Rearrangement of Genes Located On

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REARRANGEMENT OF GENES LOCATED ON HOMOLOGOUS CHROMOSOMAL SEGMENTS IN MOUSE AND MAN: THE LOCATION OF GENES FOR ALPHA- AND BETA-INTERFERON, ALPHA-1 ACID GLYCOPROTEIN-1 AND -2, AND AMINOLEVULINATE DEHYDRATASE ON MOUSE CHROMOSOME 4

JOSEPH H. NADEAU,*.’ FRANKLIN G. BERGER? KEVIN A. KELLEY,” PAULA M. PITHA,” CHARLES L. SIDMAN* AND NEIL WORRALLZ *The Jackson Laboratory, Bar Harbor, Maine 04609, +Biology Department, University of South Carolina, Columbia, South Carolina 29208, and ZOncology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Manuscript received April 28, 1986 Revised copy accepted September 8, 1986

ABSTRACT Gene mapping studies to determine the order of alpha- and beta-interferon (If., Ijb), aminolevulinate dehydratase (Lv), and alpha- J acid glycoprotein-J and -2 (Om-I, Orm-2) relative to each other and to the reference genes brown (b), B- cell maturation factor responsiveness (Bmfr-I), and major urinary protein- J (Mup-I) are reported. The most likely order was Mup-I-Lv-b-Orm-I, Orm-P--lfa, Ijb-Bmfr- 1. This order suggested that two chromosomal segments located on chromosome 4 in the mouse and chromosome 9 in man have been conserved since divergence of lineages leading to man and mouse; these segments are marked by soluble aconitase-1 (Aco-I) and galactose- J phosphate uridyl transferase (Cult) and by Lv and Orm-I. This order also demonstrated that, although genes located on opposite arms of chromosome 9 in man remain syntenic in the mouse, gene order has not been conserved; If. and Zjb are not located in their expected locations near Aco-I and Galt. The position of If. and Ifb between Orm-J and Bmfr- J could not be determined with certainty because of apparent heterogeneity in recombination frequencies between crosses involving conventional laboratory strains of mice and crosses involving interspecific matings between laboratory mice and Mus spretus. This result suggests that caution must be exercised when using M. spretus in linkage crosses.

OMPARISON of the genomic location of homologous genes in different C species, such as mouse and man, provides a powerful means for studying genome organization and evolution. For example, conserved synteny of genes located on the X chromosome among mammals provides strong evidence that chromosomal rearrangements between the X chromosome and autosomes are highly deleterious (OHNO1969). Numerous autosomal segments have also been conserved since divergence of lineages leading to man and mouse. The length

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Genetics 104: 1239-1255 December, 1986. 1240 J. H. NADEAU ET AL. of these segments is considerably shorter than the length of the X chromosome, however. The average length is estimated to be 8.1 rt 1.6 cM (NADEAUand TAYLOR1984). The number of autosomal linkage disruptions that have occurred since divergence of lineages leading to mouse and man is estimated to be 178 f 39, and these disruptions appear to be randomly distributed within the genome (NADEAUand TAYLOR1984). Because this analysis was based on 13 segments only, additional conserved segments must be identified to provide more accurate estimates of these important measures of the pattern and rate of genomic evolution. Synteny of two or more pairs of homologous genes in two or more species is evidence that the chromosomal segment on which these genes are located has been conserved since divergence of lineages leading to these species. (Syn- teny homology is defined as location of two or more pairs of homologous genes on the same chromosome in two or more species; the genes need not occur in the same order. By contrast, linkage homology is defined as conservation not only of synteny but also of gene order.) It was thought that syntenic relationships would probably not be conserved across the centromere, especially when chromosomes in one species are acrocentric and the homologous chromosomes in the other species are metacentric. Several examples of syntenic homologies across the centromere have been identified, however (LALLEYand MCKUSICK1985; NADEAUand REINER1986). These include genes on human chromosomes 3, 6, 9, 12, I7 and 20. Detailed mapping to determine whether gene order has also been conserved has not been done for any of these examples. These mapping studies are important because they provide insight into the kinds of chromosomal rearrangements that disrupted linkage groups during evolution. Although it is usually assumed that segments marked by pairs of homologous genes in different species are conserved and are not interrupted by unrelated genes, several examples of interrupted segments have been identified. One example involves lactate dehydrogenase-1 (Ldh-I)and hemoglobin beta-chain (Hbb), which are located on chromosome 7 in the mouse and on chromosome I Ip in man (NADEAUand TAYLOR1984). These genes do not mark a conserved segment, because isocitrate dehydrogenase-2 (Idh-2), which is located on chromosome 15q in man, is located between Ldh-1 and Hbb in the mouse and therefore interrupts this segment (BRITTON-DAVIDIANand PASTEUR1983). An- other example is the X chromosome in man and mouse, for which synteny but not gene order has been conserved (FRANCKEand TAGGART1980). The frequency of these interrupted segments among conserved syntenies is not known. As a result, synteny homologies may overestimate the extent of linkage conservation. An example of conservation of synteny across a centromere, and a potential example of an interrupted segment, involves aconitase-1 (Aco-I), alpha-l acid glycoprotein-1 or orosumucoid-I (Orm-I), galactose-l-phosphate uridyl transferase (Cult), alpha- and beta-interferon (vu and I@), and aminolevulinate dehydratase (Lv) (NADEAUand EICHER1982; KELLEY et al. 1983; KELLEY, KOZAK and PITHA1985; BAUMANN,HELD and BERGER1984; BAUMANNand BERGER1985; $a, $3, LV AND orm-1 MAPPING 1241

