- Email: [email protected]
t complex
Genetic Analysis of the T/t Complex K a r e n Artzt
ABSTRACT: The T/t-complex has hem considerable interest for immunologists, primarily because of its close genetic linkage to the major histocompatibility complex (MHC) on mouse chromosome 17. This interest has been heightened recently u'ith the discorery that the M H C is fully contained u'ithin the t-complex and that tu,o regions of the MHC. Qa and K. contain t-lethal genes. For a long time, T/t has been an enigmatic system, mainly because classical genetfi analysis u as not possible. Here the system is defined, recent in~rmation is presented, and our understanding of the mouse data to at'ailable information about the human MHC is correlated. ABBREVIATIONS Ce-2 kidney catalase old combined lipase deficiency
fu
fused
glo- 1 qk tf
glyoxylase- 1 quaking tufted
P R O P E R T I E S OF T H E T/t C O M P L E X
Studies of the t-complex address two major questions: first, the mechanisms of embryonic cell commitment at the cell-cell interaction level, and second, the orchestrated genetic control of a seemingly complicated developmental program. t-haplotypes are traditionally found in wild populations of mice the world over. Any particular haplotype may carry one or two different recessive t-lethal mutations, or none. O f the 16 known lethals, six have been analyzed embryologically and all of these exhibit their lethal effects early in gestation, from shortly after the zygote begins cell division (16-32 cells) until just prior to organ formation (9'/2 days gestation). This is precisely the period in development when cell commitment is taking place and when classical MHC products (K and D antigens) are not expressed. (The possible exception to the latter statement is class I expression on the extraembryonic trophoblast [ 1].) Also, these six different lethals affect the embryo at different stages of development and in a specific tissue, seemingly by a failure of cell-cell interactions [2]. Thus, t-haplotypes represent a collection of genetically linked natural tools dissecting early development. (The embryology has been reviewed extensively by Bennett [3].) In addition to their linkage, another aspect that makes these lethal genes particularly attractive to study is that they appear to be functionally related even though they act at different developmental stages. Early on, it was appreciated that these mutations exhibited interesting complementation interactions. Although the different lethals were known to complement one another (i.e., a mouse that was t ' / t ~' could be constructed and proceed normally through the
From the Memorial SIoan-Kettering Cancer Center and S/oan-Kettering Dirision. Gradnate School of Medicine Sciences, Cornell University, Neu, York. Neu' York. Address correspondence to Karen Artzt, Memorial S/oan-Kettering Cancer Center and Sloan-Kettering Dirision. Graduate School of Medical Sciences. Cornell Uniz,ersiO ~, New York. N Y 10021.
374 0198-8859/86/$3.50
Human Immunology 15, 3 v 4 - 3 8 0 (1986) ~) Elsevier Science Publishing Co., Inc., 1986 52 Vanderbilt Ave., New York, NY 10017
Genetic Analysis of the T/t Complex
375
developmental arrest characteristic o f t ~ and t'), the viability of such heterozygotes was very variable and d e p e n d e n t to a certain extent on which two mutations were involved [3]. Moreov.er, a thorough embryological study [4] showed that heterozygotes for two different t-lethals that did not survive, often died resembling a third t-lethal morphologically. These were some of the first clues to the idea that different t-lethals were related. A second and seemingly trivial property of wild derived t-haplotypes is that they all contain the same recessive mutation, "the tail interactor factor" (t T) which, in fact, was the basis of their detection. The recessive t r mutation interacts with Brachyury (T), a semidominant laboratory-derived point mutation, causing a short tail. W h e n a mouse is heterozygous for these two mutations (T/t r) it has no tail. The traditional test for the presence of t-haplotypes is to mate a wild derived normal tailed male (+/t r) to a short-tailed (T/+) female; tailless mice (T/t r) among the progeny indicate the presence of a t-haplotype. A third, and still very perplexing property of t-haplotypes is the p h e n o m e n o n known as transmission ratio distortion. Males heterozygous for a t-haplotype violate Mendel's rule and transmit the t-haplotype to more than 9 8 % of their progeny rather than the expected half. Transmission ratio distortion has been studied in detail and reviewed [5,6] and discussion of its mechanisms is outside the scope of this paper. Accepted at face value as a p h e n o m e n o n , however, it explains the worldwide prevalence of t-haplotypes because, at the population level, transmission distortion must certainly counterbalance the loss of t-chrom o s o m e s due to homozygous lethality. Another salient outcome of transmission ratio distortion is that it must make t-haplotypes a significant regulator of H-2 polymorphism, at least in the sense that in any population containing one t-lethal, many individuals are forced heterozygotes for the M H C of that t-chromosome. A final unique property of these chromosomes, and one that is now understood, is their "ability" to suppress recombination when heterozygous with a wild type chromosome. Genetic distances, such as that between T and H-2 ( 1 6 - 2 0 cM when measured in heterozygotes for two non-t-bearing chromosomes), are reduced to 0.1 cM when one of the two homologues is a t-haplotype. This recombination suppression virtually ensures that a t-haplotype will travel genetically intact and isolated from normal chromosomes in wild populations. Thus a naturally occurring t-haplotype appears to be a collection of qualitatively different kinds of mutations: genes affecting embryonic development, tail length, and male germ cells all locked together by recombination suppression.
