| Human Molecular Genetics | Pages |
Introduction
Results
Cloning and characterization of murine Mid1
Mid1 developmental expression analysis
Mid1 maps to the proximal PAR in Mus musculus
High frequency of unequal crossovers involving Mid1
Discordancy in the X-inactivation status of MID1 in man and mouse
Discussion
Mid1 is expressed in tissues affected in OS
Mid1 and evolution of the mammalian pseudoautosomal region
Genetic instability of the murine PAR involves Mid1
Materials And Methods
cDNA identification and sequence analysis
RNA in situ hybridization
Fluorescence in situ hybridization
Genetic mapping
Mouse X-inactivation studies
Human X-inactivation studies
Note added in proof
Acknowledgements
References
The mouse Mid1 gene: implications for the pathogenesis of Opitz syndrome and the evolution of the mammalian pseudoautosomal region
DDBJ/EMBL/GenBank accession no. Y14848
We have recently reported isolation of the gene responsible for X-linked Opitz G/BBB syndrome, a defect of midline development. MID1 is located on the distal short arm of the human X chromosome (Xp22.3) and encodes a novel member of the B box family of zinc finger proteins. We have now cloned the murine homolog of MID1 and performed preliminary expression studies during development. Mid1 expression in undifferentiated cells in the central nervous, gastrointestinal and urogenital systems suggests that abnormal cell proliferation may underlie the defect in midline development characteristic of Opitz syndrome. We have also found that Mid1 is located within the mouse pseudoautosomal region (PAR) in Mus musculus, while it seems to be X-specific in Mus spretus. Therefore, Mid1 is likely to be a recent acquisition of the M.musculus PAR. Genetic and FISH analyses also demonstrated a high frequency of unequal crossovers in the murine PAR, creating spontaneous deletion/duplication events involving Mid1. These data provide evidence for the first time that genetic instability of the PAR may affect functionally important genes. In addition, we show that MID1 is the first example of a gene subject to X-inactivation in man while escaping it in mouse. These data contribute to a better understanding of the molecular content and evolution of the rodent PAR.
INTRODUCTION
Opitz G/BBB syndrome (OS) (Mc Kusick 145410 and 300000) is an inherited disorder affecting primarily midline structures. Clinical findings in OS patients include hypertelorism, clefts of lip, palate and uvula, laryngo-trachea-oesophageal abnormalities leading to swallowing difficulty and hoarse cry, genitourinary defects such as hydronephrosis and hypospadias, imperforate anus, developmental delay and congenital heart defects (1-4). Recently we have isolated the gene responsible for the X-linked form of OS (5). This gene, MID1 (midline1), maps to the OS critical region in Xp22.3 and was found to be disrupted in affected individuals from a 20 member family in which the disease phenotype co-segregated with an X chromosome pericentric inversion with breakpoints in Xp22 and Xq26. Furthermore, preliminary mutation analysis has revealed MID1 mutations in three independent OS pedigrees (5).
MID1 encodes a putative 667 amino acid protein, containing a RING finger motif, followed by two B boxes and a coiled coil domain (5). This tripartite motif characterizes a subfamily of zinc finger proteins, called the B box family (6,7). The B box family genes encode proteins whose function is still largely unknown. Three of them, PML, RFP and Tif1[alpha], have oncogenic potential when involved in specific chromosomal translocations which result in fusion of the tripartite motif with other proteins (8-10). Another member of the B box family is the Xenopus laevis Xnf7 gene, a maternally derived transcriptional regulator involved in body axis formation during early development (11), suggesting a role of these genes in developmental processes. Recently the gene involved in familial Mediterranean fever (FMF) has been cloned (12,13) and found to contain a B box, a coiled coil and a C-terminal conserved domain, named Rfp-like. This C-terminal region, first identified in Rfp (9), was also noted in MID1 and other B box proteins (5). This motif is also present in butyrophilin, a component of bovine milk lacking all of the characteristic elements of the tripartite motif (14). The functional significance of this region has yet to be elucidated.
