BRIEF COMMUNICATION

Hong Xiao ? Elsy P. Jones? Ziyi ZhuKirsten Fischer Lindahl

Fine mapping of 12 microsatellites and two new recombinants

in the distal H2 complex on mouse chromosome 17

Received: 3 September 1996

Simple sequence length polymorphisms (SSLPs) or micro-satellites occur at high frequency throughout mammaliangenomes and have revolutionized linkage analysis andgenome mapping. Dietrich and co-workers (1994) publish-ed a map of the mouse genome containing 4006 SSLPs, andan update added another 2177 (Murine MapPairs , April1995, Research Genetics, Huntsville, AL). These SSLPswere mapped by analysis of 46 F2 animals (92 meioses), somany could not be separated and were assigned to bins orclusters. Two of these bins are located at the distal end ofthe mouse major histocompatibility complex (MHC),H2,on chromosome 17. We have now taken advantage ofestablished and new recombinant haplotypes and two con-tigs of yeast artificial chromosomes (YACs) to map theseD17Mit markers. Eight SSLPs from the proximal bin fellbetweenH2-K andH2-D and will be reported elsewhere (K.Blomer and co-workers, in preparation); here we describethe fine mapping distal ofH2-D of 12 SSLPs, all from thedistal bin.

The recombinant haplotypes are listed in Table 1; theydefine theH2-T andH2-M regions and involve two haplo-types, cas3 and cas4, from Mus musculus castaneus(Fischer Lindahl 1986; Steinmetz et al. 1986; Richards etal. 1989). The YACs are derived from strains C3H/HeJ (H2k) and C57BL/6 (H2b; Jones et al. 1995); thecontigs cover41.5 megabases (Mb) from theH2-M regionand link it to theH2-T region (Fig. 1).D17Mit primer pairsfor the polymerase chain reaction (PCR) were purchasedfrom Research Genetics; isolated DNA from recombinantmice and YAC clones was amplified under conditionsdescribed by Dietrich and co-workers (1992). Table 2 liststhe SSLPs tested and their polymorphisms within ourparental strains.

None of the markers were mapped to theH2-D or -Qregions. The R1 recombinant in our panel separates the

H2-Q and H2-T regions (Table 1); these regions have notbeen bridged by molecular cloning, and the length of thegap between them is unknown. Seven of the SSLPs fell intothe H2-Tand-M regions and four of them could be placedon the YACs (Fig. 1). The combined length of our contigsgreatly exceeds 1.5 Mb; the presence of four SSLPs is thusin good agreement with the goal of the MIT group to find amarker every 500 kilobases (kb; Dietrich et al. 1994).D17Mit47, from the 59 upstream region of theH2-T1gene (Fisher et al. 1989; Dietrich et al. 1992) is presenton YAC E8, which also contains multipleH2-M genes(Jones et al. 1995). Class I genes from theH2-T region ofstrain BALB/c (H2d) were cloned in three cosmid contigs;T1–T11 in cluster A, T16–T23 in cluster B, andT24 incluster C (Fisher et al. 1985).H2-T1 was mapped as themost distal gene in theH2-T region by one recombinantanalyzed for restriction fragment length polymorphism(RFLP; Passmore and Romano 1987); our result confirmsthis orientation of cluster A withT1 closest to theH2-Mregion (Jones et al. 1995), and we corrected the uncertainplacement ofD17Mit47 (LOD score#1.0) in the originalmap (Dietrich et al. 1994).

D17Mit125displayed the C3H/HeJ pattern in the R1 andR4 recombinants (Table 3), placing it in theH2-T region,proximal of the R4-early breakpoint (R4-e) by which wedefined the proximal end of theH2-M region (Richards etal. 1989).Mit125 is present on YACs E8, CO9, and E1(Fig. 1), placing theR4-ebreakpoint betweenMit125 andH2-M7 and-M8 [mapped distal ofR4-e(Wang and FischerLindahl 1993)] in a 400 kb interval (Jones et al. 1995).

D17Mit24 is from intron 3 of theH2-M2 gene (Brorsonet al. 1989; Dietrich et al. 1992), which is detected on YACsE2, E5, and E11 by hybridization with both 59 and 39 probes(Jones et al. 1995). PCR withMit24 yielded a product fromE2 and E11, but not from E5; we do not know the reason forthis, as the Southern blots are similar for all three YACs.D17Mit markers 64, 148, 232, and 234 were all mapped totheH2-M region by theR4-eandR4-late(R4-l) haplotypessharing the CAS3 pattern, which differs from C3H/HeJ andC3H.SW (Table 3).Mit148was found in YAC clones J1, J2,E11, and E5 (Fig. 1); it is therefore proximal toH2-M2

H. Xiao ? E. P. Jones? Z. Zhu ? K. Fischer Lindahl ( )Howard Hughes Medical Institute, Departments of Microbiology andBiochemistry, University of Texas Southwestern Medical Center,5323 Harry Hines Blvd., Dallas, TX 75235-9050, USA

Immunogenetics (1997) 45: 274–277 Springer-Verlag 1997

(Mit24) andD17Leh525, within 120 kb ofH2-M3 (Jones etal. 1995).

