Hum Genet (1992) 90:174-176
human .. generics 9 Springer-Verlag 1992
Mapping of the human COL5A1 gene to chromosome 9q34.3 G. Caridi l, Annalisa Pezzolo -~, Roberta Bertelli 1, G. Gimelli 2, A. DiDonato 3, G. Candiano l, and G. M. Ghiggeri 1 t Divisione di Nefrologia, 2Laboratorio di Citogenetica. and 3Servizio di Genetica Molecolare, lstituto G. Gaslini. Largo G. Gaslini 5, 1-16148 Genova, Italy Received January 21, 1992 / Revised March 5, 1992
Summary. A 353-bp region encoding for the NH2 terminus of the noncollagenic part of the ctl(V) chain was amplified by the polymerase chain reaction (PCR), subcloned and sequenced. The subcloned PCR product (pGC1) presented the same nucleotide sequence as the original fragment from the published sequence of COL5A1. In situ hybridization, using pGC1 as a probe, mapped the COL5A1 gene to chromosome 9q34.3. This assignement shows that COL5A1 is not synthenic with COL5A2, which is localized together with other collagen genes on chromosome 2.
Introduction Collagen V is an extracellular component of unknown function in several tissues and organs, including blood vessels, bone, nerve, muscle and tendon. Structurally, it occurs in various combination of chains, such as heterotrimers ctl(V)a2(V)c~3(V) or cd(V)2ct2(V), or more simply as a homotrimer of the cd(V) chain (Miller and Gay 1987; Niyibizi et al. 1984). The nucleotide sequence of c D N A encoding for human ct2(V) procollagen was first reported by Woodbury et al. (1989), and the C O L 5 A 2 gene was mapped to chromosome 2q24.3---,q31 (Vuorio and de Cronbrugghe 1990). Subsequently, Takahara et al. (1991) reported the c D N A sequence encoding the complete human prepro-ctl(V) chain, but its chromosome localization has not been identified. Information on the chromosome localization of C O L 1 A 5 is required in order to attempt a pathogenetic interpretation of those genetic diseases in which the extracellular matrix and, in particular, collagen (V) seem to be implicated (Candiano et al. 1991; Pienta et al. 1991; Nerlich and Schleicher 1991). In the present study, a 353-bp specific fragment of the COL5A1 gene coding for the NH2 terminus of the non-collagenic segment was amplified by the polymerase chain reaction (PCR), subcloned (pGC1), Correspondence to: G. M. Ghiggeri
sequenced, and was finally localized by fluorescent in situ hybridization (FISH).
Materials and methods R N A preparation
RNA was extracted from primary cultures of renal tubule epithelia in 6M guanidine isothiocyanate followed by centrifugation in a 5.7 M CsCI gradient for 20 h at 32000 rpm (Beckman SW 55 rotor, Palo Alto, Calif.). The pellet was then extracted with chloroform/ 1-butanol (4:1 vol/vol) (Maniatis et al. 1982). c D N A synthesis and P C R
From the published COL5AI eDNA sequence, two 21-met primers were chemically synthesized (PCR-MATE DNA SynthesizerApplyed Biosystem, Foster City, Calif.). The sense primer was igg 634 (5'-CTCAGCGTCCACAAGAAAAAT-3')and the antisense primer was igg 988 (5'-CCTAGG TCTTCG GGG TCTTCG-3'). First strand eDNA synthesis was performed by priming with random hexamers for 40 rain at 42~ in a final volume of 20 ~tl with 3.5 ~,tg RNA according to the instructions of the supplier of the eDNA Synthesis System Plus kit (Amersham, Little Chalfont, UK). PCR was performed in a Perkin Elmer DNA thermal cycler. The reaction mixture contained 50 pmoles primers, 10~_tlcDNA and 2.5 U Taq DNA polymerase (Gent Amp Kit-Perkin Elmer, Norwalk. Conn.). The reaction was performed under the following conditions: denaturation at 92~ for I rain, annealing at 55~ for 1rain, and extension
Fig. 1. Agarose electrophoresis of a PCR product before and after BamHI digestion. Lanes a, d pBR322 BstnI: lane b 353-bp PCR product; lane c BamHl digestion of PCR products (121 bp and 232bp)
175
Fig. 2. A Partial metaphase stained by DAPI; arrows indicate chromosomes 9. B Same partial metaphase hybridized with pGC1; arrows indicate hybrization signals on the long arms of both chromosomes 9
at 72~ for 2 min. After 35 cycles of gene amplification, the products were submitted to one final extension at 72~ for 6 rain and analyzed in a 2% agarose gel with ethidium bromide staining. BamHI digestion was carried out with 20UBamHI in buffer B (Boehringer, Mannheim, FRG) at 37~ for 1 h.
