The Murine Tcl1 Oncogene: Embryonic and Lymphoid Cell Expression

The murine Tcl1 oncogene: embryonic and lymphoid cell expression

Maria Grazia Narducci1, Laura Virgilio2, Julie B Engiles2, Arthur M Buchberg2, Linda Billips3, Antonio Facchiano1, Carlo M Croce2, Giandomenico Russo1 and Jay L Rothstein2

1Istituto Dermopatico dell' Immacolata-IRCCS, Via dei Monti di Creta 104, 00167 Rome; 2Departments of Microbiology /Immunology and Otolaryngology-Head and Neck Surgery, Kimmel Cancer Institute, Thomas Jeerson Medical College, 233 S. 10th Street, BLSB 909, Philadelphia, Pennsylvania 19107 USA; 3Howard Hughes Medical Institute, University of Alabama, Birmingham, Alabama 35294, USA

In human leukemias and lymphomas nonrandom chro- Rowley et al., 1990; Croce, 1991; Cimino et al., 1991; mosomal rearrangements cause changes in cell growth Rowley, 1992). Analysis of the chromosomal altera- and/or survival in such a way as to promote malignancy. tions from tumors revealed that breakpoints were The detailed study of the biochemical and genetic clustered at a de®ned loci suggesting that proto- pathways altered in human cancer requires the identi®ca- oncogene(s) resided at or near these positions (Croce, tion or development of models to allow the study and 1991). Speci®c chromosomal rearrangements involving manipulation of cancer gene function. Recently, the human chromosome 14q32 have been found in T- breakpoint gene TCL1, involved in chromosome translo- prolymphocytic leukemia (T-PLL) in 28% of adult T cations observed mostly in mature T-cell proliferations cell leukemias (ATL) which are associated with and chronic lymphocytic leukemias (CLL), was isolated infection by human T-lymphotropic virus type I and characterized, and showed to be part of a new gene (HTLV-I) or chronic T cell leukemias in patients with family of proteins involved in these tumors. The murine ataxia telangiectasia (AT; Taylor, 1992; Savitsky et al., Tcl1 gene, is similar in sequence to the murine and 1995). The most common rearrangement is an human MTCP1 gene also involved in T cell leukemias. inversion involving q11 and q32 or reciprocal The murine Tcl1 gene was shown to reside on mouse translocation between chromosomes 14q11 and 14q32 chromosome 12 in a region syntenic to human chromo- (Brito-Babapulle and Catovsky, 1991). some 14. Furthermore, we show that the murine Tcl1 The breakpoint region of these chromosomal altera- gene is expressed early in mouse embryonic development tions was cloned and a gene found adjacent to these and demonstrates expression in fetal hematopoietic altered regions was subsequently isolated, sequenced and organs as well as in immature T and B cells. named TCL1 (Virgilio et al., 1994). Interestingly, the Characterization of the murine Tcl1 gene will help in only other gene with signi®cant amino acid or nucleotide developing a mouse model of CLL and would provide the sequence identity with TCL1 is a gene named mature T best opportunity to study and decipher the role of TCL1 cell proliferation-1 (MTCP1; Stern et al., 1993) which in malignant transformation. showed 61% similarity at the amino acid level. MTCP1 is a recently cloned chromosomal translocation Keywords: ATM; ataxia-telangiectasia; TCL1; chronic t(X;14)(q28;q11) breakpoint gene involved in a case of lymphocytic leukemia; MTCP1; T-PLL benign clonal proliferation in an AT patient (Stern et al., 1993). The TCL1 gene was shown to be expressed in T cell acute leukemias and high grade lymphomas carrying rearrangements at 14q32.1 (Virgilio et al., 1993, 1994). Introduction The gene is also expressed in pre B cells, in B cells expressing surface IgM, and in the related B cell tumors The molecular basis of cancer is steadily being (Virgilio et al., 1994). Analysis of TCL1 mRNA in uncovered due to the wealth of investigations focusing normal tissue revealed expression in pre-B cells and on genes whose altered expression leads to abnormal CD47CD87 fetal thymocytes (Virgilio et al., 1994), but cellular dierentiation and/or proliferation. Identifica- not in poly(A)+ RNA derived from any of the adult tion of the genes responsible has been possible due to organs tested. Moreover, cellular localization studies the large number of chromosomal rearrangements that revealed that the Tcl1 protein was restricted to the have been found in speci®c types of hematopoietic microsomal fraction suggesting that TCL1 performs its tumors (Isobe et al., 1985; Baer et al., 1987; Bertness et function as an intracellular protein (Fu et al., 1994). al., 1990; Cleary, 1991; Croce, 1991). These chromo- Thus, the newly identi®ed TCL1 gene and the related somal rearrangements have provided cytogenetic land- MTCP1 gene, represent two members of a new marks for studies directed at identifying and cloning lymphoid-speci®c gene family which show restricted the genes involved in cancer. The characterized cellular expression in developing lymphocytes and chromosomal translocations have been shown to cause tumors. Further, these ®ndings strongly suggest that tumorigenesis through the activation of cellular proto- the TCL1 gene functions to regulate the development, oncogenes or through the formation of novel chimeric dierentiation or survival of lymphoid cells. genes capable of transforming hematopoietic cells Although the exact function of TCL1 is not known, (Heim and Mitelman, 1987; Savage et al., 1988; aberrant gene expression likely results in alterations in cell survival or growth rate in a subset of lymphoid cells harboring an inversion or translocation on Correspondence: JL Rothstein chromosome 14. This may result in the observed T Received 7 March 1997; revised 30 April 1997; accepted 1 May 1997 cell clonal expansion that precedes malignancy in AT Embryonic and lymphoid expression of the murine Tcl1 gene MG Narducci et al 920 patients (Narducci et al., 1995). One hypothesis is that cDNA library. This sequence comprises the 5' this proliferative event is accompanied by additional untranslated (UTR), coding and the 3' UTR regions genetic changes leading to a more progressive of the murine Tcl1 gene (Figure 1). Using these newly malignancy. However, without an appropriate animal developed probes the entire genomic region of the Tcl1 model it would be dicult to decipher the role of locus was isolated from a mouse 129/SvJ lambda TCL1 in cellular transformation. Thus, to provide a library. The genomic clones were ordered and partially better understanding of TCL1 function we sought to sequenced to deduce the Tcl1 genomic structure identify and map the murine Tcl1 gene and evaluate (Figure 2a). The intron/exon organization was mRNA expression during mammalian embryogenesis. determined from identi®ed canonical splice site The isolation of the murine gene and the elucidation of acceptor and donor sequences (Figure 2b) and these data is a prerequisite to the development of a through the cross hybridization of the murine cDNA mouse model of chronic lymphocytic leukemia. clone.

