Detlev Jähner

Chromosomal position and activation of retroviral genomes inserted into the germ line of mice

The exogenous Moloney leukemia virus (M-MuLV) was inserted into the germ line of mice by exposing embryos to virus at different stages of embryogenesis. Mice derived from exposed embryos were mosaics with respect to integrated virus. Nine new substrains, designated Mov-5 to Mov-13, were derived, each of which carries a single M-MuLV genome at a different chromosomal position in its germ line. Four substrains, Mov-1 to Mov-4, were derived previously. Restriction enzyme analyses demonstrated that, with the exception of Mov-4 and Mov-6 mice, no major rearrangements or deletions have occurred in the integrated proviral genomes. Infectious virus is not activated in the majority of substrains (Mov-4 to Mov-8 and Mov-10 to Mov-12), whereas the other mice develop viremia. A detailed comparison between Mov-1 and Mov-13 mice demonstrated that the time of virus activation is different. Mov-13 mice activate infectious virus during embryogenesis, leading to a distinct pattern of virus expression in all tissues of the adult, but the viral genome in Mov-1 mice is activated only during the first two weeks after birth, leading to virus expression predominantly in lymphatic organs. Together with previous observations, at least four different phenotypes of virus expression-that is, early virus activation during embryogenesis, virus activation after birth, virus activation late in life and no expression of infectious virus at all-can be distinguished among the 13 substrains. Our results suggest that the chromosomal region at which a viral genome is integrated influences its expression during development and differentiation.

Germline integration of Moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death

Thirteen mouse substrains genetically transmitting the exogenous Moloney murine leukemia virus (M-MuLV) at a single locus (Mov locus) have been derived previously. Experiments were performed to investigate whether homozygosity at the Mov loci would be compatible with normal development. Animals heterozygous at an Mov locus were mated, and the genotype of the offspring was analyzed. From parents heterozygous at the loci Mov1 to Mov12, respectively, homozygous offspring were obtained with the expected Mendelian frequency. In contrast, no homozygous offspring or embryos older than day 15 of gestation were obtained from parents heterozygous at the Mov13 locus. When pregnant Mov13 females at day 13 and day 14 of gestation were analyzed, approximately 25% of the embryos were degenerated. Genotyping revealed that these degenerated embryos were invariably homozygous and the normal appearing embryos were either heterozygous or negative for M-MuLV. These results suggest that integration of M-MuLV at the Mov13 locus leads to insertion mutagenesis, resulting in embryonic arrest between day 12 and day 13 of gestation. It is possible that the Mov13 locus represents a gene or gene complex involved in the early embryonic development of the mouse.

DNA Methylation in Early Mammalian Development

The genomic DNAs of vertebrates and many invertebrates contain 5-methylcytosine (5-meCyt) as the only modified base (Table 10.1). Considerable interest in DNA methylation has been created by increasing evidence that links methylation patterns to patterns of gene expression in differentiation. An inverse correlation between methylation and transcriptional activity has been found for a number of developmentally regulated genes; it will be reviewed elsewhere (see Chapter 8).

Inhibitory effects of TNFα on mouse tumor Leydig cells: possible role of ceramide in the mechanism of action

TNF alpha is reported to inhibit steroidogenesis in mouse Leydig cells. In primary cells this inhibition resulted mainly from a reduced expression of Cyp-17 gene. Mouse tumor Leydig cells, MA-10, being free of macrophages and lacking Cyp-17, appear to be an excellent model to investigate those effects of TNF alpha which are independent of either macrophages or Cyp-17. We report here that TNF alpha receptors are expressed in this cell line. Treatment of the cells with TNF alpha had no effect on basal progesterone production. In contrast, LH-, 8Br-cAMP and forskolin-stimulated progesterone production was inhibited by TNF alpha. Neither enzymes involved in the conversion of cholesterol to pregnenolone nor hormone-induced hydrolysis of [14C] cholesterol-ester were affected by TNF alpha. The hormone-induced expression of StAR protein was diminished in mitochondrial fractions from TNF alpha-treated cells. Also cell permeable ceramides markedly inhibited StAR protein levels. We show further that TNF alpha was able to induce [14C]-ceramide accumulation in MA-10 cells and suggest that this sphingolipid may be considered as a transmitter of TNF alpha signals to the StAR protein.

Conformation of free and of integrated Moloney leukemia virus proviral DNA in preleukemic and leukemic BALB/Mo mice