MENDELIAN
GENETIC ANALYSIS
Since it was discovered [7] that t-haplotypes recombine normally with other complementing t-haplotpes, over 7000 mice derived from nine different haplotypes have been analyzed in order to understand the genetic structure of the T/t-complex. T h e major foci of these studies were to map the positions of the lethal genes and to determine the structural differences that might explain recombination suppression. It was immediately apparent that the different lethal genes were nonallelic and spread over a large portion (20 cM) of c h r o m o s o m e 17 [8,9]. They were roughly organized into three clusters; one lethal was near the tail interaction factor, two others were near the locus of the marker gene tf [10], and four were intimately associated with the M H C . In the M H C cluster, two have been located m o r e precisely: t 12 is between H-2D and TL, presumably in the Qa region, and t ~'5 has not been separated from H-2K in m o r e than 1000 mice analyzed ([11], and unpublished data). The structural difference turned out to be a simple but quite large distal
376
K. Artzt inversion of 15 cM that includes the M H C and tf[12,13]. The gene order, for the markers studied, in wild type is T t f K I D TL Ce-2, whereas in all of the complete t-haplotypes studied it is t T TL D I K tf Ce-2. It remains unclear why this inversion was cytologically undetectable [ 14,15]. Nevertheless, it provides an adequate explanation for recombination suppression in the distal portion of t-haplotypes. In all likelihood, the proximal portion of the t-complex contains a companion inversion that is responsible for recombination suppression in the proximal region. It should be emphasized that throughout this large region of chromosome in mutant haplotypes there are wild type genes functioning normally. A few defined examples are: + qk, +fu, + tf, glo~I, + cldand the entire MHC. T o return to the issue of relatedness of the t-lethals, once we were able to obtain recombinants that carry two or more lethal mutations, and others that are devoid of specific lethals, we could examine complementation in cis vs. trans for the first time. Whereas the complementation in trans (t " + / + t ~') is usually poor, complementation in cis (t" t~/+ +) can be perfectly normal. This means that, although the complementing genes may map over l0 cM apart, they constitute some type of functional unit [16].
SEROLOGICAL A N D MOLECULAR ANALYSIS OF THE t - M H C Since the inversion in t-haplotypes is a simple one and not a scrambling of genetic material, the necessary wild type alleles defined by these mutations must occupy the same relative position in normal chromosomes, i.e., some of them should be in and surrounding the MHC. Thus their intimate association prompted us to investigate this region in greater detail. Analysis of t-haplotype class I genes by Southern blotting with c D N A probes shows they diverge from one another by an average of only 3.5% compared to 4 7 % for inbred strains [17]. This confirms earlier work [18,19] showing t-associated H-2 haplotypes, although derived from highly polymorphic wild populations, share a limited pool of H-2 haplotypes and, taken together with other similarities between different t-haplotypes, argues strongly for their derivation from a single ancestral haplotype (see also Nizetic et al. for discussion [20]). Although the H-2 polymorphism is thus restricted, at both the protein and D N A levels, there are enough differences to utilize for genetic dissection of the region. Using nine different serological reagents to class I antigens and eight D N A probes to class I and class II genes, we have analyzed 14 intra-H-2 recombinants derived from three t-haplotypes. Surprisingly, we can only define two breakpoints among the 14 recombinants, one between H-2D and a "middle region" containing the immune response genes and some class I specificities, and another between this middle region and H-2K (manuscript in preparation). These results suggest that there are hot spots of recombination similar to those defined for inbred strains of mice [21]. The limited origin and restricted chances for recombination make t-chromosomes a valuable tool for examining the relative contributions of mutation and recombination to the generation of diversity, especially with respect to MHC. Although the chances for recombination are restricted, on very rare occasions two different t-haplotypes may meet in a wild population. Under these circumstances a compound female (compound males are sterile) may survive and in her gametes, homozygous for the inversion, recombination can occur normally. Thus one can examine the extent to which the various t~H-2 haplotypes are related. Using 13 different serological reagents to class I antigens, and studying restriction enzyme polymorphisms detected with three molecular probes for class II genes examined with three endonucleases, we found that the major factor responsible
Genetic Analysis of the T/t Complex
377
for the diversity of class I antigens is recombination, but that for class II genes, mutation must play an important role in addition to recombination [22].