We report here cloning of the murine homolog of MID1 and a preliminary analysis of its expression pattern during development. During early development Mid1 is widely expressed; however, at later developmental stages highest Mid1 levels are present in proliferating cells of affected tissues in OS patients. In addition, we show that the Mid1 gene is located within the pseudoautosomal region (PAR) in Mus musculus, showing recombination between the X and Y copies. Genetic and FISH analyses also demonstrated a high frequency of unequal crossover in the murine PAR, creating spontaneous deletion/duplication events involving Mid1. Consistent with its pseudoautosomal location, Mid1 escapes X-inactivation. In contrast, human MID1 was found to be subject to X-inactivation.
RESULTS
Cloning and characterization of murine Mid1
A hybridization probe derived from the human MID1 cDNA (nt 1218-3452) was used to screen mouse embryo (E11.5) and adult brain cDNA libraries. One of the partial murine Mid1 cDNA clones recovered (ME10.2) was subsequently used to re-screen the mouse embryo cDNA library. A total of 71 primary positive clones were isolated, of which 13 were further characterized by restriction mapping, PCR and sequencing using both vector and human MID1 primers. A consensus cDNA sequence of 2670 bp was assembled (GenBank accession no. Y14848) containing a coding region of 2040 bp producing a predicted protein product of 680 residues. The murine Mid1 gene shows 87% identity at the nucleotide level and 95% identity at the amino acid level with human MID1. The only notable difference between the murine and human MID1 is the presence in some mouse cDNAs of an additional 39 nt, encoding 13 amino acids (positions 429-441). This insertion occurs in-frame at a characterized MID1 splice site, thus representing an alternatively spliced exon in murine Mid1. The stretch of 13 additional amino acids showed no significant homology to any known protein motifs.
Mid1 developmental expression analysis
Northern blot analysis showed human MID1 expression in all adult and fetal tissues analyzed. Previous whole mount expression studies showed that during early development (E9-E10.5) murine Mid1 is ubiquitously transcribed. However, Mid1 expression is higher within the frontonasal processes, the branchial arches and the central nervous system (CNS). At these stages the only tissue devoid of Mid1 expression is the developing heart (5). We now report preliminary RNA in situ hybridization studies at later stages of development.
From E12.5 to E16.5 high levels of Mid1 expression were found in the rostral part of the CNS. Mid1 is highly transcribed in the proliferating neuroepithelium of the telencephalic vesicles, while it is down-regulated in the outer differentiating fields where post-mitotic neurons are located (Fig. 1A and B). Mid1 expression in proliferating neurons is also detected in the neural retina at E16.5. At this stage the neural retina shows increased evidence of stratification, consisting of an outer nuclear layer, which contains proliferating neuroblasts, a transient anuclear layer (of Chevitz) and an inner nuclear layer in which ganglion cells are differentiating (15). High levels of Mid1 are present in the outer neuroblastic layer (Fig. 1C and D). In the eye Mid1 is also highly expressed in the anterior germinal epithelium of the lens (Fig. 1C and D). This epithelium contains dividing cells, which then move towards the equatorial region of the vesicle and begin to elongate. As this differentiation process occurs Mid1 expression is characteristically down-regulated.
Figure
Mid1 begins to be highly expressed in the developing kidney at E14.5 (Fig. 1B). By this stage aggregates from the condensed metanephric mesenchyme are being formed in the cortical region of the kidney. These aggregates undergo a mesenchyme-epithelial transformation and differentiate into nephrons. At E14.5 there are immature nephrons near the periphery of the cortex and more mature ones near the cortico-medullary border. Notably, metanephric mesenchyme-derived stem cells, which continue to produce nephrons, are present at the periphery of the renal cortex (16). At both E14.5 and E16.5 Mid1 is highly expressed in the periphery of the cortex and into the condensed mesenchymal aggregates, corresponding to immature nephrons (Fig. 1B, E and F). At E16.5 high levels of Mid1 are also detected in the epithelia of the urethra and of the two paramesonephric ducts (Fig. 1G and H). During development these ducts will form the two uterine horns and the upper part of the vagina, as well as the ovarian capsule in the female (15).
