scholarly journals Genomic organization, chromosomal localization, and developmentally regulated expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei.

1990 ◽  
Vol 10 (2) ◽  
pp. 720-726 ◽  
Author(s):  
K Mensa-Wilmot ◽  
D Hereld ◽  
P T Englund
Keyword(s):  
Phospholipase C ◽  
Blot Analysis ◽  
Developmental Regulation ◽  
Mrna Level ◽  
Immunoblot Analysis ◽  
Single Copy ◽  
Tsetse Fly ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Glycosyl Phosphatidylinositol

The surface of the bloodstream form of the African trypanosome, Trypansoma brucei, is covered with about 10(7) molecules of the variant surface glycoprotein (VSG), a protein tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) membrane anchor. This anchor is cleavable by an endogenous GPI-specific phospholipase C (GPI-PLC). GPI-PLC activity is down regulated when trypanosomes differentiate from the bloodstream form to the procyclic form found in the tsetse fly vector. We have mapped the GPI-PLC locus in the trypanosome genome and have examined the mechanism for this developmental regulation in T. brucei. Southern blot analysis indicates a single-copy gene for GPI-PLC, with two allelic variants distinguishable by two NcoI restriction fragment length polymorphisms. The gene was localized solely to a chromosome in the two-megabase compression region by contour-clamped homogeneous electric field gel electrophoresis. No rearrangement of the GPI-PLC gene occurs during differentiation to procyclic forms, which could potentially silence GPI-PLC gene expression. Enzymological studies give no indication of a diffusible inhibitor of GPI-PLC activity in procyclic forms, and Western immunoblot analysis reveals no detectable GPI-PLC polypeptide in these forms. Therefore, it is highly unlikely that the absence of GPI-PLC activity in procyclic forms is due to posttranslational control. Northern (RNA) blot analysis reveals barely detectable levels of GPI-PLC mRNA in procyclic forms; therefore, regulation of GPI-PLC activity in these forms correlates with the steady-state mRNA level.

Genomic organization, chromosomal localization, and developmentally regulated expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei

1990 ◽  
Vol 10 (2) ◽  
pp. 720-726
Author(s):  
K Mensa-Wilmot ◽  
D Hereld ◽  
P T Englund
Keyword(s):  
Phospholipase C ◽  
Blot Analysis ◽  
Developmental Regulation ◽  
Mrna Level ◽  
Immunoblot Analysis ◽  
Single Copy ◽  
Tsetse Fly ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Glycosyl Phosphatidylinositol

The surface of the bloodstream form of the African trypanosome, Trypansoma brucei, is covered with about 10(7) molecules of the variant surface glycoprotein (VSG), a protein tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) membrane anchor. This anchor is cleavable by an endogenous GPI-specific phospholipase C (GPI-PLC). GPI-PLC activity is down regulated when trypanosomes differentiate from the bloodstream form to the procyclic form found in the tsetse fly vector. We have mapped the GPI-PLC locus in the trypanosome genome and have examined the mechanism for this developmental regulation in T. brucei. Southern blot analysis indicates a single-copy gene for GPI-PLC, with two allelic variants distinguishable by two NcoI restriction fragment length polymorphisms. The gene was localized solely to a chromosome in the two-megabase compression region by contour-clamped homogeneous electric field gel electrophoresis. No rearrangement of the GPI-PLC gene occurs during differentiation to procyclic forms, which could potentially silence GPI-PLC gene expression. Enzymological studies give no indication of a diffusible inhibitor of GPI-PLC activity in procyclic forms, and Western immunoblot analysis reveals no detectable GPI-PLC polypeptide in these forms. Therefore, it is highly unlikely that the absence of GPI-PLC activity in procyclic forms is due to posttranslational control. Northern (RNA) blot analysis reveals barely detectable levels of GPI-PLC mRNA in procyclic forms; therefore, regulation of GPI-PLC activity in these forms correlates with the steady-state mRNA level.

scholarly journals Insulin stimulates the release of the glycosyl phosphatidylinositol-anchored membrane dipeptidase from 3T3-L1 adipocytes through the action of a phospholipase C

