Chromosome engineering: generation of mono- and dicentric isochromosomes in a somatic cell hybrid system

The most common isochromosome found in humans involves the long arm of the X, i(Xq), and is associated with a subset of Turner syndrome cases. To study the formation and behavior of isochromosomes in a more tractable experimental system, we have developed a somatic cell hybrid model system that allows for the selection of mono- or dicentric isochromosomes involving the short arm of the X, i(Xp). Simultaneous positive and negative counterselection of a mouse/human somatic cell hybrid containing a human X chromosome, selecting for retention of the UBE1 locus in Xp but against the HPRT locus in Xq, results in a variety of abnormalities of the X chromosome involving deletions of Xq. We have generated 70 such ”Pushmi-Pullyu” hybrids derived from seven independent X chromosomes. Cytogenetic analysis of these hybrids using fluorescence in situ hybridization showed i(Xp) chromosomes in ∼19% of the hybrids. Southern blot and polymerase chain reaction analyses of the Pushmi-Pullyu hybrids revealed a distribution of breakpoints along Xq. The distance between the centromeres of the dicentric i(Xp)s generated ranged from ∼2 Mb to ∼20 Mb. To examine centromeric activity in these dicentric i(Xp)s, we used indirect immunofluorescence with antibodies to centromere protein E (CENP-E). CENP-E was detected at only one of the centromeres of a dicentric i(Xp) with ∼2–3 Mb of Xq DNA. In contrast, CENP-E was detected at both centromeres of a dicentric i(Xp) with ∼14 Mb of Xq DNA. Two other dicentric i(Xp) chromosomes were heterogeneous with respect to centromeric activity, suggesting that centromeric activity and chromosome stability of dicentric chromosomes may be more complicated than previously thought. The Pushmi-Pullyu model system presented in this study may provide a tool for examining the structure and function of mammalian centromeres. Received: 15 December 1998; in revised form: 2 March 1999 / Accepted: 5 April 1999     

A possible active segment on the inactive human X chromosome

An idic(Xp-) in which the two X chromosomes are attached short arm to short arm, and which thus has two b regions (the Q-dark segment next to the centromere on Xp) between the inactivation centers, assumed to be situated on the Q-dark region next to the centromere on Xq, showed 63.8% bipartite Barr bodies as compared with 22.2% formed by idic(Xq-). In addition, the mean distance of the two parts of the Barr bodies in the fibroblasts of a patient with idic(Xp-) is significantly greater than in the cases with one or no b region. Contrary to the other patients with abnormal X chromosomes, the buccal cells of a woman idic(Xp-) showed a number of bipartite Barr bodies. — To explain these observations we have put forward the hypothesis that the b region on the Xp always remains active and thus, when the rest of the chromosome forms a Barr body, this segment is extended, allowing the two parts of the X chromatin to get farther apart and at the same time increasing the percentage of bipartite bodies.     

Heterozygous expression of X-linked chondrodysplasia punctata

Summary Two females showing partial expression of X-linked chondrodysplasia punctata were identified in a family. Bone dysplasia was caused by an aberrant X chromosome that had an inverse duplication of the segment Xp21.2–Xp22.2 and a deletion of Xp22.3-Xpter. To characterise the aberrant X chromosome, dosage blots were performed on genomic DNA from a carrier using a number of X-linked probes. Anonymous sequences from Xp21.2–Xp22.2 to which probes D2, 99.61, C7, pERT87-15, and 754 bind were duplicated on the aberrant X chromosome. The proposita was heterozygous for all these markers. Dosage blots also showed that the loci for steroid sulfatase and the cell surface antigen 12E7 (MIC2) were deleted as expected from the cytogenetic results. Mouse human cell hybrids were constructed that retained the normal X in the active state. Analysis of these hybrid clones for the markers from Xp21.2–Xp22.2 revealed that all the alleles of the informative markers, present in a single dosage in the genomic DNA, were carried on the normal X chromosome of the proposita. The duplicated X chromosome therefore had two identical alleles, indicating that the aberration resulted from an intrachromosomal rearrangement.     

