Single amino acid substitutions disrupt tetramer formation in the dihydroneopterin aldolase enzyme of Pneumocystis carinii

In the opportunistic pathogen Pneumocystis carinii, dihydroneopterin aldolase function is expressed as the N-terminal portion of the multifunctional folic acid synthesis protein (Fas). This region encompasses two domains, FasA and FasB, which are 27% amino acid identical. FasA and FasB also share significant amino acid sequence similarity with bacterial dihydroneopterin aldolases. In the present study, this enzyme function has been overproduced as an independent monofunctional activity in Escherichia coli. Recombinant FasAB-Met23 (amino acids 23-290 of the predicted open reading frame) was purified and shown to contain dihydroneopterin aldolase activity. The native FasAB-Met23 is a tetramer of the 30-kDa subunit, demonstrating characteristics of an associating-dissociating equilibrium system in which only the multimeric form of the enzyme is active. Multiple sequence alignment of FasA and FasB with other dihydroneopterin aldolases highlights only three positions where the amino acid is invariable between all the predicted proteins. The role of these conserved amino acid residues in enzyme function was investigated using site-directed mutagenesis. Mutant FasAB-Met23 species were overproduced and purified to near homogeneity. Three FasA domain mutants and two FasB domain mutants had little or no detectable dihydroneopterin aldolase activity, implicating both FasA and FasB in the catalytic mechanism. We show that each mutant protein containing an inactivating amino acid substitution has lost its ability to form stable tetramers.     

A genetic screen for mutations that disrupt an auditory response in Drosophila melanogaster

Hearing is one of the last sensory modalities to be subjected to genetic analysis in Drosophila melanogaster. We describe a behavioral assay for auditory function involving courtship among groups of males triggered by the pulse component of the courtship song. In a mutagenesis screen for mutations that disrupt the auditory response, we have recovered 15 mutations that either reduce or abolish this response. Mutant audiograms indicate that seven mutants reduced the amplitude of the response at all intensities. Another seven abolished the response altogether. The other mutant, 5L3, responded only at high sound intensities, indicating that the threshold was shifted in this mutant. Six mutants were characterized in greater detail. 5L3 had a general courtship defect; courtship of females by 5L3 males also was affected strongly. 5P1 males courted females normally but had reduced success at copulation. 5P1 and 5N18 showed a significant decrement in olfactory response, indicating that the defects in these mutations are not specific to the auditory pathway. Two other mutants, 5M8 and 5N30, produced amotile sperm although in 5N30 this phenotype was genetically separable from the auditory phenotype. Finally, a new adult circling behavior phenotype, the pirouette phenotype, associated with massive neurodegeneration in the brain, was discovered in two mutants, 5G10 and 5N18. This study provides the basis for a genetic and molecular dissection of auditory mechanosensation and auditory behavior.     

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.     

Single amino acid insertion in loop 4 confers amphotropic murine leukemia virus receptor function upon murine Pit1

Pit1 is the human receptor for gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B), while the related human protein Pit2 is a receptor for amphotropic murine leukemia virus (A-MuLV). The A-MuLV-related isolate 10A1 can utilize both Pit1 and Pit2 as receptors. A stretch of amino acids named region A was identified in Pit1 (residues 550 to 558 in loop 4) as critical for GALV and FeLV-B receptor function. We have here investigated the role of region A in A-MuLV and 10A1 entry. Insertion of a single amino acid in region A of mouse Pit1 resulted in a functional A-MuLV receptor, showing that region A plays a role in A-MuLV infection. Moreover, the downregulation of 10A1 receptor function by changes in region A of human Pit1 indicates that this region is also involved in 10A1 entry. Therefore, region A seems to play a role in infection by all viruses utilizing Pit1 and/or Pit2 as receptors.     