The expression pattern of Mid1 also suggests a role in development of the respiratory and digestive systems. At E14.5 Mid1 is highly expressed in the lungs (Fig. 1B). Between E15 and E16 the final stages of palatal closure occur, so that the nasopharynx is completely separated from the oropharynx. At this stage Mid1 was found to be expressed in the oropharynx (Fig. 1I), in the common entrance from the pharynx to the oesophagus and the trachea and into the larynx (data not shown). Beginning at E15.5 several modifications also occur in the gastrointestinal tract. In the mouse, by virtue of the difference in the mucosal lining, it is possible to distinguish two functionally different parts in the stomach. This `compound stomach' is formed by a cutaneous portion and a glandular region, which constitutes most of the body of the stomach (15). At E16.5 an increased degree of differentiation is observed in the glandular portion of the stomach. Differentiation of the gut proceeds in a rostral-caudal mode, from the small intestine to the large intestine. In fact, early evidence of differentiation with the first indication of villi and intestinal glands is already present at E15.5 in the duodenum and in the midgut (15). At E16.5 Mid1 is highly expressed in the mucosa of the cutaneous region of the stomach and of the hindgut (data not shown), while it is down-regulated in the glandular region of the stomach and in the epithelium of the villi of the small intestine (Fig. 1L and M). In the small intestine Mid1 expression appears to be restricted to the submucosa and to the region where crypts, containing regenerating cells, will develop (Fig. 1L and M).
Mid1 maps to the proximal PAR in Mus musculus
Sequence analysis of Mid1 revealed that DXYRp1, a 215 bp C57BL/6 genomic probe, constitutes part of the murine Mid1 genomic locus (17). DXYRp1 contains nt 1687-1878 of the Mid1 cDNA and an additional 23 bp of divergent sequence. This divergent sequence is assumed to be intronic, as nt 1878 in murine Mid1 corresponds to a characterized splice site in human MID1 (data not shown). DXYRp1 has previously been shown to map to the C57BL/6 PAR but to be absent from the Mus spretus genome (17). Consequently, Mid1 was also expected to map to the PAR in C57BL/6. Fluorescence in situ hybridization (FISH) and genetic mapping experiments were therefore performed to confirm the Mid1 map position in the C57BL/6 PAR and to ascertain the presence and genomic localization of Mid1 in other strains/species of mouse.
Metaphase chromosomes from 11 C57BL/6J male mice were hybridized in situ using probe ME10.2 containing the 3[prime]-region of the Mid1 cDNA. The results are summarized in Table 1. Signals on the X and Y chromosomes were recorded as single signals (one dot on each chromatid) or double signals (two or more dots on each chromatid). All mice showed positive hybridization to both the X and Y chromosomes, although individual mice differed in the distribution of signals. Mice 1-6 and 10 show a fairly equal distribution of signals between the sex chromosomes with a large number of double signals on both sex chromosomes, suggesting that the gene may be duplicated on those chromosomes. However, mice 7-9 and 11 have a clearly unequal distribution of signals, with mice 7 and 11 showing an apparent duplication of the gene only on the Y chromosome and mice 8 and 9 an apparent duplication only on the X chromosome (Fig. 2). FISH analysis of one M.musculus musculus from Skive (from now on referred to as Skive) male, using a Mid1 probe corresponding to the 3[prime]-region (ME10.2), revealed signals on both sex chromosomes but with strong double signals on the X chromosome. Hybridization of the 3[prime] probe (ME10.2) to an M.spretus male showed no apparent signals, whereas hybridization using a 5[prime] probe (ME5A) showed weak signals at the distal end of the X chromosome and no apparent signals on the Y chromosome (data not shown).
Figure
Both the 5[prime] and 3[prime] Mid1 probes demonstrated a PvuII restriction fragment length variation (RFLV) between C57BL/6 and M.spretus. This PvuII RFLV was exploited to perform genetic mapping analysis of the Mid1 locus using the Jackson BSS backcross panel (18). Haplotype analysis of the segregating C57BL/6 allele placed the Mid1 gene telomeric of Amel in the distal region of the X chromosome. Furthermore, Mid1 co-segregated with Clc4 and Sts in all 96 backcross progeny (these data were submitted to the Jackson web site). Sts maps to the PAR in M.musculus (19), while Clc4 maps to the distal X chromosome in M.spretus but to chromosome 7 in C57BL/6 (20,21). The 3[prime] Mid1 probe hybridized strongly to C57BL/6-derived genomic fragments but extended exposure also revealed weak hybridization to M.spretus-derived fragments. However, the 5[prime] Mid1 probe hybridized to C57BL/6- and M.spretus-derived bands with equal intensity (data not shown).