Homology within the new family Results Analysis of the nucleotide and deduced amino acid sequence of the murine Tcl1 gene shows that it shares Cloning of the murine Tcl1 gene 55% nucleotide and 50% amino acid sequence Initial attempts to obtain a murine cDNA directly homology to the human TCL1 gene (Figure 3) and from a mouse embryonic library using the human lower, but signi®cant, homology to the related murine TCL1 cDNA did not yield sequences corresponding to and human MTCP1 genes (Figure 3). Analysis of the the murine Tcl1 gene. Consequently, a TCL1 cDNA murine Tcl1 coding region revealed signi®cant nucleo- probe was used to screen a mouse 129/SVJ genomic tide and amino acid similarity with the human Tcl1 library to obtain murine genomic sequences. From this gene as well as to the human and murine Mtcp1 genes primary screening a clone was identi®ed that showed (Figure 3). Comparison of the mouse and human signi®cant sequence homology (*60%) to exon 1 of TCL1 and Mtcp1 sequences also showed the presence the human TCL1 gene (data not shown). From these of several high homology domains containing up to and other exon sequences, PCR primers were designed 87% amino acid similarity within the gene family to amplify the entire Tcl1 coding region from mouse (Figure 3, boxed regions). These data suggest that the embryonic RNAs. A full length 1.3 kb cDNA clone Tcl1 and Mtcp1 genes are members of a novel was isolated from a mouse embryonic stem (ES) cell lymphocyte-speci®c gene family that may share a

