A gene, ATM, that is mutated in the autosomal recessive disorder ataxia telangiectasia (AT) was identified by positional cloning on chromosome 11q22-23. AT is characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, cancer predisposition, radiation sensitivity, and cell cycle abnormalities. The disease is genetically heterogeneous, with four complementation groups that have been suspected to represent different genes. ATM, which has a transcript of 12 kilobases, was found to be mutated in AT patients from all complementation groups, indicating that it is probably the sole gene responsible for this disorder. A partial ATM complementary DNA clone of 5.9 kilobases encoded a putative protein that is similar to several yeast and mammalian phosphatidylinositol-3' kinases that are involved in mitogenic signal transduction, meiotic recombination, and cell cycle control. The discovery of ATM should enhance understanding of AT and related syndromes and may allow the identification of AT heterozygotes, who are at increased risk of cancer.

Velo-cardio-facial syndrome (VCFS) is a relatively common developmental disorder characterized by craniofacial anomalies and conotruncal heart defects. Many VCFS patients have hemizygous deletions for a part of 22q11, suggesting that haploinsufficiency in this region is responsible for its etiology. Because most cases of VCFS are sporadic, portions of 22q11 may be prone to rearrangement. To understand the molecular basis for chromosomal deletions, we defined the extent of the deletion, by genotyping 151 VCFS patients and performing haplotype analysis on 105, using 15 consecutive polymorphic markers in 22q11. We found that 83% had a deletion and >90% of these had a similar approximately 3 Mb deletion, suggesting that sequences flanking the common breakpoints are susceptible to rearrangement. We found no correlation between the presence or size of the deletion and the phenotype. To further define the chromosomal breakpoints among the VCFS patients, we developed somatic hybrid cell lines from a set of VCFS patients. An 11-kb resolution physical map of a 1,080-kb region that includes deletion breakpoints was constructed, incorporating genes and expressed sequence tags (ESTs) isolated by the hybridization selection method. The ordered markers were used to examine the two separated copies of chromosome 22 in the somatic hybrid cell lines. In some cases, we were able to map the chromosome breakpoints within a single cosmid. A 480-kb critical region for VCFS has been delineated, including the genes for GSCL, CTP, CLTD, HIRA, and TMVCF, as well as a number of novel ordered ESTs.

We have used a kinetic approach to construct cDNA libraries containing approximately equal representations of all sequences in a preparation of poly(A)+ RNA. Randomly primed cDNA fragments of a selected size range were cloned in A phage vector. Inserts were amplified by the polymerase chain reaction (PCR), denatured, and selfannealed under optimized conditions. After extensive but incomplete reannealing, the single-stranded fraction was relatively depleted of more abundant species of cDNA. Libraries of these fragments are suitable for cDNA subtraction, screening, or selection by hybridization and make it possible to detect and analyze cDNA corresponding to species of mRNA present at a low level in a small fraction of the cells in a complex tissue.

Identification of coding segments in large fragments of genomic DNA is a recurrent problem in genome mapping and positional cloning studies. We have developed a rapid and efficient protocol to achieve this goal, based on hybridization of cDNA fragments to immobilized DNA and recovery of the selected cDNAs by the PCR. Immobilization of Genomic Cosmid or YAC DNA on Nylon Discs. Cosmid DNA (0.2 ng) or YAC DNA (1-2 ng) was digested with EcoPJ, heat-denatured at 950C for 3 min along with 50 ng ofan Hae III digest ofbacteriophage 4X174 DNA, as carrier DNA, and loaded in a 0.5-,ul volume onto 6.25-mm2 nylon discs (Hybond, Amersham), in the presence of 1Ox SSC (lx SSC = 0.15 M NaCl/0.015 M sodium citrate, pH 7.0). The DNA on discs was denatured in 0.5 M NaOH containing 1.5 M NaCl for 5 min. The discs were neutralized with 0.5 M Tris Cl (pH 7.2) containing 1.5 M NaCl for 3 min, either exposed to UV irradiation in a Stratalinker (Stratagene) for 1 min (autolink mode) or baked at 80'C in vacuum for 2 hr, and stored dry at 40C until used. Quenchers of Nonspecific Hybridization to Nylon Discs. (i)Human chromosome 15 library. DNA from a human chromosome 15 genomic library (15 NSO3, ATCC 57740) was digested with EcoRI and 0.8-to 6-kb (size range) DNA was cloned into the pTZ18 vector (Pharmacia) at the EcoRI site. A total of 3 x 105 recombinants was generated after transformation and ampicillin selection.(ii) Genomic repetitive sequence library (XRLI). XRLI was prepared from the human X chromosome library (LAOXNLO1, ATTC 57750) by digestion of various DNA samples with several single restriction enzymes, pooling, cloning 0.3-to 2-kb (size fraction) DNA in Charon BS vector (22), and probing the resulting library with 32P-labeled total genomic DNA (23). Five hundred positive plaques were processed for isolation of phage DNA and subsequent plasmid preparation (22).(iii) rRNA-specific clones. Plasmid clones containing human rRNA gene EcoRI fragments of 7.3 kb and 5.8 kb cloned into pBR322 at the EcoRI site were kindly provided by D. Ward (Yale University) (24).(iv) Poly(dI)-poly(dC). Material of 724 base pairs (average length; Pharmacia) was used without any further treatment.(v) Yeast DNA. Yeast DNA from strain AB1380, which is the parent host of YAC B30H3, was prepared (20) and sonicated to an average size of 0.7-1 kb. Plasmid DNA from the sources i-iii was sonicated, digested with EcoRI, and treated with 1 unit of mung bean nuclease per pug of DNA at Abbreviations: MHC, major histocompatibility complex; PFGE, pulsed-field gel electrophoresis; YAC, yeast artificial chromosome.TTo whom reprint requests should be addressed. 9623The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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