Introduction

Deletions and other aberrations on the short arm of human chromosome 3 occur frequently in renal cell carcinoma [1, 2], lung cancer, breast and other malignancies [3–7]. Cancer epidemiologic data suggest that these malignancies develop because the deletions remove one or more tumor suppressor genes (TSG) located in this region. The search for this TSG or TSGs is, however, hampered by the large size of the deletions which cover practically the whole of the short arm of chromosome 3, that is about 100 Mb.

A detailed physical and genetic map of this region would greatly facilitate these studies, and help to understand the molecular mechanisms of carcinogenesis. Several different types of genetic markers are presently used for obtaining such maps. Many so-called anonymous markers represent randomly cloned DNA fragments. Other DNA markers possess specific features, such as the presence of known genes or expressed sequences of unknown function, CpG islands, etc. The ideal marker should contain: (1) a gene (expressed sequence); (2) a CpG island, which has been shown to be conserved in the genome and can be used for comparative gene mapping in different species; (3) a rare cutting enzyme restriction site useful for physical mapping; (4) polymorphic sequences, such as micro-satellites.

NotI-linking clones, located at CpG islands, fulfil these criteria, as a majority of them mark transcribed sequences which are easily detected by Northern blot hybridization [8]. Thus NotI-linking clones can link physical and transcriptional maps. Many of these clones contained repetitive sequences and may represent microsatellite loci. The majority of these NotI-linking clones represent evolutionary conserved DNA fragments which can be used for comparative genome mapping of other mammalian species. We have isolated over 2,000 chromosome-3-specific NotI-linking clones [9] and used them to establish a NotI map. It is estimated that this chromosome contains around 250 NotI sites.

There are several steps in the construction of a NotI map. The first is low-resolution mapping of NotI-linking clones using a panel of interspecific somatic cell hybrids and chromosomal localization using fluorescence in situ hybridization (FISH). The second step is fine mapping and ordering of probes using two or three colour FISH on metaphase (prometaphase) chromosomes and interphase nuclei, with a resolution up to 50–100 kb [10]. The third step combines several approaches, including fiber FISH [11] connection of NotI-linking clones to YAC, P1 and cosmid contigs, and to a genetic and transcription map and finally, pulsed-field gel electrophoresis. In this work, we present the ordering, regional localization and characterization of 23 unique NotI-linking clones that should facilitate the construction of a comprehensive physical and genetic map of human chromosome 3.

Materials and Methods

Isolation and Sequencing of Chromosome-3-Specific NotI-Linking Clones

Most of the clones used here were isolated from the chromosome-3-specific NotI-EcoRI (NRL1) library, while the others were derived from the chromosome-3-specific NotI-BamHI NL1 and NL2 libraries. The construction of these libraries and isolation of clones have been described previously [9, 12]. Preliminary mapping of all clones utilized a panel of somatic cell hybrids [13].

Sequencing was done using A.L.F. sequenator according to the manufacturers’ protocols. Sequencing was done from both sides of NotI sites using reverse (r) and SK-direct (d) primers.

  • Reverse primer: 5′-CAGGAAACAGCTATGAC-3′,

  • SK-direct primer: 5′-GGATGTGCTGCATCGACTCTA-3′.

Fluorescence in situ Hybridization

Metaphase spreads were obtained by standard techniques from phytohemagglutinin-stimulated human lymphocytes.

In situ hybridization and detection were performed as described previously [10, 11, 14]. For NotI clones we used 80–160 ng labelled DNA probe and 4–10 µg Cot1 DNA (BRL) per 11 µl hybridization mixture (50% formamide, 2 × SSC, 10% dextran sulfate, 0.1% Tween 20). For reverse painting we used 1 µg biotin-labelled total MCH 903.1 DNA and 20 µg Cot1 DNA per 6 µl hybridization mixture. DAPI or propidium iodide were used for chromosome counter-staining and banding. Sometimes, chromosomes were restained by the GTG-banding method for detailed signal mapping as described previously [15]. The signals were visualized with a Zeiss Axiophot fluorescence microscope equipped with a cooled CCD camera (Photometrics or Hamamatsu), and analyzed using the Smartcapture software (Digital Scientific, Cambridge, UK).

At least 20 metaphase spreads with paired signals on each copy of chromosome 3 were analysed for each probe.

In cases when several NotI-linking clones were mapped to the same chromosomal band, two- or three-color FISH on metaphase chromosomes was used to establish the correct order of the clones along the chromosome.