Figure 1 Nucleotide sequence and deduced amino acid sequence of the murine Tcl1 cDNA. Nucleotide and amino acid positions are numbers in the left and right hand margins. The nucleotide sequence was derived from the full length Tcl1 cDNA clone derived from the ES cell cDNA library and the partial (ORF only) cDNA clone isolated from 15.5 day murine embryo total RNA. Boxed sequences represent ATG start and TAA stop codons. Underline regions denote sequences involved in mRNA stability (Chen et al., 1994) Embryonic and lymphoid expression of the murine Tcl1 gene MG Narducci et al 921 common primordial ancestor as indicated by and Mus spretus strains (not shown). Thus, DNA from CLUSTAL analysis of evolutionary conservation AEJ and Spretus backcross progeny were compared by (Figure 4). Indeed this similarity may suggest even Southern analysis using a mouse Tcl1 probe. Results though the human and murine Tcl1 genes are not from the analysis of 195 backcross progeny yielded a 490% identical, they may share a high amount of concordant map location on mouse chromosome 12 in structural similarity. To investigate this notion, we close proximity to the murine immunoglobulin locus and evaluated the hydrophilicity for the Tcl1 protein. to the murine B94 TNF response gene, tnfb94 (Wolf et Hydrophilicity pro®le generally indicates the behavior al., 1994). The syntenic region of human chromosome of dierent regions of the proteins in solution. This 14q32 is the telomeric region of murine chromosome 12 pro®le is similar to that of the human Tcl1 gene (proximal to the immunoglobulin locus). Thus, the previously described (Virgilio, 1994) and suggests that murine Tcl1 clone maps to the region in the mouse Tcl1 is a hydrophilic molecule, much like the human genome that is syntenic to human chromosome 14q32 Tcl1 protein (Figure 5). (Figure 6). These data, together with the high nucleotide and amino acid sequence homology, indicate that the isolated cDNA and genomic clones represent the Chromosomal localization of the murine Tcl1 gene authentic murine homologue of the human TCL1 gene. To map the murine Tcl1 cDNA we have made use of an interspeci®c backcross panel that has been typed for Expression of Tcl1 in embryonic tissues many markers (Staats, 1981; Green, 1989; Dietrich et al., 1992; Nadeau et al., 1992). Restriction enzyme digestion The partial homology of human TCL1 to human of chromosomal DNA using KpnI demonstrated a clear MTCP1 and the high homology of the murine Tcl1 polymorphic fragment (RFLP) between Mus musculus with the human TCL1 suggest that it is part of a conserved gene family potentially important in lymphoid development. Consequently, we sought to investigate the pattern of TCL1 expression during hematopoiesis. Using a speci®c RT ± PCR assay, we analysed the expression of Tcl1 at several stages of murine development and in neonatal liver and adult hematopoietic organs. Figure 7 shows (and summar- ized in Tables 1 and 2) the result of agarose gel (Figure 4a) and Southern hybridizations (Figure 7b and c) of PCR products derived from murine mRNA. These results suggest that Tcl1 is expressed early in mouse embryogenesis with expression detected as early as 7.5 days (at the onset of blood development). Although expression of Tcl1 was detected in 15.5 day fetal liver, this product was only visible after Southern analysis suggesting a low level of mRNA expression Figure 2 (a) Restriction map of the genomic murine lambda at this stage. Tcl1 was detected in 15.5 day yolk sac, phage lfXIT22 containing the murine Tcl1 gene derived from a a tissue highly enriched for hematopoietic progenitors 129SVJ library. Restriction enzyme recognition sites are indicated: in the blood islands, suggesting that Tcl1 is expressed X=XbaI, P=PstI, H=HindIII, E=EcoRI, B=BamHI, in primitive hematopoietic organs. To con®rm this Hc=HincII (incomplete) S=SalI, N=NotI (the SalI and NotI sites pictured at the ends are from vector sequences). (b) Exon we evaluated Tcl1 expression in embryonic thymi intron structure and sequence boundaries at 3' and 5' splicing derived from 18.5 day embryos. We ®nd that signals (lowercase) are represented Tcl1 mRNA is expressed in the prenatal mouse