Localization of the gene for Wieacker-Wolff syndrome in the pericentromeric region of the X chromosome

The Wieacker-Wolff syndrome (WWS, MIM* 314580), first described clinically in 1985, is an X-linked recessive disorder. In earlier studies, linkage between the WWS gene and DXYS1 at Xq21.2 and DXS1 at Xq11 as well as AR at Xq12 was reported. Here we report on a linkage analysis using highly polymorphic, short terminal repeat markers located in the segment from Xp21 to Xq24. No recombination between the WWS locus and ALAS2 or with AR (z = 4.890 at θ = 0.0) was found. Therefore, the WWS locus was assigned to a segment of approximately 8 cM between PFC (Xp11.3–Xp 11.23) and DXS339 (Xq11.2–Xq13). Received: 14 March 1997 / Accepted: 9 April 1997     

Assignment of the Nance-Horan syndrome to the distal short arm of the X chromosome

Summary There are three types of X-linked cataracts recorded in Mendelian Inheritance in Man (McKusick 1988): congenital total, with posterior sutural opacities in heterozygotes: congenital, with microcornea or slight microphthalmia; and the cataract-dental syndrome or Nance-Horan (NH) syndrome. To identify a DNA marker close to the gene responsible for the NH syndrome, linkage analysis on 36 members in a three-generation pedigree including seven affected males and nine carrier females was performed using 31 DNA markers. A LOD score of 1.662 at 0=0.16 was obtained with probe 782 from locus DXS85 on Xp22.2–p22.3. Negative LOD scores were found at six loci on the short arm, one distal to DXS85, five proximal, and six probes spanning the long arm were highly negative. These results make the assignment of the locus for NH to the distal end of the short arm of the X chromosome likely.     

Exclusion mapping of the gene for X-linked neural tube defects in an Icelandic family

Various polymorphic markers with a random distribution along the X chromosome were used in a linkage analysis performed on a family with apparently Xlinked recessive inheritance of neural tube defects (NTD). The lod score values were used to generate an exclusion map of the X chromosome; this showed that the responsible gene was probably not located in the middle part of Xp or in the distal region of Xq. A further refining of these results was achieved by haplotype analysis, which indicated that the gene for X-linked NTD was located either within Xp21.1-pter, distal from the DMD locus, or in the region Xq12–q24 between DXS106 and DXS424. Multipoint linkage analysis revealed that the likelihood for gene location is highest for the region on Xp. The region Xq26–q28, which has syntenic homology with the segment of the murine X chromosome carrying the locus for bent tail (Bn), a mouse model for X-linked NTD, is excluded as the location for the gene underlying X-linked NTD in the present family. Thus, the human homologue of the Bn gene and the present defective gene are not identical, suggesting that more than one gene on the X chromosome plays a role in the development of the neural tube.     

An interstitial duplication of the X chromosome in a male allows physical fine mapping of probes from the Xq13-q22 region

Summary An insertional translocation into the proximal long arm of the X chromosome in a boy showing muscular hypotony, growth retardation, psychomotor retardation, cryptorchidism, and Pelizaeus-Merzbacher disease (PMD) was identified as a duplication of the Xq21–q22 segment by employing DNA probes. With densitometric scanning for quantitation of hybridization signals, 15 Xq probes were assigned to the duplicated region. Analysis of the duplication allowed us to dissect the X-Y homologous region physically at Xq21 and to refine the assignments of the loci for DXYS5, DXYS12, DXYS13, DXS94, DXS95, DXS96, DXS111, and DXS211. Furthermore, we demonstrated the presence of two different DXYS13, and DXS17 alleles in genomic DNA of our patient, suggesting that the duplication resulted from a meiotic recombination event involving the two maternal X chromosomes.     

