PROJECT NUMBER e DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl HI, 0155-01 LBG 3ERIOD COVERED October 1, 1992 - September 30. 1993 TITLE OF PROJECT (80 characters or IeSS rifle musf fir on one line bet'ween rhe borders ) Regulation of Gene Expression DR~NCIPAL INVESTIGATOR (LISI olher prolesstona/ persoone/ be/ow rhe Pnncrpal [nvesrfgafor ) (Name. fr*e. laborarory, and insrifute atl,/iaf#on) P.1: Marshall Nirenberg Chief LBG, NHLBI 3thers: Sadamitsu Asoh Visiting Associate Bruce Bethke LBG, NHLBI IRTA Yoshinobu Hara LBG, NHLBI Visiting Associate Adil Nazarali LBG, NHLBI Visiting Associate Alessandra Rovescalli LBG, NHLBI Visiting Fellow Dong-Ping Tan LBG, NHLBI Visiting Associate Wu-Hong Tsai LBG, NHLBI Visiting Scientist Vicki Guo LBG, NHLBI Chemist LBG, NHLBI COOPERATING UNITS (ii any) Scott Young, LCB, NIMH . Adam Hartman, LCB, NIMH Yongsok Kim, LMC, NHLBI J\BIBRANCH Laboratory of Biochemical Genetics SECTION Section on Molecular Biology INSTITUTE AND LOCATION NHLBI, NIH, Bethesda, MD rOTAL MAN-YEARS: PROFESSIONAL. OTHER: 8.5 7.5 1.0 5HECK APPROPRIATE BOX(ES) 0 (a) Human subjects 0 (b) Human tissues 0 (c) Neither 0 (al) Minors 0 (a2) Interviews 3JMMARY OF WORK (Use standard unreduced type Do no1 exceed fhe space provided.) A nucleotide se e cDNA clones were ___--_-.~ ~.--~ .-~ -~ REGULATION OF GENE EXPRESSION ZOl HL 00155-01 LBG REGULATION OF GENE EXPRESSION PROJECT DESCRIPTION MAJOR FINDINGS Mouse Homeobox And POU-Domain Genes . Approximately 8 kb of a novel mouse homeobox gene, NKx-1, a homolog of the Drosophila NK-1 homeobox gene, was sequenced. The amino acid sequences of the NKx-1 and NK-1 homeodomains differ by only 3 of the 60 amino acid residues present. Both proteins also contain an acidic domain. However, most of the other regions of the protein that have been defined differ markedly. NKx-1 poly A+ RNA was found to be most abundant in lo-day mouse embryos; the abundance progressively decreases thereafter. Northern analysis of poly A+ RNA from adults revealed 1 major band of NKx-1 poly A+ RNA in brain and trace bands in RNA from testes or spleen. The NKx-1 gene is expressed in discrete regions of the 14- day old mouse embryo mesencephalon and myelencephalon and also in spinal cord, vertebrae, and ribs. A mouse genomic DNA library was screened with oligodeoxynucleotide probes for novel homeobox genes. Seventy-two positive recombinants were cloned. Thus far five novel homeobox genes have been found. Restriction site analysis of the 72 clones revealed additional classes of clones that have not yet been identified. In addition, a novel mouse POU-domain gene related to Brain-3 POU-domain cDNA was cloned and 3 kb was sequenced. Sites of expression of Brain-l, Brain-2, Brain-4, and SKIP POU-domain genes in the mouse nervous system were determined by in situ hybridization as a function of mouse embryo developmental age and also were defined in the adult mouse. Hox 4.1 cDNA and genomic DNA were cloned and the complete Hox 4.1 open reading frame was sequenced. Regulation of a Calcium Channel a-l Subunit Gene . The Cl-1 subunit of a voltage-sensitive calcium channel previously was shown to be inducible in NG108-15 cells and the expression of the gene was shown to control the ability of the cells to form synapses with striated muscle cells. The 5' -upstream regulatory region of the calcium channel gene was cloned and sequenced. A nucleotide sequence was found that activates an enhancerless chloramphenicol acetyltransferase reporter gene. A protein was found in NG108-15 nuclei that specifically binds to this sequence. A cDNA expression library in hgtll was screened for recombinants that direct the synthesis of proteins that bind to the nucleotide sequence and 35 positive clones were obtained. Seven kinds of clones were found that encode proteins that bind to oligonucleotides with appropriate sequence specificity but differ in specificity for double-stranded DNA, or (+) or (-) single-stranded DNA. Further work is needed to determine whether one or more of the proteins regulate the expression of the Ca2+ gene channel. Enhancer and Promotor Selection. During the past year further work has been done on the selective amplification of DNA clones that contain enhancer or promoter nucleotide sequences that activate gene expression. The method is based on the observation that the Zol HL 00155-01 LBG synthesis of polyoma virus DNA in mouse cells requires viral enhancer sequences that also are required for the synthesis of mRNA from polyoma genes. Mouse genomic DNA fragments were ligated to polyoma DNA that lack the enhancer region of the virus. The E. coli origin of replication and P-lactamase gene also were inserted in the polyoma coat protein gene. Promoters or enhancers in the mouse genomic DNA inserts that activate plasmid DNA synthesis in mouse cells are able to replicate and hence are selectively amplified; whereas, plasmids that lack functional enhancer sequences do not replicate. Plasmid DNA was harvested from mouse cells that had been transfected and incubated for several days. Recovered DNA then was amplified in E;. &. The selection method is highly effective; some clones were shown to increase in abundance more than lOO,OOO-fold. Fragments of the recovered DNA inserts were shown to bind nuclear protein and activate the expression of an enhancerless chloramphenicol acetyltransferase reporter gene. Previously, cDNA clones were obtained that code for proteins that specifically bind to oligonucleotide sequences that were identified by the oligonucleotide selection method. Partial sequences of some of the cDNA clones were obtained.