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A Physical Map of the 6q14–q15 Region Harboring the Locus for the Lysosomal Membrane Sialic Acid Transport Defect
GENOMICS
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ARTICLE NO.
A Physical Map of the 6q14–q15 Region Harboring the Locus for the Lysosomal Membrane Sialic Acid Transport Defect PIRJO LEPPA¨NEN,* JUHA ISOSOMPPI,† JOHANNA SCHLEUTKER,* PERTTI AULA,*
AND
LEENA PELTONEN†,1
*Department of Medical Genetics, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland; and †Department of Human Molecular Genetics, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland Received April 29, 1996; accepted July 23, 1996
Obligatory recombination events observed in family material have defined the critical chromosomal region for SD to an approximately 2-cM region between the flanking markers D6S280 and D6S456. Strong linkage disequilibrium has been identified with markers D6S1596 and D6S1622 in Finnish SD chromosomes. This points to one major mutation in this isolated population and further suggests that the SD gene is situated in the immediate vicinity of these markers (Schleutker et al., 1995b). Here we have utilized analyses of extended haplotypes to monitor ancient recombination events in Finnish SD chromosomes to restrict the critical DNA region further. Furthermore we report the construction of a physical map over the SD region flanked by markers D6S280 and D6S1622. P1 and P1-derived artificial chromosome (PAC) clones covering the critical DNA region were positioned and ordered by visualizing the clones by fiber-FISH technique. We also report exclusion of seven known ESTs and nine cDNAs tentatively assigned to this chromosomal region as candidate genes, whereas two CpG islands were tentatively identified in the region and represent potential positional candidates for the gene, mutated in Salla disease and resulting in the sialic acid transport defect.
Two phenotypic presentations of excessive accumulation of free sialic acid in lysosomes, Salla disease and infantile sialic acid storage disease, have been assigned to the same locus at 6q14– q15. Here we have restricted the critical DNA region by analyses of extended haplotypes and constructed a long-range physical contig over the critical 200-kb chromosomal region flanked by the markers D6S280 and D6S1622. The efficient fiber-FISH technique was applied to order and orient the clones, and this facilitated avoidance of the tedious restriction mapping by pulsed-field gel electrophoresis. We excluded all seven known ESTs and nine cDNAs assigned to this DNA region and tentatively identified two potential CpG islands within the region, which now represent positional candidate genes for the sialic acid storage disorders. q 1996 Academic Press, Inc.
INTRODUCTION
Salla disease (SD, MIM 269920) is a recessively inherited lysosomal-free sialic acid storage disease enriched in the genetically isolated population of Finland, particularly in the northeastern part of the country (Aula et al., 1979). The phenotypic manifestation of SD is characterized by early-onset psychomotor retardation and ataxia with only a slightly reduced life span. In previous studies the Salla disease locus has been assigned to a restricted chromosomal region at 6q14– q15 by linkage analyses in Finnish family material (Haataja et al., 1994; Schleutker et al., 1995a). We have also previously reported that the strikingly different phenotype of free sialic acid storage, ISSD, represents the same allelic disorder (Schleutker et al., 1995b). Both SD and ISSD show lysosomal storage of free Nacetyl neuraminic acid due to impaired transport of sialic acid across the lysosomal membrane, but no putative transport protein(s) has yet been isolated (Mancini et al., 1989).
MATERIALS AND METHODS Family material and detection of polymorphism. The Finnish SD family material and the polymorphic markers, conditions for PCR, and polyacrylamide gel electrophoresis have been published previously (Haataja et al., 1994; Schleutker et al., 1995). Genomic YAC, P1 clones, and PAC clones. The CEPH YAC library (Albertsen et al., 1990) was screened for STSs D6S280, D6S456, D6S284, D6S286, and D6S460 by PCR. All positive clones were colony-purified and verified by hybridization with relevant probes. Cultures of yeast clones in AHC medium were used to prepare liquid DNA. The STS content of each YAC was determined by PCR screening. The P1 clones 4272, 4273, and 4274 were obtained from Genome Systems Inc. (St. Louis, MO) and were screened by PCR with the marker D6S1596. Cultures of P1 clones in TB supplemented with 25 mg/ml kanamycin and induced by IPTG to a final concentration of 1 mM were used to prepare liquid DNA by the alkaline lysis method (Sambrook et al., 1989). For screening PAC clones we used the PAC library provided by Professor Pieter J. de Jong (Roswell Park Cancer Institute, Buffalo, NY). The library was screened by
1
To whom correspondence should be addressed. Telephone: 3580-4744393. Fax: 358-0-4744480. 0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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PHYSICAL MAP OF THE SALLA REGION PCR for STSs D6S280, D6S1596, and D6S1622. Positive clones were cultured in the same way as the P1 clones and DNA extraction was performed by the alkaline lysis method. Sizing of P1 and PAC clones. The sizes of the P1s and PACs were determined by PFGE using an LKB Pulsaphor device. The clones were digested with NotI and electrophoresed in 1% agarose gels for 18 h with pulse times increasing linearly from 0.9 to 29 s in 0.51 TBE buffer. The sizes of the clones were determined from ethidium bromide-stained gels by comparing them to molecular weight markers. The sizes were also determined by restriction enzyme digestions using EcoRI and BamHI (New England Biolabs). One percent agarose gels were electrophoresed in 1.01 TBE with constant voltage and ethidium bromide-stained digests of DNA were compared to molecular weight markers. Fiber-FISH. Fiber-FISH analysis was performed as previously described (Heiskanen et al., 1994, 1995). Briefly, probes (P1 and PAC clones) were labeled by nick-translation with biotin-11– dUTP (Sigma) or digoxigenin-11 – dUTP (Boehringer Mannheim). Target DNA and slides with extended normal genomic DNA were prepared as described (Heiskanen et al., 1994). The slides were denatured in 70% formamide/21 SSC at 747C for 3 min followed by dehydration in a cold ethanol series. FISH was performed using modifications of standard procedures (Pinkel et al., 1986; Lichter et al., 