368 Nucleic Acids Research, Vol. 20, No. 2
Molecular cloning of the chicken SCL cDNA G.Goodwin, A.MacGregor, J.Zhu and M.R.Crompton Institute of Cancer Research, Chester Beatty Laboratories, Fulham Road, London SW3 6JB, UK Submitted December 3, 1991 The human stem cell leukaemia gene SCL (alternatively named TAL) was first identified at a translocation between chromosomes 1 and 14 in several cases of T-cell acute lymphoblastic leukaemia (1, 2). As a result of this translocation the gene is expressed in such leukaemia cells although it is not expressed in normal Tcells. The gene was found to be expressed in a number of other haemopoietic cells of early erythroid and myeloid origin, in megakaryocytes and mast cells, but not B-cells (1, 3). The protein encoded by the SCL gene is a DNA-binding protein containing a basic region and a helix-loop-helix (HLH) domain similar to that found in a number of transcription factors (reviewed in (4)). We have cloned a cDNA encoding the chicken homologue of the human SCL gene and this report describes the encoded protein. Redundant oligonucleotide primers designed to hybridize to the DNA encoding the two helices of the human SCL helixloop-helix were used to PCR amplify the HLH region from cDNA synthesized from poly A+ RNA from chicken monoblastic cells (the BM2 cell line (5)). This product was used to screen a BM2 Xgtl 1 cDNA library (6). The insert from one clone was sequenced on both strands. The insert of 2 kb has a poly A tail at its 3' end and an open reading frame, the amino acid sequence of which is shown in the figure (upper line) and is compared with the human SCL sequence (lower line). Amino acids that are either identical or have similar side chains in the two proteins are underlined. It can be seen that the basic region and the helix-loop-helix domains of the chicken and human proteins are identical. Throughout the rest of the proteins there is considerable homology, though the chicken protein is less similar to the human SCL than is the mouse SCL protein, the cDNA of which has recently been cloned (7). The mouse protein has 94% identical amino acid sequence to the human SCL sequence, whereas the chicken protein is 68% identical to the human protein. The main difference between the chicken and human SCL proteins is that the chicken protein lacks the long poly-glycine and poly (alanine-proline) stretches in the N-terminal and C-terminal regions. These stretches of amino acids are probably flexible linkers between domains.
ACKNOWLEDGEMENTS We would like to thank U. Kruse and A. Sippel for generously supplying BM2 cells and the cDNA library. GHG, AM and JZ are supported by the Cancer Research Campaign. MRC is supported by the Leukaemia Research Fund.
EMBL accession no. X63371
REFERENCES 1. Begley,C.G., Aplan,P.D., Denning,S.M., Haynes,B.F., Waldmann,T.A. and Kirsch,I.R. (1989) Proc. Natl. Acad. Sci. USA 86, 10128-10132. 2. Chen,Q., Cheng,J.-T., Tsai,L.-H., Schneider,N., Buchanan,G., Carroll,A., Crist,W., Ozanne,B., Siciliano,M.J. and Baer,R. (1990) EMBO J. 9, 415-424. 3. Green,A.R., Salvaris,E. and Begley,C.G. (1991) Oncogene 6, 475-479. 4. Visvader,J. and Begley,G. (1991) Trends in Biochem Sciences 16, 330-333. 5. Moscovici,C., Zeller,N. and Moscovici,M.G. (1982) In Revoltella,R.F., Pontieri,G.M., Basilico,C., Rovera,G., Gallo,R.C. and Subak-Sharpe,J.H. (eds.), Expression of Differentiated Functions in Cancer Cells. Raven Press, N.Y., pp. 435-449. 6. Rupp,R.A.W., Kruse U., Multhaup,G., Goekel,U., Beyrenthier,K. and
Sippel,A.E. (1990) Nucl. Acids Res. 18, 2607-2616. 7. Begley,C.G., Visvader,J., Green,A.R., Aplan,P., Metcalf,D., Kirsch,I. and Gough,N.M. (1991) Proc. Natl. Acad. Sci. USA 88, 869-873.
40 20 CH ... .M2MPAPPPPSSDPRDAREH2PESEPDSSRGGHEEfAEP H-HU... Ml-EEUSEAAR-SDP QLEGR-2A-A-
60 80 OTLLNGAAKFAGfPSPGPPA&&V_-YIELVRBXSL --------OV.LTNGVAKETSE ---- AAAAEEPPVTEFLGABQPGGGPAGGGGA 100 --DIK0SBRAAG-- MOQ5.&GAiPCR -------------AAEAA
ARDKGRDATA,ZARHRPTTF,LUPPGPAPAPAPASVTALLP 120
140
CF.EMYDLS2jALP LQPkE&AXNGQGT T rGS,FFCFP D GAGRMVOLSPPALAAPAAEfBALLXSLSciLASLGSaEE= ---BASIC----
160
SESXG SN-R PSPYEMFITDGPHTKVVRRIFTNSRERR AUPBETINRVKRRPSPYEMETTDCPHTKVVRRTFTNSRERWR ----HELIX----
LOOP
----HELIX---
OONVNGAFAELRKLIPTHPPDKKLSKNEITLRLAMYYINFLAKL
260 240 LNDOEEEGNQEGlVNDSGIVQ---D-----------TDMT NDOEEEGTOSA,TGEMPVVGAGGGGGGGGGGAPPDDLLTVT.P
280
300
NSSCGSSLDGAASPDSFTEEHDTLDSJI-ARNLlHAILUEGS0Q-B NSSCGSRT0D
ASPDRYTEE --- PAPETASIiPAMIEAADGAGPB
Figure 1. Comparison of the amino acid sequences of the chicken and human SCL (7) proteins.
