Cloning and functional expression of a Shaker-related voltage-gated potassium channel gene from Schistosoma mansoni (Trematoda: Digenea)

Parasitology ◽  
1995 ◽  
Vol 110 (2) ◽  
pp. 171-180 ◽  
Author(s):  
E. Kim ◽  
T. A. Day ◽  
J. L. Bennett ◽  
R. A. Pax
Keyword(s):  
Schistosoma Mansoni ◽  
Blot Analysis ◽  
Functional Expression ◽  
Single Copy ◽  
Sequence Identity ◽  
Highly Sensitive ◽  
Membrane Spanning ◽  
Membrane Spanning Domains ◽  
Potassium Channel Gene ◽  
Mast Cell Degranulating

SUMMARYWe have isolated a cDNA (SKvl. 1) encoding a Shaker-related K+ channel from an adult cDNA library of the human parasitic trematode Schistosoma mansoni. The deduced amino acid sequence (512 aa, 56·5 kDa) contains 6 putative membrane-spanning domains (S1–S6) and a pore-forming domain (H5). SKv1.1 is grouped in the Shaker family, but forms a unique branch within this family, on the basis of dendrogram analysis. SKv1.1 shows significant sequence identity with most other Shaker channels, with 64—74% identity in the core region (S1–S6). It has the highest sequence identity with the K+ channel (Ak01a) from Aplysia. Northern blot analysis detected a single primary transcript of 2·8 kb. Southern blot analysis indicated that SKv1.1 is present as a single copy in the genomic DNA of S. mansoni. Expression of SKv1. 1 in Xenopus oocytes produced a rapidly activating and inactivating outward K+ current which is highly sensitive to 4-aminopyridine, but is insensitive to tetraethylammonium, mast cell degranulating peptide, dendrotoxin and charybdo-toxin. The presence of a Shaker homologue in Schistosoma suggests that Sh subfamilies may exist in other lower invertebrates as well as platyhelminths.

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.

scholarly journals TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (8) ◽  
pp. 4266-4273 ◽  
Author(s):  
C H Ko ◽  
R F Gaber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid Sequence Identity ◽  
Functional Independence ◽  
Structural Features ◽  
Low Ph ◽  
Sequence Identity ◽  
Hydrophilic Region ◽  
Membrane Spanning ◽  
Delta Cells ◽  
Membrane Spanning Domains

We describe the cloning and molecular analysis of TRK2, the gene likely to encode the low-affinity K+ transporter in Saccharomyces cerevisiae. TRK2 encodes a protein of 889 amino acids containing 12 putative membrane-spanning domains (M1 through M12), with a large hydrophilic region between M3 and M4. These structural features closely resemble those contained in TRK1, the high-affinity K+ transporter. TRK2 shares 55% amino acid sequence identity with TRK1. The putative membrane-spanning domains of TRK1 and TRK2 share the highest sequence conservation, while the large hydrophilic regions between M3 and M4 exhibit the greatest divergence. The different affinities of TRK1 trk2 delta cells and trk1 delta TRK2 cells for K+ underscore the functional independence of the high- and low-affinity transporters. TRK2 is nonessential in TRK1 or trk1 delta haploid cells. The viability of cells containing null mutations in both TRK1 and TRK2 reveals the existence of an additional, functionally independent potassium transporter(s). Cells deleted for both TRK1 and TRK2 are hypersensitive to low pH; they are severely limited in their ability to take up K+, particularly when faced with a large inward-facing H+ gradient, indicating that the K+ transporter(s) that remains in trk1 delta trk2 delta cells functions differently than those of the TRK class.

TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (8) ◽  
pp. 4266-4273 ◽  
Author(s):  
C H Ko ◽  
R F Gaber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid Sequence Identity ◽  
Functional Independence ◽  
Structural Features ◽  
Low Ph ◽  
Sequence Identity ◽  
Hydrophilic Region ◽  
Membrane Spanning ◽  
Delta Cells ◽  
Membrane Spanning Domains