1988). The hybridization mixture contained 50% formamide, 10% dextran sulfate, 21 SSC, 0.2 mg/ml herring sperm DNA and 2.5– 7.5 ng/ml of labeled probes. Cot-1 DNA 0.25 mg/ml (BRL, Gaithersburg, MD) was added to suppress the binding of repetitive sequences (15 min at 377C). Posthybridization washes were performed in 50% formamide/ 21 SSC (31 5 min), 21 SSC (21 5 min) and 0.51 SSC (11 5 min), all at 457C. Biotinylated probes were detected using TRITC-conjugated avidin D, and the signal was amplified by biotinylated goat anti-avidin D and another layer of avidin-TRITC (all from Vector, Burlingname, CA). For digoxigenin-labeled probes, mouse anti-digoxigenin antibodies (Boehringer Mannheim) and fluorescein-conjugated sheep anti-mouse and donkey anti-sheep antibodies (both from Sigma Chemical) were used. Slides were counterstained with 5 mg/ml DAPI (Sigma) and mounted with antifade solution (Vectashield, Vector). Fluorescence microscopy, digital image analysis, and quantitation of physical distances. The system is based on the Olympus BX50 microscope and the Photometrics PXL camera (Photometrics Inc., Tucson, AZ) attached to a PowerMac 7100/Av workstation. IP Lab software (Signal Analytics Corp., Vienna, VA) controls the camera operation, image acquisition, and the Ludl Filter Wheel (Ludl Electronics, Hawthorne, NY) equipped with Chromatechnology multiband pass filters. Data analysis is also performed on a Macintosh System using the IP Lab software option. In the calculation of probe distances in kilobases, the measurements were calibrated based on the known size of the probe inserts. The gap measurements were normalized based on the signal lengths of probes on both sides to compensate optimally for the variation in the level of DNA stretching. Probe ordering and quantitative measurements were performed from a minimum of 10 different microscope fields. ESTs and cDNAs. Seven ESTs assigned to 6q14 –q16.2 and their PCR conditions have been previously published (Polymeropoulos et al., 1993, Pappas et al., 1995). The sequence information for nine cDNAs was kindly provided by R. Berry (Berry et al., 1995; pers. comm., Denver, 1995). CpG islands. The enzymes EagI, BssHII, and SacII (New England Biolabs) were selected to identify potential CpG islands in the P1 and PAC clones. The restriction digests were run on a 0.8% agarose gel, and DNA fragments were visualized under UV. If two or more restriction sites were locally identified, the region was considered to contain a potential CpG island.
RESULTS
Restricting the Critical DNA Region by Ancient Recombinations in Finnish Families We constructed extended haplotypes by genotyping several polymorphic markers from a 2-cM region
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FIG. 1. Extended haplotypes around the Salla region restricting the area by obligatory centromeric recombination (family 1) and ancient recombinations (families 2 and 3). Affected chromosomes are indicated as squares.
flanking the SD locus. As shown in Fig. 1, two Finnish families revealed ancient recombinations that further restricted the critical SD region originally limited by obligatory recombinations to a 2-cM region between markers D6S280 and D6S456 in our family material. Family 3 is a first-cousin marriage with two affected children, both of whom had inherited the SD linked alleles for D6S280 and D6S1596. However, for markers distal to D6S1622 they had inherited the alleles located on the non-disease-bearing chromosomes in the Finnish family material. Haplotype analysis in family 2, which originated from the same geographical area as family 3 (outside the Salla region in Northern Finland), revealed a similar finding. The affected child from this family inherited the same haplotype as the children in family 3, also indicating an ancient recombination that had taken place distal to D6S1622. This information on the extended haplotypes from these two families thus placed the SD locus proximal to marker D6S1622. The genetic distance between D6S280 and D6S1622 in the Ge´ne´thon map (Dib et al., 1996) is 1 cM, but D6S1622 is positioned much closer to D6S280 as we were able to show by fiber-FISH (see below). Constructing the Long-Range Clone Contig Nine microsatellite loci, D6S280, D6S1596, D6S1622, D6S456, D6S1625, D6S1589, D6S284, D6S286, and D6S460, were used to identify clones from long-range genomic libraries and to construct a DNA contig spanning the region predicted to contain the SD gene (Schleutker et al., 1995b). Oligonucleotide primers at each locus were first used to screen the CEPH YAC libraries. With the exception of two markers (D6S1622 and D6S1625) all STSs were detected in one or more YACs. The final contig summarized in Table 1 consisted of 21 YACs. The YAC clones did not completely cover the critical chromosomal region, and a gap remained between markers D6S1596 and D6S456. D6S280 and D6S1596 were detected in one YAC, 691e5, which was common to both of them. Three different sizes were offered for this YAC
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TABLE 1 STS Content Mapping in the Salla Region
YAC691e5 PAC141B1 PAC224H23 PAC286E21 P4272 P4273 P4274 PAC109C16 PAC202M22 PAC252H1 YAC743e4 YAC810b5 YAC890c10 YAC678e7 YAC788g7 YAC878c7 YAC902f12 YAC948c1 YAC949d4 YAC632a8 YAC676h4 YAC725a10 YAC756b12 YAC846e5 YAC873c4 YAC901e12 YAC939a11 YAC939f3 YAC963a5 YAC826c1
D6S280
D6S1596
D6S1622
D6S456
D6S1625
D6S1589
D6S284
D6S286
D6S460
/ / / / 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
/ 0 0 0 / / / / / 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 / 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 / / / / / / / / / 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 / 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 / / / / / / / / / / 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 / / 0 / / / / / / / 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 /
clone, suggesting its instability. To avoid problems associated with YAC clones (Ioannou et al., 1994), we proceeded to screen P1 and PAC libraries to identify clones between markers D6S280 and D6S1622. The P1 library was first screened by using D6S1622 but no clones could be identified. D6S1596 was then used and three clones, 4272, 4273, and 4274, were obtained (Genome Systems Inc.). The screening of a PAC library with D6S280 resulted in three positive clones, 286E21, 224H23, and 141B1; screening with D6S1596 resulted in two clones, 109C16 and 202M22, and screening with D6S1622 resulted in one clone, 252H1. None of these clones was PCR-positive with any other STS.
