No abstract

The cloning and characterization of the common fragile site (CFS) FRA6E (6q26) identified Parkin, the gene involved in the pathogenesis of many cases of juvenile, early-onset and, rarely, late-onset Parkinson's disease, as the third large gene to be localized within a large CFS. Initial analyses of Parkin indicated that in addition to playing a role in Parkinson's disease, it might also be involved in the development and/or progression of ovarian cancer. These analyses also indicated striking similarities among the large CFS-locus genes: fragile histidine triad gene (FHIT; 3p14.2), WW domain-containing oxidoreductase gene (WWOX; 16q23), and Parkin (6q26). Analyses of FHIT and WWOX in a variety of different cancer types have identified the presence of alternative transcripts with whole exon deletions. Interestingly, various whole exon duplications and deletions have been identified for Parkin in juvenile and early-onset Parkinson's patients. Therefore, we performed mutational/exon rearrangement analysis of Parkin in ovarian cancer cell lines and primary tumors. Four (66.7%) cell lines and four (18.2%) primary tumors were identified as being heterozygous for the duplication or deletion of a Parkin exon. Additionally, three of 23 (13.0%) nonovarian tumorderived cell lines were also identified as having a duplication or deletion of one or more Parkin exons. Analysis of Parkin protein expression with antibodies revealed that most of the ovarian cancer cell lines and primary tumors had diminished or absent Parkin expression. While functional analyses have not yet been performed for Parkin, these data suggest that like FHIT and WWOX, Parkin may represent a tumor suppressor gene.

LacI/GalR transcription regulators have extensive, non-conserved interfaces between their regulatory domains and the 18 amino acids that serve as ‘linkers’ to their DNA-binding domains. These non-conserved interfaces might contribute to functional differences between paralogs. Previously, two chimeras created by domain recombination displayed novel functional properties. Here, we present a synthetic protein family, which was created by joining the LacI DNA-binding domain/linker to seven additional regulatory domains. Despite ‘mismatched’ interfaces, chimeras maintained allosteric response to their cognate effectors. Therefore, allostery in many LacI/GalR proteins does not require interfaces with precisely matched interactions. Nevertheless, the chimeric interfaces were not silent to mutagenesis, and preliminary comparisons suggest that the chimeras provide an ideal context for systematically exploring functional contributions of non-conserved positions. DNA looping experiments revealed higher order (dimer–dimer) oligomerization in several chimeras, which might be possible for the natural paralogs. Finally, the biological significance of repression differences was determined by measuring bacterial growth rates on lactose minimal media. Unexpectedly, moderate and strong repressors showed an apparent induction phase, even though inducers were not provided; therefore, an unknown mechanism might contribute to regulation of the lac operon. Nevertheless, altered growth correlated with altered repression, which indicates that observed functional modifications are significant.

Characterization of FRA6E (6q26), the third most frequently observed common fragile site (CFS) in the human population, determined that aphidicolin-induced instability at FRA6E extends over a very large region (3.6 Mb). Sequence analysis identified eight genes (IGF2R, SLC22A1, SLC22A2, SLC22A3, PLG, LPA, MAP3K4, and PARK2) as mapping within the large FRA6E region. PARK2, the gene associated with autosomal recessive juvenile parkinsonism (ARJP), accounts for more than half of the CFS. Homozygous deletions and large heterozygous deletions have been observed in PARK2 in ARJP patients. RT-PCR analysis of the eight genes localizing to FRA6E indicated that 50% of the genes, including PARK2, were down-regulated in one or more of the primary ovarian tumors analyzed. PARK2 expression was down-regulated in 60.0% of the primary ovarian tumors analyzed. Additionally, we found tumor-specific alternative transcripts of PARK2. Loss of heterozygosity analysis of primary ovarian tumors by use of polymorphic markers in the 6q26 region demonstrated 72% LOH in the center of the PARK2 gene, the highest of any of the markers tested. FRA6E shares many similarities with FRA3B (3p14.2) and FRA16D (16q23.2) in representing a large region of genomic instability and containing an extremely large gene that may play a role in the development of ovarian and many other cancers.