DNA ligase I is required for fetal liver erythropoiesis but is not essential for mammalian cell viability.

Four distinct DNA ligase activities (I-IV) have been identified within mammalian cells. Evidence has indicated that DNA ligase I is central to DNA replication, as well as being involved in DNA repair processes. A patient with altered DNA ligase I displayed a phenotype similar to Bloom's syndrome, being immunodeficient, growth retarded and predisposed to cancer. Fibroblasts isolated from this patient (46BR) exhibited abnormal lagging strand synthesis and repair deficiency. It has been reported that DNA ligase I is essential for cell viability, but here we show that cells lacking DNA ligase I are in fact viable. Using gene targeting in embryonic stem (ES) cells, we have produced DNA ligase I-deficient mice. Embryos develop normally to mid-term when haematopoiesis usually switches to the fetal liver. Thereupon acute anaemia develops, despite the presence of erythroid-committed progenitor cells in the liver. Thus DNA ligase I is required for normal development, but is not essential for replication. Hence a previously unsuspected redundancy must exist between mammalian DNA ligases.

Development of B lymphocytes in mice heterozygous for the X-linked immunodeficiency (xid) mutation. xid inhibits development of all splenic and lymph node B cells at a stage subsequent to their initial formation in bone marrow.

CBA/N mice were crossed with CBA/Ca-Pgk-1a to produce female F1 hybrids that were heterozygous for both xid and the phosphoglycerate kinase 1 (PGK-1) allozymes. PGK acted as a quantifiable marker for the frequency of cells in which the xid-bearing X chromosome was active in lymphocytic and other cell populations. In adults, such cells (termed xid cells) were virtually absent in FACS-sorted splenic and lymph node B cells, and in all three splenic subpopulations distinguished on the basis of their relative expression of membrane mu and delta chains. Thus, the xid mutation appeared to compromise the development of all B cells. Erythrocytes, thymocytes, T cells, and granulocytes were unaffected. Selection against xid cells was less pronounced in the spleens of 2-6-wk-old mice. In the bone marrow, there was evidence for selection against xid in the production of B cells (except at 2 wk of age), but not at the pre-B cell level. These data suggest that, in competition with normal non-xid cells, newly-formed xid B cells were less likely to be incorporated into the peripheral B cell pool.

Differentiation and delayed cell death in embryonal stem cells exposed to low doses of ionising radiation.

Embryonal stem cells have been used to study the effects of environmentally relevant doses of radiation on cell death and differentation. The ES cells were found to have a greater than 60% chance of surviving the traversal of a single alpha-particle, the lowest possible dose of high linear energy transfer radiation a cell may receive. The ES cells appeared to possess the cell cycle checkpoints believed to prevent the transmission of the radiation damage. However, delayed effects were observed in the progeny. An increased incidence of apoptosis and haempoietic differentiation capacity was found to persist in the ES cell population over many cell divisions. Since both cell death and differentiation are known to play a key role in tissue kinetics, an ES cell model will provide a valuable and versatile cell system for studying the role of cell death and differentiation in the pathology of radiogenic diseases.

Numbers and dispersion of repopulating hematopoietic cell clones in radiation chimeras as functions of injected cell dose.

Lethally irradiated mice were repopulated with low (10(5)), medium (10(6)) or high (10(7)) doses of congenic bone marrow cells. Marrow donors were heterozygous for the X-chromosome-encoded allozyme marker phosphoglycerate kinase (PGK-1). A second allozyme marker, phosphoglucose isomerase (GPI-1), distinguished between donor and radioresistant host cells. Use of these markers allowed the numbers and dispersion of repopulating hematopoietic clones to be estimated by binomial statistics. The number of major repopulating clones was related to the injected cell dose in a linear fashion, the inferred frequency of clonogenic cells in donor bone marrow being about 1:40,000. In high-dose recipients, the clones grew locally, with little or no dispersion between bones. Low-dose recipients, in contrast, carried widely dispersed clones; these tended to become reduced in number with increasing time after repopulation. Most of the (few) bone marrow clones present in low-dose recipients were also present in the thymus. In contrast, only about 10% of bone marrow clones in high-dose recipients were substantially represented in the thymus at any one time--about 16 clones in each lobe.

Test for fused-cell origin of tumors with Mus caroli carrying glucose-6-phosphate dehydrogenase cellular mosaicism.

A test system for examining the fused cell-origin of tumors was developed by application of Mus caroli carrying the X-chromosome inactivation cellular mosaicism for G6PD. A G6PD heterodimer pattern is expected if a tumor is initiated from a fused cell. Among tumors induced by subcutaneous injection of a high dose of MCA, three tumors exhibited a single G6PD phenotype and one tumor exhibited a multiple G6PD phenotype; however, the G6PD heterodimer pattern was not found. Although the results obtained were not conclusive, this system is thought to be useful for detecting a possible fused cell-origin of tumors.

