Zinc fingers 1 and 7 of yeast TFIIIA are essential for assembly of a functional transcription complex on the 5 S RNA gene

The binding of transcription factor (TF) IIIA to the internal control region of the 5 S RNA gene is the first step in the assembly of a DNA–TFIIIA–TFIIIC– TFIIIB transcription complex, which promotes accurate transcription by RNA polymerase III. With the use of mutations that are predicted to disrupt the folding of a zinc finger, we have examined the roles of zinc fingers 1 through 7 of yeast TFIIIA in the establishment of a functional transcription complex both in vitro and in vivo. Our data indicate that, in addition to their role in DNA binding, the first and seventh zinc fingers contribute other essential roles in the assembly of an active transcription complex. Alanine-scanning mutagenesis identified residues within zinc finger 1 that are not required for DNA binding but are required for incorporation of TFIIIC into the TFIIIA–DNA complex. Although disruption of zinc finger 2 or 3 had a deleterious effect on the activity of TFIIIA both in vitro and in vivo, we found that increasing the level of their in vivo expression allowed these mutant proteins to support cell viability. Disruption of zinc fingers 4, 5 or 6 had minimal effect on the DNA binding and TF activities of TFIIIA

Social approach and repetitive behavior in eleven inbred mouse strains

Core symptoms of autism include deficits in social interaction, impaired communication, and restricted, repetitive behaviors. The repetitive behavior domain encompasses abnormal motoric stereotypy, an inflexible insistence on sameness, and resistance to change. In recent years, many genetic mouse models of autism and related disorders have been developed, based on candidate genes for disease susceptibility. The present studies are part of an ongoing initiative to develop appropriate behavioral tasks for the evaluation of mouse models relevant to autism. We have previously reported profiles for sociability, preference for social novelty, and resistance to changes in a learned pattern of behavior, as well as other functional domains, for 10 inbred mouse strains of divergent genetic backgrounds. The present studies extend this multi-component behavioral characterization to several additional strains: C58/J, NOD/LtJ, NZB/B1NJ, PL/J, SJL/J, SWR/J, and the wild-derived PERA/EiJ. C58/J, NOD/LtJ, NZB/B1NJ, SJL/J, and PERA/EiJ demonstrated low sociability, measured by time spent in proximity to an unfamiliar conspecific, with 30% to 60% of mice from these strains showing social avoidance. In the Morris water maze, NZB/B1NJ had a persistent bias for the quadrant where the hidden platform was located during acquisition, even after nine days of reversal training. A particularly interesting profile was found for C58/J, which had low social preference, poor performance in the T-maze, and overt motoric stereotypy. Overall, this set of tasks and observational methods provides a strategy for evaluating novel mouse models in behavioral domains relevant to the autism phenotype

Role of Ndt80, Sum1, and Swe1 as Targets of the Meiotic Recombination Checkpoint That Control Exit from Pachytene and Spore Formation in Saccharomyces cerevisiae

The meiotic recombination checkpoint, which is triggered by defects in recombination or chromosome synapsis, arrests sporulating cells of Saccharomyces cerevisiae at pachytene by preventing accumulation of active Clb-Cdc28. We compared the effects of manipulating the three known targets of the meiotic recombination checkpoint, NDT80, SWE1, and SUM1, in dmc1-arrested cells. Ndt80 is an activator of a set of middle sporulation-specific genes (MSGs), which includes CLB genes and genes involved in spore wall formation; Swe1 inhibits Clb-Cdc28 activity; and Sum1 is a repressor of NDT80 and some MSGs. Activation of the checkpoint leads to inhibition of Ndt80 activity and to stabilization of Swe1 and Sum1. Thus, dmc1-arrested cells fail to express MSGs, arrest at pachytene, and do not form spores. Our study shows that dmc1/dmc1 sum1/sum1 cells expressed MSGs prematurely and at high levels, entered the meiotic divisions efficiently, and in some cases formed asci containing mature spores. In contrast, dmc1/dmc1 swe1/swe1 cells expressed MSGs at a very low level, were inefficient and delayed in entry into the meiotic divisions, and never formed mature spores. We found that cells of dmc1/dmc1 sum1/sum1 ndt80/ndt80 and dmc1/dmc1 swe1/swe1 ndt80/ndt80 strains arrested at pachytene and that dmc1/dmc1 or dmc1/dmc1 swe1/swe1 cells overexpressing NDT80 were less efficient in bypassing checkpoint-mediated arrest than dmc1/dmc1 sum1/sum1 cells. Our results are consistent with previous suggestions that increased Clb-Cdc28 activity, caused by mutation of SWE1 or by an NDT80-dependent increase in CLB expression, allows dmc1/dmc1 cells to exit pachytene and that subsequent upregulation of Ndt80 activity by a feedback mechanism promotes entry into the meiotic divisions. Spore morphogenesis, however, requires efficient and timely activation of MSGs, which we speculate was achieved in dmc1/dmc1 sum1/sum1 cells by premature expression of NDT80

Regulation of the Premiddle and Middle Phases of Expression of the NDT80 Gene during Sporulation of Saccharomyces cerevisiae

The NDT80 gene of Saccharomyces cerevisiae, which encodes a global activator of transcription of middle sporulation-specific genes, is first expressed after the activation of early meiotic genes but prior to activation of middle sporulation-specific genes. Both upstream repression sequence 1 (URS1) and mid-sporulation element (MSE) sites are present in the promoter region of the NDT80 gene; these elements have been shown previously to contribute to the regulation of expression of early and middle sporulation-specific genes, respectively, by mediating repression in growing cells and activation at specific times during sporulation. In this study, we have shown that the overlapping windows of URS1- and MSE-mediated repression and activation are responsible for the distinctive premiddle expression pattern of the NDT80 gene. Our data suggest that a Sum1-associated repression complex bound at the NDT80 MSE sites prevents Ime1 tethered at the NDT80 URS1 sites from activating transcription of the NDT80 gene at the time that Ime1-dependent activation of early URS1-regulated meiotic genes is occurring. We propose that a decrease in the efficiency of Sum1-mediated repression as cells progress through the early events of the sporulation program allows the previously inactive Ime1 tethered at the URS1(NDT80) sites to promote a low level of expression of the NDT80 gene. This initial phase of URS1-dependent NDT80 expression is followed by Ndt80-dependent upregulation of its own expression, which requires the MSE(NDT80) sites and occurs concomitantly with Ndt80-dependent activation of a set of middle MSE-regulated sporulation-specific genes. Mutation of IME2 prevents expression of NDT80 in sporulating cells. We show in this study that NDT80 is expressed and that middle genes are activated in cells of an Δime2/Δime2 Δsum1/Δsum1 strain in sporulation medium. This suggests that Ime2 activates expression of NDT80 by eliminating Sum1-mediated repression