scholarly journals Sequence and biochemical similarities between the luciferases of the glow-worm Lampyris noctiluca and the firefly Photinus pyralis

1996 ◽  
Vol 313 (3) ◽  
pp. 761-767 ◽  
Author(s):  
Graciela B. SALA-NEWBY ◽  
Catherine M. THOMSON ◽  
Anthony K. CAMPBELL
Keyword(s):  
Amino Acids ◽  
Sequence Similarity ◽  
Biochemical Properties ◽  
Cdna Expression Library ◽  
Photinus Pyralis ◽  
Expression Library ◽  
Ph Optimum ◽  
Click Beetle ◽  
Light Organs

A full-length clone encoding Lampyris noctiluca (British glow-worm) luciferase was isolated from a complementary DNA (cDNA) expression library constructed with mRNA extracted from light organs. The luciferase was a 547-residue protein, as deduced from the nucleotide sequence. The protein was closely related to those of other lampyrid beetles, the similarity to Photinus pyralis luciferase being 84% and to Luciola 67%. In contrast, Lampyris luciferase had less sequence similarity to the luciferases of the click beetle Pyrophorus, at 48%. Engineering Lampyris luciferase in vitro showed that the C-terminal peptide containing 12 amino acids in Photinus and 9 amino acids in Lampyris was essential for bioluminescence. The pH optimum and the Km values for ATP and luciferin were similar for both Photinus and Lampyris luciferases, although the light emitted by the latter shifted towards the blue and was less stable at 37 °C. It was concluded that the molecular and biochemical properties were not sufficient to explain the glowing or flashing of the two beetles Lampyris and Photinus.

Prokaryotic expression cloning of a novel human tyrosine kinase

1994 ◽  
Vol 14 (2) ◽  
pp. 982-988
Author(s):  
J F Beeler ◽  
W J LaRochelle ◽  
M Chedid ◽  
S R Tronick ◽  
S A Aaronson
Keyword(s):  
Tyrosine Kinase ◽  
Biochemical Properties ◽  
Serine Phosphorylation ◽  
Kinase Assay ◽  
Expression Cloning ◽  
Cdna Expression Library ◽  
Human Embryonic Lung ◽  
Expression Library ◽  
Cdna Insert

Screening of a human embryonic lung fibroblast cDNA expression library with antiphosphotyrosine antibodies led to isolation of a novel protein kinase. A clone, designated A6, contained a 3-kb cDNA insert with a predicted open reading frame of 350 amino acids. DNA sequence analysis failed to reveal any detectable similarity with previously known genes, and the predicted A6 protein lacked any of the motifs commonly conserved in the catalytic domains of protein kinases. However, the bacterially expressed beta-galactosidase-A6 fusion protein demonstrated both tyrosine and serine phosphorylation in an in vitro kinase assay and phosphorylated exogenous substrates including myelin basic protein specifically on tyrosine residues. The enzyme also displayed biochemical properties analogous to those of other protein tyrosine kinases. The A6 gene was found to be expressed widely at the transcript level in normal tissues and was evolutionarily conserved. Thus, A6 represents a novel tyrosine kinase which is highly divergent from previously described members of this important class of regulatory molecules.

scholarly journals Prokaryotic expression cloning of a novel human tyrosine kinase.

1994 ◽  
Vol 14 (2) ◽  
pp. 982-988 ◽  
Author(s):  
J F Beeler ◽  
W J LaRochelle ◽  
M Chedid ◽  
S R Tronick ◽  
S A Aaronson
Keyword(s):  
Tyrosine Kinase ◽  
Biochemical Properties ◽  
Serine Phosphorylation ◽  
Kinase Assay ◽  
Expression Cloning ◽  
Cdna Expression Library ◽  
Human Embryonic Lung ◽  
Expression Library ◽  
Cdna Insert