DANDOYet al. 1984; LOVETTet al. 1984; VANDER KORPUT et al. 1985; CHENC et al. 1986). (The symbols for alpha-1 acid glycoprotein-1 and -2 have been changed from Agp-1 and Agp-2 to Orm-1 and Orm-2 because of conflicting symbols.) Although these genes are all located on chromosome 4 of the mouse, they are located on opposite arms of chromosome 9 in man (OWERBACHet al. 1981; TRENT,OLSEN and LAWN 1982; CAHILLYet al. 1985; LALLEYand MCKUSICK 1985; SMITHand SPENCE1985; VANDER KORPUT et al. 1985; DIAZ et aE. 1986). It is well-established that the chromosomal segment marked by Aco-1 and Galt has been conserved in mouse and man (NADEAUand EICHER 1982), but it is not known whether the segment marked by Ifa, Zfb, Lv and Orm-1 has been conserved. The resolution of this problem is important because Orm-1 and Lv would mark a conserved segment if Orm-1 is located between Ifa and Lv. To resolve this problem, we used both conventional linkage crosses and recombinant inbred strains to determine the order of Ifa, Ifb, Lv and Orm- 1 relative to each other and relative to reference genes on mouse chromosome 4. These studies showed that the most likely order is Mup-1-Lv-b-Orm-1, Orm-2-Ifa, Ifb and suggested that these genes identify two segments that have been conserved and at least one gene order that has been rearranged since divergence of lineages leading to mouse and man.

