J Peters

To understand how neurons control the expression of the AMPA receptor subunit GluR2, we cloned the 5' proximal region of the rat gene and investigated GluR2 promoter activity by transient transfection. RNase protection and primer extension of rat brain mRNA revealed multiple transcription initiation sites from -340 to -481 bases upstream of the GluR2 AUG codon. The relative use of 5' start sites was different in cortex and cerebellum, indicating complexity of GluR2 transcript expression among different sets of neurons. When GluR2 promoter activity was investigated by plasmid transfection into cultured cortical neurons, cortical glia, and C6 glioma cells, the promoter construct with the strongest activity, per transfected cell, was 29.4-fold (+/- 3.7) more active in neurons than in non-neural cells. Immunostaining of cortical cultures showed that >97% of the luciferase-positive cells also expressed the neuronal marker MAP-2. Evaluation of internal deletion and substitution mutations identified a functional repressor element I RE1-like silencer and functional Sp1 and nuclear respiratory factor-1 (NRF-1) elements within a GC-rich proximal GluR2 promoter region. The GluR2 silencer reduced promoter activity in glia and non-neuronal cell lines by two- to threefold, was without effect in cortical neurons, and could bind the RE1-silencing transcription factor (REST) because cotransfection of REST into neurons reduced GluR2 promoter activity in a silencer-dependent manner. Substitution of the GluR2 silencer by the homologous NaII RE1 silencer further reduced GluR2 promoter activity in non-neuronal cells by 30-47%. Maximal positive GluR2 promoter activity required both Sp1 and NRF-1 cis elements and an interelement nucleotide bridge sequence. These results indicate that GluR2 transcription initiates from multiple sites, is highly neuronal selective, and is regulated by three regulatory elements in the 5' proximal promoter region.

The combination of various concentrations of acyclovir and vidarabine or its 5'-monophosphate usually produced an additive interaction with various strains of herpes simplex virus types 1 and 2 in Vero cells. Similarly, certain combinations of these drugs were more effective than the individual drugs in decreasing the mortality and increasing the mean day of death of mice inoculated intracerebrally with herpes simplex virus type 2. Neither antagonism nor interference was noted for any of the in vitro or in vivo combinations. The increased antiviral activity was determined not to be secondary to toxic effects of the drugs. Although viruses resistant to either vidarabine or acyclovir developed readily in cell culture, no evidence of cross-resistance was obtained. Furthermore, in the presence of the two drugs, mutants resistant to vidarabine, acyclovir, or vidarabine/acyclovir could not be isolated. These findings suggest that combinations with these antivirals, which are currently being evaluated singly for the therapy of severe forms of herpetic infection, could prove clinically useful if increasing numbers of resistant viral strains are observed.

Fragile X syndrome is a common form of mental retardation associated with a fragile site on the human X chromosome. Although fragility at this site is usually evident as a nonstaining chromatid gap, it remains unclear whether or not actual chromosomal breakage occurs. By means of somatic cell hybrids containing either a normal human X or a fragile X chromosome and utilizing two genes that flank the fragile site as markers of chromosome integrity, segregation of these markers was shown to be more frequent if they encompass the fragile site under appropriate culture conditions. Hybrid cells that reveal marker segregation were found to contain rearranged X chromosomes involving the region at or near the fragile site, thus demonstrating true chromosomal breakage within this area. Two independent translocation chromosomes were identified involving a rodent chromosome joined to the human X at the location of the fragile site. DNA analysis of closely linked, flanking loci was consistent with the position of the breakpoint being at or very near the fragile X site. Fragility at the translocation junctions was observed in both hybrids, but at significantly lower frequencies than that seen in the intact X of the parental hybrid. This observation suggests that the human portion of the junctional DNA may contain part of a repeated fragility sequence. Since the translocation junctions join heterologous DNA, the molecular cloning of the fragile X sequence should now be possible.

To understand how neurons control the expression of the AMPA receptor subunit GluR2, we cloned the 5 � proximal region of the rat gene and investigated GluR2 promoter activity by transient transfection. RNase protection and primer extension of rat brain mRNA revealed multiple transcription initiation sites from �340 to �481 bases upstream of the GluR2 AUG codon. The relative use of 5 � start sites was different in cortex and cerebellum, indicating complexity of GluR2 transcript expression among different sets of neurons. When GluR2 promoter activity was investigated by plasmid transfection into cultured cortical neurons, cortical glia, and C6 glioma cells, the promoter construct with the strongest activity, per transfected cell, was 29.4-fold ( � 3.7) more active in neurons than in non-neural cells. Immunostaining of cortical cultures showed that �97 % of the luciferase-positive cells also expressed the neuronal marker

The chromosomal fragile-site mapping to Xq27.3 is associated with a frequent form of mental retardation and is prone to breakage after induced deoxyribonucleotide pool perturbation. The human hypoxanthine phosphoribosyltransferase (HPRT) and glucose-6-phosphate dehydrogenase (G6PD) genes flank the fragile X chromosome site and can be used to monitor integrity of the site in human-hamster somatic cell hybrids deficient in the rodent forms of these activities. After induction of the fragile X site, negative selection for HPRT and positive enrichment for G6PD resulted in 31 independent colonies of HPRT-,G6PD+ phenotype. Southern blot analysis demonstrated the loss of all tested markers proximal to the fragile X site with retention of all tested human Xq28 loci in a majority of the hybrids. In situ hybridization with a human-specific probe demonstrated the translocation of a small amount of human DNA to rodent chromosomes in these hybrids, suggesting chromosome breakage at the fragile X site and the subsequent translocation of Xq28. Southern blot hybridization of hybrid-cell DNA, resolved by pulsed-field gel electrophoresis, for human-specific repetitive sequences revealed abundant CpG-islands within Xq28, consistent with its known gene density. The electrophoretic banding patterns of human DNA among the hybrids were remarkably consistent, suggesting that fragile X site breakage is limited to a relatively small region in Xq27-28. These somatic cell hybrids, containing Xq27.3-qter as the sole human DNA, will aid the search for DNA associated with the fragile X site and will augment the high resolution genomic analysis of Xq28, including the identification of candidate genes for genetic-disease loci mapping to this region.