Screening of a human embryonic lung fibroblast cDNA expression library with antiphosphotyrosine antibodies led to isolation of a novel protein kinase. A clone, designated A6, contained a 3-kb cDNA insert with a predicted open reading frame of 350 amino acids. DNA sequence analysis failed to reveal any detectable similarity with previously known genes, and the predicted A6 protein lacked any of the motifs commonly conserved in the catalytic domains of protein kinases. However, the bacterially expressed beta-galactosidase-A6 fusion protein demonstrated both tyrosine and serine phosphorylation in an in vitro kinase assay and phosphorylated exogenous substrates including myelin basic protein specifically on tyrosine residues. The enzyme also displayed biochemical properties analogous to those of other protein tyrosine kinases. The A6 gene was found to be expressed widely at the transcript level in normal tissues and was evolutionarily conserved. Thus, A6 represents a novel tyrosine kinase which is highly divergent from previously described members of this important class of regulatory molecules.

Paramyosin from the parasitic miteSarcoptes scabiei: cDNA cloning and heterologous expression

Parasitology ◽  
2001 ◽  
Vol 122 (5) ◽  
pp. 555-562 ◽  
Author(s):  
J. G. MATTSSON ◽  
E. L. LJUNGGREN ◽  
K. BERGSTRÖM
Keyword(s):  
High Performance ◽  
Sequence Similarity ◽  
Sarcoptes Scabiei ◽  
Cdna Expression Library ◽  
Terminal Part ◽  
Red Foxes ◽  
Expression Library ◽  
E Coli ◽  
Similarity Searches

The burrowing miteSarcoptes scabieiis the causative agent of the highly contagious disease sarcoptic mange or scabies. So far, there is noin vitropropagation system forS. scabieiavailable, and mites used for various purposes must be isolated from infected hosts. Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity. It has also hampered the development of high performance serological assays. We have now constructed anS. scabieicDNA expression library with mRNA purified from mites isolated from red foxes. Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein. Sequence similarity searches identified the protein as a paramyosin. RecombinantS. scabieiparamyosin expressed inEscherichia coliwas recognized by sera from dogs and swine infected withS. scabiei. We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule. The miniaturized protein was efficiently expressed inE. coliand was recognized by sera from immunized rabbits. These data demonstrate that the cDNA library can assist in the isolation of importantS. scabieiantigens and that recombinant proteins can be useful for the study of scabies.

scholarly journals Firefly genomes illuminate parallel origins of bioluminescence in beetles

2017 ◽  
Author(s):  
Timothy R. Fallon ◽  
Sarah E. Lower ◽  
Ching-Ho Chang ◽  
Manabu Bessho-Uehara ◽  
Gavin J. Martin ◽  
...  
Keyword(s):  
Firefly Luciferase ◽  
Chemical Defenses ◽  
Photinus Pyralis ◽  
Click Beetles ◽  
Origin And Evolution ◽  
The North ◽  
Click Beetle ◽  
Light Organs ◽  
The Caribbean ◽  
Shed Light

AbstractFireflies and their fascinating luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of their bioluminescence remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years-ago: the North AmericanPhotinus pyralisand JapaneseAquatica lateralis.We also sequenced the genome of a related click-beetle, the CaribbeanIgnelater luminosus,with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing but contentious hypothesis of parallel gains of bioluminescence. Our analyses support two independent gains of bioluminescence between fireflies and click-beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle.One Sentence Summary:Comparative analyses of the first linkage-group-resolution genomes of fireflies and related bioluminescent beetles address long-standing questions of the origin and evolution of bioluminescence and its associated traits.

scholarly journals A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette.

1993 ◽  
Vol 13 (12) ◽  
pp. 7625-7635 ◽  
Author(s):  
P D Walden ◽  
N J Cowan
Keyword(s):  
Protein Complex ◽  
Catalytic Domain ◽  
Signal Transduction Pathway ◽  
Kinase Domain ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Serine Threonine Kinase ◽  
Expression Clone

To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules.

scholarly journals Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells.

