Biomedical subjects
P Avner
Publications and source records attributed to P Avner.
Genetics. Sweet mice, sugar daddies.
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Characterization of a murine gene expressed from the inactive X chromosome.
In mammals, equal dosage of gene products encoded by the X chromosome in male and female cells is achieved by X inactivation. Although X-chromosome inactivation represents the most extensive example known of long range cis gene regulation, the mechanism by which thousands of genes on only one of a pair of identical chromosomes are turned off is poorly understood. We have recently identified a human gene (XIST) exclusively expressed from the inactive X chromosome. Here we report the isolation and characterization of its murine homologue (Xist) which localizes to the mouse X inactivation centre region and is the first murine gene found to be expressed from the inactive X chromosome. Nucleotide sequence analysis indicates that Xist may be associated with a protein product. The similar map positions and expression patterns for Xist in mouse and man suggest that this gene may have a role in X inactivation.
Human variable number of tandem repeat probes as a source of polymorphic markers in experimental animals.
Human VNTR (Variable Number of Tandem Repeat) markers are examined as a source of polymorphism for linkage studies in inbred strains of mice and rats. High frequencies of cross-hybridization are found under fingerprinting conditions that detect many distinct minisatellite loci in these species. Linkage studies suggest that minisatellite markers are widely distributed in the mouse genome, in contrast to humans where they are clustered, particularly in telomeric regions. Human VNTR probes can be used to screen in mouse genomic libraries to isolate mouse specific VNTR sequences. Some of these sequences reveal fingerprint patterns under stringent hybridization conditions.
Adaptation of the interspersed repetitive sequence polymerase chain reaction to the isolation of mouse DNA probes from somatic cell hybrids on a hamster background.
A strategy for the rapid isolation of DNA probes from radiation-fusion Chinese hamster cell hybrids containing overlapping portions of the murine X chromosome based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) previously used with human somatic cell hybrids has been developed. This specific amplification of mouse DNA on a hamster background depends on the use of primers directed to the B2 short interspersed repeat element family and the R repeat, from the long interspersed repeat element family, L1. Two sets of amplification conditions, which gave specific amplification of mouse DNA from either a mouse X-monochromosomal hybrid or irradiation-fusion hybrids having reduced X content, were defined. The mouse X-only chromosome hybrid yielded approximately 20 discrete reproducible bands, while the irradiation-fusion hybrids yielded between 1 and 10 discrete products. Comparison of different irradiation-fusion hybrids has allowed the definition of both specific and shared products corresponding to different regions within the overlapping X-chromosome fragments present within these hybrids. Use of such hybrids and the IRS-PCR technique has allowed the isolation of probes corresponding to the central region of the mouse X chromosome that contains the X-inactivation center. The method should be widely applicable to the isolation of mouse DNA sequences from mouse hybrid cell lines on either human or Chinese hamster backgrounds.
Genetic and molecular evidence of an X-chromosome deletion spanning the tabby (Ta) and testicular feminization (Tfm) loci in the mouse.
A new radiation-induced mutation in the mouse, tabby-25H (Ta25H), has proved to be a deletion which spans both the tabby and testicular feminization (Tfm) loci on the X chromosome. The Ta phenotype closely resembles that of the original TaFa mutation in both the heterozygous and hemizygous conditions but Ta25H/Y animals additionally show the Tfm/Y phenotype, being externally female but possessing abdominally located testes. There is a shortage of both Ta25H/+ and Ta25H/Y classes relative to their normal sibs among the progeny of Ta25H/+ females at weaning age and this was indicated to be due to prenatal or neonatal losses. Exencephaly was observed in some members of both classes prior to birth. Both Ta25H classes tend to be runted at weaning but, remarkably, Ta25H/+ females often show a range of abnormalities not evident in Ta25H/Y animals. When probes for the Zfx, Ccg-1, Phk, and DXPas19 loci, which lie close to Ta, were hybridised to DNAs from Ta25H hemizygotes, the profiles of the X-linked bands were similar to those of control DNAs, suggesting these loci lie outside the deletion. However, a clear absence of an X-linked band was found with human androgen receptor probes, indicating that the Tfm locus is indeed missing. The deletion, therefore, extends a minimum of 1.5 cM and, with its proximal and distal boundaries partially defined, it could be as large as 4 cM. As Ta25H/+ females show the striped X-inactivation coat pattern, the putative X-inactivation centre, Xce, which lies close to Ta, cannot be located within the region deleted. The greasy (Gs) locus similarly appears to lie outside the deletion.
Minisatellite linkage maps in the mouse by cross-hybridization with human probes containing tandem repeats.
Tests of 29 human variable number of tandem repeat probes in inbred mouse lines showed that 80% (23/29) cross-hybridize, and 48% (14/29) produce multiple band, minisatellite polymorphisms (fingerprint patterns). Mini-satellite-type polymorphisms detected by 11 probes were characterized in eight different strains; on average, 240 polymorphic differences were detected between pairs of strains. Reproducible fingerprint patterns permit the study of the segregation of the minisatellite polymorphisms in experimentally designed crosses. As an example, we constructed primary minisatellite genetic linkage maps containing 346 polymorphic bands, distributed in 101 groups of closely linked systems, from genotypes on a recombinant inbred panel (C57BL/6J X DBA/2J); 38 of the groups were assigned by linkage to 15 autosomal chromosomes. The minisatellite genetic maps of C57BL/6J and DBA/2J can be applied in other linkage studies involving these strains.