MATERIALS AND METHODS Mice: All mice except Mus spretus were obtained from the research and production colonies of the Jackson Laboratory. Mus spretus were obtained from a randomly breed- ing colony maintained by VERNECHAPMAN. Maximum likelihood analysis: Standard maximum likelihood methods were used to calculate recombination frequencies. Methods described by BISHOP(1985) were used to calculate the likelihood for each of the alternative gene orders (equation 1, BISHOP 1985). We assumed that interference did not occur and that recombination frequencies did not differ significantly between sexes. LOD scores for order were also calculated (BISHOP1985). Crosses Both conventional linkage crosses and recombinant inbred strains were used: The Bmfr cross: (C57BL/6J X DBA/2HaSmn)FI and (DBA/2HaSmn X C57BL/6J)F1 males and females were backcrossed to DBA/2HaSmn mice. Backcross progeny were typed for b, Bmfr-1, If., Lv and Mup-1 (SIDMANet al. 1986). BXD and CXB recombinant inbred (RI) strains: The progenitors of the BXD strains are C57BL/6J and DBA/2J, and the progenitors of the CXB strains are BALB/cBy and C57BL/6By. The M. spretus cross: (AKR/J X M. spretus [Spain]F1 females were backcrossed to AKR/J males (BAUMANNand BERCER1985). Backcross progeny were typed for Ifa, I@, Mup-I, Orm-1 and Orm-2. Genetic markers and their assays B-cell maturation factor responsiveness (Bmfr-I):Bmfr- 1 controls the responsiveness of B-lymphocytes to B-cell maturation factors (BMFs) (SIDMANet al. 1986). Methods described by SIDMANet al. (1986) were used for typing Bmfr-1. DBAIPHaSmn mice are nonresponsive and have the Bmfr-1 nr allele, whereas C57BL/6J mice are responsive and have the Bmfr-1. allele. The Bmfr-I’ allele is dominant. Alpha- and beta-interferon (Zfu and ZP):Interferons are a family of proteins that inhibit the growth of many viruses, restrict tumor growth and influence the expression 1242 J. H. NADEAU ET AL. of a nuniber of genes including H-2, HLA and metallothionein (FINTER1973; DE- MAEYER,GALLASSO and SCHELLEKENS198 1; FRIEDMANand STARK1985). IFA and IFB are secreted by a variety of cell types after viral infection. Ifa is a family of closely related genes located near b on mouse chromosome 4 (DANDOYet al. 1984, 1985; CAHILLYet al. 1985); I' is a single gene that is also located near b on mouse chro- niosome 4 (VAN DER KORPUT et al. 1985; KELLEY, KOZAK and PITHA1985). Genomic DNAs were isolated from spleen cell nuclei as described previously (NADEAU,PHILLIPS and EGOROV1985). Southern blot analysis (SOUTHERN1975) was done as described previously (DANDOYet al. 1984). Briefly, high molecular weight genomic DNA (10 pg) was digested with Hind111 restriction endonuclease (Bethesda Research Laboratories, Gaithersburg, Maryland), electrophoresed through 0.5% agarose gels and transferred to nitrocellulose membranes. To type IFA, a 292-bp PstI/HincII fragment of plasmid phIF1204 IFNa:! DNA was used as probe (KELLEYet al. 1983). To type IFB, the PstI/ BglII fragment of the IFB cDNA probe pM3 was used (HIGASHIet al. 1983). The method of RICBYet al. (1977) was used for nick-translating the fragment. Prehybridi- zation, hybridization in 50% forrnamide and washing were done as described previously (DANDOYet al. 1984). Alpha-1 acid glycoprotein-1 and -2 (Om-I, Om-2): ORM-1 and ORM-2 are liver- derived serum proteins produced in response to a variety of systemic injuries, such as infection or inflammation (KUSHNER 1982). BAUMANNand BERCER(1 985) described the typing of ORM-1 and ORM-2. The human homologue of Orm-2 has not been identified. Aminolevulinate dehydratase (Lu;EC 4.2.1.24): LV catalyzes the condensation of 2 mol of delta-aminolevulinate into 1 mol of porphobilinogen. The method described by COLEMAN(1970) was used for typing LV. DBA/2HaSmn and BALB/cBy mice have high LV activity and have the Lu" allele, whereas C57BL/6J mice have low activity and have the Luh allele; Lv"/Ld mice have intermediate levels of activity (HUTTONand COLEMAN1969). At least two samples from each of the backcross progeny of the Bmfr cross and at least two mice from each of the BXD and CXB strains were typed. Major urinary protein-1 (Mup-I):MUP- 1 is an androgen-inducible protein synthe- si7ed in the liver and found abundantly in the urine of mice older than 5-6 wk of age (FINLAYSONet al. 1963; KRAUTERet al. 1982). The method described by NADEAUand EICHER(1982) and modified by SIDMANet al. (1986) was used to type MUP-1 in the Bmfr cross and in the BXD and CXB recombinant inbred strains. DBA/2HaSmn mice have the Mup-1" allele, whereas C57BL/6J mice have the Mup-lb allele. Mup-1' and Mup-lh alleles are codominant.

RESULTS The following strategy was used to determine gene order and recombination frequencies between Ifa, I@, Lv and Orm-1. b and Mup-1 were used as reference genes because their location on mouse chromosome 4 is well-established (DAV- ISSON and RODERICK1981; EICHER1981). The location of Lv relative to b and Mup-1 was then determined in the Bmfr cross and the BXD and CXB recombinant inbred strains. The same crosses were used to determine the position of Z'a relative to b and Lv. The M. spretus cross was used to test cosegregation of Ifa and Ifb. Finally, Mup-1 and Orm-1 were used as reference genes to determine the location of Zfu and Ifb in the M. spretus cross. Lv mapping: To determine whether Lv is located in the expected position between b and Mup-1 (HUTTONand COLEMAN1969), progeny of the Bmfr cross and samples of the BXD and CXB recombinant inbred strains were typed for La. Thirteen backcross progeny in which a crossover occurred between b and Mu$-I in the Bmfr cross (Table I), the 26 BXD strains (Table 2), and the If., I@, LV AND orm-1 MAPPING 1243 TABLE 1

