Vol. 3, 143-148, March 1992 Cell Growth & Differentiation 143 NSCL-2: A Basic Domain Helix-Loop-Helix Gene Expressed in Early Neurogenesis Verena G#{246}bel, Stanley Lipkowitz, Christine A. Kozak, and Ilanj. Kirsch’ Navy Medical Oncology [S. L., I. R. K.], and Metabolism Branches [V. G.], National Cancer Institute, and Laboratory of Molecular Microbiology [C. A. K.], National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland 20892 Abstrad We have identified a new basic domain helix-loop- helix (bHLH) gene, NSCL-2, which was cloned because of its homology to the previously described putative hematopoietic transcription fador, SCL NSCL-2 has been identified in both human and murine DNA. NSCL- 2 complementary DNA clones were obtained from an 1 1.5-day murine embryo library. The coding region is 405 base pairs and encodes a predided protein of 15.6 kilodaltons. There is 74% homology at the nucleotide level with the coding region of the murine SCL and 27% protein homology. Unlike the majority of previously described bHLH genes, the NSCL-2 coding region ends only six amino acids beyond the second amphipathic helix of the HLH domain. The NSCL-2 gene shows a markedly restrided pattern of expression predominantly confined to murine embryos at days 11- 13 of development, although low level expression can be deteded in murine embryos flanking this time point. Examination of 1 1- and 1 2-day mouse embryos by tissue in situ hybridization reveals expression of NSCL- 2 in the developing nervous system, most likely in developing neurons. The NSCL-2 gene maps to murine chromosome 3. The temporally and tissue restrided pattern of expression of this gene and its identification as a member of a family of transcription fadors relevant to growth and development in a wide variety of species suggest a role for NSCL-2 in the development of the eukaryotic nervous system. Introdudion Within the past 3 years, a new family of transcription factors has been defined relevant to a wide spectrum of growth and developmental systems. This family has a characteristic motif within the primary amino acid se- quence, consisting of sequences predicted to form an amphipathic a-helix, followed by a loop, then a second a-helix, the so called HLH2 domain (1). This HLH domain is often preceded by a run of basic amino acids. In those members of the family amenable to such analyses, it has Received 9/18/91. 1 To whom requests for reprints should be addressed, at National Cancer Institute-Navy Medical Oncology Branch, Building 8, Room 5101, Na- tional Naval Medical Center, Bethesda, MD 20889-5105. 2 The abbreviations used are: HLH, helix-loop-helix; b, basic domain; cDNA, complementary DNA; poIy(A), polyadenylated; Gba, glucocere- brosidase; Ngfb, nerve growth factor fi; SSC, standard saline citrate. been determined that the basic domain confers DNA binding capability on the protein and that the affinity of this DNA binding can be modulated by protein-protein interactions between the HLH domains of one family member and another (2-4). Some members of this family appear to be essentially ubiquitously expressed in a large number of tissues regardless of stage of development. Other members of the family show a temporally or spatially restricted pattern of expression (2, 5-8). We have previously reported the discovery and sub- sequent characterization of a “bHLH” putative hemato- poietic transcription factor, SCL. It was originally identi- fied because of its disruption by a t(1;14) translocation associated with the development of a leukemia in which the leukemic blasts had properties of hematopoietic pro- genitor cells (8, 9). A closely related gene, LYL1, was discovered because of its disruption by a different chro- mosomal translocation, t(7;19), associated with the de- velopment of a T-ceIl leukemia (7), and is also a bHLH gene expressed in hematopoietic tissues. Using an SCL probe, we became aware of two other genes that showed a high level of nucleotide homology. One of these genes, NSCL-1, is the subject ofa separate report (10). Here, we report the characterization of the second gene, which we call NSCL-2, because it is an SCL-related bHLH gene predominantly expressed in developing neural tissue. Results Cloning of a Murine NSCL-2 cDNA. A human SCL probe includingthe bHLH domain was used to screen a murine genomic placental library. In addition to the murine SCL gene, a different related gene, NSCL-1, was identified. Expression studies identified 10-14-day murine embry- onic development as a source of NSCL-1 messenger RNA. Therefore, an 11.5-day murine embryo cDNA Ii- brary was screened with an NSCL-1 probe. In addition to the NSCL-1 clones that are the subject of a separate report (10), two additional clones (from 10 phages screened), representing two unique overlapping inserts, were identified that were distinct but related to NSCL-1. Therefore, we called this new gene NSCL-2. The nucleotide sequence and predicted protein of the NSCL-2 gene are shown in Fig. 1 . The open reading frame begins with an ATG at nucleotide 525 and terminates at nucleotide 929. The ATG represents the first in frame ATG, and stop condons in all three reading frames 5’ of this ATG confirm it as a start codon. The stretch of A residues at the end of the 3’ untranslated region is not preceded by a polyadenylation signal and therefore may not represent the bona fide poly(A) tail of the message. The predicted protein consists of 1 35 amino acids and would have a molecular weight of 1 5,600. The protein terminates six amino acids after the end of the second amphipathic helix. There is a distinctive stretch of 25 basic amino acids on the amino terminal side of the helix- loop-helix domain. All of the invariant residues formerly