The herpes simplex virus type 1 (HSV-1)a sequence is present as a direct repeat at the two termini of the 152-kilobase viral genome and as an inverted repeat at the junction of the two unique components L and S. During replication, the HSV-1 genome undergoes inversion of L and S, producing an equimolar mixture of the four possible isomers. Isomerization is believed to result from recombination triggered by breakage at the a sequence, a recombinational hot spot. We have identified an enzyme in HeLa cell extracts that preferentially cleaves the a sequence and have purified it to near homogeneity. Microsequencing showed it to be human endonuclease G, an enzyme with a strong preference for G+C-rich sequences. Endonuclease G appears to be the only cellular enzyme that can specifically cleave the a sequence. Endonuclease G also showed the predicted recombination properties in an in vitro recombination assay. Based on these findings, we propose that endonuclease G initiates the a sequence-mediated inversion of the L and S components during HSV-1 DNA replication.
The a sequences of herpes simplex virus type 1 are believed to be the cis sites for inversion events that generate four isomeric forms of the viral genome. Using an assay that measures deletion of a beta-galactosidase gene positioned between two directly repeated sequences in plasmids transiently maintained in Vero cells, we had found that the a sequence is more recombinogenic than another sequence of similar size. To investigate the basis for the enhanced recombination mediated by the a sequence, we examined plasmids containing direct repeats of approximately 350 bp from a variety of sources and with a wide range of G+C content. We observed that all of these plasmids show similar recombination frequencies (3 to 4%) in herpes simplex virus type 1-infected cells. However, recombination between directly repeated a sequences occurs at twice this frequency (6 to 10%). In addition, we find that insertion of a cleavage site for an a-sequence-specific endonuclease into the repeated sequences does not appreciably increase the frequency of recombination, indicating that the presence of endonuclease cleavage sites within the a sequence does not account for its recombinogenicity. Finally, by replacing segments of the a sequence with DNA fragments of similar length, we have determined that only the 95-bp Uc-DR1 segment is indispensable for high-level a-sequence-mediated recombination.
In congenital stationary night blindness type 2 (CSNB2), a disorder involving the Cav1.4 (L-type) Ca2+ channel, visual impairment is mild considering that Cav1.4 mediates synaptic release from rod and cone photoreceptors. Here, we addressed this conundrum using a Cav1.4 knockout (KO) mouse and a knock-in (G369i KI) mouse expressing a non-conducting Cav1.4. Surprisingly, Cav3 (T-type) Ca2+ currents were detected in cones of G369i KI mice and Cav1.4 KO mice but not in cones of wild-type mouse, ground squirrel, and macaque retina. Whereas Cav1.4 KO mice are blind, G369i KI mice exhibit normal photopic (i.e., cone-mediated) visual behavior. Cone synapses, which fail to form in Cav1.4 KO mice, are present, albeit enlarged, and with some errors in postsynaptic wiring in G369i KI mice. While Cav1.4 KO mice lack evidence of cone synaptic responses, electrophysiological recordings in G369i KI mice revealed nominal transmission from cones to horizontal cells and bipolar cells. In CSNB2, we propose that Cav3 channels maintain cone synaptic output provided that the nonconducting role of Cav1.4 in cone synaptogenesis remains intact. Our findings reveal an unexpected form of homeostatic plasticity that relies on a non-canonical role of an ion channel.
General molecular engineering rules for the optimization of two-photon sensitive cages are presented and examples for nitrobenzyl, indole and nitrophenethyl platforms are highlighted. The efficiency of electron donor and acceptor groups in dipolar structures and the length of the conjugated system in the photolabile protecting group on two-photon uncaging efficiency will be discussed, as well as the emergence of symmetrical platform based on bis-electron donor or on quadrupolar architectures. We will then present our first results on the nano-structuration of donor-acceptor systems based on nitrophenetyl platform using diethyleneglycol or pentaerythritol cores.
