having conservation score of ≥6. Analysis of secondary structure preference of the energetically significant cation–π residues in different solvent accessible range indicates that most of the π residues are found buried or partially buried whereas cationic residues were found mostly at the protein surface. The results presented in this study will be useful for structural stability studies in translation elongation factors.     

Rapid fold and structure determination of the archaeal translation elongation factor 1β from Methanobacterium thermoautotrophicum

The tertiary fold of the elongation factor, aEF-1, from Methanobacterium thermoautotrophicum was determined in a high-throughput fashion using a minimal set of NMR experiments. NMR secondary structure prediction, deuterium exchange experiments and the analysis of chemical shift perturbations were combined to identify the protein fold as an alpha-beta sandwich typical of many RNA binding proteins including EF-G. Following resolution of the tertiary fold, a high resolution structure of aEF-1 was determined using heteronuclear and homonuclear NMR experiments and a semi-automated NOESY assignment strategy. Analysis of the aEF-1 structure revealed close similarity to its human analogue, eEF-1. In agreement with studies on EF-Ts and human EF-1, a functional mechanism for nucleotide exchange is proposed wherein Phe46 on an exposed loop acts as a lever to eject GDP from the associated elongation factor G-protein, aEF-1. aEF-1 was also found to bind calcium in the groove between helix 2 and strand 4. This novel feature was not observed previously and may serve a structural function related to protein stability or may play a functional role in archaeal protein translation.     

The complete amino acid sequences of the β1- and β2-subunits of the isolectins LoL1 and LoL11 from seeds of Lathyrus ochrus (L.) DC

The complete amino acid sequences of the β₁- and β₂-subunits of the isolectins (LoL1 and LoL11) from seeds of Lathyrus ochrus were determined by analysis of peptides derived from the proteins by digestion with trypsin, chymotrypsin, pepsin and the S. aureus V₈ protease, as well as fragments produced by cleavage with iodosobenzoic acid. Both β-subunits consisted of singlepolypeptide chains of 181 amino acids, which differed from one another in only 3 positions. The homology of the Lathyrus ochrus isolectins with the other two-chain lectins of the tribe Vicieae, and the single-chain lectins of other tribes of the Leguminosae is discussed.     

A common theme in the amino acid sequences of actin and many actin-binding proteins?

The amino acid sequences of several actin regulatory proteins have recently been determined. Do these proteins function by mimicking actin-actin interaction sites?     

Synergistic roles of eukaryotic translation elongation factors 1Bγ and 1A in stimulation of tombusvirus minus-strand synthesis

Host factors are recruited into viral replicase complexes to aid replication of plus-strand RNA viruses. In this paper, we show that deletion of eukaryotic translation elongation factor 1Bgamma (eEF1Bγ) reduces Tomato bushy stunt virus (TBSV) replication in yeast host. Also, knock down of eEF1Bγ level in plant host decreases TBSV accumulation. eEF1Bγ binds to the viral RNA and is one of the resident host proteins in the tombusvirus replicase complex. Additional in vitro assays with whole cell extracts prepared from yeast strains lacking eEF1Bγ demonstrated its role in minus-strand synthesis by opening of the structured 3' end of the viral RNA and reducing the possibility of re-utilization of (+)-strand templates for repeated (-)-strand synthesis within the replicase. We also show that eEF1Bγ plays a synergistic role with eukaryotic translation elongation factor 1A in tombusvirus replication, possibly via stimulation of the proper positioning of the viral RNA-dependent RNA polymerase over the promoter region in the viral RNA template.These roles for translation factors during TBSV replication are separate from their canonical roles in host and viral protein translation.     

Close phylogenetic relationship between vestimentifera (tube worms) and annelida revealed by the amino acid sequence of elongation factor-lα

To clarify the phylogenetic position of Vestimentifera (tube worms), 346-bp fragments of the elongation factor-l (EF-l) gene (939–1286 according to the numbering of the human gene) of a vestimentiferan, Lamellibrachia sp., a sternaspid polychaete, Sternaspis scutata, an earthworm, Pheretima sp., and a gastropod, Alviniconcha hessleri, were sequenced. From the amino acid sequences of these EF-l, and those of two other vertebrates and two arthropods, phylogenetic relationships were deduced by the maximum likelihood (ML) method, by which the phylogenetic tree can be inferred without assuming constancy of the molecular evolutionary rate. For the ML tree and all of seven alternative trees, whose log-likelihoods could not be discriminated from that of the ML tree by the criterion of the standard error, the vestimentiferan, the polychaete, and the oligochaete formed a clade, excluding the arthropods and the gastropod as outgroups. This result is convincing evidence that Vestimentifera are protostomes that are closely related to Annelida. The ML tree suggests that Vestimentifera are more closely related to Polychaeta than to Oligochaeta, though the data were not sufficient to discriminate these three groups at a significant level. From recent evidence such as morphological characteristics and molecular information, it may safely be said that vestimentiferans should be included in the Annelida provided this phylum contains polychaetes and oligochaetes.Correspondence to: S. Kojima     

