scholarly journals Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product.

1989 ◽  
Vol 9 (2) ◽  
pp. 406-414 ◽  
Author(s):  
H Romanczuk ◽  
W M Wormington
Keyword(s):  
Dna Replication ◽  
Xenopus Laevis ◽  
Gene Product ◽  
Mammalian Cells ◽  
Xenopus Laevis Oocytes ◽  
Bovine Papillomavirus ◽  
In Vitro Synthesis ◽  
E6 Gene ◽  
E6 Protein

Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication.

Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product

1989 ◽  
Vol 9 (2) ◽  
pp. 406-414
Author(s):  
H Romanczuk ◽  
W M Wormington
Keyword(s):  
Dna Replication ◽  
Xenopus Laevis ◽  
Gene Product ◽  
Mammalian Cells ◽  
Xenopus Laevis Oocytes ◽  
Bovine Papillomavirus ◽  
In Vitro Synthesis ◽  
E6 Gene ◽  
E6 Protein

Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication.

Uptake of biotin by native Xenopus laevis oocytes

1990 ◽  
Vol 259 (3) ◽  
pp. C397-C401 ◽  
Author(s):  
H. M. Said ◽  
L. Polenzani ◽  
S. Khorchid ◽  
D. Hollander ◽  
R. Miledi
Keyword(s):  
Xenopus Laevis ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Sulfhydryl Group ◽  
Maximum Velocity ◽  
Significant Inhibition ◽  
Metabolic Inhibitors ◽  
Xenopus Laevis Oocytes ◽  
Uptake System

The present study examined biotin uptake by Xenopus laevis oocytes in vitro. Uptake of low (0.03 microM) and high (10 microM) concentrations of biotin was linear with time for up to 4 h of incubation and occurred with little initial binding to oocytes. Uptake of biotin was dependent on extracellular Na+ concentration [Na+]o and was severely inhibited when Na+ was replaced by other monovalent cations [choline, tetraethylammonia, Li+, and tris(hydroxymethyl)aminomethane]. The initial rate of biotin uptake was saturable as a function of concentration with an apparent Michaelis constant of 3.9 +/- 0.5 microM and maximum velocity of 1,559 +/- 70 fmol.oocyte-1.h-1. Addition to the incubation medium of biotin structural analogues desthiobiotin and thioctic acid caused significant and concentration-dependent inhibition in the uptake of [3H]biotin. This inhibition was found to be competitive in nature with inhibition constant values of 9 and 17.5 microM. In contrast, neither the structural analogue biocytin nor biotin methyl ester (compounds in which the carboxyl group of the valeric acid moiety is blocked) showed any effect on the uptake of [3H]biotin. Biotin uptake was significantly blocked by the metabolic inhibitors dinitrophenol, cyanide, and azide and by incubation at 4 degrees C. Also, the sulfhydryl group blocker p-(chloromercuri)phenylsulfonate caused significant inhibition in biotin uptake. These results demonstrate that Xenopus oocytes possess an uptake system for biotin in its cell membrane that is Na+, energy, and temperature dependent. These characteristics of biotin uptake are similar to those reported in mammalian cells. It is suggested that Xenopus oocytes might be a useful in vitro model system to study the details of the mechanisms and regulation of biotin movement across biological membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Neurotoxin Merging: A Strategy Deployed by the Venom of the Spider Cupiennius salei to Potentiate Toxicity on Insects

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 250 ◽  
Author(s):  
Benjamin Clémençon ◽  
Lucia Kuhn-Nentwig ◽  
Nicolas Langenegger ◽  
Lukas Kopp ◽  
Steve Peigneur ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Direct Interaction ◽  
Structural Type ◽  
Structure Type ◽  
Open Loop ◽  
Cytolytic Activity ◽  
Xenopus Laevis Oocytes ◽  
Cupiennius Salei

The venom of Cupiennius salei is composed of dozens of neurotoxins, with most of them supposed to act on ion channels. Some insecticidal monomeric neurotoxins contain an α-helical part besides their inhibitor cystine knot (ICK) motif (type 1). Other neurotoxins have, besides the ICK motif, an α-helical part of an open loop, resulting in a heterodimeric structure (type 2). Due to their low toxicity, it is difficult to understand the existence of type 2 peptides. Here, we show with the voltage clamp technique in oocytes of Xenopus laevis that a combined application of structural type 1 and type 2 neurotoxins has a much more pronounced cytolytic effect than each of the toxins alone. In biotests with Drosophila melanogaster, the combined effect of both neurotoxins was enhanced by 2 to 3 log units when compared to the components alone. Electrophysiological measurements of a type 2 peptide at 18 ion channel types, expressed in Xenopus laevis oocytes, showed no effect. Microscale thermophoresis data indicate a monomeric/heterodimeric peptide complex formation, thus a direct interaction between type 1 and type 2 peptides, leading to cell death. In conclusion, peptide mergers between both neurotoxins are the main cause for the high cytolytic activity of Cupiennius salei venom.

scholarly journals Early promoters of genital and cutaneous human papillomaviruses are differentially regulated by the bovine papillomavirus type 1 E2 gene product