The development of trophoblast in vitro from blastocysts containing varying amounts of inner cell mass.

When intact mouse blastocysts are cultured in vitro in medium supplemented with foetal calf serum, trophoblast cells proliferate and undergo giant cell transformation such as occurs in vivo. If the amount of inner cell mass in the blastocyst is decreased by culture with [3H]-thymidine then giant cell transformation occurs normally but proliferation is reduced. In the absence of inner cell mass no proliferation occurs, and giant cell transformation is more rapid than in undamaged blastocysts.

Establishment of Theileria-infected bovine cell lines in scid mice.

Bovine cells transformed by infection with the protozoan parasite Theileria annulata were inoculated subcutaneously or intraperitoneally into C.B.-17 scid mice. Mice injected subcutaneously developed solid tumours at the injection site, whilst those injected intraperitoneally developed ascites. Schizont-infected cells were found in other tissues: infected cells spread much more easily from the intraperitoneal site. Karyotyping of cells isolated from tumours showed no evidence of transfer of parasites to murine cells. These results show that the scid mouse can be used as a host for Theileria-infected bovine cells.

Further evidence for the importance of parental source of the Xce allele in X chromosome inactivation.

Using mice that were mosaics for both Xce and phosphoglycerate kinase (Pgk-1) alleles, we present further evidence that the parental source of the X chromosome may affect the probability of that X chromosome remaining active. The reciprocal cross differences in PGK-1 activity described here are intermediate between those published previously for other alleles of Xce.

Expression of glucose phosphate isomerase in interspecific hybrid (Mus musculus x Mus caroli) mouse embryos.

Hybrid Mus musculus x Mus caroli embryos were produced by inseminating M. musculus (C57BL/OlaWs) females with M. caroli sperm. Control M. caroli embryos developed more rapidly than did control M. musculus embryos and implanted approximately 1 day earlier. At 1 1/2 days, both the hybrid embryos and those of the maternal species (M. musculus) had cleaved to the 2-cell stage. By 2 1/2 days some of the hybrids were retarded compared to M. musculus, and by 3 1/2 days most were lagging behind. This is consistent with the idea that the rate of development of hybrid embryos declines once it becomes dependent on embryo-coded gene products. We have used this difference in rate of preimplantation development, between hybrid and M. musculus embryos, to try to determine whether the activation of embryonic Gpi-1s genes, that encode glucose phosphate isomerase (GPI-1), is age-related or stage-related. In control M. musculus embryos (both mated and Al groups), the GPI-1AB and GPI-1A allozyme, indicative of paternal gene expression, were detected in 7 of 9 samples of 3 1/2-day compacted morula stage embryos and were seen in all 19 samples of 3 1/2-day blastocysts. In hybrid embryos, these allozymes were detected 1 day later. They were not detected in any 3 1/2-day samples (12 samples of compacted morulae) but were consistently detected at 4 1/2 days (4 samples of blastocysts and 2 samples of uncompacted morulae). Our interpretation of the results is that gene activation in hybrid embryos is stage-specific, rather than age-specific, and probably begins around the 8-cell stage, with detectable levels of enzyme accumulating later. Analysis of GPI-1 electrophoresis indicated that both the paternal (M. caroli) and maternal (M. musculus) Gpi-1s alleles were equally expressed in hybrid embryos and that the paternally derived allele was not activated before the maternally derived allele.

The number of clonal precursors of the follicular epithelium in the mouse ovary.

The number of clonal precursors of granulosa cells in mouse ovarian follicles has been estimated using a technique based on the phenomenon of random X-chromosome inactivation of somatic cells and the use of an X-linked alloenzyme variant of the glycolytic enzyme PGK-1. The granulosa cells of follicles were oligoclonal in origin and founded by a small number of cells (about 5) which was consistent with histological observations. When the analysis was extended to two subcompartments of the follicle, the mural and cumulus granulosa cells, the results indicated that the cumulus and mural granulosa cells had a common origin.

Age-dependent selection against hypoxanthine phosphoribosyl transferase-deficient cells in mouse haematopoiesis.