Molecular cloning of the chicken SCL cDNA G.Goodwin, A.MacGregor, J.Zhu and M.R.Crompton Institute of Cancer Research, Chester Beatty Laboratories, Fulham Road, London SW3 6JB, UK Submitted December 3, 1991 The human stem cell leukaemia gene SCL (alternatively named TAL) was first identified at a translocation between chromosomes 1 and 14 in several cases of T-cell acute lymphoblastic leukaemia (1, 2). As a result of this translocation the gene is expressed in such leukaemia cells although it is not expressed in normal Tcells. The gene was found to be expressed in a number of other haemopoietic cells of early erythroid and myeloid origin, in megakaryocytes and mast cells, but not B-cells (1, 3). The protein encoded by the SCL gene is a DNA-binding protein containing a basic region and a helix-loop-helix (HLH) domain similar to that found in a number of transcription factors (reviewed in (4)). We have cloned a cDNA encoding the chicken homologue of the human SCL gene and this report describes the encoded protein. Redundant oligonucleotide primers designed to hybridize to the DNA encoding the two helices of the human SCL helixloop-helix were used to PCR amplify the HLH region from cDNA synthesized from poly A+ RNA from chicken monoblastic cells (the BM2 cell line (5)). This product was used to screen a BM2 Xgtl 1 cDNA library (6). The insert from one clone was sequenced on both strands. The insert of 2 kb has a poly A tail at its 3' end and an open reading frame, the amino acid sequence of which is shown in the figure (upper line) and is compared with the human SCL sequence (lower line). Amino acids that are either identical or have similar side chains in the two proteins are underlined. It can be seen that the basic region and the helix-loop-helix domains of the chicken and human proteins are identical. Throughout the rest of the proteins there is considerable homology, though the chicken protein is less similar to the human SCL than is the mouse SCL protein, the cDNA of which has recently been cloned (7). The mouse protein has 94% identical amino acid sequence to the human SCL sequence, whereas the chicken protein is 68% identical to the human protein. The main difference between the chicken and human SCL proteins is that the chicken protein lacks the long poly-glycine and poly (alanine-proline) stretches in the N-terminal and C-terminal regions. These stretches of amino acids are probably flexible linkers between domains.
ACKNOWLEDGEMENTS We would like to thank U. Kruse and A. Sippel for generously supplying BM2 cells and the cDNA library. GHG, AM and JZ are supported by the Cancer Research Campaign. MRC is supported by the Leukaemia Research Fund.
EMBL accession no. X63371
REFERENCES 1. Begley,C.G., Aplan,P.D., Denning,S.M., Haynes,B.F., Waldmann,T.A. and Kirsch,I.R. (1989) Proc. Natl. Acad. Sci. USA 86, 10128-10132. 2. Chen,Q., Cheng,J.-T., Tsai,L.-H., Schneider,N., Buchanan,G., Carroll,A., Crist,W., Ozanne,B., Siciliano,M.J. and Baer,R. (1990) EMBO J. 9, 415-424. 3. Green,A.R., Salvaris,E. and Begley,C.G. (1991) Oncogene 6, 475-479. 4. Visvader,J. and Begley,G. (1991) Trends in Biochem Sciences 16, 330-333. 5. Moscovici,C., Zeller,N. and Moscovici,M.G. (1982) In Revoltella,R.F., Pontieri,G.M., Basilico,C., Rovera,G., Gallo,R.C. and Subak-Sharpe,J.H. (eds.), Expression of Differentiated Functions in Cancer Cells. Raven Press, N.Y., pp. 435-449. 6. Rupp,R.A.W., Kruse U., Multhaup,G., Goekel,U., Beyrenthier,K. and
Sippel,A.E. (1990) Nucl. Acids Res. 18, 2607-2616. 7. Begley,C.G., Visvader,J., Green,A.R., Aplan,P., Metcalf,D., Kirsch,I. and Gough,N.M. (1991) Proc. Natl. Acad. Sci. USA 88, 869-873.
40 20 CH ... .M2MPAPPPPSSDPRDAREH2PESEPDSSRGGHEEfAEP H-HU... Ml-EEUSEAAR-SDP QLEGR-2A-A-
60 80 OTLLNGAAKFAGfPSPGPPA&&V_-YIELVRBXSL --------OV.LTNGVAKETSE ---- AAAAEEPPVTEFLGABQPGGGPAGGGGA 100 --DIK0SBRAAG-- MOQ5.&GAiPCR -------------AAEAA
ARDKGRDATA,ZARHRPTTF,LUPPGPAPAPAPASVTALLP 120
140
CF.EMYDLS2jALP LQPkE&AXNGQGT T rGS,FFCFP D GAGRMVOLSPPALAAPAAEfBALLXSLSciLASLGSaEE= ---BASIC----
160
SESXG SN-R PSPYEMFITDGPHTKVVRRIFTNSRERR AUPBETINRVKRRPSPYEMETTDCPHTKVVRRTFTNSRERWR ----HELIX----
LOOP
----HELIX---
OONVNGAFAELRKLIPTHPPDKKLSKNEITLRLAMYYINFLAKL
260 240 LNDOEEEGNQEGlVNDSGIVQ---D-----------TDMT NDOEEEGTOSA,TGEMPVVGAGGGGGGGGGGAPPDDLLTVT.P
280
300
NSSCGSSLDGAASPDSFTEEHDTLDSJI-ARNLlHAILUEGS0Q-B NSSCGSRT0D
ASPDRYTEE --- PAPETASIiPAMIEAADGAGPB
Figure 1. Comparison of the amino acid sequences of the chicken and human SCL (7) proteins.
