Salamander rods and cones contain distinct transducin alpha 
subunits

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.

Download Full-text

The expression of rat homeobox-containing genes is developmentally regulated and tissue specific

Development ◽

10.1242/dev.103.3.601 ◽

1988 ◽

Vol 103 (3) ◽

pp. 601-610

Author(s):

M. Falzon ◽

S.Y. Chung

Keyword(s):

Amino Acid ◽

Blot Analysis ◽

Genomic Library ◽

Sequence Similarity ◽

Amino Acid Level ◽

Single Copy ◽

Developmentally Regulated ◽

Tissue Specific ◽

Rna Transcripts ◽

Flanking Sequences

Seven rat homeobox-containing sequences have been isolated by screening a genomic library with a probe derived from a Drosophila antennapedia cDNA clone. The characterization of two of these homeobox-containing clones has been described (Falzon, M., Sanderson, N.D. and Chung, S. Y. (1987) Gene 54, 23–32). Sequence analysis of the remaining five homeobox regions reveals a 180 bp domain sharing 70–95% identity at the amino acid level with the homeodomain from the Drosophila antennapedia gene and with the homeodomains from other metazoan species. Genomic blot analysis shows that each of the homeobox-containing DNA segments is probably present in a single copy per haploid genome. Northern blot analysis of RNA transcripts indicates that the rat homeobox-containing sequences are expressed during embryogenesis and in newborn and adult tissues in a tissue-specific manner; RNA expression is predominantly detected in spinal cord and kidney. Moreover, the pattern of RNA transcripts observed is distinct for each homeobox sequence, indicating differential regulation. Among the seven rat homeobox-containing sequences, the flanking sequences of five of the clones have no obvious sequence similarity with previously published sequences of homeobox-containing genes from other species. Two of the rat clones have been identified as potential homologues to mouse homeobox-containing sequences. For both pairs, a high degree of amino acid conservation (greater than 95%) is observed within the homeodomain and its immediate flanking regions between the putative homologous genes in mouse and rat. This strengthens the assertion that some of the mammalian homeobox-containing genes encode highly conserved proteins and may serve important biological functions.

Download Full-text

Cloning and Characterization of the Vitamin D Receptor from Xenopus laevis*

Endocrinology ◽

10.1210/endo.138.6.5210 ◽

1997 ◽

Vol 138 (6) ◽

pp. 2347-2353 ◽

Cited By ~ 25

Author(s):

Yan Chun Li ◽

Clemens Bergwitz ◽

Harald Jüppner ◽

Marie B. Demay

Keyword(s):

Vitamin D ◽

Amino Acid ◽

Xenopus Laevis ◽

Vitamin D Receptor ◽

Messenger Rna ◽

Mammalian Cells ◽

Amino Acid Level ◽

Ion Homeostasis ◽

Northern Analysis ◽

Lower Vertebrate

Abstract The Vitamin D receptor (VDR), a member of the nuclear receptor superfamily, mediates the effects of 1,25-dihydroxyvitamin D3 on mineral ion homeostasis. Although the mammalian and avian VDRs have been extensively studied, little is known about the VDR in lower vertebrate species. To address this, we have isolated the Xenopus laevis VDR (xVDR) complementary DNA. Overall, the xVDR shares 79%, 73%, 73%, and 75% identity at the amino acid level with the chicken, mouse, rat, and human VDRs, respectively. The amino acid residues and subdomains important for DNA binding, hormone binding, dimerization, and transactivation are mostly conserved among all VDR species. The xVDR polypeptide can heterodimerize with the mouse retinoid X receptor α, bind to the rat osteocalcin vitamin D response element (VDRE), and induce vitamin D-dependent transactivation in transfected mammalian cells. Northern analysis reveals two xVDR messenger RNA species of 2.2 kb and 1.8 kb in stage 60 Xenopus tissues. In the adult, xVDR expression is detected in many tissues including kidney, intestine, skin, and bone. During Xenopus development, xVDR messenger RNA first appears at developmental stage 13 (preneurulation), increasing to maximum at stages 57–61 (metamorphosis). Our data demonstrate that, in Xenopus, VDR expression is developmentally regulated and that the vitamin D endocrine system is highly conserved during evolution.