1997 ◽  
Vol 326 (2) ◽  
pp. 531-537 ◽  
Author(s):  
Sara MOVAHEDI ◽  
Nigel M. HOOPER
Keyword(s):  
Cell Surface ◽  
Phospholipase C ◽  
Blot Analysis ◽  
Enzymic Activity ◽  
Time Dependent ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Cyclic Monophosphate ◽  
Glut 4

Membrane dipeptidase (MDP; EC 3.4.13.19) enzymic activity that was inhibited by cilastatin has been detected on the surface of 3T3-L1 cells. On differentiation of the cells from fibroblasts to adipocytes the activity of MDP increased 12-fold. Immunoelectrophoretic blot analysis indicated that on adipogenesis the increase in the amount of MDP preceded the appearance of GLUT-4. MDP on 3T3-L1 adipocytes was anchored in the bilayer by a glycosyl phosphatidylinositol (GPI) moiety as evidenced by its release into the medium in a hydrophilic form on treatment of the cells with bacterial phosphatidylinositol-specific phospholipase C and the appearance of the inositol 1,2-cyclic monophosphate cross-reacting determinant. Incubation of 3T3-L1 adipocytes with either insulin or the sulphonylurea glimepiride led to a rapid concentration- and time-dependent release of MDP from the cell surface. The hydrophilic form of MDP released from the cells on stimulation with insulin was recognized by antibodies against the inositol 1,2-cyclic monophosphate cross-reacting determinant, indicating that it had been generated by cleavage of its GPI anchor through the action of a phospholipase C.

scholarly journals Developmentally regulated, phospholipase C-mediated release of the major surface glycoprotein of amastigotes of Trypanosoma cruzi.

1988 ◽  
Vol 167 (2) ◽  
pp. 300-314 ◽  
Author(s):  
N W Andrews ◽  
E S Robbins ◽  
V Ley ◽  
K S Hong ◽  
V Nussenzweig
Keyword(s):  
Trypanosoma Cruzi ◽  
Phospholipase C ◽  
Myristic Acid ◽  
Surface Glycoprotein ◽  
Developmentally Regulated ◽  
African Trypanosomes ◽  
Glycosyl Phosphatidylinositol ◽  
Major Surface Glycoprotein ◽  
Major Stage ◽  
Major Surface

The surface of amastigotes of Trypanosoma cruzi is covered by Ssp-4, a major stage-specific glycoprotein. Ssp-4 is anchored to the cell membrane by GPI. It can be metabolically labeled with [3H]myristic acid, and is converted into a hydrophilic form by treatment with the glycan-specific phospholipase C of T. brucei, or after lysis of the parasites in non-ionic detergents. The hydrophilic form of Ssp-4 is recognized by antibodies to the cross-reactive determinant of the variant surface glycoprotein of African trypanosomes. Ssp-4 is progressively shed during the intra- or extracellular development of amastigotes preceding their transformation into epi- and trypomastigotes. We show here that T. cruzi contains a phospholipase C and that most shed Ssp-4 is hydrophilic, does not contain myristic acid, and reacts with anti-CRD. These observations provide strong evidence that phospholipase C mediates the release of this glycosyl-phosphatidylinositol-anchored protein under physiological conditions, as the parasite undergoes differentiation.

scholarly journals TbSAP is a novel chromatin protein repressing metacyclic variant surface glycoprotein expression sites in bloodstream form Trypanosoma brucei

2021 ◽  
Vol 49 (6) ◽  
pp. 3242-3262
Author(s):  
Carys Davies ◽  
Cher-Pheng Ooi ◽  
Georgios Sioutas ◽  
Belinda S Hall ◽  
Haneesh Sidhu ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Nuclear Periphery ◽  
Tsetse Fly ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Mammalian Host ◽  
Unicellular Eukaryote ◽  
Chromatin Protein ◽  
Protective Variant

