Temperature dependency of induction of sexual agglutinability by α pheromone in the yeast Saccharomyces cerevisiae

When pheromone-pretreated cells of an inducible a strain of Saccharomyces cerevisiae carrying the inducible gene saa1 were incubated in a growth medium at 28°C, induction of sexual agglutinability began after a 10 min lag period. If the cells were incubated at 38°C during the lag period, no induction occurred even after incubation at 28°C. Contrary to this, if the cells were incubated at 28°C during the lag period, almost complete induction occurred, even after transfer to 38°C. Temperature shift experiments revealed that 5 min incubation at 28°C was necessary for the initiation of the temperature-sensitive period and further 5 min incubation for the completion of the period. The temperature-sensitive period was sensitive to phenylmethylsulfonyl fluoride.Non-common abbreviations PBS 10^-2 M phosphate buffer solution, pH 5.5 - PMSF phenylmethylsulfonyl fluoride     

The complete amino acid sequence of a mouse κ light chain

The complete amino acid sequence of the kappa-chain of the mouse myeloma protein MOPC 21 was established. The protein was reduced and alkylated with iodo[2-(14)C]acetic acid, and 21 tryptic peptides were isolated, mainly by paper electrophoresis and paper chromatography. Three large tryptic peptides (of 35, 36 and 42 residues), which were difficult to isolate in this manner, were obtained pure and in excellent yields by a combination of Sephadex G-50 gel filtration in 1% (w/v) NH(4)HCO(3) and chromatography on a DEAE-cellulose column in ammonium acetate buffer, pH8.1. Peptides overlapping the tryptic peptides were isolated from a chymotryptic digest. The chain is 214 residues long. Microheterogeneity of two peptides was observed and is believed to be due to deamidation. It was not excluded that such deamidation could occur in serum from which the protein was isolated. The sequence is compared with the sequences of two other mouse kappa-chains, and with the human kappa-chain basic sequences.     

Isolation and Structure Elucidation of α Substance-IB,a Hexapeptide Inducing Sexual Agglutination in Saccharomyces cerevisiae

Cellulose triacetate prepared from bacterial cellulose of Acetobacter xylinum subsp. sucrofermentans BPR3001A showed a higher degree of polymerization and higher mechanical strength than that from the cotton linter. The fine fibrils of bacterial cellulose required only a short time for acetylation which preserved the high degree of polymerization.     

Immunoglobulin λ-chains. The complete amino acid sequence of a Bence-Jones protein

The total amino acid sequence of a lambda Bence-Jones protein has been established. The protein contains 211 residues, which include two methionine residues. Splitting with cyanogen bromide gave three fragments, the largest of which included the C-terminal half, which is common to other Bence-Jones proteins of the same type. The peptides obtained by tryptic, chymotryptic and peptic digestion were isolated and purified by paper-electrophoretic and chromatographic techniques. Reduction followed by carboxymethylation of the cysteine residues with radioactive iodoacetate was found to be a powerful tool in the isolation of some insoluble peptides. Unusual features of the molecule are the fact that it contains six cysteine residues and not five as observed in both kappa and lambda Bence-Jones proteins studied previously, and its size, which seems two residues smaller than the smallest Bence-Jones protein studied hitherto. The similarities and differences between this and other Bence-Jones proteins are discussed.     

Isolation and characterization of the mitochondrial ATP synthase fromChlamydomonas reinhardtii. cDNA sequence and deduced protein sequence of the α subunit