Table 1
In order to refine Mid1 map position the segregation of Mid1 alleles was analyzed in 54 progeny of the Skive backcross, using probe ME10.2. To obtain this backcross C57BL/6J females were mated with Skive males and the resulting F1 male hybrids were mated with C57BL/10 females. Backcrossing to C57BL/10 allows haplotype analysis of both the C57BL/6J- andSkive-derived alleles in the backcross progeny. This cross has been typed for a number of pseudoautosomal loci, including DXYRp1, DXYRp3, DXYHgu1 and DXYHrb6 (17,22-24). Segregation of the C57BL/6J and Skive Mid1 alleles is presented separately (Fig. 3A and B). In fact, attempts to combine the two maps by minimizing the number of crossovers indicated that the relative loci order within the PAR was not conserved between C57BL/6J and Skive (Fig. 3). This is likely to be an artifact of the high level of multiple recombination events known to occur in the murine PAR (24-26). The order of DXYRp1 and DXYRp3is consistent with other data (V.Chapman, R.Elliott, I.Kalcheva and C.-H.Yen, unpublished data). Furthermore, the PAR4 probe, which corresponds to the DXYHgu locus, detects multiple copies (24). Thus it is not surprising to obtain a copy of DXYHgudistal of Mid1 in C57BL/6 and a second that is proximal to Mid1 in Skive. These copies are referred to as DXYHgu1 and DXYHgu2 respectively. Mid1 was seen to map to the proximal PAR in both M.musculus strains. However, probe ME10.2 also hybridized to non-variant fragments, which we were not able to type.
Figure
Mouse
X chromosome signals
Y chromosome signals
Singlea
Doubleb
Single
Double
1
15% (20)c
85% (20)
68% (19)
32% (19)
2
59% (32)
41% (32)
75% (24)
25% (24)
3
44% (25)
56% (25)
25% (24)
75% (24)
4
13% (23)
87% (23)
26% (19)
74% (19)
5
26% (19)
74% (19)
37% (19)
63% (19)
6
55% (11)
45% (11)
17% (12)
83% (12)
7
94% (16)
6% (16)
42% (26)
58% (26)
8
4% (25)
96% (25)
95% (20)
5% (20)
9
55% (20)
45% (20)
100% (14)
0% (14)
10
83% (12)
17% (12)
35% (17)
65% (17)
11
71% (14)
29% (14)
27% (22)
73% (22)
High frequency of unequal crossovers involving Mid1
Of the 54 backcross progeny analyzed, paternal recombination within the PAR was detected in 11 (Table 2). Analysis of the segregation of Skive alleles suggests that recombination has occurred between Mid1 and DXYHrb6 in four animals (3, 8, 20 and 32), and close to the pseudoautosomal boundary in animal 26 (Table 2). Female progeny 36 lacked paternally derived alleles for all loci analyzed, suggesting that this entire region, including the Mid1 locus, is deleted on the X chromosome derived from this parent. Thus animal 36 is monosomic for the Mid1 locus. Five animals (27, 28, 34, 40 and 45) appear to have paternally transmitted duplications of Mid1, resulting in trisomy for the Mid1 locus. This is consistent with the unequal FISH signals on the X and Y chromosomes of different C57BL/6J mice (see above).