Three distinct F-actin binding sites in the Dictyostelium discoideum 34,000 dalton actin bundling protein

The Dictyostelium 34 kDa protein is an actin bundling protein composed of 295 amino acids. However, the region(s) of the molecule that bind actin filaments is (are) unknown. Studies of the cosedimentation of 125I-34 kDa protein and F-actin show that the 34 kDa protein binds to F-actin with positive cooperativity and Hill coefficients of 1.9 and 3.0, for filaments 4.9 microm and 0.6 microm, respectively. The Hill coefficient is larger for short filaments that are more efficiently bundled than long filaments, suggesting that one of the binding sites is used in interfilament contacts or contributes to filament orientation within the bundle. Three distinct actin binding sites were identified using a synthetic peptide, protein truncations, and a novel epitope library screening method. The ability to bind actin was assessed by 125I-F-actin overlays under denaturing and nondenaturing conditions, cosedimentation, viscometry, and pyrene-labeled actin disassembly. The three actin binding domains were identified as amino acids 1-123, 193-254, and 279-295. The 62 amino acid domain (193-254) can cosediment with F-actin. The estimated Kapp obtained by the disassembly of pyrene-labeled actin was 0.11 microM and 2.7 microM for the amino acids 1-123 and 279-295, respectively. These results identify three distinct regions of the 34 kDa protein that may contribute to the positive cooperative formation of F-actin bundles.     

A study of branched-chain amino acid aminotransferase and isolation of mutations affecting the catabolism of branched-chain amino acids in Saccharomyces cerevisiae

The specific activity of branched-chain amino acid aminotransferase was highest when S. cerevisiae was grown in minimal medium containing a branched-chain amino acid as nitrogen source. Growth in complex media with glycerol or ethanol gave moderately high levels, whereas with glucose and fructose the specific activity was very low. Mutagenesis defined three genes (BAA1 to BAA3) required for branched-chain amino acid catabolism. The baa1 mutation reduced the specific activity of the aminotransferase, the stationary phase density in YEPD and caused gross morphological disturbance. Branched-chain amino acid aminotransferase is essential for sporulation.     

A genetic analysis of aromatic amino acid hydroxylases involvement in DOPA synthesis during Drosophila adult development

This study was designed to examine whether cyclosporine (CyA) acts on the endocrine system by modifying the pulsatile secretion pattern of prolactin and LH. Both pituitary-grafted and sham-operated rats were submitted to a subcutaneous vehicle or CyA (5 mg/kg) treatment daily for 10 days beginning on the day of surgery. Pituitary grafting and/or CyA administration changed the pulsatile pattern of prolactin observed in sham-operated animals. The mean values of serum prolactin were significantly increased by pituitary grafting and the treatment with CyA further increased them. The mean half-life of prolactin was significantly increased in pituitary-grafted rats and was not changed by CyA administration, although it was decreased in sham-operated rats. The frequency of prolactin pulses was significantly decreased in pituitary-grafted as compared to sham-operated controls and was not further modified by CyA administration. However, in sham-operated rats a significant decrease of this parameter was observed. Duration of the prolactin peaks was significantly increased by pituitary grafting, and was not modified by CyA administration in any of the groups studied. The absolute amplitude of the prolactin peaks was significantly increased in pituitary-grafted as compared to sham-operated animals, and the treatment with CyA further increased this parameter in both groups. Mean values of LH were significantly increased in pituitary-grafted as compared to control rats. CyA administration significantly increased LH levels in sham-operated rats and decreased them in pituitary-grafted animals. The mean half-life, the pulse frequency and the duration of LH peaks were not modified by either pituitary grafting or CyA administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

A functional analysis of Notch mutations in Drosophila

In 1900, the world population was less than 1.7 billion people; the United Nations projects that in 2000 it will be 6.2, and in 2020, 7.9 billion. The proportion of the elderly (65 years and over), will increase from 5.1% (1950) to 6.8% by the year 2000 and to 8.8% by 2020, when out of an elderly population of 796 million people, 124 million are projected to be 80 years and over. Due to an increasing gender inequality in life expectation, the majority of the elderly will be women. An aged population is a basically new feature in the history of humanity, the implications of which are-as yet-incompletely understood. It is clear, however, that the last years of life are accompanied by an increase in disability and sickness, with very high demands for health and social services. Hence, the soaring elderly population will raise major social, economic and ethical issues worldwide and may strain to the limit the ability of health, social and economic infrastructures of many countries. It may also result in an increasingly large proportion of humanity (the elderly in general and elderly women, in particular) living in absolute poverty. The demographic, health, socioeconomic and ethical dimensions of the problem are discussed with particular emphasis on the situation of elderly women and a plea is made for greatly increased medical and socioeconomic research.     

