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Partial cloning of the class I gene-encompassing regions in the rat major histocompatibility complex
Experimental Animal Science
Partial cloning of the class I gene-encompassing regions in the rat major histocompatibility complex LUTZ WALTER, SOFIA IOANNIDU,and EBERHARDGONTHER Division of Immunogenetics,University of G6ttingen, G6ningen, Germany
Summary Two contigs of overlapping PAC clones mapping to the centromeric and telomeric parts of the rat major histocompatibility complex (MHC) are described. Number and organization of class I genes in these contigs are delineated and compared to the corresponding regions in the mouse MHC. The evolutionary history of these class I genes is discussed.
Key words:MHC, class I genes,
evolution
Introduction Class I genes can be found in the centromeric as well as the telomeric parts of the rat major histocompatibility complex (MHC), representing the RT1-A and RT1-C regions. A similar organization is found in the mouse MHC, where class I genes map to the centromeric H2-K region and the telomeric H2-D, Q, T, and M regions. The centromeric class I gene-containing region is absent from the human MHC. It is generally assumed that class I genes present in the RT1-A and H2-K regions originated from a translocation of class I genes from the RT1-C and H2-D/Q regions, respectively, into the Rpsl8 - Ringl intervall. The Rpsl8 and Ringl genes show clear orthology in human, rat and mouse and are anchor genes in this part of the MHC (WALTERand G/2NTHER 1998). It remains uncertain so far, whether the translocation occurred in a common ancestor of rat and mouse, or happened independently and even repeatedly in these species. In order to analyse the evolution of the RT1-A region in detail, we have cloned this region and are in the progress of cloning the RT1-C region. The Batl gene marks the
J. Exp. Anim. Sci. 2000; 41:91-94 Urban & Fischer Verlag http://www.urbanfischer.de/journals/jeansc 0939-8600/00/41/01-02-091 $12.00/0
92
L. WALTER,S. IOANNIDUand E. GONTHER
start of the RT1-C region at the boundary of the class III/RT1-C regions and is also regarded as an anchor gene due to its orthology in human, mouse and rat.
Materials and Methods A PAC library of the BN rat strain (WooN et al. 1998) was screened with probes of the rat Sacra21, Ring1, and Batl genes. The PAC DNA was digested with different restriction enzymes,
electrophoresed in 0.8% agarose gels and blotted on positively charged nylon membranes according to standard procedures. Overlapping PAC clones were identified by common restriction fragments and crosshybrization to various probes.
Results and Discussion In order to clone the RT1-A region we screened a PAC library of the BN rat (RT1 haplotype n) with probes derived from the Sacra21 and R i n g l genes (WALTER and G~)NTH~R 1998, WAI~TEa et al. 1996). Three overlapping clones could be established which contain the complete RT1-A region. In addition to other genes like R p s l 8 , Ring2, Ke4, Rxrb, C o l l l a 2 , and R T 1 - H b three class I genes were detected in this contig. The following gene order was establisehd: cen - R p s 1 8 - Sacra21 - R T 1 - A 1 - R T 1 - A 2 - R T 1 - A 3 - Ring1 R i n g 2 - K e 4 - R x r b - C o l l l a 2 - R T 1 - H b - tel. Preliminary evidence suggests that all R T 1 - A genes of the RT1 n haplotype are expressed. In contrast, mouse H2 haplotypes investigated so far exhibit only two class I genes in the corresponding centromeric MHC region, H 2 - K and H 2 - K 1 . Whereas H 2 - K is functional, H 2 - K 1 is a pseudogene, and the other expressed class I genes map to the telomeric part of the mouse MHC, the H2-D/Q/T regions. Since the Bat1 gene maps only 10 kb proximal to the H - 2 D gene in the mouse, we screened the BN PAC library with a probe from the rat B a t l gene in order to isolate class I genes mapping to a similar position as H - 2 D . Two overlapping PAC clones were identified which contain the T n f a n d B a t l genes at a distance of about 40 kb and in addition two class I genes preliminarily designated 3. 