We describe the cloning and molecular analysis of TRK2, the gene likely to encode the low-affinity K+ transporter in Saccharomyces cerevisiae. TRK2 encodes a protein of 889 amino acids containing 12 putative membrane-spanning domains (M1 through M12), with a large hydrophilic region between M3 and M4. These structural features closely resemble those contained in TRK1, the high-affinity K+ transporter. TRK2 shares 55% amino acid sequence identity with TRK1. The putative membrane-spanning domains of TRK1 and TRK2 share the highest sequence conservation, while the large hydrophilic regions between M3 and M4 exhibit the greatest divergence. The different affinities of TRK1 trk2 delta cells and trk1 delta TRK2 cells for K+ underscore the functional independence of the high- and low-affinity transporters. TRK2 is nonessential in TRK1 or trk1 delta haploid cells. The viability of cells containing null mutations in both TRK1 and TRK2 reveals the existence of an additional, functionally independent potassium transporter(s). Cells deleted for both TRK1 and TRK2 are hypersensitive to low pH; they are severely limited in their ability to take up K+, particularly when faced with a large inward-facing H+ gradient, indicating that the K+ transporter(s) that remains in trk1 delta trk2 delta cells functions differently than those of the TRK class.

scholarly journals GIT1, a Gene Encoding a Novel Transporter for Glycerophosphoinositol in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 149 (4) ◽  
pp. 1707-1715 ◽  
Author(s):  
J L Patton-Vogt ◽  
S A Henry
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Gene ◽  
Sugar Transport ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Reading Frame ◽  
Membrane Spanning ◽  
Membrane Spanning Domains ◽  
Uptake And Metabolism ◽  
Cells Cultured

Abstract Phosphatidylinositol catabolism in Saccharomyces cerevisiae cells cultured in media containing inositol results in the release of glycerophosphoinositol (GroPIns) into the medium. As the extracellular concentration of inositol decreases with growth, the released GroPIns is transported back into the cell. Exploiting the ability of the inositol auxotroph, ino1, to use exogenous GroPIns as an inositol source, we have isolated mutants (Git−) defective in the uptake and metabolism of GroPIns. One mutant was found to be affected in the gene encoding the transcription factor, SPT7. Mutants of the positive regulatory gene INO2, but not of its partner, INO4, also have the Git− phenotype. Another mutant was complemented by a single open reading frame (ORF) termed GIT1 (glycerophosphoinositol). This ORF consists of 1556 bp predicted to encode a polypeptide of 518 amino acids and 57.3 kD. The predicted Git1p has similarity to a variety of S. cerevisiae transporters, including a phosphate transporter (Pho84p), and both inositol transporters (Itr1p and Itr2p). Furthermore, Git1p contains a sugar transport motif and 12 potential membrane-spanning domains. Transport assays performed on a git1 mutant together with the above evidence indicate that the GIT1 gene encodes a permease involved in the uptake of GroPIns.

scholarly journals Spatial expression pattern of serine proteases in the blood fluke Schistosoma mansoni determined by fluorescence RNA in situ hybridization

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lenka Ulrychová ◽  
Pavel Ostašov ◽  
Marta Chanová ◽  
Michael Mareš ◽  
Martin Horn ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Schistosoma Mansoni ◽  
Rna Sequencing ◽  
Serine Proteases ◽  
Expression Patterns ◽  
High Sensitivity ◽  
Host Parasite Interactions ◽  
Highly Sensitive ◽  
Host Parasite

Abstract Background The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host–parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay. Methodology Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques. Results FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface. Conclusions The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host–parasite interactions. Graphic abstract

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.

scholarly journals Structure and expression of human IgG FcRII(CD32). Functional heterogeneity is encoded by the alternatively spliced products of multiple genes.

1989 ◽  
Vol 170 (4) ◽  
pp. 1369-1385 ◽  
Author(s):  
D G Brooks ◽  
W Q Qiu ◽  
A D Luster ◽  
J V Ravetch
Keyword(s):  
Structural Heterogeneity ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Membrane Glycoproteins ◽  
Igg Binding ◽  
Genes Expression ◽  
Alternatively Spliced ◽  
Membrane Spanning ◽  
High Degree ◽  
Membrane Spanning Domains