Differential epitope tagging of actin in transformed Drosophila produces distinct effects on myofibril assembly and function of the indirect flight muscle

We have tested the impact of tags on the structure and function of indirect flight muscle (IFM)-specific Act88F actin by transforming mutant Drosophila melanogaster, which do not express endogenous actin in their IFMs, with tagged Act88F constructs. Epitope tagging is often the method of choice to monitor the fate of a protein when a specific antibody is not available. Studies addressing the functional significance of the closely related actin isoforms rely almost exclusively on tagged exogenous actin, because only few antibodies exist that can discriminate between isoforms. Thereby it is widely presumed that the tag does not significantly interfere with protein function. However, in most studies the tagged actin is expressed in a background of endogenous actin and, as a rule, represents only a minor fraction of the total actin. The Act88F gene encodes the only Drosophila actin isoform exclusively expressed in the highly ordered IFM. Null mutations in this gene do not affect viability, but phenotypic effects in transformants can be directly attributed to the transgene. Transgenic flies that express Act88F with either a 6x histidine tag or an 11-residue peptide derived from vesicular stomatitis virus G protein at the C terminus were flightless. Overall, the ultrastructure of the IFM resembled that of the Act88F null mutant, and only low amounts of C-terminally tagged actins were found. In contrast, expression of N-terminally tagged Act88F at amounts comparable with that of wild-type flies yielded fairly normal-looking myofibrils and partially reconstituted flight ability in the transformants. Our findings suggest that the N terminus of actin is less sensitive to modifications than the C terminus, because it can be tagged and still polymerize into functional thin filaments.     

Effect of alpha 2-adrenergic drugs dexmedetomidine and atipamezole on extracellular amino acid levels in vivo

alpha 2-Adrenoceptors are known to be involved in a variety of physiological functions and pathological conditions, including epilepsy and the extent of excitotoxin-induced cell death. In this study we evaluated whether selective alpha 2-adrenergic drugs can modulate the release of neurotransmitter amino acids. The effect of the alpha 2-adrenoceptor agonist dexmedetomidine (5 micrograms/kg, s.c.) and the alpha 2-adrenoceptor antagonist atipamezole (0.1 mg/kg and 1 mg/kg, s.c.) on the release of extracellular glutamate, aspartate and gamma-aminobutyric acid (GABA) was studied with microdialysis in the hippocampus of freely moving rats under basal and K(+)-evoked conditions. Atipamezole (1 mg/kg) decreased K(+)-evoked glutamate efflux by 30% compared to the control group (P < 0.05) but did not affect significantly the effluxes of aspartate and GABA. Dexmedetomidine and the lower dose of atipamezole (0.1 mg/kg) did not significantly alter the evoked overflow of amino acids. The results suggest that alpha 2-adrenergic drugs have only modest effects on the K(+)-stimulated overflow of extracellular neurotransmitter amino acids in rat hippocampus.     

Tetanus toxin-induced effects on extracellular amino acid levels in rat hippocampus: an in vivo microdialysis study

Tetanus toxin is a potent neurotoxin that is widely considered to produce its effect through impairment of inhibitory neurotransmission. We report the effect of a single unilateral intrahippocampal injection of tetanus toxin on extracellular levels of neuroactive amino acids in freely moving rats, at times ranging between 1 and 7 days posttreatment. Tetanus toxin treatment did not alter extracellular levels of aspartate, glutamate, and taurine at any time during the study. However, although extracellular GABA levels were unaffected by toxin injection 1, 2, and 3 days after treatment, they were reduced (45 +/- 8% of contralateral vehicle-injected level) at day 7. Challenge with a high K+ concentration, 7 days after treatment, produced elevations in extracellular levels of taurine and GABA in both vehicle- and toxin-injected hippocampi, with evoked levels of GABA being lower in the toxin-treated side (39 +/- 16% of contralateral vehicle-injected level). Aspartate and glutamate levels were not increased by high-K+ infusion. These findings are discussed in relation to the possible role that an imbalance in excitatory/inhibitory tone may play in the production of tetanus toxin-induced neurodegeneration.     