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p
Subcloning of the PCR product The PCR product was cloned in a pCR 1000 vector using the TA Cloning System (Invitrogen Corporation, San Diego, Calif.), and named pGC1. The cloned DNA fragment was separated from the vector by EcoRI-HindIII digestion.
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Sequencing pGC1 was sequenced using a T7 Sequencing Kit (Pharmacia, Uppsala, Sweden), following the method described by Sanger et al. (1977).
Metaphase chromosome preparations Normal human metaphase chromosomes were prepared from phytohemagglutinin-stimulated lymphocyte cultures. For precise mapping, the technique of methotrexase synchronization at prometaphase (Yunis et al. 1978) was used.
Fluorescent in situ hybridization Complete pGCI plasmid DNA was labeled by nick translation with biotin-16-dUTP (Boehringer). In situ hybridization was performed as described by Pinkel et al. (1986) on unbanded metaphases; for probe detection, the slides were incubated with fluorescein-conjugated (FITC) avidin. Two signal amplification cycles were achieved by subsequent incubation in anti-avidin antibody, followed by incubation in FITC avidin. Chromosomes were stained with both 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide. Slides were mounted in antifade solution and screened with a Zeiss fluroescent microscope.
Results and discussion The primers igg 634 and igg 988 were selected to amplify a 353-bp region of COL5A1, by P C R , on the basis of its low homology with other sequenced collagen genes and the presence of a BamHI at position 122. The results obtained by P C R are shown in Fig. 1 where the product was run in lane b and yielded a fragment of the expected size. Lane c shows the two fragments of 122bp and 231 bp, which established, as predicted, the presence of a BamHI site. In order to confirm these results, the frag-
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Fig. 3. Distribution of labeled sites on chromosome 9
ment was subcloned in a specific plasmid for P C R products, and n a m e d pGC1. It was completely sequenced and the results showed a precise homology with the corresponding published sequence. This probe was finally localized by F I S H on the terminal band of the long arm of c h r o m o s o m e 9q34.3 (Fig. 2). The combination of dual staining with a simple change of filter allowed the simultaneous identification of the c h r o m o s o m e according to the banding pattern and the visualization of t h e hybridization signal on the same metaphase image, with an unambiguous assignment of the probe to a specific chromosome. Following hybridization, the analysis of 80 prometaphase plates showed that 17.6% (26/147) of hybridization signals were located on the telomeric region of the long arm of one or two chromosomes 9 (Fig. 3). The salient point emerging from the present study is that the COL5A1 gene does not co-localize with any of the other genes coding for fibrillar collagens that are structurally similar to it, such as C O L 5 A 2 , COL3A1 and C O L l l A 1 (Vourio and de Crombrugghe 1990). This could have some pathogenetic importance in those genetic diseases in which collagen V, or more generally, the extracellular matrix seems to be involved. It is remarkable that, in a case of 9q32-qter m o n o s o m y (Zuffardi et al. 1989), osteopathia striata at the metaphyses of the femurs and the tibiae was reported.
176
Acknowledgements. The authors thank Professor Marco Fraccaro for helpful discussions, and Mr. R. Stubinski for his secretarial support. This work was carried out with a grant from the Italian Ministry of Health to the G. Gaslini Institute.