Bmfr cross: recombination between b, Bmfr-1, If., Lv and Mup-1

Mup-1 LlJ b rfa Bmfr-I Animal b b B b r C57BL/6J a a b a nr DBA/2HaSmn b xu b a nr 179A-26 a xb B b r 190B-46 b bxb a nr 190B-65 a axB b r 179A-24, 191A-21, 199B-14 a axB bxnr 190B-72 b bxb xbxnr 179A- 1 a x B b r 201B-20, 201B-24 a b axr 190B-50, 190B-52, 199B-23 b B bxnr 20 1A-14, 201B-26, 20 1B-29 Data for Mup-I, b and Bmfr-I are from SIDMANet al. (1986). x indicates the inferred location of a crossover. seven CXB strains (Table 3) were typed. Results for the two sets of RI strains were pooled. (Sample sizes were too small to test reliably for heterogeneity; therefore, it was assumed that recombination frequencies between the genes of interest were not different in these recombinant inbred strains.) Maximum likelihood methods (BISHOP1985) were used to identify the most likely gene order and recombination frequencies. Log likelihoods were summed for each cross and for each gene order (see Table 5). The most likely gene order was Mup-1-Lv-b. These results confirm the location of Lv between Mup-1 and b (HUTTONand COLEMAN1969). Zfu mapping: Genomic DNAs from progenitors of the Bmfr cross and of the BXD RI strains were digested with various restriction endonucleases and probed with the PstI/HincIII fragment of pMFl204. HindIII digests revealed restriction fragment variants consisting of 2.2- and 1 1.O-kb fragments in C57BL/6J and 2.1- and 9.4-kb fragments in DBAIPHaSmn (Figures 1 and 2). These variants were indistinguishable from those described by DANDOYet al. (1984) for C57BL/6J and BALB/cBy, respectively. Other fragments were observed, but their sizes were not detectably different in genomic DNAs from progenitor mice. HindIII digests of genomic DNA from (C57BL/6J X DBA/ 2HaSmn)Fl mice revealed all four variant fragments as well as the invariant fragments. To determine the location of rf. relative to b and Lv, nine of the 13 backcross animals with crossovers between b and Mup-1, six of the 17 animals with crossovers between b and Bmfr-I, and one with a double crossover were typed for Zfu (Table 1). (Genomic DNAs from the other mice were lost.) The 26 BXD RI strains were also typed (Table 2). Zfu data for the CXB RI strains are also presented (Table 3; cJ: DANDOYet al. 1984). Log likelihoods for each of the three alternative gene orders were calculated for each cross. The likelihoods for each gene order were then summed (see Table 5). The most likely order was Lv-b-Zfu. Although this order was not much more likely than the 1244 J. H. NADEAU ET AL.

I 214 Q 4 Q &:s .z w2 74 4 4 42 8 9 % 4 4xQ QZ & 72 $4 4 rq q3g 2- % 4 4x9 Q$ 2 9sE % 4 4xs 9 6 6 $2 2Q 9 9 Q..y“9 g, 9 soS. XQ g .c.’ 3 ’F: 8 89 4 Eg 41% *il -t *?I n 2 4 QXrqXQ Q - 81 9sPi3 .*c S QXCSXQ 9 b S6 0 ‘&5 * “ 9 rq 4 3 0 8%.- .E i$ c%4 rq 4XQ e4 4 m $2 w9 Q3 -1. g:rqci rq rq rqs- 6 2s m :E 13 s- 214 rq rq Q ~G* $i au .soc3 B 24. s Q 4.5 s2 Q 3;; 8 z9 4 4 9.-6 sc .**g .eY 3.9 2 2s 9 9 Qg2Z 3 TQ 214 4 4XQ E .LIZ EJ 0s gI ‘ TT, .% z9 rq 4 Q 43 2- r~ a2 “S o\ 9 rqxs Q U, EQ -5 F: wrq 4 rq ‘&a 4 4 5.2 ’94 rq 9 rqT)3: .$2e, 2 br.3 ~ b-7 =I rq Q rq -mmb: z.5 2 >c CY rq 4 4 4 sgjug 3z>m * c;bE -9 Q Q (33-0 8 sZ2 a &a+ h c ox .-am?-.5 23&P % y*g $a, rfb, LV AND orm-1 MAPPING 1245 TABLE 3 Recombination between b, Zfa, Lv and Mup-1 in CXB strains

