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The human immunoglobulin variable lambda locus IGLV9 gene is a monomorphic marker in the urban Brazilian population
Immunology Letters 69 (1999) 369 – 370
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Rapid note
The human immunoglobulin variable lambda locus IGLV9 gene is a monomorphic marker in the urban Brazilian population Maria Isabel da Silva a, Geraldo A.S. Passos a,b,* a
Grupo de Imunogene´tica Molecular (GIM), Departamento de Gene´tica, Faculdade de Medicina de Ribeira˜o Preto, Uni6ersidade de Sa˜o Paulo, 14040 -900 Ribeira˜o Preto, SP, Brazil b Uni6ersidade de Sa˜o Paulo, Faculdade de Odontologia de Ribeira˜o Preto, Departamento de Morfologia, 14040 -904 Ribeira˜o Preto, SP, Brazil
Abstract The physical map of the human immunoglobulin variable lambda locus (IGLV) located on chromosome 22q11.1-q11.2 shows the existence of 52 functional V-lambda genes distributed among three V-clusters. The IGLV9S1 gene, located in the V – B cluster, is a sequence tagged site and is a useful marker for restriction fragment length polymorphism (RFLP) population studies. The V-lambda genes are associated in the genome with EcoRI fragments detectable in Southern blots of genomic DNA samples. We have analysed DNA samples of an urban Brazilian population by Southern-EcoRI-RFLP using an IGLV9 gene segment. Among 75 unrelated individuals analysed, we detected a single 6.0 kb EcoRI fragment containing the IGLV9 gene at 100% frequency. Reverse transcription followed by polymerase chain reaction (RT-PCR) of peripheral blood leukocyte total RNA from unrelated individuals showed that IGLV9S1 is a functional gene contributing to the B lymphocyte repertoire. These data represent evidence for monomorphism of the IGVL9S1 gene in this urban population. We demonstrate that IGLV9S1 is a functional single copy gene and is an important marker in the IGLV locus. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Immunoglobulin lambda variable locus; Human V-lambda 9 gene; Human chromosome 22q11; Restriction fragment length polymorphism
The human immunoglobulin lambda variable locus (IGLV), which codes for the V-lambda light chains, is located on chromosome 22q11 [1]. The physical map of this region has shown the existence of at least 52 variable lambda gene segments with open reading frames. Of these, 31 are functional V-lambda genes, which have been subgrouped into 11 families (IGLV1– IGLV11): five VlI, five VlII, eight VlIII, three VlIV, three VlV, one VlVI, two VlVII, one VlVIII, one VlIX, one VlX, and one VlXI [2 – 4]. The TL6 Vl gene (IGLV8 family), implicated in an autoimmune disease of myelin protein, was located outside the IGLV locus, translocated on the chromosome 8q11.2 [5,6]. This project was approved by the Ethical Commission, Hospital das Clı´nicas de Ribeira˜o Preto, USP (4953/98). * Corresponding author. Fax: +55-16-633-0069. E-mail address: [email protected] (G.A.S. Passos)
Recently, about one megabase of human chromosome 22q11 including the IGLV locus was totally mapped and sequenced, representing the largest contiguous human DNA fragment sequenced to date [7–9]. Due to the high homology between the different germline Vl sequences, it is difficult to find specific single copy sequences in the IGLV locus. Only two sequences have been described so far in 800 kb of the variable locus: the IGLVA pseudogene [10] and the IGLV9S1 gene [11]. These are single copy sequences conferring sequence-tagged sites, which represent useful markers for restriction fragment length polymorphism population studies. The V-lambda genes are associated in the genome with EcoRI fragments detectable in Southern blots of genomic DNA samples [2,12]. We evaluated the IGLV9S1 gene polymorphism in an urban Brazilian population of Ribeira˜o Preto region, Sa˜o Paulo State, south-eastern Brazil by Southern hybridization.
