Definition of the limits of the Wilms tumor locus on human chromosome 11p13

In a previous report, we described a contiguous restriction map of chromosome band 11p13 that localized the Wilms tumor locus to a small group of NotI fragments. In an effort to identify and isolate the 11p13-associated sporadic Wilms tumor locus, we developed a panel of NotI fragment-specific DNA probes. These probes were selected from genomic libraries constructed using the Chinese hamster ovary-human somatic cell hybrid carrying only human 11p. The libraries were prepared from NotI-digested DNA after size selection by pulsed-field gel electrophoresis. The selected NotI fragments had been previously targeted on the basis of deletion mapping as having a high probability of containing the Wilms tumor locus. We used these newly identified 11p13-specific probes to improve the resolution of the restriction map spanning the Wilms tumor locus. The locus has been defined by a homozygous deletion in a sporadic Wilms tumor. Using these probes, the region of homozygous deletion in this tumor and presumably all or part of the Wilms tumor gene have been confined to two small SfiI fragments spanning less than 350 kb.     

Human chromosome 17 NotI linking clones and their use in long-range restriction mapping of the Miller-Dieker chromosome region (MDCR) in 17p13.3

A NotI linking library constructed from flow-sorted human chromosome 17 material was screened to aid in construction of a long-range restriction map of the Miller-Dieker chromosome region (MDCR) in 17p13.3. A total of 66 clones were mapped to one of eight regions of chromosome 17 using a somatic cell hybrid panel, and 44/66 (67%) of these clones cross-hybridized to rodent DNA on Southern blots. Of these, 24 clones were tested and all mapped to mouse chromosome 11, the homolog of human chromosome 17. Four linking clones mapped to 17p13.3 and were used for pulsed-field gel electrophoresis studies along with six other anonymous probes previously mapped to this region. Clone L132 was found to be deleted in all Miller-Dieker patients tested (n = 15) and therefore lies within the critical region for this disorder. It detects two NotI fragments (180 and 320 kb), one of which (320 kb) was shared by YNZ22 and YNH37, two probes previously shown to be co-deleted in all patients with the Miller-Dieker syndrome (MDS). These results indicate that all MDS patients share a minimum deletion region of greater than 370 kb. Two other NotI clones, L53 and L125, mapped telomeric to the MDS critical region and share a 600-kb MluI fragment with each other and with YNZ22/YNH37. This provides a 930-kb MluI map that encompasses the distal boundary of the MDS critical region but does not include the proximal boundary. A total of over 2 Mbp is represented in the MluI fragments by probes in subband p13.3, a cytogenetic region estimated to be 3-4 Mbp.     

Structural organization of the human p53 gene. I. Molecular cloning of the human p53 gene

Human p53 gene was cloned from the normal human placenta DNA and DNA from the strain of human kidney carcinoma transplanted into nude mice. Representative gene library from tumor strain of human kidney carcinoma and library of 15 kb EcoRI fragments of DNA from normal human placenta were constructed. Maniatis gene library was also used. Five clones were isolated from kidney carcinoma library; they covered 27 kb and included full-length p53 gene of 19.5 kb and flanking sequences. From normal placenta libraries three overlapped clones were obtained. Restriction map of cloned sequences was constructed and polarity of the p53 gene determined. The first intron of the gene is large (10.4 kb); polymorphic BglII site was observed in this intron, which allows to discriminate between allelic genes. One of these (BglII-) is ten times more abundant that the other (BglII+). Both allelic genes are able to synthesize the 2.8 kb p53 gene.     

Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation.

Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation. The tumor suppressor gene, p53, is mutated in approximately 50% of human sporadic cancers and in an inherited cancer predisposition (Li-Fraumeni syndrome). We have analyzed the status of the wild-type p53 allele in tumors taken from p53-deficient heterozygous (p53+/-) mice. These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild-type p53. We present evidence that a high proportion of the tumors from the p53+/- mice retain an intact, functional, wild-type p53 allele. Unlike p53+/- tumors which lose their wild-type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following gamma-irradiation, activates p21(WAF1/CIP1) and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild-type p53), shows high levels of binding to oligonucleotides containing a wild-type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization. These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis.     

Isolation and characterization of a human p53 cDNA clone: expression of the human p53 gene.