Ordering and Orientation of Genomic Clones by Fiber-FISH We systematically utilized the fiber-FISH on extended DNA fibers to position and order the obtained clones. Fiber-FISH was also adopted to estimate the distances between individual clones by comparing the sizes of gaps between hybridization signals to the fluorescent signals obtained with the known sized clones used in hybridization. The precise lengths of the inserts of the P1 and PAC clones were determined by restriction digestion followed by gel electrophoresis (data not shown). The sizes of individual clones are shown in
TABLE 2 Estimated Sizes for P1s and PACs between D6S280 and D6S1622 Clone
Size (kb)
PAC141B1 PAC224H23 PAC286E21 P4272 P4273 P4274 PAC109C16 PAC202M22 PAC252H1
70 65 75 50 45 50 145 130 60
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FIG. 2. A physical map over the Salla region based on STS content mapping and fiber-FISH. Potential CpG islands are indicated by thick, solid vertical bars.
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PHYSICAL MAP OF THE SALLA REGION
FIG. 3. Fiber-FISH image: Three PACs spanning the critical Salla region between markers D6S280 and D6S1622 are visualized by fiber-FISH. PACs were labeled either with biotin (red) or digoxigenin (green), and the overlapping regions between the clones are visualized as yellow signals. The ends of the overlapping clones are indicated by arrows.
Table 2. The PCR analyses of polymorphic markers and the data obtained by fiber-FISH proved that we have constructed an uninterrupted contig between markers D6S280 and D6S1622. The contig consists of three P1 clones and six PAC clones. Many of these overlap at significant distances and thus this critical DNA region is more than adequately covered as shown in Fig. 2. The physical distance between D6S280 and D6S1622 is maximally 200 kb as measured from the distal ends of the clones, but it is probably less because the precise positions of markers D6S280 and D6S1622 on the corresponding long-range clones are not known. The fiber-FISH data indicate that the critical chromosomal region can actually be covered by only three PAC clones, e.g., 141B1, 202M22, and 252H1, as shown in Fig. 3. To minimize the problems of potential clonal rearrangements, different combinations of genomic clones can be used to cover our region; 109C16 and 202M22 overlap with three PAC clones from the proximal boundary at D6S280 and with one PAC clone from the distal boundary at D6S1622. The fiber-FISH results showed that the gap between the cluster of clones 286E21, 224H23, and 141B1 and the clone 252H1 is maximally about 40 kb, indicating that all these PAC clones originate from the immediate vicinity of marker D6S1596. This 40-kb gap is in our contig covered by P1 clones as shown in Fig. 2.
NIB1138, have been assigned to chromosome 6 but a more precise localization is reported only for NIB1138, which is assigned to 6q12–q14.1. Also seven ESTs, FB2C12, EST00125, EST00356, EST00760, EST00783, EST01129, and EST01147, are listed in databases for the chromosomal region 6q14– q16.2 (Pappas et al., 1995). All these sequences were used to screen by PCR the genomic clones, providing multiple coverage of the SD region, but no positive clones were identified. Consequently these cDNAs and ESTs cannot be considered positional candidate genes for SD. Identification of Potential CpG Islands in Long-Range Clones By rare-cutting restriction mapping of the P1 and PAC clones (data not shown), we were able to identify two potential CpG islands within our critical DNA region. Comparison of the data obtained from the overlapping P1 and PAC clones confirmed the regional assignment of these CpG islands. Since the methylation pattern in long-range clones does not necessarily reflect that in genomic DNA, these CpG islands are only suggestive ones. However, the number of CpG islands is relatively low in our long-range clones covering the critical region, so they do represent potential markers for the SD gene. The orientation of the CpG islands is shown in Fig. 2.