CXB strain D E G H I J K Mup- 1 B C C C B C B X Lv B B c C B B X b B B C C C C B X rf. C B C C C C B B indicates alleles derived from the C57BL/6By progenitor; C indicates alleles from the BALB/ cBy progenitor. Lv was not typed in CXBJ. Data for Mup-I and b are from BAILEY (1981); data for rfa are from DANWYet al. (1984). alternative order Lv--lfa--b, results and arguments presented below suggest that the former is indeed the most likely order. Cosegregation of Zfu and Zfl in the M. spretus cross: Genomic DNAs from progeny of the M. spretus cross were digested with a variety of restriction endonucleases and probed either with the PstI/HincII fragment of the If. probe pMF1204 or with the PstIIBglII fragment of the ZJb probe pM3 (HI- GASHI et al. 1983). Hind111 digests reveal several If. restriction fragment variants with fragment sizes of 17.2, 14.8, 8.7, 6.7 and 4.2 kb in genomic DNA from AKR/J (Figure 3, lane 1) and of 28, 13.0, 11.0, 6.3, 1.5 and 1.1 kb in genomic DNA from M. spretus (Figure 3, lane 2). Other fragments were observed, but their sizes were not detectably different. HindIII digests of genomic DNA from (AKR X M. spretus)F1 mice revealed all 11 polymorphic fragments as well as the invariant fragments. Several differences between these results and those reported by DANDOYet al. (1985) were detected. These include 14.5- and 5.3-kb fragments present in their strain of M. spretus and absent in our strain, and 13.0- and 11.0-kb fragments present in our strain and absent in theirs. These differences probably result from variation in the If. genes between the strains of M. spretus that were used. HindIII digests also revealed ZJb restriction fragment variants consisting of a 26-kb fragment present in M. spretus and a 14-kb fragment present in AKR/J (Figure 4). (AKR X M. spretus)F1 mice showed both fragments. An invariant 6.0-kb fragment was also detected. Complete cosegregation of If. and ZJb variant was observed in the 23 backcross progeny (Table 4), demonstrating that these genes are closely linked. DANDOYet al. (1985) also showed complete cosegregation of Ifa and Zfb in 18 animals from a comparable backcross involving M. spretus. In the combined data for the two backcrosses, the upper 95% confidence limit for the recombination frequency between If. and ZJb was 7.1 %. Zfu and Zfl mapping in the M. spretus cross: The Zfa and ZJb typing data described in the previous section were used to determine the location of Mup- 1 and Orm-1 (Table 4). The most likely gene order was Mup-l-Orm-l--lfa, 1246 J. H. NADEAU ET AL.

f 9A- c 68 -

123456 7 FIGURE1 .-Southern blot analysis of Ifa in parental strains and nonrecombinant backcross progeny of the Bmfr cross. Ten micrograms of high molecular weight DNA that was isolated from 201A-13 (lane 1). 201A-15 (lane 2), 190B-49 (lane 3). 191A-25 (lane 4), 191A-27 (lane 5), C57BL/ 6J (lane 6) and DBA/PHaSmn (lane 7) were digested with Hindlll, electrophoresed through 0.5% agarose, transferred to nitrocellulose and hybridized to the PstIIHinclI fragment of pMFl204, as described in MATERIALS AND METHODS. Variant fragments of 1 1.O, 9.4, 2.2 and 2.1 kb are indicated on the right. The positions of lambda and phiX174 DNA size markers (kilobases) are indicated on the left. Z' Z' (Table 5), where the relative position of If. and Zfb relative to each other is uncertain.

DISCUSSION Although previous studies clearly demonstrated that If. is located on mouse chromosome 4 (KELLEYet al. 1983; KELLEY, KOZAKand PITHA1985; DANDOY et al. 1984, 1985; LOVETTet al. 1984; VAN DER KORPUT et al. 1985), its location relative to other genes was uncertain. For example, DANDOYet al. (1 984) showed that Zja is closely linked to the minor histocompatibility locus If, rf, LV AND orm-I MAPPING 1247

c 9.4. t

6.6<

4.4.

2.0. f;

1.1 .

1 2 3 4 56 7 8 91011 FIGURE2.-Southern blot analysis of If. in recombinant progeny of the Bmfr cross. Ten micrograms of high molecular weight DNAs that was isolated from 109B-46 (lane l), 191A-21 (lane 2), 179A-24 (lane 3). 179A-26 (lane 4). 190B-65 (lane 5), 199B-23 (lane 6), 201A-14 (lane 7), 190B-50 (lane 8), 190B-52 (lane 9). C57BL/6J (lane 10) and DBA/ZHaSmn (lane 11) were digested with Hind111 and analyzed as described in Figure 1. Variant fragment of 11.0, 9.4, 2.2 and 2.1 kb are indicated on the right. The positions of several lambda and phiX174 DNA size markers (kilobases) are indicated on the left.

H-15.The location of H-15 on chromosome 4 is uncertain, however (D. BAI- LEY, personel communication). In addition, in situ hybridization has been used to determine the physical location of Zj?a on mouse chromosome 4 (CAHILLYet al. 1985; CHENGet al. 1986); Zj?a is located near bands 4C3-4C7. However, because the correspondence between the physical and recombinational maps is sometimes vague (SEARLEand BEECHEY1981), it is impossible to use physical mapping to determine the precise location of genes such as Zj?a on the linkage map. Finally, although Orm-I is located distal to Mup-1 and brown b (BAU- MANN, HELDand BERGER1984; BAUMANNand BERGER1985) and vu and I’ are located near b (DANDOYet al. 1984, 1985; CHENGet al. 1986), the gene 1248 J. H. NADEAU ET AL.

