M a m m a l i a n G e n o m e 2." 260--268, 1992
~enome 9 Springer-VerlagNew York Inc. 1992
The mouse plasminogen locus maps to the recombination breakpoints of the tLub2 and T t ~ partial t haplotypes but is not at the t w73 locus Norbert Schweifer and Denise P. Barlow Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria Received July 29, 1991; accepted September 9, 1991
Abstract. The mouse plasminogen (Plg) locus maps to
a region of chromosome (Chr) 17 which is inverted in the t haplotype Chromosomal variant. Here we investigate the genomic organization of the Pig locus in structurally variant forms of Chr 17; wild-type (+), t haplotype (t), and two partial t haplotypes Tt ~ and tL"b2 which arose by recombination between + and t chromosomes. Our analysis suggests that the t haplotype chromosomal variant contains extra, inverted copies of the Plg locus, and that a single locus is present in the wild-t~cpe variant. Changes in the Plg locus in Tt Orl and tLuoz suggest that they arose by homologous recombination across elements in the Plg locus having the same orientation in the wild-type and t haplotype chromosomes. One hundred ten kb around the wild-type Pig genomic locus have been cloned and the proximal breakpoint of a deletion in the ~,b2 chromosome has been localized to a fragment 30 kb downstream of the Pig gene. The tz"b2 deletion has been shown to delete a gene named t w73 that affects blastocyst implantation, a process probably requiring proteases such as plasminogen. However, the mapping of Pig relative to the tL"b2 deletion and mRNA analysis of plasminogen in t w73 heterozygotes suggests that Plg does not lie at the t w73 locus.
Introduction
Natural populations of many species of the house mouse contain a structurally variant form of Chr 17 known as a t haplotype [for reviews on t haplotypes see Silver (1985), Frischauf (1985) and Barlow (1992)]. The structure of the t haplotype variant differs from the wild-type chromosome by the presence of four neighboring inversions, named In(17)l to In(17)4. The chromosomal region containing these inversions spans O f f p r i n t r e q u e s t s to: D.P. Barlow
20 cM and has been named the "mouse t complex." Normally, the inversions suppress meiotic recombination in mice heterozygous for both chromosomal variants (t/+). Surprisingly, a low level of recombinant chromosomes can be found in which the recombination event occurred across the inversions, between regions apparently aligned in opposite directions. The resultant chromosomes are called partial t haplotypes and carry deletions and duplications of sequences (Silver et al. 1980; Herrmann et al. 1986; Sarvetnick et al. 1986; Bucan et al. 1986; Committee for Mouse Chromosome 17 1991). Molecular and physical mapping analyses of two partial t haplotypes, tAE5 and t h45 has, however, shown that these were the products of homologous recombination events. In both cases recombination occurred across an additional inversion on the wild-type chromosome within inversion In(17)2, which generated a region With the same orientation as found in the t haplotype chromosome (Herrmann et al. 1987). Two partial t haplotype chromosomes Tt ~ and tz"b2 represent another example of products which have arisen by recombination between apparently oppositely-oriented regions within inversion In(17)2. In this case the crossover between the parental wild-type and t haplotype chromosomes occurred between the loci D17Rpl7 and Top-1 (Fig. 1), and the Tt ~ and tL"b2 chromosomes appear to represent reciprocal chromosomes from a similar recombination event, t L"be contains a duplication including T, qk and D17Rpt7, and a deletion including Tree, Tcp-I and t w73. Tt ~ contains a duplication including Tme, Top-1 and t wz3, and a deletion including T, qk and D17RpI7 (Sarvetnick et al. 1986; Fig. 1). Recently it has been shown that the mouse plasminogen (Plg) locus maps to the proximal end of Chr 17 within the t complex and between the loci D17Rpl7 and D17Leh66D (Degen et al. 1990). These data placed Plg in the region of the ~,b2 deletion. We have more recently shown that Plg is duplicated and rearranged in the tL"b2 chromosome (Barlow et al. 1991), which suggests that the Pig locus was
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Fig. 1. The proximal part of mouse Chr 17 in wild-type t haplotype and two partial t haplotype chromosomes. Schematic representation of the structure of the proximal part of wild-type (wt, white line), t haplotype (t, heavy stippled line) and partial t haplotype (t z"b2 and Tt ~ chromosomes, centromere is on the left. The numbers give the
distance in cM from the centromere, t Lub2 and Tt ~ were generated following recombination between the wild-type and t haplotype chromosome between the loci D I 7 R p 1 7 and Tcp-1. Both t Lub2 and Tt TM contain duplicated (loci enclosed in the lightly stippled area) and deleted (black line) chromosomal regions.
involved in the recombination event that generated Tt ~ and t L"b2. In this report the relationship of the P l g locus to the t L"b2 deletion was investigated. We have cloned 110 kb of the genomic region, including the proximal breakpoint of the tL"b2 deletion, and analyzed the locus in the wild-type, t haplotype, and in both Tt ~ and t L"b2. We propose a model whereby multiple, inverted copies of P l g ( t ) allowed homologous recombination with P l g ( + ) despite the fact that this locus is contained within In(17)2, a region that has an opposite orientation in wild-type and t haplotype chromosomes. The products of this recombination event were the partial t haplotypes T t ~ and t L"b2. We further examined whether plasminogen, a protease precursor, could be a candidate gene for t w73, an implantation defective mutant which maps into the t z"~ deletion (Spiegelman et al. 1976; Babiarz et al. 1980; Sarvetnick et al. 1986). Our analysis of mRNA expression in two t w73 mutant mice suggest that plasminogen does not map to the t wz3 locus.
using the alkaline-lysis method as described (Maniatis et al. 1982). Total cellular RNA was prepared by the guanidinium/cesium chloride method and electrophoresed in formaldehyde containing gels as described (Maniatis et al. 1982). The R N A was blotted without pretreatment onto nylon filters by capillary transfer using 50 mM sodium phosphate pH 7. Hybridization analysis of D N A / R N A blots was as described (Church and Gilbert 1984) using 5 • 106 cpm/ml of random primed probe (Feinberg and Vogelstein 1983).
Materials and methods
Amplification of the Y end of Plg-cDNA Total RNA was extracted, converted to cDNA, and amplified by polymerase chain reaction (PCR) using the cDNA Cycle Kit from Invitrogen. For cDNA synthesis 5 I~g total liver R N A were used. The 800 bp fragment was synthesized using the primer PlgMP33B-3' (5'-GGGTTCTGACTGCTGCCCACTGTT-Y) and the primer Plg 3'rev ( 5 ' - A C A A A G G T C A C A G C A G T G A T G G T C - 3 ' ) . The 257 bp fragment was synthesized using the primer Ptg-CR (5'A T T G A A A G G G A G A T G A G G A A T A A C - Y ) and primer Pig 3'-rev (5'-ACAAAGGTCACAGCAGTGATGGTC-3'). The PCR was performed to the following protocol: denaturation at 94~ annealing at 60~ and extension at 72~ each step for 45 s.