Whereas results withMit232 have been too weak andinconsistent to allow a conclusion,Mit64 and234were notpresent in these YACs. BecauseMit64 has been mappeddistal ofMit24 in crosses with thep haplotype (Heine et al.1994), it must also be distal ofMit148. Mit232 andMit234could be either in the gap between the two contigs or, mostlikely, distal of Mit24. Mit64, 232, and 234 are thereforegood markers for extending our contig.

D17Mit markers 11, 104, 105, 124, and 126 weremapped distal to theH2-M region. They all gave the B6pattern in theR10-late(R10-l) haplotype (see below) andthe C3H.SW pattern in theR4-l haplotype (124 did notdistinguish C3H.SW from CAS3). A probe forTpx1 (Ka-sahara et al. 1989) gives the same result in RFLP analysis(Richards et al. 1989; Zhu 1990), showing that a secondrecombination took place betweenMit64 and these markersduring the backcrossing of both theR4-e haplotype toC3H.SW and of theR10-early (R10-e) haplotype toC57BL/6J (Table 1).

H. Xiao et al.: Microsatellites nearH2-M region 275

Table 1mRecombinant strains used in mapping of SSLPs and the origin of theirH2 regions

Strain Parents Ref. H2-D H2-Q H2-T H2-M Tpx1

B10.CAS3(R1) CAS3/C3H 1 CAS3 CAS3 // C3H C3H C3HB10.CAS4(R2-l) R2-e/B10 2 CAS4 CAS4 CAS4 // B10 B10C3H.CAS3(R4-e) C3H/CAS3 1 C3H C3H C3H // CAS3 CAS3B6.CAS3(R4-l) R4-e/C.SW 1 C3H C3H C3H / CAS3 // C.SWB6.CAS3(R10-l) R10-e/B6 3 CAS3 CAS3 CAS3 CAS3 // B6

// This marks the crossover that created the particular recombinanthaplotype

/ This marks crossover in parent haplotypeReferences: 1 = Richards et al. 1989; 2 = this report; 3 = Zhu 1990

Table 2mD17Mit markers tested and their polymorphisms

Locus Relative size of PCR product

11 CAS35 CAS4 = C3H.SW5 B6 = B10 = C3H/HeJ47 C3H.SW5 CAS45 CAS3a = C3H/HeJ5 B6 = B1064 CAS35 C3H/HeJ5 C3H.SW = CAS45 B6 = B10104b C3H.SW5 C3H/HeJ5 B6 = B105 CAS4105c C3H.SW = B6 = B105 C3H/HeJ5 CAS3124 B6 = B105 CAS3 = C3H.SW5 CAS45 C3H/HeJ125 B6 = B10 = CAS45 C3H.SW5 CAS35 C3H/HeJ126 CAS3 = CAS45 C3H/HeJ5 C3H.SW = B6 = B10148 CAS3 = CAS45 C3H/HeJ5 B6 = B10 = C3H.SW232 C3H/HeJ = C3H.SW5 B6 = B105 CAS3 = CAS4234 CAS35 C3H.SW5 C3H/HeJ = B6 = B105 CAS4

a CAS3 type inferred from B6.CAS3(R10)b CAS3 gave no PCR product with104c CAS4 gave no PCR product with105 Fig. 1mSchematic presentation of theH2 complex (top part), position

of recombinational breakpoints (nameand downward arrows), YACcontigs from theH2-M region (bottom part), and order ofD17Mitmarkers (upward arrows). A detailed restriction map of the YACs hasbeen published (Jones et al. 1995). The position ofM region probes isindicated above the YACs under the scale bar (kb), andhorizontalarrowheadsmark chimeric ends of YACs. The distal contig is in theopposite orientation relative to our first report (Jones et al. 1995),because new clones, currently being characterized, have bridged therelatively short gap from G11 to E11 (M. Yoshino and H. Xiao,unpublished data). The ‘+’ symbol indicates that an SSLP is presenton the YAC. Dotted linesshow the limits within which the markersmust lie. Double slashmarks indicate gaps between cloned regions.G11, J1, and J2 clones are of B6 origin, all other clones are of C3H/HeJ origin

A similar secondary recombination occurred during thebackcrossing to C57BL/10SnJ of theR2-early (R2-e) re-combinant haplotype (Steinmetz et al. 1986). The congenicB10.CAS4(R2)/Kfl strains has retained expression of theTL antigen of CAS4, detected on thymocytes with mono-clonal antibody 18/20 (Fischer Lindahl 1985), but hasgained M3wt (B. E. Loveland and K. Fischer Lindahl,unpublished observations) from B10 together withMit148(Table 3). The breakpoint could not be mapped with respectto Mit125, due to lack of polymorphism between B10 andCAS4.

