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Construction of Full-Length cDNAs

      Construction of full-length cDNAs is a central focus in the post-sequence era of the various genome projects, including Arabidopsis. Kazuo Shinozaki of the Laboratory of Plant Molecular Biology, Tsukuba Life Science Center, The Institute for Physical and Chemical Research (RIKEN), has constructed full-length cDNAs for 10,000 genes of Arabidopsis. We are planning to coordinate our efforts with those of Dr. Shinozaki in order to deliver the entire set of full-length cDNAs for the Arabidopsis genome to the Plant Biology community. A number of critical issues pertaining to synthesis and cloning of full-length cDNAs have been recently identified. Most important is the purity and integrity of the starting material. mRNA is often contaminated with hnRNA due to the complexity of isolation of exclusive cytoplasmic RNA from plant tissues. Cytoplasmic mRNA to be used for construction of full-length cDNA libraries will be tested for the presence of contaminating nuclear RNA and DNA. This will be done by performing a test for the presence of introns of a ubiquitously expressed gene using PCR. The use of total cellular (rather than cytoplasmic) RNA is not desirable due to the expected contamination with nuclear transcripts, which will be particularly significant in the upper molecular weight range.

      What do we consider a full-length cDNA? A full-length cDNA should encompass all sequences from the CAP site to the poly (A) addition site. However, it is generally agreed upon that a cDNA comprising the entire coding sequence of a protein should be considered worthy for full-length sequencing at high accuracy. We will make every effort to obtain truly full-length cDNAs, so that sequence information could be obtained from both 5' and 3' noncoding regions as well. In this regard, we will be able to assess the correspondence between the results obtained from cDNA construction/ sequencing vs. mRNA hybridization to the 5' end mapping chip (see below). Such information will be extremely valuable for defining the promoter regions for all the Arabidopsis genes.

During our studies, we will:

  1. Assess the abundance of the mRNAs (prevalent, intermediate and rare) by hybridization to the Affymetrix Arabidopsis genome chip.
  2. Utilize the state-of-the-art methodology for cloning full-length cDNAs. We will determine the purity and integrity of the starting material as well as the quality of first strand synthesis.
  3. Construct full-length cDNAs from limited source material.
  4. Develop a unique set of full-length cDNAs for Arabidopsis.

      All three investigators have constructed numerous cDNA libraries during the last 20 years in a variety of vectors that are available to the Arabidopsis community.

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