A human intracellular apyrase-like protein, LALP70, localizes to lysosomal/autophagic vacuoles

1999 ◽  
Vol 112 (15) ◽  
pp. 2473-2484 ◽  
Author(s):  
A. Biederbick ◽  
S. Rose ◽  
H.P. Elsasser
Keyword(s):  
Fluorescent Protein ◽  
Signal Sequence ◽  
In Vitro Transcription ◽  
Autophagic Vacuole ◽  
Reading Frame ◽  
Vacuole Membrane ◽  
Conserved Regions ◽  
Punctate Pattern ◽  
Green Fluorescent

Using antibodies against autophagic vacuole membrane proteins we identified a human cDNA with an open reading frame of 1848 bp, encoding a protein of 70 kDa, which we named lysosomal apyrase-like protein of 70 kDa (LALP70). Sequence analysis revealed that LALP70 belongs to the apyrase or GDA1/CD39 family and is almost identical to a human uridine diphosphatase, with the exception of nine extra amino acids in LALP70. Members of this family were originally described as ectoenzymes, with some intracellular exceptions. Transfected LALP70 fused to the green fluorescent protein localized in the cytoplasm with a punctate pattern in the perinuclear space. These structures colocalized with the autophagic marker monodansylcadaverine and the lysosomal protein lamp1. Hydrophobicity analysis of the encoded protein revealed a transmembrane region at the N and C termini. Most of the sequence is arranged between these transmembrane domains, and contains four apyrase conserved regions. In vitro transcription/translation in the presence of microsomes showed that no signal sequence is cleaved off and that the translation product is protected from trypsin treatment. Our data indicate that LALP70 is a type III lysosomal/autophagic vacuole membrane protein with the apyrase conserved regions facing the luminal space of the vacuoles.

scholarly journals Double-Labeled Rabies Virus: Live Tracking of Enveloped Virus Transport

2007 ◽  
Vol 82 (1) ◽  
pp. 237-245 ◽  
Author(s):  
Yvonne Klingen ◽  
Karl-Klaus Conzelmann ◽  
Stefan Finke
Keyword(s):  
Cell Surface ◽  
Rabies Virus ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Surface Membrane ◽  
Fusion Construct ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Green Fluorescent

ABSTRACT Here we describe a strategy to fluorescently label the envelope of rabies virus (RV), of the Rhabdoviridae family, in order to track the transport of single enveloped viruses in living cells. Red fluorescent proteins (tm-RFP) were engineered to comprise the N-terminal signal sequence and C-terminal transmembrane spanning and cytoplasmic domain sequences of the RV glycoprotein (G). Two variants of tm-RFP were transported to and anchored in the cell surface membrane, independent of glycosylation. As shown by confocal microscopy, tm-RFP colocalized at the cell surface with the RV matrix and G protein and was incorporated into G gene-deficient virus particles. Recombinant RV expressing the membrane-anchored tm-RFP in addition to G yielded infectious viruses with mosaic envelopes containing both tm-RFP and G. Viable double-labeled virus particles comprising a red fluorescent envelope and a green fluorescent ribonucleoprotein were generated by expressing in addition an enhanced green fluorescent protein-phosphoprotein fusion construct (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004). Individual enveloped virus particles were observed under live cell conditions as extracellular particles and inside endosomal vesicles. Importantly, double-labeled RVs were transported in the retrograde direction over long distances in neurites of in vitro-differentiated NS20Y neuroblastoma cells. This indicates that the typical retrograde axonal transport of RV to the central nervous system involves neuronal transport vesicles in which complete enveloped RV particles are carried as a cargo.

scholarly journals Feline Calicivirus: Recovery of Wild-Type and Recombinant Viruses after Transfection of cRNA or cDNA Constructs

2002 ◽  
Vol 76 (12) ◽  
pp. 6398-6407 ◽  
Author(s):  
Jörg Oliver Thumfart ◽  
Gregor Meyers
Keyword(s):  
Fluorescent Protein ◽  
In Vitro Transcription ◽  
Feline Calicivirus ◽  
Nucleotide Sequencing ◽  
Structural Protein ◽  
Recombinant Viruses ◽  
Rna Transfection ◽  
Phage T7 ◽  
Green Fluorescent

ABSTRACT The RNA genome of the vaccine strain 2024 of feline calicivirus was cloned as cDNA and analyzed by nucleotide sequencing. A full-length DNA copy of the viral genome was established and proved to be a source of infectious cRNA after in vitro transcription and RNA transfection. Virus could also be recovered when the DNA construct was introduced into cells containing phage T7 RNA polymerase that was provided by vaccinia virus MVA-T7. After insertion of the sequence encoding the green fluorescent protein into the structural protein-encoding region of the infectious cDNA clone, a defective replicon was recovered that was able to replicate autonomously and was packaged into virus particles when the structural proteins were provided in trans.