Results and Discussion

Three hundred thousand clones from the NRL1 library were screened with total human DNA labeled with 32P. This library was constructed using the restriction enzymes NotI and EcoRI, in order to increase the representation of human chromosome-3-specific NotI-linking clones. All previous human NotI-linking clones from chromosome 3 had been isolated from libraries constructed using the restriction enzymes NotI and BamHI [8, 12, 13, 15]. One hundred clones hybridizing strongly to human DNA were selected and sequenced as described previously [16]. Sixteen unique NotI-linking clones, not present in previous screens, were selected. These clones were mapped first using somatic cell hybrids and then by FISH (table 1).

Table 1 General information about NotI-linking clones used in this work
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The clones were localized fairly evenly on chromosome 3, but three regions were underrepresented: 3p21.3, 3p13-q13 and 3q26. To exclude the possibility of deletions of chromosome 3 in the MCH903.1 cell line [13], reverse FISH painting was performed using DNA from the cell line on normal metaphase chromosomes. No visible deletions of chromosome 3 were found (data not shown).

Since region 3p14-22 is believed to contain TSGs, we decided to include additional NotI-linking clones from this region into the study.

We previously isolated NotI-linking clones from bands 3p14-22, using modified Alu-PCR approaches [9, 17]. Seven of these clones (AP20, NL1-232, NL1-041, NL1-320, NL2-157, NL1- 210 and NL1-358) were then incorporated into this work.

Thus all these 23 NotI-linking clones have been mapped on human chromosome 3 (table 1). Then they were ordered using two- or three-color FISH on chromosomes (tables 2, 3, fig. 1A, B). Final results of the ordering experiments are presented in figure 2 and tables 1–3.

Fig. 1
figure 1

Ordering of NotI-linking clones using two- and three-color FISH. a Two-color hybridization on metaphase chromosomes. The probe NL1-041 was labeled with digoxigenin and detected using FITC conjugated anti-digoxigenin antibody (green signal), NL2-157 and human chromosome 3 specific centromere probes were labeled with biotin and detected using Texas Red-conjugated avidin (red signals), chromosomes were counterstained with DAPI. (Original color photograph.) b Three-color hybridization on metaphase chromosomes. NL1-320 probe was labeled with digoxigenin and detected using FITC-conjugated anti-digoxigenin antibody (green signal), NL2-157 probe was labeled with biotin and detected using Texas Red conjugated avidin (red signal), NL1-210 probe was labeled with biotin and digoxigenin and detected using FITC-conjugated anti-digoxigenin antibody and Texas-Red-conjugated avidin (yellow signal), chromosomes were counterstained with DAPI (images from a CCD camera).

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Fig. 2
figure 2

Mapping and ordering of NotI clones.

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Table 2 Two-color FISH analysis of NotI-linking clones on metaphase chromosomes
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Table 3 Three probes FISH ordering on metaphase chromosomes
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The EMBL nucleotide database (all divisions) was searched with partial sequences derived from all these NotI-linking clones using the FASTA program [16] (table 4). This search revealed that the NRL-090 clone (at 3q24) contains the gene encoding guanosine 5′-mono-phosphate synthetase (GDB name: GMPS-PEN, table 4). To our knowledge, this is the first time that this gene has been localized [18]. Clone NL1-320 contains a gene encoding tRNA-Arg (97.3% homology in 73 bp). This gene is highly conserved and reveals 81 % identity even to the yeast tRNA-Arg gene. Three other clones, NRL-063, NRL-097 and NRL-143, probably contain expressed genes since their sequences are 96–100% identical to cDNAs, from genes of unknown function, in the database [19].

Table 4 NotI-linking clones containing gene-coding or gene-similar sequences
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Other clones displayed 60–85% similarities to cDNAs and different genes. For example, clone NRL-094 contains sequences homologous (71.4% identity in 91 bp) to a human cDNA with unknown function (accession number: R27083). Clone NL1-358 shows homology (61.2% identity in 103 bp) to the sheep IGF-II gene, while clone NRL-097 shows homology (60.1% identity in 148 bp) to the gene encoding human triiodothyronine receptor (THRA1 gene). Additional experiments are needed to evaluate the significance of these homologies.

Data from this work contribute to the physical and transcriptional mapping of human chromosome 3, and should facilitate the isolation of genes that play a role in pathogenesis. For example, as mentioned above, deletions and aberrations in 3p21–22 are a characteristic feature of many solid tumors including renal cell carcinoma and lung cancers [3, 4, 5, 20]. Moreover, it has been shown that this region possesses tumor suppressor activity in mice and is regularly eliminated in hybrid cell lines containing human chromosome 3 after progressive growth in SCID mice [21, 22]. It thus seems likely that a tumor suppressor gene is located in the 3p21-p22 region. The availability of a NotI restriction map of this region could be very useful in the search for the relevant TSG because many YACs isolated from this region are extremely unstable [23].