Transient expression assays in PC12 cells showed that the cAMP response element (CRE) and the TATA box of the herpes simplex virus type 1 latency-associated transcripts (LATs) promoter are essential for basal expression. Recombinant viruses were generated containing site-specific mutations in these motifs. The abilities of these recombinants to replicate, express LATs and reactivate from latency were compared with wild-type and marker-rescued viruses in a murine ocular model. The acute replication of these TATA and CRE mutant viruses was at a level equivalent to their respective marker-rescued viruses. The reactivation of virus was unaffected by mutation in the TATA box as compared with wild-type or marker-rescued viruses. In situ hybridization of TATA box mutant virus-infected ganglia, however, showed threefold fewer LAT-positive neurons than wild-type virus-infected ganglia, with consistently weaker hybridization signals. Thus, this TATA box is required for normal expression of the LATs but not for efficient reactivation. The LATs CRE mutant reactivated with slightly but reproducibly reduced frequency and delayed kinetics relative to markerrescued virus. By in situ hybridization, however, the percentage and intensity of LATs-positive neurons were found to be comparable for the CRE mutant-and wildtype virus-infected ganglia, suggesting that the CRE is dispensable for abundant LATs expression but that a reactivation function of the LATs may depend upon the presence of the CRE. Finally, using a modified assay for examining the timing of reactivation, we showed that the induction of viral reactivation by addition of exogenous cAMP can occur independently of the LATs.

Some, perhaps all, G protein-coupled receptors form homo- or heterodimers. We have shown that metabotropic glutamate receptors are covalent dimers, held together by one or more disulfide bonds near the N terminus. Here we report how mutating cysteines in this region affect dimerization and function. Covalent dimerization is preserved when cysteines 57, 93, or 99 are mutated but lost with replacement at 129. Coimmunoprecipitation under nondenaturing conditions indicates that the C[129]S mutant receptor remains a dimer, via noncovalent interactions. Both C[93]S and C[129]S bind [3H]quisqualate, whereas binding to C[57]S or C[99]S mutants is absent or greatly attenuated. The C[93]S and C[129]S receptors have activity similar to wild-type when assayed by fura-2 imaging of intracellular calcium in human embryonic kidney cells or electrophysiologically in Xenopus laevis oocytes. In contrast, C[57]S or C[99]S are less active in both assays but do respond with higher glutamate concentrations in the oocyte assay. These results demonstrate that 1) covalent dimerization is not critical for mGlu5 binding or function; 2) mGlu5 remains a noncovalent dimer even in the absence of covalent dimerization; and 3) high-affinity binding requires Cys-57 and Cys-99.

Host and reovirus mRNAs compete with one another for translation in infected cells. Kinetic analysis has suggested that the site of competition is a message discriminatory initiation factor which must bind to the mRNA before it can interact with the 40S ribosomal subunit. The present communication describes an in vitro assay which can detect message discriminatory activities. A competitive situation is established by using reovirus and globin mRNAs, and then the specificity with which this competition is relieved by added components is measured. Among the various initiation factors surveyed with this assay, two have the properties expected ofthe mRNA discriminatory factor. These are eukaryotic initiation factor 4A and a "cap binding protein" complex. Inasmuch as the cap binding protein complex contains a subunit similar or identical to the initiation factor eIF-4A, it seems likely that only one form of the latter factor may be active in vivo. In vitro, both factors relieve competition among both capped and uncapped reovirus mRNAs according to similar hierarchies. These results suggest that some feature other than the m7G cap, such as nucleotide sequence or secondary structure, is recognized by the discriminatory factor.The fact that different mRNAs may be translated at different rates in eukaryotic cells has been firmly established (1-3). However, the molecular mechanisms that determine translation rates ofindividual mRNAs are not well understood. To account for mRNA specificity in the initiation step, it has been proposed that mRNAs must compete for a limiting message-discriminatory initiation factor in order to be translated and that competitive inhibition of translation of one mRNA by other mRNAs may be an important factor in regulating their initiation rates (4, 5). It is now clear that mRNA competition plays a central role in the replication of a number of animal viruses, notably encephalomyocarditis virus, vesicular stomatitis virus, and reovirus (4-12). This concept has been applied to uninfected cells as well (13)(14)(15)(16)).The precise identification of the message discriminatory factor has remained elusive. Early work using partially purified initiation factor preparations, called IF-M3 and IF-M4, suggested that both were involved in specific mRNA recognition (5, 13 In the present communication we describe experiments designed to answer this question. Highly purified initiation factors (>70% pure) were prepared from mouse Krebs ascites tumors, HeLa cells, or rabbit reticulocytes. They were tested for the ability to relieve competition between reovirus mRNAs and globin mRNA in vitro according to the specific patterns previously described (19,20). Our results show that both eIF-4A and CBP II relieve competition specifically and so are candidates for the message discriminatory factor. MATERIALS AND METHODSTranslation Initiation Factors. Various eukaryotic initiation factors used in the present study have been purified according to published procedures (21-24)-eIF-2, eIF-3, eIF-4A, eIF-4B, and C...

Strains of Volvox carteri forma nagariensis derived from Japanese and Indian isolates ("J" and "I" strains, respectively) exhibited length differences (RFLPs) for approximately 90% of the restriction fragments detected by hybridization with a variety of unique-sequence, small-gene-family and repetitive-element probes, including heterologous probes of chloroplast and mitochondrial origin. Extensive post-zygotic mortality was observed among the zygotes produced by crossing J and I strains, suggesting some form of genetic incompatability between them. Most of the viable progeny exhibited recombinant patterns of nuclear inheritance and maternal inheritance of mitochondrial and chloroplast markers. However, many progeny exhibited exclusively uniparental (usually maternal, but in one case paternal) inheritance of both nuclear and organellar markers. Some of these non-recombinant individuals may be derived from "parthenospores" (dormant asexual cells resembling zygospores). Others may be a result of "pseudogamy," in which one of the parental pronuclei is excluded from the zygote, followed by selective exclusion of both the mitochondrial and the chloroplast genomes derived from that same parent. When segregation patterns for 44 nuclear markers were analyzed in 90 recombinant progeny, statistically significant, locus-specific deviations from expected Mendelian transmission ratios were observed for a sizeable fraction of all markers in both reciprocal crosses: some markers were preferentially transmitted by the J strain, while others were preferentially transmitted by the I strain. It is speculated that these transmission distortions may be related to the regions of inter-isolate genetic incompatibility, and may complicate the use of J x I crosses to establish a RFLP-based linkage map for the species.