The XLR sequence family: dispersion on the X and Y chromosomes of a large set of closely related sequences, most of which are pseudogenes.
The XLR sequence family encodes RNA transcripts specific to late-stage T and B cells and their neoplasms. Only one apparently functional mRNA has been identified thus far and this encodes a novel 25 kDa nuclear protein. In this report, we find that the XLR gene family is composed of 50-75 copies per haploid genome which localize to at least two different portions of the mouse X chromosome. Neither of these locations are near the xid mutation that earlier work had correlated with XLR. In addition, some members of this family are also on the Y chromosome. Another surprising finding is that while the fourteen genomic clones examined to date have the same exon-intron structure and are closely related with respect to sequence conservation (90%), all appear (in most cases by multiple criteria) to be non-functional, raising the possibility that all but one of the members of this large semi-dispersed family are pseudogenes.
Localization of murine X and autosomal sequences homologous to the human Y located testis-determining region.
Recently a candidate gene for the primary testis-determining factor (TDF) encoding a zinc finger protein (ZFY) has been cloned from the human Y chromosome. A highly homologous X-linked copy has also been identified. Using this human sequence it is possible to identify two Y loci, an X and an autosomal locus in the mouse (Zfy-1, Zfy-2, Zfx and Zfa, respectively). Suprisingly ZFY is more homologous to the mouse X and autosomal sequences than it is to either of the Y-linked loci. Both Zfy-1 and Zfy-2 are present in the Sxr region of the Y but Zfy-2 is absent in the Sxr deletion variant Sxrb (or Sxr") suggesting it is not necessary for male determination. Extensive backcross analyses map Zfa to mouse chromosome 10 and Zfx to a 5-cM interval between anonymous X probe MDXS120 and the tabby locus (Ta). We also show that the mouse androgen receptor locus (m-AR) believed to underlie the testicular feminization mutation (Tfm) shows complete linkage to Zfx. Comparative mapping indicates that in man these genes lie in separate conserved DNA segments.
Molecular cloning and sequencing of a murine pgk-1 pseudogene family.
Seven genomic mouse DNA fragments carrying pgk-1-homologous regions have been cloned and sequenced. They have to be classified as processed genes because intervening sequences, present in their productive counterpart, are absent. Four pseudogenes (I-IV) represent nearly the complete sequence of pgk-1 cDNA. Two of these genes (I and II), although rather different from the published mouse pgk-1 cDNA in the 3'-untranslated region, represent the actual mouse pgk-1 cDNA sequence in the coding part except for substitutions in the third position of three codons. These genes can code for a functional PGK protein but, lacking as they do classical promoter structures, are probably not expressed. They show the typical characteristics of retroposons, being flanked by A-rich regions and direct repeats which are localized at the positions where the homology with the mouse pgk-1 cDNA is interrupted. Pseudogenes III and IV have numerous mutations. Gene III is also flanked by direct repeats, whereas gene IV is flanked by inverted repeats. The other three genes are flanked by direct repeats localized further inside the target sites. They are truncated and mutated extensively as usually observed with pseudogenes.
Genetic analysis of the mouse using interspecific crosses.
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Conservation and reorganization of loci on the mammalian X chromosome: a molecular framework for the identification of homologous subchromosomal regions in man and mouse.
By means of cross-reacting molecular probes, some 18 loci specific for the X chromosome of both man and mouse have been localized on the mouse X chromosome using an interspecific mouse cross involving the inbred SPE/Pas strain derived from Mus spretus. Comparison of the localizations of these loci on the mouse X with their positions on the human X chromosome suggests that intrachromosomal rearrangements involving at least five X chromosome breakage events must have occurred during the period of evolutionary divergence separating primates from rodents. Within the five blocks of chromosomal material so defined, there is for the moment little or no evidence that either chromosomal inversion events or extensive rearrangements have occurred. These data confirm the remarkable evolutionary conservation of the X chromosome apparent in mammalian species, compared to autosomal synteny groups in which both inter- and intrachromosomal rearrangement events appear to have occurred frequently. The breakage events described here for the X chromosome should therefore provide a minimal estimate for the frequency of chromosomal rearrangement events, such as breakage and inversion, which have affected autosomal synteny groups during the evolutionary period separating man from mouse. The definition of the number of chromosome breakage events by which the X chromosomes of these species differ, together with their localization, provides a framework for the use of interspecies mouse crosses for further detailed mapping of particular subchromosomal regions of the human X chromosome and for defining loci in the mouse homologous to those implicated in human congenital diseases.
A-raf oncogene localizes on mouse X chromosome to region some 10-17 centimorgans proximal to hypoxanthine phosphoribosyltransferase gene.