T/t-LIKE GENES IN MAN
The search for t-like mutations in man has attracted much effort and has been fraught with considerable difficulty not the least of which has been the absence of a tail! Since T/t T tailless mice frequently have less than no tail, resulting in spina bifida, sacro-caudal defects have been taken to represent the phenotype that may be expected from such mutations in man. Both Amos et al. [23] and Fellous et al. [24] have studied multigeneration pedigrees of families in which spina bifida appeared frequently and found that the condition shows linkage disequilibrium with specific HLA haplotypes segregating in those families. Thus by analogy, since T is linked to H-2, they proposed that a T-like gene was responsible for the dysraphic defects. The criterion of fetal wastage is not useful as the timing of human development would mean that homozygosity for a t-lethal would result in first trimester abortion, probably before the first 5 weeks of pregnancy. Linkage disequilibrium does, however, provide a promising criterion. Awdeh and Alper and their colleagues [25,26] have found strong evidence for linkage disequilibrium between specific combinations of human M H C components, and they suggest that the linkage disequilibrium is maintained by cross-over suppression that is analogous to mouse t-haplotypes. As a second convincing point they also showed that a specific human haplotype (HLA-BS, DR3, SCO1, Glo 1.2) is transmitted by males to 8 3 % of their offspring, suggesting that this human haplotype results in the high transmission through males, which is typical of mouse t-haplotypes [27]. Thus, there is substantial evidence that they have uncovered human analogues of a murine t-mutation. Another suggestion for t-haplotypes in man comes from Degos et al. [28]. Their demographic studies of a North African tribe, the Tuareg, showed them to practice a rigid system of inbreeding which results in the tribe having only a very limited polymorphism for HLA haplotypes, in striking contrast to the extensive HLA polymorphism typical of most human populations. But surprisingly the most common HLA type in the Tuareg population was never found to occur in homozygous condition; thus this haplotype was assumed to be associated with a t-like lethal mutation in a region of recombination suppression typical of t-haplotypes, and to be maintained in the population by transmission distortion. In considering these human population studies, it is worth pointing out that the situation in man may not be completely analogous to the mouse complete t-haplotypes we have discussed up to now. It is not necessary to find sacro-caudal shortening factors, large stretches of recombination suppression, or enormously high transmission ratios. In the laboratory, when rare exceptional crossovers occur (0.1%) in ÷ / t heterozygotes, partial t-haplotypes are generated. All distal partial haplotypes maintain the lethal mutations in linkage disequilbrium with the M H C because they maintain the distal inversion but they have lost the tail interaction factor, and have a normal or moderately distorted transmission ratio. One such possible partial haplotype has been described in the rat [29]. Such partial t-haplotypes, with a less extensive region of recombination suppression, could well exist in man and other species. In any case, it would be hard to conceive of a chordate species without functioning wild type counterparts of the t-lethal mutations and, given what is known about the conservation of this linkage group, it may be predicted that some of them will be very closely associated with the MHC.
378
K. Artzt Is there any lesson we can learn from our understanding of the genetic structure of t-haplotypes that may apply to the linkage disequilibrium associated with H L A in man? By analogy, two major components of linkage disequilibrium could be recombination suppression due to inversions and nonrandom recombination. " H o t spots" of recombination have been blamed for nonrandom recombination as they would make markers appear much farther apart than they are in reality. Unfortunately, the physical basis of recombination hot spots is still not understood. It could be due to repetitive features of the D N A sequence ("minisatellite" regions as has been suggested by Jeffreys [30]) or a combination of recombination suppression due to inversions and the inevitable compensation that surrounds an inversion.
SPECULATIONS W e do not yet know the nature of the gene products of the t-lethal mutations. Although in serological studies on male germ cells [31] and appropriate embryos [32] the mutants have been associated with cell surface antigens, these studies have been controversial [33]. The antigens are, operationally speaking, a very weak serological system, probably because of the autoantigenic nature of male germ cells and also because the immunodominant determinants implicated are sugar [ 34,35 ]. But, there is a growing body of evidence implicating carbohydrates in embryonic cell surface recognition [36]. Abnormalities of surface glycosyltransferases have also been demonstrated for t-haplotypes [37,38]. Nevertheless, it is still tempting to speculate that the t-complex may represent an embryonic analogue of the adult M H C with both systems operating as mediators of cell-cell recognition. O t h e r than close linkage, the true nature of the relationship will have to await the cloning of the t-lethal genes.
REFERENCES 1. Wiley LM: Early embryonic cell surface antigens as developmental probes. Curr Topics Devel Biol 13:167, 1979. 2. Bennett D: Genetically programmed abnormalities of cell interactions. In: RA Lerner, Ed. The molecular basis of cell-cell interaction, birth defects: original article series. New York, AR Liss, 1978. 3. Bennett D: The T-locus of the mouse: a review. Cell 6:441, 1975. 4. Silagi S: A genetical and embryological study of partial complementation between lethal alleles at the T locus of the house mouse. Devel Biol 5:35, 1962. 5. Bennett D, Alton AK, Artzt K: Genetic analysis of transmission ratio distortion by t-haplotypes in the mouse. Genet Res Camb 41:29, 1983. 6. Lyon MF: Transmission ratio distortion in mouse t-haplotypes is due to multiple distorter genes acting on a responder locus. Cell 37:621, 1984. 7. Silver LM, Artzt K: Recombination suppression of mouse t-haplotypes is due to chromatin mismatching. Nature 290:68, 1981. 8. Artzt K, McCormick P, Bennett D: Gene mapping within the T/t-complex of the mouse. I. t-lethal genes are nonallelic. Cell 28:463, 1982. 9. Condamine H, GuenetJ-L, Jacob F: Recombination between two mouse t-haplotypes (tw~2tf and tL"6-~): segregation of lethal factors relative to centromere and tufted (tf) locus. Genet Res, Camb 42:335, 1983.
