An EST-enriched comparative map of Brassica oleracea and Arabidopsis thaliana.

A detailed comparative map of Brassica oleracea and Arabidopsis thaliana has been established based largely on mapping of Arabidopsis ESTs in two Arabidopsis and four Brassica populations. Based on conservative criteria for inferring synteny, "one to one correspondence" between Brassica and Arabidopsis chromosomes accounted for 57% of comparative loci. Based on 186 corresponding loci detected in B. oleracea and A. thaliana, at least 19 chromosome structural rearrangements differentiate B. oleracea and A. thaliana orthologs. Chromosomal duplication in the B. oleracea genome was strongly suggested by parallel arrangements of duplicated loci on different chromosomes, which accounted for 41% of loci mapped in Brassica. Based on 367 loci mapped, at least 22 chromosomal rearrangements differentiate B. oleracea homologs from one another. Triplication of some Brassica chromatin and duplication of some Arabidopsis chromatin were suggested by data that could not be accounted for by the one-to-one and duplication models, respectively. Twenty-seven probes detected three or more loci in Brassica, which represent 25.3% of the 367 loci mapped in Brassica. Thirty-one probes detected two or more loci in Arabidopsis, which represent 23.7% of the 262 loci mapped in Arabidopsis. Application of an EST-based, cross-species genomic framework to isolation of alleles conferring phenotypes unique to Brassica, as well as the challenges and opportunities in extrapolating genetic information from Arabidopsis to Brassica and to more distantly related crops, are discussed.

Long range cooperative interactions regulate the initiation of replication in the Tetrahymena thermophila rDNA minichromosome.

The Tetrahymena thermophila rDNA exists as a 21 kb palindromic minichromosome with two initiation sites for replication in each half palindrome. These sites localize to the imperfect, repeated 430 bp segments that include the nucleosome-free domains 1 and 2 (D1 and D2). To determine if the D1 and D2 segments act independently or in concert to control initiation, stable DNA transformation assays were performed. Single domain derivatives of the plasmid prD1 failed to support autonomous replication in Tetrahymena. Instead, such constructs propagated exclusively by integration into endogenous rDNA minichromosomes and displayed weak origin activity as detected by 2D gel electrophoresis. D1/D1 and D2/D2 derivatives also transformed Tetrahymena poorly, showing similar replication defects. Hence, the D1 and D2 segments are functionally non-redundant and cooperate rather than compete to control initiation. The observed replication defect was greatly reduced in a plasmid derivative that undergoes palindrome formation in Tetrahymena, suggesting that a compensatory mechanism overcomes this replication block. Finally, using a transient replication assay, we present evidence that phylogenetically-conserved type I elements directly regulate DNA replication. Taken together, our data support a model in which cooperative interactions between dispersed elements coordinately control the initiation of DNA replication.

Conserved cis- and trans-acting determinants for replication initiation and regulation of replication fork movement in tetrahymenid species.

The rDNA minichromosomes of Tetrahymena thermophila and Tetrahymena pyriformis share a high degree of sequence similarity and structural organization. The T.thermophila 5' non-transcribed spacer (5' NTS) is sufficient for replication and contains three repeated sequence elements that are conserved in T.pyriformis , including type I elements, the only known determinant for replication control. To assess the role of conserved sequences in replication control, structural and functional studies were performed on T.pyriformis rDNA. Similar to T.thermophila , replication initiates exclusively in the 5' NTS, localizing to a 900 bp segment. Elongating replication forks arrest transiently at one site which bears strong similarity to a tripartite sequence element present at fork arrest sites in T.thermophila rDNA. An in vitro type I element binding activity indistinguishable from the T.thermophila protein, ssA-TIBF, was detected in T.pyriformis extracts. The respective TIBF proteins bind with comparable affinity to type I elements from both species, suggesting that in vivo recognition could cross species boundaries. Despite these similarities, the T.pyriformis 5' NTS failed to support replication in transformed T.thermophila cells, suggesting a more complex genetic organization than previously realized.