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LOYOLA UNIVERSFFY CI-IICAGO STRITCH SCHOOL OF MEDICINE l)eparLn~ent ol .Mc)Iccular aml C:t'll~flnr l~iochcnfi.,~t~" Fax: [70SI 21~,-~5L~ January 25, 1992 Dr. Arthur D. Eisenberg Associate Research Director The Council for Tobacco Research-U.S.A., Inc. 900 Third Avenue New York, NY 10022 Dear Dr. Eisenberg: Thank you for sending your Statement of Policy, Information for Applicants, and Annual Report from The Council for Tobacco Research. I wish to submit the enclosed Preliminary Application for review, and am pleased to supply the following additional information: 1) Name, degrees and academic title: Sally A. Amero, Ph.D. Assistant Professor Molecular and Cellular Biochemistry 2) Project title: "Molecular Targets for the PEP Protein in Drosophila" 3) Phone number: (708)-216-3365 Project duration: Three years 5) Estimated direct costs - year 1 : $72,023. We are eager to begin this investigation, and I look forward to hearing from you. Sinc.erely, 7 Sally A. Amero. Ph.D. 40000004

PROJECT DESCRIPTION "Molecular Targets for the PEP Protein in Drosophi/a" Background and Significance The Drosophila system offers a unique opportunity to study gene expression, due to the powerful combination of cytology and genetics it provides. The major developmental transitions in the fly are accompanied by a rise in the titre of the steroid hormone ecdysone, and the pulse of ecdysone that occurs during the second larval intermolt induces puffing patterns on polytene chromosomes during the third larval inster; puffs are sites of intense transcriptional activity of the ecdysone-regulated genes and are readily visible under the light microscrope. The major features of the phenomenon were accommodated by Michael Ashburner and colleagues in a transcriptional cascade model, whereby the products of the early genes were proposed as trans-activators of the late genes and their products in turn were proposed to autoregulate their own synthesis (1). New molecular information about the ecdysone response shows the Ashburner model to be essentially correct (2). The entire cascade leads to metamorphosis, which entails histolysis of the salivary gland, but also may represent a model of the events which occur in imaginai disks where ecdy- sone is believed to initiate differentiati6n into adult structures. Little is known concerning the initial fate of newly-synthesized pre-mRNA as it emerges from the transcription complex (see 3). The management of nascent transcripts may be particular- ly important for genes such as the ecdysone-induced genes that are experiencing enhanced rather than basal levels of transcription, since by definition their polymerase density is high and many of these genes produce exceptionally long primary transcripts (often _> 100 kb)o The Drosophila PEP ~rotein on Ecdysone Puffs) protein displays a noticeable preference in binding to ecdysone-in- duced puffs on polytene chromosomes, in an indirect immunostaining assay, as may be expected for a regulator in the ecdysone response (4). Unlike a typical transcription factor, however, PEP appears highly concentrated at these sites, possibly present in multiple copies along each tran- scription unit. (Pep is neither an early nor late gene; it is constitutive). Cloning and sequencing of one Pep gene cDNA product revealed a most unusual protein sequence containing motifs charac- teristic of both typical transcriptional activators and of certain RNA-binding proteins (4). Recent work places the PEP protein in an unusual ribonucleoprotein complex associated with the hnRNP complex that assembles on Pol II transcripts (5). Therefore, it is reasonable to suggest a need for the orderly coupling of RNA synthesis to protein deposition onto the transcripts of the ecdysone- induced genes, and that PEP may contribute to this process. Specific Aims and Experimental Design My broad goals for the PEP project are 1) elucidation of PEP's role in gene expression in Drosophila as a model for transcription-coupled protein deposition onto nascent transcripts, through a combined molecular and genetic approach, and 2) determination of the importance of this role in Drosophila development. Eventually we will turn our focus to transcriptional enhance- ment in mammalian systems, and look for similar mechanisms in their hormone-inducible systems. My goal for this award is to identify the molecular target for PEP's binding on polytene chromo- somes. The gene of choice is the Sgs-4 glue gene, since it has been studied extensively at the molecular, developmental, and cytological levels (6,7), and immunostaining experiments suggest this as a target for PEP binding. These studies will be conducted in a continuing collaboration with Drs. Laurie VonKalm and Steven Beckendorf (UC, Berkeley). My specific aims are: 1. to test PEP as a DNA-binding protein on Sgs-4 gene sequences in vitro. To test for specific PEP-DNA binding interactions, affinity-purified PEP will be tested in footprinting assays with cloned Sgs-4 sequences; this gene is sufficiently short (1 kb) to make 40000005