C o s m i d libraries A genomic cosmid library, constructed from the wild-type strain 129Sv, in the vector pcos2EMBL, was a gift from A.-M. Frischauf (London, UK) and was screened as described (Herrmann et al. 1987).
Isolation and analysis of DNA/RNA Genomic DNAs were isolated from various mouse organs (Herrmann and Frischauf 1987), and D N A blots prepared as described (Herrmann et al. 1987). Cosmid and plasmid DNAs were isolated
Hybridization probes The plasminogen cDNA clone MP33B used in this work was a gift from S.J.F. Degen. It is a 1920 bp fragment of the plasminogen
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N. Schweifer and D.P. Barlow: The plasminogen locus in wild-type and t haplotype mice
cDNA that has a total length of 2.72 kb. The absent 3' sequences were amplified by PCR. Fragments from cosmids and plasmids were isolated as described (Herry et al. 1990).
A. Restriction Map of Pig cDNA clone MP33B
Mice Mice and genomic DNA were kindly provided as listed: TtY/tw73and Tt~ ~ strains from J.-L. Gu6net, T22~/+ and tL"b2/+~from M. Lyon, ThP/Spe from E. Eicher and wild-type mice from The Jackson Laboratory. The tw12/t w12 cell line was provided by H. Axelrod.
Results
The Plg locus in the wild-type t haplotype and in the partial t haplotypes tLub2 and Tt ~ P r e v i o u s w o r k ( B a r l o w et al. 1991) h a s s h o w n t h a t ~ub2 c o n t a i n s an a p p a r e n t l y c o m p l e t e P i g ( + ) l o c u s a n d a n a p p a r e n t l y i n c o m p l e t e Plg(t) l o c u s . This p a t tern had not previously been described for other markers f r o m this r e g i o n a n d s u g g e s t e d t h a t Pig w a s r e a r r a n g e d in ~,b2. I n o r d e r to d e f i n e t h e e x t e n t o f this r e a r r a n g e m e n t , 400 b p f r o m t h e 5' e n d a n d 400 b p f r o m t h e 3' e n d o f t h e p l a s m i n o g e n c D N A M P 3 3 B (5' p r o b e a n d 3' p r o b e , F i g . 2A) w e r e i s o l a t e d a n d u s e d to a n a l y z e tr"b2 a n d Tt ~ T h e d a t a (Fig. 2) s u g g e s t e d t h a t t h e Plg w i l d - t y p e l o c u s is c o m p l e t e in ~,b2, b u t t h e Plg t h a p l o t y p e l o c u s h a s 5' a n d 3' s e q u e n c e s b o t h p r e s e n t a n d a b s e n t ( s e e f i g u r e l e g e n d f o r details). W e h a v e i n t e r p r e t e d this i n i t i a l l y p u z z l i n g r e s u l t as s u g g e s t i n g that the t haplotype normally has multiple copies of Plg(t) a n d t h a t o n e is p r e s e n t in t L"b2 b u t o n e h a s b e e n d e l e t e d . T h i s i n t e r p r e t a t i o n s u g g e s t e d t h a t t h e Plg loc u s is v e r y c l o s e t o t h e p r o x i m a l b r e a k p o i n t o f t h e t c"b2 d e l e t i o n . T h e s a m e a n a l y s i s o f Tt ~ s h o w e d t h a t the wild-type locus was completely deleted but the t haplotype was completely present.
Cosmid walk around the Plg locus Initial analysis using the MP33B cDNA clone showed that, although the coding region of plasminogen was n o t d e l e t e d in t c"b2, t h e Plg l o c u s m u s t lie c l o s e to t h e deletion. In order to isolate the proximal deletion b r e a k p o i n t o f ~ub2, t h e g e n o m i c r e g i o n a r o u n d t h e Plg l o c u s w a s o b t a i n e d f r o m a m o u s e c o s m i d l i b r a r y (prep a r e d f r o m s t r a i n 129/Sv b y A . - M . F r i s c h a u f , L o n d o n , U K ) u s i n g t h e M P 3 3 B c D N A c l o n e as a s t a r t i n g p o i n t . Two pairs of overlapping cosmids were obtained and m a p p e d u s i n g t h e e n z y m e Sca I, w i t h r e s p e c t to t h e partial cDNA MP33B. The cloned region spans about 110 k b (Fig. 3), h o w e v e r , a c o s m i d c l o n e c o n t a i n i n g 200 b p f r o m t h e m i d d l e o f t h e c D N A (1.3-1.5 kb) w a s n o t f o u n d in f i v e d i f f e r e n t c o s m i d l i b r a r i e s . It h a s b e e n shown by genomic mapping, using the ends of the f l a n k i n g c D N A , t h a t this r e g i o n is c o n t a i n e d o n a genomic fragment maximally 5 kb long (data not shown).
Fig. 2. Analysis of the plasminogen locus in wild-type t haplotype and partial t haplotype mice. (A) Restriction map of the plasminogen cDNA MP33B. A Pst I restriction map of the cDNA clone MP33B is shown (Degen et al. 1990) which contained 1920 bp of the plasminogen cDNA and lacked 800 bp of the 3' end. EcoR I indicates the cloning site. The positions of the 5' and 3' probes used in (B) are indicated by the lines under the restriction map. (B) Mapping analysis of MP33B. A DNA panel composed of the partial t haplotypes tzub2/Spe, tL"b2/+ and Tt~ (Fig. 1), wild-type chromosomes C57BL/6 (BI6), Mus spretus (Spe) and DBA, one homozygous t haplotype twJ2/tw12, [a cell line derived by Axelrod and co-workers (1981)], one proximal partial t haplotype lh44/t h44, and two deletion chromosomes (T~P/Spe and T2214/Spe)was digested either with Taq I (I_MS panel) or Pvu II (RHS panel). 7~p (a gift from E. Eicher) contains a deletion from D17Leh66EII to D17Leh66D (Herrmann et al. 1987; Fig. 1), T22H (Howard et al. 1990) contains a deletion including D17Lehll91 to sequences proximal to D17Rpl7. The Taq I digested panel was hybridized with a probe from the 5' end of the plasminogen cDNA and the Pvu II panel was hybridized with a probe from the 3' end (Fig. 2A). The 5' probe recognized three wild-type bands, which were polymorphic for all the DNAs and it can be seen that rc"b2 contains all 5' wild-type bands, 2.4 kb( + ), 2.5 kb(+), and 3.3 kb(+). The 5' probe recognized four t haplotype bands which were polymorphic for all DNAs and it can be seen that one of these bands 5.3 kb (t) is absent from tLubz but the remainder are present. Tt~ contains no wild-type bands but all four t haplotype bands are present. The 3' probe recognized four wild-type bands but only the 5.0 kb (+) band was polymorphic, this was retained in tr"b2. In the t haplotype the 3' probe recognized six bands, two were polymorphic, the 2.75 kb (t) was deleted and the 4.0 kb (t) was present in tL"b2. Tt~ did not contain the polymorphic wild-type fragment 5.0 kb (+) but contained both polymorphic t haplotype fragments 2.75 kb (t) and 4.0 kb (t).
