GENOMICS 50, 34–43 (1998)ARTICLE NO. GE985270

The Human Homologue of the Drosophila tailless Gene (TLX):Characterization and Mapping to a Region of Common Deletion

in Human Lymphoid Leukemia on Chromosome 6q21

Amanda Jackson,* Panayiotis Panayiotidis,† and Letizia Foroni*,1

*Academic Department of Haematology, Royal Free Hospital Medical School, Pond Street, London, NW3 2QG, United Kingdom;and †Department of Medicine, Kapodistrian University of Athens, Agian Toma No. 17, Goudi, Athens, Greece

Received September 9, 1997; accepted February 18, 1998

some 13q14, for example, uncovers a recessive alleleDeletion of the long arm of chromosome 6 (6q) is one leading to retinoblastoma (Cavenee et al., 1983). Loss

of the most common chromosomal abnormalities in of the WT1 gene on chromosome 11p13 is involved inhuman hematological malignancies. Two distinct re- the pathogenesis of Wilms tumor (Call et al., 1990; Rosegions of minimal deletion have been identified by loss et al., 1990), and deletion of the DCC gene on 18q21of heterozygosity studies at 6q25 to 6q27 (RMD-1) and (Fearon et al., 1990) is implicated in colon carcinoma.at 6q21 to 6q23 (RMD-2), suggesting the presence of The p53 gene on chromosome 17p13 is mutated and/orone or more tumor suppressor genes. We have cloned lost in a variety of human malignancies, includingsequences within RMD-2 and screened for novel genes

breast cancer (Mackay et al., 1988), astrocytomasusing a combination of direct sequencing, cDNA li-(James et al., 1989), and colon cancer (Baker et al.,brary screening, and exon trapping. Sequences gener-1989). The possibility of similar mechanisms underly-ated from a cosmid fragment, mapping within RMD-2,ing hematological malignancies is suggested by the het-showed homology to the Drosophila tailless gene (tll).erozygous loss of material from chromosomes 6 (ThirdThe human homologue of the Drosophila tailless geneInternational Workshop on Chromosomes in Leukae-(human tlx; MGMW-approved symbol, TLX) was subse-mia, 1983), 13 (Brown et al., 1993), and 18 (Porfiri etquently cloned from a fetal brain cDNA library. The

gene is a member of the steroid nuclear receptor su- al., 1993).perfamily and is homologous to tll genes from other Cytogenetic studies have shown that deletions of thespecies that are involved in brain development. TLX long arm of chromosome 6 represent common chromo-is predominately expressed in the brain and maps to somal changes in non-Hodgkin lymphoma (NHL) (OffitRMD-2 at 6q21 between DNA markers FYN and et al., 1993) and acute lymphoblastic leukemia (ALL)D6S447, in a YAC clone that also contains marker (Third International Workshop on Chromosomes inD6S246. The contributions of this gene to human B- Leukemia, 1983; Hayashi et al., 1990). Several commoncell leukemia and to brain development are unknown regions of deletion have been reported on 6q includingat present. q 1998 Academic Press

(1) two regions of minimal deletion (RMDs) determinedby loss of heterozygosity (LOH) studies in NHL (Gai-dano et al., 1992). RMD-1 spans bands 6q25 to 6q27,

INTRODUCTION and RMD-2 is localized to 6q21–q23. These RMDs havebeen correlated with three regions of minimal cytoge-

The occurrence of specific, nonrandom chromosomal netic deletions (RCDs) identified by karyotypic analysisdeletions has long been demonstrated in the pathogen- of NHL patients: RCD1 encompasses 6q25–q27, RDC2esis of human malignancies. Deletion of genetic infor- at 6q21, and RCD3 at 6q23 (Offit et al., 1993). (2) Fluo-mation suggests the presence of one or more tumor rescence in situ hybridization (FISH) analysis with asuppressor genes, the loss of which may be of pathoge- series of yeast artificial chromosome (YAC) clones map-netic importance. A number of tumor suppressor genes ping to 6q defined a region of deletion in ALL at 6q14–have been identified through the study of tumor-associ- q21 (Menasce et al., 1993). (3) The combination of chro-ated deletions. Deletion of the RB1 gene on chromo- mosome microdissection and FISH (MICROFISH)

