Chimeric proteins tagged with specific 3xHA cassettes may present instability and functional problems.
Ontology highlight
ABSTRACT:
SUBMITTER: Saiz-Baggetto S
PROVIDER: S-EPMC5553802 | biostudies-other | 2017
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Cyanobacteria of the Synechococcus and Prochlorococcus genera are important contributors to photosynthetic productivity in the open oceans. Recently, core photosystem II (PSII) genes were identified in cyanophages and proposed to function in photosynthesis and in increasing viral fitness by supplementing the host production of these proteins. Here we show evidence for the presence of photosystem I (PSI) genes in the genomes of viruses that infect these marine cyanobacteria, using pre-existing metagenomic data from the global ocean sampling expedition as well as from viral biomes. The seven cyanobacterial core PSI genes identified in this study, psaA, B, C, D, E, K and a unique J and F fusion, form a cluster in cyanophage genomes, suggestive of selection for a distinct function in the virus life cycle. The existence of this PSI cluster was confirmed with overlapping and long polymerase chain reaction on environmental DNA from the Northern Line Islands. Potentially, the seven proteins encoded by the viral genes are sufficient to form an intact monomeric PSI complex. Projection of viral predicted peptides on the cyanobacterial PSI crystal structure suggested that the viral-PSI components might provide a unique way of funnelling reducing power from respiratory and other electron transfer chains to the PSI.
| S-EPMC4605144 | biostudies-literature
Project description:Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.
| S-EPMC2957861 | biostudies-literature
Project description:During the last decade, there was a marked increase in the development of tools and techniques to study the molecular mechanisms of the HIV replication cycle by using fluorescence microscopy. Researchers often apply the fusion of tags and fluorophores to viral proteins, surrogate proteins, or dyes to follow individual virus particles while they progress throughout infection. The inclusion of such fusion motifs or surrogates frequently disrupts viral infectivity or results in a change of the wild-type phenotype. Here, we detail the construction and functional characterization of two new constructs where we fused fluorescent proteins to the N-terminus of HIV-1 Integrase. In the first, IN is recruited into assembling particles via a codon optimized Gag to complement other viral constructs, while the second is fused to a Gag-Pol expression vector fully capable of integration. Our data shows that N-terminal tagged IN is functional for integration by both recovery of integration of catalytically inactive IN and by the successful infectivity of viruses carrying only labeled IN. These tools will be important to study the individual behavior of viral particles and associate such behavior to infectivity.
| S-EPMC7997544 | biostudies-literature
Project description:ObjectivesThe transposon Tn1331 possesses a region including three antibiotic resistance genes with the structure aac(6')-Ib-attC-aadA1-attI1*-bla(OXA-9)-attC, which potentially includes four gene cassettes. Experimental data on the mobility of fusion cassettes as well as those on mobility of cassettes in a genetic environment such as Tn1331, which lacks an integrase gene, are limited. Therefore, experiments using pJHCMW1, a plasmid harbouring this transposon, in the presence of IntI1 supplied in trans were carried out to define which cassettes are mobile in vivo.MethodsIn vivo excision of resistance genes was investigated in Escherichia coli cells harbouring pJHCMW1 and in a recombinant clone that included the intI1 gene under the control of the P(tac) promoter. Plasmid DNA was purified and subjected to PCR analysis, and DNA sequencing of PCR products was performed to determine whether excision had occurred.Results and conclusionsIn vivo recombination experiments showed that the fused aadA1-attI1*-bla(OXA-9)-attC gene cassette was excised in the presence of IntI1. The excision of a DNA fragment including aadA1-attI1* was also detected but at a lower frequency. The analysis of the latter recombination reaction showed that, although attI1* includes only a small fraction of the complete attI1 sequence, it is still used as a substrate by IntI1, albeit in a very inefficient manner.