Identification o f the proximal breakpoint o f the ~ub2 deletion DNA fragments from both ends of the cosmid walk, '5.8 k b Sca I/Pvu I I ' f r o m c o s m i d 3 A (5' to t h e Plg
N. Schweifer and D.P. Barlow: The plasminogen locus in wild-type and t haplotype mice 0
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Fig. 3. Physical map of cosmids enlarging the Pig locus and bordering the deletion breakpoint. A restriction map spanning 110 kb is shown containing four cosmid clones, cos3A, cos5B, cos2A and cos4A. (A) The complete plasminogen cDNA of 2.72 kb (scale above), the diagonally striped box indicates the coding region, the vertical striped box at the 3' end indicates the poly(A) tail (Degen et al. 1990). Triangles show the position of PCR primers used for amplifying the 3' end. (B) Sca I restriction map of the genomic region (scale underneath), cos3A and cos5B are overlapping clones, as are
cos2A and cos4A. The size of the region separating these two pairs of overlapping clones has been calculated as maximally 5 kb by genomic mapping (not shown). The relationship between cDNA fragments and the genomic fragments is shown. The mouse Plg genomic locus is estimated to be 43--47 kb long. The black boxes show the position of the probes that were used for mapping: 5.8 kb-Sca I/Pvu II for the 5' end of the cosmid cluster, 2.3 kb-BamH I for the 3' end of the cluster, and the 6.5 kb BamH I fragment that identifies the deletion breakpoint of tL'b2.
gene) and ' B a m H I 2.3 kb f r o m cosmid 4A (3' to the plasminogen gene) were examined for deletion from the ~ , b 2 c h r o m o s o m e (Fig. 3). The D N A end-fragment from cosmid 3A was duplicated in t L~b2 identifying b o t h wild-type and t h a p l o t y p e specific fragments (data not shown). The D N A end-fragment from cosmid 4A identified a B a m H I wild-type specific fragment ( B a m H I-2.3 kb ( + ) , Fig. 4A, lane 1) as deleted from t Lub2. The use of Taq I identified a t-specific fragment (Taq 1-2.5 kb (t), Fig. 4A, lane 10) as present in ~,b2. The absence of the wild-type fragment suggests that one end of cosmid 4A m a p s into the tLub2 deletion. The presence of the t-specific fragment provides an additional a r g u m e n t that the region containing the Plg(t) locus is present in multiple copies on the t haplotype c h r o m o s o m e (see following section; Fig. 6). The exact location of the proximal breakpoint within cosmid 4A was identified by analyzing which of the B a m H I fragments within cosmid 4A showed a size p o l y m o r p h i s m in the wild-type allele present in t L"b2. A 6.5 kb B a m H I fragment located approximately in the center of cosmid 4A (details in Fig. 3) was identified in this m a n n e r as being close to the breakpoint (Fig. 4B).
suits r e v e a l e d that the 3' end of the p l a s m i n o g e n c D N A was contained on cosmid 2A and that no part m a p s into the t L"b2 deletion (Fig. 3).
Is the p l a s m i n o g e n g e n e d e l e t e d in lLub2?
The c D N A clone MP33B lacked the last 3' 800 bp of the plasminogen c D N A . In order to localize the position of the complete c D N A with respect to the t Lub2 deletion 257 bp f r o m the e x t r e m e 3' end (Degen et al. 1990) were amplified using the P C R method. The re-
P l a s m i n o g e n e x p r e s s i o n in t h a p l o t y p e s , a n d in l Lub2 a n d t w73
T h e a b o v e data suggested that the P i g locus was present in multiple copies in t haplotypes and that the locus was very close to the t L"b2 deletion. We have analyzed plasminogen m R N A levels in these chromosomal variants to check, firstly, if the p r o p o s e d multiple P l g ( t ) loci w e r e functional and, secondly, to check if plasminogen could lie at the t w73 locus. T w o reasons p r o m p t e d us to check the possibility that plasminogen could be a candidate for the t w73 mutation. Firstly, the t w73 mutation and P l g m a p to the t Lub2 deletion and the mutant p h e n o t y p e of t w73, a failure of the blastocyst to implant after a t t a c h m e n t to the uterine endometrium (Spiegelman et al. 1976; Babiarz et al. 1982; Axelrod 1985), could be explained b y lack of a protease such as plasmin, the active f o r m of plasminogen. Secondly, although our analysis had shown that all of the plasminogen c D N A sequences were present in the t Lub2 c h r o m o s o m e , it remained formally possible that necessary regulatory sequences had b e e n deleted leading to deregulation of expression, m R N A plasminogen levels were therefore examined in wildtype t haplotype, and in two t w73 mutants (t w73 and tLub2). H o m o z y g o u s t w73 and t Lub2 e m b r y o s die at day 6 of d e v e l o p m e n t (Babiarz et al. 1982; Axelrod 1985) but direct analysis of this stage is not possible because
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N. Schweifer and D.P. Barlow: The plasminogen locus in wild-type and t haplotype mice
of the small size of the embryo (approximately 0.1 mm in length). Therefore, we have examined plasminogen expression in the livers of adult mice heterozygous for t w73 and t Lub2. The results in Fig. 5 show that plasminogen expression in livers from strains homozygous for the t haplotype chromosome (th2tfand t h44tf) is similar to wild-type mice (BALB/c and C57BL/6). This result suggests that the multiple copies of the Plg locus in the t haplotype do not contain functional plasminogen genes. It is unlikely that the Plg locus is subject to feedback controls that maintain mRNA levels, because we have shown (Barlow et al. 1991) that in animals heterozygous for a deletion of Pig (I~P/+) the plasminogen mRNA levels are half that of control wild-type mice. When expression in tw73/+ and ~.b2/+ livers is compared to that in wild-type livers, the expression levels appear to be similar, suggesting that the Plg locus is functional in both these mutant chromosomes. Discussion
The Plg locus in the wild-type t haplotype and in the partial t haplotypes Tt ~ and t Lub2
Fig. 4. Cosmid 4A spans the proximal breakpoint of the tz"b2 deletion. (A) The distal end of cosmid 4A is deleted in t':"b2. The DNA panel shown in Fig. 2B was digested with BarnH I (LHS panel) or with Taq I (RHS panel) and both panels hybridized with fragment 2.3 k b - B a m H I from the end of cosmid 4A (Fig. 3). The B a m H I digest shows that the wild-type band [2.3 kb (+)] was polymorphic but the t-specific band [6.0 kb (t)] couId not be distinguished from the spretus band. This panel shows that the wild-type fragment was deleted from tL"b2 and Tt ~ The Taq I digest allows observation of the t-specific band and shows that this band is present in both ~ub2 and Tt TM. The absence in tr"b2 of the wild-type specific band identified by fragment 2.3 k b - B a m H I identifies cosmid 4A as spanning