Figure 3 Comparison of the murine Tcl1 deduced amino acid sequence with the human TCL1, murine Mtcp1 and human MTCP1 sequences. Light blue, white lettered, regions represent complete identity for all family members (conserved domains); light blue, black lettered, regions represent homology within the TCL1 and MTCP1 genes and yellow regions represent amino acid sequences conserved in 75% of the sequences. Boxed regions represent TCL1 high-homology domains conserved between mouse Tcl1 and human TCL1 (*80% similarity) and the dotted boxes represent the corresponding regions in the MTCP1 genes Embryonic and lymphoid expression of the murine Tcl1 gene MG Narducci et al 922 a

Figure 4 CLUSTAL genetic evolutionary analysis of human (Hu) TCL1, human MTCP1, murine (Mu) Tcl1 and murine Mtcp1 sequences using the DNASTARTM program. Forks in the tree indicate likely branch points during evolution suggesting a common ancestor b

c Figure 5 Hydrophilicity pro®le of the deduced Tcl1 amino acid sequence. The Tcl1 deduced amino acid sequence was analysed for hydrophilicity using the MacVectorTM program (Kodak Scienti®c Imaging Systems, New Haven, CT)

M12345678910 11 Figure 7 Tcl1 expression in murine embryonic RNA as measured by nested RT ± PCR. PCR reactions were performed on cDNA synthesized from RNA extracted from: (a) Lane 1, 15.5 day fetal liver, lane 2, 15.5 day yolk sac, lane 3, 15.5 day whole embryo, lane 4, 13.5 day fetal liver, lane 5, 13.5 day whole embryo, lane 6, 12.5 day whole embryo, lane 7, 11.5 day whole embryo, lane 8, 10.5 day whole embryo, lane 9, 7.5 day whole embryo, lane 10, empty well, lane 11, water (negative control). All Tcl1-speci®c products are 360 bp in length. (b) Represents the same gel as a blotted and hybridized with the Tcl1 cDNA probe. Lane M is fX174 digested DNA as molecular weight marker. (c) Represents the corresponding cDNA samples ampli®ed with a G3PDH control primers set (1035 bp product). Lane M is fX174 digested DNA as molecular weight marker

Table 1 Whole embryo expression of murine Tcl1 and MTCP1 genes Murine RNA derived from embryo at days post coituma Gene 7.5 9.5 10.5 11.5 12.5 13.5 15.5 Tcl1 +b + + + + + + Mtcp1 NDc ND ± ND + + + aEmbryos were isolated and RNA extracted as described in Materials and methods and using standard protocols (Sambrook et al., 1989). b+ Indicates positive PCR product as visualized on ethidium bromide stained gel and veri®ed by Southern hybridization (Sambrook et al., 1989). cND; not determined