0165-2478/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 9 9 ) 0 0 1 0 9 - 1
370
M.I. da Sil6a, G.A.S. Passos / Immunology Letters 69 (1999) 369–370
This gene is located within a 6.0 kb EcoRI fragment [2] and we used a 177 base pair DNA fragment as a probe, including a portion of the IGLV9S1 gene, nucleotides 137–312, covering part of the FR2, CDR2, FR3 and CDR3 regions (probe pVl9M177, EMBL accession No. 75389) [13]. Among 75 unrelated individuals analysed, we demonstrated an invariable 6.0 kb EcoRI genomic fragment consisting of an important monomorphic marker (Fig. 1). The expression of the IGLV9S1 gene was investigated by reverse transcription-polymerase chain reaction (RT-PCR) analysis of leukocytes total RNA samples from 15 unrelated individuals. Total cDNA was used in PCR with specific primers for the IGLV9S1 gene exon [11]. We obtained an expected PCR product of 400 base pairs, demonstrating that the IGLV9S1 is a functional gene in all the individuals analysed (Fig. 2). Our results agree with the physical and functional maps of the IGLV locus [2,14]. We concluded that IGLV9S1 is an expressed single copy gene and represents an important tool for further analysis of the IGLV locus.
Acknowledgements
Fig. 2. RT-PCR of total RNA from peripheral leukocytes of two unrelated individuals. Total cDNA was used in PCR with primers covering the entire IGLV9S1 exon [11]. The PCR conditions were 94°C for 3 min, 60°C for 10 min (one cycle), 72°C for 2 min, 94°C for 1.5 min, 60°C for 2 min (30 cycles), 72°C for 10 min, and 10°C for 5 min. The expected 400 base pair PCR product was resolved by 1.5% agarose gel electrophoresis and blotted to a Hybond N + membrane. The blot was hybridized to the pVl9M177 – 32P-labeled probe and washed under high stringency conditions. (typical result). Lane 1, control PCR with genomic DNA; lane 2, negative control PCR without DNA; lanes 3 and 4, IGLV9S1 cDNAs.
(98/05584-9; 98/09789-4) and CNPq-FAPESP (96/58422). We thank Leonardo Medeiros for technical assistance (FAPESP fellow 98/12904-0).
MIS received a predoctoral fellow from CNPq-Brasil (141317/95-7). GASP received grants from FAPESP References
Fig. 1. Southern blot of seven samples of human genomic DNA digested with EcoRI and hybridized with the pVl9M177 probe. All unrelated individuals presented a unique 6.0 kb monomorphic fragment. Membrane Hybond N + (Amersham) was washed under high stringency conditions. (typical result).
[1] A. de la Chapelle, A. Lenoir, G. Boue, P. Gallano, C. Huerre, M.F. Szajnert, M. Jeanpierre, M. Laloel, J.C. Kaplan, Nucleic Acids Res. 11 (1983) 1133 – 1142. [2] J.-P. Frippiat, S.C. Williams, I.M. Tomlinson, G.P. Cook, D. Cherif, D. Le Paslier, J.E. Collins, I. Dunhan, G. Winter, M.-P. Lefranc, Hum. Mol. Genet. 4 (1995) 983 – 991. [3] K. Kawasaki, S. Minoshima, K. Schooler, J. Kudoh, S. Asakawa, P.J. de Jong, N. Shimizu, Genome Res. 5 (1995) 125– 135. [4] S.C. Williams, J.-P. Frippiat, I.M. Tomlinson, O. Ignatovich, M.-P. Lefranc, G. Winter, J. Mol. Biol. 264 (1996) 220–232. [5] J.-P. Frippiat, P. Dard, S. Marsh, G. Winter, M.-P. Lefranc, Eur. J. Immunol. 27 (1997) 1260 – 1265. [6] R.G.P. Queiroz, A. Carrier, G. Victorero, B. Jordan, G.A.S. Passos, Immunol. Lett. 59 (1997) 177 – 180. [7] K. Kawasaki, S. Minoshima, E. Nakato, K. Shibuia, A. Shintani, J.L. Schmeits, J. Wang, N. Shimizu, Genome Res. 7 (1997) 250 – 261. [8] G.A.S. Passos, Genet. Mol. Biol. 21 (1998) 281 – 286. [9] P. Chuchana, D. Alexandre, G. Lefranc, M.-P. Lefranc, Nucleic Acids Res. 19 (1991) 4786. [10] G.A.S. Passos, J.-P. Frippiat, M.-P. Lefranc, Clin. Exp. Immunogenet. 11 (1994) 222 – 226. [11] S.C. Williams, G. Winter, Eur. J. Immunol. 23 (1993) 1456– 1461. [12] O. Ignatovich, I.M. Tomlinson, P.T. Jones, G. Winter, J. Mol. Biol. 268 (1997) 69 – 77.