The claustrum is a small subcortical nucleus that has extensive excitatory connections with many cortical areas. While the anatomical connectivity from the claustrum to the cortex has been studied intensively, the physiological effect and underlying circuit mechanisms of claustrocortical communication remain elusive. Here we show that the claustrum provides strong, widespread, and long-lasting feedforward inhibition of the prefrontal cortex (PFC) sufficient to silence ongoing neural activity. This claustrocortical feedforward inhibition was predominantly mediated by interneurons containing neuropeptide Y, and to a lesser extent those containing parvalbumin. Therefore, in contrast to other long-range excitatory inputs to the PFC, the claustrocortical pathway is designed to provide overall inhibition of cortical activity. This unique circuit organization allows the claustrum to rapidly and powerfully suppress cortical networks and suggests a distinct role for the claustrum in regulating cognitive processes in prefrontal circuits.
In congenital stationary night blindness type 2 (CSNB2)—a disorder involving the Ca v 1.4 (L-type) Ca 2+ channel—visual impairment is mild considering that Ca v 1.4 mediates synaptic release from rod and cone photoreceptors. Here, we addressed this conundrum using a Ca v 1.4 knockout (KO) mouse and a knock-in (G369i KI) mouse expressing a non-conducting Ca v 1.4. Surprisingly, Ca v 3 (T-type) Ca 2+ currents were detected in cones of G369i KI mice and Ca v 1.4 KO mice but not in cones of wild-type mouse, ground squirrel, and macaque retina. Whereas Ca v 1.4 KO mice are blind, G369i KI mice exhibit normal photopic (i.e., cone-mediated) visual behavior. Cone synapses, which fail to form in Ca v 1.4 KO mice, are present, albeit enlarged, and with some errors in postsynaptic wiring in G369i KI mice. While Ca v 1.4 KO mice lack evidence of cone synaptic responses, electrophysiological recordings in G369i KI mice revealed nominal transmission from cones to horizontal cells and bipolar cells. In CSNB2, we propose that Ca v 3 channels maintain cone synaptic output provided that the nonconducting role of Ca v 1.4 in cone synaptogenesis remains intact. Our findings reveal an unexpected form of homeostatic plasticity that relies on a non-canonical role of an ion channel.
Abstract Receptive field (RF) size and preferred spatial frequency (SF) vary greatly across the primary visual cortex (V1), increasing in a scale invariant fashion with eccentricity. Recent studies reveal that preferred SF also forms a fine-scale periodic map. A fundamental open question is how local variability in preferred SF is tied to the overall spatial RF. Here, we use two-photon imaging to simultaneously measure maps of RF size, phase selectivity, SF bandwidth, and orientation bandwidth—all of which were found to be topographically organized and correlate with preferred SF. Each of these newly characterized inter-map relationships strongly deviate from scale invariance, yet reveal a common motif—they are all accounted for by a model with uniform spatial pooling from scale invariant inputs. Our results and model provide novel and quantitative understanding of the output from V1 to downstream circuits.
We have partially purified an activity from extracts of cells infected with herpes simplex virus type 1 that mediates recombination between repeated copies of the 317-base-pair a sequence of herpes simplex virus type 1. Recombination leads to deletion of a lacZ indicator gene situated between two directly repeated copies of the a sequence and is scored by transformation of lacZ- Escherichia coli. The two products of the reaction can be observed directly by restriction enzyme digestion and Southern blot analysis. The recombinase activity is also detectable, but at a lower level, in uninfected cell extracts. The DNA substrate must contain the two a sequences arranged in direct orientation to generate the lacZ deletion. However, when the a sequences are arranged in inverted orientation, an inversion results. A substrate with two homologous sequences of size and G + C content similar to the a sequence undergoes recombination at a much lower frequency. The reaction requires a divalent cation (Mg2+ or Mn2+) but not ATP or any other nucleoside triphosphate. The simple requirements and specificity for the a sequence suggest that the recombination may proceed by a site-specific mechanism.
