Werner's syndrome (WS) is an inherited disease with clinical symptoms resembling premature aging. Early susceptibility to a number of major age-related diseases is a key feature of this disorder. The gene responsible for WS (known as WRN) was identified by positional cloning. The predicted protein is 1432 amino acids in length and shows significant similarity to DNA helicases. Four mutations in WS patients were identified. Two of the mutations are splice-junction mutations, with the predicted result being the exclusion of exons from the final messenger RNA. One of the these mutations, which results in a frameshift and a predicted truncated protein, was found in the homozygous state in 60 percent of Japanese WS patients examined. The other two mutations are nonsense mutations. The identification of a mutated putative helicase as the gene product of the WS gene suggests that defective DNA metabolism is involved in the complex process of aging in WS patients.

Sclerosteosis is an autosomal recessive sclerosing bone dysplasia characterized by progressive skeletal overgrowth. The majority of affected individuals have been reported in the Afrikaner population of South Africa, where a high incidence of the disorder occurs as a result of a founder effect. Homozygosity mapping in Afrikaner families along with analysis of historical recombinants localized sclerosteosis to an interval of approximately 2 cM between the loci D17S1787 and D17S930 on chromosome 17q12-q21. Here we report two independent mutations in a novel gene, termed "SOST." Affected Afrikaners carry a nonsense mutation near the amino terminus of the encoded protein, whereas an unrelated affected person of Senegalese origin carries a splicing mutation within the single intron of the gene. The SOST gene encodes a protein that shares similarity with a class of cystine knot-containing factors including dan, cerberus, gremlin, prdc, and caronte. The specific and progressive effect on bone formation observed in individuals affected with sclerosteosis, along with the data presented in this study, together suggest that the SOST gene encodes an important new regulator of bone homeostasis.

This work describes a multifunctional phage A expression vector system, AYES, designed to facilitate gene isolation from eukaryotes by complementation of Escherichia coli and Saccharomyces cerevisiae mutations. AYES vectors have a selection for cDNA inserts using an oligo adaptor strategy and are capable of expressing genes in both E. coli and S. cerevisiae. They also allow conversion from phage A to plasmid clones by using the cre-lox site-specific recombination system, referred to here as automatic subcloning. A simple method has been developed for the conversion of any plasmid into a phage A cDNA cloning vector with automatic subcloning capability. cDNA libraries constructed in these vectors were used to isolate genes from humans and Arabidopsis thaliana by complementation of yeast and bacterial mutations, respectively.Cloning of genes by complementation is a powerful method for gene isolation in prokaryotic and lower eukaryotic systems. This technique has not been used extensively in higher eukaryotes because of the difficulty of introducing large numbers of clones into a mutant organism-e.g., humans, mice, fruit flies, and plants-and the general lack of mutations in the systems where complementation is possiblee.g., mammalian tissue culture. One approach which circumvents some of these problems is expression of genes from higher eukaryotes in organisms more amenable to complementation, using selections and screens to identify the genes of interest. Interspecies complementation has been used previously to isolate genes (1-3). The first gene from yeast was isolated by complementation of an amino acid biosynthetic mutation in Escherichia coli (4). The Drosophila phosphoribosylglycinamide formyltransferase (GART) gene was isolated by complementation of the yeast ade8 mutation (5). A human gene complementing the cdc2 mutation of Schizosaccharomyces pombe (6), a cAMP phosphodiesterase from rat brains (7), and the human GART gene (8) were isolated by using heterologous cDNA libraries in yeast expression vectors. Although interspecies complementation cannot isolate every gene of interest from higher organisms, a very large number of genes are potentially accessible using this approach, including many of the general biosynthetic genes, genes involved in conserved cellular processes such as protein sorting and transport, and cell cycle regulatory genes. A fertile ground for gene isolation using heterologous cDNA expression libraries lies in the cloning of sequence specific DNA-binding proteins and transcription factors. The basic mechanism of transcriptional activation and repression seems to be conserved in yeast and other eukaryotes, suggesting that simple artificial systems could be built to select for genes encoding proteins that activate or repress transcription in a sequence-specific fashion. We have previously devised a genetic selection for genes encoding sequencespecific DNA-binding proteins that function in E. coli (9, 10).This paper describes the construction and successful testing of a multifuncti...

The soil bacterium Rhizobium meliloh invades and establishes a symbiosis with host plants such as alfalfa. Bacterial nodulation (nod) genes are required for this invasion, but their mechanism of action and the timing of their expression are not known. We have used translational lacZ fusions to monitor expression of nodD and nodC, which are located in the cluster of four nod genes on the R. melUoh megaplasmid (pSym). nodD is expressed at comparable levels by broth-grown bacterial cells and by cells exposed to exudates from aseptically grown plants. Activity of the nodC-lacZ protein fusion in broth-grown bacterial cells is very low. nodC-lacZ activity is increased -30-fold by plant exudate when nodD is expressed at a high level but not when nodD expression is low. Both fusions show differences in expression when borne on inc-P vectors as compared to when located on the pSym megaplasmid. nodD expression from vector-borne copies of the nod segment and response of nodC to plant exudate appear to require additional loci on the megaplasmid. Our results suggest that regulation of bacterial nod gene expression is an important control mechanism early in the symbiosis, and that the biochemical nature of some nod gene products may be cryptic except in cells grown in the presence of plant exudate.

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