(Meltzer et al., 1992) has determined a RMD at 6q11–Sequence data from this article have been deposited with the q21 in B-cell lymphoma (Guan et al., 1996). Final proofEMBL/GenBank Data Libraries under Accession No. Y13276.

of tumor suppressor activity of a candidate gene on1 To whom correspondence should be addressed. Telephone: 0171794 0500, x4472. Fax: 0171 830 2092. E-mail: letizia@rfhsm.ac.uk. chromosome 6 has been provided by Sandhu and col-

340888-7543/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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HUMAN HOMOLOGUE OF Drosophila tailless GENE (TLX) 35

obtain fragments between 35 and 45 kb. The DNA was ligated withleagues (Sandhu et al., 1994). They have shown thatthe arms of the cosmid vector Lorist 6 and transfected into the bacte-introduction of a normal human chromosome 6 or 6qrial strain XL-1 blue (Stratagene) using an in vitro packaging extractcan suppress the immortal phenotype of simian virus (Stratagene). A total of 2000 colonies were spread, replicated, and

40-transformed human fibroblasts and proposed that screened as described (Sambrook et al., 1989).a gene for cellular senescence (SEN6) in human fibro- Restriction mapping of cosmid clones. Cosmids containing human

DNA were identified by screening the cosmid library filters againstblasts is present on 6q. Similar results were also ob-total human DNA, resulting in a cosmid library containing 45 cos-tained by Gualandi and colleagues (Gualandi et al.,mids. Positive clones were picked and grown overnight in LB medium1994). They discovered that introduction of normal hu-containing 30 mg/ml kanamycin. Cosmid DNA was prepared using

man chromosome 6 into human papovavirus trans- an alkaline lysis protocol (Sambrook et al., 1989). Restriction enzymeformed mouse cells reverts the immortal phenotype, digestion was carried out according to the manufacturer’s recommen-

dations.with the exception of several clones showing deletionscDNA isolation. A human fetal brain bacteriophage cDNA libraryinvolving the 6q21 cytogenetic band.

was used for screening (a kind gift from Dr. Michael Goedert, MRC,Notably, many of these RMDs encompass the 6q21Cambridge, UK; Goedert et al., 1989). Approximately 106 plaquesband, suggesting that this might be the locus of a tumor were screened each time. The initial screening was performed by

suppressor gene, the absence of which contributes to hybridization to a 317-bp probe generated by RT-PCR on a mouseneoplasia. A recent FISH study has narrowed the locus brain cDNA sample using two primers (MNR1: 5* GGATCAACA-

AGCCGCATTTTAGATC 3* and MNR2 5* TTGTGTCCACGGAAG-to a 2-Mb interval between markers D6S447 andTAGAGAGCCACC 3*) designed from the mouse Tlx sequence (bpD6S246 on band q21 (Sherratt et al., 1997).780–804 and bp 970–996, respectively). Subsequent screenings wereIn this study we have identified and characterized performed using PCR products generated by exon trapping (Nehls

three YAC clones mapping to the deleted area. YAC et al., 1994). The bacteriophage inserts were subcloned from lgt10into the plasmid vector Bluescript SK/. Nucleotide sequences of the12BB4 contains the marker ROS and maps to the te-positive clones were determined as described below.lomeric part of 6q21 (data not shown). YAC 28CD7

Exon trapping. Exon trapping was carried out on cosmid insertscontains the marker D6S246 (Menasce et al., 1994),using the l GET vector (for genomic exon trapping; Nehls et al.,and YAC 17DA11 contains the marker FYN (Popescu1994). Briefly, cosmid DNA fragments of up to 19 kb were subclonedet al., 1987). Preliminary analysis using YAC 28CD7 into the l GET vector. l GET recombinants were converted to plas-

showed its deletion in all cases of ALL and NHL (Men- mids by infecting the cre-recombinase expressing Escherichia colistrain BNN132. Purified plasmids were transfected into COS-7 cells,asce et al., 1993). Through pulse-field gel analysis andand trapped exons were isolated by RNA-based PCR. The resultingcosmid library characterization of clusters of CpG sites,exon fragments were cloned into the Bluescript SK/ plasmid forspecific probes were generated. Combined sequencesubsequent analysis.