Excluding Seven ESTs and Nine cDNAs as Possible Candidate Genes In the databases nine cDNAs, IB1004, IB1284, IB3683, IB707, IB877, IB926, NIB1314, NIB1335, and
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DISCUSSION
After initial positioning of the SD locus by linkage analyses in Finnish families we proceeded to construct
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a physical contig over the critical DNA region since no physical mapping data have been available for this chromosomal region. Based on the data by Dib et al. (1996) the markers D6S280 and D6S1596 could not be separated on the genetic map whereas a 1-cM distance was estimated between D6S1596 and D6S1622. The genetic distance between the flanking markers, D6S280 and D6S1622, could thus be approximated to be 1 cM. Due to the ancient recombination events detectable in extended haplotype analyses in two Finnish SD families we were able to restrict this initial critical DNA region further and to determine a new flanking marker, D6S1622, on the telomeric side of the SD locus. This again emphasizes the power of extended haplotype analyses in refinement of the locus assignment in isolated populations exposing one founder mutation behind a disease (Sulisalo et al., 1994; Peltonen et al., 1995). We began our contig assembly with YAC clones but proceeded by using P1s and PACs. The inherent problems with YAC clones, such as chimerism, deletions, and large insert sizes (Jones et al., 1994; Neuhausen et al., 1994), could be avoided with P1s and PACs, which carry a smaller insert size and smaller copy number and are therefore more stable and less prone to rearrangements (Smoller et al., 1991; Shizuya et al., 1992; Ioannou et al., 1994). The genomic clones were first ordered by STS-content mapping immediately followed by the fiber-FISH technique, which greatly facilitated the efficient construction of a complete genomic contig for the SD region. The fiber-FISH technique was utilized both to confirm the order and orientation of the genomic clones and to determine the physical distances between them. The linear range of the fiber-FISH method in experienced hands ranges from 2 to 500 kb with a measuring accuracy similar to that of pulsedfield gel electrophoresis (Heiskanen et al., 1995). We had no difficulties in determining that the different hybridization signals were located on the same DNA strand since most of our clones were overlapping or gaps between them were very small. Occasional DNA breaks were encountered but misinterpretations could be avoided by repeated analysis (Heiskanen et al., 1995). Comparing the fiber-FISH and conventional contig construction methods, the definitive advantages of fiber-FISH are feasibility and saving of time. Due to avoidance of end cloning and PFGE-based contig construction, fiber-FISH also avoids the handling of radioactivity required in traditional contig construction methods. The concordance of size determination of physical clones obtained by fiber-FISH and other physical mapping methods was recently shown to be very good (Heiskanen et al., 1995). The only disadvantage of the fiber-FISH technique is the need for the digital imaging facility, at least if one aims at precise length determinations of hybridization signals and gaps between the signals (Heiskanen et al., 1996).
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On the genetic map the distance between the two flanking markers of the SD locus was about 1 cM, in general, equivalent to approximately 1 Mb. Our contig data demonstrated that the distance between the flanking markers is actually smaller. The fiber-FISH data and PFGE-confirmed sizes of the PAC clones both indicate that the critical DNA region for the SD locus is approximately only 200 kb. The constructed physical map of the region harboring the SD gene now forms a solid basis for future efforts to isolate the gene itself. We already know that the region contains at least two potential CpGs, highly probable markers for the gene causing the deficient free sialic acid transport on the lysosomal membrane and leading to SD or alternatively to ISSD. Elucidation of these genes and their products will eventually clarify the pathogenesis of these diseases and throw light on the structure and functional properties of the lysosomal membrane. ACKNOWLEDGMENTS We thank Professor Pieter J. de Jong for kindly providing the PAC library. We thank Aarno Palotie, Mervi Heiskanen, and Nina HorelliKuitunen for helpful discussions. This study was supported financially by the Academy of Finland, the Hjelt Foundation, and the Maud Kuistila Foundation.
REFERENCES Albertsen, H. M., Abderrahim, H., Cann, H. M., Dausset, J., LePaslier, D., and Cohen, D. (1990). Construction and characterization of a yeast artificial chromosome library containing seven haploid human genome equivalents. Proc. Natl. Acad. Sci. USA 87: 4256 – 4260. Aula, P., Autio, S., Raivio, K. O., Rapola, J., Thoden, C. J., Koskela, S. L., and Yamashina, I. (1979). ‘‘Salla disease’’: A new lysosomal storage disorder. Arch. Neurol. 36: 88–94. Berry, R., Stevens, T. J., Walter, N. A. R., Wilcox, A. S., Rubano, T., Hopkins, J. A., Weber, J., Goold, R., Bento Soares, M., and Sikela, J. M. (1995). Gene-based sequence-tagged-sited (STSs) as the basis for a human gene map. Nature Genet. 10: 415 –423. Dib, C., Faure´, S., Fizames, C., Samson, D., Drout, N., Vignal, A., Millasseau, P., Marc, S., Hazan, J., Seboun, E., Lathrop, M., Gyapay, G., Morissette, J., and Weissenbach, J. (1996). The Ge´ne´thon human genetic linkage map. Nature (Suppl.) 380. Haataja, L., Schleutker, J., Laine, A-P., Renlund, M., Savontaus, M-L., Dib, C., Weissenbach, J., Peltonen, L., and Aula, P. (1994). The genetic locus for free sialic acid storage disease maps to the long arm of chromosome 6. Am. J. Hum. Genet. 54: 1042 – 1049. Heiskanen, M., Karhu, R., Hellsten, E., Peltonen, L., Kallioniemi, O. P., and Palotie, A. (1994). High resolution mapping using fluorescence in situ hybridization to extended DNA fibers prepared from agarose-embedded cells. BioTechniques 17: 928– 934. Heiskanen, M., Hellsten, E., Kallioniemi, O. P., Ma¨kela¨, T. P., Alitalo, K., Peltonen, L., and Palotie, A. (1995). Visual mapping by fiber-FISH. Genomics 30: 31– 36. Heiskanen, M., Kallioniemi, O. P., and Palotie, A. (1996). FiberFISH: Experiences and a refined protocol. Genet. Anal. Biomol. Eng. 12: 179–184. Ioannou, P. A., Amemiya, C. T., Garnes, J., Kroisel, P. M., Shizuya, H., Chen, C., Batzer, M. A., and de Jong, P. J. (1994). A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nature Genet. 6: 84–89.