The data presented in Fig. 2B shows that in the tzub2 chromosome t haplotype specific sequences at both ends of the plasminogen cDNA are both present and absent. In contrast, the wild-type specific fragments were all present. In the Tt ~ chromosome t haplotype specific sequences from both ends of the cDNA were present and the locus apparently complete; the wildtype locus was totally absent. An explanation consistent with these data is that the Plg locus is present in multiple copies in the t haplotype chromosome, and that one or more copies were deleted during the formation of the tLub2 chromosome but none were deleted during the formation of the Tt ~ chromosome. Using this information we propose a model for the organization of the Plg locus in the wild-type and the t haplotype chromosome, and show the most likely recombination event that created the partial t haplotypes Tt ~ and tLub2 (Fig. 6). The existence of a single wild-type Plg locus has been confirmed by identifying the three wild-type Taq I fragments seen in lane 7, Fig. 2B in cosmid 5B (data not shown). Additional support for the existence of multiple loci in the t haplotype comes from two sources. Firstly, in Fig. 2B, it can be seen that the size of the genomic region recognized by different parts of the plasminogen cDNA is from 50-80% larger in t haplotypes than in wild types (summarized the deletion breakpoint in this chromosome. (B) Mapping of the deletion breakpoint. The DNA panel shown in the RHS of Fig. 4A, was hybridized with a 6.5 kb B a m H I fragment from the central part of cosmid 4A. This probe recognizes a 7.8 kb ( + ) specific band, a 4.5 kb (Spe) specific band and a 2.3 kb (t) specific band. The t-specific band is present in both tLub2 and Tt ~ the wild-type specific band was absent in Tt ~ and present as a differently sized fragment of 5.0 kb (+) in tL"bz. The changed size of the wild-type band in tLub2 indicates that this Taq I fragment contains the deletion breakpoint.
N. Schweifer and D.P. Barlow: The plasminogen locus in wild-type and t haplotype mice
265
Fig. 5. PlasminogenRNA analysis in wild-type t haplotype and partial t haplotype mice. Panel A shows RNA blot analysis and panel B shows a genotype analysis of various Chr 17 variants, The mice analyzed were: t~73/I~f (a complete t haplotype carrying the t~73 embryonic lethal gene), partial t haplotypes th2/t h2, lh44/l h44 (which contain the genomic region including the Pig locus in the t haplotype form), tL~b2/+t: (which contains Plg at the proximal deletion breakpoint, A, B and C are three different animals) and wild-type strains (BALB/c and C57BL/6). A blot prepared using 10 Ixgtotal cell RNA from liver was hybridized with a probe from the 3' of the plasminogen cDNA. The filter was stripped and rehybridized using the fox gene (Van Beveren et al. 1984), a constitutively expressed transcript, to quantitate the RNA loading. Actin expression in the liver
could not be used for this purpose since it was found to vary as much as 50% between different inbred wild-type strains. These results show that plasminogen mRNA levels in t haplotypes and wild-type mice are similar (compare lanes 2, 3 with lanes 4, 5), and that the chromosomes which do not contain a functioning tW73gene do not show a decrease in plasminogenmRNA levels compared to the wild type (lanes 1, 6, 7, 8 compared to lanes 2, 3). The genotype analysis in 5B used Taq I to identify a wild-type-t haplotype polymorphism using a probe containing 200 bp from the 5' end of the plasminogen cDNA MP33B. The results confirm that tw73, the and I h44 contain t haplotype specific DNA at the Plg locus and that the tL"b2heterozygotes show the expected +/t patterns as described in this report.
in Table 1). Secondly, the results in Fig. 4A show that the distal end of the cosmid cluster (defined by probe B a m H 1-2.3 kb; Fig. 3) has crossed the recombination breakpoint that generated tL"b2 but is still contained within sequences present in multiple copies in t haplotypes. This is deduced because loci such as D17Rp17 that map proximal to Pig in the wild-type c h r o m o s o m e are duplicated in t Lub2 (both t and wild-type loci are present) and loci such as Igf2-r, Tcp-1 and Sod-2, that map distal to Pig in the wild-type c h r o m o s o m e (Fig. 1), are completely deleted from t Lub2 (Sarvetnick et al. 1986; Barlow et al. 1991). A probe from the proximal end of the plasminogen cosmid cluster (5.8 kb Sac UPvu II) behaves as the D 1 7 R P I 7 locus (is duplicated) while the probe from the distal end of the cosmid cluster (2.3 kb B a m H I) recognizes only t-specific bands. The only c h r o m o s o m a l position consistent with this data is at the recombination breakpoint (Fig. 6). Continuation of the cosmid walk distal to Plg will eventually lead to sequences, like the Igf2r locus, that are completely deleted in t Lub2. Two other duplicated regions, associated with partial t h a p l o t y p e b r e a k p o i n t s , have been o b s e r v e d within the t complex region on the wild-type chromo-
some. These are the 650 kb D 1 7 L e h l 1 9 - D 1 7 L e h 6 6 E region (Herrmann et al. 1987) and the 110 kb elements in the D I 7 L e h 6 6 D region (Bullard and Schimenti 1990). In the former case an inverted duplication occurred permitting homologous recombination across inversion In(17)2. In the latter case it is proposed that the D1766D locus is contained between In(17)2 and In(17)3 (Hammer et al. 1989), and thus, is in the same orientation in both c h r o m o s o m e variants. Here we identify a duplicated region on the t haplotype chrom o s o m e that we propose also permitted homologous recombination across inversion In(17)2 and is associated with the creation of the two partial t haplotypes, Tt ~ and tLub2. It is k n o w n that Mus spretus shares In(17)2 with the t haplotypes ( H a m m e r et al. 1989), however, it is not clear from our analyses whether or not Mus spretus has multiple copies o f the Pig locus. The extra loci proposed to exist in the t haplotype do not result in an increase in steady state levels of plasminogen m R N A relative to the wild-type (Fig. 5), because data from analysis of deletion mutants (Barlow et al. 1991) does not support the existence of feedback controls; this suggests that these extra loci do not encode functioning genes.
