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1746 Humań Molecular Genetics. 1999. Vol. S. No. 9

Figurę 4. Localization of the STAT55 gene in relation to the RARA loctii b> FISH on normai prometaphases. PAC I96pl 7 was biotin labeled and co-hybridized with digoxigenin-iabeled Smith-Magenis/D17S25S tl”pi 1.2i and RARA (17q21.1) probes (Oncor) on norma) prometaphases and nuclet. On proir.etaphase ehromosome 17. the STAT5b green signal. obsened alter FnrC-avidin detection. co-localizcs with the RARA red signal visualized after rhodammc-anti-dieoxigenin detection. as demonstrated by yellow sienahs on 17q21.1—q21No background was noted at any other ehromosome location using the PAC clone 196pI7 as a probe lor FISH analysis.

Tobie 1. OiigomicleocKics used in this study

Name	Location	Sequencc p —3')
RARA R2	E.\on 5	GCACCTCCTTCTTCTTCTTG
R3	Exon 3	CAGCCCTCACAGGCGCTGAC
R4	Exon 3	CTGGGC ACTATCTCTT CAG A ACT
STATSb Stgn	Exon n	TGCTTGGAAGTTTGArrCTC
StgU	E.\on n - 1	GTTTGACGGTGTGATGGAAGTG
StgL2	Exon n+ 1	aagtctctggtggtaaaagg
Sićx5’	First e.\on	ctcagcagctccaaggagaagc
Sil65U	Last exon	AGCTTCTTCATC TTCACCA
Sil-8	Intron //	CCAGCACTTTGGGAGGCC
St 165L	3-UTR	AAACACATACTCGCACTCG

MATERIALS AND METHODS Patient

DetaiJs of tlie patient. a 67-year-old man. have been previously pub-lished (5). He had acute myeloid leukaemia (AML Ml in the French/American/British classificationj. However. in the bonę marrow a minority of blast cells showed morphological features sugges-tive of ihe microgranular variant of APL. M3v. Blas: cells faiJed to respond tc ATRA in ritro. Cytogenetic analysis of bonę marrow cells revealed a derivative ehromosome i 7 larger :han a norma] ehromosome 17. Chromosome microdissection (5) and CGH analysis allowed us to demonstrate that extra materiał on den 171 was the result of a duplication of ihe 17q21.3-q23 region (data not shown).

5’-RACE PCR

Total RN As from patient bonę marrow cells were exiractcd according to the TRIzoi protocol (Sigma;. Amplification of the unknown 5'-end of the chimeric mRNA was perfonned with a 573'-RACE kit (Boehringer Mannheim). Tlić ineubation step for first strand cDNA synthesis was performed at 55^CC. Gene-specific first strand cDNA symhesis was pcrfomied using primer R2 (for oligonucieotides used in this study see Tablc I) localized in exon 4 of the RARA gene (accession no. X066I4). After dA tailing oi' the punfied chimeric cDNA. a first PCR with an oligo(dT) anchor primer .ind R3 followed by a second semi-nesied PCR using an anchor primer and R4 allowed us loobtain a 1.2 kb PCR product. Boih PCRs were car-ncd out willi the Expand Long Tcmplatc system (Boehringer Mannheim) at an anncaiing temperaturę of 63°C. Secondary PCR products were cioned using the PCR-Scnpt A.MP SK<+jcloning kit (Siratagenej. After transformation of Epicurian Coli Xl.l-Bluc MRF Kan supercompeient ce!K. plasmid DNA was cxtracietl from while colomes with the Qiagen Plasmid Minikit and suhsequent]y sequenccd using primer. T3 and T7

Construction of the patient genomie library

A phage library was constructed from patiem genomie DNA extracted from bonę marrow leukaemic cells by inserting partialiy .V//wJ-dige$ted DNA into the Xho\ site of the a Fi.\Il vector (Stratagene) and the library' was packaged with Gigapack II Plus Packaging extracts according to the manufacturers* protocols. The total phage library (10⁶ phages) was screened with a 3.5 kb £a>RI genomie probe used to confirm an RARA gene rearrangement in the patient (1). A total of five bacteriophages were isolated after three rounds of screening. Bacteriophage DNAs were prepured with the Qiagen Lambda Maxi kit. Hybridization of the RARA 5.5 kb £o>RI genomie probe on £o)RI-dige$ted phage DNA identified two phages (Ha and Da) spanning the junction fragment. Hvbridization of chemiluminescence-labeled R4 and Sten oliso-

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probes confirmed the presence of exonic sequences derived from botłi STAT5b and RARA in the 6.5 kb junction fragment.

Sequencing of the junction fragment was performed on the R4/ Stgn PCR product of DNA from phage ciones Ea and Da. PCR products were analysed by electrophoresis on a 0.8^ agarose gel. The 2.4 kb band was punfied with a Gel Extraction kit (Qiagem. Initially. sequencing was performed with R4 and Stgn: subsc-quently nested primers were designed to reach the joining site.

$equencing reaction

Purified PCR products (30-100 ng) or cioned DNA (800 ng) were Cycle sequenced using dve terminator chemistry with the AmpiiTaq FS enzyme (Applied Biosystems) and were run on an ABI 373 DNA sequencer.

Isolation of a genomie clone containing the STAT5b gene

PAC 196p 17 containing the entirc coding region of the STAT5H ecne was obiained al ter PCR screening of the RPCI1 PAC librurv constructed by loannou et ul. (60). Primary and secondary pools as well as individual microliire plates were purchased Irom the UK HGMP Rcsourcc Centro (Cambridge. UK). Initial PCR screening was performed with primers Si 165U and Si 1651. Banking the STAT5b stop codon. DNA Irom PAC I96pl7 was pre-pared with the Qiagcn Maxi-prep kit. The presence ofthc 5 -eml ot

STAT5H was shown bv Southern biot analvsis with ihe Stex5

• *

oligoprobc. Labeiling by terminal transferase with digo\igenin-