The localization of the A-raf cellular oncogene on the mouse X chromosome has been determined using Xbal-restricted DNAs prepared from progeny of an interspecies backcross between the B6.CBA.R1 and the Spe/Pas mouse strains. This localization to the proximal part of the mouse X chromosome has been confirmed by the use of somatic cell hybrids, carrying partially deleted X chromosomes and suggests that the A-raf oncogene localizes to a region lying some 10-17 centimorgans proximal to the hypoxanthine phosphoribosyltransferase (Hprt) gene between the locus DXPas4 and the locus DXPas7 defined by the cross-reacting human X chromosome-specific probe DXS32 (M2C). This localization on the mouse X chromosome is compatible with the presence of the A-raf oncogene on the short arm of the human X chromosome between the centromere and Xp21.
Characterization of a panel of somatic cell hybrids for regional mapping of the mouse X chromosome.
A panel of five hybrid cell lines containing mouse X chromosomes with various deletions has been obtained by fusing splenocytes from male mice carrying one of a series of reciprocal X-autosome translocations with the azaguanine-resistant Chinese hamster cell line CH3g. These hybrids have been extensively characterized by using the allozymes hypoxanthine/guanine phosphoribosyltransferase (encoded by the Hprt locus) and alpha-galactosidase (Ags) and a series of 11 X-chromosome-specific DNA probes whose localization had been previously established by linkage studies. Such studies have established the genetic breakpoints of the T(X;12)13Rl and T(X;2)14Rl X-autosome translocations on the X chromosome and provided additional information as to the X-chromosome genetic breakpoints of the T(X;16)16H, T(X;4)7Rl, and T(X;7)6Rl translocations. The data establish clearly that both the T(X;4)7Rl and T(X;12)13Rl X-chromosome breakpoints are proximal to Hprt, the breakpoint of the former being more centromeric, lying as it does in the 9-centimorgan interval between the ornithine transcarbamoylase (Otc) and DXPas7 (M2C) loci. Similarly, it is now clear that the T(X;16)16H X-autosome translocation breakpoint lies distal to the DXPas8 (St14-1) locus, narrowing the X-chromosome breakpoint down to a region flanked proximally by this marker and representing, as expected from previous data, the distal quarter of the Hprt-Ta subchromosomal span. These five hybrid cell lines provide, with the previously characterized EBS4 hybrid cell line, a nested series of seven mapping intervals distributed along the length of the mouse X chromosome. Their characterization not only allows further correlation of the genetic and cytological X-chromosome maps but also should permit the rapid identification of DNA probes specific for particular regions of the mouse X chromosome.
Detailed ordering of markers localizing to the Xq26-Xqter region of the human X chromosome by the use of an interspecific Mus spretus mouse cross.
Five probes localizing to the Xq26-Xqter region of the human X chromosome have been genetically mapped on the mouse X chromosome using an interspecific cross involving Mus spretus to a contiguous region lying proximally to the Tabby (Ta) locus. Pedigree and recombinational analysis establish the marker order as being Hprt-FIX-c11-G6PD-St14-1. The size of this contiguous region is such that the X-linked muscular dystrophy (mdx) mouse mutation probably maps within this segment. This in turn suggests that it is highly improbable that the mouse mdx locus represents a model for Duchenne muscular dystrophy (DMD). It is, however, compatible with the idea that this mutation may correspond in man to Emery Dreifuss muscular dystrophy. The high frequency of restriction fragment length polymorphisms found in this interspecific system for all the human cross-reacting probes examined up until now, using only a limited number of restriction enzymes, suggests that the Mus spretus mapping system may be of great potential value for establishing the linkage relationships existing in man when conserved chromosomal regions are concerned and human/mouse cross-reacting probes are available or can be obtained.
Mapping the mouse X chromosome: possible symmetry in the location of a family of sequences on the mouse X and Y chromosomes.
Major advances in our knowledge of the genetic organization of the mouse X chromosome have been obtained by the use of interspecific crosses involving Mus spretus-derived strains. This system has been used to study sequences detected by three probes 80Y/B, 302Y/B and 371Y/B isolated from a mouse Y-chromosome library which have been shown to recognize both male-female common and male-female differential sequences. These patterns are due to the presence of a family of cross-reacting sequences on the mouse X and Y chromosomes. Detailed genetic analysis of the localization of the X-chromosome-specific sequences using both a somatic cell hybrid panel and an interspecific mouse cross has revealed the presence of at least three discrete clusters of loci (X-Y)A, (X-Y)B and (X-Y)C. Two of these clusters, (X-Y)B and (X-Y)C, lie distally on the mouse X chromosome, the other cluster (X-Y)A being situated close to the centromere. In situ hybridization shows a striking symmetry in the localization of the major sequences on both the X and Y chromosomes detected by these probes, hybridization being preferentially localized to a subcentromeric and subtelomeric region on each chromosome. This striking localization symmetry between the X and Y chromosome sequences is discussed in terms of the extensive pairing of the X-Y chromosomes noted during meiosis.