We have isolated the F₀F₁-ATP synthase complex from oligomycin-sensitive mitochondria of the green algaChlamydomonas reinhardtii. A pure and active ATP synthase was obtained by eans of sonication, extraction with dodecyl maltoside and ion exchange and gel permeation chromatography in the presence of glycerol, DTT, ATP and-21. The enzyme consists of 14 subunits as judged by SDS-PAGE. A cDNA clone encoding the ATP synthase subunit has been sequenced. The deduced protein sequence contains a presequence of 45 amino acids which is not present in the mature protein. The mature protein is 58–70% identical to corresponding mitochondrial proteins from other organisms. In contrast to the ATP synthase subunit fromC. reinhardtii (Franzen and Falk, Plant Mol Biol 19 (1992) 771–780), the protein does not have a C-terminal extension. However, the N-terminal domain of the mature protein is 15–18 residues longer than in ATP synthase subunits from other organisms. Southern blot analysis indicates that the protein is encoded by a single-copy gene.Abbreviations DM dodecyl--D-maltoside - OSCP oligomycin sensitivity conferring protein - PMSF phenyl-methylsulfonylfluoride - DTT dithiothreitol - EDTA ethylenediaminotetraacetic disodium salt     

Isolation of a Peptidyl Factor, α Substance-IA,Inducing Sexual Agglutinability in Saccharomyces cerevisiae

After a eleven-steps purification, a peptidyl factor named α substance-Ia was isolated in pure form from a culture filtrate of α type cells of the heterothallic Saccharomyces cerevisiae. The substance induced the sexual agglutinability in a haploid cells belonging to the opposite mating type at concentrations from 0.4 to 0.8 ng/ml.     

A reevaluation of the amino acid sequence of human follitropin β-subunit

A collaborative study from two laboratories has been undertaken to re-evaluate the human follitropin β-subunit sequence (hFSHβ), since areas of uncertainty remain in the wake of two earlier reports. The first report was by Shome and Parlow (1974). The second, by Saxena and Rathnam (1976), proposed revisions for sequence not definitively placed in the first study, as well as some differences in other placements. We have re-examined the sequence of the hFSHβ with more recent methodology. This has led to revision of certain areas of the sequence and resolution of differences between the two earlier proposals. Specifically, an-Ile-Ser- is established at 21–22, Asp at 41, Arg at 44, Lys at 46, and Glu at 111. These were areas of disagreement in the earlier proposals. A definitive placement of the residues around tryptophan-27 has now been obtained by three laboratories. C-terminal heterogeneity was observed with subunits ending at residue 107, 109, or 111. N-terminal heterogeneity has been observed in all preparations examined to date. A significant population of molecules with a proteolytic nick between residues 38–39 is noted. This is very likely an artifact of the collection and processing. The preparations examined in the present studies showed no evidence of residues 112–118 proposed by Saxena and Rathnam.     

Human immunoglobulin subclasses. Partial amino acid sequence of the constant region of a γ4 chain

The heavy chain of a human myeloma protein (Vin) belonging to the gamma4 subclass was subjected to tryptic digestion after reduction and carboxymethylation. Cyanogen bromide fragments were also prepared and all 19 tryptic peptides that account for one of them (the Fc-like fragment) were studied. Selected peptic peptides were isolated and provided evidence for the order of 15 of the tryptic peptides. In addition the sequence of two large peptic peptides derived from two sections of the molecule including all the interchain bridges is presented. Comparison with published data on other chains allows us to propose a sequence of gamma4 chains that extends from just before the presumed starting point of the invariable region (at about residue 113) to the C-terminal end of the chain (approx. residue 446), except for a section of about 50 residues. The results of the comparison suggest that the immunoglobulin subclasses have a recent independent evolutionary origin in different species. Implications for complement fixation and for the evolutionary origin of antibody diversity are also discussed.     

Isolation and partial sequence of goat spleen prothymosin α

Goat prothymosin , a highly acidic polypeptide of pl 3.5, 109 amino acid residues, has been isolated from lymphoid and non-lymphoid tissues of young female goats. Unlike rat, murine and porcine prothymosins , goat prothymosin appears at a higher concentration in the spleen compared with the thymus. The sequence of segments of the polypeptide involving known mutations has been determined, by automatic sequencing of its tryptic peptide fragments. The acidic amino acid-rich segment in the middle of the molecule, including residues 49–83, has not been sequenced. Goat prothymosin closely resembles bovine prothymosin , with only one substitution, proline for alanine at position 85. It also resembles human prothymosin , with only three substitutions. It differs more significantly from rat and murine prothymosins , by two deletions and three substitutions. The results show the highly conserved nature of the molecule, with substitutions at given positions only.Abbreviations ProT Prothymosin - T₁ Thymosin ₁ - MLR Mixed Lymphocyte Response - HPLC High Performance Liquid Chromatography - RIA Radioimmunoassay - B Aspartic acid or Asparagine - Z Glutamic acid or Glutamine     