Two type II keratin genes are localized on human chromosome 12

Summary Human genomic DNA containing two type II keratin genes, one coding for keratin 1 (K1, a 68-kD basic protein) and another closely linked type II gene 10–15 kb upstream (K?, gene product unknown), was isolated on a single cosmid clone. EcoRI restriction fragments of the cosmid were subcloned into pGEM-3Z, and specific probes comprising the C-terminal coding and 3 noncoding regions of the two genes were constructed. The type II keratin genes were localized by in situ hybridization of the subcloned probes to normal human lymphocyte chromosomes. In a total of 70 chromosome spreads hybridized with the K? probe (gHK?-3, PstI, 800 bp), 36 of the 105 grains observed were on chromosome 12, and 32 of these were clustered on the long arm near the centromere (12q11–13). In 100 labeled metaphases hybridized with the K1 probe (gHK1–3, BamHI-PstI, 2100 bp), 53 grains localized to chromosome 12 and 46 of these were found in the same region (q11–13). Therefore, both the gene for human keratin 1, a specific marker for terminal differentiation in mammalian epidermis, and another closely linked unknown type II keratin gene (K?, 10–15 kb upstream of K1) are on the long arm (q11–13) of human chromosome 12.     

Localization of G6PD and HPRT to different arms of the X chromosome of the North American marsupial (Didelphis virginiana) by in situ hybridization and delection mapping: Evolutionary significance

We have mapped HPRT and G6PD loci on the X chromosome in the American opossum, Didelphis virginiana, by in situ hybridization to cells derived from two females by using genomic opossum DNA as probes. The localizations (G6PD to Xp13 and HPRT to Xq21), indicating that the two genes are separated by the centromere, were confirmed by results of hybridization to X chromosomes with deletions that include the HPRT locus and opossum-mouse cell hybrids containing the relevant fragment of the opossum X chromosome.     

The Candidate Sex-ReversingDAX1Gene Is Autosomal in Marsupials: Implications for the Evolution of Sex Determination in Mammals

The human X-linkedDAX1gene was cloned from the region of the short arm of the human X found in duplicate in sex-reversed X_dupY females (E. Zanariaet al.,1994,Nature372: 635–641).DAX1is suggested to be required for ovarian differentiation and to play an important role in mammalian sex determination or differentiation pathways. Its proposed dose-dependent effect on sexual development suggests thatDAX1could represent an evolutionary link with an ancestral sex-determining mechanism that depended on the dosage of an X-linked gene. Furthermore,DAX1could also represent the putative X-linked switch gene, which independently controls sexual dimorphisms in marsupial mammals in an X-dose-dependent manner (D. W. Cooperet al.,1993,Semin. Dev.4: 117–128). IfDAX1has a present role in marsupial sexual differentiation or had an ancestral role in mammalian sex determination, it would be expected to lie on the marsupial X chromosome, despite the autosomal localization of other human Xp genes. We therefore cloned and mapped theDAX1gene in the tammar wallaby (Macropus eugenii).DAX1was located on wallaby chromosome 5p near other human Xp genes, indicating that it was originally autosomal and that it is not involved in X-linked dose-dependent sex determination in an ancestral mammal nor in marsupial sexual differentiation.     

Molecular definition of breakpoints associated with human Xq isochromosomes: implications for mechanisms of formation.

