Mutants of SACCHAROMYCES CEREVISIAE Resistant to the α Mating-Type Factor

Mutants that are resistant to α-factor have been isolated from a mating-type haploid strains of yeast by direct selection on agar medium containing partially purified α-factor. All resistant mutants isolated were found to be sterile. They were characterized and compared with mutants previously isolated as nonmating. Among 93 able to mate at low frequency and to sporulate, none showed linkage to the mating-type locus. The results support the hypothesis that the response to α-factor by cells of mating-type a is essential for mating.     

Structural Analysis of Mutations in the Drosophila β2-Tubulin Isoform Reveals Regions in the β-Tubulin Molecule Required for General and for Tissue-Specific Microtubule Functions

We have determined the lesions in a number of mutant alleles of βTub85D, the gene that encodes the testis-specific β2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the β2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all β-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t(6) contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate β-tubulins. Correspondingly, B2t(6) disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that β3, a developmentally regulated Drosophila β-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type β2-tubulin. We show here by complementation analysis that β3 and the B2t(6) product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the β2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the β2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the β2 variant lacking the carboxy terminus and the B2t(6) variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from comparisons of vertebrate β-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type β-tubulins. We propose that the integrity of this structure in the Drosophila testis β2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtubule morphology as a function of tubulin assembly kinetics.     

Isolation and Characterization of Two β-d-Glucosidases from Bifidobacterium breve 203

Two β-d-glucosidases were purified to homogeneity from Bifidobacterium breve 203: one ( β-d-glucosidase I; molecular weight, 96,000) showed reactivity toward p-nitrophenyl (p-NP) β-d-fucoside, 74% of that to p-NP β-d-glucoside, and the other ( β-dglucosidase II; molecular weight, 450,000) did not. They also differed in their thermal and pH stabilities. Laminaribiose, cellobiose and gentiobiose were hydrolyzed by β-d-glucosidase I, with 53%, 34% and 3% of the reactivity in the case of p-NP β-d-glucoside, and by β-dglucosidase II, with 53%, 6% and 107% of the reactivity. The reaction of β-dglucosidase I with p-NP β-dfucoside was enhanced by the addition of glucose and other monosaccharides to the reaction mixture, whereas that with p-NP β-dglucoside was not affected. The activity of β-dglucosidase II with p-NP β-dglucoside was inhibited by glucose.     

Mutations of the Microsomal Triglyceride-Transfer–Protein Gene in Abetalipoproteinemia

Elevated plasma levels of apolipoprotein B (apoB)–containing lipoproteins constitute a major risk factor for the development of coronary heart disease. In the rare recessively inherited disorder abetalipoproteinemia (ABL) the production of apoB-containing lipoproteins is abolished, despite no abnormality of the apoB gene. In the current study we have characterized the gene encoding a microsomal triglyceride-transfer protein (MTP), localized to chromosome 4q22-24, and have identified a mutation of the MTP gene in both alleles of all individuals in a cohort of eight patients with classical ABL. Each mutant allele is predicted to encode a truncated form of MTP with a variable number of aberrant amino acids at its C-terminal end. Expression of genetically engineered forms of MTP in Cos-1 cells indicates that the C-terminal portion of MTP is necessary for triglyceride-transfer activity. Deletion of 20 amino acids from the carboxyl terminus of the 894-amino-acid protein and a missense mutation of cysteine 878 to serine both abolished activity. These results establish that defects of the MTP gene are the predominant, if not sole, cause of hereditary ABL and that an intact carboxyl terminus is necessary for activity.     

Structural and Functional Characterization of the α-Tubulin Acetyltransferase MEC-17

Tubulin protomers undergo an extensive array of post-translational modifications to tailor microtubules to specific tasks. One such modification, the acetylation of lysine 40 of α-tubulin, located in the lumen of microtubules, is associated with stable, long-living microtubule structures. MEC-17 was recently identified as the acetyltransferase that mediates this event. We have determined the crystal structure of the catalytic core of human MEC-17 in complex with its cofactor acetyl-CoA at 1.7 Å resolution. The structure reveals that the MEC-17 core adopts a canonical Gcn5-related N-acetyltransferase (GNAT) fold that is decorated with extensive surface loops. An enzymatic analysis of 33 MEC-17 surface mutants identifies hot-spot residues for catalysis and substrate recognition. A large, evolutionarily conserved hydrophobic surface patch that is critical for enzymatic activity is identified, suggesting that specificity is achieved by interactions with the α-tubulin substrate that extend outside of the modified surface loop. An analysis of MEC-17 mutants in Caenorhabditis elegans shows that enzymatic activity is dispensable for touch sensitivity.     

Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin

Proteolytic digestion of bovine β-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15–20), AASDISLLDAQSAPLR (residues 25–40), IPAVFK (residues 78–83) and VLVLDTDYK (residues 92–100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55–64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of β-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of β-lactoglobulin could be useful to increase its antimicrobial function.     