Download Full-text

Cloning of Beauveria bassiana Chitinase Gene Bbchit1 and Its Application To Improve Fungal Strain Virulence

Applied and Environmental Microbiology ◽

10.1128/aem.71.1.363-370.2005 ◽

2005 ◽

Vol 71 (1) ◽

pp. 363-370 ◽

Cited By ~ 185

Author(s):

Weiguo Fang ◽

Bo Leng ◽

Yuehua Xiao ◽

Kai Jin ◽

Jincheng Ma ◽

...

Keyword(s):

Amino Acid ◽

Beauveria Bassiana ◽

Entomopathogenic Fungi ◽

Pathogenic Fungus ◽

Amino Acid Level ◽

Single Copy ◽

Chitinase Gene ◽

Streptomyces Avermitilis ◽

Regulatory Sequence ◽

Significant Similarity

ABSTRACT Entomopathogenic fungi can produce a series of chitinases, some of which act synergistically with proteases to degrade insect cuticle. However, chitinase involvement in insect fungus pathogenesis has not been fully characterized. In this paper, an endochitinase, Bbchit1, was purified to homogeneity from liquid cultures of Beauveria bassiana grown in a medium containing colloidal chitin. Bbchit1 had a molecular mass of about 33 kDa and pI of 5.4. Based on the N-terminal amino acid sequence, the chitinase gene, Bbchit1, and its upstream regulatory sequence were cloned. Bbchit1 was intronless, and there was a single copy in B. bassiana. Its regulatory sequence contained putative CreA/Crel carbon catabolic repressor binding domains, which was consistent with glucose suppression of Bbchit1. At the amino acid level, Bbchit1 showed significant similarity to a Streptomyces avermitilis putative endochitinase, a Streptomyces coelicolor putative chitinase, and Trichoderma harzianum endochitinase Chit36Y. However, Bbchit1 had very low levels of identity to other chitinase genes previously isolated from entomopathogenic fungi, indicating that Bbchit1 was a novel chitinase gene from an insect-pathogenic fungus. A gpd-Bbchit1 construct, in which Bbchit1 was driven by the Aspergiullus nidulans constitutive promoter, was transformed into the genome of B. bassiana, and three transformants that overproduced Bbchit1 were obtained. Insect bioassays revealed that overproduction of Bbchit1 enhanced the virulence of B. bassiana for aphids, as indicated by significantly lower 50% lethal concentrations and 50% lethal times of the transformants compared to the values for the wild-type strain.

Download Full-text

Molecular cloning, sequencing and expression of the cDNA of the mitochondrial form of phosphoenolpyruvate carboxykinase from human liver

Biochemical Journal ◽

10.1042/bj3150807 ◽

1996 ◽

Vol 315 (3) ◽

pp. 807-814 ◽

Cited By ~ 16

Author(s):