To test the centromere misdivision model of isochromosome formation, we have defined the breakpoints of cytogenetically monocentric and dicentric Xq isochromosomes (i(Xq)) from Turner syndrome probands, using FISH with cosmids and YACs derived from a contig spanning proximal Xp. Seven different pericentromeric breakpoints were identified, with 10 of 11 of the i(Xq)s containing varying amounts of material from Xp. Only one of the eight cytogenetically monocentric i(Xq)s demonstrated a single alpha-satellite (DXZ1) signal, consistent with classical models involving centromere misdivision. The remaining seven were inconsistent with such a model and had breakpoints that spanned proximal Xp11.21: one was between DXZ1 and the most proximal marker, ZXDA; one occurred between the duplicated genes, ZXDA and ZXDB; two were approximately 2 Mb from DXZ1; two were adjacent to ALAS2 located 3.5 Mb from DXZ1; and the largest had a breakpoint just distal to DXS1013E, indicating the inclusion of 8 Mb of Xp DNA between centromeres. The three cytologically dicentric i(Xq)s had breakpoints distal to DXS423E in Xp11.22 and therefore contained > or = 12 Mb of DNA between centromeres. These data demonstrate that the majority of breakpoints resulting in i(Xq) formation are in band Xp11.2 and not in the centromere itself. Therefore, we hypothesize that the predominant mechanism of i(Xq) formation involves sequences in the proximal short arm that are prone to breakage and reunion events between sister chromatids or homologous X chromosomes.     

Replication variants of the human inactive X chromosome

Replication variants of the inactive X chromosome were investigated in lymphocytes from six donors by means of terminal BrdU or thymidine incorporation. There were interindividual differences in the incidence of particular variants. In endoreduplicated and tetraploid cells both allocyclic X chromosomes showed the same replication sequence. The Xp22 band of the allocyclic X chromosome seemed to replicate later than the homologous material in some cells. Initiation time of DNA synthesis within the inactive X chromosome was found to be stable; termination time, however, varied greatly relative to the other chromosomes. Early completion of replication within the heterochromatic X chromosome could be demonstrated preferentially for the Xq25–27 terminal sequence, but other variants expressed the phenomenon also. A variable replication rate of the inactive X chromosome is believed to be responsible for its asynchronous, independent replication. The biological significance of the phenomenon is discussed with respect to cell differentiation.     

Molecular definition of Xq common-deleted region in patients affected by premature ovarian failure

High-resolution cytogenetic analysis of a large number of women with premature ovarian failure (POF) identified six patients carrying different Xq chromosome rearrangements. The patients (one familial and five sporadic cases) were negative for Turner's stigmata and experienced a variable onset of menopause. Microsatellite analysis and fluorescent in situ hybridization (FISH) were used to define the origin and precise extension of the Xq anomalies. All of the patients had a Xq chromosome deletion as the common chromosomal abnormality, which was the only event in three cases and was associated with partial Xp or 9p trisomies in the remaining three. Two of the Xq chromosome deletions were terminal with breakpoints at Xq26.2 and Xq21.2, and one interstitial with breakpoints at Xq23 and Xq28. In all three cases, the del(X)s retained Xp and Xq specific telomeric sequences. One patient carries a psu dic(X) with the deletion at Xq22.2 or Xq22.3; the other two [carrying (X;X) and (X;9) unbalanced translocations, respectively] showed terminal deletions with the breakpoint at Xq22 within the DIAPH2 gene. Furthermore, the rearranged X chromosomes were almost totally inactivated, and the extent of the Xq deletions did not correlate with the timing of POF. In agreement with previous results, these findings suggest that the deletion of a restricted Xq region may be responsible for the POF phenotype. Our analysis indicates that this region extends from approximately Xq26.2 (between markers DXS8074 and HIGMI) to Xq28 (between markers DXS 1113 and ALD) and covers approximately 22 Mb of DNA. These data may provide a starting point for the identification of the gene(s) responsible for ovarian development and folliculogenesis.     