A cDNA clone for human p53 cellular tumor antigen has been isolated and characterized. This clone contains the complete 3'-untranslated region and most of the open reading frame for the protein. Nucleotide sequence analysis revealed that p53 mRNA contains an Alu repeat in the 3'-untranslated region. Hybridization selection experiments showed this clone was capable of selectively binding p53 mRNA. In vitro translation of SV80 mRNA resulted in the synthesis of two immunoreactive p53 polypeptide species. Northern blot analysis showed that human p53 mRNA was 2.8 kb in length and was present in cell lines containing high and low levels of p53 protein. There appears to be only a single p53 gene in human cells and Southern blot analysis demonstrated no major genomic rearrangements or amplification of the p53 gene in the transformed cell lines examined.     

A sequence-ready BAC clone contig of human chromosome 10p15 spanning the loss of heterozygosity region in glioma

Deletion of chromosome 10 is one of the most common chromosomal alterations in glioma. At 10p15, the telomeric region of the short arm of chromosome 10, loss of heterozygosity (LOH) has been frequently observed by microsatellite analysis, suggesting the presence of a tumor suppressor gene. We examined LOH in 34 gliomas on chromosome 10, and frequent LOH on 10p was detected on 10p15, in agreement with deletion mapping studies on chromosome 10. We then constructed a bacterial artificial chromosome (BAC) clone contig covering the critical region, which spanned the interval between D10S249 and D10S533 on 10p15. The map contained 68 BAC clones connected by 74 sequenced tag sites (STSs) and covered approximately 2.7 Mb, with one gap. A total of 74 STSs, including 6 microsatellite markers, 29 expressed sequenced tags (ESTs), and 39 BAC end STSs, were physically arranged. Twenty-eight ESTs were mapped in the interval between D10S249 and D10S559 (approximately 1200 kb), and another EST was mapped in the interval between D10S559 and D10S533 (approximately 1300 kb). This sequence-ready BAC clone contig map will be a basic resource for high-quality sequencing and positional cloning of the putative tumor suppressor gene at 10p15 in glioma.     

The structure and expression of the murine wildtype p53-induced phosphatase 1 (Wip1) gene

The human wildtype p53-induced phosphatase 1 (Wip1; GenBank symbol Ppm1d) gene encodes a type 2C protein phosphatase (PP2C) that is induced by ionizing radiation in a p53-dependent manner. We have cloned and sequenced the mouse Wip1 gene and its encoded mRNA. The mouse Wip1 gene is composed of six exons and spans over 36 kb of DNA. The mouse cDNA sequence predicts a 598-amino-acid protein with a molecular mass of roughly 66 kDa. Comparison of human and mouse Wip1 sequences revealed 83% overall identity at the amino acid level. The 5'-flanking region of exon 1 had promoter elements characteristic of a housekeeping gene. The Wip1 coding sequences share conserved functional regions with other PP2Cs from a diverse array of species. Expression of Wip1 mRNA was detected ubiquitously in adult and embryonic tissues, though expression in the testis was much higher than in other tissues. Wip1 has been mapped near the p53 gene on mouse chromosome 11.     

The telomeric 60 kb of chromosome arm 4p is homologous to telomeric regions on 13p, 15p, 21p, and 22p.

A telomere YAC clone containing the most distal 115 kb of chromosome arm 4p has been previously isolated. This clone is of particular interest as it spans a potential candidate region for the Huntington disease gene. The YAC was subcloned into a phage vector, and a high-resolution restriction map extending to within 13 kb of the telomere was constructed. In situ hybridization of the YAC to human metaphase spreads gives a peak of hybridization on 4pter but also an increase in the number of signals close to several other telomeres. Where possible, these results were investigated further by the hybridization of probes from the YAC to somatic cell hybrids containing single human chromosomes. This analysis indicates that the most telomeric 60 kb of chromosome arm 4p is homologous to telomeric regions on 13p, 15p, 21p, and 22p. The extent of this homology makes it less likely that the mutation for Huntington's disease is located within the telomere YAC clone.     