Said MODARESSI ◽

Bruno CHRIST ◽

Jutta BRATKE ◽

Stefan ZAHN ◽

Tilman HEISE ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Human Liver ◽

Phosphoenolpyruvate Carboxykinase ◽

Amino Acid Level ◽

Cdna Probe ◽

Rat Hepatocytes ◽

Eukaryotic Expression ◽

Amino Acid Sequences ◽

Sequence Identity

In human liver, phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.32) is about equally distributed between cytosol and mitochondria in contrast with rat liver in which it is essentially a cytosolic enzyme. Recently, the isolation of the gene and cDNA of the human cytosolic enzyme has been reported [Ting, Burgess, Chamberlain, Keith, Falls and Meisler (1993) Genomics 16, 698–706; Stoffel, Xiang, Espinosa, Cox, Le Beau and Bell (1993) Hum. Mol. Genet. 2, 1–4]. It was the goal of this investigation to isolate the cDNA of the human mitochondrial form of hepatic PCK. A human liver cDNA library was screened with a rat cytosolic PCK cDNA probe comprising sequences from exons 2 to 9. A cDNA clone was isolated which had overall a 68% DNA sequence and a 70% deduced amino acid sequence identity with the human cytosolic PCK cDNA. Without the flanking 270 bases (=90 amino acids) each at the 5´ and 3´ end, the sequence identity was 73% on the DNA and 78% on the amino acid level. The isolated cDNA had an open reading frame of 1920 bp; it was 54 bp (equivalent to 18 amino acids) longer than that of human or rat cytosolic PCK cDNA. The isolated cDNA was cloned into the eukaryotic expression vector pcDNAI and transfected into human embryonal kidney cells HEK293; PCK activity was increased by 3-fold in the mitochondria, which normally contain 70% of total PCK activity, but not in the cytosol. The isolated cDNA was also transfected into cultured rat hepatocytes; again, PCK activity was enhanced by about 40-fold in the mitochondria, which normally possess only 10% of total PCK activity, but not in the cytosol. In the rat hepatocytes only the endogenous cytosolic PCK and not the transfected mitochondrial PCK was induced 3-fold with glucagon. Comparison of the amino acid sequences deduced from the isolated cDNA with human and rat cytosolic PCK showed that the additional 18 amino acids were located at the N-terminus of the protein and probably constitute a mitochondrial targeting signal. Northern-blot analyses revealed the human mitochondrial PCK mRNA to be 2.25 kb long, about 0.6 kb shorter than the mRNA of the cytosolic PCK. Primer extension experiments showed that the 5´-untranslated region of mitochondrial PCK mRNA was 134 nucleotides in length.

Download Full-text

A family of dynein genes in Drosophila melanogaster.

Molecular Biology of the Cell ◽

10.1091/mbc.5.1.45 ◽

1994 ◽

Vol 5 (1) ◽

pp. 45-55 ◽

Cited By ~ 54

Author(s):

K Rasmusson ◽

M Serr ◽

J Gepner ◽

I Gibbons ◽

T S Hays

Keyword(s):

Amino Acid ◽

Heavy Chain ◽

Amino Acid Level ◽

Single Copy ◽

Cytoplasmic Dynein ◽

Amino Acid Identity ◽

Chain Gene ◽

Phosphate Binding ◽

Atp Binding Site ◽

Dynein Heavy Chain

We report the identification and initial characterization of seven Drosophila dynein heavy chain genes. Each gene is single copy and maps to a unique genomic location. Sequence analysis of partial clones reveals that each encodes a highly conserved portion of the putative dynein hydrolytic ATP-binding site in dyneins that includes a consensus phosphate-binding (P-loop) motif. One of the clones is derived from a Drosophila cytoplasmic dynein heavy chain gene, Dhc64C, that shows extensive amino acid identity to cytoplasmic dynein isoforms from other organisms. Two other Drosophila dynein clones are 85 and 90% identical at the amino acid level to the corresponding region of the beta heavy chain of sea urchin axonemal dynein. Probes for all seven of the dynein-related sequences hybridize to transcripts that are of the appropriate size, approximately 14 kilobases, to encode the characteristic high molecular weight dynein heavy chain polypeptides. The Dhc64C transcript is readily detected in RNA from ovaries, embryos, and testes. Transcripts from five of the six remaining genes are also detected in much lesser amounts in tissues other than testes. All but one of the dynein transcripts are expressed at comparable levels in testes suggesting their participation in flagellar axoneme assembly and motility.