Interferon-γ induces a tryptophan-selective amino acid transporter in human colonic epithelial cells and mouse dendritic cells

IDO1, which encodes the immunosuppressive and tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase-1 (IDO1), is a target for interferon-γ (IFN-γ). IDO1-mediated tryptophan catabolism in dendritic cells and macrophages arrests T cell proliferation, thereby providing a molecular basis for the immunosuppressive function of IDO1. Whether the entry of tryptophan into IDO1-expressing cells is also regulated by IFN-γ is not known. Here we used a human colonic epithelial cell line (CCD841) and a mouse dendritic cell line (DC2.4) to test the hypothesis that IFN-γ, which induces IDO1, also induces a tryptophan transporter to promote substrate availability to IDO1. Upon treatment with IFN-γ, there was a marked increase in IDO1 mRNA and a concomitant increase in tryptophan uptake in both cell lines. The induced uptake system was selective for tryptophan and saturable with a Michaelis constant of 36 ± 3 μM in CCD841 cells and 0.5 ± 0.1 μM in DC2.4 cells. The induction by IFN-γ and the tryptophan-selectivity of the induced transport system were demonstrable even in the presence of physiologic concentrations of all other amino acids. Since kynurenine, the catabolic end product of IDO1, is a signaling molecule as an agonist for the aryl hydrocarbon receptor (AhR), we examined if AhR signaling induces the tryptophan-selective transporter. Treatment of the cells with kynurenine and other AhR agonists increased tryptophan uptake. The present studies demonstrate that IFN-γ coordinately induces IDO1 and a tryptophan-selective transporter to maximize tryptophan depletion in IDO1-expressing cells and that the process involves a positive feedback mechanism via kynurenine-AhR signaling.     

Isolation and sequence analysis of a β-tubulin gene from arbuscular mycorrhizal fungi

A full-length β-tubulin gene has been cloned and sequenced from Gigaspora gigantea and Glomus clarum, two arbuscular mycorrhizal fungi (AMF) species in the phylum Glomeromyota. The gene in both species is organized into five exons and four introns. Both genes are 94.9% similar and encode a 447 amino acid protein. In comparison with other fungal groups, the amino acid sequence is most similar to that of fungi in the Chytridiomycota. The codon usage of the gene in both AMF species is broad and biased in favor of an A or a T in the third position. The four introns varied in length from 87 to 168 bp for G. gigantea and from 90 to 136 bp for G. clarum. Of all fungi in which full-length sequences have been published, only AMF do not have an intron before codon 174. The introns positioned at codons 174 and 257 in AMF match the position of different introns in β-tubulin genes of some Zygomycete, Basidiomycete, and Ascomycete fungi. The 5′ and 3′ splice site consensus sequences are similar to those found in introns of most fungi. Sequence analysis from single-strand conformation polymorphism analysis confirmed the presence of two β-tubulin gene copies in G. clarum, but only one copy was evident in G. gigantea based on Southern hybridization analysis.     

