National Institutes of Health, National Heart, Lung, and Blood Institute, Contracts Operations Branch, 6701 Rockledge Drive, Room 6100, MSC 7902, Bethesda, MD 20892

B -- SPECIAL STUDIES AND ANALYSIS -- NOT R&D SOL NHLBI-PS-2000-645 DUE 080400 POC Deborah Coulter, Purchasing Agent (301) 435-0368 Fax (301) 480-3345 The National Heart, Lung, and Blood Institute intends to issued a non-competitive modification to an existing purchase order, number 26-MJ-007302, with Dr. Dong Ping Tan, 11400 Saddleview Place, North Potomac, Maryland, 20878. Through non-competitive procedures Dr. Tan, was issued a purchase order to complete studies on the Mouse HOXA6 Gene Regulation Project for publication. The nature of the work to be performed involves computer analysis of the previously generated sequence data, together with construction and analysis of an array of putative regulatory gene constructs to determine the nature of the Hoxa6 regulatory gene. Dr. Tan's, studies has made some important discoveries which requires additional time for the completion of the services. Therefore, an award to any other source for this extension would result in a substantial duplication of cost to the Government which would not be expected to be recovered through competition. The Statement of Work for this requirement is as follows: Background: Dr. Dong-Pin Tan, previously generated excess of 6kb of DNA sequence data describing the Hoxa6 mouse homebox gene as well as the putative upstream regulatory region. He is prepared to complete the characterization of this gene with respect to its regulation. Contractor Requirements: The specific tasks to be performed are as follows: (1) Complete and refine the 6.3 kb Hoxa6 genomic sequence by using primer walking, compile the sequence into a contig using the GCG programs. Use the enhancer computer program to search the 3 kb Hoxa6 upstream region for candidate cis-elements that are binding sites for transcription factors. (2) For Hoxa6 promoter analysis, select suitable cell lines in which the Hoxa6 promoter can be activated. Use RT-PCR or Northern analysis to detect endogenous Hoxa6 expression in those cells. The candidate cell lines are the p19 embryonal carcinoma cell line and NIH3T3, in which some other Hox genes can be induced. (3) Construct a series of Hoxa6- luciferase fusion genes by subcloning restriction fragments or PCR fragments of the 3 kb Hoxa6 5'-upstream region into the polylinker region of the luciferase vectors pGL3 or pGL2. (4) Transfect cell lines with Hoxa6-reporter fusion genes and measure the luciferase activity of each construct to pinpoint the region corresponding to the Hoxa6 minimal promoter. Compare the promoter activities of different fragments and correlate these activities with the candidate transcription factor sites obtained from the computer data searches. (5) Select candidate transcription factors and make expression vectors of those transcription factors by subcloning their cDNAs mammalian expression vectors. (6) Co-transfect Hoxa6 promoter fusion genes with the expression vector(s) encoding transcription factor(s) to measure changes of promoter activity by detecting increases or decrease of luciferase activity. Test the dose-responses of these transcription factor(s). (7) Mutate the domains of transcription factors to decipher which domain is essential for the activation/inhibition activity of the transcription factor on the Hoxa6 promoter. (8) Use site-directed mutagenesis to mutate candidate cis-elements to detect which site is necessary for the transcription factor to activate or inhibit the Hoxa6 promoter. (9) Confirm the binding of the transcription factor to the candidate cis-elements, using electrophoretic mobility shift assays. The transcription factor protein or nuclear extract will be incubated with end-labeled oligonucleotides, synthesized to match the cis-elements. The desired results, functions, or end items required to accomplish those results are to: Assemble all data, make illustrations and write a manuscript for publication. The technical specifications and applicable standards or methodologies to be used are: Genetic computer analysis, cell culture, PCR and Northern analysis, gene cloning and fusion gene construction, DNA transfection, site-directed mutagenesis and electrophoretic mobility shift assays. Government Responsibilities: Will provide laboratory space inbuilding 36 room 4C-11 comprising computers, PCR equipment, DNA sequencing equipment, incubators, electrophoresis instruments and all appropriate disposable materials for carrying out the studies. The Government will be responsible for reviewing and approving reports and similar matters generated include periodic review of Dr. Tan's experimental data together with final assembly of the accumulate data for publication purposes. Reporting Requirements and Deliverables: The reporting requirements involve daily progress reports to Dr. Peterkofsky. Period of Performance: Extended through September 30, 2000. This acquisition is being conducted under simplified acquisition procedures. The Standard Industrial Classification (SIC) Code 8999, Size Standard $5.0M is applicable to this requirement. This notice of intent is not a request for competitive proposals. Interested parties may identify their interest and capabilities in response to this requirement. The determination by the Government not to compete the proposed contract based upon responses to this notice is solely within the discretion of the Government. Information received will normally be considered solely for the purpose of determining whether to conduct future competitive procurement. Responses to this announcement, referencing synopsis number NHLBI-PS-2000-645 may be submitted to the National Heart, Lung, and Blood Institute, Contracts Operations Branch, Procurement Section, Building RKL2, Room 6143, 6701 Rockledge Drive, Bethesda, MD 20892-7902, Attention Deborah Coulter, Purchasing Agent. All responsible sources may submit a quotation, which if timely received, shall be considered by the agency. Posted 08/01/00 (W-SN480578). (0214)

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