Consistent with our results, Jiang and co-workers (1995)could not separateMit24, Mit47, andMit64 in their analysisof congenic intervals. Vernet and Artzt (1995) recentlymapped 12D17Mit microsatellites from chromosome 17by typing recombinants betweent haplotypes. They sepa-rated Mit47 from Mit64 by one recombinant and placedthem in the same order relative to theH2 complex as wedid; they also could not separateMit104 and105, and theyplaced them withtf, at the opposite end of Inversion 4[In(17)4] in t haplotypes fromMit64 andH2, separated byseven recombinants fromMit124 insideIn(17)4. Two of ourrecombinants involving wild-type chromosomes,R4-l andR10-l, separateMit64 from Mit104, 105, and124; they aretherefore consistent with the interpretation offered byVernet and Artzt (1995) that the distal breakpoint ofIn(17)4 is betweenMit64 and the other three markers.However, some additional rearrangement or scrambling atthe end of the inversion must be postulated to account forthe location ofMit124 proximal to tf in t haplotypes.

In summary, the order of the 12 SSLPs fromthe centromere is D17Mit47-Mit125-Mit148-Mit24-[Mit64, 232, 234]-[Mit11, 104, 105, 124, 126]with thecaveat thatMit232 could be betweenMit125 and Mit148(Fig. 1). The presence ofMit47 on YAC E8 confirms thatT1 is the most distal of theH2-Tclass I genes.Mit64, 232,and 234 are in an uncloned part of theH2-M region. TheR4-e recombinational breakpoint is betweenMit125 andH2-M7 and M8, in a 400 kb interval. Two new recombi-nants,R2-l (CAS4/B10) on the proximal side andR10-l

(CAS3/B6) on the distal side, provide additional tools fordefinition and mapping of theH2-M region (Nataraj et al.1996; Morse et al. 1996).

AcknowledgmentsmWe thank Dr. Lorraine Flaherty for helpful dis-cussions and gifts of DNA from recombinant strains, used in earlyphases of this work, and Irene Carlo for preparations of DNA. Thiswork was supported in part by NIH grant AI37818.

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H. Xiao et al.: Microsatellites nearH2-M region276

Table 3mAlleles of D17Mit markes in parental and recombinant haplotypes

Straina 47 125 148 64 232 234 11 104 105 124 126

C3H/HeJ 3b 4 2 2 1 3 3 2 2 4 2B10.CAS3(R1) 3 4 2 2 1 3 3 2 2 4 2B6.CAS3 ? 3 1 1 3 1 1 0 3 2 1R4-early 3 4 / 1 ? 3 1 1 0 3 2 1R4-late 3 4 1 1 3 1 / 2 1 1 2 3C3H.SW 1 2 3 3 1 2 2 1 1 2 3B6 4 1 3 4 2 3 3 3 1 1 3R10-early 3 3 1 1 3 1 1 0 3 2 1B6.CAS3(R10-l) 3 3 1 1 3 1 / 3 3 1 1 3B10.CAS4 2 1 1 3 3 4 2 4 0 3 1R2-early 2 1 1 ? 3 4 2 4 0 3 1B10.CAS4(R2-l) 2 /? 1 /? 3 4 2 3 3 3 1 1 3B10 4 1 3 4 2 3 3 3 1 1 3

a Recombinant strains and their differential segments are boldfaced;the parent strains are listed in Table 1; / marks the crossovers

b Alleles are numbered according to the size of the PCR product, 1being the smallest, 0 meaning that no PCR product was obtained, and ?that type was not determined

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Note added in proof: The gap between YACs G11 and E11 is spannedby a 130 kb BAC clone from strain 129/J.D17Mit232is present on twoBAC clones that containH2-M3 but not Mit148; we therefore placeMit232 proximal of Mit148, nearH2-M3. Multiple new YAC cloneshave established the order from the centromere of the remainingmarkers asD17Mit64-Mit234-Mit104-Mit126-Mit11-Mit105, leavingMit124 as the most distal by default (M. Yoshino and H. Xiao,unpublished data).

H. Xiao et al.: Microsatellites nearH2-M region 277