Abstract: In higher eukaryotes, E2F transcription factors often drive expression of genes necessary for the cell cycle, notably the G1/S phase transition. With conventional transcriptional reporter systems, expression of a reporter gene from an E2F-responsive promoter would allow one to identify the fraction of cells making this transition. Here, we have engineered an E2F-responsive genetic reporter system that outputs the proliferation rate. The system takes advantage of the long half-lives of fluorescent protein reporters and output signal normalization. By doing so, it converts dynamic pulses of E2F activity into an analog output proportional to the proliferation rate. Such a system should be useful for applications involving high-throughput drug or genetic screens, investigation of cellular environment, and biological engineering.
Abstract: Expression of the autoimmune regulator (Aire) protein in mice and humans is thought to be restricted to the medullary epithelial and monocyte-dendritic cells of the thymus. There it mediates expression and presentation of a large variety of proteins, including those that are peripheral organ-specific and are not expressed by other thymocytes. In this way, self-reactive T lymphocytes that would attack peripheral cells producing these proteins are confronted with the self-Ags and, as a consequence, are deleted. In this study, we show that Aire mRNA is also expressed in the testis--another tissue with promiscuous gene expression. Aire protein, however, is expressed only sporadically in spermatogonia and spermatocytes. Transcription of genes that are under Aire control in the thymus is unaffected by Aire in the testis. However, in mice with a disrupted Aire gene, the scheduled apoptotic wave of germ cells, which is necessary for normal mature spermatogenesis, is reduced, and sporadic apoptosis in adults is increased. Because Rag-1 deficiency does not abolish the effect, the adaptive immune system is not involved. We suggest that there is a link between the scheduled and sporadic apoptotic processes and propose that scheduled apoptosis provides a counterselection mechanism that keeps the germline stable.
Abstract: We have developed a modular method of plasmid construction that can join multiple DNA components in a single reaction. A nicking enzyme is used to create 5' and 3' overhangs on PCR-generated DNA components. Without the use of ligase or restriction enzymes, components are joined using oligonucleotide linkers that recognize the overhangs. By specifying the sequences of the linkers, desired components can be assembled in any combination and order to generate different plasmid vectors.
Abstract: It is unknown how activation-induced cytidine deaminase (AID) targets immunoglobulin (Ig) genes during somatic hypermutation. Results to date are difficult to interpret: while some results argue that Ig genes have special sequences that mobilize AID, other work shows that non-Ig transgenes mutate. In this report, we have examined the effects of the intronic mu enhancer on the somatic hypermutation rates of a retroviral vector. For this analysis, we used centroblast-like Ramos cells to capture as much of the natural process as possible, used AIDhi and AIDlow Ramos variants to ensure that mutations are AID induced, and measured mutation of a GFP-provirus to achieve greater sensitivity. We found that mutation rates of the non-Ig provirus were AID-dependent, were similar at different genomic loci, but were approximately 10-fold lower than the V-region suggesting that AID can mutate non-Ig genes at low rates. However, the intronic mu enhancer did not increase the mutation rates of the provirus. Interestingly, exogenous over-expression of AID revealed that the V-region mutation rate can be saturated by lower levels of AID than the provirus, suggesting that selective mutation of Ig sequences is compromised in cells that over-express AID.
Abstract: When gene expression varies unpredictably between genetically identical organisms, this is sometimes ascribed as stochastic. With the prevalence of retroviral vectors, stochastic repression is often observed and can complicate the interpretation of outcomes. But it may also faithfully reflect characteristics of sites in the genome.
Abstract: Retroviruses can cause tumors when they integrate near a protooncogene or tumor suppressor gene of the host. We infected >2,500 mice with the SL3-3 murine leukemia virus; in 22 resulting tumors, we found provirus integrations nearby or within the gene that contains the mir-17-92 microRNA (miRNA) cistron. Using quantitative real-time PCR, we showed that expression of miRNA was increased in these tumors, indicating that retroviral infection can induce expression of oncogenic miRNAs. Our results demonstrate that retroviral mutagenesis can be a potent tool for miRNA discovery.