| S-EPMC2536708 | biostudies-literature
Project description:A region in the first intron of a metallothionein-encoding gene of the sea urchin Strongylocentrotus purpuratus (SpMTA gene) regulates its 5' promoter activity. Within this region is a 290-bp cassette of six sequence motifs that are present in other genes in this species and posited to operate as regulatory elements. The cassette, present at high multiplicity in the genome, was used to screen genomic DNA clones. Of these, six diverse individuals were partially sequenced and found to have segments 94% identical to the 290-bp cassette in the SpMTA gene. Their next 80 bp diverged from the SpMTA sequence but were highly identical among the six non-SpMTA clones and contained an additional regulatory motif. These diverse clones thus contained 370-bp cassettes of an overall 94% sequence identity and an apparent content of seven regulatory elements. The regulatory cassettes were transposon-like, insofar as the termini of the highly identical regions consisted of 24- to 25-bp inverted repeats, bracketed by 6- to 9-bp direct repeats in the divergent regions. In addition to being in transcripts of the SpMTA intron, the cassette was found in other sea urchin embryo poly(A)+ RNAs, in eggs and embryos, and enriched in pluteus ectoderm. The cassette sequence was present in moderate abundance in transcripts in both sense and antisense orientation. We report the presence of a transposon-like cassette of regulatory elements that is also represented in RNA, which potentially could function differently from previously described transposons.
| S-EPMC47876 | biostudies-other
Project description:BackgroundThe Drosophila SXL protein controls sex determination and dosage compensation. It is a sex-specific factor controlling splicing of its own Sxl pre-mRNA (auto-regulation), tra pre-mRNA (sex determination) and msl-2 pre-mRNA plus translation of msl-2 mRNA (dosage compensation). Outside the drosophilids, the same SXL protein has been found in both sexes so that, in the non-drosophilids, SXL does not appear to play the key discriminating role in sex determination and dosage compensation that it plays in Drosophila. Comparison of SXL proteins revealed that its spatial organisation is conserved, with the RNA-binding domains being highly conserved, whereas the N- and C-terminal domains showing significant variation. This manuscript focuses on the evolution of the SXL protein itself and not on regulation of its expression.MethodologyDrosophila-Sciara chimeric SXL proteins were produced. Sciara SXL represents the non-sex-specific function of ancient SXL in the non-drosophilids from which presumably Drosophila SXL evolved. Two questions were addressed. Did the Drosophila SXL protein have affected their functions when their N- and C-terminal domains were replaced by the corresponding ones of Sciara? Did the Sciara SXL protein acquire Drosophila sex-specific functions when the Drosophila N- and C-terminal domains replaced those of Sciara? The chimeric SXL proteins were analysed in vitro to study their binding affinity and cooperative properties, and in vivo to analyse their effect on sex determination and dosage compensation by producing Drosophila flies that were transgenic for the chimeric SXL proteins.ConclusionsThe sex-specific properties of extant Drosophila SXL protein depend on its global structure rather than on a specific domain. This implies that the modifications, mainly in the N- and C-terminal domains, that occurred in the SXL protein during its evolution within the drosophilid lineage represent co-evolutionary changes that determine the appropriate folding of SXL to carry out its sex-specific functions.
| S-EPMC3677924 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2023-05-01 | GSE220678 | GEO
Project description:Chromosomal translocations encoding chimeric fusion proteins constitute one of the most common mechanisms underlying oncogenic transformation in human cancer. Fusion peptides resulting from such oncogenic chimeric fusions, though unique to specific cancer subtypes, are unexplored as cancer biomarkers. Here we show, using an approach termed fusion peptide multiple reaction monitoring mass spectrometry, the direct identification of different cancer-specific fusion peptides arising from protein chimeras that are generated from the juxtaposition of heterologous genes fused by recurrent chromosomal translocations. Using fusion peptide multiple reaction monitoring mass spectrometry in a clinically relevant scenario, we demonstrate the specific, sensitive, and unambiguous detection of a specific diagnostic fusion peptide in clinical samples of anaplastic large cell lymphoma, but not in a diverse array of benign lymph nodes or other forms of primary malignant lymphomas and cancer-derived cell lines. Our studies highlight the utility of fusion peptides as cancer biomarkers and carry broad implications for the use of protein biomarkers in cancer detection and monitoring.
| S-EPMC3790285 | biostudies-literature
Project description:Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats, which may expand the coding capacity of mRNA and diversify the regulation in human.
2023-05-01 | GSE220677 | GEO
Project description:Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats,which may expand the coding capacity of mRNA and diversify the regulation in human.
2023-05-01 | GSE220676 | GEO