1992 ◽  
Vol 73 (6) ◽  
pp. 1395-1400 ◽  
Author(s):  
M. C. Guido ◽  
R. Zamorano ◽  
E. Garrido-Guerrero ◽  
P. Gariglio ◽  
A. Garcia-Carranca
Keyword(s):  
Gene Product ◽  
Human Papillomaviruses ◽  
Bovine Papillomavirus ◽  
E2 Gene

scholarly journals Xenopus laevis oocytes infected with multi-drug–resistant bacteria: implications for electrical recordings

2011 ◽  
Vol 138 (2) ◽  
pp. 271-277 ◽  
Author(s):  
Denice O'Connell ◽  
Karen Mruk ◽  
Jessica M. Rocheleau ◽  
William R. Kobertz
Keyword(s):  
Xenopus Laevis ◽  
Mammalian Cells ◽  
Resistant Bacteria ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
Drug Resistant ◽  
Animal Pole ◽  
Antibiotic Cocktail ◽  
Storage Media ◽  
Drug Resistant Bacteria

The Xenopus laevis oocyte has been the workhorse for the investigation of ion transport proteins. These large cells have spawned a multitude of novel techniques that are unfathomable in mammalian cells, yet the fickleness of the oocyte has driven many researchers to use other membrane protein expression systems. Here, we show that some colonies of Xenopus laevis are infected with three multi-drug–resistant bacteria: Pseudomonas fluorescens, Pseudomonas putida, and Stenotrophomonas maltophilia. Oocytes extracted from infected frogs quickly (3–4 d) develop multiple black foci on the animal pole, similar to microinjection scars, which render the extracted eggs useless for electrical recordings. Although multi-drug resistant, the bacteria were susceptible to amikacin and ciprofloxacin in growth assays. Supplementing the oocyte storage media with these two antibiotics prevented the appearance of the black foci and afforded oocytes suitable for whole-cell recordings. Given that P. fluorescens associated with X. laevis has become rapidly drug resistant, it is imperative that researchers store the extracted oocytes in the antibiotic cocktail and not treat the animals harboring the multi-drug–resistant bacteria.

scholarly journals Competition for DNA Binding Sites between the Short and Long Forms of E2 Dimers Underlies Repression in Bovine Papillomavirus Type 1 DNA Replication Control

1998 ◽  
Vol 72 (3) ◽  
pp. 1931-1940 ◽  
Author(s):  
Daniel A. Lim ◽  
Manfred Gossen ◽  
Chris W. Lehman ◽  
Michael R. Botchan
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Short Form ◽  
Bovine Papillomavirus ◽  
Binding Studies ◽  
Proliferating Cells ◽  
Dna Binding Sites ◽  
Replication Control

ABSTRACT Papillomaviruses establish a long-term latency in vivo by maintaining their genomes as nuclear plasmids in proliferating cells. Bovine papillomavirus type 1 encodes two proteins required for viral DNA replication: the helicase E1 and the positive regulator E2. The homodimeric E2 is known to cooperatively bind to DNA with E1 to form a preinitiation complex at the origin of DNA replication. The virus also codes for two short forms of E2 that can repress viral functions when overexpressed, and at least one copy of the repressor is required for stable plasmid maintenance in transformed cells. Employing a tetracycline-regulated system to control E1 and E2 production from integrated loci, we show that the short form of E2 negatively regulates DNA replication. We also found that the short form could repress replication in a cell-free replication system and that the repression requires the DNA binding domain of the protein. In contrast, heterodimers of the short and long forms were activators and, by footprint analysis, were shown to be as potent as homodimeric E2 in loading E1 to its cognate site. DNA binding studies show that when E1 levels are low and are dependent upon E2 for occupancy of the origin site, the repressor can block E1-DNA interactions. We conclude that DNA replication modulation results from competition between the different forms of E2 for DNA binding. Given that heterodimers are active and that the repressor form of E2 shows little cooperativity with E1 for DNA binding, this protein is a weak repressor.

scholarly journals Interactions of the Papovavirus DNA Replication Initiator Proteins, Bovine Papillomavirus Type 1 E1 and Simian Virus 40 Large T Antigen, with Human Replication Protein A

1999 ◽  
Vol 73 (6) ◽  
pp. 4899-4907 ◽  
Author(s):  
YuFeng Han ◽  
Yueh-Ming Loo ◽  
Kevin T. Militello ◽  
Thomas Melendy
Keyword(s):  
Dna Replication ◽  
Protein A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Bovine Papillomavirus ◽  
Replication Proteins ◽  
E1 Protein ◽  
Cellular Dna

ABSTRACT Papovaviruses utilize predominantly cellular DNA replication proteins to replicate their own viral genomes. To appropriate the cellular DNA replication machinery, simian virus 40 (SV40) large T antigen (Tag) binds to three different cellular replication proteins, the DNA polymerase α-primase complex, the replication protein A (RPA) complex, and topoisomerase I. The functionally similar papillomavirus E1 protein has also been shown to bind to the DNA polymerase α-primase complex. Enzyme-linked immunoassay-based protein interaction assays and protein affinity pull-down assays were used to show that the papillomavirus E1 protein also binds to the cellular RPA complex in vitro. Furthermore, SV40 Tag was able to compete with bovine papillomavirus type 1 E1 for binding to RPA. Each of the three RPA subunits was individually overexpressed in Escherichia colias a soluble fusion protein. These fusion proteins were used to show that the E1-RPA and Tag-RPA interactions are primarily mediated through the 70-kDa subunit of RPA. These results suggest that different viruses have evolved similar mechanisms for taking control of the cellular DNA replication machinery.

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