Abstract: Retroviral activation of the AP-1/ATF super family member Jdp2 was recently reported to be a common event in M-MLV-induced T cell lymphoma in p27-null C57x129 mice as compared to wild type-inoculated mice but has not been found important in other models. On the basis of retroviral tag retrieval from 1190 individual Akv- and SL3-3-induced lymphomas, we here report that insertional mutagenesis into the 250-kb Fos/Jdp2/Batf locus is associated with SL3-3 MLV-induced T but not Akv-induced B cell lymphomas of NMRI and SWR mice. Integration pattern and clonality analyses suggest that Jdp2 participates in SL3-3-induced tumorigenesis distinctly as compared to the M-MLV setting. Northern blot analysis showed Jdp2 to be alternatively spliced in various normal tissues as well as MLV-induced lymphomas. Interestingly, in some tumors, proviral insertion seems to activate different mRNA sub-species. Whereas elevated mRNA levels of the Fos gene could not be correlated with provirus presence, in one case, Northern blot analysis as well as quantitative real-time PCR indicated proviral activation of the AP-1 super family member Batf, a gene not previously reported to be a target of insertional mutagenesis. A novel integration cluster between Jdp2 and Batf apparently did not influence the expression level of either gene, underscoring the importance of addressing expression effects to identify target genes of insertion. Altogether, such distinct insertion patterns point to different mechanism of activation of specific proto-oncogenes and are consequently of importance for the understanding of proviral activation mechanisms as well as the specific role of individual oncogenes in tumor development.
Abstract: It is generally believed that in cells undergoing Ig somatic hypermutation, more cell divisions result in more mutations. This is because DNA synthesis and replication is thought to play roles in the known mechanisms-cytidine deamination and subsequent conversion to thymidine, uracil-DNA glycosylase-mediated repair, mismatch repair, and DNA synthesis by error-prone polymerases. In this study, we manipulated the number of cell generations by varying the rate at which cultures of a mouse cell line were replenished with fresh medium. We found that the frequency of mutants does not necessarily increase with the number of cell generations. On the contrary, a greater number of divisions can lead to a lower frequency of mutants, indicating that cell division is not a rate-limiting step in the hypermutation process. Thus, when comparing mutation rates, we suggest that rates are more appropriately expressed as mutations per day than per cell generation.
Abstract: The cell line WEHI-231 expresses activation-induced cytidine deaminase (AID), the enzyme that mediates hypermutation and immunoglobulin class switch recombination in activated B cells. Although both the cDNA sequence and protein expression of AID appear normal, the frequency of mutation at the endogenous immunoglobulin locus is low. In this report, we have tested the mutational activity of the cell line with three different indicator constructs. The first construct measures a composite rate of transversions of C to G and C to A, respectively. The second construct measures only transversion from C to G. The third measures the canonical AID activity, from C to U, which after cell replication can result in a C to T transition. We found that in WEHI-231, the C to G activity is 32- to 37-times lower than in the hypermutating cell line 18-81. The C to T activity is also much reduced, but only 12-fold. We suggest that the WEHI-231 lacks an activity that subverts the faithful repair of incipient C to U mutations.
Abstract: DNA mutagenesis is generally considered harmful. Yet activated B cells normally mutate the Ig loci. Because this somatic hypermutation is potentially dangerous, it has been hypothesized that mutations do not occur throughout the genome but instead are actively targeted to the Ig loci. Here we challenge this longstanding and widely accepted hypothesis. We demonstrate that hypermutation requires no Ig gene sequences. Instead, activation-induced cytidine deaminase and other trans-acting hypermutation factors may function as general mutators.
Abstract: To generate a replication-competent retrovirus that could be conditionally inactivated, we flanked the viral genes of the Akv murine leukemia virus with LoxP sites. This provirus can delete its envelope gene by LoxP/Cre mediated recombination and thereby allow superinfection of Cre recombinase expressing cells.
Abstract: Small resting B lymphocytes all start out producing IgM Abs. Upon encountering Ag, the cells become activated and make a switch from IgM to other Ig classes. This class switch serves to distribute a particular V region to different Ig C regions. Each C region mediates a specialized effector function, and so, through switching, an organism can guide its Abs to various sites. Creating the new H chain requires loop-out and deletion of DNA between switch regions. These DNA acrobatics require transcription of the switch regions, presumably so that necessary factors can gain access to the DNA. These requisite switching factors include activation-induced cytidine deaminase and components of general DNA repair, including base excision repair, mismatch repair, and double-strand break repair. Despite much recent progress, not all important factors have been discovered, especially those that may guide recombination to a particular subclass.
Abstract: Four decades ago, it was hypothesized that lymphocytes mutate the immunoglobulin loci at a rate much greater than the spontaneous rate. Over time this Ig somatic hypermutation became the prime example of in vivo, site-directed mutagenesis. But recent studies have demonstrated that this is not necessarily the case, and that hypermutation occurs not only at the Ig locus but also at various locations throughout the genome. Here, we propose that hypermutation can be genome-wide with minimal injury, if a sufficient mutation rate is coupled with strong clonal selection.