thymus (not shown). The expression of Tcl1 in adult tissue was examined to investigate the expression in adult hematopoietic organs. Tcl1 expression was found in adult thymus and spleen but not heart, liver, or kidney (Table 2). Figure 6 Schematic of updated mouse chromosome 12. Results of RI mapping using a murine Tcl1 probe and DNAs derived The human TCL1 gene is related to the T cell from 195 AEJ x Spretus backcross progeny. Position of the proliferation gene MTCP1 at the nucleotide, amino acid murine Tcl1 gene is placed along the distal region of the and in mRNA expression (Stern et al., 1993). This chromosome in close proximity to the immunoglobulin locus. similarity is also seen with the murine homologue Numbers on left indicate approximate distance (in CM) from Igh locus. Tcl1 map distance is *6.4 cM from Igh and 5.4 cM from (Madani et al., 1996). Thus, to compare the expression the chromosomal marker d12mit8. The tnfb94 gene is *3.5 cM patterns of the murine Tcl1 gene with the related murine from the Igh locus. No recombinations were identi®ed between Mtcp1 gene (Madani et al., 1996) we performed an RT ± Tcl1 and the d12mit28 marker (linked loci). Distances shown on PCR analysis from embryonic and adult tissues. Interest- schematic are approximate and Figure is not drawn to scale. This ingly, both of these related genes demonstrated early region of the murine chromosome is syntenic to the distal region of human chromosome 14 where the human TCL1 gene has been expression in the embryos (as early as 10.5 days of localized (Virgilio et al., 1993, 1994) development), although Tcl1 was expressed one day Embryonic and lymphoid expression of the murine Tcl1 gene MG Narducci et al 923 Table 2 Embryonic and adult organ expression data for the murine Tcl1 and MTCP1 genes Murine RNA derived from embryo at days post coitum Gene 13.5FLa 14.5FL 15.5FL 14.5YS 15.5YS Heart Kidney Liver Spleen Thymus Tcl1 7 +b + + + 7 7 7 + + Mtcp1 + NDc 7 ND +/7 7 + + + 7 aFL; fetal liver, YS; yolk sac, Heart, Kidney, Liver, Spleen and thymus represent organs taken from 8 week old adult mice. b+ Indicates positive PCR product as visualized on ethidium bromide stained gel and veri®ed by Southern hybridization (Sambrook et al., 1989). cND; not done, see text for details