analysis and cDNA library screening using such probesDNA sequencing and sequence analysis. cDNAs and cosmid in-

led to the identification of the human homologue of serts were gel-purified and shotgun-sequenced in M13mp18 by thethe Drosophila steroid nuclear receptor tailless gene dideoxynucleotide method (Bankier et al., 1987). Sequences were

assembled into contigs using Lasergene software (DNASTAR).(Pignoni et al., 1990) on 6q21, defining a new memberSearches for homologous sequences were carried out using theof the nuclear receptor superfamily. In Drosophila, theBLAST analysis program (BLASTX, BLASTN, BLASTP) using thetailless gene is involved in the differentiation of the BLAST remote server at the National Center for Biotechnology

brain, foregut, midgut, and hindgut. The human tlx Information.gene2 is predominately expressed in the brain and Expression analysis. Northern blot filters containing 2 mg ofmaps to RMD-2, an area of minimal deletion in ALL poly(A)/ RNA from a variety of different human tissues were used

(Clontech). TLX cDNA probes were hybridized in ExpressHyb hy-and NHL.bridization solution (Clontech) for 2 h at 657C. The final washingstringency was 0.11 SSC, 0.1% SDS at 507C, and filters were exposed

MATERIALS AND METHODS for between 2 and 21 days.

Nucleic acid techniques. DNA probes were purified as described RESULTSby He and associates (He et al., 1992). They were labeled using arandom priming kit (Amersham). Hybridization to filters was carried

Physical Mapping of YAC 28CD7out overnight in hybridization buffer: 0.5 M sodium phosphate buffer,pH 7.2, 1 mM EDTA, 1% BSA, and 7% SDS. Filters were usually

Preliminary PFGE analysis indicated the presencewashed to 0.51 SSC, 0.1% SDS at 657C except when using the mu-of a cluster of BssHII and MluI sites on YAC 28CD7.rine-derived RT-PCR probe, P317, when the filters were washed to

a stringency of 31 SSC, 0.1% SDS at 657C. The filters were exposed Such restriction enzymes may have ú75% of their rec-to Kodak XAR X-ray film at 0807C for between 12 h and 21 days. ognition sequences within CpG islands (Craig and

Pulsed-field gel electrophoresis (PFGE) analysis. YAC DNA was Bickmore, 1994). Our results were indicative of the ex-prepared as described previously (Sambrook et al., 1989). The DNA istence of at least one cluster of CpG sites on 28CD7.was separated in an LKB 2015 apparatus under the following condi-

For more detailed physical mapping and sequencing,tions: 120 V, 60-s pulse for 24 h, 90-s pulse for 48 h at 147C.a YAC-specific cosmid library was constructed fromSubcloning of the YAC 28CD7 into cosmids. The total YAC 28CD728CD7 (see Materials and Methods). Cosmid LO9 con-DNA was partially digested with the restriction enzyme HindIII totained three BssHII sites and one MluI site and wasfurther characterized. Partial DNA sequences gener-2 The HGMW-approved symbol for the gene described in this paper

is TLX. ated from BssHII- and MluI-containing fragments were

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JACKSON, PANAYIOTIDIS, AND FORONI36

FIG. 1. Transcript map showing probes used for fetal brain cDNA library screening and position of products generated by lGET exontrapping relative to the P317-2 and Q9 cDNAs. The complete TLX cDNA sequence was obtained from two overlapping bacteriophage clones:P317-2 and Q9. P317 corresponds to a 317-bp mouse RT-PCR probe (see Materials and Methods). All probes with the prefix LO9 correspondto PCR products generated by exon trapping.