Gene Conversion and Functional Divergence in the β-Globin Gene Family

Different models of gene family evolution have been proposed to explain the mechanism whereby gene copies created by gene duplications are maintained and diverge in function. Ohta proposed a model which predicts a burst of nonsynonymous substitutions following gene duplication and the preservation of duplicates through positive selection. An alternative model, the duplication–degeneration–complementation (DDC) model, does not explicitly require the action of positive Darwinian selection for the maintenance of duplicated gene copies, although purifying selection is assumed to continue to act on both copies. A potential outcome of the DDC model is heterogeneity in purifying selection among the gene copies, due to partitioning of subfunctions which complement each other. By using the d_N/d_S () rate ratio to measure selection pressure, we can distinguish between these two very different evolutionary scenarios. In this study we investigated these scenarios in the -globin family of genes, a textbook example of evolution by gene duplication. We assembled a comprehensive dataset of 72 vertebrate -globin sequences. The estimated phylogeny suggested multiple gene duplication and gene conversion events. By using different programs to detect recombination, we confirmed several cases of gene conversion and detected two new cases. We tested evolutionary scenarios derived from Ohtas model and the DDC model by examining selective pressures along lineages in a phylogeny of -globin genes in eutherian mammals. We did not find significant evidence for an increase in the ratio following major duplication events in this family. However, one exception to this pattern was the duplication of -globin in simian primates, after which a few sites were identified to be under positive selection. Overall, our results suggest that following gene duplications, paralogous copies of -globin genes evolved under a nonepisodic process of functional divergence.[Reviewing Editor: Martin Kreitman]     

Genetic Variability of the β-Tubulin Genes in Benzimidazole-Susceptible and -Resistant Strains of Haemonchus Contortus

Benzimidazole anthelmintics are the most common chemotherapeutic agents used to remove intestinal helminths from farm animals. The development of drug resistance within helminth populations is wide-spread and can render these drugs essentially useless. The mechanism of benzimidazole resistance appears to be common to many species ranging from fungi to nematodes and involves alterations in the genes encoding β-tubulin. During the selection process resulting in resistance, there must be quantitative changes in the population gene pool. Knowledge of these changes would indicate the mechanisms underlying the spread of resistance in the population, which in turn could be used to design more effective drug administration strategies. To this end we have identified allelic variation at two β-tubulin genes in Haemonchus contortus using restriction map analysis of individual adults. Extremely high levels of variation were identified at both loci within a susceptible strain. In two independently derived benzimidazole resistant strains, allele frequencies at both loci were significantly different from the susceptible strain but not from each other. The same alleles at both loci, in both resistant strains, were favored by selection with benzimidazoles, suggesting that both loci are involved in determining benzimidazole resistance. These data confirm that changes in allele frequency, rather than novel genetic rearrangements induced by exposure to the drug, explain the changes associated with benzimidazole resistance. These results also show that any DNA based test for the development of benzimidazole resistance must take into account the frequency of alleles present in the population and not simply test for the presence or absence of specific allelic types.     

Selective Abortion of Two Nonsister Nuclei in a Developing Ascus of the hfd1–1 Mutant in SACCHAROMYCES CEREVISIAE

A recessive mutation, hfd1–1, in strain SOS4 of Saccharomyces cerevisiae leads the mutant cells to produce predominantly two-spored asci. Light microscopical examination of Giemsa-stained cells revealed no significant differences in the meiotic figures between mutant and wild-type strains. However, only two of the four meiotic products in a developing ascus matured to ascospores in SOS4. Dyad analysis was carried out on an hfd1–1 mutant strain heterozygous for three markers, asp5, gal1 and arg4, which are closely linked to their centromeres, and for his4, which is loosely linked to its centromere. The twospored asci produced by the hfd1–1 mutant segregated dominant (+) and recessive (-) alleles of each marker in a 1:1 ratio; they generally contained one + and one - spore for any given marker. The occurrence of rare dyads with two + or two - spores can be explained quantitatively by recombination between the marker and its centromere. From the results of these cytological and genetical analyses, we infer that, in the mutant strain, one genome set is partitioned to each of the four second-meiotic division poles, but only two nonsister genomes are incorporated into mature spores. Thus, the hfd1–1 mutation in SOS4 blocks incorporation of two nonsister nuclei into mature ascospores, but does not block enclosure of the remaining two nonsister nuclei.     

Isolation and Characterization of β-1,4- Endoxylanase from Trichoderma pseudokonigi

13-1,4-endoxylanase from Triehoderma pseudokonigi Rifai has been purified by anion-exchange chromatography on DEAE-Sephadex A50, DEAE-Sepharose CL-6B and mono Q. The endoxylanase was shown to be homogeneous by Native-PAGE and SDS-PAGE. This endoxylanase is a single-peptide chain protein with a molecular weight estimated as 66 kD. The endoxylanase was purified by 10-fold with a specific activity of 15.87 U·mg－1 Optimum endoxylanase activity was obtained when the enzyme was incubated at pH 4.5, 55 ℃ with a Km of 20 mg/mL and Vmax of 3.3 μmol·min－1·mg－1. Hg2 + and Cu2 + have a strong inhibition while Fe2 + and Mn2 + have a increasing effect on the enzymatic reaction rate.     