Abstract The African trypanosome Trypanosoma brucei is a unicellular eukaryote, which relies on a protective variant surface glycoprotein (VSG) coat for survival in the mammalian host. A single trypanosome has >2000 VSG genes and pseudogenes of which only one is expressed from one of ∼15 telomeric bloodstream form expression sites (BESs). Infectious metacyclic trypanosomes present within the tsetse fly vector also express VSG from a separate set of telomeric metacyclic ESs (MESs). All MESs are silenced in bloodstream form T. brucei. As very little is known about how this is mediated, we performed a whole genome RNAi library screen to identify MES repressors. This allowed us to identify a novel SAP domain containing DNA binding protein which we called TbSAP. TbSAP is enriched at the nuclear periphery and binds both MESs and BESs. Knockdown of TbSAP in bloodstream form trypanosomes did not result in cells becoming more ‘metacyclic-like'. Instead, there was extensive global upregulation of transcripts including MES VSGs, VSGs within the silent VSG arrays as well as genes immediately downstream of BES promoters. TbSAP therefore appears to be a novel chromatin protein playing an important role in silencing the extensive VSG repertoire of bloodstream form T. brucei.

scholarly journals Molecular cloning and sequence analysis of the Chlamydomonas gene coding for radial spoke protein 3: flagellar mutation pf-14 is an ochre allele.

1989 ◽  
Vol 109 (1) ◽  
pp. 235-245 ◽  
Author(s):  
B D Williams ◽  
M A Velleca ◽  
A M Curry ◽  
J L Rosenbaum
Keyword(s):  
Blot Analysis ◽  
Immunoblot Analysis ◽  
Single Copy ◽  
Mutant Protein ◽  
Coding Region ◽  
Expression Library ◽  
Translational Initiation ◽  
Radial Spoke ◽  
Cell Biol

Chlamydomonas reinhardtii flagellar motility mutant pf-14 fails to assemble radial spokes because of a deficiency for assembly-competent radial spoke protein 3 (Huang, B., G. Piperno, Z. Ramanis, and D. J. L. Luck. 1981. J. Cell Biol. 88:80-88). Here, we raise an antiserum to protein 3 and use it to isolate the corresponding structural gene from an expression library. Southern blot analysis indicates that the gene is single copy and has not undergone major rearrangement in mutant pf-14 cells. Northern blot analysis suggests that wild-type amounts of an apparently normal 2.3-kb transcript accumulate in mutant cells during flagellar regeneration. When this mutant RNA is hybrid selected and translated in vitro, however, it produces a slightly truncated polypeptide 3 with an altered charge. The mutant protein 3 fails to assemble into pf-14 flagella and is maintained within a cytoplasmic pool of unassembled radial spoke polypeptides, as indicated by immunoblot analysis of proteins from whole cells and isolated axonemes using antisera to several radial spoke polypeptides. Interestingly, amounts of the mutant protein are greatly diminished relative to other spoke components. Complete genomic and cDNA nucleotide sequences were determined, and the pf-14 mutation was identified. It is a C-to-T transition near the 5' end of the protein coding region, which changes codon 21 to the ochre termination signal UAA. The size and charge of the mutant protein, and its reduced levels in cells, suggest that it is produced by relatively inefficient translational initiation as codon 42. The unphosphorylated isoform of radial spoke protein 3 is identified, and the sequence similarities between intervening sequences of the radial spoke protein 3 gene and a conserved intervening sequence of the two Chlamydomonas beta-tubulin genes (Youngblom, J., J. A. Schloss, and C. D. Silflow. 1984. Mol. Cell. Biol. 4:2686-2696) are reported.

Intracellular localization of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei

1989 ◽  
Vol 93 (2) ◽  
pp. 233-240
Author(s):  
R. Bolow ◽  
G. Griffiths ◽  
P. Webster ◽  
Y.D. Stierhof ◽  
F.R. Opperdoes ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Trypanosoma Brucei ◽  
Cellular Localization ◽  
Intracellular Localization ◽  
Surface Glycoprotein ◽  
Glycosyl Phosphatidylinositol ◽  
Membrane Bound ◽  
Cyclic Phosphate ◽  
Inositol Ring ◽  
Cytoplasmic Side