Genetic Analysis of the T/t Complex
379
10. Artzt K: Gene mapping within the T/t-complex of the mouse. III. t-lethal genes are arranged in three clusters on chromosome 17. Cell 39:565, 1984. 11. Shin H-S, Bennett D, Artzt K: Gene mapping within the T/t-complex of the mouse. IV. The inverted MHC is intermingled with several t-lethal genes. Cell 39:573, 1984. 12. Artzt K, Shin H-S, Bennett D: Gene mapping within the T/t-complex of the mouse. II. Anomalous position of the H-2 complex in t-haplotypes. Cell 28:471, 1982. 13. Pla M, Condamine H: Recombination between two mouse t haplotypes (t'<~tf and tLub-l): mapping of the H-2 complex relative to centromere and tufted (tf) locus. Immunogenetics 20:277, 1984. 14. Womack JE, Roderick TH: T-alleles in the mouse are probably not inversions. J Hered 65:308, 1974. 15. Tres LL, Erickson RP: Electron microscopy of t-allele synamptonemal complexes discloses no inversions. Nature 299:752, 1982. 16. Shin H-S, McCormick P, Artzt K, Bennett D: Cis-trans test shows a functional relationship between non-allelic lethal mutations in the T/t-complex. Cell 33:925, 1983. 17. Shin H-S, Stavnezer J, Artzt K, Bennett D: The genetic structure and origin of thaplotypes of mice, analyzed with H-2 cDNA probes. Cell 29:969, 1982. 18. Hammerberg C, Klein J: Linkage disequilibrium between H-2 and t complexes in chromosome 17 of the mouse. Nature 258:296, 1975. 19. Levinson JR, McDevitt HO: Murine t factors: an association between alleles at t and at H-2. J Exp Med 144:834, 1976. 20. Nizetic D, Figueroa F, Klein J: Evolutionary relationships between the t and H-2 haplotypes in the house mouse. Immunogenetics 19:3ll, 1984. 21. Steinmetz M, Minard K, Horvath S, McNicholas J, Srelinger J, Wake C, Long E, Mach B, Hood L: A molecular map of the immune response region from the major histocompatibility complex of the mouse. Nature 300:35, 1982. 22. Artzt K, Shin H-S, Bennett D, DiMeo-Talento A: Analysis of H-2 haplotypes of tchromosomes reveals the majority of diversity is generated by recombination. J Exp Med 162:93, 1985. 23. Amos DB, Ruderman RJ, Mendell NP, Johnson AH: Linkage between HLA and spinal development. Transplant Proc 7:93, 1975. 24. Fellous M, Boue J, Malbrunot C, Wollman E, Sasportes J, Van Cong N, Marcelli A, Rebourcet R, Hubert Ch (Clinical and Immunogenetic Studies)//Demenais F, Elston RC, Namboodiri KK, Kaplan EB, Fellous M (Segregation and Linkage Analyses): a five generation family with saral agenesis and spina bifida: possible similarities with the mouse T-locus. Am J Med Genet 12:465, 1982, 25. Alper CA, Awdeh ZL, Raum DD, Yunis EJ: Extended major histocompatibility complex haplotypes in man: role of alleles analogous to murine t-mutant. Clin Immunol Immunopathol 24:276, 1982. 26. Fleischnick E, Awdeh ZL, Raum D, Granados J, Alosco SM, Crigler JF, Gerald PS, Giles CM, Yunis E, Alper CA: Extended MHC haplotypes in 2 l-hydroxylase deficiency congenital adrenal hyperplasia: shared genotypes in unrelated patients. Lancet i: 152, 1983. 27. Awdeh ZL, Raum D, Yunis EJ, Alper CA: Extended HLA/complement allele haplotypes: evidence for T/t-like complex in man. Proc Natl Acad Sci USA 80:259, 1983. 28. Degos L, Colombani J, Chaventre A, Bengtson B, Jacquard A: Selective pressure on HL-A polymorphism. Nature 249:62, 1974.
380
K. Artzt 29. Artzt K, Lockwood M, Bennett D, Kunz HW, Gill TJ: Serological evidence for a partial t-haplotype in the rat. J Immunogenet 9:371, 1982. 30. Jeffreys AJ, Wilson V, Thein SL: Hypervariable 'minisatellite' regions in human DNA. Nature 314:67. 1985. 31. Artzt K, Bennett D: Serological analysis of sperm of antigenically cross-reacting T/thaplotypes and their recombinants. Immunogenetics 5:97, 1977. 32. Marticorena P, Artzt K, Bennett D: Relationship of F9 antigen and genes of the T/t complex. Immunogenetics 7:337, 1978. 33. Gable RJ, Levinson JR, McDevitt HO, Goodfellow PN: Assay for antibody mediated cytotoxicity of mouse spermatozoa by ~'rubidium release. Tissue Antigens 13:177, 1979. 34. Cheng CC, Bennett D: Nature of the antigenic determinants ofT-locus antigens. Cell 19:537, 1980. 35. Cheng C, Sege K, Alton AK, Bennett D, Artzt K: Characterization of an antigen present on testicular cells and pre-implantation embryos whose expression is modified by the t ~e haplotype. J Immunogenet 10:465, 1983. 36. Ivatt RJ: Role ofglycoproteins during early mammalian embryogenesis. In: RJ Ivatt, Ed. The biology of glycoproteins. New York, Plenum Press, 1984. 37. Shur BD: Galactosyltransferase activities on mouse sperm bearing multiple t ]~th~Eand t '~hjL`haplotypes of the T/t complex. Genet Res, Camb 38:225, 1981. 38. Shut BD: Evidence that galactosyltransferase is a surface receptor for poly(N)-acetyllactosamine glycoconjugates on embryonal carcinoma cells. J Biol Chem 257:6871, 1982.