– – +

– signal appears most intense, however, in the B220 – – lo hi

αµ αµ population expressing low levels of m suggesting higher αµ αµ CD8 CD4 CD8 lo hi + + CD8 – + + + expression levels of the Tcl1 mRNA. The persistence of expression at a low level in B220+mhi cells may be due B220 B220 B220 CD4 CD8 CD4 B220 CD4 to contaminating B220+mlo cells or to authentic low level expression in the B220+mhi population. This pattern of expression is similar to that observed for human TCL1 in fetal B-cells (Virgilio et al., 1994). The expression of murine TCL1 was also evaluated in the in fetal T cell populations. cDNAs were prepared from ¯ow sorted thymocytes and semi-nested PCR was performed using Tcl1 speci®c primers. The results of these analysis are shown in Figure 8 indicating that Tcl1 is expressed in CD4+CD8+ (double positive) and Figure 8 Expression of Tcl1 in ¯ow sorted bone marrow and CD47CD87 (double negative) but not CD4+ CD87 fetal thymic cell populations. Tcl1 RT ± PCR products were cells. A faint, but discernible, band was observed in the resolved on a 2% agarose gel and transferred to nylon membrane. lane containing CD8+CD47 cells suggesting low level The membrane was hybridized with an internal Tcl1 speci®c probe and exposed to X-ray ®lm for 24 h. The PCR results are expression in this population or possible residual shown. Lanes are labeled with phenotype of ¯ow sorted cell double positive cells contaminating this ¯ow sorted populations. For example, lanes 1 ± 4 correspond to RNA derived population. from ¯ow sorted B lymphocytes which stained low (lo) or high (hi) with antibodies (a) speci®c for B220 and or surface IgM (m). Lanes 5 ± 8 represent T lymphocytes sorted with CD4 or CD8 speci®c antibodies and separated by the surface phenotype Discussion indicated In this report we have presented the cloning, mapping and the embryonic expression pattern of the murine earlier than MTCP1. In contrast to Tcl1, we ®nd that homologue of the T-cell leukemia/lymphoma 1 gene MTCP1 is expressed in adult kidney and liver (Table 1). In TCL1. This gene is known to be involved in the addition, while Tcl1 is expressed in yolk sac, only faint pathogenesis of human mature T-cell proliferations, bandsareobserved in13.5and15.5dayfetalliver; revealed such as T-PLL and in similar leukemias originating only after Southern analysis of the PCR gel (Figure 7b). with high frequency in patients with AT (Taylor, This reduced expression coincides with the emigration of 1982; Baer et al., 1987; Croce et al., 1985; Heim and thymocytes out of the liver and into the fetal thymus. We Mitelman, 1987; Russo et al., 1988; Bertness et al., ®ndthatMTCP1isalsoexpressedin13.5dayfetalliverbut 1990; Wei et al., 1990; Brunning, 1991; Croce, 1991). not 15.5 day fetal liver (not shown). The summary of This gene is also overexpressed in Adult T-cell expression data related to murine Tcl1 and murine leukemias, a T-lymphoproliferation that shares many MTCP1 are shown in Tables 1 and 2. similarities to T-PLL and associated with endemic infection of the HTLV-I virus (Narducci et al., unpublished results). Further this gene is transcrip- Expression of Tcl1 in fetal B and T lymphocytes tionally regulated in the lymphoid compartment, The expression of Tcl1 in hematopoietic organs in the suggesting that TCL1 could play a signi®cant role in developing mouse embryo suggested that Tcl1 may be the development of lymphocytes much like that restricted to hematopoietic cells. Since Tcl1 is observed in other leukemia-causing genes which lead dysregulated in lymphocytes we sought to determine to altered growth or dierentiation of lymphocytes if Tcl1 was physiologically expressed in isolated (see Croce, 1991 for review). Since sequence of this lymphocytes from embryonic thymus. Fetal B and T gene does not reveal known structural similarities with lymphocytes were isolated from 12 day fetal bone other proteins that could provide insight into the marrow and thymus as described (Virgilio et al., 1994). function of the Tcl1 protein, it is important to develop B lymphocytes were identi®ed by ¯ow cytometry using animal models to evaluate the eects of Tcl1 antibodies to surface IgM and the B220 marker perturbation. expressed on immature B cells. T lymphocytes were The genomic structure of the murine and human genes isolated on the basis of their CD4 and CD8 positively. demonstrates the similarity of the two genes. By RT ± PCR was performed from cDNA derived from comparison, the intron/exon structure (boundaries) are these sorted populations. The results of a representa- identical to those in the human gene and the extent of 5' tive experiment show that Tcl1 mRNA can be detected and 3' UTR regions are the same. This is particularly in B cells expressing low to high levels of the B220 important since TCL1 and MTCP sequences do not marker and negative for surface IgM(m) (Figure 8). The show signi®cant similarity to other sequences in PIR Embryonic and lymphoid expression of the murine Tcl1 gene MG Narducci et al 924 database. This, and the information related to the 12 (Barlow et al., 1996). These data suggest that structural analyses, strongly support the hypothesis leukemias may be developing at a high rate in these that TCL1 and MTCP1 sequences belong to a newly ATM7/7 mice due to the transcriptional activation of identi®ed gene family. The overall high similarity Tcl1, much like that observed in human CLL. Future between TCL1 and MTCP1 require only minimal gap analysis of the leukemias in ATM7/7 mice as well as insertion. The analysis revealed that both TCL1 and other in vivo models of Tcl1 function (e.g. Tcl1 MTCP are proteins rich in tryptophans. Interestingly, transgenics and Tcl17/7 mice) will assist in studying almost all