Table 2
| Progeny | Sex | C57BL/6 alleles | M.m.musculus Skive alleles | Conclusion | ||||
| Mid1 | DXYHgu1 | DXYRp3 | DXYHgu2 | Mid1 | DXYHrb6 | |||
| 3 | F | + | + | + | - | - | + | NR |
| 20 | F | + | + | + | - | - | + | NR |
| 27 | F | + | + | + | - | + | + | Duplication |
| 28 | F | + | + | + | - | + | + | Duplication |
| 36 | F | - | - | - | - | - | - | Deletion |
| 45 | F | + | + | - | + | + | + | Duplication |
| 8 | M | - | - | - | + | + | - | NR |
| 26 | M | + | + | + | - | - | - | NR |
| 32 | M | - | - | - | + | + | - | NR |
| 34 | M | + | + | + | + | + | + | Duplication |
| 40 | M | + | + | + | + | + | + | Duplication |
Discordancy in the X-inactivation status of MID1 in man and mouse
As pseudoautosomal genes are represented on both the X and Y chromosomes, there is no requirement for dosage compensation between the sexes and, consequently, pseudoautosomal genes are observed to escape X-inactivation (27). To determine whether Mid1 escapes X-inactivation in mouse an RT-PCR analysis was done on cell lines derived from F1 female mice, obtained by crossing a laboratory strain of mouse deficient in Hprt (hypoxanthine-phosphoribosyl transferase) and M.spretus. Cell lines were grown either in 6-thioguanine, to select for cells with an active laboratory strain X chromosome (28), or in HAT, to select for cells with an inactive laboratory strain X chromosome. In addition, Mid1 expression was also examined in tissues from T(X;16)16H × M.spretus female mice, in which the M.spretus X chromosome is always inactive (29), and from parental strains. Mid1 was found to be expressed in the two cell lines with opposite X-inactivation patterns using primers for the 3[prime]-end of the gene (Fig. 4A), indicating that the gene does escape X-inactivation. PCR reactions using RT minus samples were negative, indicating that there was no DNA contamination. A control Smcx gene was found to be expressed in all cell lines and tissues, indicating that the RNA was amplifiable (Fig. 4A).
Figure
The X-inactivation status of human MID1 was tested by RT-PCR of somatic cell hybrids retaining either an active or inactive human X chromosome (30). MID1 expression is seen in three independent somatic cell hybrids carrying an active X chromosome, but not in any of the five independent hybrids carrying an inactive X chromosome, demonstrating that MID1 is subject to X-inactivation (Fig. 4B). Control RT-PCR experiments using primers derived from SMCX, a gene known to escape X-inactivation (31), were performed in parallel to ensure that all of the somatic cell hybrid samples contained amplifiable cDNA.
DISCUSSION
Mid1 is expressed in tissues affected in OS
We present preliminary data on the expression pattern of Mid1 during mouse development. Although Mid1 appears to be transcribed in most tissues during early development (5), high levels of expression were found at later stages in the central nervous system, in the mucosa of the oropharynx, oesophagus, trachea and larynx, in the stomach, in the gut and in the urogenital system. Patterning of all these tissues is abnormal in OS patients. OS is characterized by defective fusion of midline structures (1-4). Abnormal medio-lateral patterning of the orbits causes hypertelorism, a defect in closure of the facial and pharyngeal processes underlies the labio-palatoschisis and the abnormalities of the trachea/oesophagus, and defective fusion of urethral folds produces hypospadias. However, very little is known about the molecular mechanisms which govern these processes during normal development. Furthermore, little inference on the role of MID1 can be made from the finding that the gene encodes a novel member of the B box family of zinc finger proteins. In fact, this family comprises molecules with very different functions, which are generally thought to be indirect transcriptional regulators (6,7).
In many tissues Mid1 expression is confined to proliferating cells. In the brain Mid1 is up-regulated in the ventricular zone, where proliferating neurons are located, while it is excluded in terminally differentiated neurons. Similarly, at E16.5 Mid1 is expressed in the inner neuroblastic layer of the neural retina, which contains proliferating undifferentiated neurons. An analogous situation occurs in organs of ectodermal, endodermal and mesodermal origin. At E16.5 Mid1 is expressed in the anterior epithelium of the lens, which contains proliferating cells, while it is excluded from the posterior epithelium, where crystallin-producing, differentiated cells are located. In the gut down-regulation of Mid1 expression parallels terminal differentiation of the mucosa. In the kidney Mid1 expression is restricted to the cortical region, where stem cells and mesenchymal aggregates, forming immature nephrons, are located. Taken together, these data raise the possibility that the midline defects observed in OS patients may be due to abnormal cell proliferation in affected tissue during development.