Glycosyl-phosphatidylinositol-specific phospholipase C (GPI-PLC) is a membrane-bound enzyme of bloodstream forms of Trypanosoma brucei, which cleaves the GPI-membrane anchor of the variant surface glycoprotein forming diacylglycerol and 1,2-cyclic phosphate on the inositol ring. The cellular localization of the enzyme was studied by fractionation of sub-cellular organelles and immunofluorescence microscopy and was found to be primarily cytoplasmic. This was confirmed by immuno-electron microscopy using cryo-sections, which showed that the labelling was predominantly on the cytoplasmic side of intracellular membranes but was absent from the plasma membrane including the region lining the flagellar pocket. The significance of these results for the possible function of the phospholipase is discussed.

scholarly journals Molecular Characterization of a cDNA that Encodes Glutamate-1-semialdehyde-2,1-aminomutase in Tomato

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 877F-877
Author(s):  
Gary F. Polking ◽  
David J. Hannapel ◽  
Richard J. Gladon
Keyword(s):  
Biosynthetic Pathway ◽  
Northern Blot Analysis ◽  
Aminolevulinic Acid ◽  
Developmental Regulation ◽  
Tomato Fruit ◽  
Transit Peptide ◽  
Immunoblot Analysis ◽  
Single Copy ◽  
Fruit Development And Ripening

Our recent research has focused on the control of genes and enzymes involved with the synthesis of chlorophyll, especially as it relates to tomato fruit development and ripening. Glutamate-1-semialdehyde-2,1-aminomutase (GSAAM) is one of the first committed enzymes in the chlorophyll biosynthetic pathway, and it is one of three enzymes that catalyze the conversion of glutamate into 5-aminolevulinic acid. We have isolated a full-length cDNA clone of GSAAM from a tomato fruit library. The tomato primary sequence shows extensive homology to GSAAM sequences found in other plant species. The primary structure also predicts a 46.7-kDa, 437-amino acid, mature protein and a transit peptide of 44 amino acids. Southern analysis indicated that GSAAM was present as a single copy. Northern blot analysis showed that GSAAM was expressed differentially in various tomato organs and that GSAAM transcripts decreased with increased fruit age. Immunoblot analysis also indicated that GSAAM protein decreased dramatically with increased fruit age. These results show that there is developmental regulation of the expression of GSAAM in tomato fruits.

scholarly journals Essential Roles for GPI-anchored Proteins in African Trypanosomes Revealed Using Mutants Deficient in GPI8

2003 ◽  
Vol 14 (3) ◽  
pp. 1182-1194 ◽  
Author(s):  
Simon Lillico ◽  
Mark C. Field ◽  
Pat Blundell ◽  
Graham H. Coombs ◽  
Jeremy C. Mottram
Keyword(s):  
Trypanosoma Brucei ◽  
Cell Cycle Progression ◽  
Tsetse Fly ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Surface Coat ◽  
Immune Challenge ◽  
Gene Encoding ◽  
Cycle Progression ◽  
African Trypanosomes

The survival of Trypanosoma brucei, the causative agent of Sleeping Sickness and Nagana, is facilitated by the expression of a dense surface coat of glycosylphosphatidylinositol (GPI)-anchored proteins in both its mammalian and tsetse fly hosts. We have characterized T. brucei GPI8, the gene encoding the catalytic subunit of the GPI:protein transamidase complex that adds preformed GPI anchors onto nascent polypeptides. Deletion ofGPI8 (to give Δgpi8) resulted in the absence of GPI-anchored proteins from the cell surface of procyclic form trypanosomes and accumulation of a pool of non–protein-linked GPI molecules, some of which are surface located. Procyclic Δgpi8, while viable in culture, were unable to establish infections in the tsetse midgut, confirming that GPI-anchored proteins are essential for insect-parasite interactions. Applying specific inducible GPI8 RNAi with bloodstream form parasites resulted in accumulation of unanchored variant surface glycoprotein and cell death with a defined multinuclear, multikinetoplast, and multiflagellar phenotype indicative of a block in cytokinesis. These data show that GPI-anchored proteins are essential for the viability of bloodstream form trypanosomes even in the absence of immune challenge and imply that GPI8 is important for proper cell cycle progression.

scholarly journals Characterization of the Major Antigenic Protein 2 of Ehrlichia canis and Ehrlichia chaffeensis and Its Application for Serodiagnosis of Ehrlichiosis

