Pleiotropy in microdeletion syndromes: neurologic and spermatogenic abnormalities in mice homozygous for the p6H deletion are likely due to dysfunction of a single gene.

Variability and complexity of phenotypes observed in microdeletion syndromes can be due to deletion of a single gene whose product participates in several aspects of development or can be due to the deletion of a number of tightly linked genes, each adding its own effect to the syndrome. The p6H deletion in mouse chromosome 7 presents a good model with which to address this question of multigene vs. single-gene pleiotropy. Mice homozygous for the p6H deletion are diluted in pigmentation, are smaller than their littermates, and manifest a nervous jerky-gait phenotype. Male homozygotes are sterile and exhibit profound abnormalities in spermiogenesis. By using N-ethyl-N-nitrosourea (EtNU) mutagenesis and a breeding protocol designed to recover recessive mutations expressed hemizygously opposite a large p-locus deletion, we have generated three noncomplementing mutations that map to the p6H deletion. Each of these EtNU-induced mutations has adverse effects on the size, nervous behavior, and progression of spermiogenesis that characterize p6H deletion homozygotes. Because EtNU is thought to induce primarily intragenic (point) mutations in mouse stem-cell spermatogonia, we propose that the trio of phenotypes (runtiness, nervous jerky gait, and male sterility) expressed in p6H deletion homozygotes is the result of deletion of a single highly pleiotropic gene. We also predict that a homologous single locus, quite possibly tightly linked and distal to the D15S12 (P) locus in human chromosome 15q11-q13, may be associated with similar developmental abnormalities in humans.

Effect of 2′-O-methyl antisense ORNs on expression of thymidylate synthase in human colon cancer RKO cells

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein end-product TS in a negative autoregulatory manner. Disruption of this regulation results in increased synthesis of TS and may lead to the development of cellular drug resistance to TS-directed anticancer agents. As a strategy to inhibit TS expression, antisense 2′-O-methyl RNA oligoribonucleotides (ORNs) were designed to directly target the 5′ upstream cis-acting regulatory element (nucleotides 80–109) of TS mRNA. A 30 nt ORN, HYB0432, inhibited TS expression in human colon cancer RKO cells in a dose-dependent manner but had no effect on the expression of β-actin, α-tubulin or topoisomerase I. TS expression was unaffected by treatment with control sense or mismatched ORNs. HYB0504, an 18 nt ORN targeting the same core sequence, also repressed expression of TS protein. However, further reduction in oligo size resulted in loss of antisense activity. Following HYB0432 treatment, TS protein levels were reduced by 60% within 6 h and were maximally reduced by 24 h. Expression of p53 protein was inversely related to that of TS, suggesting that p53 expression may be directly linked to intracellular levels of TS. Northern blot analysis demonstrated that TS mRNA was unaffected by HYB0432 treatment. The half-life of TS protein was unchanged after antisense treatment suggesting that the mechanism of action of antisense ORNs is mediated through a process of translational arrest. These findings demonstrate that an antisense ORN targeted at a critical cis-acting element on TS mRNA can specifically inhibit expression of TS protein in RKO cells.

Cone photoreceptors respond to their own glutamate release in the tiger salamander.

Pulse-like currents resembling miniature postsynaptic currents were recorded in patch-clamped isolated cones from the tiger salamander retina. The events were absent in isolated cones without synaptic terminals. The frequency of events was increased by either raising the osmotic pressure or depolarizing the cell. It was decreased by the application of either glutamate or the glutamate-transport blockers dihydrokainate and D,L-threo-3-hydroxyaspartate. The events required external Na+ for which Li+ could not substitute. The reversal potential of these currents followed the equilibrium potential for Cl- when internal Cl- concentration was changed. Thus, these miniature currents appear to represent the presynaptic activation of the glutamate receptor with glutamate transporter-like pharmacology, caused by the photoreceptor's own vesicular glutamate release. Using a noninvasive method to preserve the intracellular Cl- concentration, we showed that glutamate elicits an outward current in isolated cones. Fluorescence of the membrane-permeable form of fura-2 was used to monitor Ca2+ entry at the cone terminal as a measure of membrane depolarization. The increase in intracellular Ca2+ concentration, elicited by puff application of 30 mM KCl, was completely suppressed in the presence of 100 microM glutamate. Puff application of glutamate alone had no measurable depolarizing effect. These results suggest that the equilibrium potential for Cl-, ECl, was more negative than the activation range for Ca2+ channels and that glutamate elicited an outward current, hyperpolarizing the cones.