1993 ◽  
Vol 123 (2) ◽  
pp. 357-371 ◽  
Author(s):  
D Masson ◽  
T E Kreis
Keyword(s):  
Epithelial Cells ◽  
Molecular Characterization ◽  
Hela Cells ◽  
Binding Proteins ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Microtubule Binding ◽  
Terminal Domain

A novel microtubule-associated protein (MAP) of M(r) 115,000 has been identified by screening of a HeLa cell cDNA expression library with an anti-serum raised against microtubule-binding proteins from HeLa cells. Monoclonal and affinity-purified polyclonal antibodies were generated for the further characterization of this MAP. It is different from the microtubule-binding proteins of similar molecular weights, characterized so far, by its nucleotide-insensitive binding to microtubules and different sedimentation behavior. Since it is predominantly expressed in cells of epithelial origin (Caco-2, HeLa, MDCK), and rare (human skin, A72) or not detectable (Vero) in fibroblastic cells, we name it E-MAP-115 (epithelial MAP of 115 kD). In HeLa cells, E-MAP-115 is preferentially associated with subdomains or subsets of perinuclear microtubules. In Caco-2 cells, labeling for E-MAP-115 increases when they polarize and form blisters. The molecular characterization of E-MAP-115 reveals that it is a novel protein with no significant homologies to other known proteins. The secondary structure predicted from its sequence indicates two domains connected by a putative hinge region rich in proline and alanine (PAPA region). E-MAP-115 has two highly charged regions with predicted alpha-helical structure, one basic with a pI of 10.9 in the NH2-terminal domain and one neutral with a pI of 7.6 immediately following the PAPA region in the acidic COOH-terminal half of the molecule. A novel microtubule-binding site has been localized to the basic alpha-helical region in the NH2-terminal domain using in vitro microtubule-binding assays and expression of mutant polypeptides in vivo. Overexpression of this domain of E-MAP-115 by transfection of fibroblasts lacking significant levels of this protein with its cDNA renders microtubules stable to nocodazole. We conclude that E-MAP-115 is a microtubule-stabilizing protein that may play an important role during reorganization of microtubules during polarization and differentiation of epithelial cells.

scholarly journals Anti-osteosarcoma effect of antiserum against cross antigen TPD52 between osteosarcoma and Trichinella spiralis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Tao-Tao Yue ◽  
Nan Zhang ◽  
Jian-Hua Li ◽  
Xiang-Yun Lu ◽  
Xiao-Cen Wang ◽  
...  
Keyword(s):  
Trichinella Spiralis ◽  
Serum Levels ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cdna Expression Library ◽  
Dependent Manner ◽  
Expression Library ◽  
Muscle Larvae ◽  
In Vivo Animal Models

Abstract Background Trichinella spiralis (T. spiralis) is a parasite occurring worldwide that has been proven to have antitumour ability. However, studies on the antitumour effects of cross antigens between the tumour and T. spiralis or antibodies against cross antigens between tumours and T. spiralis are rare. Methods To study the role of cross antigens between osteosarcoma and T. spiralis, we first screened the cDNA expression library of T. spiralis muscle larvae to obtain the cross antigen gene tumour protein D52 (TPD52), and prepared fusion protein TPD52 and its antiserum. The anti-osteosarcoma effect of the anti-TPD52 antiserum was studied using cell proliferation and cytotoxicity assays as well as in vivo animal models; preliminary data on the mechanism were obtained using western blot and immunohistochemistry analyses. Results Our results indicated that TPD52 was mainly localized in the cytoplasm of MG-63 cells. Anti-TPD52 antiserum inhibited the proliferation of MG-63 cells and the growth of osteosarcoma in a dose-dependent manner. The tumour inhibition rate in the 100 μg treatment group was 61.95%. Enzyme-linked immunosorbent assay showed that injection of anti-TPD52 antiserum increased the serum levels of IFN-γ, TNF-α, and IL-12 in nude mice. Haematoxylin and eosin staining showed that anti-TPD52 antiserum did not cause significant pathological damage. Apoptosis of osteosarcoma cells was induced by anti-TPD52 antiserum in vivo and in vitro. Conclusions Anti-TPD52 antiserum exerts an anti-osteosarcoma effect by inducing apoptosis without causing histopathological damage. Graphical abstract