Download Full-text

A Rab4-like GTPase in Dictyostelium discoideum colocalizes with V-H(+)-ATPases in reticular membranes of the contractile vacuole complex and in lysosomes

Journal of Cell Science ◽

10.1242/jcs.107.10.2801 ◽

1994 ◽

Vol 107 (10) ◽

pp. 2801-2812 ◽

Cited By ~ 9

Author(s):

J. Bush ◽

K. Nolta ◽

J. Rodriguez-Paris ◽

N. Kaufmann ◽

T. O'Halloran ◽

...

Keyword(s):

Dictyostelium Discoideum ◽

Blot Analysis ◽

Mammalian Cells ◽

Amino Acid Level ◽

Single Copy ◽

Contractile Vacuole ◽

Western Blots ◽

Atpase Subunit ◽

Cell Extracts ◽

Gradient Fractionation

In the course of screening a cDNA library for ras-related Dictyostelium discoideum genes, we cloned a 0.7 kb cDNA (rabD) encoding a putative protein that was 70% identical at the amino acid level to human Rab4. Rab4 is a small M(r) GTPase, which belongs to the Ras superfamily and functions to regulate endocytosis in mammalian cells. Southern blot analysis indicated that the rabD cDNA was encoded by a single copy gene while Northern blot analysis revealed that the rabD gene was expressed at relatively constant levels during growth and differentiation. Affinity-purified antibodies were prepared against a RabD fusion protein expressed in bacteria; the antibodies recognized a single 23 kDa polypeptide on western blots of cell extracts. Density gradient fractionation revealed that the RabD antigen co-distributed primarily with buoyant membranes rich in vacuolar protons pumps (V-H(+)-ATPases) and, to a lesser extent, with lysosomes. This result was confirmed by examining cell lines expressing an epitope-tagged version of RabD. Magnetically purified early endocytic vesicles and post-lysosomal vacuoles reacted more weakly with anti-RabD antibodies than did lysosomes. Other organelles were negative for RabD. Double-label indirect immunofluorescence microscopy revealed that RabD and the 100 kDa V-H(+)-ATPase subunit colocalized in a fine reticular network throughout the cytoplasm. This network was reminiscent of spongiomes, the tubular elements of the contractile vacuole system. Immunoelectron microscopy confirmed the presence of RabD in lysosome fractions and in the membranes rich in V-H(+)-ATPase. We conclude that a Rab4-like GTPase in D. discoideum is principally associated with the spongiomes of contractile vacuole complex.

Download Full-text

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

Parasitology ◽

10.1017/s0031182000063939 ◽

1995 ◽

Vol 110 (2) ◽

pp. 171-180 ◽

Cited By ~ 16

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.

Download Full-text

Molecular cloning, expression and characterization of a novel class glutathione S-transferase from the fungus Cunninghamella elegans

Biochemical Journal ◽

10.1042/bj20020400 ◽

2002 ◽

Vol 368 (2) ◽

pp. 589-595 ◽

Cited By ~ 12

Author(s):

Chang-Jun CHA ◽

Seong-Jae KIM ◽

Yong-Hak KIM ◽

Robin STINGLEY ◽

Carl E. CERNIGLIA

Keyword(s):