N. Schweifer and D.P. Barlow: The plasminogen locus in wild-type and t haplotype mice
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The mouse plasminogen locus maps to the recombination breakpoints of the tLub2 and T t ~ partial t haplotypes but is not at the t w73 locus Norbert Schweifer and Denise P. Barlow Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria Received July 29, 1991; accepted September 9, 1991
Abstract. The mouse plasminogen (Plg) locus maps to
a region of chromosome (Chr) 17 which is inverted in the t haplotype Chromosomal variant. Here we investigate the genomic organization of the Pig locus in structurally variant forms of Chr 17; wild-type (+), t haplotype (t), and two partial t haplotypes Tt ~ and tL"b2 which arose by recombination between + and t chromosomes. Our analysis suggests that the t haplotype chromosomal variant contains extra, inverted copies of the Plg locus, and that a single locus is present in the wild-t~cpe variant. Changes in the Plg locus in Tt Orl and tLuoz suggest that they arose by homologous recombination across elements in the Plg locus having the same orientation in the wild-type and t haplotype chromosomes. One hundred ten kb around the wild-type Pig genomic locus have been cloned and the proximal breakpoint of a deletion in the ~,b2 chromosome has been localized to a fragment 30 kb downstream of the Pig gene. The tz"b2 deletion has been shown to delete a gene named t w73 that affects blastocyst implantation, a process probably requiring proteases such as plasminogen. However, the mapping of Pig relative to the tL"b2 deletion and mRNA analysis of plasminogen in t w73 heterozygotes suggests that Plg does not lie at the t w73 locus.
Introduction
Natural populations of many species of the house mouse contain a structurally variant form of Chr 17 known as a t haplotype [for reviews on t haplotypes see Silver (1985), Frischauf (1985) and Barlow (1992)]. The structure of the t haplotype variant differs from the wild-type chromosome by the presence of four neighboring inversions, named In(17)l to In(17)4. The chromosomal region containing these inversions spans O f f p r i n t r e q u e s t s to: D.P. Barlow
20 cM and has been named the "mouse t complex." Normally, the inversions suppress meiotic recombination in mice heterozygous for both chromosomal variants (t/+). Surprisingly, a low level of recombinant chromosomes can be found in which the recombination event occurred across the inversions, between regions apparently aligned in opposite directions. The resultant chromosomes are called partial t haplotypes and carry deletions and duplications of sequences (Silver et al. 1980; Herrmann et al. 1986; Sarvetnick et al. 1986; Bucan et al. 1986; Committee for Mouse Chromosome 17 1991). Molecular and physical mapping analyses of two partial t haplotypes, tAE5 and t h45 has, however, shown that these were the products of homologous recombination events. In both cases recombination occurred across an additional inversion on the wild-type chromosome within inversion In(17)2, which generated a region With the same orientation as found in the t haplotype chromosome (Herrmann et al. 1987). Two partial t haplotype chromosomes Tt ~ and tz"b2 represent another example of products which have arisen by recombination between apparently oppositely-oriented regions within inversion In(17)2. In this case the crossover between the parental wild-type and t haplotype chromosomes occurred between the loci D17Rpl7 and Top-1 (Fig. 1), and the Tt ~ and tL"b2 chromosomes appear to represent reciprocal chromosomes from a similar recombination event, t L"be contains a duplication including T, qk and D17Rpt7, and a deletion including Tree, Tcp-I and t w73. Tt ~ contains a duplication including Tme, Top-1 and t wz3, and a deletion including T, qk and D17RpI7 (Sarvetnick et al. 1986; Fig. 1). Recently it has been shown that the mouse plasminogen (Plg) locus maps to the proximal end of Chr 17 within the t complex and between the loci D17Rpl7 and D17Leh66D (Degen et al. 1990). These data placed Plg in the region of the ~,b2 deletion. We have more recently shown that Plg is duplicated and rearranged in the tL"b2 chromosome (Barlow et al. 1991), which suggests that the Pig locus was
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distance in cM from the centromere, t Lub2 and Tt ~ were generated following recombination between the wild-type and t haplotype chromosome between the loci D I 7 R p 1 7 and Tcp-1. Both t Lub2 and Tt TM contain duplicated (loci enclosed in the lightly stippled area) and deleted (black line) chromosomal regions.
involved in the recombination event that generated Tt ~ and t L"b2. In this report the relationship of the P l g locus to the t L"b2 deletion was investigated. We have cloned 110 kb of the genomic region, including the proximal breakpoint of the tL"b2 deletion, and analyzed the locus in the wild-type, t haplotype, and in both Tt ~ and t L"b2. We propose a model whereby multiple, inverted copies of P l g ( t ) allowed homologous recombination with P l g ( + ) despite the fact that this locus is contained within In(17)2, a region that has an opposite orientation in wild-type and t haplotype chromosomes. The products of this recombination event were the partial t haplotypes T t ~ and t L"b2. We further examined whether plasminogen, a protease precursor, could be a candidate gene for t w73, an implantation defective mutant which maps into the t z"~ deletion (Spiegelman et al. 1976; Babiarz et al. 1980; Sarvetnick et al. 1986). Our analysis of mRNA expression in two t w73 mutant mice suggest that plasminogen does not map to the t wz3 locus.
using the alkaline-lysis method as described (Maniatis et al. 1982). Total cellular RNA was prepared by the guanidinium/cesium chloride method and electrophoresed in formaldehyde containing gels as described (Maniatis et al. 1982). The R N A was blotted without pretreatment onto nylon filters by capillary transfer using 50 mM sodium phosphate pH 7. Hybridization analysis of D N A / R N A blots was as described (Church and Gilbert 1984) using 5 • 106 cpm/ml of random primed probe (Feinberg and Vogelstein 1983).
Materials and methods
Amplification of the Y end of Plg-cDNA Total RNA was extracted, converted to cDNA, and amplified by polymerase chain reaction (PCR) using the cDNA Cycle Kit from Invitrogen. For cDNA synthesis 5 I~g total liver R N A were used. The 800 bp fragment was synthesized using the primer PlgMP33B-3' (5'-GGGTTCTGACTGCTGCCCACTGTT-Y) and the primer Plg 3'rev ( 5 ' - A C A A A G G T C A C A G C A G T G A T G G T C - 3 ' ) . The 257 bp fragment was synthesized using the primer Ptg-CR (5'A T T G A A A G G G A G A T G A G G A A T A A C - Y ) and primer Pig 3'-rev (5'-ACAAAGGTCACAGCAGTGATGGTC-3'). The PCR was performed to the following protocol: denaturation at 94~ annealing at 60~ and extension at 72~ each step for 45 s.
