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.

Wilson disease (WD) is an autosomal recessive disorder of copper transport which map to chromosome 13q14.3. In pursuit of the WD gene, we developed yeast artificial chromosome and cosmid contigs, and microsatellite markers which span the WD gene region. Linkage disequilibrium and haplotype analysis of 115 WD families confined the disease locus to a single marker interval. A candidate cDNA clone was mapped to this interval which, as shown in the accompanying paper, is very likely the WD gene. Our haplotype and mutation analyses predict that approximately half of all WD mutations will be rare in the American and Russian populations.

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IntroductionB-cell chronic lymphocytic leukemia (B-CLL) represents the most common leukemia in the Western countries with an estimated incidence of 1 per 100 000 per year. The disease is characterized by the monoclonal expansion of B lymphocytes expressing the CD5 marker and exhibiting a long life span, possibly because of a perturbed apoptotic program. 1 Current knowledge of the molecular pathogenesis of B-CLL is limited because no specific genetic alteration has yet been associated with this disease. In particular, B-CLL is not associated with reciprocal balanced chromosomal translocations. 2 Accordingly, none of the proto-oncogenes involved in chromosomal translocations in other mature B cell malignancies, including cyclin D1, 4 Although in a fraction of B-CLL cases, inactivation of the tumor suppressor gene p53 (on chromosome 17p13) and deletions or mutations of the ATM gene (on chromosome 11q22-23) have been reported, 5-9 such lesions were observed in late stages of the disease and may not represent primary tumorigenic events.Chromosome 13q14 deletions (approximately 50% of the patients) 2,10,11 are the commonest chromosomal alterations associated with B-CLL, followed by structural aberrations of chromosome 11q (19% of the cases) and trisomy of chromosome 12 (15%). 2 Moreover, chromosome 13 deletions can be the only cytogenetically detectable abnormality, suggesting an early role in B-CLL pathogenesis. Together, these findings point to the presence in this region of a tumor suppressor gene whose loss or inactivation may be crucial for the leukemogenesis.Deletions of chromosome 13q14 have also been reported in a variety of human tumors, including various other types of lymphoid tumors 12-17 and myeloid leukemias, 18 as well as prostate, 19,20 head and neck, 21 and non-small-cell lung cancers. 22 Although the deleted region of chromosome 13q14 has not yet been defined precisely in most of these neoplasms, these observations suggest that a common tumor suppressor gene may reside on this chromosomal segment.To identify the B-CLL-associated tumor suppressor gene, we previously constructed a high-density contiguous cosmid-based physical map encompassing the deleted interval. 10 This map allowed us to demonstrate that somatic loss at 13q14 occurs in 54% of the patients 10 (and unpublished results) and that the loss is monoallelic in 81% of such cases and biallelic in the remaining 19%. A minimal deleted region (MDR) in B-CLL, spanning less than 300 kilobase (kb) and representing the site of the B-CLLassociated putative tumor suppressor gene, was assigned on our For personal use only. on April 29, 2019. by guest www.bloodjournal.org From physical map in the interval between markers 173a12-82 and 138G4/1.3R. 10 This interval has also been confirmed by other studies. 13 In fact, the MDR exhibits a partial overlap with the regions of deletion as assigned in additional reports, [23][24][25][26] and it includes a locus of 10 kb that was previously defined as a minimal consensus for 13q14 deletion in B-CLL, based on the ...