Size Lane no. Ifa marker frogment (kb) I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 size(kb1

23.7- - 17.2 14.8 I I3 -11 U 9.5- I *8.7 6.7 - I t6.7 I -6.3

4.3- ; I *4.2 I

2.3- 4

2.0- I

- 1.1 FIGURE3.--Southern blot analysis of Ifa in the M. spretus cross. Ten micrograms of high molecular weight DNA that was isolated from AKR/J (lane l), M. sprefus (lane 2) and backcross progeny numbers 46 (lane 3). 47 (lane 4), 48 (lane 5). 49 (lane 6), 50 (lane 7), 51 (lane 8). 52 (lane 9), 53 (lane lo), 54 (lane Il), 55 (lane 12), 56 (lane 13). 57 (lane 14). 58 (lane 15) and (AKR X M. sprefus)F, (lane 16) was digested with Hind111 and analyzed as described in Figure 1. Variant fragments of 28, 13.0, 1 1.O, 6.3, 1.5 and 1.1 kb characteristic of M. sfrefus and fragments of 17.2, 14.8, 8.7, 6.7 and 4.2 kb characteristic of AKR/J are indicated on the right. The positions of several lambda and phiX174 DNA size markers (kilobases) are indicated on the left. Faint 28- kb fragments were seen in lanes 6, 9 and 12. None of the other fragments characteristic of M. spretus were detected in these samples. The most likely explanation is a partial digest of AKR/J- derived DNA in samples 49, 52 and 55. The 13-kb band is absent in lane 11. The most likely explanation is recombination between this fragment and other Ifa related fragments. DANIIOYef al. (1985) reported similar fragments. Finally, the 4.3-kb fragment present in lane 16 of the Ifa blot probably results from plasmid contamination of the (AKR X M. sprefus)F, DNA; this fragment was also observed in the Ijb blot (not shown). rfa, I@, LU AND orm-J MAPPING 1249

9.5+

6.7 + -6

FIGURE4.-Southerii blot analysis of I' in the M. sprefus cross. Blots were prepared and analyzed as described in Figure 3, with the exception that the cDNA probe for I' (HIGASHIet al. 1983) was used. Variant fragments of 26 and 14 kb are indicated on the right. The positions of several lambda and phiX174 DNA size markers (kilobases) are indicated on the left.

TABLE 4

M. spretu cross: recombination between Om-I, Zfa, Zfb, and Mup-1

Zlupl" Orm-1" IJa Ifs Animal

a a a a 30, 34, 36, 44, 45, 46, 47, 49, 50, 52. 53, 55, 56 UC ab aC ac 31, 39. 40, 43, 51, 54, 57, 58 a x ab ac aC 38 ac x a a a 42 a a x ac aC 37, 48

a Data for Orm-1 and Mup-1 are from BAUMANN,HELD and BERGER(1984). order of Ijiu, Ifb, Lu and Orm-J has not been determined previously; therefore, the location of Ijiu and Ifb relative to other markers of interest on mouse chromosome 4 was uncertain. Results of the present study conclusively demonstrated the following gene order: Mup-J-Lu-b (Table 4). The recombination percentage between b and Lu was previously estimated to be 5 & 2% (HUTTONand COLEMAN1969). In the present study, this percentage was estimated to be 7.3 & 2.5% in the Bmfr cross and 8.5 & 4.3% in the RI strains, with an average of 7.9%. The recombination percentage between Lu and Mup-J does not appear to have been estimated previously. The average percentage of 3.2% for the Bmfr cross and the RI strains was used in the map (Figure 5). The other gene order that was conclusively demonstrated in the present study was Mup-J-Orm-J-Ijiu, Ifb (Table 5). This gene order is important because it demonstrates that Ijiu and Ifb are distal both to Lu and to Orm-J. By contrast, it was not possible to conclusively identify the most likely order for b and Vu, Ifb because the order Lu-b-Ijiu, Ifb was only slightly more TABLE 5 Maximum likelihood analysis of gene order and recombination percentages for genes on mouse chromosome 4

a. b. Lu, Mup-1

Log likelihood for order

Cross" Mup-1-Lu-b Lu-MuQ-1-b Mup-1-b-Lu Bmfr -2 1.24 -26.13 -29.77 BXD, CXB -1 1.96 -11.96 -16.61 Pooled -33.20 -38.09 -46.38 Most likely gene order and recombination percentages ( %)b: M~p-I-3.2-L~-7.9--6 LOD score for order': 4.89 Comment: The order Mup-I-Lv--b is more than 7 X lo4 times more likely than the order with the next largest likelihood