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PHYSICAL MAP OF THE SALLA REGION Jones, K. A., Black, D. M., Brown, M. A., Griffiths, B. L., Nicolai, H. M., Chambers, J. A., Bonjardim, M., Xu, C.-F., Boyd, M., McFarlane, R., Korn, B., Poutska, A., North, M. A., Schalkwyk, L., Lehrach, H., and Solomon, E. (1994). The detailed characterisation of a 400 kb cosmid walk in the BRCA1 region: Identification and localisation of 10 genes including a dual-specificity phosphatase. Hum. Mol. Genet. 3: 1927 – 1934. Lichter, P., Cremer, T., Borden, J., Manuelidis, I., and Ward, D. C. (1988). Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80: 224– 234. Mancini, G. M. S., de Jonge, H. R., Galjaard, H., and Verheijen, F. W. (1989). Characterization of a proton-driven carrier for sialic acid in the lysosomal membrane. J. Biol. Chem. 264: 15247 – 15254. Neuhausen, S. L., Swensen, J., Miki, Y., Liu, Q., Tavtigian, S., Shattuck-Eidens, D., Kamb, A., Hobbs, M. R., Gingrich, J., Shizuya, H., Kim, U.-J., Cochran, C., Futreal, P. A., Wiseman, R. W., Lynch, H. T., Tonin, P., Narod, S., Cannon-Albright, L., Skolnick, M. H., and Goldgar, D. E. (1994). A P1-based physical map of the region from D17S776 to D17S78 containing the breast cancer susceptibility gene BRCA1. Hum. Mol. Genet. 3: 1919– 1926. Pappas, G. J., Polymeropoulos, M. H., Boyle, J. M., and Trent, J. M. (1995). Regional assignment by hybrid mapping of 36 expressed sequence tags (ESTs) on human chromosome 6. Genomics 25: 124– 129. Peltonen, L., Pekkarinen, P., and Aaltonen, J. (1995). Messages from an isolate: Lessons from the Finnish gene pool. Biol. Chem. 376: 697–704. Pinkel, D., Straume, T., and Gray, J. (1986). Cytogenetic analysis
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using quantitative, high sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. USA 83: 2934 – 2938. Polymeropoulos, M. H., Xiao, H., Sikela, J. M., Adams, M., Venter, J. C., and Merrill, C. R. (1993). Chromosomal distribution of 320 genes from a brain cDNA library. Nature Genet. 4: 381–386. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schleutker, J., Laine, A.-P., Haataja, L., Renlund, M., Weissenbach, J., Aula, P., and Peltonen, L. (1995a). Linkage disequilibrium utilized to establish a refined genetic position of the Salla disease locus on 6q14 – q15. Genomics 27: 286 –292. Schleutker, J., Leppa¨nen, P., Ma˚nsson, J.-E., Erikson, A., Weissenbach, J., Peltonen, L., and Aula, P. (1995b). Lysosomal free sialic acid storage disorders with different phenotypic presentations, ISSD and Salla disease, represent allelic disorders on 6q14 –15. Am. J. Hum. Genet. 57: 893– 901. Shizuya, H., Birren, B., Kim, U.-J., Mancino, V., Slepak, T., Tachiiri, Y., and Simon, M. (1992). Cloning and stable maintenance of 300kilobasepair fragments of human DNA in Escherichia coli using an F-factor based vector. Proc. Natl. Acad. Sci. USA 89: 8794 – 8797. Smoller, D. A., Petrov, D., and Hartl, D. L. (1991). Characterization of bacteriophage P1 library containing inserts of Drosophila DNA of 75–100 kilobase pairs. Chromosoma 100: 487 –494. Sulisalo, T., Klockars, J., Ma¨kitie, O., Francomano, C. A., de la Chapelle, A., Kaitila, I., and Sistonen, P. (1994). High-resolution linkage-disequilibrium mapping of the cartilage-hair hypoplasia gene. Am. J. Hum. Genet. 55: 937– 945.
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ARTICLE NO.
A Physical Map of the 6q14–q15 Region Harboring the Locus for the Lysosomal Membrane Sialic Acid Transport Defect PIRJO LEPPA¨NEN,* JUHA ISOSOMPPI,† JOHANNA SCHLEUTKER,* PERTTI AULA,*
AND
LEENA PELTONEN†,1
*Department of Medical Genetics, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland; and †Department of Human Molecular Genetics, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland Received April 29, 1996; accepted July 23, 1996
Obligatory recombination events observed in family material have defined the critical chromosomal region for SD to an approximately 2-cM region between the flanking markers D6S280 and D6S456. Strong linkage disequilibrium has been identified with markers D6S1596 and D6S1622 in Finnish SD chromosomes. This points to one major mutation in this isolated population and further suggests that the SD gene is situated in the immediate vicinity of these markers (Schleutker et al., 1995b). Here we have utilized analyses of extended haplotypes to monitor ancient recombination events in Finnish SD chromosomes to restrict the critical DNA region further. Furthermore we report the construction of a physical map over the SD region flanked by markers D6S280 and D6S1622. P1 and P1-derived artificial chromosome (PAC) clones covering the critical DNA region were positioned and ordered by visualizing the clones by fiber-FISH technique. We also report exclusion of seven known ESTs and nine cDNAs tentatively assigned to this chromosomal region as candidate genes, whereas two CpG islands were tentatively identified in the region and represent potential positional candidates for the gene, mutated in Salla disease and resulting in the sialic acid transport defect.
Two phenotypic presentations of excessive accumulation of free sialic acid in lysosomes, Salla disease and infantile sialic acid storage disease, have been assigned to the same locus at 6q14– q15. Here we have restricted the critical DNA region by analyses of extended haplotypes and constructed a long-range physical contig over the critical 200-kb chromosomal region flanked by the markers D6S280 and D6S1622. The efficient fiber-FISH technique was applied to order and orient the clones, and this facilitated avoidance of the tedious restriction mapping by pulsed-field gel electrophoresis. We excluded all seven known ESTs and nine cDNAs assigned to this DNA region and tentatively identified two potential CpG islands within the region, which now represent positional candidate genes for the sialic acid storage disorders. q 1996 Academic Press, Inc.