The structural heterogeneity of the human low affinity receptor for IgG, FcRII(CD32), has been elucidated through the isolation, characterization, and expression of cDNA clones derived from myeloid and lymphoid RNA. These clones predict amino acid sequences consistent with integral membrane glycoproteins with single membrane spanning domains. The extracellular domains display sequence homology to other Fc gamma Rs and members of the Ig supergene family. A minimum of three genes (Fc gamma RIIa, IIa', and Fc gamma RIIb) encode these transcripts, which demonstrate highly related extracellular and membrane spanning domains. IIa/IIa' differ substantially in the intracytoplasmic domain from IIb. Alternative splicing of the IIb gene generates further heterogeneity in both NH2- and COOH-terminal domains of the predicted proteins. Comparison to the murine homologues of these molecules reveals a high degree of conservation between the products of one of these genes, Fc gamma RIIb, and the murine beta gene in primary sequence, splicing pattern, and tissue distribution. In contrast, the sequence of IIa' indicates its relationship to the beta-like genes, with mutation giving rise to a novel cytoplasmic domain, while IIa is a chimera of both alpha- and beta-like genes. Expression of these cDNA molecules by transfection results in the appearance of IgG binding molecules that bear the epitopes defined by the FcRII(CD32) mAbs previously described.

NHE3 contains a cleavable signal peptide followed by 11 membrane spanning domains: New insights into topology

2000 ◽  
Vol 118 (4) ◽  
pp. A871
Author(s):  
Mirza Zizak ◽  
M E Cavet ◽  
D. Bayle ◽  
C M Tse ◽  
S. Hallen ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

Cloning, Heterologous Expression and Kinetic Analysis of Glycerol Kinase (TbGLK1) from Trypanosoma brucei

2000 ◽  
Vol 381 (11) ◽  
pp. 1071-1077 ◽  
Author(s):  
K. Steinborn ◽  
A. Szallies ◽  
D. Mecke ◽  
M. Duszenko
Keyword(s):  
Heterologous Expression ◽  
Kinetic Analysis ◽  
Trypanosoma Brucei ◽  
Blot Analysis ◽  
Spatial Separation ◽  
Single Copy ◽  
Glycerol Kinase ◽  
Mrna Levels ◽  
Atp Production ◽  
C Terminus

Abstract We have cloned and sequenced the gene for the glycerol kinase of Trypanosoma brucei (TbGLK1), obtained by RT-PCR. The corresponding mRNA is 2.3 kb in size and contains an ORF encoding a protein with high homology to known glycerol kinases of other organisms. It is 512 amino acids in length with a PTS1-like targeting sequence (AKL) at its C-terminus, suggesting glycosomal compartmentalization of this enzyme. Although Northern blot analysis revealed higher mRNA levels in slender bloodstream forms than in the procyclic insect forms, specific glycerol kinase activities were found to be virtually identical in both life stages. Southern blot analysis suggested a single copy gene, but we were able to clone two alleles utmost similar to each other. Heterologous expression of the trypanosomal glycerol kinase in E. coli enabled us to perform a kinetic analysis of this enzyme. In particular, we have been able to monitor ATP production from glycerol-3-phosphate and ADP, a reaction which, although thermodynamically very unfavorable, is regarded essential for the survival of Trypanosoma brucei under anoxic conditions. Since the unique spatial separation of glycolysis in the kinetoplastida imposes important consequences for the regulation of the energy metabolism in these organisms, we discuss the observed differences between TbGLK1 and glycerol kinases from other organisms in view of its physiological relevance.

Overexpression of a truncated cyclin B gene arrests Dictyostelium cell division during mitosis

1994 ◽  
Vol 107 (11) ◽  
pp. 3105-3114 ◽  
Author(s):  
Q. Luo ◽  
C. Michaelis ◽  
G. Weeks
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Growth ◽  
Dictyostelium Discoideum ◽  
Single Copy ◽  
Cyclin B ◽  
Dictyostelium Cell ◽  
Maximum Accumulation ◽  
Sequence Identity ◽  
Cyclin Gene

A cyclin gene has been isolated from Dictyostelium discoideum and the available evidence indicates that the gene encodes a B type cyclin. The cyclin box region of the protein encoded by the gene, clb1, has the highest degree of sequence identity with the B-type cyclins of other species. Levels of cyclin B mRNA and protein oscillate during the cell cycle with maximum accumulation of mRNA occurring prior to cell division and maximum levels of protein occurring during cell division. Overexpression of a N-terminally truncated cyclin B protein lacking the destruction box inhibits cell growth by arresting cell division during mitosis. The gene is present as a single copy in the Dictyostelium genome and there is no evidence for any other highly related cyclin B genes.

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