C o s m i d libraries A genomic cosmid library, constructed from the wild-type strain 129Sv, in the vector pcos2EMBL, was a gift from A.-M. Frischauf (London, UK) and was screened as described (Herrmann et al. 1987).
Isolation and analysis of DNA/RNA Genomic DNAs were isolated from various mouse organs (Herrmann and Frischauf 1987), and D N A blots prepared as described (Herrmann et al. 1987). Cosmid and plasmid DNAs were isolated
Hybridization probes The plasminogen cDNA clone MP33B used in this work was a gift from S.J.F. Degen. It is a 1920 bp fragment of the plasminogen
262
N. Schweifer and D.P. Barlow: The plasminogen locus in wild-type and t haplotype mice
cDNA that has a total length of 2.72 kb. The absent 3' sequences were amplified by PCR. Fragments from cosmids and plasmids were isolated as described (Herry et al. 1990).
A. Restriction Map of Pig cDNA clone MP33B
Mice Mice and genomic DNA were kindly provided as listed: TtY/tw73and Tt~ ~ strains from J.-L. Gu6net, T22~/+ and tL"b2/+~from M. Lyon, ThP/Spe from E. Eicher and wild-type mice from The Jackson Laboratory. The tw12/t w12 cell line was provided by H. Axelrod.
Results
The Plg locus in the wild-type t haplotype and in the partial t haplotypes tLub2 and Tt ~ P r e v i o u s w o r k ( B a r l o w et al. 1991) h a s s h o w n t h a t ~ub2 c o n t a i n s an a p p a r e n t l y c o m p l e t e P i g ( + ) l o c u s a n d a n a p p a r e n t l y i n c o m p l e t e Plg(t) l o c u s . This p a t tern had not previously been described for other markers f r o m this r e g i o n a n d s u g g e s t e d t h a t Pig w a s r e a r r a n g e d in ~,b2. I n o r d e r to d e f i n e t h e e x t e n t o f this r e a r r a n g e m e n t , 400 b p f r o m t h e 5' e n d a n d 400 b p f r o m t h e 3' e n d o f t h e p l a s m i n o g e n c D N A M P 3 3 B (5' p r o b e a n d 3' p r o b e , F i g . 2A) w e r e i s o l a t e d a n d u s e d to a n a l y z e tr"b2 a n d Tt ~ T h e d a t a (Fig. 2) s u g g e s t e d t h a t t h e Plg w i l d - t y p e l o c u s is c o m p l e t e in ~,b2, b u t t h e Plg t h a p l o t y p e l o c u s h a s 5' a n d 3' s e q u e n c e s b o t h p r e s e n t a n d a b s e n t ( s e e f i g u r e l e g e n d f o r details). W e h a v e i n t e r p r e t e d this i n i t i a l l y p u z z l i n g r e s u l t as s u g g e s t i n g that the t haplotype normally has multiple copies of Plg(t) a n d t h a t o n e is p r e s e n t in t L"b2 b u t o n e h a s b e e n d e l e t e d . T h i s i n t e r p r e t a t i o n s u g g e s t e d t h a t t h e Plg loc u s is v e r y c l o s e t o t h e p r o x i m a l b r e a k p o i n t o f t h e t c"b2 d e l e t i o n . T h e s a m e a n a l y s i s o f Tt ~ s h o w e d t h a t the wild-type locus was completely deleted but the t haplotype was completely present.
Cosmid walk around the Plg locus Initial analysis using the MP33B cDNA clone showed that, although the coding region of plasminogen was n o t d e l e t e d in t c"b2, t h e Plg l o c u s m u s t lie c l o s e to t h e deletion. In order to isolate the proximal deletion b r e a k p o i n t o f ~ub2, t h e g e n o m i c r e g i o n a r o u n d t h e Plg l o c u s w a s o b t a i n e d f r o m a m o u s e c o s m i d l i b r a r y (prep a r e d f r o m s t r a i n 129/Sv b y A . - M . F r i s c h a u f , L o n d o n , U K ) u s i n g t h e M P 3 3 B c D N A c l o n e as a s t a r t i n g p o i n t . Two pairs of overlapping cosmids were obtained and m a p p e d u s i n g t h e e n z y m e Sca I, w i t h r e s p e c t to t h e partial cDNA MP33B. The cloned region spans about 110 k b (Fig. 3), h o w e v e r , a c o s m i d c l o n e c o n t a i n i n g 200 b p f r o m t h e m i d d l e o f t h e c D N A (1.3-1.5 kb) w a s n o t f o u n d in f i v e d i f f e r e n t c o s m i d l i b r a r i e s . It h a s b e e n shown by genomic mapping, using the ends of the f l a n k i n g c D N A , t h a t this r e g i o n is c o n t a i n e d o n a genomic fragment maximally 5 kb long (data not shown).
Fig. 2. Analysis of the plasminogen locus in wild-type t haplotype and partial t haplotype mice. (A) Restriction map of the plasminogen cDNA MP33B. A Pst I restriction map of the cDNA clone MP33B is shown (Degen et al. 1990) which contained 1920 bp of the plasminogen cDNA and lacked 800 bp of the 3' end. EcoR I indicates the cloning site. The positions of the 5' and 3' probes used in (B) are indicated by the lines under the restriction map. (B) Mapping analysis of MP33B. A DNA panel composed of the partial t haplotypes tzub2/Spe, tL"b2/+ and Tt~ (Fig. 1), wild-type chromosomes C57BL/6 (BI6), Mus spretus (Spe) and DBA, one homozygous t haplotype twJ2/tw12, [a cell line derived by Axelrod and co-workers (1981)], one proximal partial t haplotype lh44/t h44, and two deletion chromosomes (T~P/Spe and T2214/Spe)was digested either with Taq I (I_MS panel) or Pvu II (RHS panel). 7~p (a gift from E. Eicher) contains a deletion from D17Leh66EII to D17Leh66D (Herrmann et al. 1987; Fig. 1), T22H (Howard et al. 1990) contains a deletion including D17Lehll91 to sequences proximal to D17Rpl7. The Taq I digested panel was hybridized with a probe from the 5' end of the plasminogen cDNA and the Pvu II panel was hybridized with a probe from the 3' end (Fig. 2A). The 5' probe recognized three wild-type bands, which were polymorphic for all the DNAs and it can be seen that rc"b2 contains all 5' wild-type bands, 2.4 kb( + ), 2.5 kb(+), and 3.3 kb(+). The 5' probe recognized four t haplotype bands which were polymorphic for all DNAs and it can be seen that one of these bands 5.3 kb (t) is absent from tLubz but the remainder are present. Tt~ contains no wild-type bands but all four t haplotype bands are present. The 3' probe recognized four wild-type bands but only the 5.0 kb (+) band was polymorphic, this was retained in tr"b2. In the t haplotype the 3' probe recognized six bands, two were polymorphic, the 2.75 kb (t) was deleted and the 4.0 kb (t) was present in tL"b2. Tt~ did not contain the polymorphic wild-type fragment 5.0 kb (+) but contained both polymorphic t haplotype fragments 2.75 kb (t) and 4.0 kb (t).