Molecular evolution inferred from small subunit rRNA sequences: what does it tell us about phylogenetic relationships and taxonomy of the parabasalids?

The Parabasala are a primitive group of protists divided into two classes: the trichomonads and the hypermastigids. Until recently, phylogeny and taxonomy of parabasalids were mainly based on the comparative analysis of morphological characters primarily linked to the development of their cytoskeleton. Recent use of molecular markers, such as small subunit (SSU) rRNA has led to now insights into the systematics of the Parabasala and other groups of prolists. An updated phylogeny based on SSU rRNA is provided and compared to that inferred from ultrastructural data. The SSU rRNA phylogeny contradicts the dogma equating simple characters with pumitive characters. Hypermastigids, possessing a hyperdeveloped cytoskeleton, exhibit the most basal emergence in the parabasalid lineage. Other observations emerge from the SSU rRNA analysis, such as the secondary loss of some cytoskeleton structures in all representatives of the Monocercomonadidae, the existence of secondarily free living taxa (reversibility of parasitism) and the evidence against the co-evolution of the endobiotic parabasalids and their animal hosts. According to phylogenies based on SSU rRNA, all the trichomonad families are not monophyletic groups, putting into question the validity of current taxonomic assignments. The precise branching order of some taxa remains unclear, but this issue can possibly be addressed by the molecular analysis of additional parabasalids. The goal of such additional analyses would be to propose, in a near future, a revision of the taxonomy of this group of protists that takes into account both molecular and morphological data.     

Structure of α-keratin: Structural implication of the amino acid sequences of the type I and type II chain segments

Two amino acid sequences from potentially helical fragments of low-sulphur proteins from α-keratin have been analysed computationally and periods 9.4 and 28 residues long noted in the axial disposition of charged residues. Ionic interactions between chains have also been calculated and these indicate a preference for the helical fragments to aggregate in parallel with zero shift between chains in a manner essentially identical to that found for α-tropomyosin.     

Origin and production of phosphatases in the acid Lake Gårdsjön

The activity of acid phosphatases was followed for one year in Lake Gårdsjön as well as in the inlet and the outlet of the lake. A budget of the phosphatases was calculated, including an estimation of the production of phosphatases. The phosphatase activity was also measured in two basins upstream of L. Gårdsjön: the north basin and the south basin of L. Stora Hästevatten.The acid phosphatase activity was very high compared with reported alkaline phosphatase activities in other lakes. About 95% of the phosphatases in L. Gårdsjön was produced in the lake, and the production was highest in early summer.Small Chrysophyceae (< 10 µm) probably produced the majority of the acid phosphatases in the investigated lakes, and accordingly could be favoured in environments with low phosphorus supply due to their ability to produce large amounts of phosphatases.     

Biosynthesis,cDNA and amino acid sequences of a precursor of conglutin δ, a sulphur-rich protein from Lupinus angustifolius

The biosynthesis of conglutin has been studied in developing cotyledons of Lupinus angustifolius L. Precursors of conglutin formed the major sink for [³⁵S]-cysteine incorporated by developing lupin cotyledons, and these precursors were rapidly sequestered into the endoplasmic reticulum. The sequence of a cDNA clone coding for one such precursor of conglutin was determined. The structure of the precursor polypeptide for conglutin predicted from the cDNA sequence contained an N-terminal leader peptide of 22 amino acids directly preceding a subunit polypeptide of M _r 4520, together with a linking region of 13 amino acids and a subunit polypeptide of M _r 9558 at the C-terminus. The amino acid sequence predicted from the cDNA sequence showed minor variations from that established by sequencing of the protein purified from mature dried seeds (Lilley and Inglis, 1986). These were consistent with the existence of a multi-gene family coding for conglutin . Comparison of the sequences of conglutin with those of other 2S storage proteins showed that the cysteines involved in internal disulphide bridges between the mature subunits of conglutin , were maintained throughout this family of proteins but that little else was conserved either at the protein or DNA level.     

