Loss of heterozygosity on the long arm of human chromosome 7 in sporadic renal cell carcinomas.

Cytogenetic and molecular analysis of DNA sequences with highly polymorphic microsatellite markers have implicated allele loss in several chromosomal regions including 3p, 6p, 6q, 8p, 9p, 9q, 11p and 14q in the pathogenesis of sporadic renal cell carcinomas (RCCs). Deletions involving the long arm of chromosome 7 have not been described in RCCs although they have been seen in several other tumor types. However, there have been no detailed analysis of loss of heterozygosity (LOH) of 7q sequences in sporadic RCCs. We therefore studied LOH for DNA sequences on 7q with 10 highly polymorphic markers in 92 matched normal/tumor samples representing sporadic RCCs including papillary, nonpapillary, and oncocytomas in order to determine whether allelic loss could be detected in a tumor type with no visible 7q rearrangements at the cytogenetic level. We found chromosome 7q allele loss in 59 of 92 cases (64%) involving one, two, or more microsatellite markers. The most common allele loss included loci D7S522 (24%) and D7S649 (30%) at 7q31.1-31.2, a region that contains one of the common fragile sites, FRA7G. By comparative multiplex PCR analysis, we detected a homozygous deletion of one marker in the 7q 31.1-31.2 region in one tumor, RC21. These results support the idea that a tumor suppressor gene in 7q31 is involved in the pathogenesis of sporadic renal cell carcinomas.

Mutations in the arginine-rich protein gene (ARP) in pancreatic cancer.

The ARP gene encodes a highly conserved arginine-rich protein from chromosomal band 3p21.1. At the cytogenetic level this region is frequently deleted in a variety of different solid tumors, although not in pancreatic cancer. We have reported the presence of a specific mutation (ATG50-->AGG) or deletion of codon 50 of the ARP gene in different tumor types (Shridhar et al., 1996, 1996a). In the present study, we have observed mutations involving codon 50 in 11 of 37 pancreatic tumors. The frequency of codon 50 mutation is roughly the same in pancreatic tumors as in the other types of tumors previously examined. In addition, we have detected mutations at codon 51 in multiple PCR subclones in two other pancreatic tumors. Mutations in the ARP gene are thus commonly observed in pancreatic cancer, as well as many other cancers.

Frequent breakpoints in the region surrounding FRA3B in sporadic renal cell carcinomas.

The constitutive fragile site at chromosomal band 3p14.2, FRA3B, is the most active common fragile site in the human genome. We have localized aphidicolin-induced breakpoints to two distinct clusters, separated by 200 Kb, in FRA3B (Paradee et al., 1996). Sequence analysis of these regions identified two polymorphic microsatellite markers immediately adjacent to each of these breakpoint clusters. In this report we have used these two new microsatellites and 14 additional 3p microsatellites to analyse chromosome 3p breakage and loss in 94 sporadic RCC samples, including nonpapillary, papillary and oncocytomas. We have found heterozygous loss of 3p14 sequences in >60% of the RCC samples, including both clear cell and papillary renal cell carcinomas. We have found frequent breakage in the region immediately surrounding FRA3B, demonstrating that FRA3B does play a role in chromosome breakage and loss in RCC. In contrast to other reports, >50% of the papillary tumors also showed LOH of 3p markers. We also observed microsatellite instability (MIN) with most of the tested markers in seven of eight oncocytomas and one of 69 clear cell carcinomas. The MIN in some oncocytomas was of the RER+ (replication error) type I phenotype. None of the five 3p14.2 markers detected any homozygous deletions in tumor samples, but 69/94 (73%) of the tumors had LOH for the region, which includes the recently identified FHIT gene.

Mutations in the arginine-rich protein gene, in lung, breast, and prostate cancers, and in squamous cell carcinoma of the head and neck.