Abstract Restriction enzyme analysis has been used to characterize the structure of Moloney leukemia virus (M-MuLV) DNA in preleukemic and leukemic BALB/Mo mice. Leukemogenesis in these mice is accompanied by a somatic amplification of M-MuLV-specific DNA sequences which are derived from the germ line-transmitted Moloney leukemia virus genome. Quantitation by DNA-DNA annealing in solution has shown that this increase of M-MuLV-specific DNA sequences occurs in two steps, a first step from 1 to approximately 2 copies in spleen and thymus of preleukemic mice and a second increase to 3–4 copies following leukemic transformation. Using a specific cDNA probe for restriction enzyme analysis, no somatically acquired M-MuLV copies are detectable in tissues of preleukemic BALB/Mo mice. This indicates that virus integrated at many different chromosomal sites in individual cells. In contrast, restriction enzyme analysis of DNA from leukemic tissues shows 5–8 integrated copies in addition to the endogenous M-MuLV genome. Identical integration patterns are observed in leukemic tumors arising at different anatomical sites in individual animals. Tumors from different animals, however, have distinct integration patterns and no specific integration site common to all tumors can be identified. These results suggest that in BALB/Mo mice transformed cells originate among a population of preleukemic target cells which is heterogenous with respect to integration sites of newly acquired proviral copies. Selection of one transformed cell clone leads to monoclonal disease. Similar results are obtained with BALB/c mice infected with virus as newborns. Gel analysis indicated that unintegrated forms of M-MuLV viral DNA appear in target tissues of preleukemic BALB/Mo mice. Quantitation by quantitative hybridization in solution showed that up to 0.5 copy per cell can be present in thymic DNA from 1− to 5-month-old mice but not in spleens of the same animals. Small amounts of unintegrated proviral DNA are occasionally detected in thymus, spleen, and bone marrow of animals younger than 4 weeks of age. These results provide the first evidence for occurrence of free proviral DNA in animals and suggest that superinfection of target cells with endogenous virus is involved in the process of leukemic transformation.

Novel splicing variants of the human thyrotropin receptor encode truncated polypeptides without a membrane-spanning domain.

The thyrotropin receptor is of fundamental importance to normal thyroid function and is considered to be the predominant antigen affected by the autoantibodies of Graves’ autoimmune hyperthyroidism. The identification of the epitopes on the receptor to which the autoantibodies bind or the mechanism by which the autoantibodies arise remain to be established. In this report we have analysed in detail thein vivo transcription of the human TSH receptor gene (hTSH-R), demonstrating the presence of numerous novel TSH receptor transcripts. Northern blot analysis of mRNA from human thyroid tissue using a radiolabelled cDNA probe specific for the extracellular domain of the hTSH-R revealed the presence of small polyadenylated mRNAs, in addition to the full-length hTSH-R mRNA. A PCR strategy devised to clone transcripts with 3′ polyadenylation and 5′ hTSH-R specific sequences was used to clone five different hTSH-R transcripts (hTSH-R. ST1 to ST5; 250bp-1.7 kb) from human thyroid tissue. Sequence analysis demonstrated that the small transcripts arose by alternative splicing of the hTSH-R mRNA. The transcripts were associated with polysomes and were demonstrated in human thyroid tissue from patients suffering from Graves’ disease, sporadic goiter as well as in healthy lobes of thyroid tissue.In situ hybridization demonstrated that two of the alternative transcripts adopted a tissue distribution pattern identical to that of the full-length hTSH-R transcript. The two major truncated transcripts ST4 and ST5 contained unique sequences at the 3′ end of the mRNAs and thus potentially represent the molecular origin of soluble TSH receptor variants which have been postulated on numerous occasions.

Molecular cloning and testicular expression of the gene transcripts encoding the murine multiubiquitin-chain-binding protein (Mcb1)

A murine cDNA encoding a homolog of the human multiubiquitin-chain-binding protein Mcb1 was isolated and sequenced from a mouse testis cDNA library. The encoded Mcb1 protein is highly conserved between mouse and human. Northern hybridisation showed expression of Mcb1 transcripts in all examined mouse tissues. In the testis, however, there is additionally a second, longer Mcb1 transcript in wild-type mice that is absent in the azoospermic W/Wv mutant mice, suggesting expression of this transcript in association with germ cell differentiation.

DNase I sensitivity of endogenous and exogenous proviral genome copies in M-MuLV-induced tumors of Mov-3 Mice.

Abstract Limited DNase I digestion and subsequent restriction enzyme analysis were used to compare the chromatin conformation of different proviral genome copies in M-MuLV-induced tumors of Mov-3 mice. The results show that the endogenous, genetically transmitted viral genome copy is highly resistant, whereas the somatically acquired genome copies are sensitive to digestion with DNase I.

Differential Display PCR Cloning of W/Wv-Mutant Testis Specific Genes

The W/Wv mouse is an important model with which to investigate azoospermia and its consequences in a mammalian system. The W locus encodes the transmembrane tyrosine kinase receptor c-kit (Chabot et al., 1988). Mutations in the c-kit gene affect the differentiation of hematopoetic stem cells, melanocyte progenitors and primordial germ cells. The latter results in infertility due to azoospermia.

DERIVATION OF THREE MOUSE STRAINS CARRYING MOLONEY LEUKEMIA VIRUS IN THEIR GERM LINE AT DIFFERENT GENETIC LOCI

The exogenous Moloney leukemia virus (=M-MuLV) was integrated experimentally into the germ line of mice. Substrains of mice were derived carrying three new genetic loci which were designated as Mov-2, Mov-3 and Mov-4, and represent M-MuLV genes integrated into the mouse genome. Mice carrying the Mov-3 gene developed early viremia and died rapidly of leukemia, whereas animals transmitting the Mov-2 gene activated virus only occasionally late in life. The M-MuLV provirus integrated at the Mov-2 and Mov-3 locus was identical by restriction enzyme analysis and the virus activated from Mov-2, Mov-3 and BALB/Mo mice (=Mov-1) was identical by biological and biochemical criteria. The M-MuLV genome in Mov-4 mice was shown to have a partial deletion and no virus expression was observed in these animals. Together with previous results obtained in the BALB/Mo system, our data suggest that the chromosomal location of an endogenous virus influences its tissue-specific expression.