Deletion of one copy of the p16INK4A tumor suppressor gene is implicated as a predisposing factor in pediatric leukemia

The p16INK4A tumor suppressor gene is frequently disrupted by mutation or deletion in a wide range of cancer types, ranging from leukemia to cancers of the bladder, skin, lung, liver, and spleen. We have previously shown that deletion of at least one copy of the p16INK4A gene is associated with an increased risk of relapse in pediatric leukemia. Our data suggest that hemizygous p16INK4A deletion may be constitutional, conferring susceptibility to leukemia. Confirmation of this association is worthy of a larger study. Data from primary leukemia specimens are also presented here which examined the possibility that the remaining allele of the gene was inactivated by another mechanism such as mutation or was silenced by methylation. These possibilities were formally excluded in a case of hemizygous loss of the p16INK4A gene in leukemia, establishing that in this case the p16INK4A deletion was either semidominant or fully haploinsufficient for relapse susceptibility in this disease. Implementation of high throughput methods such as those used here for detecting hemizygous loss of tumor suppressor genes will become increasingly important for molecular diagnosis of cancer. This is particularly true for the emerging class of tumor suppressor genes where deletion of one allele is sufficient to confer cancer susceptibility or poor prognosis with standard treatment.     

Human breast cancer: frequent p53 allele loss and protein overexpression

A sample of 114 primary breast tumors and corresponding constitutional DNA were tested for loss of heterozygosity (LOH) of the YNZ22 and p53 genes, both located in the 17p13 region. Loss of the p53 allele was found in 28 of 44 primary breast carcinomas (64%). In contrast LOH in only 26 of 61 tumors (43%) was detected with the variable number of tandem repeats (VNTR) probe YNZ22 mapping at 17p13.3 close to the p53 locus at 17p13.1. Among 19 tumors informative for both probes allele loss at 17p13.3 never occurred without p53 involvement. These data suggest, that p53 is the target of 17p13 allelic deletions in human breast cancer. Immunohistochemistry showed overexpression of the p53 protein in 25 of 50 cases (50%) presumably reflecting activating point mutations. Overexpression was not correlated with allele loss but seemed to be closely related to the presence of point mutations in this study. No homozygous deletions or rearrangements of the p53 gene were detected. This would argue for an important role of heterozygous p53 mutations in human breast cancer.     

Analysis of the gene coding for the murine cellular tumour antigen p53

A genomic clone containing the functional gene for the murine p53 cellular tumour antigen was isolated and structurally characterised. The gene contains at least 11 exons and 10 introns, the first intron possessing a length of 6.1 kb. Attempts to determine the exact 5' end of p53 mRNA were inconclusive, probably due to the presence of a remarkable stem and loop structure (delta G degrees approximately equal to -56 kcal/mol) in the 5' region of the gene. Suggestive similarities were found to exist between p53 and the protein product of the myc oncogene.     

An integrated physical map of 8q22-q24: use in positional cloning and deletion analysis of Langer-Giedion syndrome

We have developed an integrated map for a 35-cM area of human chromosome 8 surrounding the Langer-Giedion syndrome deletion region. This map spans from approximately 8q22 to 8q24 and includes 10 hybrid cell intervals, 89 polymorphic STSs, 118 ESTs, and 37 known genes or inferred gene homologies. The map locations of 25 genes including osteoprotegerin, syndecan-2, and autotaxin have been refined from the general locations previously reported. In addition, the map has been used to indicate the location of nine deletions in patients with Langer-Giedion syndrome and trichorhinophalangeal syndrome type I to demonstrate the potential usefulness of the map in the analysis of these complex syndromes. The map will also be of interest to anyone trying to clone positionally disease genes in this region, such as Cohen syndrome (8q22-q23), Klip-Feil syndrome (8q22.2), hereditary spastic paraplegia (8q24), and benign adult familial myoclonic epilepsy (8q23.3-q24.1).     

Vectorettes for long chromosome walking in genomic DNA of the human p53 gene

Chromosome walking in mammalian DNA by vectorette PCR is not always very specific, and the walks have been limited to distances <1 kb. To improve the method, we have designed new vectorettes, which unlike the currently used ones have very little repetitive sequences or homology with known DNA sequences of various origins in the data banks. We have tested these new vectorettes for chromosome walking in human p53 tumor suppressor gene, human tissue-type plasminogen activator gene, and mouse stanniocalcin gene with good success. In chromosome walking of the human p53 gene, we isolated gene-specific fragments of 2.4. kb, and by walking in a bacterial artificial chromosome (BAC) clone carrying the mouse stanniocalcin gene, we isolated fragments up to about 7 kb in size. We further sequenced the 5' region of the p53 gene and found that the nucleotides upstream of -1009 are transcribed in antisense orientation into a messenger RNA (mRNA) (flj10385) encoding a putative serine/threonine kinase.     