Arginine-rich protein (ARP) is a highly conserved gene that maps to human chromosomal band 3p21.1. This gene contains an imperfect trinucleotide repeat which encodes a string of arginines. We previously detected a specific mutation (ATG50-->AGG) within this region of the gene in 10 of 21 sporadic renal cell carcinomas. Here, we report the detection of the same mutation in 5 of 21 squamous cell carcinomas of the head and neck, 1 of 2 small cell lung cancer cell lines, 6 of 18 non-small cell lung carcinomas, 9 of 22 breast tumors, and 5 of 13 prostate tumors. This mutation was seen in several early stage tumors and may thus be an early event in tumorigenesis. We also detected a mutation at codon 53 of this gene in both primary and metastatic tumors from one patient. Other nucleotide changes were observed in a few PCR subclones, but their frequency was the same in both tumor and control samples, suggesting that many of these changes were PCR or subcloning artifacts rather than mutations in the tumor cells themselves.

A gene from human chromosomal band 3p21.1 encodes a highly conserved arginine-rich protein and is mutated in renal cell carcinomas.

We have identified a gene, called ARP for Arginine-rich protein, in human chromosomal band 3p21. It is approximately 600 Kb telomeric to the ACY1 locus (Miller et al., 1989) and encodes a previously unidentified 234 amino acid long, highly basic protein. This gene is highly conserved at the DNA and RNA level. It is found in all species including hamster, rat, mouse, bovine and yeast. We have detected a point mutation (ATG50 to AGG) or deletion of ATG50 in 10 of 21 sporadic renal cell carcinomas. The mutable region is in an imperfect trinucleotide repeat in the coding region which is non-polymorphic among 50 normal individuals examined. The point mutation (ATG50 to AGG) or deletion of codon 50 removes a methionine and increases the stretch of arginines encoded by the AGG repeats in the ARP gene.

Isolation of two contigs of overlapping cosmids derived from human chromosomal band 3p21.1 and identification of 5 new 3p21.1 genes.

Consistent loss of DNA sequences from several regions on the short arm of human chromosome 3 has suggested that multiple tumor suppressor genes reside on chromosome 3p in various types of cancer cells. We have focused our efforts on an analysis of chromosomal band 3p21.1 since aminoacylase-1 (ACY1), which is localized to this band, has been shown to have lower levels of expression in several small cell and non-small cell lung cancer cell lines. Starting with two cosmids within 3p21.1, D3S92 and D3S93, we have isolated two separate contigs of overlapping cosmids within 3p21.1, by screening a library of 5700 chromosome 3-specific cosmid clones. Detailed restriction maps for these two contigs show that they contain multiple clusters of rare cutting restriction endonuclease sites. One contig extends for 100 kb and encompassed both ACY1 and D3S92, and the other extends about 80 kb around the D3S93 locus. Many different restriction fragments derived from these two contigs were found to be evolutionarily conserved and hybridized to distinct message transcripts. These fragments were used to identify homologous cDNAs from an adenogastric cDNA library, and several of these cDNAs were partially sequenced. We have identified five new genes from these two contigs and there is evidence to suggest that several additional genes reside within these cosmid contigs. The genes identified from 3p21.1 were then hybridized to DNA, isolated from a series of lung cancer cell lines and matched normal and tumor DNA from lung cancer patients. No alterations were detected with any of these probes, both at the DNA or RNA levels. A similar analysis with DNA fragments derived from these two genomic regions also failed to detect any alterations.

Seven genes on the short arm of human chromosome 3 map to two regions on Macropus eugenii (tammar wallaby) chromosome 2.

Seven genes were mapped by in situ hybridization to metaphase chromosomes of the marsupial species Macropus eugenii, using a series of human-derived cloned probes (six cosmids and one cDNA). The genes were located in two widely separated clusters on the long arm of M. eugenii chromosome 2, in contrast to their location in a single cluster on the distal half of the short arm of human chromosome 3. Multiple rearrangements had to be involved in the evolutionary divergence of these chromosome segments from the unknown arrangement in the common ancestor.

Lamin A/C gene and a related sequence map to human chromosomes 1q12.1-q23 and 10.

Lamins A and C are products of alternate splicing of one transcript from a single gene. We have isolated a partial cDNA, cE1-2, whose 800-bp sequence is 99% identical to the 3' untranslated region of the lamin A/C gene. We report here the mapping of this gene and a closely related sequence to human chromosomes 1 and 10, more specifically, to 1q12.1-q23. The localization of cE1-2 hybridizing sequences to two different chromosomes suggests that one of these loci represents an as yet unknown member of the lamin gene family, either a pseudogene or an expressed gene.