CLONING OF HUMAN INTERLEUKIN-10 GENE AND TRANSGENE EXPRESSION IN RABBIT SYNOVIAL CELLS IN VITRO

2008 ◽  
Vol 11 (01) ◽  
pp. 1-7 ◽  
Author(s):  
Xiangping Liu ◽  
Haibo Wang ◽  
Kun Yang ◽  
Aihua Sui ◽  
Yanming Wang ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Fluorescent Protein ◽  
Interleukin 10 ◽  
Eukaryotic Expression ◽  
Cell Culture Supernatant ◽  
Enzyme Linked Immunosorbent Assay ◽  
Synovial Cells ◽  
Reading Frame ◽  
Green Fluorescent

In this work, the full-length open reading frame of the human interleukin-10 (hIL-10) gene was amplified through reverse transcription–polymerase chain reaction (RT-PCR), and then PCR products were inserted into pcDNA4/HisMax to construct an eukaryotic expression vector. After optimization by green fluorescent protein (GFP), recombinant hIL-10 genes were transfected and expressed in rabbit synovial cells compounded with liposome in vitro. In cell culture supernatant, rhIL-10 was detected using enzyme-linked immunosorbent assay (ELISA) at time intervals of 12 hours, 24 hours, 48 hours, 72 hours, 7 days, and 14 days. After 12 hours of transfection, ELISA showed that transgene expression of hIL-10 in rabbit synovial cells was elevated; at 72 hours, hIL-10 expression reached its peak value; and then it declined gradually until 7 days, compared with the control. After 14 days, transgene expression ceased. Gene cloning of hIL-10 and its transgene expression in synovial cells therefore gives a basis for the gene therapy of rheumatoid arthritis.

scholarly journals Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’

2001 ◽  
Vol 82 (5) ◽  
pp. 1013-1025 ◽  
Author(s):  
Michelle L. L. Donnelly ◽  
Garry Luke ◽  
Amit Mehrotra ◽  
Xuejun Li ◽  
Lorraine E. Hughes ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Foot And Mouth Disease ◽  
Peptide Bond ◽  
Open Reading Frame ◽  
Translation Product ◽  
Reading Frame ◽  
C Terminus ◽  
Mouth Disease Virus ◽  
Green Fluorescent

The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A ‘primary’ intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational ‘cleavage’ products quantified. The processing products from our artificial polyprotein systems showed a molar excess of ‘cleavage’ product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control construct encoding the P1P2 region of the human rhinovirus polyprotein, known to be proteolytically processed, were quantified and found to be equimolar. Translation of a construct encoding green fluorescent protein (GFP), FMDV 2A and β-glucuronidase, also in a single open reading frame, in the presence of puromycin, showed this antibiotic to be preferentially incorporated into the [GFP2A] translation product. We conclude that the discrete translation products from our artificial polyproteins are not produced by proteolysis. We propose that the FMDV 2A sequence, rather than representing a proteolytic element, modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNAGly ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows the synthesis of a discrete downstream translation product to proceed. This process produces a ribosomal ‘skip’ from one codon to the next without the formation of a peptide bond.

185 CELLULAR VIABILITY AND EXPRESSION OF GREEN FLUORESCENT PROTEIN IN BOVINE FETAL FIBROBLASTS FOLLOWING TRANSFECTION OF SYNTHETIC mRNA INCLUDING MODIFIED BASES

2014 ◽  
Vol 26 (1) ◽  
pp. 207
Author(s):  
T. L. Adams ◽  
S. E. Farmer ◽  
J. A. Sarmiento-Guzmán ◽  
K. R. Bondioli
Keyword(s):  
Fluorescent Protein ◽  
In Vitro Transcription ◽  
Cellular Reprogramming ◽  
Cellular Viability ◽  
Treatment Groups ◽  
Fetal Fibroblasts ◽  
Green Fluorescent ◽  
Synthetic Mrna ◽  
Number Of Cells

