Genomic structure and promoter characterization of an imprinted tumor suppressor gene ARHI.

We have recently identified a maternally imprinted tumor suppressor gene, ARHI (aplysia ras homolog I), the expression of which is lost in ovarian and breast cancers. We have now characterized the genomic structure of the gene including its promoter and the methylation status of its upstream CpG islands. The ARHI gene spans approximately 8 kb containing two exons and one intron. Exon 1 contains 81 non-translated nucleotides, connected to exon 2 with a 3.2-kb intron. The entire protein-coding region is located within exon 2 and encodes a 229-residue small GTP-binding protein belonging to the Ras superfamily. Genomic structure analysis has identified three potential CpG islands. Two of them (CpG island I and II) are located within the promoter and adjacent exon 1 of the ARHI gene. Aberrant methylation of these CpG islands has been detected in breast cancer cells but not in normal epithelial cells, supporting the possibility that appropriate methylation status of the CpG islands in the promoter region may play a role in the downregulation of ARHI gene expression. A TATA box is found 27 bp upstream of the transcription start site associated with several putative transcription factor binding sites. Transient transfection with nested deletion constructs of the 2-kb ARHI promoter regions fused to a luciferase reporter indicated a 121-bp sequence upstream of the transcription initiation site is required for basal promoter activity. Interestingly, this is the region where lower promoter activity has been observed in cancer cells than in normal cells.

Gene expression in the developing mouse retina by EST sequencing and microarray analysis.

Retinal development occurs in mice between embryonic day E11.5 and post-natal day P8 as uncommitted neuroblasts assume retinal cell fates. The genetic pathways regulating retinal development are being identified but little is understood about the global networks that link these pathways together or the complexity of the expressed gene set required to form the retina. At E14.5, the retina contains mostly uncommitted neuroblasts and newly differentiated neurons. Here we report a sequence analysis of an E14.5 retinal cDNA library. To date, we have archived 15 268 ESTs and have annotated 9035, which represent 5288 genes. The fraction of singly occurring ESTs as a function of total EST accrual suggests that the total number of expressed genes in the library could approach 27 000. The 9035 ESTs were categorized by their known or putative functions. Representation of the genes involved in eye development was significantly higher in the retinal clone set compared with the NIA mouse 15K cDNA clone set. Screening with a microarray containing 864 cDNA clones using wild-type and brn-3b (-/-) retinal cDNA probes revealed a potential regulatory linkage between the transcription factor Brn-3b and expression of GAP-43, a protein associated with axon growth. The retinal EST database will be a valuable platform for gene expression profiling and a new source for gene discovery.

ARHI is the center of allelic deletion on chromosome 1p31 in ovarian and breast cancers.

In our previous work, we had characterized ARHI as an imprinted putative tumor-suppressor gene in ovarian and breast cancers. ARHI is expressed in primary breast and ovarian cell lines but largely absent from the corresponding malignant tumors. Moreover, the non-imprinted functional allele is typically deleted in malignant cells. Since ARHI had been mapped to 1p31, a common deletion site in breast and ovarian cancer and male germ-cell tumors, in this study, we set out to define precisely the physical location of ARHI at 1p31 and to determine if this location lies within the smallest common region of deletion in breast and ovarian cancers. To this end, we first carried out radiation hybrid mapping of ARHI and surrounding markers, followed by a high-resolution study of loss of heterozygosity at 1p31 in 49 ovarian and breast cancers. Combining a radiation hybrid map and a physical map of the region encompassing ARHI, 3 discrete regions of minimal deletion were found at 1p31 in breast and ovarian cancers. ARHI is the most common deletion region at 1p31. Two other less common regions of deletion were found centromeric to this gene. One of them centered on D1S207 and the other one included and was proximal to D1S488. We also confirmed the preferential loss of non-imprinted functional allele in 7 of 9 tumor specimens. These data support the possibility that ARHI is a tumor-suppressor gene and suggest that additional tumor-suppressor genes may lie proximal to ARHI at 1p31. The data obtained from our study should aid in the identification and characterization of genes in this novel imprinted region.

The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development.

A wild-type sasA locus is critical for Myxococcus xanthus multicellular development. Mutations in the sasA locus cause defective fruiting body formation, reduce sporulation, and restore developmental expression of the early A-signal-dependent gene 4521 in the absence of A signal. The wild-type sasA locus has been located on a 14-kb cloned fragment of the M. xanthus chromosome. The nucleotide sequence of a 7-kb region containing the complete sasA locus was determined. Three open reading frames encoded by the genes, designated rfbA, B and C were identified. The deduced amino acid sequences of rfbA and rfbB show identity to the integral membrane domains and ATPase domains, respectively, of the ATP-binding cassette (ABC) transporter family. The highest identities are to a set of predicted ABC transporters required for the biosynthesis of lipopolysaccharide O-antigen in certain gram-negative bacteria. The rfbC gene encodes a predicted protein of 1,276 amino acids. This predicted protein contains a region of 358 amino acids that is 33.8% identical to the Yersinia enterocolitica O3 rfbH gene product, which is also required for O-antigen biosynthesis. Immunoblot analysis revealed that the sasA1 mutant, which was found to encode a nonsense codon in the beginning of rfbA, produced less O-antigen than sasA+ strains. These data indicate that the sasA locus is required for the biosynthesis of O-antigen and, when mutated, results in A-signal-independent expression of 4521.