2003 ◽  
Vol 10 (4) ◽  
pp. 520-524 ◽  
Author(s):  
Tamece T. Knowles ◽  
A. Rick Alleman ◽  
Heather L. Sorenson ◽  
David C. Marciano ◽  
Edward B. Breitschwerdt ◽  
...  
Keyword(s):  
Blot Analysis ◽  
Single Copy ◽  
Ehrlichia Canis ◽  
Specific Method ◽  
Ehrlichia Chaffeensis ◽  
Antigenic Protein ◽  
Canine Monocytic Ehrlichiosis ◽  
Copy Gene ◽  
Species Specific

ABSTRACT Canine monocytic ehrlichiosis, caused by Ehrlichia canis or Ehrlichia chaffeensis, can result in clinical disease in naturally infected animals. Coinfections with these agents may be common in certain areas of endemicity. Currently, a species-specific method for serological diagnosis of monocytic ehrlichiosis is not available. Previously, we developed two indirect enzyme-linked immunosorbent assays (ELISAs) using the major antigenic protein 2 (MAP2) of E. chaffeensis and E. canis. In this study, we further characterized the conservation of MAP2 among various geographic isolates of each organism and determined if the recombinant MAP2 (rMAP2) of E. chaffeensis would cross-react with E. canis-infected dog sera. Genomic Southern blot analysis using digoxigenin-labeled species-specific probes suggested that map2 is a single-copy gene in both Ehrlichia species. Sequences of the single map2 genes of seven geographically different isolates of E. chaffeensis and five isolates of E. canis are highly conserved among the various isolates of each respective ehrlichial species. ELISA and Western blot analysis confirmed that the E. chaffeensis rMAP2 failed to serologically differentiate between E. canis and E. chaffeensis infections.

Salamander rods and cones contain distinct transducin alpha subunits

2000 ◽  
Vol 17 (6) ◽  
pp. 847-854 ◽  
Author(s):  
JAMES C. RYAN ◽  
SERGEY ZNOIKO ◽  
LIN XU ◽  
ROSALIE K. CROUCH ◽  
JIAN-XING MA
Keyword(s):  
Amino Acid ◽  
Blot Analysis ◽  
Cellular Localization ◽  
Amino Acid Level ◽  
Single Copy ◽  
Lower Vertebrate ◽  
Rods And Cones ◽  
Sequence Identity ◽  
Outer Segments ◽  
Cone Pigments

The mammalian retina is known to contain two distinct transducins that interact with their respective rod and cone pigments. However, there are no reports of a nonmammalian species having two distinct transducins. In the present study, we report the cloning and cellular localization of two transducin α subunits (Gαt) from the tiger salamander. Through degenerate polymerase chain reaction (PCR) and subsequent screening of a salamander retina cDNA library, we have identified two forms of Gαt. When compared to existing sequences in GenBank, the cloned subunits showed high similarity to rod and cone transducins. The salamander Gαt-1 has 91.2–93.7% amino acid sequence identity to mammalian rod Gαt subunits and 79.7–80.9% to mammalian cone Gαts. The salamander Gαt-2 has 86.2–87.9% sequence identity to mammalian cone Gαts and 78.9–80.9% to mammalian rod Gαts at the amino acid level. The Gαt-1 cDNA encodes 350 amino acids while the Gαt-2 cDNA encodes 354 residues, which is typical for rod and cone Gαts, respectively, and we thus identified the Gαt-1 as rod and Gαt-2 as cone Gαt. Sequences identified as effector binding sites and GTPase activity regions are highly conserved between the two subunits. Genomic Southern blot analysis showed that rod and cone Gαt subunits are both encoded by single-copy genes. Northern blot analysis identified retina-specific transcripts of 3.0 kb for rod Gαt and 2.6 kb for cone Gαt. Immunohistochemistry in the flat-mounted salamander retina demonstrated that rod Gαt is localized to rods, predominantly in the outer segments; similarly, cone Gαt is localized to cone outer segments. The results confirm that the two sequences encode rod and cone transducins and demonstrate that this lower vertebrate contains two distinct transducins that are localized specifically to rod and cone photoreceptors.

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