Enhancers, or activators, dramatically increase the transcriptional activity of certain eukaryotic genes. A series of multiple point mutations affecting the simian virus 40 (SV40) enhancer-activator region were generated in order to define the nucleotide sequence required for this function. Three independent assays provided information leading to the identification of nucleotides essential for enhancer function. One class leads to a decrease in gene expression, while the second completely abolishes functional activity. One critical replacement appears to be the first G (guanine) in a sequence TGGAAAG (T, thymine, A, adenine) located in the 5' region of the 72 base-pair repeat of SV40. Comparison of this sequence with nucleotide sequences in other known enhancers leads to the identification of potential related core elements.
Enkephalins are endogenous opioid peptides that are derived from a pre-proenkephalin precursor protein. They are thought to be vital in regulating many physiological functions, including pain perception and analgesia, responses to stress, aggression and dominance. Here we have used a genetic approach to study the role of the mammalian opioid system. We disrupted the pre-proenkephalin gene using homologous recombination in embryonic stem cells to generate enkephalin-deficient mice. Mutant enk-/- animals are healthy, fertile, and care for their offspring, but display significant behavioural abnormalities. Mice with the enk-/- genotype are more anxious and males display increased offensive aggressiveness. Mutant animals show marked differences from controls in supraspinal, but not in spinal, responses to painful stimuli. Unexpectedly, enk-/- mice exhibit normal stress-induced analgesia. Our results show that enkephalins modulate responses to painful stimuli. Thus, genetic factors may contribute significantly to the experience of pain.
The antidiuretic effect of arginine vasopressin (AVP) is mediated by renal-type (V2) receptors linked to adenylyl cyclase. We report here the cloning of the rat kidney V2 AVP receptor complementary DNA that encodes a 370-amino-acid protein with a transmembrane topography characteristic of G protein-coupled receptors, and with similarity to the V1a (hepatic) AVP receptor in its seven membrane-spanning domains. Expression of the cloned cDNA in mammalian cells showed specific ligand binding and activity characteristic of the native V2 AVP receptor. The receptor messenger RNA is detected only in the kidney. The human V2 receptor gene has been localized to the long arm of the X chromosome close to the locus for nephrogenic diabetes insipidus, an X-linked recessive disorder characterized by renal resistance to the antidiuretic action of AVP.
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...
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