References
Candiano G, Altieri P, Oleggini R, Ginevri F, Ghiggeri GM (1991) Identification and characterization by CNBr peptide mapping of two collagen chains in cyst epithelia from polycystic kidney (abstract). J Am Soc Nephrol 2 : 250 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY Miller E J, Gay S (1987) The collagens: an overview and update. Method Enzymo[ 144 : 3-41 Nerlich A, Schleicher E (1991) Immunohistochemical localization of extracellular matrix components in human diabetic glomerular lesion. Am J Pathol 139 : 889-899 Niyibizi C, Fietzek PP, Van der Rest M (1984) Human placenta type V collagens. Evidence for existence of an ~.I(V)ct2(V)~3(V) collagen molecule. J Biol Chem 259 : 14170-14174 Pienta K, Murphy BC, Getzenberg RH, Coffey DS (1991) The effect of extracellular matrix interaction on morphologic transformation in vitro. Biochem Biophys Res Comm 179 : 333-339
Note added in proof. After submission and acceptance of this paper, the same localization of COL5A1 on 9q34.2-~q34.3 was reported by Greespan et al. [Genomics (1992) 12:836-837].
Pinkel D, Straume T. Gray J (1986) Cytogenetic analysis using quantitative high sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83 : 2934-2938 Sanger F, Nicklen S, Coulsen A R (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467 Takahara K, Sato Y, Okazawa K, Okamoto N, Noda A, Yaoi Y. Kato Y (1991) Complete primary structure of human collagen cd(V) chain. J Biol Chem 266 : 13124-13129 Vuorio E, Crombrugghe D de (1990) The family of collagen genes. Annu Rev Biochem 59 : 837-872 Woodbury D, Benson-Chanada V, Ramirez F (1989) Amino-terminal propeptide of human pro-c~2(V) collagen conforms to the structural criteria of a fibrillar procollagen molecule. J Biol Chem 264 : 2735-2738 Yunis J J, Sawyer JR, Ball DW (1978) Characterization of banding patterns of metaphase-profase G banded chromosomes and their use in gene mapping. Cytogenet Cell Genet 22 : 678-683 Zuffardi O, Caiulo A, Maraschio P, Tupler R, Bianchi E. Amisano P, Beluffi G, Moratti R, Liguri G (1989) Regional assignment of the loci for adenylate kinase to 9q32 and for alacid glycoprotein to 9q31-q32: a lkocus for Goltz syndrome in region 9q32-qter? Hum Genet 82:17-19
human .. generics 9 Springer-Verlag 1992
Mapping of the human COL5A1 gene to chromosome 9q34.3 G. Caridi l, Annalisa Pezzolo -~, Roberta Bertelli 1, G. Gimelli 2, A. DiDonato 3, G. Candiano l, and G. M. Ghiggeri 1 t Divisione di Nefrologia, 2Laboratorio di Citogenetica. and 3Servizio di Genetica Molecolare, lstituto G. Gaslini. Largo G. Gaslini 5, 1-16148 Genova, Italy Received January 21, 1992 / Revised March 5, 1992
Summary. A 353-bp region encoding for the NH2 terminus of the noncollagenic part of the ctl(V) chain was amplified by the polymerase chain reaction (PCR), subcloned and sequenced. The subcloned PCR product (pGC1) presented the same nucleotide sequence as the original fragment from the published sequence of COL5A1. In situ hybridization, using pGC1 as a probe, mapped the COL5A1 gene to chromosome 9q34.3. This assignement shows that COL5A1 is not synthenic with COL5A2, which is localized together with other collagen genes on chromosome 2.
Introduction Collagen V is an extracellular component of unknown function in several tissues and organs, including blood vessels, bone, nerve, muscle and tendon. Structurally, it occurs in various combination of chains, such as heterotrimers ctl(V)a2(V)c~3(V) or cd(V)2ct2(V), or more simply as a homotrimer of the cd(V) chain (Miller and Gay 1987; Niyibizi et al. 1984). The nucleotide sequence of c D N A encoding for human ct2(V) procollagen was first reported by Woodbury et al. (1989), and the C O L 5 A 2 gene was mapped to chromosome 2q24.3---,q31 (Vuorio and de Cronbrugghe 1990). Subsequently, Takahara et al. (1991) reported the c D N A sequence encoding the complete human prepro-ctl(V) chain, but its chromosome localization has not been identified. Information on the chromosome localization of C O L 1 A 5 is required in order to attempt a pathogenetic interpretation of those genetic diseases in which the extracellular matrix and, in particular, collagen (V) seem to be implicated (Candiano et al. 1991; Pienta et al. 1991; Nerlich and Schleicher 1991). In the present study, a 353-bp specific fragment of the COL5A1 gene coding for the NH2 terminus of the non-collagenic segment was amplified by the polymerase chain reaction (PCR), subcloned (pGC1), Correspondence to: G. M. Ghiggeri
sequenced, and was finally localized by fluorescent in situ hybridization (FISH).