The Localization of a Gene Causing X-Linked Cleft Palate and Ankyloglossia (CPX) in an Icelandic Kindred Is between DXS326 and DXYS1X

The locus responsible for X-linked, nonsyndromic cleft palate and/or ankyloglossia (CPX) has previously been mapped to the proximal long arm of the human X chromosome between Xq21.31 and q21.33 in an Icelandic kindred. We have extended these studies by analyzing an additional 14 informative markers in the family as well as including several newly investigated family members. Recombination analysis indicates that the CPX locus is more proximal than previously thought, within the interval Xq21.1-q21.31. Two recombinants place DXYS1X as the distal flanking marker, while one recombinant defines DXS326 as the proximal flanking marker, an interval of less than 5 cM. Each of the flanking markers recombines with the CPX locus, giving 2-point lod scores of Z_max = 4.16 at θ = 0.08 (DXS326) and Z_max = 5.80 at θ = 0.06 (DXYS1X).     

The similarity of phenotypic effects caused by Xp and Xq deletions in the human female: a hypothesis

Summary We have collected from the literature adult nonmosaic women with the following aberrant X chromosomes: Xp- (52), Xq- (67), idic(Xp-)(10), idic(Xq-)(9), and interstitial deletions (12). Lack of Xp, and especially Xcen-Xp11 (b region), may cause full-blown Turner syndrome. However, individual Turner symptoms, including gonadal dysgenesis, otherwise seem to be randomly distributed with respect to the different Xp and Xq deletions, although breakpoints distal to Xq25 do not give rise to any phenotypic anomalies except in a few cases of secondary amenorrhea or premature menopause. Of the carriers of an Xp- or Xq- chromosome, 65% and 93%, respectively, suffer from ovarian dysgenesis, whereas all idic(Xp-) and idic(Xq-) chromosomes cause primary or secondary amenorrhea. Xq deletions do not induce specific symptoms different from those caused by Xp deletions. Lack of the tip of Xp has led in 46/52 cases to short stature, but 43% of the Xq- carriers are also short. To explain these observations, we propose the following hypothesis. Since deletions of truly inactivated regions do not seem to cause any symptoms, we assume that the b region (Xcen-p11) always stays active in a normal inactive X, but is inactivated in deleted X chromosomes, especially in Xq- chromosomes. In some cases, inactivation may spread to the tip of Xp; this would explain the apparently variable behavior of the Xg and STS genes, and the short stature of some Xq- carriers. Full chromosome pairing seems to be a prerequisite for the viability of oocytes and thus for gonadal development. Deleted X chromosomes necessarily leave a portion of the normal X unpaired and isodicentrics probably interfere with pairing, resulting in atresia of oocytes. The role played by the critical region (Xq13–q24) in ovarian development is still unclear.     

Deletion (X)(q26.1-->q28) in a proband and her mother: molecular characterization and phenotypic-karyotypic deductions.

During a routine prenatal diagnosis we detected a female fetus with an apparent terminal deletion of an X chromosome with a karyotype 46,X,del(X)(q25); the mother, who later underwent premature ovarian failure, had the same Xq deletion. To further delineate this familial X deletion and to determine whether the deletion was truly terminal or, rather, interstitial (retaining a portion of the terminal Xq28), we used a combination of fluorescence in situ hybridization (FISH) and Southern analyses. RFLP analyses and dosage estimation by densitometry were performed with a panel of nine probes (DXS3, DXS17, DXS11, DXS42, DXS86, DXS144E, DXS105, DXS304, and DXS52) that span the region Xq21 to subtelomeric Xq28. We detected a deletion involving the five probes spanning Xq26-Xq28. FISH with a cosmid probe (CLH 128) that defined Xq28 provided further evidence of a deletion in that region. Analysis with the X chromosome-specific cocktail probes spanning Xpter-qter showed hybridization signal all along the abnormal X, excluding the possibility of a cryptic translocation. However, sequential FISH with the X alpha-satellite probe DXZ1 and a probe for total human telomeres showed the presence of telomeres on both the normal and deleted X chromosomes. From the molecular and FISH analyses we interpret the deletion in this family as 46,X,del(X) (pter-->q26::qter). In light of previous phenotypic-karyotypic correlations, it can be deduced that this region contains a locus responsible for ovarian maintenance.     