used to search the GenBank database using the BLAST the gene, exon LO9/18 was used as a probe to screen thefetal brain cDNA library, resulting in the identificationsearch program (Altschul et al., 1990, 1994). This

search demonstrated strong DNA and protein sequence of cDNA clone Q9. Q9 showed homology between aminoacids 1 and 200 when compared to the mouse and chicksimilarity between a 98-bp stretch of sequence and the

chick and mouse homologues of the Drosophila gene Tlx proteins and contains the proposed initiator methio-nine (Fig. 1). In addition, the complete nucleotide se-tailless (Pignoni et al., 1990; Yu et al., 1994; Monaghan

et al., 1995). As the chick and mouse nuclear receptor quence of TLX, contained in overlapping clones P317-2and Q9, was identical to three human expressed se-genes are specifically expressed in embryonic brain, a

human fetal brain cDNA library in the lgt10 bacterio- quence tags (ESTs) (GenBank Accession Nos. R18964,D80939, and W96098).phage vector was screened to isolate the human tailless

homologue.Sequence Analysis of the TLX cDNA

cDNA IsolationThe cDNA sequence of the human TLX gene that we

isolated consists of 1593 bp and defines an open read-To identify the human TLX cDNA, we screened a hu-man fetal brain cDNA library with a mouse RT-PCR ing frame of 1158 bp, encoding 386 amino acids (Fig.

2). The AUG nucleotide at position 169 is likely toprobe, P317 (see Materials and Methods), resulting inthe isolation of clone P317-2. The P317-2 cDNA clone be the initiator methionine, which is located 168 bp

downstream of an in-frame termination codon. The se-showed homology between amino acids 38 and 386 whencompared to the mouse and chick Tlx protein sequences quence surrounding the potential initiation site shows

good homology to the Kozak consensus sequence (Ko-and included the termination codon. Further screeningwith probe P317-2 failed to identify any new positive zak, 1987) with the critical purine residue at position

03. The cDNA contains a short 3 * untranslated regionclones. Therefore, a combination of exon trapping andfetal brain cDNA library screening was performed to of 266 bp but does not represent a full-length tran-

script as neither a polyadenylation site nor a poly(A)/obtain the full-length cDNA sequence (Fig. 1). Exon trap-ping and PCR amplification (see Materials and Methods) tail is present within the cDNA clones thus far identi-

fied. The open reading frame predicts a basic proteinwere carried out on cosmid LO9 using lGET as the clon-ing vector (Nehls et al., 1994). Five putative exons were sequence (pI Å 8.95) with a calculated molecular mass

of 42.6 kDa.isolated: LO9/18, LO9/24, LO9/4, LO9/6, and LO9/11. Se-quence analysis allowed the positioning of these exons The cDNA and predicted protein sequences were

used to search the GenBank, SwissProt, and PIR data-relative to the P317-2 and Q9 cDNAs (Fig. 1). Subse-quent analysis demonstrated that LO9/18 corresponded bases using the BLAST homology search programs

(Altschul et al., 1994). Comparison of the deducedto TLX amino acids 10–37 and LO9/24 to amino acids38–67, LO9/4 and LO9/6 were identical and corre- amino acid sequences of the mouse, chick, and human

TLX genes showed remarkable interspecies conserva-sponded to TLX amino acids 228–312, and LO9/11 corre-sponded to amino acids 228–277. Exon LO9/18 extended tion with human and mouse sharing 99% overall simi-

larity and human and chick sharing 97% (Fig. 3A). Thethe already available sequence from clone P317-2 by 147bp. To isolate a full-length cDNA encoding the 5* end of human, mouse, and chick TLX genes are homologues

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HUMAN HOMOLOGUE OF Drosophila tailless GENE (TLX) 37

FIG. 2. Nucleotide and predicted amino acid sequence of the human TLX cDNA. The proposed initiation codon and termination siteare underlined. BLASTN searches showed that portions of the full-length cDNA sequence are identical to the following three ESTs (searchof April 28, 1997): R18964, D80939, and W96098.