The basis of a previously observed difference in the level of contribution of hypoxanthine phosphoribosyltransferase-deficient cells between the haematopoietic and non-haematopoietic tissues of chimaeric and heterozygous mice has been clarified by studying two populations of female mice that differ only in that one is heterozygous for a null allele at the hprt locus and the other is wild type at this locus. Both populations are heterozygous for an electrophoretic variant allele at the X-linked Pgk-1 locus, so that X-chromosome inactivation generates cells expressing different isozymes of phosphoglycerate kinase which can be assayed to monitor cell selection. The results show that hypoxanthine phosphoribosyltransferase deficiency itself, rather than an effect of another X-linked gene, causes a reduced level of contribution to haematopoietic tissues. Further, the extent of the depletion increases significantly with age, and this effect is due to a progressive reduction in the level of contribution to haematopoietic tissues rather than to an increase in the level of contribution to non-haematopoietic tissues.

Oligoclonal tumours.

Tumours are commonly classified as monoclonal or polyclonal. The question of how many clones are present in a polyclonal tumour is seldom asked; it is important, however, because the answer may show whether or not clones arise and develop independently, and whether the number of clones in tumours of a particular kind tends to increase or decrease with time. We have used two procedures to assess the clonality of chemically-induced murine fibrosarcomas, one based on the heterozygosity of the tumour hosts for an X-linked marker, the other on the expression of tumour-associated transplantation antigens (TATA) by the tumours. As we have reported previously, many of these tumours are pleoclonal. Evidence now presented suggests that the clones do not develop independently and that many of the tumours are biclonal.

Polycystic kidney disease in the CBA/N immunodeficient mouse.

We describe a polycystic lesion of the kidney in the CBA/N mouse with an X-linked recessive immunodeficient syndrome. There is progressive cystic dilatation affecting all parts of the nephron. The cyst lining is composed of a single layered epithelium with focal nuclear crowding and the formation of micropapillary structures. The cystic epithelial cells show subnuclear vacuolation. Focal basement membrane thickening is also a feature. There is no significant inflammatory infiltrate present within these kidneys. Electron microscopic examination reveals that the subnuclear vacuolation is due to loss of the membrane infoldings at the basal pole of the epithelial cell with fluid accumulation within the extracellular space. The basement membrane thickening is due to expansion of the lamina densa. These changes are not present at birth but develop progressively with age. The finding of a polycystic kidney lesion in these mice offers an opportunity to investigate the relationship between the immune system and renal cyst formation.

A limited temporal window for the derivation of multilineage repopulating hematopoietic progenitors during embryonal stem cell differentiation in vitro.

Embryonal stem cells have been shown to differentiate in vitro into all hematopoietic lineages. This has been used successfully as one approach to the study of genetic events occurring during haematopoiesis. However, studies on the commitment of mesodermal precursors to the hematopoietic lineage have been limited due to the inability to define a system in which embryonal stem (ES) cells will give rise to primitive hematopoietic stem cells in vitro. Using a colony forming assay (CFU-A), we determined that the earliest time point at which primitive multilineage hematopoietic precursors can be detected during ES cell differentiation in vitro in the absence of exogenous conditioned medium or stromal cell culture is 4 days. Lethally irradiated adult recipient mice that received differentiated ES cells from this time point survived for more than 3 weeks; and in two out three experiments, peripheral blood from these animals contained ES-derived progeny. Fluorescence activated cell sorting (FACS) found ES-derived CD45+ hematopoietic cells in both lymphoid and myeloid compartments at 12 weeks posttransplantation, suggesting that the population of day 4 differentiated ES cells contains primitive hematopoietic precursors. A preliminary RT-PCR analysis of gene expression around this time point suggests that there are very few hematopoietic cells present. This approach should prove useful in studies of genetic control of commitment to and maintenance of hematopoietic lineages in vitro and in vivo.

Radiation-induced genomic instability and persisting oxidative stress in primary bone marrow cultures.

There is accumulating evidence that cells exposed to low and often environmentally relevant doses of ionizing radiation survive the initial insult, but transmit genomic instability to their progeny. The underlying mechanism of radiation-induced genomic instability is unknown. We present bio-chemical evidence consistent with the hypothesis that enhanced and persistent oxy-radical activity may be responsible.

Suppressive effects of Corynebacterium parvum on primary and secondary antibody responses in mice.

The splenic plaque forming cell (PFC) response of mice to an intraperitoneal injection of sheep erythrocytes was severely depressed by prior treatment with Corynebacterium parvum given four days beforehand by the same route. However, total antibody levels were less affected, and soon attained near normal titres. This implied that the effects of C. parvum were limited to the spleen, and that other tissues gave a substantial response during the period when the splenic response was suppressed. Equally, this apparently local immunosuppressive effect of C. parvum failed to inhibit the eventual development of a normal memory cell pool. It was also shown that primed cells in the spleen, challenged during the period when C. parvum inhibited primary IgG responses, were relatively refractory to its suppressive effects.