The isolation and amino acid sequence of the β- and γ-subunits of the lectin from the seeds of Dioclea Grandiflora

Although the two smaller β- and γ- subunits of the lectin from Dioclea grandiflora were clearly resolved by sodium dodecyl sulphate (SDS) gel electrophoresis, the concensus of other techniques including ultracentrifugation, isoelectric focusing in 8 M urea, size-exclusion chromatography in dissociating solvents and amino acid and sequence analysis indicated that they were similar in molecular size and that they had arisen either by a single enzymic cleavage at Asn¹¹⁸-Ser¹¹⁹ in the middle of the 237 residue-long mature α-subunit or by multiple cleavages occurring during post-translational processing of intermediates. The existence of minor forms of the β- and γ- subunits resulting from a cleavage at Asn¹²⁴-Ser¹²⁵ of the α-subunit was also recognized. The results indicated that the apparent difference in molecular size of the β- and γ-subunits deduced from SDS-gel electrophoresis could be explained by the anomalous behaviour of both subunits in this separation technique. The structural features of the D. grandiflora lectin are compared with those of concanavalin A obtained from seeds of the botanically related Canavalia ensiformis.     

Comparison of the carbohydrate and amino acid composition of normal and S-variant α-1-antitrypsin

α-1-Antitrypsin has been isolated and purified from the serum of an individual with the Pi S phenotype whose serum contains only 50–60% as much α-1-antitrypsin as normal M-type serum. The preparation was homogeneous by the criteria of sodium dodecyl sulfate polyacrylamide gel electrophoresis and sedimentation equilibrium ultracentrifugation. When analyzed in the ultracentrifuge, the S-type α-1-antitrypsin exhibited a molecular weight of 47500 which was essentially the same as that of the M-type (47300) and the Z-type (47500) α-1-antitrypsin. The S-type α-1-antitrypsin contains 15.2% carbohydrate consisting of 16.4 residues/mol of N-acetylglucosamine, 7.8 residues/mol of mannose. 6.7 residues/mol of galactose and 7.1 residues/mol of sialic acid which is essentially the same as the carbohydrate composition of the M-type α-1-antitrypsin. In addition, M- and S-type α-1-antitrypsin have very similar amino acid compositions.     

An aromatic amino acid within intracellular loop 2 of the prostaglandin EP2 receptor is a prerequisite for selective association and activation of Gαs

We previously demonstrated that the aromatic moiety of Tyr¹⁴³ within the intracellular loop 2 (ICL2) region of the prostaglandin EP2 receptor plays a crucial role in Gs coupling. Here we investigated whether the ICL2 of the EP2 receptor directly binds to Gαs and whether an aromatic moiety affects this interaction. In Chinese hamster ovary cells, mutations of Tyr¹⁴³ reduced the ability of the EP2 receptor to interact with G proteins as demonstrated by GTPγS sensitivity, as well as the ability of agonist-induced cAMP formation, with the rank order of Phe > Tyr (wild-type) = Trp > Leu > Ala (= 0). We found that the wild-type ICL2 peptide (i2Y) and its mutant with Phe at Tyr¹⁴³ (i2F) inhibited receptor-G protein complex formation of wild-type EP2 in membranes, whereas the Ala-substituted mutant (i2A) did not. Specific interactions between these peptides and the Gαs protein were detected by surface plasmon resonance, but Gαs showed different association rates, with a rank order of i2F > i2Y ≫ i2A, with similar dissociation rates. Moreover, i2F and i2Y, but not i2A activated membrane adenylyl cyclase. These results indicate that the ICL2 region of the EP2 receptor is its potential interaction site with Gαs, and that the aromatic side chain moiety at position 143 is a determinant for the accessibility of the ICL2 to the Gαs protein.     

The nucleotide and deduced amino acid sequences of a cDNA encoding a new species of Pregnancy-Specific β1-Glycoprotein (PSβG)

We screened a cDNA library of a human placenta with cDNA for nonspecific cross-reacting antigen, a member of the carcinoembryonic antigen gene family. One of the positive clones, PS34, was found to encode a 426 amino acid protein belonging to pregnancy-specific β₁-glycoprotein (PSβG). The mature PS34 protein consisted of domains, N, A1, A2, B2 and C. The domain-N of PS34 showed sequence similarities of 79.8–83.5% to those of the PSβG members so far reported, indicating PS34 is a new member of PSβG and also of the carcinoembryonic antigen gene family.