ABSTRACT: The T/t-complex has hem considerable interest for immunologists, primarily because of its close genetic linkage to the major histocompatibility complex (MHC) on mouse chromosome 17. This interest has been heightened recently u'ith the discorery that the M H C is fully contained u'ithin the t-complex and that tu,o regions of the MHC. Qa and K. contain t-lethal genes. For a long time, T/t has been an enigmatic system, mainly because classical genetfi analysis u as not possible. Here the system is defined, recent in~rmation is presented, and our understanding of the mouse data to at'ailable information about the human MHC is correlated. ABBREVIATIONS Ce-2 kidney catalase old combined lipase deficiency
fu
fused
glo- 1 qk tf
glyoxylase- 1 quaking tufted
P R O P E R T I E S OF T H E T/t C O M P L E X
Studies of the t-complex address two major questions: first, the mechanisms of embryonic cell commitment at the cell-cell interaction level, and second, the orchestrated genetic control of a seemingly complicated developmental program. t-haplotypes are traditionally found in wild populations of mice the world over. Any particular haplotype may carry one or two different recessive t-lethal mutations, or none. O f the 16 known lethals, six have been analyzed embryologically and all of these exhibit their lethal effects early in gestation, from shortly after the zygote begins cell division (16-32 cells) until just prior to organ formation (9'/2 days gestation). This is precisely the period in development when cell commitment is taking place and when classical MHC products (K and D antigens) are not expressed. (The possible exception to the latter statement is class I expression on the extraembryonic trophoblast [ 1].) Also, these six different lethals affect the embryo at different stages of development and in a specific tissue, seemingly by a failure of cell-cell interactions [2]. Thus, t-haplotypes represent a collection of genetically linked natural tools dissecting early development. (The embryology has been reviewed extensively by Bennett [3].) In addition to their linkage, another aspect that makes these lethal genes particularly attractive to study is that they appear to be functionally related even though they act at different developmental stages. Early on, it was appreciated that these mutations exhibited interesting complementation interactions. Although the different lethals were known to complement one another (i.e., a mouse that was t ' / t ~' could be constructed and proceed normally through the
From the Memorial SIoan-Kettering Cancer Center and S/oan-Kettering Dirision. Gradnate School of Medicine Sciences, Cornell University, Neu, York. Neu' York. Address correspondence to Karen Artzt, Memorial S/oan-Kettering Cancer Center and Sloan-Kettering Dirision. Graduate School of Medical Sciences. Cornell Uniz,ersiO ~, New York. N Y 10021.
374 0198-8859/86/$3.50
Human Immunology 15, 3 v 4 - 3 8 0 (1986) ~) Elsevier Science Publishing Co., Inc., 1986 52 Vanderbilt Ave., New York, NY 10017
Genetic Analysis of the T/t Complex
375
developmental arrest characteristic o f t ~ and t'), the viability of such heterozygotes was very variable and d e p e n d e n t to a certain extent on which two mutations were involved [3]. Moreov.er, a thorough embryological study [4] showed that heterozygotes for two different t-lethals that did not survive, often died resembling a third t-lethal morphologically. These were some of the first clues to the idea that different t-lethals were related. A second and seemingly trivial property of wild derived t-haplotypes is that they all contain the same recessive mutation, "the tail interactor factor" (t T) which, in fact, was the basis of their detection. The recessive t r mutation interacts with Brachyury (T), a semidominant laboratory-derived point mutation, causing a short tail. W h e n a mouse is heterozygous for these two mutations (T/t r) it has no tail. The traditional test for the presence of t-haplotypes is to mate a wild derived normal tailed male (+/t r) to a short-tailed (T/+) female; tailless mice (T/t r) among the progeny indicate the presence of a t-haplotype. A third, and still very perplexing property of t-haplotypes is the p h e n o m e n o n known as transmission ratio distortion. Males heterozygous for a t-haplotype violate Mendel's rule and transmit the t-haplotype to more than 9 8 % of their progeny rather than the expected half. Transmission ratio distortion has been studied in detail and reviewed [5,6] and discussion of its mechanisms is outside the scope of this paper. Accepted at face value as a p h e n o m e n o n , however, it explains the worldwide prevalence of t-haplotypes because, at the population level, transmission distortion must certainly counterbalance the loss of t-chrom o s o m e s due to homozygous lethality. Another salient outcome of transmission ratio distortion is that it must make t-haplotypes a significant regulator of H-2 polymorphism, at least in the sense that in any population containing one t-lethal, many individuals are forced heterozygotes for the M H C of that t-chromosome. A final unique property of these chromosomes, and one that is now understood, is their "ability" to suppress recombination when heterozygous with a wild type chromosome. Genetic distances, such as that between T and H-2 ( 1 6 - 2 0 cM when measured in heterozygotes for two non-t-bearing chromosomes), are reduced to 0.1 cM when one of the two homologues is a t-haplotype. This recombination suppression virtually ensures that a t-haplotype will travel genetically intact and isolated from normal chromosomes in wild populations. Thus a naturally occurring t-haplotype appears to be a collection of qualitatively different kinds of mutations: genes affecting embryonic development, tail length, and male germ cells all locked together by recombination suppression.