Relationship between structure and amino acid sequence of strongly twisted and coiled β-hairpins in globular proteins

β-Hairpins are widespread in proteins, and it is possible to find them both within β-sheets and separately. In this work, a comparative analysis of amino acid sequences of β-strands within strongly twisted β-hairpins from different structural protein subclasses has been conducted. Strongly twisted and coiled β-hairpin generates in the space a right double helix out of β-strands that are connected by a loop region (connections). The frequencies of amino acid residues on the internal (concave) and external (convex) surfaces of strongly twisted β-hairpins have been determined (220 β-hairpins from nonhomologous proteins were studied). The concave surface of these β-hairpins is mainly generated by hydrophobic residues, while the convex surface by hydrophilic residues; accordingly, the alternation of hydrophobic internal and hydrophilic external residues is observed in their amino acid sequences. Amino acid residues of glycine and alanine (especially in places of the largest twisting of the strands) were anomalously frequently found in internal positions of strongly twisted and coiled β-hairpins. It was established that internal positions never contain the proline residues, while external positions in the twisting region contain them in a relatively large amount. It was demonstrated that at least one amino acid residue in α_L- or ε-conformation is required for generation of relatively short (up to 7 amino acid residues) connection. As a rule, these positions are occupied by glycines. Thus, not only the alternation of hydrophobic and hydrophilic amino acid residues, but also the presence of one or two glycine residues in the connection region and the excess of glycines and alanines in the places of the largest strand twisting on the concave surface, as well as the presence of prolines on the convex surface, are required to generate a strongly twisted and coiled β-hairpin.     

Isolation of monotreme T-cell receptor α and β chains

Monotremes are an ancient mammalian lineage that last shared a common ancestor with the marsupial and eutherian (placental) mammals about 170 million years ago. Characterization of their immune genes is allowing us to gain insights into the evolutionary processes that lead to the mammalian immune response. Here we describe the characterization of the first cDNA clones encoding T-cell receptors from a monotreme. Two TCR -chain cDNAs (TCRA) from the short-beaked echidna, Tachyglossus aculeatus, containing complete variable, joining and constant regions were isolated. The echidna TCRA constant region shares approximately 37% amino acid identity with other mammalian TCRA constant region sequences. The two variable regions belong to the TCRAV group C, which also contains V genes from humans, mice, cattle and chickens. One echidna TCR -chain cDNA (TCRB) containing the entire constant region was isolated and sequenced. It shares about 63% identity with other mammalian TCRB constant region sequences. The echidna TCRBV belongs to TCRBV group A, which also contains V genes from various eutherian species. Southern blot analysis indicates that, like in other mammalian species, there is only one TCRA constant region copy in the echidna genome, but at least two TCRB constant regions.     

Complete amino acid sequence and location of the five disulfide bridges in porcine pancreatic α-amylase

Porcine pancreatic α-amylase (1,4-α-d-glucan glucanohydrolase EC 3.2.1.1), a single polypeptide chain, contains nine residues of methionine. Eight different fragments resulting from cleavage of this molecule by cyanogen bromide were characterized. The sequences of six of them have previously been reported. Two missing fragments, CN2 (82 residues) and CN3b1 (76 residues) were purified after breaking of the interpeptidic disulfide bridge and their complete sequence as well as that of the previously purified CN1 peptide (102 residues) are reported here. The location of the three disulfide bridges present in these peptides was determined. Ordering of the carboxymethylated cyanogen bromide fragments was carried out by pulse labeling the amylase chain in vivo. The complete sequence of the porcine pancreatic amylase chain (496 residues) and the location of its five disulfide bridges is presented. Comparison with human and mouse pancreatic and salivary α-amylases and with rat pancreatic amylase obtained from the corresponding cDNA nucleotidic sequences shows a high degree of homology between mammalian α-amylases.     

Reinvestigation of the N-terminal amino acid sequence of β-lipotropin from human pituitary glands

The N-terminal sequence of the first 60 amino acid residues of human β-LPH was reinvestigated by automated Edman degradation on the intact peptide. The result shows some differences with the recently proposed data. We have established the amino acid sequence of residues 9–26 to be Glu-Gly-Asp-Gly-Pro-Asp-Gly-Pro-Ala-Asp-Asp-Gly-Ala-Gly-Ala-Gln-Ala-Asp. From residues 1–8 and 27–60 the sequence is exactly identical with that of recently reported data.     

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