Eukaryotic gene invasion by a bacterial mobile insertion sequence element IS2 during cloning into a plasmid vector

Escherichia coli (E. coli) are commonly used as hosts for DNA cloning and sequencing. Upon transformation of E. coli with recombined vector carrying a gene of interest, the bacteria multiply the gene of interest while maintaining the integrity of its content. During the subcloning of a mouse genomic fragment into a plasmid vector, we noticed that the size of the insert increased significantly upon replication in E. coli. The sequence of the insert was determined and found to contain a novel DNA sequence within the mouse genomic insert. A BLAST search of GenBank revealed the novel sequence to be that of the Insertion Sequence 2 (IS2) element from E. coli that was likely inserted during replication in that organism. Importantly, a detailed search of GenBank shows that the IS2 is present within many eukaryotic nucleotide sequences, and in many cases, has been annotated as being part of the protein. The results of this study suggest that one must perform additional careful analysis of the sequence results using BLAST comparisons, and further verification of gene annotation before submission into the GenBank

The intein of the Thermoplasma A-ATPase A subunit: Structure, evolution and expression in E. coli

BACKGROUND: Inteins are selfish genetic elements that excise themselves from the host protein during post translational processing, and religate the host protein with a peptide bond. In addition to this splicing activity, most reported inteins also contain an endonuclease domain that is important in intein propagation. RESULTS: The gene encoding the Thermoplasma acidophilum A-ATPase catalytic subunit A is the only one in the entire T. acidophilum genome that has been identified to contain an intein. This intein is inserted in the same position as the inteins found in the ATPase A-subunits encoding gene in Pyrococcus abyssi, P. furiosus and P. horikoshii and is found 20 amino acids upstream of the intein in the homologous vma-1 gene in Saccharomyces cerevisiae. In contrast to the other inteins in catalytic ATPase subunits, the T. acidophilum intein does not contain an endonuclease domain. T. acidophilum has different codon usage frequencies as compared to Escherichia coli. Initially, the low abundance of rare tRNAs prevented expression of the T. acidophilum A-ATPase A subunit in E. coli. Using a strain of E. coli that expresses additional tRNAs for rare codons, the T. acidophilum A-ATPase A subunit was successfully expressed in E. coli. CONCLUSIONS: Despite differences in pH and temperature between the E. coli and the T. acidophilum cytoplasms, the T. acidophilum intein retains efficient self-splicing activity when expressed in E. coli. The small intein in the Thermoplasma A-ATPase is closely related to the endonuclease containing intein in the Pyrococcus A-ATPase. Phylogenetic analyses suggest that this intein was horizontally transferred between Pyrococcus and Thermoplasma, and that the small intein has persisted in Thermoplasma apparently without homing

Conservation of intron and intein insertion sites: implications for life histories of parasitic genetic elements

<p>Abstract</p> <p>Background</p> <p>Inteins and introns are genetic elements that are removed from proteins and RNA after translation or transcription, respectively. Previous studies have suggested that these genetic elements are found in conserved parts of the host protein. To our knowledge this type of analysis has not been done for group II introns residing within a gene. Here we provide quantitative statistical support from an analyses of proteins that host inteins, group I introns, group II introns and spliceosomal introns across all three domains of life.</p> <p>Results</p> <p>To determine whether or not inteins, group I, group II, and spliceosomal introns are found preferentially in conserved regions of their respective host protein, conservation profiles were generated and intein and intron positions were mapped to the profiles. Fisher's combined probability test was used to determine the significance of the distribution of insertion sites across the conservation profile for each protein. For a subset of studied proteins, the conservation profile and insertion positions were mapped to protein structures to determine if the insertion sites correlate to regions of functional activity. All inteins and most group I introns were found to be preferentially located within conserved regions; in contrast, a bacterial intein-like protein, group II and spliceosomal introns did not show a preference for conserved sites.</p> <p>Conclusions</p> <p>These findings demonstrate that inteins and group I introns are found preferentially in conserved regions of their respective host proteins. Homing endonucleases are often located within inteins and group I introns and these may facilitate mobility to conserved regions. Insertion at these conserved positions decreases the chance of elimination, and slows deletion of the elements, since removal of the elements has to be precise as not to disrupt the function of the protein. Furthermore, functional constrains on the targeted site make it more difficult for hosts to evolve immunity to the homing endonuclease. Therefore, these elements will better survive and propagate as molecular parasites in conserved sites. In contrast, spliceosomal introns and group II introns do not show significant preference for conserved sites and appear to have adopted a different strategy to evade loss.</p