of the Drosophila gene tailless, which is required for (DBD) and the ligand-binding domain (LBD), whichshow varying degrees of similarity. The highest degreepattern formation at the anterior and posterior poles

of the Drosophila embryo and is involved in the devel- of conservation is observed in the DBD, consisting of a66-to 68-amino-acid domain with invariantly spacedopment of the nervous system. Tailless encodes a mem-

ber of the steroid nuclear receptor gene family and cysteine residues that are involved in the formation ofDNA-binding zinc fingers. The homology between theprobably functions as a transcription factor (Pignoni et

al., 1990). DBD of TLX and the DBD of tailless is 81% over 86amino acids. The similarity to other nuclear receptorsComparison of the predicted TLX protein with the

tailless protein showed that they share 60% overall ranges from 100% with mouse and chick Tlx (Mo-naghan et al., 1995; Yu et al., 1994), 99% with Xenopussimilarity. All members of this superfamily are com-

posed of two main domains, the DNA-binding domain Tlx (Hollemann et al., 1997), 60% with human avian

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JACKSON, PANAYIOTIDIS, AND FORONI38

FIG. 3. (A) Comparison of chick, human, and mouse TLX amino acid sequences using the MEGALIGN sequence analysis program.Conserved sequences are indicated by gray boxes and single amino acid differences by white boxes. One amino acid, at position 149, differsamong all three species and is indicated by a black box. Humans and mice share 98.7% overall similarity; humans and chicks share 96.7%overall similarity. (B) Comparison of the putative htlx DBD and LBD (C) with the mouse (mtll), chick (ctlx), and Xenopus (xtll) Tlx proteins;the Drosophila tailless protein (dtll), human chicken ovalbumin upstream promoter (COUP) protein, the Drosophila seven-up protein (dsvp),and the human retinoic acid receptor a (RXRa) protein showing amino acid differences. Identities are underlined, and dots indicate gaps.The percentage identity is indicated after the aligned sequences. The P, D, T, and A boxes are outlined in boldface type.

erythroblastosis virus-related gene 3/chicken oval- 54% with human retinoic acid receptor a (RXRa)(Mangelsdorf et al., 1990) (Fig. 3B).bumin upstream promoter (EAR3/COUP) (Miyajima et

al., 1988; Ritchie et al., 1990), 58% with the Drosophila Two discrete regions within the DBD are critical fortarget specificity, the P box and the D box (Schwabe etseven-up protein (dsvp) (Mlodzik et al., 1990), and to

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FIG. 3—Continued

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JACKSON, PANAYIOTIDIS, AND FORONI40

FIG. 4. Northern blot analysis of the TLX mRNA transcript. A 1256-bp partial TLX cDNA (cDNA clone P317-2) was radiolabeled andhybridized to total human (A) and brain (B) multiple tissue Northern blots (Clonetech). A 3.9-kb band (arrow) was visualized in those lanescontaining brain tissue.

al., 1993). In these positions the fly tll protein differs tissue. This transcript was present in all brain sectionstested, being most strongly detected in the caudate nu-from all other nuclear receptors. A conserved lysine

residue is replaced by an alanine in the P box cleus and hippocampus. The weakest expression wasobserved in the thalamus.(DGCAG), and while the D box contains five amino

acids in all other receptors, there are seven in the tll Since ALL and NHL are diseases of the lymphoidsystem, we were interested in investigating expressionprotein (Pignoni et al., 1990). Both the altered lysine

and the expanded D box are also found in the mouse, of human TLX in such cells and tissues. A multitissueimmune system Northern blot (Clontech) was probedchick, and human TLX proteins (Fig. 3B) (Monaghan

et al., 1995; Yu et al., 1994). Two domains separate with a partial human TLX cDNA. No expression wasdetected, after a 3-week exposure, in spleen, lymphfrom the P and D boxes, the T and A boxes, which are

thought to be involved in dimerization and sequence node, thymus, appendix, peripheral blood leukocytes,bone marrow, or fetal liver (data not shown).recognition, respectively, are also conserved (Wilson et

al., 1992).Genomic Organization of the TLX GeneIn the LBD, human TLX shows 100% similarity to

chick tlx, 99.6% to murine tll, 95% to Xenopus tll, and The intron/exon boundaries of exons 1–9 of the TLX41% to tll (Fig. 3C). The homology to other nuclear gene were determined by sequence comparisons ofreceptors ranges from 41% with EAR3/COUP, to 39% cDNA and genomic clones obtained from cosmid LO9with dsvp, and to 36% with RXRa. All 12 of the amino (Table 1). The sequences of all splice sites conformedacid changes between human and chick TLX genes and to the GT/AG rule (Breathnach and Chambon, 1981).4 of 5 changes between mouse and human TLX genes All nine TLX exons are contained within cosmid LO9are located in the hinge region between the DBD and and span 24 kb of genomic distance.the LBD.