INTRODUCTION
Salla disease (SD, MIM 269920) is a recessively inherited lysosomal-free sialic acid storage disease enriched in the genetically isolated population of Finland, particularly in the northeastern part of the country (Aula et al., 1979). The phenotypic manifestation of SD is characterized by early-onset psychomotor retardation and ataxia with only a slightly reduced life span. In previous studies the Salla disease locus has been assigned to a restricted chromosomal region at 6q14– q15 by linkage analyses in Finnish family material (Haataja et al., 1994; Schleutker et al., 1995a). We have also previously reported that the strikingly different phenotype of free sialic acid storage, ISSD, represents the same allelic disorder (Schleutker et al., 1995b). Both SD and ISSD show lysosomal storage of free Nacetyl neuraminic acid due to impaired transport of sialic acid across the lysosomal membrane, but no putative transport protein(s) has yet been isolated (Mancini et al., 1989).
MATERIALS AND METHODS Family material and detection of polymorphism. The Finnish SD family material and the polymorphic markers, conditions for PCR, and polyacrylamide gel electrophoresis have been published previously (Haataja et al., 1994; Schleutker et al., 1995). Genomic YAC, P1 clones, and PAC clones. The CEPH YAC library (Albertsen et al., 1990) was screened for STSs D6S280, D6S456, D6S284, D6S286, and D6S460 by PCR. All positive clones were colony-purified and verified by hybridization with relevant probes. Cultures of yeast clones in AHC medium were used to prepare liquid DNA. The STS content of each YAC was determined by PCR screening. The P1 clones 4272, 4273, and 4274 were obtained from Genome Systems Inc. (St. Louis, MO) and were screened by PCR with the marker D6S1596. Cultures of P1 clones in TB supplemented with 25 mg/ml kanamycin and induced by IPTG to a final concentration of 1 mM were used to prepare liquid DNA by the alkaline lysis method (Sambrook et al., 1989). For screening PAC clones we used the PAC library provided by Professor Pieter J. de Jong (Roswell Park Cancer Institute, Buffalo, NY). The library was screened by
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To whom correspondence should be addressed. Telephone: 3580-4744393. Fax: 358-0-4744480. 0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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PHYSICAL MAP OF THE SALLA REGION PCR for STSs D6S280, D6S1596, and D6S1622. Positive clones were cultured in the same way as the P1 clones and DNA extraction was performed by the alkaline lysis method. Sizing of P1 and PAC clones. The sizes of the P1s and PACs were determined by PFGE using an LKB Pulsaphor device. The clones were digested with NotI and electrophoresed in 1% agarose gels for 18 h with pulse times increasing linearly from 0.9 to 29 s in 0.51 TBE buffer. The sizes of the clones were determined from ethidium bromide-stained gels by comparing them to molecular weight markers. The sizes were also determined by restriction enzyme digestions using EcoRI and BamHI (New England Biolabs). One percent agarose gels were electrophoresed in 1.01 TBE with constant voltage and ethidium bromide-stained digests of DNA were compared to molecular weight markers. Fiber-FISH. Fiber-FISH analysis was performed as previously described (Heiskanen et al., 1994, 1995). Briefly, probes (P1 and PAC clones) were labeled by nick-translation with biotin-11– dUTP (Sigma) or digoxigenin-11 – dUTP (Boehringer Mannheim). Target DNA and slides with extended normal genomic DNA were prepared as described (Heiskanen et al., 1994). The slides were denatured in 70% formamide/21 SSC at 747C for 3 min followed by dehydration in a cold ethanol series. FISH was performed using modifications of standard procedures (Pinkel et al., 1986; Lichter et al., 1988). The hybridization mixture contained 50% formamide, 10% dextran sulfate, 21 SSC, 0.2 mg/ml herring sperm DNA and 2.5– 7.5 ng/ml of labeled probes. Cot-1 DNA 0.25 mg/ml (BRL, Gaithersburg, MD) was added to suppress the binding of repetitive sequences (15 min at 377C). Posthybridization washes were performed in 50% formamide/ 21 SSC (31 5 min), 21 SSC (21 5 min) and 0.51 SSC (11 5 min), all at 457C. Biotinylated probes were detected using TRITC-conjugated avidin D, and the signal was amplified by biotinylated goat anti-avidin D and another layer of avidin-TRITC (all from Vector, Burlingname, CA). For digoxigenin-labeled probes, mouse anti-digoxigenin antibodies (Boehringer Mannheim) and fluorescein-conjugated sheep anti-mouse and donkey anti-sheep antibodies (both from Sigma Chemical) were used. Slides were counterstained with 5 mg/ml DAPI (Sigma) and mounted with antifade solution (Vectashield, Vector). Fluorescence microscopy, digital image analysis, and quantitation of physical distances. The system is based on the Olympus BX50 microscope and the Photometrics PXL camera (Photometrics Inc., Tucson, AZ) attached to a PowerMac 7100/Av workstation. IP Lab software (Signal Analytics Corp., Vienna, VA) controls the camera operation, image acquisition, and the Ludl Filter Wheel (Ludl Electronics, Hawthorne, NY) equipped with Chromatechnology multiband pass filters. Data analysis is also performed on a Macintosh System using the IP Lab software option. In the calculation of probe distances in kilobases, the measurements were calibrated based on the known size of the probe inserts. The gap measurements were normalized based on the signal lengths of probes on both sides to compensate optimally for the variation in the level of DNA stretching. Probe ordering and quantitative measurements were performed from a minimum of 10 different microscope fields. ESTs and cDNAs. Seven ESTs assigned to 6q14 –q16.2 and their PCR conditions have been previously published (Polymeropoulos et al., 1993, Pappas et al., 1995). The sequence information for nine cDNAs was kindly provided by R. Berry (Berry et al., 1995; pers. comm., Denver, 1995). CpG islands. The enzymes EagI, BssHII, and SacII (New England Biolabs) were selected to identify potential CpG islands in the P1 and PAC clones. The restriction digests were run on a 0.8% agarose gel, and DNA fragments were visualized under UV. If two or more restriction sites were locally identified, the region was considered to contain a potential CpG island.
