Identification and analysis of the first 2009 pandemic H1N1 influenza virus from U.S. feral swine.

The first case of pandemic H1N1 influenza (pH1N1) virus in feral swine in the United States was identified in Texas through the United States Department of Agriculture (USDA) Wildlife Services' surveillance program. Two samples were identified as pandemic influenza by reverse transcriptase quantitative PCR (RT-qPCR). Full-genome Sanger sequencing of all eight influenza segments was performed. In addition, Illumina deep sequencing of the original diagnostic samples and their respective virus isolation cultures were performed to assess the feasibility of using an unbiased whole-genome linear target amplification method and multiple sample sequencing in a single Illumina GAIIx lane. Identical sequences were obtained using both techniques. Phylogenetic analysis indicated that all gene segments belonged to the pH1N1 (2009) lineage. In conclusion, we have identified the first pH1N1 isolate in feral swine in the United States and have demonstrated the use of an easy unbiased linear amplification method for deep sequencing of multiple samples.

High-speed, multicolor fluorescent two-dimensional gene scanning.

Two-dimensional gene scanning (TDGS) is a method for analyzing multiple DNA fragments in parallel for all possible sequence variations, using extensive multiplex PCR and two-dimensional electrophoretic separation on the basis of size and melting temperature. High throughput application of TDGS is limited by the prolonged time periods necessary to complete the second-dimension electrophoretic separation step--denaturing gradient gel electrophoresis--and the current need for gel staining. To address these problems, we constructed a high-voltage, automatic, two-dimensional electrophoresis system and used this in combination with thinner gels to reduce two-dimensional electrophoresis time about 80%. Instead of gel staining, we used three different fluorophores to simultaneously analyze three samples in the same gel. These improvements greatly increase TDGS speed and throughput and make the method highly suitable for large-scale single-nucleotide polymorphism discovery and genetic testing.

Negative regulation of selected bHLH proteins by eHAND.

The bHLH protein eHAND plays an important role in the development of extraembryonic, mesodermal, and cardiac cell lineages, presumably through heterodimerization with other HLH proteins and DNA binding. In this study, we have identified a novel transcriptional activity of eHAND. In transient transfection assays, eHAND is a potent inhibitor of activation by some but not all bHLH proteins. eHAND can prevent E-box DNA binding by these bHLH proteins. Interestingly, eHAND can also strongly inhibit transactivation activity by a MyoD approximately E47 tethered dimer, which suggests a distinct mechanism of action. eHAND also inhibits MyoD-dependent skeletal muscle cell differentiation and expression of the muscle-specific myosin heavy chain protein. In addition, we show that eHAND can repress activity of the natural p75LNGFR promoter, whose expression overlaps that of eHAND and dHAND. The inhibitory activity of eHAND may be attributed to multiple mechanisms, such as the ability to act as a corepressor, the presence of a repression domain, and its ability to sequester E proteins in an inactive complex. Based upon its inhibitory effect on bHLH proteins and cellular differentiation, we propose that eHAND may function by several mechanisms to promote placental giant cell proliferation by negatively regulating the activities of the bHLH protein MASH-2.

Characterization of the mouse JAB1 cDNA and protein.

JAB1 was originally described as a transcriptional coactivator of c-Jun and Jun D. Recent data suggests that JAB1 is a component of a large protein complex, the JAB1 signalosome in mammals and the COP9 complex in plants. The JAB1 signalosome is implicated in the phosphorylation of selected transcription factors, while the COP9 complex is involved in repression of photomorphogenesis in Arabidopsis. In this study, we describe the partial characterization of mouse JAB1 (mJAB1). The murine JAB1 protein is encoded by a gene located on mouse chromosome 1. mJAB1 mRNA is abundantly expressed in a variety of adult tissues as well as in mouse embryos. The JAB1 protein was readily detectable in many cell types and localized to both the nucleus and cytoplasm. Endogenous JAB1 protein is relatively stable and its degradation is not perturbed by blocking 26S proteasome activity, suggesting that this protein is not degraded by the ubiquitin-mediated proteolytic pathway.

Degradation of Id proteins by the ubiquitin-proteasome pathway.

Id proteins act as negative regulators of bHLH transcription factors by forming transcriptionally inactive protein complexes. The proposed function of these proteins includes promotion of cell growth and cell cycle progression, induction of apoptosis, and inhibition of cellular differentiation. We investigated the role of the ubiquitin-mediated proteolytic pathway in the degradation of the Id3 protein. We found Id3 to be a short-lived protein and estimated the half-life to be approximately 20 min in 293 cells. Using specific inhibitors of the 26S proteasome and mutant fibroblast cells with a temperature-sensitive defect in the essential E1 ubiquitin-activating enzyme, we show that Id3 and the related Id1 and Id2 proteins are degraded through the ubiquitin-proteasome pathway. We found the Id4 protein to be much less sensitive to inhibitors of the 26S proteasome, but its degradation was dependent on the E1 enzyme. In addition, we observed that coexpression of the bHLH protein E47 with Id3 significantly reduced the rate of degradation of Id3, suggesting that Id3 is less susceptible to degradation by the 26S proteasome when complexed to a bHLH protein. -Bounpheng, M. A., Dimas, J. J., Dodds, S. G., Christy, B. A. Degradation of Id proteins by the ubiquitin-proteasome pathway.

Identification of a novel transcriptional activity of mammalian Id proteins.

The Id proteins are a family of related mammalian helix-loop-helix (HLH) proteins which can interact with other HLH proteins but lack a basic region and are thus not thought to bind to DNA. Instead, they are hypothesized to act as dominant negative regulators of DNA-binding basic HLH (bHLH) proteins, by forming inactive heterodimers with these proteins. All four Id family proteins possess related HLH dimerization domains and can interact with similar bHLH proteins, although with differing affinities. The functions of the largely unrelated N- and C-terminal regions of the proteins are unknown. In this study, we have identified a novel transcriptional activity of the mammalian Id proteins. We show that when fused to the heterologous GAL4 DNA-binding domain, all four of the mammalian Id proteins can activate GAL4-dependent transcription. The HLH domain is necessary for the transactivation activity observed, suggesting that interaction with a cellular HLH protein is required. Co-transfection with exogenous Class A bHLH proteins (E-proteins) greatly potentiates the transactivation, which is abolished upon co-transfection with Class B bHLH proteins. These results are consistent with the idea that the Id proteins have a transcriptional activity when present in a DNA-binding complex.

Differential biological activities of mammalian Id proteins in muscle cells.

Id proteins are helix-loop-helix (HLH) transcription factors that lack DNA-binding domains. These proteins form inactive heterodimers with basic HLH (bHLH) factors, inhibiting their DNA-binding and transcriptional activities. Consistent with a proposed role for Id proteins as inhibitors of terminal differentiation, Id1 and Id3 have been shown to negatively regulate myogenesis in cultured muscle cells. Here we have investigated the possibility that Id2 and/or Id4 can act in a similar manner. Surprisingly, while overexpression of Id2 resulted in inhibition of differentiation of Sol 8 myoblast cells, overexpression of Id4 did not. Sol 8 cells stably transfected with Id4 showed no apparent changes in expression of muscle-specific genes upon differentiation. DNA-binding activities present at the muscle creatine kinase (MCK) enhancer E-box and transcription of the MCK enhancer were not altered in Id4-overexpressing cells, compared with vector-transfected cells. Id2 is also a more potent inhibitor of protein/DNA complex formation at the MCK-R enhancer E-box than Identified in vitro. Therefore, our data support the notion that members of the Id family might be involved in the regulation of distinct developmental pathways.