DISCUSSIONExpression Analysis

The chick nuclear receptor TLX is expressed exclu- We have described here a novel human gene, desig-nated human TLX, that is the human homologue ofsively in the neuroepithelium of the embryonic brain

(Yu et al., 1994) and mouse tll is specifically localized the Drosophila gene tailless and belongs to the steroidnuclear receptor superfamily of genes. A combinationto the developing forebrain and dorsal midbrain. Mu-

rine tll is also present in adult brain (Monaghan et of direct sequencing and cDNA library screening led tothe isolation of a partial cDNA that corresponded toal., 1995). Expression of human TLX was analyzed by

probing human multitissue Northern blots (Clontech) the chick, mouse, and Drosophila nuclear receptorgenes. The entire coding sequence of 1593 bp was iso-with a partial human TLX cDNA (Figs. 4A and 4B). A

single transcript of Ç3.9 kb was detected only in brain lated from a fetal brain cDNA library using probes gen-

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HUMAN HOMOLOGUE OF Drosophila tailless GENE (TLX) 41

erated by exon trapping. Like the mouse and chick Also, the pattern of TLX expression in chick embryosgenes, the human gene exhibits 60% overall similarity is similar to that observed in the mouse. It has beento tailless, which is required for the differentiation of suggested that chick TLX and mouse TLX are involvedthe brain, foregut, midgut, and hindgut in flies (Pignoni in transcriptional control in undifferentiated neuroepi-et al., 1990). The chick and Drosophila genes are exclu- thelial cells in the developing vertebrate brain (Mo-sively expressed in the embryonic brain. However, the naghan et al., 1995). It is possible, based on the strikingmouse gene can also be found in adult brain, where the homology between TLX and both these genes, that hu-human gene has been detected so far in this study. man TLX may share a similar role. The human tailless

The DBDs of the chick and mouse proteins show high gene may be required for brain development. However,sequence conservation to the Drosophila protein (81%), functional studies will be required to understand thesuggesting that they might bind to similar DNA se- biological role of human TLX. Recently, such functionalquences. This was confirmed by in vitro binding assays studies were carried out in the mouse (Monaghan et al.,that demonstrated that the Drosophila, chick, and 1997). Using homologous recombination, the Tlx locusmouse proteins can bind to the same, specific target was disrupted, resulting in defective development of theDNA sequences (Yu et al., 1994). rhinencephalic and limbic structures and behavioral

It is usual for vertebrate and Drosophila nuclear hor- changes in the mutant animals.mone receptors to show high sequence homology in the Evidence from various cytogenetic studies suggestsLBDs. For example, the human avian erythroblastosis that deletions of the long arm of chromosome 6 repre-virus-related gene 3/chicken ovalbumin upstream pro- sent recurrent lesions in various lymphoid malignan-moter (EAR3/COUP) shows 92% homology to the Dro- cies, specifically in ALL and NHL. Several commonlysophila seven-up protein, suggesting that these recep- deleted bands along 6q have been reported, notably thetors may bind a similar ligand. However, there is lim- 6q21 band. A recent report has further narrowed theited similarity (41%) between the human, mouse, and deleted region to 2 Mb between the markers D6S447chick proteins and tailless. This suggests that there and D6S246 on 6q21 (Sherratt et al., 1997). This sug-may be a common ligand for the human, chick, and gests the presence of a tumor suppressor gene, the ab-mouse, different from that bound by the fly, or that all sence of which is relevant for malignant transformation.four receptors are ligand independent. Alternatively, We have isolated a novel gene, human TLX, mappingthe human, chick, and mouse genes may have gained to YAC 28CD7. This YAC contains the marker D6S246or lost the ability to bind ligands since the divergence and lies within the RMD implicated in ALL and NHLof insects and vertebrates. on chromosome 6q21 (Menasce et al., 1993, 1994). OneRecently, it has been demonstrated that overex- other gene has been localized to this region: the cellpression of the chick tailless homologue in Drosophila surface antigen CD24 (Hough et al., 1994). CD24 ismimics the action of tailless in vivo. It is likely, based

