Familial primary pulmonary hypertension is a rare autosomal dominant disorder that has reduced penetrance and that has been mapped to a 3-cM region on chromosome 2q33 (locus PPH1). The phenotype is characterized by monoclonal plexiform lesions of proliferating endothelial cells in pulmonary arterioles. These lesions lead to elevated pulmonary-artery pressures, right-ventricular failure, and death. Although primary pulmonary hypertension is rare, cases secondary to known etiologies are more common and include those associated with the appetite-suppressant drugs, including phentermine-fenfluramine. We genotyped 35 multiplex families with the disorder, using 27 microsatellite markers; we constructed disease haplotypes; and we looked for evidence of haplotype sharing across families, using the program TRANSMIT. Suggestive evidence of sharing was observed with markers GGAA19e07 and D2S307, and three nearby candidate genes were examined by denaturing high-performance liquid chromatography on individuals from 19 families. One of these genes (BMPR2), which encodes bone morphogenetic protein receptor type II, was found to contain five mutations that predict premature termination of the protein product and two missense mutations. These mutations were not observed in 196 control chromosomes. These findings indicate that the bone morphogenetic protein-signaling pathway is defective in patients with primary pulmonary hypertension and may implicate the pathway in the nonfamilial forms of the disease.

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We present the genomic sequence of Legionella pneumophila, the bacterial agent of Legionnaires' disease, a potentially fatal pneumonia acquired from aerosolized contaminated fresh water. The genome includes a 45-kilobase pair element that can exist in chromosomal and episomal forms, selective expansions of important gene families, genes for unexpected metabolic pathways, and previously unknown candidate virulence determinants. We highlight the genes that may account for Legionella's ability to survive in protozoa, mammalian macrophages, and inhospitable environmental niches and that may define new therapeutic targets.

DNA fragments 536 base pairs long differing by single base-pair substitutions were clearly separated in denaturing gradient gel electrophoresis. Transversions as well as transitions were detected. The correspondence between the gradient gel measurements and the sequence-specific statistical mechanical theory of melting shows that mutations affecting final gradient penetration lie within the first cooperatively melting sequence. Fragments carrying substitutions in domains melting at a higher temperature reach final gel positions indistinguishable from wild type. The gradient data and the sites of substitution bracket the boundary between the first domain and its neighboring highermelting domain within eight base pairs or fewer, in agreement with the calculated boundary. The correspondence between the gradient displacement of the mutants and the calculated change in helix stability permits substantial inference as to the type of substitution. Excision of the lowest melting domain allows recognition *of mutants in the next ranking domain.Detection .and localization of single base substitutions within long DNA sequences may be impractical by complete sequence determination and improbable on the basis of restriction endonuclease vulnerability. We present here the results of a procedure by which DNA molecules that have minimal sequence differences are separated and by which some conclusions can be drawn as to the nature of the change. A number of samples can conveniently be examined in a single slab gel; each DNA species is focused into a sharp band-at a gel position determined by its sequence and composition. The physical separation of fragments of altered sequence provided by the denaturing gel makes possible further analysis and manipulation.Our system makes an unconventional use of electrophoresis. Where DNA molecules migrate into a gradient of ascending concentration of denaturant, they undergo an -abrupt decrease in mobility at a characteristic depth, resulting in positions and patterns that change. little if application of the field is continued. The retardation depth in the gradient is determined by the least stable part of the molecule and is relatively insensitive to other parts of the sequence or to the overall length (1).To understand the basis of the' sensitivity of the system to single base substitutions, we have undertaken a close comparison of the gel results with those of a sequence-specific statistical mechanical theory of the stability of the double helix. Experimental studies on the helix-disorder transition, melting,have not yet provided a detailed test of the theory, which pre-'dicts intricate and interesting patterns for the progression of equilibria from full helicity to separated strands as the temperature increases for molecules of different sequence. In our system, the molecule is exposed to a gradual denaturation-promoting change in the medium, linearly equivalent (2) to a gradual increase in temperature. 'The strong decrease in mobility as the helix unravels provides the basis for...

Duplex DNA fragments differing by single base substitutions can be separated by electrophoresis in denaturing gradient polyacrylamide gels, but only substitutions in a restricted part of the molecule lead to a separation (1). In an effort to circumvent this problem, we demonstrated that the melting properties and electrophoretic behavior of a 135 base pair DNA fragment containing a beta-globin promoter are changed by attaching a GC-rich sequence, called a 'GC-clamp' (2). We predicted that these changes should make it possible to resolve most, if not all, single base substitutions within fragments attached to the clamp. To test this possibility we examined the effect of several different single base substitutions on the electrophoretic behavior of the beta-globin promoter fragment in denaturing gradient gels. We find that the GC-clamp allows the separation of fragments containing substitutions throughout the promoter fragment. Many of these substitutions do not lead to a separation when the fragment is not attached to the clamp. Theoretical calculations and analysis of a large number of different mutations indicate that approximately 95% of all possible single base substitutions should be separable when attached to a GC-clamp.