Large deletions and other gross forms of chromosome imbalance compatible with viability and fertility in the mouse.

Large deletions and other gross forms of chromosome imbalance are known in man but have rarely been found in the mouse. By screening progeny of spermatogonially irradiated male mice for a combination of runting and other phenotypic effects, we have identified animals that have large deletions comprising from 2.5-30 percent of the length of individual chromosomes, or other major chromosome changes, which are compatible with viability and fertility. Certain chromosome regions appear particularly susceptible to the generation of viable deletions and this has implications for radiation mutagenesis studies. Correlations with human deletions are also indicated.

T(In1;5)44H, a complex mouse chromosomal rearrangement with a phenotypic effect.

A complex murine chromosomal rearrangement, T(In1;5)44H, was recovered after 5 Gy + 5 Gy (given 24 h apart) spermatogonial X-irradiation. T44H is a paracentric inversion of most of Chromosome (Chr) 1 (1A1-1H6), followed by splitting of the inverted segment through a reciprocal translocation with Chr 5, the latter breakpoints being in 1C2 and 5F. Linkage tests have shown that the probable order on Chr 1 is fz-ln-T44H with 2.4 +/- 2.4 crossover units between ln and T44H. On Chr 5 the probable order is W-T44H-go-bf with 7.1 +/- 4.9 crossover units between T44H and go. All heterozygotes show a marked dilution of coat colour. Heterozygotes of both sexes are fertile, producing small litters with a marked shortage of T44H carriers. The number of live embryos produced from female carriers is significantly lower than from males. Despite the complex nature of the rearrangement, complete chromosome pairing and chiasma formation occur regularly at meiosis. Depending on the strands involved, this leads to the production of either one or two dicentric chromatids per spermatocyte, and their disjunctional fate can be followed into metaphase II. Analysis of chromatid classes at this stage suggests reasons for both the high embryonic mortality and the shortage of liveborn T44H carriers.

A candidate mouse model for Prader-Willi syndrome which shows an absence of Snrpn expression.

The best examples of imprinting in humans are provided by the Angelman and Prader-Willi syndromes (AS and PWS) which are associated with maternal and paternal 15q11-13 deletions, respectively, and also with paternal and maternal disomy 15. The region of the deletions has homology with a central part of mouse chromosome 7, incompletely tested for imprinting effects. Here, we report that maternal duplication for this region causes a murine imprinting effect which may correspond to PWS. Paternal duplication was not associated with any detectable effect that might correspond with AS. Gene expression studies established that Snrpn is not expressed in mice with the maternal duplication and suggest that the closely-linked Gabrb-3 locus is not subject to imprinting. Finally, an additional new imprinting effect is described.

Adenosine deaminase, Ada, is in mouse chromosome 2H3, and is not allelic with wasted, wst.

The adenosine deaminase locus (Ada) in the mouse has been localized by in situ hybridization to band 2H3. Linkage analysis of backcross data has shown that Ada is 13.8 +/- 2.7 cM from the coat texture mutant, ragged, Ra. From the results of earlier work (Abbott, C. M., et al., Proc. Natl. Acad. Sci. USA 83:693, 1986), it had been suggested that wst was a low-activity allele of Ada, but this cannot be so because Ada and wst have been found to be nonallelic.

Isolation and characterization of a cDNA clone corresponding to the mouse t-complex gene Tcp-1x.

The mouse t complex on chromosome 17 is known to harbour many genes which have an important role in spermatogenesis. One of these, Tcp-1 has been cloned and shown to code for a protein probably essential for acrosome formation. During the isolation of a cDNA for Tcp-1 two other homologous sequences were recognized and described as Tcp-1x and Tcp-1y. In this paper we describe the isolation of a cDNA which has been shown by in situ hybridization to correspond to the Tcp-1x gene. Sequence analysis has confirmed that a 140 bp region of homology between Tcp-1 and Tcp-1x lies in the 3' portion of both genes. Northern blotting has revealed that the Tcp-1x gene is expressed abundantly in liver where two transcripts are detectable and hybrid selection shows that the gene codes for a 37 kDa protein. A search of the DNA database has failed to find any significant homology between Tcp-1x and any other sequences apart from Tcp-1.