b. b, rfa, Lu

Log likelihood for order

Cross Lu-Ifa-b Ifa-Lu-b Lu-b-Ifa Bmfr -10.95 -18.28 -12.18 BXD, CXB -15.38 -17.61 -14.37 Pooled -26.33 -35.89 -26.55 Most likely gene order and recombination percentages (%)b: Lv-7.5-b-2.5-lfa LOD score for order: 0.22 Comment: The order Lu-b-Zja is more than 1.7 times more likely than the order with the next largest likelihood

c. Orm-1, rfa, Mup-1

Log likelihood for order

Mup-1 -Om-1 Orm-1 -Mup-1 Mup- 1-rfa Cross -rfa -rfa -0rm-1 M. spretus -5.90 -7.57 -7.57 Most likely gene order and recombination percentages (%): Mup-I-8.7-0rm-I-8.7-Ifa LOD score for order: 1.67 Comment: The order Mup-I-Orm-I-Ifa is more than 46 times more likely than the order with the next largest likelihood.

See MATERIALS AND METHODS for a description of the crosses. Average (unweighted) recombination percentages for the two crosses are given. 'The LOD score for order is defined as the likelihood difference between the maximum likelihood order and the order with the next largest likelihood and is interpreted as a measure of support for the maximum likelihood order (BISHOP1985). 1250 $a, I@, LV AND orm-1 MAPPING 1251

RF= 5 9 3 7 3 13 FiGURE 5.-Provisional linkage map of If., @, Lv and Orm-1 on mouse chromosome 4.