Abstract: After rearrangement of immunoglobulin gene segments, the immune system evolves the antibody repertoire by mutating the immunoglobulin variable region at a high rate. While this somatic hypermutation was thought to occur only at the variable region, recent studies suggest that hypermutation can occur at locations throughout the genome. Building upon this notion, we sought to exploit this mechanism as a mutagenesis tool. We created a substrate based on GFP that could be screened using flow cytometry and showed that retroviral infection can deliver the transgene to genomic locations that support hypermutation. Infected cells generated various GFP mutants with increased fluorescence intensity and analysis revealed mutations not only at the chromophore, but also an unexpected mutation at a distant residue. Our results demonstrate in principle that immunoglobulin somatic hypermutation can be a potent means of mutagenesis. With appropriate selection conditions it may be utilized to evolve gene products with desired properties.
Abstract: Pseudomonas aeruginosa CW961, an isolate from the vicinity of a deep-sea hydrothermal vent, grew in the presence of 5 mM Cd(2+) and removed Cd(2+) from solution. Sulfate was sufficient for growth when Cd(2+) was not present in the culture medium; however, thiosulfate was necessary for Cd(2+) precipitation and cell survival in the presence of Cd(2+).
Abstract: We previously have genetically engineered an aerobic sulfate reduction pathway in Escherichia coli for the generation of hydrogen sulfide and demonstrated the pathway's utility in the precipitation of cadmium. To engineer the pathway, the assimilatory sulfate reduction pathway was modified so that cysteine was overproduced. Excess cysteine was then converted by cysteine desulfhydrase to an abundance of hydrogen sulfide, which then reacted with aqueous cadmium to form cadmium sulfide. In this study, observations of various E. coli clones were combined with an analysis of kinetic and transport phenomena. This analysis revealed that cysteine production is the rate-limiting step in the engineered pathway and provided an explanation for the phenomenon of cell surface precipitation. An analytical model showed that cadmium sulfide must form at the cell surface because the rate of cadmium sulfide formation is extremely fast and the rate of sulfide transport is relatively slow.
Abstract: The cysteine desulfhydrase gene of Treponema denticola was over-expressed in Escherichia coli to produce sulfide under aerobic conditions and to precipitate metal sulfide complexes on the cell wall. When grown in a defined salts medium supplemented with cadmium and cysteine, E. coli producing cysteine desulfhydrase secreted sulfide and removed nearly all of the cadmium from solution after 48 h. A control strain produced significantly less sulfide and removed significantly less cadmium. Measurement of acid-labile sulfide and energy dispersive X-ray spectroscopy indicated that cadmium was precipitated as cadmium sulfide. Without supplemental cysteine, both the E. coli producing cysteine desulfhydrase and the control E. coli demonstrated minimal cadmium removal.
Abstract: The conversion of sulfate to an excess of free sulfide requires stringent reductive conditions. Dissimilatory sulfate reduction is used in nature by sulfate-reducing bacteria for respiration and results in the conversion of sulfate to sulfide. However, this dissimilatory sulfate reduction pathway is inhibited by oxygen and is thus limited to anaerobic environments. As an alternative, we have metabolically engineered a novel aerobic sulfate reduction pathway for the secretion of sulfides. The assimilatory sulfate reduction pathway was redirected to overproduce cysteine, and excess cysteine was converted to sulfide by cysteine desulfhydrase. As a potential application for this pathway, a bacterium was engineered with this pathway and was used to aerobically precipitate cadmium as cadmium sulfide, which was deposited on the cell surface. To maximize sulfide production and cadmium precipitation, the production of cysteine desulfhydrase was modulated to achieve an optimal balance between the production and degradation of cysteine.
Abstract: A fluorescent pseudomonad (strain CW-96-1) isolated from a deep-sea vent sample grew at 30 degrees C under aerobic conditions in an artificial seawater medium and tolerated cadmium concentrations up to 5 mM. After 140 h, strain CW-96-1 removed > 99% of the cadmium from solution. Energy dispersive microanalysis revealed that the cadmium was removed by precipitation on the cell wall; sulfide production was confirmed by growth on Kligler's agar. Based on 16S ribosomal DNA sequencing and fatty acid analysis, the microorganism is closely related to Pseudomonas aeruginosa.