Mid1 and evolution of the mammalian pseudoautosomal region
Genetic and FISH mapping data, performed using a probe corresponding to the 3[prime]-region of the cDNA, indicated that the murine Mid1 gene maps to the proximal PAR. The PAR is a region of identity between the X and Y chromosomes, necessary to allow pairing and chiasma formation during male meiosis and subsequent proper segregation (32,33). An obligatory crossover event occurs within this region in any functional male meiotic division and this may account for its high evolutionary divergence in eutherian mammals (25,34). Although the size and structure of the human PAR is well established, very little information is available on the rodent PAR. To date no markers from the human PAR have been shown to have mouse pseudoautosomal homologs, or vice versa, suggesting that there is complete divergence between the PAR of mice and humans (33). The only gene so far isolated from the murine PAR is Sts (19). This gene shows atypically low sequence homology with its human ortholog (63% identity at the nucleotide level and 59% at the amino acid level). The human STS gene is located in the X-specific region of Xp22.3, has a non-functional Y homolog and escapes X-inactivation. These features are shared by other human genes located in distal Xp, such as ARSD, ARSE and KAL, leading to the hypothesis that this region of the human X chromosome was originally part of an extended ancestral PAR which was disrupted by a pericentric inversion on the Y chromosome recently in evolution (35-38). The occurrence of chromosomal rearrangements has been considered an important aspect of PAR evolution in different mammalian species. The majority of available data indicates that there is a reduction in the size of this region during evolution (39). The autosomal localization of the murine Csfgmr[alpha] and Il3r[alpha] genes, which are pseudoautosomal in human, may be regarded as further evidence (40,41).
Mid1 is the second gene to be isolated from the M.musculus PAR. We estimate that Mid1 resides in the proximal 20% of the PAR, assuming an expected genetic length for this region of at least 50 map units. However, until a physical map of the PAR becomes available it is not possible to establish the actual locus order in the PAR. We postulate that MID1 was X-linked in a common progenitor of rodent and primate lineages and has recently been acquired by the M.musculus PAR. Consistent with this, Mid1, unlike Sts, still displays very high sequence homology with its human counterpart. Furthermore, human MID1, in contrast to most genes in Xp22.3, has no detectable Y homolog and is subject to X-inactivation, so that it is unlikely to have once been part of an ancestral PAR. These data would provide the first evidence that mammalian PARs are susceptible not only to reductions but also to increases in size and gene content. Future mapping studies of Mid1 in non-eutherian mammals and primates will help clarify this issue.
The picture is further complicated by mapping inconsistencies in different Mus species. In fact, notable differences in the content of the distal X chromosome between M.musculus and M.spretus are becoming apparent. These were first demonstrated for the Clc4 gene, which maps to the distal M.spretus X chromosome, but to chromosome 7 in C57BL/6 (20,21). Our preliminary results suggest that the 3[prime]-region of the Mid1 gene might have been lost or very poorly conserved in M.spretus and that the gene may be X-linked rather than pseudoautosomal in M.spretus. Further studies are necessary to confirm these findings. Remarkably, Clc4 and Mid1 are very close to each other in human and we may postulate that complex rearrangements moved Clc4 to chromosome 7 and Mid1 to the PAR in inbred strains.
We showed that human MID1 is subject to X-inactivation. In contrast, as expected for a pseudoautosomal gene, Mid1 was found to escape X-inactivation in M.musculus. Although discrepancies in the X-inactivation status between human and murine genes have already been documented (27), Mid1 provides the first evidence of a gene escaping X-inactivation in the mouse while being inactivated in human.
Genetic instability of the murine PAR involves Mid1
High evolutionary divergence in the PAR is seen not only among different mammalian species but also among and within Mus species. Structural variation of the PAR in inbred strains has been demonstrated using several pseudoautosomal markers (17,24). In many cases polymorphisms were detected between animals of the same inbred strain, suggesting an unusually high mutation rate. Pseudoautosomal PacI fragments were shown to be unstable in C57BL/6 × C57BL/6 crosses, with new alleles occurring at a sex averaged rate of ~30% per allele (26). This genetic variation has been attributed to misalignment and unequal crossovers during meiosis of repetitive elements in the PAR (26). It has been suggested that genetic divergence in the X-Y pairing region, leading to unsuccessful male meiosis, may explain hybrid sterility in the heterogametic sex (Haldane's rule), which contributes to the establishment of reproductive barriers between species during the process of speciation (42-46). Although at least two mouse hybrid sterility loci have been mapped close to or within the PAR, it has been proposed that long range X-Y structural incompatibility within the PAR, rather than a particular gene locus, could be responsible for hybrid sterility (20,21,26,46,47).
