Finishing phase of Chromobacterium violaceum genome project 53 Genetics and Molecular Research 3 (1): 53-63 (2004) www.funpecrp.com.br Closure of rRNA related gaps in the Chromobacterium violaceum genome with the PCR-assisted contig extension (PACE) protocol Dirce Maria Carraro 1 , Anamaria Aranha Camargo 1 , Anna Christina Matos Salim 1 , Luiz Gonzaga 2 , Gisele Cavalcante da Costa 2 , Ana Tereza Ribeiro de Vasconcelos 2 and Andrew J.G. Simpson 3 1 Laboratory of Genetics, Ludwig Institute for Cancer Research, São Paulo, SP, Brasil 2 LNCC/MCT, Rio de Janeiro, RJ, Brasil 3 Ludwig Institute for Cancer Research, New York, NY, USA Corresponding author: A.J.G. Simpson E-mail: asimpson@licr.org Genet. Mol. Res. 3 (1): 53-63 (2004) Received October 13, 2003 Accepted January 12, 2004 Published March 31, 2004 ABSTRACT. In the finishing phase of the Chromobacterium viola- ceum genome project, the shotgun sequences were assembled into 57 contigs that were then organized into 19 scaffolds, using the information from shotgun and cosmid clones. Among the 38 ends resulting from the 19 scaffolds, 10 ended with sequences corresponding to rRNA genes (seven ended with the 5S rRNA gene and three ended with the 16S rRNA gene). The 28 non-ribosomal ends were extended using the PCR-assisted contig extension (PACE) methodology, which immediately closed 15 real gaps. We then applied PACE to the 16S rRNA gene containing ends, resulting in eight different sequences that were cor- rectly assembled within the C. violaceum genome by combinatory PCR strategy, with primers derived from the non-repetitive genomic region flanking the 16S and 5S rRNA gene. An oriented combinatory PCR was used to correctly position the two versions (copy A and copy B, which differ by the presence or absence of a 100-bp insert); it revealed six copies corresponding to copy A, and two to copy B. We estimate that Genetics and Molecular Research 3 (1): 53-63 (2004) FUNPEC-RP www.funpecrp.com.br