RESULTS
Restricting the Critical DNA Region by Ancient Recombinations in Finnish Families We constructed extended haplotypes by genotyping several polymorphic markers from a 2-cM region
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FIG. 1. Extended haplotypes around the Salla region restricting the area by obligatory centromeric recombination (family 1) and ancient recombinations (families 2 and 3). Affected chromosomes are indicated as squares.
flanking the SD locus. As shown in Fig. 1, two Finnish families revealed ancient recombinations that further restricted the critical SD region originally limited by obligatory recombinations to a 2-cM region between markers D6S280 and D6S456 in our family material. Family 3 is a first-cousin marriage with two affected children, both of whom had inherited the SD linked alleles for D6S280 and D6S1596. However, for markers distal to D6S1622 they had inherited the alleles located on the non-disease-bearing chromosomes in the Finnish family material. Haplotype analysis in family 2, which originated from the same geographical area as family 3 (outside the Salla region in Northern Finland), revealed a similar finding. The affected child from this family inherited the same haplotype as the children in family 3, also indicating an ancient recombination that had taken place distal to D6S1622. This information on the extended haplotypes from these two families thus placed the SD locus proximal to marker D6S1622. The genetic distance between D6S280 and D6S1622 in the Ge´ne´thon map (Dib et al., 1996) is 1 cM, but D6S1622 is positioned much closer to D6S280 as we were able to show by fiber-FISH (see below). Constructing the Long-Range Clone Contig Nine microsatellite loci, D6S280, D6S1596, D6S1622, D6S456, D6S1625, D6S1589, D6S284, D6S286, and D6S460, were used to identify clones from long-range genomic libraries and to construct a DNA contig spanning the region predicted to contain the SD gene (Schleutker et al., 1995b). Oligonucleotide primers at each locus were first used to screen the CEPH YAC libraries. With the exception of two markers (D6S1622 and D6S1625) all STSs were detected in one or more YACs. The final contig summarized in Table 1 consisted of 21 YACs. The YAC clones did not completely cover the critical chromosomal region, and a gap remained between markers D6S1596 and D6S456. D6S280 and D6S1596 were detected in one YAC, 691e5, which was common to both of them. Three different sizes were offered for this YAC
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TABLE 1 STS Content Mapping in the Salla Region
YAC691e5 PAC141B1 PAC224H23 PAC286E21 P4272 P4273 P4274 PAC109C16 PAC202M22 PAC252H1 YAC743e4 YAC810b5 YAC890c10 YAC678e7 YAC788g7 YAC878c7 YAC902f12 YAC948c1 YAC949d4 YAC632a8 YAC676h4 YAC725a10 YAC756b12 YAC846e5 YAC873c4 YAC901e12 YAC939a11 YAC939f3 YAC963a5 YAC826c1
D6S280
D6S1596
D6S1622
D6S456
D6S1625
D6S1589
D6S284
D6S286
D6S460
/ / / / 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
/ 0 0 0 / / / / / 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 / 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 / / / / / / / / / 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 / 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 / / / / / / / / / / 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 / / 0 / / / / / / / 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 /
clone, suggesting its instability. To avoid problems associated with YAC clones (Ioannou et al., 1994), we proceeded to screen P1 and PAC libraries to identify clones between markers D6S280 and D6S1622. The P1 library was first screened by using D6S1622 but no clones could be identified. D6S1596 was then used and three clones, 4272, 4273, and 4274, were obtained (Genome Systems Inc.). The screening of a PAC library with D6S280 resulted in three positive clones, 286E21, 224H23, and 141B1; screening with D6S1596 resulted in two clones, 109C16 and 202M22, and screening with D6S1622 resulted in one clone, 252H1. None of these clones was PCR-positive with any other STS.
Ordering and Orientation of Genomic Clones by Fiber-FISH We systematically utilized the fiber-FISH on extended DNA fibers to position and order the obtained clones. Fiber-FISH was also adopted to estimate the distances between individual clones by comparing the sizes of gaps between hybridization signals to the fluorescent signals obtained with the known sized clones used in hybridization. The precise lengths of the inserts of the P1 and PAC clones were determined by restriction digestion followed by gel electrophoresis (data not shown). The sizes of individual clones are shown in
TABLE 2 Estimated Sizes for P1s and PACs between D6S280 and D6S1622 Clone
Size (kb)
PAC141B1 PAC224H23 PAC286E21 P4272 P4273 P4274 PAC109C16 PAC202M22 PAC252H1
70 65 75 50 45 50 145 130 60
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FIG. 2. A physical map over the Salla region based on STS content mapping and fiber-FISH. Potential CpG islands are indicated by thick, solid vertical bars.
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PHYSICAL MAP OF THE SALLA REGION
FIG. 3. Fiber-FISH image: Three PACs spanning the critical Salla region between markers D6S280 and D6S1622 are visualized by fiber-FISH. PACs were labeled either with biotin (red) or digoxigenin (green), and the overlapping regions between the clones are visualized as yellow signals. The ends of the overlapping clones are indicated by arrows.
