The heterotrimeric guanine nucleotide-binding proteins (G proteins) act as switches that regulate information processing circuits connecting cell surface receptors to a variety of effectors. The G proteins are present in all eukaryotic cells, and they control metabolic, humoral, neural, and developmental functions. More than a hundred different kinds of receptors and many different effectors have been described. The G proteins that coordinate receptor-effector activity are derived from a large gene family. At present, the family is known to contain at least sixteen different genes that encode the alpha subunit of the heterotrimer, four that encode beta subunits, and multiple genes encoding gamma subunits. Specific transient interactions between these components generate the pathways that modulate cellular responses to complex chemical signals.

Murine G alpha 14 and G alpha 15 cDNAs encode distinct alpha subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins). These alpha subunits are related to members of the Gq class and share certain sequence characteristics with G alpha q, G alpha 11, and G alpha 16, such as the absence of a pertussis toxin ADP-ribosylation site. G alpha 11 and G alpha q are ubiquitously expressed among murine tissues but G alpha 14 is predominantly expressed in spleen, lung, kidney, and testis whereas G alpha 15 is primarily restricted to hematopoietic lineages. Among hematopoietic cell lines, G alpha 11 mRNA is found in all cell lines tested, G alpha q is expressed widely but is not found in most T-cell lines, G alpha 15 is predominantly expressed in myeloid and B-cell lineages, and G alpha 14 is expressed in bone marrow adherent (stromal) cells, certain early myeloid cells, and progenitor B cells. Polyclonal antisera produced from synthetic peptides that correspond to two regions of G alpha 15 react with a protein of 42 kDa expressed in B-cell membranes and in Escherichia coli transformed with G alpha 15 cDNA. The expression patterns that were observed in mouse tissues and cell lines indicate that each of the alpha subunits in the Gq class may be involved in pertussis toxin-insensitive signal-transduction pathways that are fundamental to hematopoietic cell differentiation and function.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are integral to the signal transduction pathways that mediate the cell's response to many hormones, neuromodulators, and a variety of other ligands. While many signaling processes are guanine nucleotide dependent, the precise coupling between a variety ofreceptors, G proteins, and effectors remains obscure. We found that the family of genes that encode the a subunits of heterotrimeric G proteins is much larger than had previously been supposed. These novel alpha subunits could account for some of the diverse activities attributed to G proteins. We have now obtained cDNA clones encoding two murine a subunits, Gaq and Gall, that are 88% identical. They lack the site that is ordinarily modified by pertussis toxin and their sequences vary from the canonical Gly-Ala-Gly-Glu-Ser (GAGES) amino acid sequence found in most other G protein a subunits. Multiple mRNAs as large as 7.5 kilobases hybridize to Gaq specific probes and are expressed at various levels in many different tissues. Gall1 is encoded by a single 4.0-kilobase message which is expressed ubiquitously. Amino acid sequence comparisons suggest that Gaq and Gall represent a third class of a subunits. A member of this class was found in Drosophila melanogaster. This a subunit, DGaq, is 76% identical to Gaq. The presence of the Gq class in both vertebrates and invertebrates points to a role that is central to signal transduction in multicellular organisms. We suggest that these a subunits may be involved in pertussis toxin-insensitive pathways coupled to phospholipase C.Ga, and Ga, subtypes in gating of specific ion channels (8,9)

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) are central to the signaling processes of multiceilular organisms. We have explored the diversity of the G protein subunits in mammals and found evidence for a large family of genes that encode the a subunits.Amino acid sequence comparisons show that the different a subunits fail into at least three classes. These classes have been conserved in animals separated by considerable evolutionary distances; they are present in mammals, DrosophUa, and nematodes. We have now obtained cDNA clones encoding two murine a subunits, Gal2 and Gal3, that define a fourth class. The translation products are predicted to have molecular masses of 44 kDa and to be insensitive to ADP-ribosylation by pertussis toxin. They share 67% amino acid sequence identity with each other and <45% identity with other a subunits. Their transcripts can be detected in every tissue examined, although the relative levels of the Gal3 message appear somewhat variable.Guanine nucleotide-binding regulatory proteins (G proteins) are heterotrimers composed of a, f3, and y subunits (for reviews see refs. 1-3). The a subunits belong to a much larger group of GTPases, including elongation factor Tu and Ras, which share similar structural elements (4,5). In all of these GTPases, a cycle of guanine nucleotide exchange and hydrolysis enables the protein to exist in two distinct states. This cycle allows G proteins to transiently relay signals from cell-surface receptors to intracellular effectors. The receptors comprise a diverse family of proteins characterized by their transmembrane structure; they have seven membranespanning domains with highly conserved amino acid sequences. Upon interaction with the appropriate agonist, the receptor serves to accelerate the exchange of GDP for GTP on the G protein a subunit. This exchange is believed to be accompanied by dissociation of the a and f3-y subunits, allowing a (and in some cases P-y) to interact with effectors.

We describe here a transposon-based DNA sequencing strategy that allows the introduction of sequencing priming sites throughout a target sequence by bacterial mating. A miniplasmid was designed to select against transposon insertions into the vector. Sites of transposon insertion are mapped by the polymerase chain reaction with bacterial overnight cultures providing the templates. A small set of plasmids with transposons spaced several hundred base pairs apart can then be sequenced. Sequencing primers corresponding to the transposon ends allow sequencing in both directions. Thus, the entire sequence of both strands can be easily determined.One ofthe major problems in DNA sequence analysis oflarge or even moderately sized fragments is how to position unsequenced regions next to known priming sites. A variety of techniques have been developed for this purpose including random shotgun subcloning, unidirectional deletions and subcloning, and the continued synthesis of additional oligodeoxynucleotide primers (1-4). These methods are expensive or require many molecular manipulations.A number of strategies employ bacterial transposons to generate priming sites within a target DNA sequence (5-10). Several criteria exist for an efficient transposon-based sequencing strategy: (i) Mobilization of the transposon must be relatively simple. (ii) Selection for transposon insertions into the plasmid as opposed to the bacterial chromosome must be efficient. (iii) The transposon must insert into the target sequence and not into the plasmid vector. (iv) The transposition sites must be easily mapped to minimize the number of required sequencing reactions. In this paper we describe a transposon-based strategy that meets these criteria.We employ y6, which belongs to the Tn3 family of transposons (11) and which has been used previously in transposon-facilitated strategies (8,20). The members of this family contain 38-base-pair (bp) terminal inverted repeats and transpose by a replicative mechanism. Donor and target sequences are joined in an intermediate structure termed a cointegrate. The cointegrate, which contains two copies of the transposon, is rapidly resolved by a site-specific recombination system. The resolvase is encoded by the transposon and acts at the 120-bp res site located within the mobile element.