7 and 18 (Fig. 1). The distance between the first class I gene and Bat1 was determined to be 10 kb, similar to the mouse. However, partial sequence analysis of exons 2 and 5 indicates that 3. 7 and 18 are more closely related to R T 1 - A class I genes than to H2-D, H 2 - L or H 2 - K class I genes of the mouse. This might indicate that R T 1 - A region class I genes could have originated from 3.7 or 18 or closely related genes, favoring independent translocation of class I genes from the telomeric to the centromeric region in rat and mouse. Otherwise, closer intra- versus interspecies relationship of class I genes could also reflect gene conversion events (RADA et al. 1990). In the meantime a third Tnf- and B a t l - p o s i t i v e PAC clone could be isolated. It contains 6 class I genes in a region 120 kb telomeric of B a t l including the 3. 7 and 18 genes. In the human M H C class I region M I C genes, which are distantly related to classical class I genes, are located between Bat1 and H L A - B (Fig. 1). Interestingly, M I C - h o m o l o g o u s gene are absent from the rat and the mouse. In the H2 d haplotype a duplication of class I
Partial cloning of the class I gene-encompassing regions
93
Sacra21 RpslS[A Ringl n
(~
'//'
RT1 n
"q! It" i
Trlf
i
40
B D
Hsp70 Tiff Batl
II
Mog
Ill
Batl 3.7 ~ 10 ~
I
//,
18
PAC-A PAC-G
TNF 40
~BATl . 30 . . . MICB40 ~
T~
40
Batl
40
~ 9
X 50~ A s o
&
90
C
HLA
H2 b
--~
H2 d
"£:2
17 D
,~ . . . . . . . . . . . D
60
D2 20 [33 20 D~loBatlb
L
Fig. 1. Schematic representation of the different MHC regions and map positions of selected anchor genes. Transcriptional orientation is indicated by an arrow. Distances between genes are given in kb. The RT1 map is according to WALTERet al. (1996) and WALTERand GONTHER(1998). The HLA map is based on CAMPBELLand TROWSDALE(1997), and the H2 maps are published by WROBLEWSKIet al. (1994).
genes as well as the Bat1 gene could be observed (WROBLEWSKIet al. 1994). The duplicated Batl gene most probably represents a pseudogene. Screening the PAC library has so far not resulted in the identification of further Bat1 genes.
Acknowledgements
The authors acknowledge the German Resource Center (RZPD) in Berlin, Germany, for supply of PAC filters and clones. This study was supported by EU Grant PL 96562.
References
CAMPBELL,D. and J. TROWSDALE.1997. Map of the human MHC. Immunol. Today 14: 43. RADA, C., R. LORENZ1,S. J. PowIs, J. VAN DEN BOGAERDE,P. PARHAM,and J. C. HOWARD. 1990. Concerted evolution of class I genes in the major histocompatibility complex of murine rodents. Proc. Natl. Acad. Sci. USA 87: 2167-2171.
94
L. WALTER,S. IOANNIDUand E. GI)NTHER
WALTER,L. and E. GUNTHER.1998. Isolation and characterization of a novel highly conserved gene in the centromeric part of the MHC. Genomics 52: 298-304. WALTER,L., K. FISCHER,and E. G12NTHER.1996. Physical mapping of the Ringl, Ring2, Ke6, Ke4, Rxrb, Collla2, and RT1-Hb genes in the rat major histocompatibility complex. Immunogenetics 44: 218-221. WOON, P. Y., K. OSOEGAWA,P. J. KAISAKI,B. ZHAO,J. J. CATANESE,D. GAUGIJIER,R. Cox, E. R. LEVY, G. M. LATHROP,A. P. MONACO,and P. J. DE JONG. 1998. Construction and characterization of a 10-fold genome equivalent rat Pl-derived artificial chromosome library. Genomics 50: 306-16. WROBLEWS~:I,J, M., S. G. KAMINS~Y,and I. NAKAMURA.1994. Bat-I genes and the origin of multiple class I loci in the H-2D region. Immunogenetics 39: 276-280.