tkese stud[e~ feasible. (I am able routinely to produce 1 - 10 pgs of purified PEP from Drosophila culture.d ceils in one day; while tedious~..this:approachis.possible. Dr. Von~Kalm is performing such footprinting analyses on the Sgs-4 sequences routinely in Dr. Beckendorf's laboratory, and he has generously offered to include purified PEP in his experiments. 2. to test PEP as an RNA-b|nding protein on Sgs-4 gene sequences [n vitro. Affinity-purified PEP will also be tested for specific interactions with RNA sequences tran- scribed from the Sgs-4 gene, utilizing Riboprobe copies synthesized and labelled by viral polymer- ase in vitro. First, the Riboprobes will be used to probe western blots bound with purified PEP in Northwestern analyses, as will appropriate antisensa controls and Riboprobes synthesized from appropriate "negative control" genes (clones are in hand). Second, RNA band shift assays will be performed with the Riboprobes and purified PEP; if necessary, we will use crude nuclear extract and our PEP-specific mAbs to effect "supershifts". Finally, it may be necessary to biotinylate the Riboprobes and assemble proteins onto them from a crude nuclear extract; using mercury affinity chromatography, we will recover the RNA and associated proteins, and look for PEP in the bound fraction. 3. to test PEP binding to Sgs-4 gene sequences in vivo, Dr. Beckendorf has engineered a large library of fly strains containing transduced copies of the Sgs-4 promoter and enhancer sequences driving.transcription of either the homologous Sgs-4 gone sequences or gene sequences from heterologous genes, such as Adh or lac Z (6,7). These fly strains are available for immunostaining with my PEP-specific monoclonal antibodies, as a direct test of PEP's binding to the sequences delineated in Specific Aims 1 and 2. Similar experiments have also delineated the heat shock-inducible gene hspTO as a second putative target for PEP binding. The appropriate clones and fly strains are in hand to duplicate the analyses above on these sequences, should unforeseen problems arise with the Sgs-4 approach. These experiments will advance the PEP project towards understanding the role this unique protein plays in gene expression and will provide a foundation for reconstructing these processes in vitro. Moreover, this work will help us build a model of the mechanisms that manage nascent transcripts as they emerge from the transcription complex, a fundamental question in eukaryotic gene expression. I discovered the PEP protein just over six years ago, and to my knowledge we are its sole investigators, save my collaborators. Supporting Data In collaboration with Dr. S. Beckendorf (UC, Berkeley), I have used the immunofloures- cence assay to map a putative target for PEP within the ecdysone-responsive Sgs-4 glue gene at locus 3C (also the site of the dunce and Pig-1 genes). Naturally-occurring, underproducing Sgs-4 mutants have been isolated and characterized (7). The Berkeley-1 mutant carries a small deletion within the promoter of the Sgs-4 gene and produces 0.01% the amount of Sgs-4 RNA as does the wild-type gene. The nonoverlapping Hikone R mutant carries another small deletion of the Sgs-4 promoter and produces = 10% of the normal levels of Sgs-4 RNA. These deletions fall into now well-defined sequence elements responsible for proper spatial and temporal expression of the Sgs-4 gene, as well as for proper down-regulation of the adjacent Pig-I gene. As shown below, I observed a drastic reduction in PEP-specific fluorescence at this site on chromosomes from the Bet strain, but PEP staining was unaffected on ci4romosomes from the Hikone R strain. These observations strongly establish the Sgs-4 gene as a probable target for PEP. (Note that neither the Pig-1 or Sgs-~- genes possesses an intron; therefore it is doubtful that PEP participates in pre- mRNA splicing.) These observations do not address, however, whether PEP binds to DNA and/or RNA at this site, necessitating the experiments outlined above. 40000008

Literature Cltml 1. Ashburnc~=. M. 1990. Cell 61: 1-3. 2. Andres, A.J. and Thummel, C.S. 1992. Trends in ,7,~'et~'s $: '3.'-138, • 3. Amero, "3.A.. Raychaudhuri, G., Cass, C.L., van Ve~\\ ,~.,. -,,~oets, W.J.. I. =.01a0.'= and Beyer, A. L. 1992. Proc Natl Acad Sci ~e^ e¢ ~4~'~$4!-" ,~;nclosod). .~. 4. Amero, S,.\., Elgnn, S.C.R., and Beyer, A.L. 1991. G;-.'es 2~:~. ,~: tS8-=00 5. Amero, S.:&.. Hockensmith, J.W., Raychaudhuri, G. ,~ \: ~.,~er. &,L. Manus{:0al'n ..... (enclosed). 6. Mougneau. E., Mort Seggern, D., Fowler, T., Rosen~,~*,t ,. - on,jens, T., Ro(Inu'~. !t ~ ~pn'! ~' '~' D., and Beck~,mtorf, S.K. 1993. Mol. Cell. Biol. 13:1,q4 195. 7. Barnett, ,~A\'., Flynn, K., Webster, M.K., and Beck~:~e,'rt. ,~ ~,. 1990. Dov. 373. 40000007