tryptophan residues (i.e. four out of ®ve) occur the mechanistic relationship between ATM and Tcl1 in matching positions. Further, the similar hydropathy gene products. Furthermore, the study of Tcl1 gene plots indicate that dierences in the sequence are almost expression may be important for the understanding of silent as the exposition to the aqueous solvent is the physiology of early T and B cell development. concerned. Indeed, the isolation of the Tcl1 gene will not only be The expression of murine Tcl1 was not evident in useful for studying the maturation of the mammalian 18 day fetal liver (not shown), a time when immune system, but also for the construction of a developing lymphocytes are emigrating out of the murine model of chronic lymphocytic leukemia. This liver and into the fetal thymus. Thus, the TCL1 gene model may someday enable the testing and analysis of may contain promoter/enhancer elements exclusive better diagnostics and treatments for patients aicted to lymphoid cells throughout development. Indeed, with chronic leukemia. tight transcriptional regulation of Tcl1 is likely required for normal Tcl1 function since even increased expression in immature lymphocytes results Materials and methods in transformation. These transformed cells would then be hypothesized to accumulate, predisposing Sample tissue, RNA and DNA isolation, Northern analysis and them to a second genetic hit, causing the leukemia RT ± PCR observed in CLL. Analysis of leukemia in humans supports this hypothesis since TCL1 dysregulation via RNA derived from mouse embryos at sequential stages translocation precedes transformation (Virgilio et al., during development were collected from 7.5 d.p.c. through neonate at daily intervals, extracted and prepared as 1994). This restricted expression may also be described (Nagasaka et al., 1983; Freeman, 1990; important for normal lymphocyte function during Rothstein et al., 1993). After RNA isolation, ®rst-strand hematopoietic development. Consistent with this is cDNA synthesis was performed as described for the the ®nding of Tcl1 expression at low levels in the construction of cDNA libraries (Rothstein et al., 1993). spleen and thymus, but no other organs of adult The synthesized single-stranded cDNA was ampli®ed in a mice (Table 2). standard PCR reaction (Innis et al., 1990; Ausubel et al., Expression of murine Tcl1 also follows a pattern 1991) containing 2 mM dNTPs, 2.5 units Taq Polymerase similar to that observed with human TCL1 (Virgilio et (Perkin-Elmer Cetus), and 2 pmole each of the 3' and 5' al., 1994). TCL1 expression in double negative gene-speci®c oligonucleotide primers, in a total volume of 50 mlin16PCR reaction buer (supplied by manufac- (CD47CD87) but not in CD8+ BALB/c thymocytes is turer; Perkin-Elmer Cetus). Following 30 cycles of consistent with the pattern of Tcl1 expression observed denaturation at 948C for 30 s, 50 ± 608C annealing for in human cells. However it must be noted that the 30 s and elongation at 728Cfor1±2min,thePCR expression in double positive cells has been observed in products were electrophoresed in a 1.5% agarose gel murine but not in human fetal double positive T-cells. and, in some cases, blotted onto nylon membranes and At the moment we do not know the signi®cance of this hybridized with 32P dCTP-labeled gene-speci®c probes as discrepancy. Identical patterns of Tcl1 expression were described (Innis et al., 1990; Ausubel et al., 1991). The observed in B-cell from mice and those from previous embryonic RNA was adjusted so that each PCR reaction reports in humans (Virgilio et al., 1994). Thus, murine contained the same amount of template for RT ± PCR Tcl1 expression parallels that observed in humans and (Innis et al., 1990). represents expression in lymphoid progenitors in the developing embryo and young mice. These data also Lymphocyte isolation and analysis suggest that the expression of Tcl1 is tightly regulated in The expression of the murine TCL1 gene was evaluated lymphoid/hematopoietic precursors by the Tcl1 gene at selected stages of normal B and T cell dierentiation. promoter. Murine fetal bone marrow B cell subpopulations The map location of the murine Tcl1 gene placed it (1.56107 cells) were separated by two color immuno- in close proximity with the B94 gene which is a tumor ¯uorescence cell sorting as described (Virgilio et al., necrosis factor-a inducible protein expressed in fetal 1994). cDNA from selected lymphocyte populations were liver, spleen and thymus (Wolf et al., 1994). In prepared and PCR was carried out with mouse Tcl1 exon addition, this gene product was found to be 1 speci®c primers as described below and previously exclusively expressed in lymphopoietic organs of the (Virgilio et al., 1994). adult (Wolf et al., 1994). Although the relationship of B94 and Tcl1 is not clear, the close proximity of these Primers and sequence analysis genes in the genome, like immunoglobulin, T cell For the analysis of lymphocyte populations Tcl1 exon 1 receptor and HOX genes, may be to control speci®c speci®c primers were used (Ex1 5' primer: CAG/GGC/AGA/ developmental pathways during mammalian embryonic GAC/ACC/TGC/ACA and Ex1 3' primer: TTA/ATG/ development. TGG/ACA/GGA/TCT/CCA) to yield a fragment of Interestingly, mice de®cient in the ataxia telangiecta- 300 bp. For all other expression analysis by RT ± PCR, the sia or ATM gene (ATM7/7) develop T cell leukemias open reading frame primers (orfp) were used 5' orfp1 primer with karyotypic abnormalities involving chromosome ATG/GCT/ACC/CAG/CGG/GCA/CAC and 3' orfp2 pri- Embryonic and lymphoid expression of the murine Tcl1 gene MG Narducci et al 925 mer TTA/TTC/ATC/GTT/GGA/CTC/CGA) which yields a 1993). Brie¯y, 6- to 8-week old male and female