Linkage analysis of two cloned DNA sequences flanking the Duchenne muscular dystrophy locus on the short arm of the human X chromosome.

The inheritance of two restriction fragment length polymorphisms (RFLPs) on the short arm of the human X chromosome has been studied relative to Duchenne muscular dystrophy. This provides a partial genetic map of the short arm of the human X chromosome between Xp110 and Xp223. The data were derived from the segregation between a RFLP located at Xp21-Xp223, the DMD locus, and a RFLP located at Xp110-Xp113. The genetic distance from Xp110 to Xp223 was found to be approximately 40 centimorgans (cM). This provides experimental confirmation that 1cM corresponds to approximately 1,000 kilobase pairs of DNA for this region of the human X chromosome. Our data confirm that the DMD mutation lies between Xp223 and Xp110. The availability of flanking probes surrounding the DMD locus will assist in the ordering of further DNA sequences relative to the mutation.     

Human and rat chromosomal localization of two genes for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by analysis of somatic cell hybrids and in situ hybridization

Two genes encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were localized in human and rat chromosomes. PFKFB1 (previously PFRX), which encodes the liver and muscle isozymes, was assigned to Xq22-q31 in the rat and to Xq27–q28 in the human by in situ hybridization using probes generated by the polymerase chain reaction. PFKFB2, which encodes the heart isozyme of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was assigned to chromosome 13 in the rat and to chromosome 1 in the human by hybridization of DNA from somatic cell hybrids. By in situ hybridization, this gene was localized to the regions 13q24–25 in the rat and 1q31 in the human.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

Salivary gland X chromosome puffing patterns are described for the Oregon stock of Drosophila melanogaster and for the Berkeley stock of D. simulans. In D. melanogaster regular phase specific puffing was recorded at 21 loci in the third larval instar and subsequent prepupal stage. A comparison of the X chromosome puffing patterns of male and female larvae failed to show any qualitative differences although in the males a group of puffs were active for a longer time during development than in females. The X chromosome puffing patterns of D. simulans are similar to those described for D. melanogaster although two puffs (4F 1–4 and 7B 1–3) were active in D. simulans but not in D. melanogaster. The sex differences in puffing observed in D. melanogaster were also observed in D. simulans.     

Enrichment of Triadic and Terminal Cisternae Vesicles from Rabbit Skeletal Muscle

An enriched triad and terminal cisternae preparation was achieved from skeletal muscle through alterations of the differential centrifugation and muscle homogenization protocols. Both yield and specific activity (pmoles of radioligand binding per mg protein) were optimized for ³H-PN200-l10 (transverse tubule marker) and ³H-ryanodine (terminal cisternae marker) binding sites. By pelleting crude microsomes between 2,000 an 12,000 × g without any rehomogenizations, we improved both the yield and specific activity of transverse tubule and terminal cisternae markers in crude microsomes by approximately 4-fold to 1000–3000 pmoles binding sites (starting material: approximately 400 grams wet weight fast twitch skeletal muscle), with 10–15 pmoles/mg. Rehomogenization of the 1,000 × g pellet, which is typically discarded, allowed recovery of an additional 5000 pmoles PN200-110 binding sites and an additional 8000 pmoles ryanodine binding sites. Crude microsomes from the rehomogenized 1,000 × g pellets typically displayed specific activities of 20–25 pmoles binding/mg for both ³H-PN200-110 and ³H-ryanodine. Separation of crude microsomes on a sucrose gradient increased specific activity up to a maximum of 50 pmoles/mg in a specific fraction, a five- to ten-fold increase over standard triadic or terminal cisternae preparations. The mean specific activity for enriched triads was 30–40 pmoles/mg for both PN200-110 and ryanodine in pooled fractions, while pooled fractions of enriched terminal cisternae displayed low ³H-PN200-110 binding (3–5 pmoles/mg) and high ³H-ryanodine-specific activity (30–40 pmoles/mg).