Localization of the properdin factor complement locus Pfc to band A3 on the mouse X chromosome.

The locus for properdin (properdin factor complement, Pfc), a plasma glycoprotein, has been mapped to band A3 of the mouse X chromosome by in situ hybridization to metaphase spreads containing an X;2 Robertsonian translocation. The X-linkage of the locus has also been confirmed by analysis of Mus musculus x Mus spretus interspecific crosses. The XA3 localization for Pfc places it in the chromosomal segment conserved between man and mouse which is known to contain at least six other homologous loci (Cybb, Otc, Syn-1 Maoa, Araf, Timp).

Illegitimate pairing of the X and Y chromosomes in Sxr mice.

X/Y male mice carrying the sex reversal factor, Sxr, on their Y chromosomes typically produce 4 classes of progeny (recombinant X/X Sxr male male and X/Y non-Sxr male male, and non-recombinant X/X female female and X/Y Sxr male male) in equal frequencies, these deriving from obligatory crossing over between the chromatids of the X and Y during meiosis. Here we show that X/Y males that, exceptionally, carry Sxr on their X chromosome, rather than their Y, produce fewer recombinants than expected. Cytological studies confirmed that X-Y univalence is frequent (58%) at diakinesis as in X/Y Sxr males, but among those cells with X-Y bivalents only 38% showed normal X-Y pseudo-autosomal pairing. The majority of such cells (62%) instead showed an illegitimate pairing between the short arms of the Y and the Sxr region located at the distal end of the X, and this can be understood in terms of the known homology between the testis-determining region of the Y short arm and that of the Sxr region. This pairing was sufficiently tenacious to suggest that crossing over took place between the 2 regions, and misalignment and unequal exchange were suggested by indications of bivalent asymmetry. Metaphase II cells deriving from meiosis I divisions in which the normal X-Y exchange had not occurred were also found. The cytological data are therefore consistent with the breeding results and suggest that normal pseudo-autosomal pairing and crossing over is not a prerequisite for functional germ cell formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and expression of a new mouse homeobox gene.

A homeobox-containing clone has been isolated from an adult mouse kidney cDNA library and shown by DNA sequence analysis to be a new isolate, Hox-6.1. A genomic clone containing Hox-6.1 has been isolated and found to contain another putative homeobox sequence (Hox-6.2), within 7 kb of Hox-6.1. In situ hybridization of mouse metaphase chromosomes shows this Hox-6 locus to be located on chromosome 14 (14E2). Hox-6.1 has been studied in detail and the predicted protein sequence of the homeobox is 100% homologous to the Xenopus Xeb1 (formally AC1) homeobox and the human c8 homeobox (Carrasco et al. 1984; Boncinelli et al. 1985; Simeone et al. 1987). Southern blotting shows that the DNA sequence encoding Hox-6.1 is single copy. Expression of Hox-6.1 has been studied in adult tissues and embryos by RNase protection assays, Northern blotting analysis and in situ hybridization. RNase protection assays show that Hox-6.1 transcripts are present in embryos between days 9 1/2 and 13 1/2 of gestation and in extraembryonic tissues at day 9 1/2. Adult expression is detectable in kidney and testis but not in liver, spleen and brain. One major transcript is detectable on Northern blots of kidney and day-13 1/2 embryo RNA. In kidney, this transcript is 2.7 kb whereas in embryos the major transcript is smaller at 1.9 kb, a much fainter band being visible at 2.7 kb. Localized expression of Hox-6.1 is observed in the spinal cord and prevertebral column of day-12 1/2 embryos, and in the posterior mesoderm and ectoderm of day-8 1/4 embryos. An anterior boundary of expression is located just behind the hindbrain whereas the boundary in the mesoderm is located at the level of the 7th prevertebra.

