An efficient and accurate method for controlled in vivo transgene modulation by site-directed recombination is described. Seven transgenic mouse founder lines were produced carrying the murine lens-specific aA-crystallin promoter and the simian virus 40 large tumor-antigen gene sequence, separated by a 1.3-kilobase-pair Stop sequence that contains elements preventing expression of the large tumorantigen gene and Cre recombinase recognition sites. Progeny from two of these lines were mated with transgenic mice expressing the Cre recombinase under control of either the murine aA-crystallin promoter or the human cytomegalovirus promoter. AU double-transgenic offspring developed lens tumors. Subsequent analysis confirmed that tumor formation resulted from large tumor-antigen activation via site-specific, Cre-mediated deletion of Stop sequences.A desired goal of transgene technology is efficient and accurate manipulation of DNA sequences after their integration in the germ line. DNA recombinases that mediate integration or excision of sequences at specific recognition sites in both prokaryotic (1-5) and eukaryotic (6-10) systems are well suited for this purpose. The bacteriophage P1 recombinase Cre catalyzes reciprocal recombination at a specific locus ofcrossing over (lox) (11-16). The lox sequence is composed of two 13-base-pair (bp) inverted repeats separated by an 8-bp spacer region. Upon binding to the inverted repeats, Cre synapses with a second lox site and then cleaves the DNA in the spacer region to initiate strand exchange with the synapsed lox partner. No additional factors are required in the recombination.In this study, we examine the potential of the cre/lox system to activate a dormant transgene in the mouse. The simian virus 40 (SV40) large tumor antigens (TAgs) directed to the lens by a murine aA-crystallin promoter (maA) cause malignant lens tumors (17). We inserted between maA and TAg a specially designed Stop sequence that prevents gene expression and is flanked by lox sequences. By crossing the dormant TAg transgenic mouse lines with Cre-expressing transgenic lines, we report here that the Cre protein recognizes the lox sites of the maA-Stop-TAg transgene and recombines the two lox sequences, thereby removing Stop and activating TAg. Our studies show that targeted transgene modification in the mouse can be performed efficiently and accurately with a prokaryotic recombinase.

The discrete anatomical distribution of arginine vasopressin and corticotropin releasing factor (CRF) immunoreactivity in the paraventricular nucleus (PVN) of the rat hypothalamus is altered after adrenalectomy. Not only is the immunostaining of both peptides enhanced, but vasopressin immunoreactivity, normally confined to the magnocellular subdivision, becomes clear in a large percentage of CRF neurones in the parvocellular subdivision. These changes in immunoreactivity may reflect changes in post-translational events, peptide metabolism or genomic activity that lead indirectly or directly to the enhanced expression of vasopressin. Here we report that levels of transcripts homologous to vasopressin messenger RNA increase in the PVN after adrenalectomy, in parallel with increases in vasopressin immunoreactivity. In fact, after adrenalectomy, vasopressin mRNA can be detected in CRF-immunoreactive neurones. These results indicate that a considerable degree of plasticity is retained by the adult neuronal genome of the rat and that this plasticity may be modulated by the endocrine environment.

Immunocytochemical studies have shown that adrenalectomy produces changes in the content and distribution of [arginine-8]vasopressin (AVP) immunoreactivity in the paraventricular nucleus of the hypothalamus. The purpose of this study was to determine whether manipulation of adrenal hormones affects the levels of AVP mRNA. In situ hybridization assays with highly specific synthetic oligodeoxyribonucleotide probes and immunocytochemistry were used to detect the distribution of AVP mRNA and AVP-immunoreactive perikarya. AVP mRNA is codistributed with AVP immunoreactivity in the posterior magnocellular subdivision of the paraventricular nucleus and its accessory nuclei, the supraoptic nucleus and the suprachiasmatic nucleus. In adrenalectomized rats, the density and distribution of the hybridization signal were increased in the paraventricular nucleus; a 2-fold increase in the area comprising the signal was observed. At the cellular level, silver grains were detected in corticotropin-releasing-factor-immunoreactive neurons throughout the medial parvocellular subdivision of the paraventricular nucleus. No changes were seen in the distribution of AVP mRNA in the supraoptic or suprachiasmatic nuclei. Treatment with dexamethasone prevented the increase in AVP mRNA produced by adrenalectomy. In contrast, adrenalectomy did not alter the hybridization signal obtained with a probe for a-tubulin mRNA. These results suggest, at the cellular level, that adrenalectomy induces a glucocorticoid-sensitive stimulation of AVP mRNA synthesis in the central nervous system. Thus, considerable plasticity in gene expression is retained in the hypothalamus of the adult rat.The discrete anatomical organization of hypothalamic neurosecretory neurons within the paraventricular nucleus (PVN) has established this region as a unique site to study the neurohypophyseal secretory system. [arginine-8]Vasopressin (AVP) and oxytocin are contained within neurons of the PVN and the supraoptic nucleus (SON) (1, 2) and are involved in both autonomic and neurosecretory functions (3, 4). The axons emanating from these perikarya form the hypothalamoneurohypophyseal tracts, which project to the pars nervosa where AVP is released from their terminal endings (5,6). However, AVP has been detected in axon terminals within the external zone of the median eminence (7), suggesting that it may be involved, to some extent, in regulating anterior pituitary functions. Unlike the neurohypophyseal projection, the majority of AVP-immunoreactive axon terminals in the median eminence originate from neurons within the medial parvocellular subdivision of the PVN (8). Biochemical studies have confirmed quantitatively that adrenalectomy increases AVP immunoreactivity (7, 9). After adrenalectomy, corticotropin-releasing factor (CRF) and AVP were reported to be colocalized within neurons in the medial parvocellular region of the PVN (10, 12). This finding is of particular interest because immunocytochemical studies have shown that adrenalectomy produces a dexamethasonesen...

Hybrid complementary metal oxide semiconductor (CMOS)/molecular memory devices are based on a dynamic random-access memory (DRAM) architecture, are fast, have high density, and exhibit low power consumption. These devices use a well-characterized charge storage mechanism to store information based on the intrinsic properties of molecules attached to a CMOS platform. The molecules are designed in a rational way to have known electrical properties and can be incorporated into CMOS devices with only minor modification of existing fabrication methods. Each memory element contains a monolayer of molecules (typically 100,000–1,000,000) to store charge; this process yields a structure that has many times the charge density of a typical DRAM capacitor, obviating the necessity for a trench or stacked capacitor geometry. The magnitude of voltage required to remove each electron is quantized (typically a few hundred millivolts per state), making it much easier to put molecules in a known state and to detect that state with low-power operation. Existing devices have charge retention times that are >1000 times that of semiconductors, and nonvolatile strategies based on simple modifications of existing systems are possible. All of these devices are ultimately scalable to molecular dimensions and will enable the production of memory products as small as state-of-the-art lithography will allow.