Nocistatin excites rostral agranular insular cortex-periaqueductal gray projection neurons by enhancing transient receptor potential cation conductance via G(alphaq/11)-PLC-protein kinase C pathway.

Rostral agranular insular cortex (RAIC) projects to periaqueductal gray (PAG) and inhibits spinal nociceptive transmission by activating PAG-rostral ventromedial medulla (RVM) descending antinociceptive circuitry. Despite being generated from the same precursor prepronociceptin, nocistatin (NST) and nociceptin/orphanin FQ (N/OFQ) produce supraspinal analgesic and hyperalgesic effects, respectively. Prepronociceptin is highly expressed in the RAIC. In the present study, we hypothesized that NST and N/OFQ modulate spinal pain transmission by regulating the activity of RAIC neurons projecting to ventrolateral PAG (RAIC-PAG). This hypothesis was tested by investigating electrophysiological effects of N/OFQ and NST on RAIC-PAG projection neurons in brain slice. Retrogradely labeled RAIC-PAG projection neurons are layer V pyramidal cells and express mRNA of vesicular glutamate transporter subtype 1, a marker for glutamatergic neurons. N/OFQ hyperpolarized 25% of RAIC-PAG pyramidal neurons by enhancing inwardly rectifying potassium conductance via pertussis toxin-sensitive G(alphai/o). In contrast, NST depolarized 33% of RAIC-PAG glutamatergic neurons by causing the opening of canonical transient receptor potential (TRPC) cation channels through G(alphaq/11)-phospholipase C-protein kinase C pathway. There were two separate populations of RAIC-PAG pyramidal neurons, one responding to NST and the other one to N/OFQ. Our results suggest that G(alphaq/11)-coupled NST receptor mediates NST excitation of RAIC-PAG glutamatergic neurons, which is expected to cause the supraspinal analgesia by enhancing the activity of RAIC-PAG-RVM antinociceptive pathway. Opposite effects of NST and N/OFQ on supraspinal pain regulation are likely to result from their opposing effects on RAIC-PAG pyramidal neurons.

Transcriptional effects on double-strand break-induced gene conversion tracts.

Transcription stimulates spontaneous homologous recombination, but prior studies have not investigated the effects of transcription on double-strand break (DSB)-induced recombination in yeast. We examined products of five ura3 direct repeat substrates in yeast using alleles that were transcribed at low or high levels. In each strain, recombination was stimulated by DSBs created in vivo at an HO site in one copy of ura3. Increasing transcription levels in donor or recipient alleles did not further stimulate DSB-induced recombination, nor did it alter the relative frequencies of conversion and deletion (pop-out) events. This result is consistent with the idea that transcription enhances spontaneous recombination by increasing initiation. Gene conversion tracts were measured using silent restriction fragment length polymorphisms (RFLPs) at approximately 100bp intervals. Transcription did not alter average tract lengths, but increased transcription in donor alleles increased both the frequency of promoter-proximal (5') unidirectional tracts and conversion of 5' markers. Increased transcription in recipient alleles increased the frequency of bidirectional tracts. We demonstrate that these effects are due to transcription per se, and not just transcription factor binding. These results suggest that transcription influences aspects of gene conversion after initiation, such as strand invasion and/or mismatch repair (MMR).

Evidence for independent mismatch repair processing on opposite sides of a double-strand break in Saccharomyces cerevisiae.

Double-strand break (DSB) induced gene conversion in Saccharomyces cerevisiae during meiosis and MAT switching is mediated primarily by mismatch repair of heteroduplex DNA (hDNA). We used nontandem ura3 duplications containing palindromic frameshift insertion mutations near an HO nuclease recognition site to test whether mismatch repair also mediates DSB-induced mitotic gene conversion at a non-MAT locus. Palindromic insertions included in hDNA are expected to produce a stem-loop mismatch, escape repair, and segregate to produce a sectored (Ura+/-) colony. If conversion occurs by gap repair, the insertion should be removed on both strands, and converted colonies will not be sectored. For both a 14-bp palindrome, and a 37-bp near-palindrome, approximately 75% of recombinant colonies were sectored, indicating that most DSB-induced mitotic gene conversion involves mismatch repair of hDNA. We also investigated mismatch repair of well-repaired markers flanking an unrepaired palindrome. As seen in previous studies, these additional markers increased loop repair (likely reflecting corepair). Among sectored products, few had additional segregating markers, indicating that the lack of repair at one marker is not associated with inefficient repair at nearby markers. Clear evidence was obtained for low levels of short tract mismatch repair. As seen with full gene conversions, donor alleles in sectored products were not altered. Markers on the same side of the DSB as the palindrome were involved in hDNA less often among sectored products than nonsectored products, but markers on the opposite side of the DSB showed similar hDNA involvement among both product classes. These results can be explained in terms of corepair, and they suggest that mismatch repair on opposite sides of a DSB involves distinct repair tracts.

Double-strand break-induced mitotic gene conversion: examination of tract polarity and products of multiple recombinational repair events.

Double-strand break (DSB)-induced gene conversion in yeast was studied in crosses between ura3 heteroalleles carrying phenotypically silent markers at approximately 100-bp intervals, which allow high-resolution analyses of tract structures. DSBs were introduced in vivo by HO nuclease at sites within shared homology and were repaired using information donated by unbroken alleles. Previous studies with these types of crosses showed that most tracts of Ura+ products are continuous, unidirectional, and extend away from frameshift mutations in donor alleles. Here we demonstrate that biased tract directionality is a consequence of selection pressure against Ura- products that results when frameshift mutations in donor alleles are transferred to recipient alleles. We also performed crosses in which frameshift mutations in recipient and donor alleles were arranged such that events initiated at DSBs could not convert broken alleles to Ura+ via a single gap repair event or a single long-tract mismatch repair event in heteroduplex DNA. This constraint led to low recombination frequencies relative to unconstrained crosses, and inhibited preferential conversion of broken alleles. Physical analysis of 51 DSB-induced products arising from multiple recombinational repair events suggested that hDNA formation is generally limiting, but that some hDNA regions may extend more than 600 bp. Among these products, markers separated by 20 bp were independently repaired about 40% of the time.