We now provide evidence for the first time that the high genetic instability of the PAR may involve a disease gene. Genetic mapping has shown that a high frequency of unequal crossovers occurs in the murine PAR, resulting in spontaneous deletion/duplication events involving Mid1. Furthermore, the finding that four out of 11 C57BL/6 mice analyzed by FISH had unequal intensity of Mid1 signal on either the X or the Y chromosome is further evidence of the frequent occurrence of non-homologous recombination in the PAR involving Mid1. A more detailed analysis of the Mid1 genomic locus will be needed to determine whether Mid1 is indeed duplicated on some of the X and Y chromosomes examined, as FISH experiments suggest, and whether this duplication involves the entire gene or only part of it. We cannot establish at this point the exact number of Mid1 copies, their relative arrangements and their functional status. Furthermore, it is still possible that a small part of the gene is X-specific. Unfortunately, the progeny of the Skive backcross, whose DNA was used for genetic mapping, are not available anymore, precluding phenotypic analysis of animals trisomic or monosomic for Mid1. This will be the subject of future studies. It may be possible, theoretically, to obtain null mutants for Mid1 by simply crossing mice monosomic for the gene, provided that they are fertile.
In conclusion, our results indicate that interspecies and intraspecies structural instability of the mouse PAR is reflected not only in changes in the physical map of the region, but in altered copy number of functionally important genes. The isolation of Mid1 from the M.musculus PAR provides a valuable reference point for constructing a comparative map of this region and may contribute to understanding of the molecular mechanisms underlying evolution of this region of the mammalian genome, inter-hybrid sterility and the speciation process.
MATERIALS AND METHODS
cDNA identification and sequence analysis
Mouse brain (Stratagene) and E11.5 total embryo (Clontech) cDNA libraries were screened using standard techniques (48). cDNA sequence analysis and database searches were performed as previously described (49). Data on similarity/identity were obtained using the PileUp program of the Wisconsin GCG software package, v.8.1.
RNA in situ hybridization
A fragment corresponding to nt 1947-2086 of the full-length Mid1 cDNA was subcloned into pBSSK- and linearized with appropriate restriction enzymes to transcribe either sense or antisense 35S-labeled riboprobes. Mouse embryo tissue sections were prepared and RNA in situ hybridization experiments were performed as previously described (50). Autoradiographs were exposed for 2 days. Slides were then dipped in Kodak NTB2 emulsion and exposed for 14-21 days. Micrographs are double exposures: red represents the in situ hybridization signal and blue shows the nuclei stained with Hoechst 33258 dye.
Fluorescence in situ hybridization
Chromosome preparations were obtained from 11 C57BL/6J male mice, from one M.spretus and from one M.m.musculus Skive. Metaphase cells were collected from short term spleen cultures as described previously (51). Mid1 gene probes ME10.2 (nt 972-2486) and ME5A (nt 1-1048) were labeled with biotin. Hybridization, probe detection and chromosome identification by banding were done as previously described (51).
Genetic mapping
Filters containing PvuII-digested DNA from progeny derived from the Jackson BSS backcross (18) were purchased from Jackson Laboratories. Hybridizations were performed using standard protocols (48) with the Mid1 probes ME5A and ME10.2 (see above).
Table 3
| Locus | Probe | Enzyme | Fragment sizea (kb) | Probe reference |
| DXYRp1 | B6-38 | PstI | 2.8 (B6) 2; 2.5 (B10) |
Kalcheva et al., 1995 (17) |
| DXYRp3 | pYS1N2.5 | BglII | 3; 7 (B6)| Yen and Elliott, 1997 (22) |
|
| DXYHgu1 | PAR4 | StuI | 1.7; 3.1 (B6) | Kipling et al., 1996 (24) |
| DXYHgu2 | PAR4 | StuI | 3.3 (Sk) | Kipling et al., 1996 (24) |
| DXYHrb6 | p15-4 | SstI | 3.5 (Sk) | Harbers et al., 1986 (23) |
| Mid1 | ME10.2 | PstI | 2.8 (B6) 13 (Sk) 2; 2.5 (B10) |
this study |
Mice were purchased from Jackson Laboratories or bred in the colony at Roswell Park Cancer Institute. Female C57BL/6J mice were mated with M.m.musculus Skive males (52) and the resulting male F1 hybrids were mated to C57BL/10J females. This cross is designated the Skive cross. Liver, spleen and kidney were dissected from the backcross progeny and DNA was isolated (53). Table 3 illustrates loci with corresponding probes for which the Skive backcross has been typed. Sizes of variant fragments are also listed. The data were entered into the Map Manager Classic Program (54).
