Sequence conservation at human and mouse orthologous common fragile regions, FRA3B / FHIT and Fra14A2 / Fhit

Abstract

It has been suggested that delayed DNA replication underlies fragility at common human fragile sites, but specific sequences responsible for expression of these inducible fragile sites have not been identified. One approach to identify such cis-acting sequences within the large nonexonic regions of fragile sites would be to identify conserved functional elements within orthologous fragile sites by interspecies sequence comparison. This study describes a comparison of orthologous fragile regions, the human FRA3B͞FHIT and the murine Fra14A2͞Fhit locus. We sequenced over 600 kbp of the mouse Fra14A2, covering the region orthologous to the fragile epicenter of FRA3B, and determined the Fhit deletion break points in a mouse kidney cancer cell line (RENCA). The murine Fra14A2 locus, like the human FRA3B, was characterized by a high AT content. Alignment of the two sequences showed that this fragile region was stable in evolution despite its susceptibility to mitotic recombination on inhibition of DNA replication. There were also several unusual highly conserved regions (HCRs). The positions of predicted matrix attachment regions (MARs), possibly related to replication origins, were not conserved. Of known fragile region landmarks, five cancer cell break points, one viral integration site, and one aphidicolin break cluster were located within or near HCRs. Thus, comparison of orthologous fragile regions has identified highly conserved sequences with possible functional roles in maintenance of fragility.C haracterization and sequencing of inherited fragile sites have determined the specific cause of fragility at a number of rare fragile sites; for example, FRA11B is caused by expansion of CGG triplets and FRA16B by AT rich minisatellite repeats (1, 2). These rare fragile sites were isolated by positional cloning of the relevant genomic loci from DNA of family members segregating these rare fragile sites. However, the common non-familial fragile sites (n Ͻ 100) are considered to be normal chromosome structures. To date, common fragile sites FRA3B (3), FRA7H (4), FRA7G (5), and FRA16D (6, 7) have been identified, cloned, and sequenced. Analyses and comparisons of these sequences have not revealed the mechanism of their fragility, although they revealed that common fragile sites are actually large fragile regions (Ϸ150 to 1,000 kbp). Expanded repeats were not found in these fragile regions (4,8,9).The human FRA3B locus at chromosome region 3p14.2 is the most inducible common fragile site, exhibiting apparent breaks in up to 50% of metaphases after exposure to aphidicolin (10). Deletions and structural rearrangements in FRA3B have been observed in a large fraction of tumor types. The tumor suppresser gene FHIT encompasses the FRA3B fragile region and is altered by deletion or translocation in many types of cancer, including those of lung, cervix, esophagus, bladder, and kidney carcinomas (11-16). We have been interested in the mechanism of fragility in this region and its contribution to cancer susceptibility. Previously, w...

Alterations in the common fragile site gene Parkin in ovarian and other cancers

“…Both FRA3B and FRA16D represent fragile regions in the mouse, Fra14A2 and Fra8E1, respectively (Shiraishi et al, 2001;Krummel et al, 2002). Additionally, the mouse orthologs of FHIT and WWOX (Fhit and Wox1) also appear to have been conserved together through evolution (Shiraishi et al, 2001;Krummel et al, 2002). The FRAXB region is distinct from the other three highly active CFS regions, as instability within this CFS region extends for only 500 kb (Arlt et al, 2002).…”

“…FRA3B and FRA16D also appear to be highly conserved between humans and mice. Both FRA3B and FRA16D represent fragile regions in the mouse, Fra14A2 and Fra8E1, respectively (Shiraishi et al, 2001;Krummel et al, 2002). Additionally, the mouse orthologs of FHIT and WWOX (Fhit and Wox1) also appear to have been conserved together through evolution (Shiraishi et al, 2001;Krummel et al, 2002).…”

Covariation in Frequencies of Substitution, Deletion, Transposition, and Recombination During Eutherian Evolution

“…As noted above, significant variation in the level of sequence conservation between human and mouse from locus to locus has been reported in several studies of long DNA sequences of single loci (Hardison et al 1991(Hardison et al , 1997Koop 1995;DeBry and Seldin 1996;Gött-gens et al 2001;Shiraishi et al 2001;Wilson et al 2001) and in comparative studies of gene sequences in these and other mammals (Wolfe et al 1989;Bernardi 1993Bernardi , 1995Casane et al 1997;Matassi et al 1999;Williams and Hurst 2000;Lercher et al 2001;Castresana 2002a,b), albeit with some dissenting analysis (Williams and Hurst 2000;Kumar and Subramanian 2002). With ∼700 4D sites/Mb and 50,000 AR sites/Mb genome-wide, we were able to do a much larger scale study of regional variation in rates of substitution, and found correlated fluctuations in regional substitution rates for both types of sites.…”

Molecular Cloning of the Canine Fragile Histidine Triad (FHIT) Gene and Fhit Protein Expression in Canine Peripheral Blood Mononuclear Cells

“…The chromosomal fragile sites varied depending on breed and age, but chromosome 20, which contains the canine FHIT gene locus, was not identified as a fragile site [20]. However, the FRA3B/Fra14A2 loci are conserved in ATrich sequences with numerous short and long repeats throughout the region [9,18]. The intronic regions of the canine FHIT gene also have AT-rich short repeat sequences (data not shown).…”

“…Moreover, its amino acid sequence was extremely similar to those of the other species. In the human and mouse, the FHIT gene is located in the unstable fragile regions of the genomes, FRA3B and Fra14A2, respectively [18,19]. A previous study reported the canine folate/thymidine depleted and aphidicolin-inducible fragile site using PBMCs from the Boxer and Doberman Pinscher breeds.…”