Synthetic RNA transfection has been an invaluable tool in understanding the mammalian genome because of its ability to deliver exogenous protein without mutagenic effects caused by double-stranded DNA. A common problem associated with the introduction of exogenous mRNA into mammalian cells is the stimulated interferon response. This innate immune response can be avoided with the addition of modified bases during the in vitro transcription process of synthetically derived mRNA. The bases cytidine triphosphate (CTP) and uridine triphosphate (UTP) are replaced with 5-methylcytidine-5′-triphosphate (5-Methyl-CTP) and pseudouridine-5′-triphosphate (Pseudo-UTP) during in vitro transcription. Cellular reprogramming is achieved by the delivery of this mRNA into the cytoplasm. Previous cellular reprogramming experiments lacking modified bases resulted in increased toxicity and a decrease in cellular viability, which lead to the incorporation of modified bases. In the first experiment, bovine fetal fibroblasts were transfected with modified synthetic mRNA encoding green fluorescent protein (GFP) to evaluate the effects on cellular viability and fluorescence. The cellular viability was measured by counting a final number of cells after seeding a constant number of cells in all treatment groups. The control group consisted of bovine fetal fibroblasts cultured in normal growth medium. A no-RNA (NR) group was held under the same conditions with the addition of the transfection reagent, Lipofectamine (Invitrogen, Carlsbad, CA, USA), to account for toxicity resulting from the transfection reagent alone. The cells were transfected every other day for 12 days and were evaluated on days 3, 6, 9, and 12 for viability and fluorescence by flow cytometry. There was no difference in viability of all cells treated with synthetic mRNA encoding GFP when compared to controls (P = 0.9). There was a significant difference in fluorescence on all time points when compared to controls (Day 3, P = 0.004; Day 6, P = 0.004; Day 9, P = 0.007; Day 12, P = 0.04). The second experiment consisted of bovine fetal fibroblasts transfected with modified synthetic mRNA encoding pluripotency factors. The controls were identical to the previous experiment, but treatment groups were transfected with modified synthetic mRNA encoding either three factors (3F: OCT4, SOX2, KLF4) or four factors (4F: OCT4, SOX2, KLF4, c-MYC). The treated cells were transfected every other day and evaluated on Day 24 for cellular viability. There was no difference in cellular viability in all treatment groups when compared to controls (P = 0.2). The introduction of synthetic mRNA containing modified bases maintains cellular viability when compared to controls. The decreased immune response by the inclusion of modified bases may be advantageous in a variety of applications from the introduction of transcription activator-like effector nuclease (TALEN) or zinc finger nucleases for genomic editing to increased efficiency of the development of induced pluripotent stem cells.

Analysis of targeting sequences demonstrates that trafficking to the Toxoplasma gondii plastid branches off the secretory system

2000 ◽  
Vol 113 (22) ◽  
pp. 3969-3977 ◽  
Author(s):  
A. DeRocher ◽  
C.B. Hagen ◽  
J.E. Froehlich ◽  
J.E. Feagin ◽  
M. Parsons
Keyword(s):  
Green Fluorescent Protein ◽  
Toxoplasma Gondii ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Transit Peptide ◽  
Green Fluorescent ◽  
Secretory System

Apicomplexan parasites possess a plastid-like organelle called the apicoplast. Most proteins in the Toxoplasma gondii apicoplast are encoded in the nucleus and imported post-translationally. T. gondii apicoplast proteins often have a long N-terminal extension that directs the protein to the apicoplast. It can be modeled as a bipartite targeting sequence that contains a signal sequence and a plastid transit peptide. We identified two nuclearly encoded predicted plastid proteins and made fusions with green fluorescent protein to study protein domains required for apicoplast targeting. The N-terminal 42 amino acids of the apicoplast ribosomal protein S9 directs secretion of green fluorescent protein, indicating that targeting to the apicoplast proceeds through the secretory system. Large sections of the S9 predicted transit sequence can be deleted with no apparent impact on the ability to direct green fluorescent protein to the apicoplast. The predicted transit peptide domain of the S9 targeting sequence directs protein to the mitochondrion in vivo. The transit peptide can also direct import of green fluorescent protein into chloroplasts in vitro. These data substantiate the model that protein targeting to the apicoplast involves two distinct mechanisms: the first involving the secretory system and the second sharing features with typical chloroplast protein import.

scholarly journals Expression of the nfa1 Gene Cloned from Pathogenic Naegleria fowleri in Nonpathogenic N. gruberi Enhances Cytotoxicity against CHO Target Cells In Vitro

2005 ◽  
Vol 73 (7) ◽  
pp. 4098-4105 ◽  
Author(s):  
Seok-Ryoul Jeong ◽  
Sang-Chul Lee ◽  
Kyoung-Ju Song ◽  
Sun Park ◽  
Kyongmin Kim ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Target Cells ◽  
Naegleria Fowleri ◽  
Specific Primers ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Nægleria Fowleri ◽  
Green Fluorescent ◽  
Naegleria Gruberi