Structurally similar Drosophila alpha-tubulins are functionally distinct in vivo

We used transgenic analysis in Drosophila to compare the ability of two structurally similar alpha-tubulin isoforms to support microtubule assembly in vivo. Our data revealed that even closely related alpha-tubulin isoforms have different functional capacities. Thus, in multicellular organisms, even small changes in tubulin structure may have important consequences for regulation of the microtubule cytoskeleton. In spermatogenesis, all microtubule functions in the postmitotic male germ cells are carried out by a single tubulin heterodimer composed of the major Drosophila alpha-84B tubulin isoform and the testis-specific beta 2-tubulin isoform. We tested the ability of the developmentally regulated alpha 85E-tubulin isoform to replace alpha 84B in spermatogenesis. Even though it is 98% similar in sequence, alpha 85E is not functionally equivalent to alpha 84B. alpha 85E can support some functional microtubules in the male germ cells, but alpha 85E causes dominant male sterility if it makes up more than one-half of the total alpha-tubulin pool in the spermatids. alpha 85E does not disrupt meiotic spindle or cytoplasmic microtubules but causes defects in morphogenesis of the two classes of singlet microtubules in the sperm tail axoneme, the central pair and the accessory microtubules. Axonemal defects caused by alpha 85E are precisely reciprocal to dominant defects in doublet microtubules we observed in a previous study of ectopic germ-line expression of the developmentally regulated beta 3-tubulin isoform. These data demonstrate that the doublet and singlet axoneme microtubules have different requirements for alpha- and beta-tubulin structure. In their normal sites of expression, alpha 85E and beta 3 are coexpressed during differentiation of several somatic cell types, suggesting that alpha 85E and beta 3 might form a specialized heterodimer. Our tests of different alpha-beta pairs in spermatogenesis did not support this model. We conclude that if alpha 85E and beta 3 have specialized properties required for their normal functions, they act independently to modulate the properties of microtubules into which they are incorporated.     

A single amino acid change in Raf-1 inhibits Ras binding and alters Raf-1 function

Ras and Raf-1 are key proteins involved in the transmission of developmental and proliferative signals generated by receptor and nonreceptor tyrosine kinases. Genetic and biochemical studies demonstrate that Raf-1 functions downstream of Ras in many signaling pathways. Although Raf-1 directly associates with GTP-bound Ras, an effect of this interaction on Raf-1 activity in vivo has not been established. To examine the biological consequence of the Ras/Raf-1 interaction in vivo, we set out to identify key residues of Raf-1 required for Ras binding. In this report, we show that a single amino acid mutation in Raf-1 (Arg89 to Leu) disrupted the interaction with Ras in vitro and in the yeast two-hybrid system. This mutation prevented Ras-mediated but not tyrosine kinase-mediated enzymatic activation of Raf-1 in the baculovirus/Sf9 expression system. Furthermore, kinase-defective Raf-1 proteins containing the Arg89-->Leu mutation were no longer dominant-inhibitory or capable of blocking Ras-mediated signal transduction in Xenopus laevis oocytes. These results demonstrate that the association of Raf-1 and Ras modulates both the kinase activity and the biological function of Raf-1 and identify Arg89 as a critical residue involved in this interaction. In addition, the finding that tyrosine kinases can stimulate the enzymatic activity of Raf-1 proteins containing a mutation at the Ras-interaction site suggests that Raf-1 can be activated by Ras-independent pathways.     

Single amino acid substitution in constant region 1 or 4 of gp120 causes the phenotype of a human immunodeficiency virus type 1 variant with mutations in hypervariable regions 1 and 2 to revert

The second major cysteine loop of human immunodeficiency virus type 1 envelope glycoprotein gp120 contains 5 to 11 consensus N-linked glycosylation sites, which is disproportionately higher than the number of such sites found in other regions of gp120. Amino acid substitutions introduced at three of six N-linked glycosylation sites in this region of an infectious molecular clone, HXB2, resulted in severe impairment of virus infectivity. Isolation and genetic characterization of a revertant of this mutant revealed an isoleucine-for-valine substitution at position 84 in constant region 1 and an isoleucine-for-methionine substitution at position 434 in constant region 4. Further mutational analysis indicated that either isoleucine substitution was sufficient to confer the revertant phenotype. These findings demonstrate that V1/V2 not only functionally interacts with C4, as previously reported, but also interacts with C1. The observation that compensatory changes do not involve regeneration of N-linked glycosylation sites in the second major cysteine loop suggests that replication of human immunodeficiency virus type 1 in vitro is independent of the presence of a disproportionate number of N-linked glycosylation sites within this loop.