Abbreviations: PP, pancreatic polypeptide; En, embryonic day n; bHLH, basic helix-loophelix; ES, embryonic stem; kb, kilobase; TUNEL, terminal deoxynucleotidyltransferasemediated UTP end labeling.

or by ␥-irradiation revealed an extreme sensitivity and a high genomic instability to both agents. Following whole body ␥-irradiation (8 Gy) mutant mice died rapidly from acute radiation toxicity to the small intestine. Mice-derived PARP ؊/؊ cells displayed a high sensitivity to MNU exposure: a G 2 ͞M arrest in mouse embryonic fibroblasts and a rapid apoptotic response and a p53 accumulation were observed in splenocytes. Altogether these results demonstrate that PARP is a survival factor playing an essential and positive role during DNA damage recovery.To protect their genome from the deleterious consequences of accumulation of unrepaired or misrepaired lesions, cells have developed an intricate DNA damage surveillance network. Through its function as a single-stranded breaks detector, poly(ADP-ribose) polymerase [PARP; NAD ϩ ADP-ribosyltransferase; NAD ϩ : poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30], a nuclear enzyme, participates to this basic process (1). PARP (113 kDa) has a modular organization (2): a N-terminal DNA-binding domain that acts as a molecular nick-sensor, encompassing two zinc-finger motifs (3) and a bipartite nuclear location signal (4), a central region bearing the auto-poly(ADP-ribosylation) sites which serves to regulate PARP-DNA interactions and a C-terminal catalytic domain involved in the nick-binding dependent poly(ADP-ribose) synthesis (5). The x-ray crystallographic structure of this domain has been recently solved revealing a surprising structural homology between the active site of PARP and that of bacterial mono-ADP-ribosylating toxins despite weak sequence homology (6).Although the physiological role of PARP is still much debated, recent molecular and genetic approaches including expression of either a dominant-negative mutant (7-10) or antisense (11) have clearly revealed the implication of PARP in the maintenance of the genomic integrity in the base excision repair pathway (7)(8)(9)(10)12). To elucidate its function we disrupted the mouse PARP gene by homologous recombination and exposed the PARP-deficient mice and derived cells to various genotoxins. MATERIALS AND METHODSGene Targeting in Embryonic Stem Cells and Generation of Mice. Mouse PARP was isolated from a 129SVJ strain genomic library. The targeting vector was constructed using a 9-kb EcoRI fragment extending from intron 2 to 7 by inserting PGK-neo (phosphoglycerate kinase promoter followed by the neo gene) in the BamHI site of the 4th exon and herpes simplex virus thymidine kinase followed by the TK gene (HSV-Tk) in the XhoI site outside the sequence of the targeting vector. Following electroporation, embryonic stem cells were selected in 200 g⅐ml Ϫ1 G418 and 2 mM of gancyclovir. A positive clone microinjected into C57BL͞6 blastocysts (13) gave rise to chimaeric offspring, which in turn were mated with C57BL͞6.

Signalling through dopamine D2 receptors governs physiological functions related to locomotion, hormone production and drug abuse. D2 receptors are also known targets of antipsychotic drugs that are used to treat neuropsychiatric disorders such as schizophrenia. By a mechanism of alternative splicing, the D2 receptor gene encodes two molecularly distinct isoforms, D2S and D2L, previously thought to have the same function. Here we show that these receptors have distinct functions in vivo; D2L acts mainly at postsynaptic sites and D2S serves presynaptic autoreceptor functions. The cataleptic effects of the widely used antipsychotic haloperidol are absent in D2L-deficient mice. This suggests that D2L is targeted by haloperidol, with implications for treatment of neuropsychiatric disorders. The absence of D2L reveals that D2S inhibits D1 receptor-mediated functions, uncovering a circuit of signalling interference between dopamine receptors.

