Corneal dystrophy and perceptive deafness (Harboyan syndrome): CDPD1 maps to 20p13.

The association of congenital corneal dystrophy with teenage onset perceptive hearing loss (Harboyan syndrome) has been reported in two sibships, one with consanguineous parents, which were consistent with autosomal recessive transmission. We have observed a Moroccan sibship where four girls and one boy were affected with this rare syndrome. The parents were first cousins once removed and unaffected. Genome wide homozygosity mapping using 386 microsatellite markers linked the locus to 20p13. A maximum multipoint lod score of 4.20 was obtained at marker D20S179. The minimal critical region is 7.73 cM between markers D20S199 and D20S437. These results confirm the syndromic association of congenital corneal dystrophy and teenage onset hearing loss, and further increase the genetic heterogeneity of recessive deafness.

Ribozyme-mediated decrease in mumps virus nucleocapsid mRNA level and progeny in infected vero cells.

The effects of endogenously expressed ribozymes directed to the mumps virus nucleocapsid (NP) mRNA were studied during viral infection. To this end, eukaryotic expression vectors encoding ribozymes or controls of passive hybridization effects were constructed and used to transfect mumps permissive Vero cells. Transcripts spanning trans-acting ribozymes of the hammerhead and hairpin types were designed to hydrolyze the first 5'GUC-3' sequence downstream from the initiation site and to hybridize to a 16 base sequence containing the putative cleavage site. Control vectors encoded mutated and catalytically inactive forms of the ribozymes or a 16 base antisense version of the target sequence. When stably expressed in cells, both ribozymes and passive control RNAs reduced viral yields. A ribozyme-mediated effect on viral growth was, however, observed, as both ribozyme types reduced viral titers by approximately 80%, well above the highest inhibition level of approximately 35% found when noncatalytic RNAs were expressed. In addition, levels of NP mRNA were generally lower in cells expressing catalytic RNAs, supporting the observed inhibition of viral growth. Although cleavage in vitro of a synthetic analog of the NP mRNA was demonstrated using RNAs isolated from ribozyme-expressing cells, in vivo cleavage products were not detectable despite the use of sensitive methods, possibly because of degradation phenomena. We also suggest here that additional controls should be conducted when semicompetitive RT-PCR methods are used to evaluate intracellular cleavage by ribozymes, as the results may depend on the initial target RNA concentration.

Contribution of Na/Ca exchange to Ca2+ outflow and entry in the rat pancreatic beta-cell: studies with antisense oligonucleotides.

To characterize the role played by Na/Ca exchange in the pancreatic beta-cell, phosphorothioated antisense oligonucleotides (AS-oligos) were used to knock down the exchanger in rat pancreatic beta-cells. Na/Ca exchange activity was evaluated by measuring cytosolic free Ca2+ concentration ([Ca2+]i) in single cells using fura-2. Exposure of beta-cells to 500 nmol/l of the AS-oligos for 24 h inhibited Na/Ca exchange activity by approximately 77%. In contrast, control oligonucleotides (scrambled and mismatched) did not affect Na/Ca exchange activity. In AS-oligo-treated cells, the increase in [Ca2+]i induced by membrane depolarization (K+ or the hypoglycemic sulfonylurea, tolbutamide) was reduced by 28 or 40%, respectively. Likewise, the rate of [Ca2+]i decrease after K+ or tolbutamide removal was reduced by 72 or 40%, respectively. AS-oligos treatment also abolished the nifedipine-resistant increase in [Ca2+]i induced by K+ and profoundly altered the oscillatory or sustained increases in [Ca2+]i induced by 11.1 mmol/l glucose. The present study shows that AS-oligos may specifically inhibit Na/Ca exchange in rat pancreatic beta-cells. In those cells, Na/Ca exchange appears to mediate Ca2+ entry in response to membrane depolarization and to be responsible for up to 70% of Ca2+ removal from the cytoplasm upon membrane repolarization.

Tailing cDNAs with terminal deoxynucleotidyl transferase in RT-PCR assays to identify ribozyme cleavage products.

Polytailing a cDNA with terminal deoxynucleotidyltransferase (TdT) results in the addition of a homopolymeric sequence at its 3'-end. Here we describe the use of tailing in competitive RT-PCR assays to evaluate cleavage efficiency of ribozymes. Using a system that perfectly mimics intracellular cleavage, we were able to detect as few as 1% of cleaved moieties. Furthermore, employing primers overlapping the junction between tails and the cleaved RNA moiety in non-competitive assays, the sensitivity of the method could be improved to <10 fg. Using the latter protocol and reactions employing a trans -acting hairpin ribozyme targeting the nucleocapsid mRNA of the mumps virus, we were able to demonstrate ribozyme-induced cleavage.

"Hairpin" and "hammerhead" ribozymes directed towards the mumps virus nucleocapsid RNA: specific cleavage of a small synthetic RNA substrate and full-length mRNA.

In an attempt to develop efficient antiviral agents against Mumps virus, we designed ribozymes targeting the nucleocapsid (NP) mRNA. Transacting catalytic RNAs of the hammerhead and hairpin types were synthesized; they contained specific motifs, shared similar flanking regions and were directed against a 5'GUC3' target immediately downstream to the initiation codon of NP mRNA. Both ribozymes were first assayed on a synthetic 16 bases target RNA and found to catalytically and efficiently cleave the substrate in a sequence specific way. No cleavage, however, occurred when mutated forms of the ribozymes were used. In addition, both ribozyme types, when tested on the full length NP mRNA, were also able to cleave the substrate although turnover could not be demonstrated. As a rule, the hammerhead ribozyme proved more efficient than its hairpin counterpart, as well on the synthetic RNA substrate as on the full length NP mRNA target.