Extent of the mouse t complex and its inversions shown by in situ hybridization.

Probes for loci situated near one end of the proximal (Tcp-1) and distal (Qa-2, 3) inversions of the mouse t complex have been hybridized to chromosomes of mice with and without t complexes and with morphologically distinguishable chromosome 17s. Both the probe for Tcp-1 and that for Qa-2, 3 hybridized to clearly different positions on t and non-t chromosomes, thus making visible the extent of the two inversions. The proximal inversion extends from roughly the junction of bands A1 and A2 to band A3, and the distal inversion from band A3 to band C. Thus, the whole t complex extends from the band A1-A2 junction to band C, and is therefore somewhat larger than previously thought, and occupies about 1.2% of the genome. A probe for complement component 3 (C3-1), genetically known to be several cM distal to the t complex, was found by in situ hybridization to lie in band E1. The proximal part of chromosome 17 is one of the best known parts of the mouse genome, at both the genetic and molecular levels. It may soon be possible to correlate the length of the t complex in terms of chromosomal distance with its physical length in megabases.

Localization of the Hprt locus by in situ hybridization and distribution of loci on the mouse X-chromosome.

The hypoxanthine phosphoribosyltransferase locus (Hprt) of the mouse has been localized by in situ hybridization to band XA6. Comparison of the distributions of known loci on the genetic and cytogenetic maps of the X-chromosome suggests some chiasma localization with a relatively high frequency of chiasmata in the F bands. In the A bands there appear to be fewer known loci than expected, but no evidence has been found so far of excessive chiasma formation.

Lack of inactivation of a mouse X-linked gene physically separated from the inactivation centre.

Previous evidence had shown that, when a mammalian X-chromosome is broken by a translocation, only one of the two X-chromosome segments shows cytological signs of X-inactivation in the form of late replication or Kanda staining. In the two mouse X-autosome translocations T(X;4)37H and T(X;11)38H the X-chromosome break is in the A1-A2 bands; in both, the shorter translocation product fails to exhibit Kanda staining. By in situ hybridization, the locus of ornithine carbamoyltransferase (OCT) was shown to be proximal to the breakpoint (i.e. on the short product) in T37H and distal to the breakpoint in T38H. Histochemical staining for OCT showed that in T38H the locus of OCT undergoes random inactivation, as in a chromosomally normal animal, whereas in T37H the OCT locus remains active in all cells. The interpretation is that, when a segment of X-chromosome is physically separated from the X-inactivation centre, it fails to undergo inactivation. This point is important for the understanding of the mechanism of X-inactivation, since it implies that inactivation is a positive process, brought about by some event that travels along the chromosome. It is also relevant to the interpretation of the harmful effects of X-autosome translocations and the abnormalities seen in individuals carrying such translocations.

Clonal analysis of X-chromosome inactivation and the origin of the germ line in the mouse embryo.

Cloning of cells from peri-implantation embryos by blastocyst injection was used to investigate the time of X-chromosome inactivation in that part of the ectoderm lineage giving rise to foetal tissues of the mouse. Matings were arranged so that the two X-chromosomes of female donor cells controlled two distinct coat colours and host blastocysts were of a third colour genotype. No coat chimaeras were obtained in experiments using donor cells from the primitive ectoderm of 6th or 7th day embryos or from lactationally delayed implanting or reactivated blastocysts. In contrast, a minimum of 80 unequivocal coat chimaeras were obtained in experiments in which primitive ectoderm cells from 5th day implanting blastocysts were used for injection. The majority of these chimaeras that had received a female cell exhibited both donor colours in addition to host colour in their coats, suggesting that the donor cell had not undergone X-inactivation until one or more cycles after transplantation. The remainder of such chimaeras exhibited only one or other donor coat colour. Determination of the parental origin of the allocyclic X-chromosome in donor metaphase preparations in internal tissues of several chimaeras revealed that the coat pattern did not always reflect the X-activity status of the donor cell clone as a whole. Nevertheless, the findings suggest that X-inactivation takes place shortly after implantation in the primitive ectoderm cell population from which the foetus is derived. Of the 68 chimaeras in which the sex of both the donor and host component was established 62 proved to be fertile. Furthermore, 21 of the 37 fertile chimaeras whose sex corresponded with that of the donor cell yielded functional gametes of donor origin. Injection of cells from a single donor blastocyst into a series of host blastocysts established that at least 2 cells in 5th day primitive ectoderm can give rise to both somatic cells and functional germ cells among their mitotic descendants.

