Mutational inactivation of the retinoblastoma susceptibility (RB) gene, a recessive cancer gene, has been implicated in the genesis of retinoblastoma and certain other human neoplasms. This gene is now shown to be inactivated in two of nine human breast cancer cell lines examined. The RB gene of one cell line had a homozygous internal duplication of a 5-kilobase region containing exons 5 and 6. The RB messenger RNA transcript was correspondingly lengthened, and its translation was probably terminated prematurely due to a shifted reading frame. The other cell line had a homozygous deletion of the RB gene that removed the entire gene beyond exon 2. The RB gene product, pp110RB, was not detectable in either cell line by immuno-precipitation with specific antibodies. These findings are significant in relation to proposed genetic mechanisms of breast cancer formation.

Complete inactivation of the human retinoblastoma gene (RB) is believed to be an essential step in tumorigenesis of several different cancers. To provide a framework for understanding inactivation mechanisms, the structure of RB was delineated. The RB transcript is encoded in 27 exons dispersed over about 200 kilobases (kb) of genomic DNA. The length of individual exons ranges from 31 to 1889 base pairs (bp). The largest intron spans >60 kb and the smallest one has only 80 bp. Deletion of exons 13-17 is frequently observed in various types of tumors, including retinoblastoma, breast cancer, and osteosarcoma, and the presence of a potential "hot spot" for recombination in the region is predicted. A putative "leucine-zipper" motif is exclusively encoded by exon 20. The detailed RB structure presented here should prove useful in defining potential functional domains of its encoded protein. Transcription of RB is initiated at multiple positions and the sequences surrounding the initiation sites have a high G+C content. A typical upstream TATA box is not present. Localization of the RB promoter region was accomplished by utilizing a heterologous expression system containing a bacterial chloramphenicol acetyltransferase gene. Deletion analysis revealed that a region as small as 70 bp is sufficient for RB promoter activity, similar to other previously characterized G+C-rich gene promoters. Several direct repeats and possible stem-and-loop structures are found in the promoter region. No enhancer element was detected within the 7.3 kb of upstream sequence studied. Several features of the RB promoter are reminiscent of the characteristics associated with many "housekeeping" genes, consistent with its ubiquitous expression pattern.

A gene in chromosome region 13q14 has been identified as the human retinoblastoma susceptibility (RB) gene on the basis of altered gene expression found in virtually all retinoblastomas. In order to further characterize the RB gene and its structural alterations, we examined genomic clones of the RB gene isolated from both a normal human genomic library and a library made from DNA of the retinoblastoma cell line Y79. First, a retiction and exon map of the RB gene was constructed by al overlapping genomic clones, yielding three contiguous regions ("contig') of 150 kilobases total length separated by two gaps. At least 20 exons were identified ip genomic clones, and these were provisionally numbered. Second, two overlapping genomic clones that demonstrated a DNA deletion of exons 2 through 6 from one RB allele were isolated from the Y79 library. To confirm and extend this result, a unique sequence probe from intron 1 was used to detect similar and possibly identical heterozygous deletions in genomic DNA from three retinoblastoma cell lines, thereby explaining the origs of their shortened RB mRNA htansripts. The same probe detecte genomic rearrangements in fibroblasts from two hereditary retinoblastoma patients, indicating that intron 1 includes a frequent site for mutations conferring predisposition to retinoblastona. Third, this probe also detete a polymorphic site for BamEil with allele frequencies near 0.5/0.5.Identification of commonly mutated regions will contribute significantly to genetic diagnosis in retinoblastoma patients and families.Retinoblastoma is an intraocular cancer of early childhood that arises from the developing retina. In a substantial proportion of cases, susceptibility to retinoblastoma can be inherited from a parent who was previously cured of the tumor (1,2 Recently, a gene in band 13q14 encoding an mRNA of 4.7 kilobases (kb) was identified as the RB gene based on tumor-specific alterations in expression and its apparent recessive nature at the cellular level (10). cDNA segments representing the RB gene transcript have been cloned (10-12) and sequenced (10). All hereditary and nonhereditary retinoblastomas examined to date have demonstrated altered RB gene expression: RB mRNA transcripts are either markedly reduced in quantity or are abnormal in length (10-12). About 40% of retinoblastomas show DNA deletions detectable by RB cDNA probes. Antibodies generated against the RB gene product, ppllORn, show complete absence of this nucleophosphoprotein in five out of five retinoblastomas, whereas it was easily detected in all normal or neoplastic cells containing a normal RB mRNA transcript (13). These data further strengthen the hypothesis that the absence of functional RB protein is potentially oncogenic.Given the different patterns of genetic alteration observed in retinoblastomas, it is evident that the RB gene is subject to a variety of mutational mechanisms, the details of which are as yet unknown. In order to further characterize the RB gene and some of its mutations, we construc...

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The gene encoding human esterase D (EsD), a member of the nonspecific esterase family, is a useful genetic marker for retinoblastoma (RB) and Wilson's disease. Previously we identified a cDNA clone from this gene and determined its chromosomal location. In this report, we present the complete cDNA sequence of the human EsD gene. A long open reading frame encoded a predicted protein of 282 amino acids with molecular weight of 30 kD. A computer-assisted search of a protein sequence data base revealed homology with two other esterases, acetylcholinesterase of Torpedo and esterase-6 of Drosophila. Homologous region were centered around presumptive active sites, suggesting that the catalytic domains of the esterases are conserved during evolution. Three genomic clones of this gene were also isolated and characterized by restriction mapping. At least ten exons were distributed over a 35-kb (kilobase pair) region; each exon contained an average of 100 basepairs (bp). A polymorphic site for Apa I, located within an intron of the esterase D gene, can be used to identify chromosome 13 carrying defective RB alleles within retinoblastoma families.

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