Current mouse gene targeting technology is unable to introduce somatic mutations at a chosen time and/or in a given tissue. We report here that conditional site-specific recombination can be achieved in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand-binding domain of the human estrogen receptor (ER) resulting in a tamoxifen-dependent Cre recombinase, Cre-ERT, which is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ERT under the control of a cytomegalovirus promoter. We show that excision of a chromosomally integrated gene flanked by loxP sites can be induced by administration of tamoxifen to these transgenic mice, whereas no excision could be detected in untreated animals. This conditional sitespecific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.The study of the genetic control of mammalian development and physiology has been revolutionized by the ability to inactivate (knockout) specific genes by homologous recombination in the mouse (1). However, using current gene targeting technology, interpretations of knockout phenotypes are often limited by several factors. First, the presence of a selection marker may influence the phenotype of the mutation (2, 3). Second, artefacts can arise due to the lack of a gene product for the whole lifetime of the animal. Third, the inactivation of a gene may result in intra-utero lethality, precluding analysis of the possible function(s) of the gene at later stages of development and/or post-natally. A conditional gene targeting method based on the inducible activity of an engineered DNA recombinase could overcome these limitations by allowing the removal of the selection cassette and the timed and tissuespecific inactivation of target genes at will during development and in the adult mouse (4). Furthermore, such an inducible system could help in certain cases to distinguish between anomalies related to a mixed genetic background and those due to mutation of the targeted gene.The bacteriophage P1 Cre recombinase efficiently excises DNA flanked by two directly repeated loxP recognition sites in mammalian cells (5, 6). We have previously reported that fusion of the ligand-binding domain (LBD) of the estrogen receptor (ER) to the Cre recombinase generates a chimeric recombinase whose activity in cultured cells is dependent on the presence of an estrogen (estradiol) or an anti-estrogen (tamoxifen) (7). To achieve conditional gene targeting in mice, where endogenous estradiol is present, we have subsequently fused Cre to a mutated LBD of the human ER (Gly 521 -> Arg, G521R) resulting in the chimeric protein Cre-ERT. Indeed, the corresponding mouse ER LBD mutant (G525R) does not bind 17,B-estradiol (E2), whereas it binds the synthetic ligands tamoxifen and 4-hydroxytamoxifen (OHT) (8). We report here that Cre-ERT is a functional tamoxifen-dependent recombinase in cultured cells and in transgenic m...

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The role of the opioid system in controlling pain, reward and addiction is well established, but its role in regulating other emotional responses is poorly documented in pharmacology. The mu-, delta- and kappa- opioid receptors (encoded by Oprm, Oprd1 and Oprk1, respectively) mediate the biological activity of opioids. We have generated Oprd1-deficient mice and compared the behavioural responses of mice lacking Oprd1, Oprm (ref. 6) and Oprk1 (ref. 7) in several models of anxiety and depression. Our data show no detectable phenotype in Oprk1-/- mutants, suggesting that kappa-receptors do not have a role in this aspect of opioid function; opposing phenotypes in Oprm-/- and Oprd1-/- mutants which contrasts with the classical notion of similar activities of mu- and delta-receptors; and consistent anxiogenic- and depressive-like responses in Oprd1-/- mice, indicating that delta-receptor activity contributes to improvement of mood states. We conclude that the Oprd1-encoded receptor, which has been proposed to be a promising target for the clinical management of pain, should also be considered in the treatment of drug addiction and other mood-related disorders.

authors request that the following corrections be noted. It was accidentally stated that the studies by Kajita et al. (1) and Lee et al. (2) dealt with cinnamoyl-CoA reductase modified plants when in fact they concerned 4-coumarate:coenzyme A ligase (4CL) transgenic plants. Lignin concentration was reduced by down-regulation of 4CL activity in both studies (1, 2). In a subsequent article, Kajita et al. (3) reported a negligible decrease in lignin concentration and a decreased syringyl-toguaiacyl ratio for lignin composition of a sense-suppressed 4CL transgenic tobacco line. Kajita et al. (1) rather than Kajita et al. (3) was inadvertently cited when this later report was contrasted with the large decreases in lignin concentration and an increased syringyl-to-guaiacyl lignin ratio for anti-sense suppressed 4CL Arabidopsis transgenics (2). The authors apologize for the confusion these errors have created for readers of their Commentary and to the authors of the cited work for misrepresenting their research. November 10, 1998, of Proc. Natl. Acad. Sci. USA (95, 13612-13617), the authors request that the following correction be noted: In Fig. 2 appearing on page 13614, the genotype identification for testicular histology in panels C and D were shown reversed. The correct identification is Ϫ͞Ϫ for panel C and ϩ͞ϩ for panel D. The fifth sentence of the figure legend should read as follows: "Histological sections at lower (E) and higher (D) magnification of the seminiferous tubuli from a wild-type and mutant (F and C) mouse."Cell Biology. In the article "Efficient construction of a large nonimmune phage antibody library: The production of highaffinity human single-chain antibodies to protein antigens" by

The neuromodulator serotonin (5-hydroxytryptamine, 5-HT) has been associated with mood disorders such as depression, anxiety, and impulsive violence. To define the contribution of 5-HT receptor subtypes to behavior, mutant mice lacking the 5-HT1B receptor were generated by homologous recombination. These mice did not exhibit any obvious developmental or behavioral defects. However, the hyperlocomotor effect of the 5-HT1A/1B agonist RU24969 was absent in mutant mice, indicating that this effect is mediated by 5-HT1B receptors. Moreover, when confronted with an intruder, mutant mice attacked the intruder faster and more intensely than did wild-type mice, suggesting the participation of 5-HT1B receptors in aggressive behavior.