Chromosomal sex and distribution of functional germ cells in a series of chimeric mice.

An unselected series of chimeric mice were test mated to determine the parental lineage of their functional gametes. The cytological sex of each animal was established and confirmed in all cases by karyological analysis of peripheral blood lymphocytes. The parental cell lineage for each cytological sex was unequivocally established by the presence or absence of the radiation-induced translocation 15(14) (T6). Eleven animals analysed, 10 of these were chimeric. Among the 10 chimeras, 3 were phenotypically female and 7 were phenotypically male. The cytological sex ratio (XY/XY:XX/XY:XX/XX) was 1:6:2. There were 646 offspring from test matings of these chimeras. The coat color analysis of these offspring demonstrated a concordance of cytological sex of the lineage resulting in functional gametes with the phenotypic sex of the animal.

A foreign beta-globin gene in transgenic mice: integration at abnormal chromosomal positions and expression in inappropriate tissues.

We have investigated the chromosomal location, inheritance, and expression of a cloned rabbit beta-globin gene introduced into the mouse germ line by microinjection into mouse eggs. Experiments utilizing in situ hybridization to metaphase chromosomes show that the gene has integrated into one or two different chromosomal loci in each of five mouse lines analyzed. Each locus contains between three and forty copies of the foreign DNA sequence arranged in a tandem array, and the sequences at each locus are stably inherited as a single Mendelian marker. Neither globin mRNA nor polypeptides encoded by the rabbit beta-globin gene are detected in erythroid cells in the seven transgenic lines examined, indicating that the expression of the foreign gene is not correctly regulated. However, in two of the mouse lines, rabbit beta-globin transcripts are found at a low level in specific, although inappropriate, tissues: skeletal muscle in one line and testis in another line. These unusual patterns of beta-globin gene transcription are heritable traits in the two mouse lines and may result from the beta-globin gene's integration at abnormal chromosomal positions.

The analysis of malignancy by cell fusion. IX. Re-examination and clarification of the cytogenetic problem.

Previous experiments with crosses between malignant and diploid mouse cells had shown that the reappearance of malignancy in hybrids in which it was initially suppressed was associated in some cases with the elimination of the chromosomes 4 derived from the diploid parent cell. In others, however, this did not appear to be so. In the present study, we have re-examined the role of the diploid chromosomes 4 in the suppression of malignancy using natural polymorphisms of the centromeric heterochromatin to identify the parental origin of the chromosomes 4 in the hybrid cells. We now find that the diploid chromosomes 4 are indeed involved in the suppression of malignancy in all the tumours that we have examined, which include a carcinoma, a melanoma, a sarcoma and a lymphoma. In all crosses between these malignant tumour cells and diploid fibroblasts, there is selective pressure in vivo against the chromosomes 4 derived from the diploid cell and in favour of the chromosomes 4 derived from the malignant cell. This indicates that the chromosomes 4 in all these tumours are in some way functionally different from the chromosomes 4 of the diploid fibroblast. Reappearance of malignancy in hybrids in which it was initially suppressed may result from a reduction in the number of diploid chromosomes 4, an increase in the number of malignant chromosomes 4, or both. The gene on the diploid chromosome 4 responsible for the suppression of malignancy acts in a dose-dependent manner.