Mouse X-inactivation studies
Expression of Mid1 was determined on cell lines Hombski and Patski and on T(X:16)16H translocation mouse tissues. Cell line Hombski and T(X;16) mice contain an active laboratory strain X chromosome and an inactive M.spretus X chromosome (28,29). Cell line Patski contains an inactive laboratory strain X chromosome and an active M.spretus X chromosome in all cells, as shown by allelic analysis of Rps4 (ribosomal protein S4 gene) (data not shown). Cytogenetic analysis showed that the Patski cell line contains two X chromosomes of normal appearance.
RNA was extracted from the cell lines and from the mouse tissues using an Ultraspec RNA isolation kit from Biotecx. All RNA samples were treated with DNase I to remove contaminating DNA. Control reactions without reverse transcriptase were done to ensure that there was no DNA contamination. Reverse transcription was carried out using random primers and standard methodology. PCR reactions were set up using primers to amplify the 5[prime]- and 3[prime]-ends of Mid1 and to amplify a control gene, Smcx, which escapes X-inactivation (31,55). Two rounds of amplification using nested primers were used for Mid1. The first set of primers was 5[prime]-GCTCAGCAGATTGCAAACTG-3[prime] and 5[prime]-CTCTTCTTAGAGGACGAGTC-3[prime] and the second set 5[prime]-CTCGCTGAAGGAAAATGACC-3[prime] and 5[prime]-TACTTGGTGCCACTTTGCAG-3[prime]. To amplify Smcx the primers 5[prime]-CTGAGGAAGTGACTGCTCTG-3[prime] and 5[prime]-AGGACTGGTCACACTGTCCC-3[prime] were used. Reaction conditions were 30 cycles of 1 min at 94°C, 1 min at 55°C and 1 min at 72°C. Products of amplification were separated on 2% agarose.
Human X-inactivation studies
RNA preparations and reverse transcription reactions were performed as described (30). SMCX was amplified with primers 1 (5[prime]-ACCTGAGG AGCCTCCTAACT-3[prime]) and 2 (5[prime]-CAGTCAACTGTGGCAACAGCG-3[prime]) (31) as described (30) for 30 cycles with a 55°C annealing temperature. MID1 was amplified with primers MID-6F (5[prime]-CTGTGTGACCGATGACCAGT-3[prime]) and MID-11R (5[prime]-AGTTTGCGAAGCCTCATCA-3[prime]) for 35 cycles with a 58°C annealing temperature.
Note added in proof
During the revision of this manuscript, Palmer et al. independently reported the isolation of the murine Mid1 gene, which they refer to as Fxy [Palmer,S., Perry,J., Kipling,D. and Ashworth,A. (1997) A gene spans the pseudoautosomal boundary in mice. Proc. Natl Acad. Sci. USA, 94, 12030-12035]. They found that the gene spans the pseudoautosomal boundary, with the first three exons being X-linked, and the last exons pseudoautosomal. Their result is consistent with our findings, since we performed genetic mapping using a cDNA probe containing exons 5-11 which are all located within the pseudoautosomal region.
ACKNOWLEDGEMENTS
We thank Cristina Ghezzi, Silvia Messali and Sara Volorio for helpful technical assistance, Germana Meroni and Sophia Colamarino for critical reading of the manuscript and Melissa Smith for manuscript preparation. This work was supported by the Italian Telethon Foundation, NIH grant GM33160 to R.E., NIH grant GM46883 to C.D. and NIH grant GM45441 to H.F.W. N.A.Q. is supported by a Marie Curie post-doctoral fellowship (ERBFMBICT960649) from the EC.
REFERENCES
*To whom correspondence should be addressed. Tel: +39 2 2156 0233; Fax: +39 2 2156 0220; Email: rugarli@tigem.it
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+The first two authors contributed equally to the work
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