Genetic Selection of Poliovirus 2Apro-Binding Peptides

1999 ◽  
Vol 73 (1) ◽  
pp. 814-818 ◽  
Author(s):  
Iván Ventoso ◽  
Angel Barco ◽  
Luis Carrasco
Keyword(s):  
Genetic Selection ◽  
Initiation Factor ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Yeast Two Hybrid System ◽  
Human Proteins ◽  
Binding Peptides ◽  
Eukaryotic Initiation Factor 4G ◽  
Selection Of

ABSTRACT The yeast two-hybrid system has been used to identify mammalian clones that interact with poliovirus 2A proteinase (2Apro). Eight clones which encode previously unidentified human proteins were selected from a HeLa cell cDNA expression library. In addition, five clones encoding short peptides that interact with poliovirus 2Apro were also identified. The lengths of these peptides range from 6 to 30 amino acids, but all of them contain the Leu-X-Thr-Z motif (X represents any amino acid; Z represents a hydrophobic residue). This sequence is invariably located just at the carboxy terminus of each peptide. This approach raises the possibility of designing substrate analogue inhibitors of 2Apro. Thus, two nonhydrolyzable peptides containing the Leu-X-Thr-Z motif prevented cleavage of eukaryotic initiation factor 4G by poliovirus 2Apro in vitro. A more general method for identifying peptides with antiproteinase activity is discussed.

Characterization of a cDNA-clone encoding Nc-p43, a major Neospora caninum tachyzoite surface protein

Parasitology ◽  
1997 ◽  
Vol 115 (6) ◽  
pp. 581-590 ◽  
Author(s):  
A. HEMPHILL ◽  
R. FELLEISEN ◽  
B. CONNOLLY ◽  
B. GOTTSTEIN ◽  
B. HENTRICH ◽  
...  
Keyword(s):  
Host Cell ◽  
Neospora Caninum ◽  
Surface Protein ◽  
Cell Types ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Cdna Insert ◽  
Veterinary Importance ◽  
Major Surface

Neospora caninum is an apicomplexan parasite of veterinary importance which invades many different cell types and tissues. N. caninum tachyzoites proliferate intracellularly by endodyogeny. Eventually the massive proliferation of tachyzoites leads to host cell lysis and the newly formed parasites are released and invade neighbouring cells. Tachyzoite cell surface molecules could serve as ligands, mediating host cell adhesion and invasion. Nc-p43 is a recently identified N. caninum tachyzoite surface protein which is functionally involved in the processes leading to host cell invasion in vitro. Affinity-purified antibodies directed against Nc-p43 were used to screen a lambda gt22A-cDNA expression library constructed from N. caninum tachyzoites. The cDNA insert of one immunoreactive clone was subcloned and expressed in E. coli as a poly-histidine fusion protein. The identity of the resulting recombinant antigen termed recNc-p43 was confirmed by immunoblotting, immunofluorescence and electron microscopy using affinity-purified antibodies. The sequence of the cDNA insert encoding recNc-p43 was determined. Analysis of the deduced amino acid sequence revealed that Nc-p43 exhibited similarity to SAG1 (p30) and SAG3 (p43), 2 major surface antigens of Toxoplasma gondii tachyzoites. These similarities were not reflected on the immunochemical level, since no cross-antigenicity between SAG1, SAG3 and Nc-p43 was observed.

scholarly journals Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance

2006 ◽  
Vol 397 (2) ◽  
pp. 305-312 ◽  
Author(s):  
G. H. Erica Law ◽  
Olga A. Gandelman ◽  
Laurence C. Tisi ◽  
Christopher R. Lowe ◽  
James A. H. Murray
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Green Light ◽  
Firefly Luciferase ◽  
Wild Type ◽  
Photinus Pyralis ◽  
Wild Type Enzyme ◽  
Ph Tolerance

Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow–green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 °C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.

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