MENDELIAN
GENETIC ANALYSIS
Since it was discovered [7] that t-haplotypes recombine normally with other complementing t-haplotpes, over 7000 mice derived from nine different haplotypes have been analyzed in order to understand the genetic structure of the T/t-complex. T h e major foci of these studies were to map the positions of the lethal genes and to determine the structural differences that might explain recombination suppression. It was immediately apparent that the different lethal genes were nonallelic and spread over a large portion (20 cM) of c h r o m o s o m e 17 [8,9]. They were roughly organized into three clusters; one lethal was near the tail interaction factor, two others were near the locus of the marker gene tf [10], and four were intimately associated with the M H C . In the M H C cluster, two have been located m o r e precisely: t 12 is between H-2D and TL, presumably in the Qa region, and t ~'5 has not been separated from H-2K in m o r e than 1000 mice analyzed ([11], and unpublished data). The structural difference turned out to be a simple but quite large distal
376
K. Artzt inversion of 15 cM that includes the M H C and tf[12,13]. The gene order, for the markers studied, in wild type is T t f K I D TL Ce-2, whereas in all of the complete t-haplotypes studied it is t T TL D I K tf Ce-2. It remains unclear why this inversion was cytologically undetectable [ 14,15]. Nevertheless, it provides an adequate explanation for recombination suppression in the distal portion of t-haplotypes. In all likelihood, the proximal portion of the t-complex contains a companion inversion that is responsible for recombination suppression in the proximal region. It should be emphasized that throughout this large region of chromosome in mutant haplotypes there are wild type genes functioning normally. A few defined examples are: + qk, +fu, + tf, glo~I, + cldand the entire MHC. T o return to the issue of relatedness of the t-lethals, once we were able to obtain recombinants that carry two or more lethal mutations, and others that are devoid of specific lethals, we could examine complementation in cis vs. trans for the first time. Whereas the complementation in trans (t " + / + t ~') is usually poor, complementation in cis (t" t~/+ +) can be perfectly normal. This means that, although the complementing genes may map over l0 cM apart, they constitute some type of functional unit [16].
SEROLOGICAL A N D MOLECULAR ANALYSIS OF THE t - M H C Since the inversion in t-haplotypes is a simple one and not a scrambling of genetic material, the necessary wild type alleles defined by these mutations must occupy the same relative position in normal chromosomes, i.e., some of them should be in and surrounding the MHC. Thus their intimate association prompted us to investigate this region in greater detail. Analysis of t-haplotype class I genes by Southern blotting with c D N A probes shows they diverge from one another by an average of only 3.5% compared to 4 7 % for inbred strains [17]. This confirms earlier work [18,19] showing t-associated H-2 haplotypes, although derived from highly polymorphic wild populations, share a limited pool of H-2 haplotypes and, taken together with other similarities between different t-haplotypes, argues strongly for their derivation from a single ancestral haplotype (see also Nizetic et al. for discussion [20]). Although the H-2 polymorphism is thus restricted, at both the protein and D N A levels, there are enough differences to utilize for genetic dissection of the region. Using nine different serological reagents to class I antigens and eight D N A probes to class I and class II genes, we have analyzed 14 intra-H-2 recombinants derived from three t-haplotypes. Surprisingly, we can only define two breakpoints among the 14 recombinants, one between H-2D and a "middle region" containing the immune response genes and some class I specificities, and another between this middle region and H-2K (manuscript in preparation). These results suggest that there are hot spots of recombination similar to those defined for inbred strains of mice [21]. The limited origin and restricted chances for recombination make t-chromosomes a valuable tool for examining the relative contributions of mutation and recombination to the generation of diversity, especially with respect to MHC. Although the chances for recombination are restricted, on very rare occasions two different t-haplotypes may meet in a wild population. Under these circumstances a compound female (compound males are sterile) may survive and in her gametes, homozygous for the inversion, recombination can occur normally. Thus one can examine the extent to which the various t~H-2 haplotypes are related. Using 13 different serological reagents to class I antigens, and studying restriction enzyme polymorphisms detected with three molecular probes for class II genes examined with three endonucleases, we found that the major factor responsible
Genetic Analysis of the T/t Complex
377
for the diversity of class I antigens is recombination, but that for class II genes, mutation must play an important role in addition to recombination [22].