Materials and methods R N A preparation
RNA was extracted from primary cultures of renal tubule epithelia in 6M guanidine isothiocyanate followed by centrifugation in a 5.7 M CsCI gradient for 20 h at 32000 rpm (Beckman SW 55 rotor, Palo Alto, Calif.). The pellet was then extracted with chloroform/ 1-butanol (4:1 vol/vol) (Maniatis et al. 1982). c D N A synthesis and P C R
From the published COL5AI eDNA sequence, two 21-met primers were chemically synthesized (PCR-MATE DNA SynthesizerApplyed Biosystem, Foster City, Calif.). The sense primer was igg 634 (5'-CTCAGCGTCCACAAGAAAAAT-3')and the antisense primer was igg 988 (5'-CCTAGG TCTTCG GGG TCTTCG-3'). First strand eDNA synthesis was performed by priming with random hexamers for 40 rain at 42~ in a final volume of 20 ~tl with 3.5 ~,tg RNA according to the instructions of the supplier of the eDNA Synthesis System Plus kit (Amersham, Little Chalfont, UK). PCR was performed in a Perkin Elmer DNA thermal cycler. The reaction mixture contained 50 pmoles primers, 10~_tlcDNA and 2.5 U Taq DNA polymerase (Gent Amp Kit-Perkin Elmer, Norwalk. Conn.). The reaction was performed under the following conditions: denaturation at 92~ for I rain, annealing at 55~ for 1rain, and extension
Fig. 1. Agarose electrophoresis of a PCR product before and after BamHI digestion. Lanes a, d pBR322 BstnI: lane b 353-bp PCR product; lane c BamHl digestion of PCR products (121 bp and 232bp)
175
Fig. 2. A Partial metaphase stained by DAPI; arrows indicate chromosomes 9. B Same partial metaphase hybridized with pGC1; arrows indicate hybrization signals on the long arms of both chromosomes 9
at 72~ for 2 min. After 35 cycles of gene amplification, the products were submitted to one final extension at 72~ for 6 rain and analyzed in a 2% agarose gel with ethidium bromide staining. BamHI digestion was carried out with 20UBamHI in buffer B (Boehringer, Mannheim, FRG) at 37~ for 1 h.
eo
p
Subcloning of the PCR product The PCR product was cloned in a pCR 1000 vector using the TA Cloning System (Invitrogen Corporation, San Diego, Calif.), and named pGC1. The cloned DNA fragment was separated from the vector by EcoRI-HindIII digestion.
oo
Sequencing pGC1 was sequenced using a T7 Sequencing Kit (Pharmacia, Uppsala, Sweden), following the method described by Sanger et al. (1977).
Metaphase chromosome preparations Normal human metaphase chromosomes were prepared from phytohemagglutinin-stimulated lymphocyte cultures. For precise mapping, the technique of methotrexase synchronization at prometaphase (Yunis et al. 1978) was used.
Fluorescent in situ hybridization Complete pGCI plasmid DNA was labeled by nick translation with biotin-16-dUTP (Boehringer). In situ hybridization was performed as described by Pinkel et al. (1986) on unbanded metaphases; for probe detection, the slides were incubated with fluorescein-conjugated (FITC) avidin. Two signal amplification cycles were achieved by subsequent incubation in anti-avidin antibody, followed by incubation in FITC avidin. Chromosomes were stained with both 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide. Slides were mounted in antifade solution and screened with a Zeiss fluroescent microscope.