PCR product 350 bp in length. Sequences of human TCL1 (B6D2)F1 mice (Jackson Laboratories, Bar Harbor, and human MTCP1 were taken from PIR database ME) were naturally mated, timed and embryos isolated (accession #'s Hstcl1sp1 and Hsmtcplf respectively). Mouse at speci®ed times after observation of a vaginal plug MTCP1 sequence (accession # Mmu32332) was derived from (day of vaginal plug was considered equal to day 0.5 of a previously published report (Madani et al., 1996). Multiple embryonic development). Isolated embryos were ®rst alignment was performed by means of the CLUSTAL placed in phosphate buered saline (PBS) and then routine from the software package PCGENE (IntelliGe- transferred into embryo lysis buer (100 mM NaCl, netics Inc.), according to Higgins and Sharp (1989). The 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.5% sodium multiple alignment obtained was carried out with the dodecyl sulfate, 5 mg E. coli tRNA, 5 mg/ml proteinase following setting parameters: k-tup 2, gap penalty 3, K), homogenized and incubated at 378Cfor30±60min. ®ltering level 2, open gap cost 15, unit gap cost 10. The Embryo lysates were then phenol-chloroform extracted hydropathy (hydrophilicity) pro®le of Tcl1 sequence was twice and chloroform extracted once, followed by performed according to Kyte and Doolittle, by using a ethanol precipitation as previously described (Sambrook shifting window of seven residues (Kyte and Doolittle, et al., 1989; Rothstein et al., 1992, 1993). RNA was 1982). quanti®ed by optical density and concentrations con- ®rmed by agarose gel electrophoresis (Sambrook et al., 1989). Isolation of murine and genomic cDNAs To isolate the murine TCL1 gene, a genomic library derived from the murine 129/SVJ library (Stratagene, Inc.) was screened with the human TCL1 cDNA at low Abbreviations stringency (26SSC) as described (Sambrook et al., ATM, ataxia telangiectasia; CLL, chronic lymphocytic 1989). Positively hybridizing clones were selected and leukemia; MTCP1, mature T cell proliferation-1; T-PLL, DNA extracted using the lambda WizardTM extraction kit T cell prolymphocytic leukemia; TCL1, T cell locus-1. (Promega, Corp.). Isolated lambda DNA was digested with appropriate restriction enzymes and overlapping clones oriented as described (Sambrook et al., 1989). Fragments containing Tcl1 exons were identi®ed by cross Acknowledgements hybridization and subcloned into the pBluescript-II SK+TM This work was supported by the Public Health Service (Stratagene) plasmid for sequencing (Innis et al., 1990; grant CA-21124 (to JLR) from the National Cancer Ausubel et al., 1991). Institute, and by the Outstanding Investigator Grant CA39869 from the National Institutes of Health (to CMC) and by the Human Frontier Grant 350/96 (GR). Embryonic RNA isolation The Kimmel Cancer Center shared research facilities were The procedures for embryo isolation and RNA extrac- supported by a Cancer Center Grant CA56336 from the tion are as outlined in previous reports (Rothstein et al., National Institutes of Health.

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