Corresponding author: Dr. LUTZ WALTER, Abteilung Immungenetik, Universit~it G6ttingen, Heinrich-Dfiker-Weg 12, 37073 G6ttingen, Germany Tel.: (49) 551-395854; Fax: (49) 551-395852; e-mail: [email protected]
Partial cloning of the class I gene-encompassing regions in the rat major histocompatibility complex LUTZ WALTER, SOFIA IOANNIDU,and EBERHARDGONTHER Division of Immunogenetics,University of G6ttingen, G6ningen, Germany
Summary Two contigs of overlapping PAC clones mapping to the centromeric and telomeric parts of the rat major histocompatibility complex (MHC) are described. Number and organization of class I genes in these contigs are delineated and compared to the corresponding regions in the mouse MHC. The evolutionary history of these class I genes is discussed.
Key words:MHC, class I genes,
evolution
Introduction Class I genes can be found in the centromeric as well as the telomeric parts of the rat major histocompatibility complex (MHC), representing the RT1-A and RT1-C regions. A similar organization is found in the mouse MHC, where class I genes map to the centromeric H2-K region and the telomeric H2-D, Q, T, and M regions. The centromeric class I gene-containing region is absent from the human MHC. It is generally assumed that class I genes present in the RT1-A and H2-K regions originated from a translocation of class I genes from the RT1-C and H2-D/Q regions, respectively, into the Rpsl8 - Ringl intervall. The Rpsl8 and Ringl genes show clear orthology in human, rat and mouse and are anchor genes in this part of the MHC (WALTERand G/2NTHER 1998). It remains uncertain so far, whether the translocation occurred in a common ancestor of rat and mouse, or happened independently and even repeatedly in these species. In order to analyse the evolution of the RT1-A region in detail, we have cloned this region and are in the progress of cloning the RT1-C region. The Batl gene marks the
J. Exp. Anim. Sci. 2000; 41:91-94 Urban & Fischer Verlag http://www.urbanfischer.de/journals/jeansc 0939-8600/00/41/01-02-091 $12.00/0
92
L. WALTER,S. IOANNIDUand E. GONTHER
start of the RT1-C region at the boundary of the class III/RT1-C regions and is also regarded as an anchor gene due to its orthology in human, mouse and rat.
Materials and Methods A PAC library of the BN rat strain (WooN et al. 1998) was screened with probes of the rat Sacra21, Ring1, and Batl genes. The PAC DNA was digested with different restriction enzymes,
electrophoresed in 0.8% agarose gels and blotted on positively charged nylon membranes according to standard procedures. Overlapping PAC clones were identified by common restriction fragments and crosshybrization to various probes.
Results and Discussion In order to clone the RT1-A region we screened a PAC library of the BN rat (RT1 haplotype n) with probes derived from the Sacra21 and R i n g l genes (WALTER and G~)NTH~R 1998, WAI~TEa et al. 1996). Three overlapping clones could be established which contain the complete RT1-A region. In addition to other genes like R p s l 8 , Ring2, Ke4, Rxrb, C o l l l a 2 , and R T 1 - H b three class I genes were detected in this contig. The following gene order was establisehd: cen - R p s 1 8 - Sacra21 - R T 1 - A 1 - R T 1 - A 2 - R T 1 - A 3 - Ring1 R i n g 2 - K e 4 - R x r b - C o l l l a 2 - R T 1 - H b - tel. Preliminary evidence suggests that all R T 1 - A genes of the RT1 n haplotype are expressed. In contrast, mouse H2 haplotypes investigated so far exhibit only two class I genes in the corresponding centromeric MHC region, H 2 - K and H 2 - K 1 . Whereas H 2 - K is functional, H 2 - K 1 is a pseudogene, and the other expressed class I genes map to the telomeric part of the mouse MHC, the H2-D/Q/T regions. Since the Bat1 gene maps only 10 kb proximal to the H - 2 D gene in the mouse, we screened the BN PAC library with a probe from the rat B a t l gene in order to isolate class I genes mapping to a similar position as H - 2 D . Two overlapping PAC clones were identified which contain the T n f a n d B a t l genes at a distance of about 40 kb and in addition two class I genes preliminarily designated 3. 