Structural stability and endonuclease activity of a PI-SceI GFP-fusion protein

<p>Homing endonucleases are site-specific and rare cutting endonucleases often encoded by intron or intein containing genes. They lead to the rapid spread of the genetic element that hosts them by a process termed 'homing'; and ultimately the allele containing the element will be fixed in the population.</p> <p>PI-<i>Sce</i>I, an endonuclease encoded as a protein insert or intein within the yeast V-ATPase catalytic subunit encoding gene (<i>vma1</i>), is among the best characterized homing endonucleases. The structures of the <i>Sce</i> VMA1 intein and of the intein bound to its target site are known. Extensive biochemical studies performed on the PI-<i>Sce</i>I enzyme provide information useful to recognize critical amino acids involved in self-splicing and endonuclease functions of the protein. Here we describe an insertion of the Green Fluorescence Protein (GFP) into a loop which is located between the endonuclease and splicing domains of the <i>Sce</i> VMA1 intein. The GFP is functional and the additional GFP domain does not prevent intein excision and endonuclease activity. However, the endonuclease activity of the newly engineered protein was different from the wild-type protein in that it required the presence of Mn²⁺ and not Mg²⁺ metal cations for activity.</p

Mutation of POLB Causes Lupus in Mice

A replication study of a previous genome-wide association study (GWAS) suggested that a SNP linked to the POLB gene is associated with systemic lupus erythematosus (SLE). This SNP is correlated with decreased expression of Pol β, a key enzyme in the base excision repair (BER) pathway. To determine whether decreased Pol β activity results in SLE, we constructed a mouse model of POLB that encodes an enzyme with slow DNA polymerase activity. We show that mice expressing this hypomorphic POLB allele develop an autoimmune pathology that strongly resembles SLE. Of note, the mutant mice have shorter immunoglobulin heavy-chain junctions and somatic hypermutation is dramatically increased. These results demonstrate that decreased Pol β activity during the generation of immune diversity leads to lupus-like disease in mice, and suggest that decreased expression of Pol β in humans is an underlying cause of SLE

PPARγ Negatively Regulates T Cell Activation to Prevent Follicular Helper T Cells and Germinal Center Formation

<div><p>Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that regulates lipid and glucose metabolism. Although studies of PPARγ ligands have demonstrated its regulatory functions in inflammation and adaptive immunity, its intrinsic role in T cells and autoimmunity has yet to be fully elucidated. Here we used CD4-PPARγ^KO mice to investigate PPARγ-deficient T cells, which were hyper-reactive to produce higher levels of cytokines and exhibited greater proliferation than wild type T cells with increased ERK and AKT phosphorylation. Diminished expression of IκBα, Sirt1, and Foxo1, which are inhibitors of NF-κB, was observed in PPARγ-deficient T cells that were prone to produce all the signature cytokines under Th1, Th2, Th17, and Th9 skewing condition. Interestingly, 1-year-old CD4-PPARγ^KO mice spontaneously developed moderate autoimmune phenotype by increased activated T cells, follicular helper T cells (T_FH cells) and germinal center B cells with glomerular inflammation and enhanced autoantibody production. Sheep red blood cell immunization more induced T_FH cells and germinal centers in CD4-PPARγ^KO mice and the T cells showed increased of Bcl-6 and IL-21 expression suggesting its regulatory role in germinal center reaction. Collectively, these results suggest that PPARγ has a regulatory role for T_FH cells and germinal center reaction to prevent autoimmunity.</p></div

PPARγ deficiency induces hyper-reactivity in T cells.

<p>CD4⁺CD25⁻ T cells from the spleens of 6- to 8-week-old female littermate control (Cre−) and CD4-PPARγ^KO (Cre+) mice were stimulated for 24 h with plate-bound anti-CD3 and soluble anti-CD28 antibodies. (A) IFN-γ, (B) IL-4, (C) IL-17, and (D) IL-2 levels in culture supernatants were measured by ELISA. (E) Proliferation of 3-day-anti-CD3/CD28 stimulated cells was measured by H³-thymidine incorporation. Values represent the mean ± SEM of counts per minute (CPM) in triplicate wells. CD4⁺25⁻ T cells were differentiated under Th1, Th2, Th17, and Th9 differentiation conditions. (F) After 5 days, the culture supernatants were collected and cytokine levels (IFN-γ, IL-13, IL-17, and IL-9) were measured by ELISA. Values represent the mean ± SEM, n = 4–6. *<i>P</i><0.05. (G) Flow cytometric analysis for lineage-specific cytokines (IFN-γ, IL-4, IL-17, and IL-9) of Th1, Th2, Th17 and Th9 were determined by intracellular staining. Representative data were shown from five independent experiments.</p