thought to be involved in signal transduction duringon the high sequence conservation between chick TLXthe differentiation and activation of granulocytes andand murine TLX (97%), that the mouse gene can alsomature B-lymphocytes (Hough et al., 1994; Stefanovasubstitute for the function of tailless in Drosophila.et al., 1991). It is possible, based on its location and

TABLE 1 function in B-cell differentiation, that CD24 is involvedin the pathogenesis of ALL and NHL. To elucidate thisIntron/Exon Boundaries of Human tlxfurther, it will be necessary to search for genetic alter-

Size 5* Splice 3 * Splice ations of CD24 in patients with 6q deletions.Exon (bp) junction junction At present it is not possible to comment further on

the function of the human tailless gene. It is likely,1 26 gccagcrATGAGC AACAAGrgtggtabut not compulsory, that the tumor suppressor gene2 145 tttcagrCCGCAT AACCAGrgtacctinvolved in ALL and/or NHL would be expressed in3 88 ttccagrGGAGGC AAGACGrgtaatc

4 236 ctgcagrCCGTGC CCCAAGrgtcagc cells or tissues of the immune system, whereas human5 147 acccagrTACCCC GACCAGrgtatga TLX is expressed only in the brain. However, deletion6 97 tgacagrCTGATG TATCTGrgtaaga

of 6q shows little lineage restriction as it has been de-7 150 ttctagrGCATGA AAGCCGrgtaagctected in myeloid, lymphoid B-cell and T-cell prolifera-8 106 ttctagrTTCCTA TACCAGrgtgacc

9 ú163 ttccagrATATCC ATCTAArgctcac tions. Therefore it is possible that an early, stage-re-stricted expression of the gene occurs in the hematopoi-

Note. For exon 1, 26 bp is the length of the protein coding portion. etic system. Consequently, it is possible to postulateIn addition, exon 1 contains 168 bp of 5* UTR sequences. For exonthat the deletion of human TLX occurring at an early9, 163 bp is the length of the protein coding sequence plus the transla-

tion stop codon. The exon contains additional 3 * UTR sequences but stage of myeloid, B-cell, or T-cell differentiation mightthe complete 3 * UTR has not yet been sequenced. Capital letters have a selective effect on cell growth restricted to oneindicate exon sequences; dots indicate exon/intron boundaries; lower- of the hematopoietic lineages. To determine whethercase letters indicate intron sequences. Letters in boldface type indi-

the human TLX gene is involved in the pathogenesiscate the translation initiation codon in exon 1 and the translationstop codon in exon 9. of lymphoid malignancies (ALL and/or NHL), we have

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JACKSON, PANAYIOTIDIS, AND FORONI42

tion of a recurrent breakpoint region on chromosome 6 in humanbegun to examine all the candidate genes for mutationsB-cell lymphoma. Blood 88: 1418–1422.in patients carrying 6q deletions.

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ACKNOWLEDGMENTS malities of the long arm of chromosome 6 in childhood acute lymph-oblastic leukeamia. Blood 76: 1626–1630.

We thank Marlene Attard for sequencing, Maria Papaioannou for He, M., Liu, H., Wang, Y., and Austen, B. (1992). Optimised centrifu-YAC probes, and Dylan Jones for exon trapping. We also thank Tim gation for rapid elution of DNA agarose gels. Gene Anal. Tech. 9:Sherratt and Christine Harrison for sharing FISH data before publi- 31–33.cation. This work was supported by the Kay Kendall Leukemia Fund. Hollemann, T., Bellefroid, E., and Pieler, T. (1998). Xtll—The Xeno-The sequence of htlx has been deposited with the EMBL/GenBank pus homologue of the Drosophila tailless gene has a function inData Libraries under Accession No. Y13276. early eye development. Dev. Biochem., in press.

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