T/t-LIKE GENES IN MAN
The search for t-like mutations in man has attracted much effort and has been fraught with considerable difficulty not the least of which has been the absence of a tail! Since T/t T tailless mice frequently have less than no tail, resulting in spina bifida, sacro-caudal defects have been taken to represent the phenotype that may be expected from such mutations in man. Both Amos et al. [23] and Fellous et al. [24] have studied multigeneration pedigrees of families in which spina bifida appeared frequently and found that the condition shows linkage disequilibrium with specific HLA haplotypes segregating in those families. Thus by analogy, since T is linked to H-2, they proposed that a T-like gene was responsible for the dysraphic defects. The criterion of fetal wastage is not useful as the timing of human development would mean that homozygosity for a t-lethal would result in first trimester abortion, probably before the first 5 weeks of pregnancy. Linkage disequilibrium does, however, provide a promising criterion. Awdeh and Alper and their colleagues [25,26] have found strong evidence for linkage disequilibrium between specific combinations of human M H C components, and they suggest that the linkage disequilibrium is maintained by cross-over suppression that is analogous to mouse t-haplotypes. As a second convincing point they also showed that a specific human haplotype (HLA-BS, DR3, SCO1, Glo 1.2) is transmitted by males to 8 3 % of their offspring, suggesting that this human haplotype results in the high transmission through males, which is typical of mouse t-haplotypes [27]. Thus, there is substantial evidence that they have uncovered human analogues of a murine t-mutation. Another suggestion for t-haplotypes in man comes from Degos et al. [28]. Their demographic studies of a North African tribe, the Tuareg, showed them to practice a rigid system of inbreeding which results in the tribe having only a very limited polymorphism for HLA haplotypes, in striking contrast to the extensive HLA polymorphism typical of most human populations. But surprisingly the most common HLA type in the Tuareg population was never found to occur in homozygous condition; thus this haplotype was assumed to be associated with a t-like lethal mutation in a region of recombination suppression typical of t-haplotypes, and to be maintained in the population by transmission distortion. In considering these human population studies, it is worth pointing out that the situation in man may not be completely analogous to the mouse complete t-haplotypes we have discussed up to now. It is not necessary to find sacro-caudal shortening factors, large stretches of recombination suppression, or enormously high transmission ratios. In the laboratory, when rare exceptional crossovers occur (0.1%) in ÷ / t heterozygotes, partial t-haplotypes are generated. All distal partial haplotypes maintain the lethal mutations in linkage disequilbrium with the M H C because they maintain the distal inversion but they have lost the tail interaction factor, and have a normal or moderately distorted transmission ratio. One such possible partial haplotype has been described in the rat [29]. Such partial t-haplotypes, with a less extensive region of recombination suppression, could well exist in man and other species. In any case, it would be hard to conceive of a chordate species without functioning wild type counterparts of the t-lethal mutations and, given what is known about the conservation of this linkage group, it may be predicted that some of them will be very closely associated with the MHC.
378
K. Artzt Is there any lesson we can learn from our understanding of the genetic structure of t-haplotypes that may apply to the linkage disequilibrium associated with H L A in man? By analogy, two major components of linkage disequilibrium could be recombination suppression due to inversions and nonrandom recombination. " H o t spots" of recombination have been blamed for nonrandom recombination as they would make markers appear much farther apart than they are in reality. Unfortunately, the physical basis of recombination hot spots is still not understood. It could be due to repetitive features of the D N A sequence ("minisatellite" regions as has been suggested by Jeffreys [30]) or a combination of recombination suppression due to inversions and the inevitable compensation that surrounds an inversion.
SPECULATIONS W e do not yet know the nature of the gene products of the t-lethal mutations. Although in serological studies on male germ cells [31] and appropriate embryos [32] the mutants have been associated with cell surface antigens, these studies have been controversial [33]. The antigens are, operationally speaking, a very weak serological system, probably because of the autoantigenic nature of male germ cells and also because the immunodominant determinants implicated are sugar [ 34,35 ]. But, there is a growing body of evidence implicating carbohydrates in embryonic cell surface recognition [36]. Abnormalities of surface glycosyltransferases have also been demonstrated for t-haplotypes [37,38]. Nevertheless, it is still tempting to speculate that the t-complex may represent an embryonic analogue of the adult M H C with both systems operating as mediators of cell-cell recognition. O t h e r than close linkage, the true nature of the relationship will have to await the cloning of the t-lethal genes.
REFERENCES 1. Wiley LM: Early embryonic cell surface antigens as developmental probes. Curr Topics Devel Biol 13:167, 1979. 2. Bennett D: Genetically programmed abnormalities of cell interactions. In: RA Lerner, Ed. The molecular basis of cell-cell interaction, birth defects: original article series. New York, AR Liss, 1978. 3. Bennett D: The T-locus of the mouse: a review. Cell 6:441, 1975. 4. Silagi S: A genetical and embryological study of partial complementation between lethal alleles at the T locus of the house mouse. Devel Biol 5:35, 1962. 5. Bennett D, Alton AK, Artzt K: Genetic analysis of transmission ratio distortion by t-haplotypes in the mouse. Genet Res Camb 41:29, 1983. 6. Lyon MF: Transmission ratio distortion in mouse t-haplotypes is due to multiple distorter genes acting on a responder locus. Cell 37:621, 1984. 7. Silver LM, Artzt K: Recombination suppression of mouse t-haplotypes is due to chromatin mismatching. Nature 290:68, 1981. 8. Artzt K, McCormick P, Bennett D: Gene mapping within the T/t-complex of the mouse. I. t-lethal genes are nonallelic. Cell 28:463, 1982. 9. Condamine H, GuenetJ-L, Jacob F: Recombination between two mouse t-haplotypes (tw~2tf and tL"6-~): segregation of lethal factors relative to centromere and tufted (tf) locus. Genet Res, Camb 42:335, 1983.