The physical organization of the human immunoglobulin heavy chain gene complex.

Two dimensional DNA electrophoresis (2D-DE) was used to map the variable (VH) region of the human heavy chain immunoglobulin gene cluster. Seventy-six VH gene segments were mapped to specific SfiI, BssHI and NotI fragments by 2D-DE. We have determined that a common insertion/deletion polymorphism of 80 kb, involving three VH gene segments, occurs in the VH region. The physical map suggests that the evolution of the human IGH gene complex involved duplication of blocks containing different VH families. This physical map will allow comparison of the usage of VH loci in human ontogeny with their proximity to the CH region. Knowledge of the germline repertoire of a particular DNA source studied in essential as the number of the dispersed VH gene segments of VH families, especially of the VH5 family, is variable. 2D-DE, as illustrated here for the IGH gene cluster, has general application in the development of large scale physical maps of gene and repeat families.     

Characterization of a 500-kb contig spanning the region between c-Ha-Ras and MUC2 on chromosome 11p15.5

The 11p15.5 region is associated with a broad range of diseases, including childhood acute myeloid leukemia; non-small cell lung carcinoma; arthrogryposis multiplex congenita, distal type 2B; and bladder cancer. Since targets for these diseases are unknown, we have constructed a physical map consisting of BAC and PAC clones spanning the region from the HRAS1 gene to the cluster of mucin genes on 11p15.5. The contig spans approximately 500 kb and includes 13 genes (9 novel), 9 STSs (5 novel), and 1 SNP and builds upon a published physical map spanning the region from the telomere to the HRAS gene. In addition, we expand the mucin gene cluster located on 11p15.5 to include a novel mucin-like gene (MUCDHL) located less than 250 kb telomeric to MUC6. The identification of potential disease genes within an organizational and evolutionary context provides valuable clues to function and as such will benefit our understanding of this region of the genome.     

A physical map of the human Y-chromosome short arm

A physical map of the Y-chromosome short arm was constructed using DNA probes p19B, Y-286/la5, pZFY, Y-280, and Y-227. These probes hybridize with four NotI fragments of 400 kb (p19B and Y-286/la5), 350 kb, 1.9 Mb, and 3.0 Mb, respectively. The restriction fragments were shown to be adjacent to each other by analysis of NotI partial digests, overlapping restriction fragments, and/or the detection of rearranged restriction fragments in a 46,XX male. The present map covers approximately 5.6 Mb of contiguous DNA of Yp. Previously, the size of the pseudoautosomal region was estimated to be 2.3 Mb, and a 5.3-Mb NotI fragment containing Y-specific repeated DNA was assigned to proximal Yp. These and the present data account for approximately 13 Mb and thus for most of the DNA content of the Y short arm.     

Loss of p53 protein during radiation transformation of primary human mammary epithelial cells.

The causative factors leading to breast cancer are largely unknown. Increased incidence of breast cancer following diagnostic or therapeutic radiation suggests that radiation may contribute to mammary oncogenesis. This report describes the in vitro neoplastic transformation of a normal human mammary epithelial cell strain, 76N, by fractionated gamma-irradiation at a clinically used dose (30 Gy). The transformed cells (76R-30) were immortal, had reduced growth factor requirements, and produced tumors in nude mice. Remarkably, the 76R-30 cells completely lacked the p53 tumor suppressor protein. Loss of p53 was due to deletion of the gene on one allele and a 26-bp deletion within the third intron on the second allele which resulted in abnormal splicing out of either the third or fourth exon from the mRNA. PCR with a mutation-specific primer showed that intron 3 mutation was present in irradiated cells before selection for immortal phenotype. 76R-30 cells did not exhibit G1 arrest in response to radiation, indicating a loss of p53-mediated function. Expression of the wild-type p53 gene in 76R-30 cells led to their growth inhibition. Thus, loss of p53 protein appears to have contributed to neoplastic transformation of these cells. This unique model should facilitate analyses of molecular mechanisms of radiation-induced breast cancer and allow identification of p53-regulated cellular genes in breast cells.