ABSTRACT The pathogenic amoeba Naegleria fowleri has a 360-bp nfa1 gene that encodes the Nfa1 protein (13.1 kDa), which is located in the pseudopodia of the amoeba, and an anti-Nfa1 antibody reduces N. fowleri-induced mammalian-cell cytotoxicity in vitro. In contrast, an anti-Nfa1 antibody cannot detect Nfa1 protein expression in the nonpathogenic amoeba Naegleria gruberi, which also possesses the nfa1 gene. In the present study, the nfa1 gene cloned from pathogenic N. fowleri was transfected into nonpathogenic N. gruberi to determine whether it was related to pathogenicity. The nfa1 gene was initially inserted into a eukaryotic transfection vector, pEGFP-C2, containing a cytomegalovirus promoter and the green fluorescent protein (GFP) gene, and was designed as pEGFP-C2/nfa1UTR (nfa1UTR contains 5′ upstream regions, the nfa1 open reading frame, and 3′ downstream regions). After transfection, the green fluorescence was observed in the cytoplasm of N. gruberi trophozoites. These transfectants were preserved for more than 9 months after selection. The transfected nfa1 gene was observed by PCR using nfa1- and vector-specific primers in the genomic DNA of N. gruberi transfected with the pEGFP-C2/nfa1UTR vector. In addition, the nfa1 and GFP genes were identified by reverse transcription-PCR in transgenic N. gruberi. The Nfa1 protein expressed in transgenic N. gruberi was identified as a 13.1-kDa band by Western blotting using an anti-Nfa1 antibody. Finally, N. gruberi transfected with the pEGFP-C2/nfa1UTR vector was found to have enhanced cytotoxicity against CHO cells compared with naïve N. gruberi.

Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase

2008 ◽  
Vol 410 (2) ◽  
pp. 291-299 ◽  
Author(s):  
Fui-Ching Tan ◽  
Qi Cheng ◽  
Kaushik Saha ◽  
Ilka U. Heinemann ◽  
Martina Jahn ◽  
...  
Keyword(s):  
Expression Vector ◽  
Fluorescent Protein ◽  
Sequence Similarity ◽  
Homology Search ◽  
Protein Database ◽  
Gene Encoding ◽  
Reading Frame ◽  
Green Fluorescent

UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.

scholarly journals Classification and Strength Measurement of Stationary-Phase Promoters by Use of a Newly Developed Promoter Cloning Vector

2004 ◽  
Vol 186 (21) ◽  
pp. 7112-7122 ◽  
Author(s):  
Tomohiro Shimada ◽  
Hideki Makinoshima ◽  
Yoshito Ogawa ◽  
Takeyoshi Miki ◽  
Michihisa Maeda ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Fluorescent Protein ◽  
In Vitro Transcription ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Promoter Cloning ◽  
Genome Transcription ◽  
Green Fluorescent

ABSTRACT When an Escherichia coli culture changes from exponential growth to the stationary phase, expression of growth-related genes levels off, while a number of stationary-phase-specific genes are turned on. To gain insight into the growth phase-dependent global regulation of genome transcription, we analyzed the strength and specificity of promoters associated with the stationary-phase genes. For the in vivo assay of promoter activity, 300- to 500-bp DNA fragments upstream from the translation initiation codon were isolated and inserted into a newly constructed doubly fluorescent protein (DFP) vector. The activity of test promoters was determined by measuring the green fluorescent protein (GFP). To avoid the possible influence of plasmid copy number, the level of transcription of reference promoter lacUV5 on the same plasmid was determined by measuring the red fluorescent protein (RFP). Thus, the activities of test promoters could be easily and accurately determined by determining the GFP/RFP ratio. Analysis of the culture time-dependent variation of 100 test promoters indicated that (i) a major group of the stationary-phase promoters are up-regulated only in the presence of RpoS sigma; (ii) the phase-coupled increase in the activity of some promoters takes place even in the absence of RpoS; and (iii) the activity of some promoters increases in the absence of RpoS. This classification was confirmed by testing in vitro transcription by using reconstituted RpoD and RpoS holoenzymes.

scholarly journals Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

2021 ◽  
Vol 9 (5) ◽  
pp. 1005
Author(s):  
Olga Chervyakova ◽  
Elmira Tailakova ◽  
Nurlan Kozhabergenov ◽  
Sandugash Sadikaliyeva ◽  
Kulyaisan Sultankulova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Viral Vector ◽  
Foot And Mouth Disease ◽  
Open Reading Frames ◽  
Foreign Gene ◽  
Mouth Disease Virus ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

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