Table 2. The PCR analyses of polymorphic markers and the data obtained by fiber-FISH proved that we have constructed an uninterrupted contig between markers D6S280 and D6S1622. The contig consists of three P1 clones and six PAC clones. Many of these overlap at significant distances and thus this critical DNA region is more than adequately covered as shown in Fig. 2. The physical distance between D6S280 and D6S1622 is maximally 200 kb as measured from the distal ends of the clones, but it is probably less because the precise positions of markers D6S280 and D6S1622 on the corresponding long-range clones are not known. The fiber-FISH data indicate that the critical chromosomal region can actually be covered by only three PAC clones, e.g., 141B1, 202M22, and 252H1, as shown in Fig. 3. To minimize the problems of potential clonal rearrangements, different combinations of genomic clones can be used to cover our region; 109C16 and 202M22 overlap with three PAC clones from the proximal boundary at D6S280 and with one PAC clone from the distal boundary at D6S1622. The fiber-FISH results showed that the gap between the cluster of clones 286E21, 224H23, and 141B1 and the clone 252H1 is maximally about 40 kb, indicating that all these PAC clones originate from the immediate vicinity of marker D6S1596. This 40-kb gap is in our contig covered by P1 clones as shown in Fig. 2.
NIB1138, have been assigned to chromosome 6 but a more precise localization is reported only for NIB1138, which is assigned to 6q12–q14.1. Also seven ESTs, FB2C12, EST00125, EST00356, EST00760, EST00783, EST01129, and EST01147, are listed in databases for the chromosomal region 6q14– q16.2 (Pappas et al., 1995). All these sequences were used to screen by PCR the genomic clones, providing multiple coverage of the SD region, but no positive clones were identified. Consequently these cDNAs and ESTs cannot be considered positional candidate genes for SD. Identification of Potential CpG Islands in Long-Range Clones By rare-cutting restriction mapping of the P1 and PAC clones (data not shown), we were able to identify two potential CpG islands within our critical DNA region. Comparison of the data obtained from the overlapping P1 and PAC clones confirmed the regional assignment of these CpG islands. Since the methylation pattern in long-range clones does not necessarily reflect that in genomic DNA, these CpG islands are only suggestive ones. However, the number of CpG islands is relatively low in our long-range clones covering the critical region, so they do represent potential markers for the SD gene. The orientation of the CpG islands is shown in Fig. 2.
Excluding Seven ESTs and Nine cDNAs as Possible Candidate Genes In the databases nine cDNAs, IB1004, IB1284, IB3683, IB707, IB877, IB926, NIB1314, NIB1335, and
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DISCUSSION
After initial positioning of the SD locus by linkage analyses in Finnish families we proceeded to construct
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a physical contig over the critical DNA region since no physical mapping data have been available for this chromosomal region. Based on the data by Dib et al. (1996) the markers D6S280 and D6S1596 could not be separated on the genetic map whereas a 1-cM distance was estimated between D6S1596 and D6S1622. The genetic distance between the flanking markers, D6S280 and D6S1622, could thus be approximated to be 1 cM. Due to the ancient recombination events detectable in extended haplotype analyses in two Finnish SD families we were able to restrict this initial critical DNA region further and to determine a new flanking marker, D6S1622, on the telomeric side of the SD locus. This again emphasizes the power of extended haplotype analyses in refinement of the locus assignment in isolated populations exposing one founder mutation behind a disease (Sulisalo et al., 1994; Peltonen et al., 1995). We began our contig assembly with YAC clones but proceeded by using P1s and PACs. The inherent problems with YAC clones, such as chimerism, deletions, and large insert sizes (Jones et al., 1994; Neuhausen et al., 1994), could be avoided with P1s and PACs, which carry a smaller insert size and smaller copy number and are therefore more stable and less prone to rearrangements (Smoller et al., 1991; Shizuya et al., 1992; Ioannou et al., 1994). The genomic clones were first ordered by STS-content mapping immediately followed by the fiber-FISH technique, which greatly facilitated the efficient construction of a complete genomic contig for the SD region. The fiber-FISH technique was utilized both to confirm the order and orientation of the genomic clones and to determine the physical distances between them. The linear range of the fiber-FISH method in experienced hands ranges from 2 to 500 kb with a measuring accuracy similar to that of pulsedfield gel electrophoresis (Heiskanen et al., 1995). We had no difficulties in determining that the different hybridization signals were located on the same DNA strand since most of our clones were overlapping or gaps between them were very small. Occasional DNA breaks were encountered but misinterpretations could be avoided by repeated analysis (Heiskanen et al., 1995). Comparing the fiber-FISH and conventional contig construction methods, the definitive advantages of fiber-FISH are feasibility and saving of time. Due to avoidance of end cloning and PFGE-based contig construction, fiber-FISH also avoids the handling of radioactivity required in traditional contig construction methods. The concordance of size determination of physical clones obtained by fiber-FISH and other physical mapping methods was recently shown to be very good (Heiskanen et al., 1995). The only disadvantage of the fiber-FISH technique is the need for the digital imaging facility, at least if one aims at precise length determinations of hybridization signals and gaps between the signals (Heiskanen et al., 1996).
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On the genetic map the distance between the two flanking markers of the SD locus was about 1 cM, in general, equivalent to approximately 1 Mb. Our contig data demonstrated that the distance between the flanking markers is actually smaller. The fiber-FISH data and PFGE-confirmed sizes of the PAC clones both indicate that the critical DNA region for the SD locus is approximately only 200 kb. The constructed physical map of the region harboring the SD gene now forms a solid basis for future efforts to isolate the gene itself. We already know that the region contains at least two potential CpGs, highly probable markers for the gene causing the deficient free sialic acid transport on the lysosomal membrane and leading to SD or alternatively to ISSD. Elucidation of these genes and their products will eventually clarify the pathogenesis of these diseases and throw light on the structure and functional properties of the lysosomal membrane. ACKNOWLEDGMENTS We thank Professor Pieter J. de Jong for kindly providing the PAC library. We thank Aarno Palotie, Mervi Heiskanen, and Nina HorelliKuitunen for helpful discussions. This study was supported financially by the Academy of Finland, the Hjelt Foundation, and the Maud Kuistila Foundation.
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