Identification o f the proximal breakpoint o f the ~ub2 deletion DNA fragments from both ends of the cosmid walk, '5.8 k b Sca I/Pvu I I ' f r o m c o s m i d 3 A (5' to t h e Plg
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Fig. 3. Physical map of cosmids enlarging the Pig locus and bordering the deletion breakpoint. A restriction map spanning 110 kb is shown containing four cosmid clones, cos3A, cos5B, cos2A and cos4A. (A) The complete plasminogen cDNA of 2.72 kb (scale above), the diagonally striped box indicates the coding region, the vertical striped box at the 3' end indicates the poly(A) tail (Degen et al. 1990). Triangles show the position of PCR primers used for amplifying the 3' end. (B) Sca I restriction map of the genomic region (scale underneath), cos3A and cos5B are overlapping clones, as are
cos2A and cos4A. The size of the region separating these two pairs of overlapping clones has been calculated as maximally 5 kb by genomic mapping (not shown). The relationship between cDNA fragments and the genomic fragments is shown. The mouse Plg genomic locus is estimated to be 43--47 kb long. The black boxes show the position of the probes that were used for mapping: 5.8 kb-Sca I/Pvu II for the 5' end of the cosmid cluster, 2.3 kb-BamH I for the 3' end of the cluster, and the 6.5 kb BamH I fragment that identifies the deletion breakpoint of tL'b2.
gene) and ' B a m H I 2.3 kb f r o m cosmid 4A (3' to the plasminogen gene) were examined for deletion from the ~ , b 2 c h r o m o s o m e (Fig. 3). The D N A end-fragment from cosmid 3A was duplicated in t L~b2 identifying b o t h wild-type and t h a p l o t y p e specific fragments (data not shown). The D N A end-fragment from cosmid 4A identified a B a m H I wild-type specific fragment ( B a m H I-2.3 kb ( + ) , Fig. 4A, lane 1) as deleted from t Lub2. The use of Taq I identified a t-specific fragment (Taq 1-2.5 kb (t), Fig. 4A, lane 10) as present in ~,b2. The absence of the wild-type fragment suggests that one end of cosmid 4A m a p s into the tLub2 deletion. The presence of the t-specific fragment provides an additional a r g u m e n t that the region containing the Plg(t) locus is present in multiple copies on the t haplotype c h r o m o s o m e (see following section; Fig. 6). The exact location of the proximal breakpoint within cosmid 4A was identified by analyzing which of the B a m H I fragments within cosmid 4A showed a size p o l y m o r p h i s m in the wild-type allele present in t L"b2. A 6.5 kb B a m H I fragment located approximately in the center of cosmid 4A (details in Fig. 3) was identified in this m a n n e r as being close to the breakpoint (Fig. 4B).
suits r e v e a l e d that the 3' end of the p l a s m i n o g e n c D N A was contained on cosmid 2A and that no part m a p s into the t L"b2 deletion (Fig. 3).
Is the p l a s m i n o g e n g e n e d e l e t e d in lLub2?
The c D N A clone MP33B lacked the last 3' 800 bp of the plasminogen c D N A . In order to localize the position of the complete c D N A with respect to the t Lub2 deletion 257 bp f r o m the e x t r e m e 3' end (Degen et al. 1990) were amplified using the P C R method. The re-
P l a s m i n o g e n e x p r e s s i o n in t h a p l o t y p e s , a n d in l Lub2 a n d t w73
T h e a b o v e data suggested that the P i g locus was present in multiple copies in t haplotypes and that the locus was very close to the t L"b2 deletion. We have analyzed plasminogen m R N A levels in these chromosomal variants to check, firstly, if the p r o p o s e d multiple P l g ( t ) loci w e r e functional and, secondly, to check if plasminogen could lie at the t w73 locus. T w o reasons p r o m p t e d us to check the possibility that plasminogen could be a candidate for the t w73 mutation. Firstly, the t w73 mutation and P l g m a p to the t Lub2 deletion and the mutant p h e n o t y p e of t w73, a failure of the blastocyst to implant after a t t a c h m e n t to the uterine endometrium (Spiegelman et al. 1976; Babiarz et al. 1982; Axelrod 1985), could be explained b y lack of a protease such as plasmin, the active f o r m of plasminogen. Secondly, although our analysis had shown that all of the plasminogen c D N A sequences were present in the t Lub2 c h r o m o s o m e , it remained formally possible that necessary regulatory sequences had b e e n deleted leading to deregulation of expression, m R N A plasminogen levels were therefore examined in wildtype t haplotype, and in two t w73 mutants (t w73 and tLub2). H o m o z y g o u s t w73 and t Lub2 e m b r y o s die at day 6 of d e v e l o p m e n t (Babiarz et al. 1982; Axelrod 1985) but direct analysis of this stage is not possible because
264
N. Schweifer and D.P. Barlow: The plasminogen locus in wild-type and t haplotype mice
of the small size of the embryo (approximately 0.1 mm in length). Therefore, we have examined plasminogen expression in the livers of adult mice heterozygous for t w73 and t Lub2. The results in Fig. 5 show that plasminogen expression in livers from strains homozygous for the t haplotype chromosome (th2tfand t h44tf) is similar to wild-type mice (BALB/c and C57BL/6). This result suggests that the multiple copies of the Plg locus in the t haplotype do not contain functional plasminogen genes. It is unlikely that the Plg locus is subject to feedback controls that maintain mRNA levels, because we have shown (Barlow et al. 1991) that in animals heterozygous for a deletion of Pig (I~P/+) the plasminogen mRNA levels are half that of control wild-type mice. When expression in tw73/+ and ~.b2/+ livers is compared to that in wild-type livers, the expression levels appear to be similar, suggesting that the Plg locus is functional in both these mutant chromosomes. Discussion
The Plg locus in the wild-type t haplotype and in the partial t haplotypes Tt ~ and t Lub2
Fig. 4. Cosmid 4A spans the proximal breakpoint of the tz"b2 deletion. (A) The distal end of cosmid 4A is deleted in t':"b2. The DNA panel shown in Fig. 2B was digested with BarnH I (LHS panel) or with Taq I (RHS panel) and both panels hybridized with fragment 2.3 k b - B a m H I from the end of cosmid 4A (Fig. 3). The B a m H I digest shows that the wild-type band [2.3 kb (+)] was polymorphic but the t-specific band [6.0 kb (t)] couId not be distinguished from the spretus band. This panel shows that the wild-type fragment was deleted from tL"b2 and Tt ~ The Taq I digest allows observation of the t-specific band and shows that this band is present in both ~ub2 and Tt TM. The absence in tr"b2 of the wild-type specific band identified by fragment 2.3 k b - B a m H I identifies cosmid 4A as spanning
