ISOLATION OF A eDNA-DERIVED EXPRESSED-SEQUENCE-TAG CLONE ON CHROMOSOME 15

Complementary DNA clones isolated from human brain cDNA libraries have been partially DNA sequenced to generate expressed sequence tags (EST) (Adams et aI" 1992), ESTs have been essential for construction of transcript maps across large chromosome regions and for identification of genes responsible for inherited diseases and cancers, A procedure for rapid chromosomal assignment of EST has been developed (Polymeropoulos et at"~ 1992) , Pairs of oligonucleotide primers for each ESTs were used to amplify by PCR DNA template human-rodent somatic cell hybrid chromosomal panels, They were able to loca li ze 46 brain EST to chromosomes, One EST (ESTOO 165) was mapped to chromosome 15, the chromosome involved in Angelman syndrome (AS), a complex disorder characterized by severe mental retardation and puppet-like upperlimb movements, which is caused by deletions of proximal 15q (Knoll et aI., 1989), In our study, a genomic clone for this EST has been isolated and used to determine whether it maps to the AS chromosome region.


INTRODUCTION
Complementary DNA clones isolated from human brain cDNA libraries have been partially DNA sequenced to generate expressed sequence tags (EST) (Adams et aI" 1992), ESTs have been essential for con struction of transcript maps across large chromosome regions and for identification of genes responsible for inherited diseases and cancers, A procedure for rapid chromosomal as signment of EST has been developed (Polymeropoulos et at"~ 1992) , Pairs of oli gonucleotide primers for each ESTs were used to amplify by PCR DNA template human-rodent somatic cell hybrid chromosomal panels, They were abl e to loca li ze 46 brain EST to chromosomes, One EST (ESTOO 165) was mapped to chromosome 15, the chromosome involved in Angelman syndrome (AS), a complex disorder characterized by severe mental retardation and puppet-like upperlimb movements, which is cau sed by deletions of proximal 15q (Knoll et aI., 1989), In our study , a genomic clone for this EST has been isolated and used to determine whether it maps to the AS chromosome region.

Cloning of phage DNA
The preparation and purification of the phage clone, and the isolation of the phage DNA were done as previously described (Sambrook et al., 1989).The phage DNA was digested with with Bam Hl and Sail (New England Biolabs).The 5.3-kb fragment was confirmed to contain the EST sequence by PCR.It was ligated to a plasmid vector, pBluescript (Stratagene), and then transformed into competent Echerichia coli XLl-Blue cells (Stratagene).The transformants containing the recombinant DNA were identified by the blue/white plate assay, and then by the EST PCR.The radioactive labelling, suppression ofrepeated sequences and Southern hybridization were done as previously described (Driscoll and Migeon, 1990).The zoo blot was prepared as previously described (Monaco eta!., 1986).The Y AC clones (Kuwano et aI. , 1992) used were from the CEPH Y AC library: 273A2 (450 kb); St. Louis Y AC library: B230E3 (250 kb), A229A2 (250 kb) and A203G I (400 kb); and the ICI Y AC library: 6GD4 (165 kb) .The Y ACs were not chimeric, except A203G 1.

RESULTS AND DISCUSSION
The titre of a human genomic library in lambda EMBL3 was 1.5x I 0 6 .It was plated at 50,000 plaque forming units (pfu) per plate on thirty 150-mm plates.The phage lysate was prepared from each plate and was used in PCR amplification with the EST primers.From the 50 phage-lysate preparations screened only one produced an amplification of the appropriately-sized product of 105 bp (Figure I a).Others produced no PCR amplification .The titre of the positive lysate was 5x 1 0 9 pfu/ml.The lysate was subsequently plated at 10,000, then 1,000 and then 100 pfu/plate.Ateach stage of plating, w.s.KIM I:T AL.
appropriate numbers of plates were used to provide three-times coverage of the number of independent phages.At 100 pfu/plate, individual plaques were picked and tested separately.A positive plaque was identified.This was plated-out once more and its progeny plaques were tested.All were positive.
The DNA from the positive phage was prepared and digested with restriction endonucleases BamHI and SaIl.These enzymes released the human insert from the lambda-vector arms, as well as, internally digesting the insert (Figure I b).The digested DNA fragments were well-separated on an agarose gel and each DNA fragment was tested for amplification with the EST PCR.Only the 5.3-kb fragment gave the 105-bp PCR product.The same-sized PCR product was obtained from both cDNA and genomic DNA, therefore there may be no intron sequences between these two primer sites.The 5.3-kb fragment was cloned into pBluescript digested with BamHI and Sal! (Figure Ic).It hybridized to a Southern blot of human genomic DNA suppressed for repeated sequences.The probe also hybridized to cow and rat genomic DNAs on a zoo blot, indicating the presence of unique sequences and evolutionary conservation.The clone was tested to determine whether it maps to our area of interest, the Angelman syndrome chromosome region.However, it did not hybridize to Y AC clones, 273A2, B230E3, 6GD4, A229A2 and A203G 1, which cover this region.Nonetheless, the clone hybridized exclusively to a mouse-human somatic cell line containing human chromosome 15.Moreover, with subsequent chromosomal sublocation, the EST can serve as an anchor point on human genome map to facilitate the ordering of large genomic clones and also as an initiation point for continuous sequencing of large genomic fragments.

Fig
Fig ure I a. Th e EST peR amplification of a human ge nomic lambda library.Lane (I) amplification of the I05-bp produ ct from a positi ve clone; (2) pBR322-Mspl DNA size marker; (3.4) negati ve clones; (5) a negati ve control -water; and (6) a positi ve control -to tal genomic DNA.

Figure
Figure I b.The rc striction digestion of the EST phage clone.Lane (I) A-Hilldlll DNA si/c marker ; (2) the EST lambda clone dige sted w ith S(/II; (3) the EST lambda clone diges ted with H(/II1H I; and (4) the EST lambda clone diges ted with S(/II and BmllHI.