Phosphorylation of RNA polymerase II is independent of P-TEFb in the C. elegans germline.

RNA polymerase II (Pol II) elongation in metazoans is thought to require phosphorylation of serine 2 (Ser2-P) of the Pol II C-terminal domain (CTD) by the P-TEFb complex, CDK-9/cyclin T. Another Ser2 kinase complex, CDK-12/cyclin K, which requires upstream CDK-9 activity has been identified in Drosophila and human cells. We show that regulation of Ser2-P in C. elegans soma is similar to other metazoan systems, but Ser2-P in the germline is independent of CDK-9, and largely requires only CDK-12. The observed differences are not due to differential tissue expression as both kinases and their cyclin partners are ubiquitously expressed. Surprisingly, loss of CDK-9 from germ cells has little effect on Ser2-P, yet CDK-9 is essential for germline development. By contrast, loss of CDK-12 and Ser2-P specifically from germ cells has little impact on germline development or function, although significant loss of co-transcriptional H3K36 trimethylation is observed. These results show a reduced requirement for Pol II Ser2-P in germline development and suggest that generating Ser2-P is not the essential role of CDK-9 in these cells. Transcriptional elongation in the C. elegans germline thus appears to be uniquely regulated, which may be a novel facet of germline identity.

Exploring symmetry as an avenue to the computational design of large protein domains.

It has been demonstrated previously that symmetric, homodimeric proteins are energetically favored, which explains their abundance in nature. It has been proposed that such symmetric homodimers underwent gene duplication and fusion to evolve into protein topologies that have a symmetric arrangement of secondary structure elements--"symmetric superfolds". Here, the ROSETTA protein design software was used to computationally engineer a perfectly symmetric variant of imidazole glycerol phosphate synthase and its corresponding symmetric homodimer. The new protein, termed FLR, adopts the symmetric (ßα)(8) TIM-barrel superfold. The protein is soluble and monomeric and exhibits two-fold symmetry not only in the arrangement of secondary structure elements but also in sequence and at atomic detail, as verified by crystallography. When cut in half, FLR dimerizes readily to form the symmetric homodimer. The successful computational design of FLR demonstrates progress in our understanding of the underlying principles of protein stability and presents an attractive strategy for the in silico construction of larger protein domains from smaller pieces.

Insulin/IGF Signaling and Vitellogenin Provisioning Mediate Intergenerational Adaptation to Nutrient Stress.

The roundworm C. elegans reversibly arrests larval development during starvation [1], but extended early-life starvation reduces reproductive success [2, 3]. Maternal dietary restriction (DR) buffers progeny from starvation as young larvae, preserving reproductive success [4]. However, the developmental basis of reduced fertility following early-life starvation is unknown, and it is unclear how maternal diet modifies developmental physiology in progeny. We show here that extended starvation in first-stage (L1) larvae followed by unrestricted feeding results in a variety of developmental abnormalities in the reproductive system, including proliferative germ-cell tumors and uterine masses that express neuronal and epidermal cell fate markers. We found that maternal DR and reduced maternal insulin/insulin-like growth factor (IGF) signaling (IIS) increase oocyte provisioning of vitellogenin lipoprotein, reducing penetrance of starvation-induced abnormalities in progeny, including tumors. Furthermore, we show that maternal DR and reduced maternal IIS reduce IIS in progeny. daf-16/FoxO and skn-1/Nrf, transcriptional effectors of IIS, are required in progeny for maternal DR and increased vitellogenin provisioning to suppress starvation-induced abnormalities. daf-16/FoxO activity in somatic tissues is sufficient to suppress starvation-induced abnormalities, suggesting cell-nonautonomous regulation of reproductive system development. This work reveals that early-life starvation compromises reproductive development and that vitellogenin-mediated intergenerational insulin/IGF-to-insulin/IGF signaling mediates adaptation to nutrient availability.

Amino Acid Substitutions in the Caenorhabditis elegans RNA Polymerase II Large Subunit AMA-1/RPB-1 that Result in α-Amanitin Resistance and/or Reduced Function.

Mutations in the Caenorhabditis elegans RNA polymerase II AMA-1/RPB-1 subunit that cause α-amanitin resistance and/or developmental defects were isolated previously. We identified 12 of these mutations and mapped them onto the Saccharomyces cerevisiae RPB1 structure to provide insight into AMA-1 regions that are essential for development in a multicellular organism.

Contrast response of temporally sparse dichoptic multifocal visual evoked potentials.

Temporally sparse stimuli have been found to produce larger multifocal visual evoked potentials than rapid contrast-reversal stimuli. We compared the contrast-response functions of conventional contrast-reversing (CR) stimuli and three grades of temporally sparse stimuli, examining both the changes in response amplitude and signal-to-noise ratio (SNR). All stimuli were presented dichoptically to normal adult human subjects. One stimulus variant, the slowest pattern pulse, had interleaved monocular and binocular stimuli. Response amplitudes and SNRs were similar for all stimuli at contrast 0.4 but grew faster with increasing contrast for the sparser stimuli. The best sparse stimulus provided an SNR improvement that corresponded to a recording time improvement of 2.6 times relative to that required for contrast reversing stimuli. Multiple regression of log-transformed response metrics characterized the contrast-response functions by fitting power-law relationships. The exponents for the two sparsest stimuli were significantly larger (P < 0.001) than for the CR stimuli, as were the mean response amplitudes and signal-to-noise ratios for these stimuli. The contrast-dependent response enhancement is discussed with respect to the possible influences of rapid retinal contrast gain control, or intracortical and cortico-geniculate feedback.