The data presented in Fig. 2B shows that in the tzub2 chromosome t haplotype specific sequences at both ends of the plasminogen cDNA are both present and absent. In contrast, the wild-type specific fragments were all present. In the Tt ~ chromosome t haplotype specific sequences from both ends of the cDNA were present and the locus apparently complete; the wildtype locus was totally absent. An explanation consistent with these data is that the Plg locus is present in multiple copies in the t haplotype chromosome, and that one or more copies were deleted during the formation of the tLub2 chromosome but none were deleted during the formation of the Tt ~ chromosome. Using this information we propose a model for the organization of the Plg locus in the wild-type and the t haplotype chromosome, and show the most likely recombination event that created the partial t haplotypes Tt ~ and tLub2 (Fig. 6). The existence of a single wild-type Plg locus has been confirmed by identifying the three wild-type Taq I fragments seen in lane 7, Fig. 2B in cosmid 5B (data not shown). Additional support for the existence of multiple loci in the t haplotype comes from two sources. Firstly, in Fig. 2B, it can be seen that the size of the genomic region recognized by different parts of the plasminogen cDNA is from 50-80% larger in t haplotypes than in wild types (summarized the deletion breakpoint in this chromosome. (B) Mapping of the deletion breakpoint. The DNA panel shown in the RHS of Fig. 4A, was hybridized with a 6.5 kb B a m H I fragment from the central part of cosmid 4A. This probe recognizes a 7.8 kb ( + ) specific band, a 4.5 kb (Spe) specific band and a 2.3 kb (t) specific band. The t-specific band is present in both tLub2 and Tt ~ the wild-type specific band was absent in Tt ~ and present as a differently sized fragment of 5.0 kb (+) in tL"bz. The changed size of the wild-type band in tLub2 indicates that this Taq I fragment contains the deletion breakpoint.
N. Schweifer and D.P. Barlow: The plasminogen locus in wild-type and t haplotype mice
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Fig. 5. PlasminogenRNA analysis in wild-type t haplotype and partial t haplotype mice. Panel A shows RNA blot analysis and panel B shows a genotype analysis of various Chr 17 variants, The mice analyzed were: t~73/I~f (a complete t haplotype carrying the t~73 embryonic lethal gene), partial t haplotypes th2/t h2, lh44/l h44 (which contain the genomic region including the Pig locus in the t haplotype form), tL~b2/+t: (which contains Plg at the proximal deletion breakpoint, A, B and C are three different animals) and wild-type strains (BALB/c and C57BL/6). A blot prepared using 10 Ixgtotal cell RNA from liver was hybridized with a probe from the 3' of the plasminogen cDNA. The filter was stripped and rehybridized using the fox gene (Van Beveren et al. 1984), a constitutively expressed transcript, to quantitate the RNA loading. Actin expression in the liver
could not be used for this purpose since it was found to vary as much as 50% between different inbred wild-type strains. These results show that plasminogen mRNA levels in t haplotypes and wild-type mice are similar (compare lanes 2, 3 with lanes 4, 5), and that the chromosomes which do not contain a functioning tW73gene do not show a decrease in plasminogenmRNA levels compared to the wild type (lanes 1, 6, 7, 8 compared to lanes 2, 3). The genotype analysis in 5B used Taq I to identify a wild-type-t haplotype polymorphism using a probe containing 200 bp from the 5' end of the plasminogen cDNA MP33B. The results confirm that tw73, the and I h44 contain t haplotype specific DNA at the Plg locus and that the tL"b2heterozygotes show the expected +/t patterns as described in this report.
in Table 1). Secondly, the results in Fig. 4A show that the distal end of the cosmid cluster (defined by probe B a m H 1-2.3 kb; Fig. 3) has crossed the recombination breakpoint that generated tL"b2 but is still contained within sequences present in multiple copies in t haplotypes. This is deduced because loci such as D17Rp17 that map proximal to Pig in the wild-type c h r o m o s o m e are duplicated in t Lub2 (both t and wild-type loci are present) and loci such as Igf2-r, Tcp-1 and Sod-2, that map distal to Pig in the wild-type c h r o m o s o m e (Fig. 1), are completely deleted from t Lub2 (Sarvetnick et al. 1986; Barlow et al. 1991). A probe from the proximal end of the plasminogen cosmid cluster (5.8 kb Sac UPvu II) behaves as the D 1 7 R P I 7 locus (is duplicated) while the probe from the distal end of the cosmid cluster (2.3 kb B a m H I) recognizes only t-specific bands. The only c h r o m o s o m a l position consistent with this data is at the recombination breakpoint (Fig. 6). Continuation of the cosmid walk distal to Plg will eventually lead to sequences, like the Igf2r locus, that are completely deleted in t Lub2. Two other duplicated regions, associated with partial t h a p l o t y p e b r e a k p o i n t s , have been o b s e r v e d within the t complex region on the wild-type chromo-
some. These are the 650 kb D 1 7 L e h l 1 9 - D 1 7 L e h 6 6 E region (Herrmann et al. 1987) and the 110 kb elements in the D I 7 L e h 6 6 D region (Bullard and Schimenti 1990). In the former case an inverted duplication occurred permitting homologous recombination across inversion In(17)2. In the latter case it is proposed that the D1766D locus is contained between In(17)2 and In(17)3 (Hammer et al. 1989), and thus, is in the same orientation in both c h r o m o s o m e variants. Here we identify a duplicated region on the t haplotype chrom o s o m e that we propose also permitted homologous recombination across inversion In(17)2 and is associated with the creation of the two partial t haplotypes, Tt ~ and tLub2. It is k n o w n that Mus spretus shares In(17)2 with the t haplotypes ( H a m m e r et al. 1989), however, it is not clear from our analyses whether or not Mus spretus has multiple copies o f the Pig locus. The extra loci proposed to exist in the t haplotype do not result in an increase in steady state levels of plasminogen m R N A relative to the wild-type (Fig. 5), because data from analysis of deletion mutants (Barlow et al. 1991) does not support the existence of feedback controls; this suggests that these extra loci do not encode functioning genes.
N. Schweifer and D.P. Barlow: The plasminogen locus in wild-type and t haplotype mice
266
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