Genetic Analysis of the T/t Complex
379
10. Artzt K: Gene mapping within the T/t-complex of the mouse. III. t-lethal genes are arranged in three clusters on chromosome 17. Cell 39:565, 1984. 11. Shin H-S, Bennett D, Artzt K: Gene mapping within the T/t-complex of the mouse. IV. The inverted MHC is intermingled with several t-lethal genes. Cell 39:573, 1984. 12. Artzt K, Shin H-S, Bennett D: Gene mapping within the T/t-complex of the mouse. II. Anomalous position of the H-2 complex in t-haplotypes. Cell 28:471, 1982. 13. Pla M, Condamine H: Recombination between two mouse t haplotypes (t'<~tf and tLub-l): mapping of the H-2 complex relative to centromere and tufted (tf) locus. Immunogenetics 20:277, 1984. 14. Womack JE, Roderick TH: T-alleles in the mouse are probably not inversions. J Hered 65:308, 1974. 15. Tres LL, Erickson RP: Electron microscopy of t-allele synamptonemal complexes discloses no inversions. Nature 299:752, 1982. 16. Shin H-S, McCormick P, Artzt K, Bennett D: Cis-trans test shows a functional relationship between non-allelic lethal mutations in the T/t-complex. Cell 33:925, 1983. 17. Shin H-S, Stavnezer J, Artzt K, Bennett D: The genetic structure and origin of thaplotypes of mice, analyzed with H-2 cDNA probes. Cell 29:969, 1982. 18. Hammerberg C, Klein J: Linkage disequilibrium between H-2 and t complexes in chromosome 17 of the mouse. Nature 258:296, 1975. 19. Levinson JR, McDevitt HO: Murine t factors: an association between alleles at t and at H-2. J Exp Med 144:834, 1976. 20. Nizetic D, Figueroa F, Klein J: Evolutionary relationships between the t and H-2 haplotypes in the house mouse. Immunogenetics 19:3ll, 1984. 21. Steinmetz M, Minard K, Horvath S, McNicholas J, Srelinger J, Wake C, Long E, Mach B, Hood L: A molecular map of the immune response region from the major histocompatibility complex of the mouse. Nature 300:35, 1982. 22. Artzt K, Shin H-S, Bennett D, DiMeo-Talento A: Analysis of H-2 haplotypes of tchromosomes reveals the majority of diversity is generated by recombination. J Exp Med 162:93, 1985. 23. Amos DB, Ruderman RJ, Mendell NP, Johnson AH: Linkage between HLA and spinal development. Transplant Proc 7:93, 1975. 24. Fellous M, Boue J, Malbrunot C, Wollman E, Sasportes J, Van Cong N, Marcelli A, Rebourcet R, Hubert Ch (Clinical and Immunogenetic Studies)//Demenais F, Elston RC, Namboodiri KK, Kaplan EB, Fellous M (Segregation and Linkage Analyses): a five generation family with saral agenesis and spina bifida: possible similarities with the mouse T-locus. Am J Med Genet 12:465, 1982, 25. Alper CA, Awdeh ZL, Raum DD, Yunis EJ: Extended major histocompatibility complex haplotypes in man: role of alleles analogous to murine t-mutant. Clin Immunol Immunopathol 24:276, 1982. 26. Fleischnick E, Awdeh ZL, Raum D, Granados J, Alosco SM, Crigler JF, Gerald PS, Giles CM, Yunis E, Alper CA: Extended MHC haplotypes in 2 l-hydroxylase deficiency congenital adrenal hyperplasia: shared genotypes in unrelated patients. Lancet i: 152, 1983. 27. Awdeh ZL, Raum D, Yunis EJ, Alper CA: Extended HLA/complement allele haplotypes: evidence for T/t-like complex in man. Proc Natl Acad Sci USA 80:259, 1983. 28. Degos L, Colombani J, Chaventre A, Bengtson B, Jacquard A: Selective pressure on HL-A polymorphism. Nature 249:62, 1974.
380
K. Artzt 29. Artzt K, Lockwood M, Bennett D, Kunz HW, Gill TJ: Serological evidence for a partial t-haplotype in the rat. J Immunogenet 9:371, 1982. 30. Jeffreys AJ, Wilson V, Thein SL: Hypervariable 'minisatellite' regions in human DNA. Nature 314:67. 1985. 31. Artzt K, Bennett D: Serological analysis of sperm of antigenically cross-reacting T/thaplotypes and their recombinants. Immunogenetics 5:97, 1977. 32. Marticorena P, Artzt K, Bennett D: Relationship of F9 antigen and genes of the T/t complex. Immunogenetics 7:337, 1978. 33. Gable RJ, Levinson JR, McDevitt HO, Goodfellow PN: Assay for antibody mediated cytotoxicity of mouse spermatozoa by ~'rubidium release. Tissue Antigens 13:177, 1979. 34. Cheng CC, Bennett D: Nature of the antigenic determinants ofT-locus antigens. Cell 19:537, 1980. 35. Cheng C, Sege K, Alton AK, Bennett D, Artzt K: Characterization of an antigen present on testicular cells and pre-implantation embryos whose expression is modified by the t ~e haplotype. J Immunogenet 10:465, 1983. 36. Ivatt RJ: Role ofglycoproteins during early mammalian embryogenesis. In: RJ Ivatt, Ed. The biology of glycoproteins. New York, Plenum Press, 1984. 37. Shur BD: Galactosyltransferase activities on mouse sperm bearing multiple t ]~th~Eand t '~hjL`haplotypes of the T/t complex. Genet Res, Camb 38:225, 1981. 38. Shut BD: Evidence that galactosyltransferase is a surface receptor for poly(N)-acetyllactosamine glycoconjugates on embryonal carcinoma cells. J Biol Chem 257:6871, 1982.