The tachykinin neuropeptides, substance P and substance K, are produced in nociceptive primary sensory neurons and in many brain regions involved in pain signaling. However, the precise role and importance of these neuropeptides in pain responses has been debated. We now show that mice that cannot produce these peptides display no significant pain responses following formalin injection and have an increased pain threshold in the hotplate test. On the other hand, the mutant mice react normally in the tail f lick assay and acetic acid-induced writhing tests. These results demonstrate that substance P and͞or substance K have essential functions in specific responses to pain.The tachykinins are a family of structurally related neuropeptides. In the mouse, they are encoded by the genes Tac1 and Tac2. Tac1 produces substance P, substance P (neurokinin A), neurokinin A (3-10), neuropeptide K, and neuropeptide ␥ as a result of differential splicing and posttranslational processing (1-4). Tac2 produces the peptide neurokinin B.The undecapeptide substance P was first detected by von Euler and Gaddum (5) in 1931. Its structure was revealed by Leeman and her coworkers (6, 7) in 1971. The Tac1 cDNA was cloned in 1983 by Nakanishi and his coworkers (2,8). The Tac1 gene is expressed in many regions in the central and peripheral nervous system, as well as in nonneuronal tissues. Substance P has been implicated in a variety of physiological processes including cardiovascular, respiratory, and gastrointestinal functions; inflammatory responses; and nociception. In addition, Hunt and coworkers have suggested that substance P may be involved in axon guidance during embryonic development (9).The precise role of substance P in these processes is unclear. For example, substance P is synthesized in nociceptive primary sensory neurons, which send C-and A␦ fibers to dorsal horn projection neurons in lamina I and IV-V, and to nociceptionspecific interneurons in lamina II-III of the spinal cord. Axons of projection neurons terminate in many supraspinal nuclei that are involved in pain transmission (10, 11). Nociceptive stimulation triggers the release of substance P from C-afferent terminals in the marginal layers of the spinal cord (12), evokes slow excitatory postsynaptic potentials in second-order sensory neurons in the dorsal horn, and facilitates their activation (13). These data, together with other functional evidence (11,14), indicated an important role for substance P in the processing of nociceptive signals.We have begun to use a genetic approach to study the functions of tachykinin peptides. As a first step, we have generated mice with a targeted mutation in the Tac1 gene. These mice are viable and fertile, but exhibit striking defects in nociceptive behaviors. MATERIALS AND METHODS Generation and Breeding of Tac1؊͞؊ Mice. Tac1 mutations were established by homologous recombination in MPI2 embryonic stem (ES) cells according to standard protocols (15). One mutant ES cell line was used to derive chimeras by morula aggrega...

Abstract. The 40-S subunit of eukaryotic ribosomes binds to the capped 5"end of mRNA and scans for the first AUG in a favorable sequence context to initiate translation. Most eukaryotic mRNAs therefore have a short 5'-untranslated region (5"UTR) and no AUGs upstream of the translational start site; features that seem to assure efficient translation. However, ,o5-10% of all eukaryotic mRNAs, particularly those encoding for regulatory proteins, have complex leader sequences that seem to compromise translational initiation. The retinoic-acid-receptor-/32 (RARB2) mRNA is such a transcript with a long (461 nucleotides) 5'-UTR that contains five, partially overlapping, upstream open reading frames (uORFs) that precede the major ORE We have begun to investigate the function of this complex 5'-UTR in transgenic mice, by introducing mutations in the start/stop codons of the uORFs in RAP-~2-1acZ reporter constructs. When we compared the expression patterns of mutant and wild-type constructs we found that these mutations affected expression of the downstream RAR~2-ORF, resulting in an altered regulation of RAP-~2-1acZ expression in heart and brain. Other tissues were unaffected. RNA analysis of adult tissues demonstrated that the uORFs act at the level of translation; adult brains and hearts of transgenic mice carrying a construct with either the wild-type or a mutant UTR, had the same levels of mRNA, but only the mutant produced protein. Our study outlines an unexpected role for uORFs: control of tissue-specific and developmentally regulated gene expression.

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Abstract. Regulation of mRNA translation and stability plays an important role in the control of gene expression during embryonic development. We have recently shown that the tissue-specific expression of the RAR[32 gene in mouse embryos is regulated at the translational level by short upstream open reading frames (uORFs) In the 5'-untranslated region (Zimmer, A., A.M. Zimmer, and K. Reynolds. 1994. J. Cell Biol. 127:1111-1119. To gain insight into the molecular mechanism, we have performed a systematic mutational analysis of the uORFs. Two series of constructs were tested: in one series, each uORF was individually inactivated by introducing a point mutation in its start codon; in the second series, all but one ORF were inactivated. Our results indicate that individual uORFs may have different functions, uORF4 seems to inhibit translation of the major ORF in heart and brain, while uORFs 2 and 5 appear to be important for efficient translation in all tissues. To determine whether the polypeptide encoded by uORF4 or the act of translating it, is the significant event, we introduced point mutations to create silent mutations or amino acid substitutions in uORF4. Our results indicate that the uORF4 amino acid coding sequence is important for the inhibitory effect on translation of the downstream major 0RF. T hE scanning model for translational initiation predicts that the translational initiation site is dictated by the location and sequence context of the initiation codon (Kozak, 1989a). The 40S preinitiation complex binds to the 5' cap-proximal region of the mRNA and migrates downstream until it encounters an AUG in a favorable context. At this point, the 60S ribosomal subunit binds to the complex and protein synthesis commences. Most eukaryotic mRNAs have a short 5'-untranslated region (UTR) 1 without stable secondary structures and no upstream AUGs. These features enable the preinitiation complex to efficiently scan the 5'-region for the translation start site. Complex 5'-UTRs with upstream AUGs and/or the ability to form stable secondary structures are found only in a small minority of transcripts (Kozak, 1989a). Strinkingly, most of these genes, which include homeobox genes, proto-oncogenes, transcription factors, and signal transduction components are thought to play an important role in the regulation of embryonic development. It has been proposed that expression of many of these genes is regulated at the translational level (Kozak, 1989a The retinoic acid receptor-~2 (RARI32) mRNA has a complex 5'-UTR. RAR[32 is a retinoic acid (RA)-dependent transcription factor that belongs to the superfamily of steroid/thyroid hormone receptors (Evans, 1988). RA plays an important role as a signaling molecule during embryoic development and homeostasis. It is a very potent teratogen and can induce a large variety of severe congenital defects, depending on the dose and time of exposure. Administration during early gastrulation, for example, results in craniofacial and heart defects (Kochhar, 1967) as well as skeletal abn...