Amino Acid ◽

Cdna Library ◽

Fasciola Hepatica ◽

Mrna Level ◽

Amino Acid Level ◽

Gene Probe ◽

Cunninghamella Elegans ◽

Glutathione S Transferase ◽

Sequence Identity ◽

Degenerate Oligonucleotide

The structural gene for glutathione S-transferase (CeGST1-1) in the fungus Cunninghamella elegans was cloned by screening a cDNA library using a degenerate oligonucleotide probe based on the N-terminal sequence of the purified protein. Open reading frame analysis indicated that the cegst1 gene encodes a protein of 210 amino acid residues. The deduced amino acid sequence showed 25% sequence identity with the sequence of the Pi-class GST from Danio rerio (zebrafish). Similarity was also shown with the Alpha-class GST from Fasciola hepatica (liver fluke; 23% identity), the Mu class from Mus musculus (22%) and the Sigma class from Ommastrephes sloani (squid; 21%). Further screening of a cDNA library with the cegst1 gene probe revealed the presence of another GST isoenzyme (CeGST2-2) in this fungus, which shows 84% sequence identity with CeGST1-1 at the amino acid level. Reverse transcription PCR revealed that cegst2 was also expressed at the mRNA level in the fungus C. elegans. Both cegst genes were overexpressed in Escherichia coli using the expression vector pQE51, displaying specific activities with 1-chloro-2,4-dinitrobenzene of 2.04 and 0.75μmol/min permg of protein respectively. Both enzymes exhibited a similar substrate specificity and inhibition profile, indicating that CeGST1-1 and CeGST2-2 belong to the same GST class. Mutagenesis analysis revealed that Tyr10 in the N-terminal region is essential for catalysis of CeGST1-1. We propose from these results that the CeGSTs are novel Gamma-class GSTs and designated as GSTG1-1 and GSTG2-2 respectively.

Download Full-text

Molecular Cloning of a Trypanosoma cruzi Cell Surface Casein Kinase II Substrate, Tc-1, Involved in Cellular Infection

Infection and Immunity ◽

10.1128/iai.00045-06 ◽

2006 ◽

Vol 74 (7) ◽

pp. 3922-3929 ◽

Cited By ~ 7

Author(s):

Swinburne A. J. Augustine ◽

Yuliya Y. Kleshchenko ◽

Pius N. Nde ◽

Siddharth Pratap ◽

Edward A. Ager ◽

...

Keyword(s):

Amino Acid ◽

Cell Surface ◽

Trypanosoma Cruzi ◽

Blot Analysis ◽

Transmembrane Domain ◽

Amino Acid Level ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Host Cells ◽

Direct Immunofluorescence

ABSTRACT In this work, we report the cloning and characterization of the first cell surface casein kinase II (CKII) substrate (Tc-1) of Trypanosoma cruzi, the causative agent of Chagas' disease. Analysis of the gene sequence revealed a 1,653-bp open reading frame coding for 550 amino acid residues. Northern blot analysis showed a 4.5-kb transcript that is expressed in invasive trypomastigotes but not in noninvasive epimastigote forms of T. cruzi. Southern blot analysis indicates that Tc-1 is a single-copy gene. At the amino acid level, Tc-1 displayed 95% and 99% identity to two hypothetical proteins recently reported by the T. cruzi genome project. Analysis of the translated amino acid sequence indicates that the Tc-1 gene has a putative transmembrane domain with multiple cytoplasmic and extracellular CKII phosphosites. Exogenous human CKII was able to phosphorylate serine residues on both recombinant Tc-1 and Tc-1 of intact trypomastigotes. This phosphorylation was inhibited by the CKII inhibitors heparin and 4,5,6,7,-tetrabromo-2-azabenzimidazole. Immunoblots of solubilized trypomastigotes, epimastigotes, and amastigotes probed with anti-recombinant Tc-1 immunoglobulin G revealed a 62-kDa protein that is expressed only in infective trypomastigotes. Immunoprecipitation of labeled surface proteins of trypomastigotes indicated that the 62-kDa protein is a surface protein, and we found that the protein is uniformly distributed on the surface of trypomastigotes by direct immunofluorescence. Antibodies to Tc-1 effectively blocked trypomastigote invasion of host cells and consequently reduced parasite load. Preincubation of either trypomastigotes or myoblasts with CKII inhibitors blocked T. cruzi infection. Thus, for the first time, we describe a cell surface CKII substrate of a protozoan parasite that is phosphorylated by human CKII and that is involved in cellular infection.

Download Full-text

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

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.10.4.520-524.2003 ◽

2003 ◽

Vol 10 (4) ◽

pp. 520-524 ◽

Cited By ~ 7

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.

Download Full-text