Galt Aco-1 Lv Orm-1 Orm-2 lfa If6 ---- Mouse Chromosome 4

Human Chromosome 9 ** 9P 9q FIGURE6.-Comparison of the location of genes on chromosome 4 in the mouse and on chromosome 9 in man. The human homologue of these genes have been assigned to a chromosome arm, but the relative position of these genes on each arm has not been determined, with the exception of rf. and lfb (cf: DIAZ et al. 1986). likely than the order Lv-Ifa, ZJb-b (Table 5). However, when the order of other genes is considered, it is very likely that the order given in Figure 5 is correct. The recombination percentage between b and Orm-I (and Orm-2) is estimated to be 3.3% (BAUMANN,HELD and BERGER1984; Figure 6). The recombination percentage between Mup-I and Orm-1 (and Orm-2) is 11 & 4% (BAUMANNand BERGER1985). These resuits strongly suggest that Orm-I is distal to b; the difference between these two percentages (1 1% - 3.3% = 7.7%) is an estimate of the recombination percentage between b and Mup-I and is consistent with estimates discussed above. The recombination percentage of 3% for the b-Orm-1 (and Orm-2) interval was used in the map (Figure 5). Thus, if Orm-I and Orm-2 are located distal to b, then Ifa and ZP must be located distal to b and, hence, distal to Lv. Although gene order appears reasonably certain, the recombination percentage between b and rfa varied considerably between crosses involving conventional laboratory strains and crosses involving M. spretus. In crosses with conventional strains, b and rfa appear to be closely linked. The recombination percentage between b and Ifa was 1.0 & 1.0% in the Bmfr cross and 4.0 & 2.3% in the RI strains, with an average percentage of 2.5% (Table 5). By contrast, the percentage was 17 & 8.8% in the M. spretus cross reported by DANDOYet al. (1985). In the M. spretus cross reported here, the recombination percentage between Ifa, I' and Orm-I was 8.7 -C 5.9% (Table 5). Because Orm-1 is located about 3 cM distal to b (BAUMANN,HELD and BERGER1984; BAUMANNand BERGER19S5), the recombination percentage between b and Ifa is estimated to be about 12% in the M. spretus cross. Thus, in conventional strains, Ifa appears to be located about 2.5 cM distal to b, whereas in the M. spretus cross reported by DANDOYet al. (1985) and in the M. spretus cross reported here, Ifa appears to be located about 15 cM distal to b. Although sample sizes for the M. spretus crosses are small (18 and 23), the recombination percentages were too heterogeneous to justify using an average percentage for 1252 J. H. NADEAU ET AL. constructing the map. The reason for the heterogeneity is unknown, but could result from a small interstitial inversion for which occurrence differs between laboratory strains and &I. spretus, or from recombination “hot spots” between If. and b in M. spretus or recombination “cold spots” between vu and b in conventional laboratory strains. Regardless of the correct interpretation, these results suggest that caution must be exercised when using M. spretus in linkage crosses. Because of heterogeneity in recombination frequencies for the b-Zfu interval between crosses involving conventional laboratory strains and crosses involving M. spretus, it is important to compare recombination frequencies estimated in the present study with published frequencies for the same intervals. b and Mup-1 are widely used genetic markers in linkage crosses (EICHER198 1). The average recombination percentage between these genes is 5.8 f 1.3% (DAVISSONand RODERICK1981). In the present study, the recombination percentage was 11.9 f 3.1% in the Bmfr cross and 8.5 f 4.3% in the RI strains, with an average of 10.2% (Table 5). Although this percentage is somewhat higher than the average for the previously published studies, the 95% confidence limits overlap. Thus, the heterogeneity in recombination frequencies observed in crosses involving conventional laboratory strains and those involving M. spretus was confined to the interval distal to b. There was no evidence for different gene orders. It is often assumed that synteny of two or more pairs of homologous genes in species such as man and mouse represents conserved linkage (OHNO 1969; LALLEY,MINNA and FRANCKE1978; LUNDIN 1979; NADEAUand TAYLOR 1984). Gene order is not necessarily conserved, however. For example, the large number of pairs of homologous genes that are located on chromosome 4 in the mouse and on chromosome 9 in man is considered to be evidence for extensive synteny conservation, if not linkage conservation. Results presented here, however, clearly demonstrate that the order of genes on chromosome 4 of the mouse and chromosome 9 in man these genes has not been conserved. In the mouse, the chromosomal segment marked by the genes Aco-I, Cult, vu and ZJb, all of which are located on chromosome arm 9p in man (SMITHand SPENCE 1985), is interrupted by the chromosomal segment marked by the genes Orm-1 and Lu, both of which are located on chromosome arm 99 in man (SMITHand SPENCE1985). CHENCet al. (1986) used in situ hybridization to map If. and concluded that If. is proximal to Lv and that vu is part of the conserved segment marked by Aco-1 and Gult. Data presented here clearly demonstrate that Zfu is distal to Lu. The discrepancy between the order obtained in the present study and the order inferred by CHENC et al. (1986) probably results from the loose correlation between the physical and recombinational maps (SEARLEand BEECHEY198 1). Other examples of interrupted linkages involving syntenic homologies in man and mouse are genes on the X chromosome (FRANCKEand TAGCART1980) and the genes Hbb, Zdh-2 and Ldh-l on chromosome 7 in the mouse (BRITTON-DAVIDIANand PASTEUR1983; NADEAUand TAYLOR1984) and on chromosomes 1 Ip and 159 in man (LALLEY and MCKUSICK1985; SMITHand SPENCE1985). These results suggest that If., ~fb,LV AND orm-1 MAPPING 1253 caution must be exercised when using synteny maps for estimating the extent of linkage homology. Although gene order was not conserved, two conserved segments were nevertheless identified. The segment marked by Aco-1 and Galt has apparently been conserved since divergence of lineages leading to man and mouse (NA- DEAU and EICHER1982; Figure 6). Estimates of the minimum and the true length are provided by NADEAUand TAYLOR(1984). The other segment that has apparently been conserved is that marked by Lv and Orm-1 (Figure 6). The minimum length of the Lv-Orm-1 segment is approximately 10 cM (Fig- ure 6); the true length is estimated to be approximately 30 cM (4equation 2, NADEAUand TAYLOR1984). Thus, these genes provide two examples of chromosomal segments that have apparently been conserved since divergence of lineages leading to man and mouse and an example of disrupted gene order. It was expected that linkage across the centromere would not be conserved during evolution, especially in species such as the mouse that have acrocentric chromosomes only. However, at least six examples of conservation across a centromere have now been identified (LALLEYand MCKUSICK 1985; NADEAU and REINER1986). The mapping studies reported here represent the first detailed analysis of one of these examples. These studies demonstrated that gene order was not conserved (Figure 6). Comparison of the order of these genes in man and mouse may provide evidence for the kinds of chromosomal rearrangements that disrupted gene order across the centromere since divergence of lineages leading to man and mouse. Reciprocal translocations are the usual and simplest explanation for linkage disruptions. A single reciprocal translocation accounts for the results described here. Alternative explanations include transpositions, inversions and combinations of Robertsonian translocations and paracentric inversions. Be- cause the order of Aco-l, Galt, If., Zfb, Lv and Orm-l is not known precisely in man, it presently impossible to determine whether more complex rearrangements may be involved.

We thank D. W. BAILEYand B. A. TAYLORfor gifts of CXB and BXD mice; D. W. BAILEY, B. A. TAYLOR,M. DAVISSONand JANAN EPPIG for their many helpful comments on an earlier draft of this paper; D. L. COLEMANfor advice with LV typing and for sharing NPand perchloric acid; Y. KAWADEfor the gift of the beta-interferon pM3 plasmid; and C. DUNBAR,J. MARSHALL and D. VARNUMfor expert technical assistance. This work was supported by grants GM32461, CA35845, A120232 and AI19737 from the National Institutes of Health. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.

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