7 and 18 (Fig. 1). The distance between the first class I gene and Bat1 was determined to be 10 kb, similar to the mouse. However, partial sequence analysis of exons 2 and 5 indicates that 3. 7 and 18 are more closely related to R T 1 - A class I genes than to H2-D, H 2 - L or H 2 - K class I genes of the mouse. This might indicate that R T 1 - A region class I genes could have originated from 3.7 or 18 or closely related genes, favoring independent translocation of class I genes from the telomeric to the centromeric region in rat and mouse. Otherwise, closer intra- versus interspecies relationship of class I genes could also reflect gene conversion events (RADA et al. 1990). In the meantime a third Tnf- and B a t l - p o s i t i v e PAC clone could be isolated. It contains 6 class I genes in a region 120 kb telomeric of B a t l including the 3. 7 and 18 genes. In the human M H C class I region M I C genes, which are distantly related to classical class I genes, are located between Bat1 and H L A - B (Fig. 1). Interestingly, M I C - h o m o l o g o u s gene are absent from the rat and the mouse. In the H2 d haplotype a duplication of class I
Partial cloning of the class I gene-encompassing regions
93
Sacra21 RpslS[A Ringl n
(~
'//'
RT1 n
"q! It" i
Trlf
i
40
B D
Hsp70 Tiff Batl
II
Mog
Ill
Batl 3.7 ~ 10 ~
I
//,
18
PAC-A PAC-G
TNF 40
~BATl . 30 . . . MICB40 ~
T~
40
Batl
40
~ 9
X 50~ A s o
&
90
C
HLA
H2 b
--~
H2 d
"£:2
17 D
,~ . . . . . . . . . . . D
60
D2 20 [33 20 D~loBatlb
L
Fig. 1. Schematic representation of the different MHC regions and map positions of selected anchor genes. Transcriptional orientation is indicated by an arrow. Distances between genes are given in kb. The RT1 map is according to WALTERet al. (1996) and WALTERand GONTHER(1998). The HLA map is based on CAMPBELLand TROWSDALE(1997), and the H2 maps are published by WROBLEWSKIet al. (1994).
genes as well as the Bat1 gene could be observed (WROBLEWSKIet al. 1994). The duplicated Batl gene most probably represents a pseudogene. Screening the PAC library has so far not resulted in the identification of further Bat1 genes.
Acknowledgements
The authors acknowledge the German Resource Center (RZPD) in Berlin, Germany, for supply of PAC filters and clones. This study was supported by EU Grant PL 96562.
References
CAMPBELL,D. and J. TROWSDALE.1997. Map of the human MHC. Immunol. Today 14: 43. RADA, C., R. LORENZ1,S. J. PowIs, J. VAN DEN BOGAERDE,P. PARHAM,and J. C. HOWARD. 1990. Concerted evolution of class I genes in the major histocompatibility complex of murine rodents. Proc. Natl. Acad. Sci. USA 87: 2167-2171.
94
L. WALTER,S. IOANNIDUand E. GI)NTHER
WALTER,L. and E. GUNTHER.1998. Isolation and characterization of a novel highly conserved gene in the centromeric part of the MHC. Genomics 52: 298-304. WALTER,L., K. FISCHER,and E. G12NTHER.1996. Physical mapping of the Ringl, Ring2, Ke6, Ke4, Rxrb, Collla2, and RT1-Hb genes in the rat major histocompatibility complex. Immunogenetics 44: 218-221. WOON, P. Y., K. OSOEGAWA,P. J. KAISAKI,B. ZHAO,J. J. CATANESE,D. GAUGIJIER,R. Cox, E. R. LEVY, G. M. LATHROP,A. P. MONACO,and P. J. DE JONG. 1998. Construction and characterization of a 10-fold genome equivalent rat Pl-derived artificial chromosome library. Genomics 50: 306-16. WROBLEWS~:I,J, M., S. G. KAMINS~Y,and I. NAKAMURA.1994. Bat-I genes and the origin of multiple class I loci in the H-2D region. Immunogenetics 39: 276-280.
Corresponding author: Dr. LUTZ WALTER, Abteilung Immungenetik, Universit~it G6ttingen, Heinrich-Dfiker-Weg 12, 37073 G6ttingen, Germany Tel.: (49) 551-395854; Fax: (49) 551-395852; e-mail: [email protected]