Synthesis of Novel Heterobranched β-Cyclodextrins from α-D-Mannosyl- Maltotriose and β-Cyclodextrin by the Reverse Action of Pullulanase,and Isolation and Characterization of the Products

α-D-Mannosyl-maltotriose (Man-G3) were synthesized from methyl α-mannoside and maltotriose by the transfer action of α-mannosidase. (Man-G3)-βCD and (Man-G3)₂-βCD were produced in about 20% and 4% yield, respectively when Aerobacter aerogenes pullulanase (160 units per 1 g of Man-G3) was incubated with the mixture of 1.6 M Man-G3 and 0.16 M βCD at 50°C for 4 days. The reaction products, (Man-G3)-βCD were separated to three peaks by HPLC analysis on a YMC-PACK A-323-3 column and (Man-G3)₂-βCD were separated to several peaks by HPLC analysis on a Daisopak ODS column. The major product of (Man-G3)-βCDs was identified as 6-O-α-(6³-O-α-D-mannosyl-maltotriosyl)-βCD by FAB-MS and NMR spectroscopies. The structures of (Man-G3)₂-βCDs were analyzed by TOF-MS and NMR spectroscopies, and confirmed by comparison of elution profiles of their hydrolyzates by α-mannosidase and glucoamylase on a graphitized carbon column with those of the authentic di-glucosyl-βCDs. The structures of three main components of (Man-G3)₂-βCDs were identified as 6¹,6²-, 6¹,6³- and 6¹,6⁴-di-O-(6³-O-α-D-mannosyl-maltotriosyl)-βCD.     

Reevaluation of the Evolutionary Position of Opalinids Based on 18S rDNA,and α- and β-Tubulin Gene Phylogenies

Opalinids are enigmatic endosymbiotic protists principally found in the large intestine of anuran amphibians. They are multinucleates and uniformly covered with numerous flagella (or cilia). Their appearance is somewhat similar to that of ciliates, leading to opalinid’s initial classification as ciliates, or later as protociliates. However, on the basis of their monomorphic nuclei, absence of a ciliate-like life cycle characterized by conjugation, and an interkinetal fission mode, opalinids were subsequently transferred in the zooflagellates. As several common ultrastructural characteristics shared with proteromonads were elucidated, in particular of the flagellar base, such as their double-stranded flagellar helix, an alliance with proteromonads was widely accepted. Thus, opalinids are currently favored to be placed in the class Opalinea, within the heterokont kingdom Chromista. However, the question of their classification has not been fully resolved, because of a lack of molecular information. Here, we report their phylogenetic position inferred from 18S rDNA, and α- and β-tubulin gene sequences. The 18S rDNA tree gives the opalinids an ancestral position in heterokonts, together with proteromonads, as suggested by the morphological studies. In great contrast, α- and β-tubulin gene analyses suggest an affiliation of opalinids to alveolates, not to heterokonts. However, the AU test implies that opalinids are not closely related with any of other three phyla in the alveolates, suggesting an occupation of an ancestral position within the alveolates. Based on the present molecular information, in particular rDNA phylogeny, and the ultrastructural character of the double helix common to heterokonts, we conclude that opalinids would have a common origin with heterokonts, although analyses based on two tubulin genes do not as yet completely deny a possible placement outside heterokonts. The ambiguity of the evolutionary position shown by the discrepancy between rDNA and tubulin genes phylogenies might reflect an early emergence of opalinids in ancestral chromalveolates, and an extreme specialization during a lengthy history of parasitism, as suggested by a long branch in the rDNA tree. Reviewing Editor: Dr. Patrick Keeling     

Isolation and Characterization of Novel β-Cyanoalanine Synthase and Cysteine Synthase Genes from Cassava

Cassava is an important staple food crop, feeding 600 million people worldwide, which produce cyanogenic glycosides. Cyanogenic glycosides in cassava are known to act as a deterrent for herbivores as well as serve as a mobile source of reduced nitrogen. Cassava is also equipped with a cyanide detoxification pathway, mediated by β-cyanoalanine synthase (β-CAS) which converts cyanide into asparagine. β-CAS, belonging to the Bsas family of enzymes, is multi functional and shares sequence homology with cysteine synthase (CS). Using rapid amplification of cDNA end-polymerase chain reaction (RACE-PCR), two cDNA sequences were isolated from cassava. The two clones named MANes;BsasA (accession no. EU350583) and MANes;BsasB (accession no. HQ257219), showed high homology to known β-CAS enzymes (80% and 75% amino acid similarity to Arabidopsis and 76% and 82% similarity to spinach, respectively). The kinetic properties of the two clones were characterized in a Escherichia coli NK3 mutant strain which lacks activity for any of the Bsas proteins. Kinetic studies showed that MANes;BsasB is a β-CAS with a CAS/CS activity ratio of 72 while MANes;BsasA is a CS showing bifunctional capabilities and with a CAS/CS activity ratio of 11. The isolation of cassava β-CAS and CS genes reported here paves the way for their utilization in genetically enhancing the cyanide detoxification potential of cassava and/or increase of the essential amino acid cysteine, which has been found to be low in nutritionally compromised individuals.