www.elsevier.com/locate/
Rapid note
The human immunoglobulin variable lambda locus IGLV9 gene is a monomorphic marker in the urban Brazilian population Maria Isabel da Silva a, Geraldo A.S. Passos a,b,* a
Grupo de Imunogene´tica Molecular (GIM), Departamento de Gene´tica, Faculdade de Medicina de Ribeira˜o Preto, Uni6ersidade de Sa˜o Paulo, 14040 -900 Ribeira˜o Preto, SP, Brazil b Uni6ersidade de Sa˜o Paulo, Faculdade de Odontologia de Ribeira˜o Preto, Departamento de Morfologia, 14040 -904 Ribeira˜o Preto, SP, Brazil
Abstract The physical map of the human immunoglobulin variable lambda locus (IGLV) located on chromosome 22q11.1-q11.2 shows the existence of 52 functional V-lambda genes distributed among three V-clusters. The IGLV9S1 gene, located in the V – B cluster, is a sequence tagged site and is a useful marker for restriction fragment length polymorphism (RFLP) population studies. The V-lambda genes are associated in the genome with EcoRI fragments detectable in Southern blots of genomic DNA samples. We have analysed DNA samples of an urban Brazilian population by Southern-EcoRI-RFLP using an IGLV9 gene segment. Among 75 unrelated individuals analysed, we detected a single 6.0 kb EcoRI fragment containing the IGLV9 gene at 100% frequency. Reverse transcription followed by polymerase chain reaction (RT-PCR) of peripheral blood leukocyte total RNA from unrelated individuals showed that IGLV9S1 is a functional gene contributing to the B lymphocyte repertoire. These data represent evidence for monomorphism of the IGVL9S1 gene in this urban population. We demonstrate that IGLV9S1 is a functional single copy gene and is an important marker in the IGLV locus. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Immunoglobulin lambda variable locus; Human V-lambda 9 gene; Human chromosome 22q11; Restriction fragment length polymorphism
The human immunoglobulin lambda variable locus (IGLV), which codes for the V-lambda light chains, is located on chromosome 22q11 [1]. The physical map of this region has shown the existence of at least 52 variable lambda gene segments with open reading frames. Of these, 31 are functional V-lambda genes, which have been subgrouped into 11 families (IGLV1– IGLV11): five VlI, five VlII, eight VlIII, three VlIV, three VlV, one VlVI, two VlVII, one VlVIII, one VlIX, one VlX, and one VlXI [2 – 4]. The TL6 Vl gene (IGLV8 family), implicated in an autoimmune disease of myelin protein, was located outside the IGLV locus, translocated on the chromosome 8q11.2 [5,6]. This project was approved by the Ethical Commission, Hospital das Clı´nicas de Ribeira˜o Preto, USP (4953/98). * Corresponding author. Fax: +55-16-633-0069. E-mail address: [email protected] (G.A.S. Passos)
Recently, about one megabase of human chromosome 22q11 including the IGLV locus was totally mapped and sequenced, representing the largest contiguous human DNA fragment sequenced to date [7–9]. Due to the high homology between the different germline Vl sequences, it is difficult to find specific single copy sequences in the IGLV locus. Only two sequences have been described so far in 800 kb of the variable locus: the IGLVA pseudogene [10] and the IGLV9S1 gene [11]. These are single copy sequences conferring sequence-tagged sites, which represent useful markers for restriction fragment length polymorphism population studies. The V-lambda genes are associated in the genome with EcoRI fragments detectable in Southern blots of genomic DNA samples [2,12]. We evaluated the IGLV9S1 gene polymorphism in an urban Brazilian population of Ribeira˜o Preto region, Sa˜o Paulo State, south-eastern Brazil by Southern hybridization.
