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Effective intracellular expression of small RNA therapeutics inclusion of specific U6 snRNA sequences from positions depends on a number of factors. The RNA, whether anti-+19 to +27. In situ localization of the transcripts shows that sense, ribozyme, or RNA aptamer, must be efficiently tranboth tRNA and U6 promoter transcripts give primarily puncscribed, stabilized against rapid degradation, folded cortate nuclear patterns, and that capping of transcripts is not rectly, and directed to the part of the cell where it can be required for nuclear retention. Several different insert most effective. To overcome a number of these problems RNAs directed against HIV-1 were tested by cotransfection we have been testing expression cassettes based on the with HIV-1 provirus and assay for subsequent viral reverse human tRNA met and U6 snRNA promoters, in which trantranscriptase production. These include antisense RNA, scripts encoding small RNA inserts are protected against hairpin and hammerhead ribozymes, and RNA ligands attack from the 3′ end. Transient expression in cultured (aptamers) for Tat and Rev RNA binding proteins. Results cells results in 10 3 -2 × 10 7 full-length transcripts per cell,show that Rev-binding RNAs efficiently block HIV-1 gene depending partially on the promoter construct used but expression, whereas other RNAs have little or no effect also on the nature of the insert RNA. 5′ ␥-Phosphate when expressed in these cassettes. methylation (capping) depended, as expected, on the

The tar and tsr genes of Escherichia coli encode homologous transducer proteins that mediate distinct chemotactic responses. We report here the construction of two tasr chimeric genes in which the 5' coding region of the tar gene is fused to the 3' coding region of the tsr gene at either of two conserved restriction sites. Both chimeric genes code for chemotactically functional proteins. Results of analyses of behavior and methylation in cells carrying the chimeric genes support existing models for the disposition of transducer domains across the cell membrane and reveal that the receptors for internal pH map in a specific region of the COOH-terminal (cytoplasmic) domain.

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