Methylation of the 5' flanking sequences of the ribosomal DNA in human cell lines and in a human-hamster hybrid cell line.

In a human lymphoblastoid cell line (Z83) in which rDNA genes on chromosome 22 are amplified but transcribed at a low level, immunocytological studies with antibodies to 5 methylcytidine provided evidence for hypermethylation of the rDNA. The extent of methylation of the 5' flanking sequences of the ribosomal DNA was examined by comparing the size of restriction fragments obtained by digestion of genomic DNA with EcoRI and HpaII or EcoRI and MspI. Southern blots indicated hypermethylation of the 5' flanking sequences of many copies of rRNA genes in these cells, but not in a control lymphoblastoid cell line without rDNA amplification. Results obtained with a somatic hybrid human-hamster cell line, in which the rRNA genes on the single human chromosome 22 are inactive, showed that only a small fraction of the CCGG sites in the 5' flanking sequences of the transcriptionally silent rRNA genes in this hybrid were methylated. Since inactive rRNA genes can show such a minimal level of methylation, it is likely that the extreme hypermethylation of the amplified rRNA genes in Z83 occurred in association with their inactivation rather than following it.

Traditional and molecular cytogenetics.

Traditional cytogenetic methods have relied on tissue culture techniques to generate adequate mitotic cells for the analysis of chromosome disorders for prenatal diagnosis. Chromosome banding techniques allow the evaluation of mitotic cells for structural and numerical aberrations and define the nature of any rearrangement. With the advent of fluorescent in situ hybridization methodology, which combines the molecular technologies of chromosome-specific probes and in situ molecular hybridization, it has become possible to analyze chromosomal numerical and structural aberrations from interphase cells. The use of molecular cytogenetic techniques should greatly increase the speed and diagnostic resolution of clinical specimens.

Isolation and characterization of a mouse subtelomeric sequence.

A mouse subtelomeric sequence, ST1, was generated from genomic DNA of the mouse HR9 (129/Sv origin) cell line by the polymerase chain reaction (PCR) using a single telomeric primer. ST1 was cloned and characterized: it is composed of 670 bp of novel DNA sequence flanked on each end by inverted telomeric hexanucleotide repeats (TTAGGG)n. PCR amplification from BALB/c mouse DNA using this single primer gave the same major product. Southern analysis and PCR using internal ST1 primers confirmed that the ST1 sequence is present in mouse genomic DNA. In situ hybridization to metaphase chromosomes of SJL origin mapped ST1 to many, if not every, mouse telomere. PCR experiments using different combinations of the telomeric, minor satellite, and ST1 primers indicated that some ST1 copies are adjacent to minor satellite sequences, that telomeric and ST1 sequences are not generally interspersed with minor satellite sequences, and that ST1 and the minor satellite have a consistent and specific orientation relative to each other and to the telomere.

The human loci DNF15S2 and D3S94 have a high degree of sequence similarity to acyl-peptide hydrolase and are located at 3p21.3.

The short arm of chromosome 3 undergoes genetic loss in most small-cell lung cancers and renal cell carcinomas. The most frequently deleted region includes the DNF15S2 locus (mapped to 3p21), suggesting that a putative recessive tumor-suppressor gene might be located nearby. A cosmid clone, cA476, contains the D3S94 locus and two HTF islands and detects a PstI RFLP. We have isolated cDNAs homologous to conserved fragments within cA476; and these cDNAs have 96% sequence similarity to a cDNA derived from the DNF15S2 locus. Sequence information from cDNAs derived from both the rat and pig acyl-peptide hydrolase (E.C.3.4.19.1) gene show that they have a high degree of sequence similarity to cDNAs derived from D3S94 and DNF15S2, suggesting that they are all the same locus. Cosmid cA476 (DNF15S2) has been mapped, by fluorescent in situ hybridization, to chromosome 3p21.3. D3S94 and DNF15S2 are quite distinct from aminoacylase 1 (ACY1), which has been physically linked to D3S2, D3S92, and D3S93, all localized within 3p21.1.

Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.

Comparative mapping of a gorilla-derived alpha satellite DNA clone on great ape and human chromosomes.

We have isolated an alpha satellite DNA clone, pG3.9, from gorilla DNA. Fluorescence in situ hybridization on banded chromosomes under high stringency conditions revealed that pG3.9 identifies homologous sequences at the centromeric region of ten gorilla chromosomes, and, with few exceptions, also recognizes the homologous chromosomes in human. A pG3.9-like alphoid DNA is present on a larger number of orangutan chromosomes, but, in contrast, is present on only two chromosomes in the chimpanzee. These results show that the chromosomal subsets of related alpha satellite DNA sequences may undergo different patterns of evolution.

Isolation of large numbers of chromosome 3-specific cosmids containing clusters of rare restriction-endonuclease sites.

We tested 519 chromosome 3-specific cosmids for the presence of rare restriction-endonuclease sites in a search for cosmids containing HTF islands. We have identified 49 cosmids (9% of those tested) that contain multiple rare restriction-endonuclease sites. The cosmids were digested with several common cutting restriction endonucleases to liberate small fragments which were tested as unique-sequence chromosome 3-specific hybridization probes and for evolutionary sequence conservation. Unique-sequence hybridization probes isolated from the cosmids were hybridized to a somatic cell hybrid deletion mapping panel to subchromosomally localize the cosmids. Fragments from many of these cosmids demonstrated conservation of sequence through evolution, and these fragments hybridize to distinct transcripts. These cosmids should therefore prove a useful resource for the identification of many chromosome 3-specific genes, in addition to having potential use as linking clones for pulsed-field gel mapping studies.

Isolation of a variant family of mouse minor satellite DNA that hybridizes preferentially to chromosome 4.

Two cosmids (HRS-1 and HRS-2) containing mouse minor satellite DNA sequences have been isolated from a mouse genomic library. In situ hybridization under moderate stringency conditions to metaphase chromosomes from RCS-5, a tumor cell line derived from the SJL strain, mapped both HRS-1 and HRS-2 to the centromeric region of chromosome 4. Sequence data indicate that these cloned minor satellite DNA sequences have a basic higher order repeat of 180 bp, composed of three diverged 60-bp monomers. Digestion of mouse genomic DNA with several restriction enzymes produces a ladder of minor satellite fragments based on a 120-bp repeat. The restriction enzyme NlaIII (CATG) digests all the minor satellite DNA into three prominent bands of 120, 240, and 360 bp and a weak band of 180 bp. Thus, the majority of minor satellite sequences in the genome are arranged in repeats based on a 120-bp dimer, while the family of minor satellite sequences described here represents a rare variant of these sequences. Our results raise the possibility that there may be other variant families of minor satellites analogous to those of alphoid DNA present in humans.

A chimpanzee-derived chromosome-specific alpha satellite DNA sequence conserved between chimpanzee and human.

We describe a cloned 2.7 kb alpha satellite sequence, Pan-3, from the pygmy chimpanzee (Pan paniscus) that specifically hybridizes in situ to chromosome 19 in the pygmy chimpanzee and to the homeologous human chromosome, no. 17. Using high stringency conditions of hybridization on Southern blots, this sequence hybridized to DNA from both species of chimpanzee (P. paniscus and P. troglodytes) and from human but not to DNA from gorilla (Gorilla gorilla) or orangutan (Pongo pygmaeus). Partial sequence analysis showed that Pan-3 and a previously described human chromosome 17-specific clone have up to 91% sequence identity. To our knowledge this is the highest sequence similarity reported between alphoid subsets from human and any other primate.

Chromosome-specific subsets of human alphoid DNA identified by a chromosome 2-derived clone.

We have cloned an alphoid DNA fragment, pBS4D, from the DNA of a human-hamster hybrid cell line containing chromosome 2 as its only cytologically detectable human component. Under high stringency conditions, pBS4D hybridized in situ mostly to chromosome 2 and to a lesser extent to chromosomes 18 and 20. Restriction analysis using the DNA from selected somatic hybrid cell lines revealed that the genomic organization of this alphoid DNA differs on each of these three chromosomes.