Results and discussion The primers igg 634 and igg 988 were selected to amplify a 353-bp region of COL5A1, by P C R , on the basis of its low homology with other sequenced collagen genes and the presence of a BamHI at position 122. The results obtained by P C R are shown in Fig. 1 where the product was run in lane b and yielded a fragment of the expected size. Lane c shows the two fragments of 122bp and 231 bp, which established, as predicted, the presence of a BamHI site. In order to confirm these results, the frag-
eoooeoooooooo ooooeoooooooo
Fig. 3. Distribution of labeled sites on chromosome 9
ment was subcloned in a specific plasmid for P C R products, and n a m e d pGC1. It was completely sequenced and the results showed a precise homology with the corresponding published sequence. This probe was finally localized by F I S H on the terminal band of the long arm of c h r o m o s o m e 9q34.3 (Fig. 2). The combination of dual staining with a simple change of filter allowed the simultaneous identification of the c h r o m o s o m e according to the banding pattern and the visualization of t h e hybridization signal on the same metaphase image, with an unambiguous assignment of the probe to a specific chromosome. Following hybridization, the analysis of 80 prometaphase plates showed that 17.6% (26/147) of hybridization signals were located on the telomeric region of the long arm of one or two chromosomes 9 (Fig. 3). The salient point emerging from the present study is that the COL5A1 gene does not co-localize with any of the other genes coding for fibrillar collagens that are structurally similar to it, such as C O L 5 A 2 , COL3A1 and C O L l l A 1 (Vourio and de Crombrugghe 1990). This could have some pathogenetic importance in those genetic diseases in which collagen V, or more generally, the extracellular matrix seems to be involved. It is remarkable that, in a case of 9q32-qter m o n o s o m y (Zuffardi et al. 1989), osteopathia striata at the metaphyses of the femurs and the tibiae was reported.
176
Acknowledgements. The authors thank Professor Marco Fraccaro for helpful discussions, and Mr. R. Stubinski for his secretarial support. This work was carried out with a grant from the Italian Ministry of Health to the G. Gaslini Institute.
References
Candiano G, Altieri P, Oleggini R, Ginevri F, Ghiggeri GM (1991) Identification and characterization by CNBr peptide mapping of two collagen chains in cyst epithelia from polycystic kidney (abstract). J Am Soc Nephrol 2 : 250 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY Miller E J, Gay S (1987) The collagens: an overview and update. Method Enzymo[ 144 : 3-41 Nerlich A, Schleicher E (1991) Immunohistochemical localization of extracellular matrix components in human diabetic glomerular lesion. Am J Pathol 139 : 889-899 Niyibizi C, Fietzek PP, Van der Rest M (1984) Human placenta type V collagens. Evidence for existence of an ~.I(V)ct2(V)~3(V) collagen molecule. J Biol Chem 259 : 14170-14174 Pienta K, Murphy BC, Getzenberg RH, Coffey DS (1991) The effect of extracellular matrix interaction on morphologic transformation in vitro. Biochem Biophys Res Comm 179 : 333-339
Note added in proof. After submission and acceptance of this paper, the same localization of COL5A1 on 9q34.2-~q34.3 was reported by Greespan et al. [Genomics (1992) 12:836-837].
Pinkel D, Straume T. Gray J (1986) Cytogenetic analysis using quantitative high sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83 : 2934-2938 Sanger F, Nicklen S, Coulsen A R (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467 Takahara K, Sato Y, Okazawa K, Okamoto N, Noda A, Yaoi Y. Kato Y (1991) Complete primary structure of human collagen cd(V) chain. J Biol Chem 266 : 13124-13129 Vuorio E, Crombrugghe D de (1990) The family of collagen genes. Annu Rev Biochem 59 : 837-872 Woodbury D, Benson-Chanada V, Ramirez F (1989) Amino-terminal propeptide of human pro-c~2(V) collagen conforms to the structural criteria of a fibrillar procollagen molecule. J Biol Chem 264 : 2735-2738 Yunis J J, Sawyer JR, Ball DW (1978) Characterization of banding patterns of metaphase-profase G banded chromosomes and their use in gene mapping. Cytogenet Cell Genet 22 : 678-683 Zuffardi O, Caiulo A, Maraschio P, Tupler R, Bianchi E. Amisano P, Beluffi G, Moratti R, Liguri G (1989) Regional assignment of the loci for adenylate kinase to 9q32 and for alacid glycoprotein to 9q31-q32: a lkocus for Goltz syndrome in region 9q32-qter? Hum Genet 82:17-19
