A bacterial coning system for mapping and analysis of complex genomes has been developed. The BAC system (for bacterial artificial chromosome) is based on Escherichia colt and its single-copy plasmid F factor. It is capable of maintaining human genomic DNA fragments of >300 kilobase pai-. Individual clones of human DNA appear to be maintained with a high degree ofstructural stability in the host, even after 100 generations of serial growth. Because of high cloning efficiency, easy manipulation of the cloned DNA, and stable maintenance of inserted DNA, the BAC system may facilitate construction of DNA libraries of complex genomes with fuller representation and subsequent rapid analysis of complex genomic structure.There is currently underway an intense effort to construct a high-resolution physical map of each of the human chromosomes. Eventually, these maps will be composed of overlapping fragments of human DNA and will allow the direct acquisition of DNA fragments that correspond to specific genes. Completion of the physical map requires the availability of comprehensive libraries of DNA clones in appropriate vectors. Furthermore, the accuracy and efficiency of physical mapping increase progressively with the size of the clone firgments in these libraries. Thus, the construction of libraries using yeast artificial chromosomes (YACs), which permit cloning of fragments of ;500 kilobase pairs (kb), represents a fundamental advance in our ability to generate physical maps that order DNA over multi-megabase distances (1). However, some difficulties have been encountered with the manipulation of YAC libraries (2-4). Thus, for example, in various libraries a fraction of the clones result from co-cloning events; i.e., they include in a single clone noncontiguous DNA fiagments. We describe here a bacterial cloning system that we refer to as BACs, bacterial artificial chromosomes. This system may provide a supplement and alternative to the YAC system for some applications requiring cloning of large fragments. The BAC system is based on the well-studied Escherichia coli F factor. Replication of the F factor in E. coli is strictly controlled (5). The F plasmid is maintained in low copy number (one or two copies per cell), thus reducing the potential for recombination between DNA fragments carried by the plasmid. Furthermore, F factors carrying inserted bacterial DNA are capable of maintaining fragments as large as 1 megabase pair, suggesting that the F factor is suitable for cloning of large DNA fragments (6). Other bacterial systems for cloning large DNA have been developed. For example, the system based on bacteriophage P1 is in use (7). However, the P1 vector has a maximum cloning capacity of 100 kb. A bacterial system based on F factors has been reported (8). However, in this system, human DNA inserts >120 kb have not been cloned and characterized. The BAC system allows us to clone large DNA from a variety of complex genomic sources into bacteria, where the DNA is stable, easy to manipulate, and represents a si...

Heterotrimeric GTP-binding proteins (G proteins) participate in cellular signaling and regulate a variety of physiological processes. Disruption of the gene encoding the G protein subunit alpha13 (Galpha13) in mice impaired the ability of endothelial cells to develop into an organized vascular system, resulting in intrauterine death. In addition, Galpha13 (-/-) embryonic fibroblasts showed greatly impaired migratory responses to thrombin. These results demonstrate that Galpha13 participates in the regulation of cell movement in response to specific ligands, as well as in developmental angiogenesis.

RGS (regulator of G protein signaling) proteins containing the G protein ␥-like (GGL) domain (RGS6, RGS7, RGS9, and RGS11) interact with the fifth member of the G protein ␤-subunit family, G␤5. This interaction is necessary for the stability of both the RGS protein and for G␤5. Consistent with this notion, we have found that elevation of RGS9-1 mRNA levels by transgene expression does not increase RGS9-1 protein level in the retina, suggesting that G␤5 levels may be limiting. To examine further the interactions of G␤5 and the GGL domain-containing RGS proteins, we inactivated the G␤5 gene. We found that the levels of GGL domain-containing RGS proteins in retinas and in striatum are eliminated or reduced drastically, whereas the levels of G␥2 and RGS4 proteins remain normal in the absence of G␤5. The homozygous G␤5 knockout (G␤5 ؊͞؊ ) mice derived from heterozygous knockout mating are runty and exhibit a high preweaning mortality rate. We concluded that complex formation between GGL domain-containing RGS proteins and the G␤5 protein is necessary to maintain their mutual stability in vivo. Furthermore, in the absence of G␤5 and all four RGS proteins that form protein complexes with G␤5, the animals that survive into adulthood are viable and have no gross defects in brain or retinal morphology.F irst discovered functionally as negative regulators of G protein signaling in Saccharomyces cerevisiae (Sst2p) (1) and Caenorhabditis elegans (EGL10) (2), RGS (regulator of G protein signaling) proteins accelerate the hydrolysis of GTP by the ␣-subunits of heterotrimeric G proteins (3). They form a large gene family with a diagnostic Ϸ120-aa RGS domain in which the GTPase-accelerating activity resides (4). In addition to the RGS domain, most RGS proteins possess additional domains that enable them to interact with other cellular signaling molecules (5). A subgroup of the RGS family, namely RGS9, RGS11, RGS7, and RGS6, possesses a G ␥-like (GGL) domain that binds the fifth member of the heterotrimeric G protein ␤-subunit (G␤5) both in vitro and in vivo (6-10). There are five known members of the G protein ␤-subunit family (11, 12). The first four members, G␤ 1-4 , are highly similar, sharing 80-90% sequence identity. G␤5 is the most divergent member of this family, sharing only 50% sequence identity with G␤ 1-4 . G␤5 exists in two forms: the common, short-splice form (G␤5-S) and a unique, long-splice form (G␤5-L) that exists exclusively in retinal photoreceptors. The long form results from the addition of an N-terminal exon through alternative splicing (11). G␤5 complexes with either GGL domain-containing RGS proteins or with certain G protein ␥-subunits such as G␥2 (12, 13). Likewise, the GGL domain-containing RGS proteins not only interact with G␤5, they also can interact with other proteins such as polycystin (for RGS7) (14) and SCG10 (for RGS6) (15). Interestingly, these RGS proteins do not interact with G␤ 1-4 , indicating that their interaction with G␤5 is selective and may be important for their in vivo function (6, 1...

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Phospholipase Cbeta (PLCbeta) isozymes represent a family of molecules that link G protein-coupled receptors (GPCRs) to an intracellular signaling network. Here, we investigated the function of PLCbeta isozymes in sensory neurons by using mutant mice deficient for specific PLCbeta family members. Expression analysis indicated that PLCbeta3, one of the four isoforms, is predominantly expressed in a subpopulation of C-fiber nociceptors. A subset of these neurons expressed the histamine H1 receptor. Ca(2+) imaging studies revealed that PLCbeta3 specifically mediates histamine-induced calcium responses through the histamine H1 receptor in cultured sensory neurons. In line with this, we found that PLCbeta3(-/-) mice showed significant defects in scratching behavior induced by histamine; histamine-trifluoromethyl-toluidine (HTMT), a selective H1 agonist; and compound 48/80, a mast cell activator. These results demonstrate that PLCbeta3 is required to mediate "itch" sensation in response to histamine acting on the histamine H1 receptor in C-fiber nociceptive neurons.