0165-2478/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 9 9 ) 0 0 1 0 9 - 1
370
M.I. da Sil6a, G.A.S. Passos / Immunology Letters 69 (1999) 369–370
This gene is located within a 6.0 kb EcoRI fragment [2] and we used a 177 base pair DNA fragment as a probe, including a portion of the IGLV9S1 gene, nucleotides 137–312, covering part of the FR2, CDR2, FR3 and CDR3 regions (probe pVl9M177, EMBL accession No. 75389) [13]. Among 75 unrelated individuals analysed, we demonstrated an invariable 6.0 kb EcoRI genomic fragment consisting of an important monomorphic marker (Fig. 1). The expression of the IGLV9S1 gene was investigated by reverse transcription-polymerase chain reaction (RT-PCR) analysis of leukocytes total RNA samples from 15 unrelated individuals. Total cDNA was used in PCR with specific primers for the IGLV9S1 gene exon [11]. We obtained an expected PCR product of 400 base pairs, demonstrating that the IGLV9S1 is a functional gene in all the individuals analysed (Fig. 2). Our results agree with the physical and functional maps of the IGLV locus [2,14]. We concluded that IGLV9S1 is an expressed single copy gene and represents an important tool for further analysis of the IGLV locus.
Acknowledgements
Fig. 2. RT-PCR of total RNA from peripheral leukocytes of two unrelated individuals. Total cDNA was used in PCR with primers covering the entire IGLV9S1 exon [11]. The PCR conditions were 94°C for 3 min, 60°C for 10 min (one cycle), 72°C for 2 min, 94°C for 1.5 min, 60°C for 2 min (30 cycles), 72°C for 10 min, and 10°C for 5 min. The expected 400 base pair PCR product was resolved by 1.5% agarose gel electrophoresis and blotted to a Hybond N + membrane. The blot was hybridized to the pVl9M177 – 32P-labeled probe and washed under high stringency conditions. (typical result). Lane 1, control PCR with genomic DNA; lane 2, negative control PCR without DNA; lanes 3 and 4, IGLV9S1 cDNAs.
(98/05584-9; 98/09789-4) and CNPq-FAPESP (96/58422). We thank Leonardo Medeiros for technical assistance (FAPESP fellow 98/12904-0).
MIS received a predoctoral fellow from CNPq-Brasil (141317/95-7). GASP received grants from FAPESP References
Fig. 1. Southern blot of seven samples of human genomic DNA digested with EcoRI and hybridized with the pVl9M177 probe. All unrelated individuals presented a unique 6.0 kb monomorphic fragment. Membrane Hybond N + (Amersham) was washed under high stringency conditions. (typical result).
[1] A. de la Chapelle, A. Lenoir, G. Boue, P. Gallano, C. Huerre, M.F. Szajnert, M. Jeanpierre, M. Laloel, J.C. Kaplan, Nucleic Acids Res. 11 (1983) 1133 – 1142. [2] J.-P. Frippiat, S.C. Williams, I.M. Tomlinson, G.P. Cook, D. Cherif, D. Le Paslier, J.E. Collins, I. Dunhan, G. Winter, M.-P. Lefranc, Hum. Mol. Genet. 4 (1995) 983 – 991. [3] K. Kawasaki, S. Minoshima, K. Schooler, J. Kudoh, S. Asakawa, P.J. de Jong, N. Shimizu, Genome Res. 5 (1995) 125– 135. [4] S.C. Williams, J.-P. Frippiat, I.M. Tomlinson, O. Ignatovich, M.-P. Lefranc, G. Winter, J. Mol. Biol. 264 (1996) 220–232. [5] J.-P. Frippiat, P. Dard, S. Marsh, G. Winter, M.-P. Lefranc, Eur. J. Immunol. 27 (1997) 1260 – 1265. [6] R.G.P. Queiroz, A. Carrier, G. Victorero, B. Jordan, G.A.S. Passos, Immunol. Lett. 59 (1997) 177 – 180. [7] K. Kawasaki, S. Minoshima, E. Nakato, K. Shibuia, A. Shintani, J.L. Schmeits, J. Wang, N. Shimizu, Genome Res. 7 (1997) 250 – 261. [8] G.A.S. Passos, Genet. Mol. Biol. 21 (1998) 281 – 286. [9] P. Chuchana, D. Alexandre, G. Lefranc, M.-P. Lefranc, Nucleic Acids Res. 19 (1991) 4786. [10] G.A.S. Passos, J.-P. Frippiat, M.-P. Lefranc, Clin. Exp. Immunogenet. 11 (1994) 222 – 226. [11] S.C. Williams, G. Winter, Eur. J. Immunol. 23 (1993) 1456– 1461. [12] O. Ignatovich, I.M. Tomlinson, P.T. Jones, G. Winter, J. Mol. Biol. 268 (1997) 69 – 77.