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Biomedical subjects

R Jaenisch

Publications and source records attributed to R Jaenisch.

277 records · Page 16Linked to original sources
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Retrovirus genomes methylated by mammalian but not bacterial methylase are non-infectious.

The biological importance of DNA methylation for gene expression in eukaryotes is becoming increasingly evident, and a direct role of methylation in gene expression has been suggested by an analysis of the infectivity of integrated retroviral genomes in a transfection assay. These studies, however, did not address whether specific methylatable residues are involved in gene regulation. Methylation by sequence-specific bacterial DNA methylases has been shown to suppress the expression of some genes, but not others. To investigate the effect of methylation on gene expression without having to rely on sequence-specific methylases, a rat liver enzyme was used to methylate in vitro all C-G dinucleotides of a proviral genomic clone. This treatment reduced the biological activity of Moloney murine leukaemia virus (M-MuLV) proviral DNA by more than three orders of magnitude, whereas complete methylation of 35 HpaII sites in the same DNA had only a marginal effect. The rat methylase-induced inactivation was reversible, as treatment of recipient cells with 5-azacytidine rendered the non-infectious viral genomes biologically active. This suggests that methylation in other C-G dinucleotides than those detectable with restriction enzymes can be crucial for gene expression.

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Embryonic lethal mutation in mice induced by retrovirus insertion into the alpha 1(I) collagen gene.

Experimental insertion of a retrovirus into the germ line of mice has resulted in an embryonic recessive lethal mutation. Integration of the proviral genome occurred at the 5' end of the alpha 1(I) collagen gene, leading to complete transcriptional block. Developmental arrest of embryos homozygous at the mutated allele coincides with high expression of the gene in normal embryogenesis. Insertion mutagenesis by retroviruses may offer a general approach to the identification and isolation of genes which are transcriptionally active during mammalian development.

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Retrovirus-induced de novo methylation of flanking host sequences correlates with gene inactivity.

The pattern of DNA methylation changes during development of eukaryotes, and hypomethylation frequently correlates with gene expression (for reviews see refs 1-4). A causal relationship between hypermethylation and gene inactivity has been established for retroviral genomes which are methylated de novo when inserted into the germ line of mice (ref. 5; for review, see ref. 6). The mutual interaction of the provirus with the host genome can influence virus expression and can result in inactivation of the host gene by insertional mutagenesis. We report here that the insertion of a provirus can change the methylation pattern of the host DNA. Sequences flanking the provirus become methylated de novo within 1 kilobase (kb) of the integration site. In Mov-13 mice, which carry a lethal mutation of the alpha 1(I) collagen gene, de novo methylation of host DNA is associated with a change in chromatin conformation. This suggests that virus-induced DNA methylation can alter DNA-protein interactions and thereby interfere with correct gene activation during embryonic development.

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Mammalian neural crest cells participate in normal embryonic development on microinjection into post-implantation mouse embryos.

The production of chimaeric mice by aggregating pre-implantation mouse embryos or by injection of cells into the blastocyst has been of great value in analysing the regulation of early mammalian development and in dissecting the relationships of early cell lineages. While the totipotent cells of the pre-implantation embryo can be grown in vitro and thus are readily accessible to experimental manipulation, this is not possible after the embryo has implanted into the uterus. This problem has severely hampered the analysis of cell migration and of cell lineage relationships in later stages of mammalian development. In contrast, the chicken embryo can be manipulated experimentally throughout embryo-genesis and this has made the bird a favourable system for studying patterns of cell migration in the development of higher vertebrates. In mammals, the introduction of retroviruses and haematopoietic cells has provided two means of probing post-implantation development by direct intervention. I report here that cultured neural crest cells, when microinjected into 9-day-old mouse embryos, can migrate over considerable distances and participate in normal development, and the resulting chimaeric animals show pigmentation derived from the donor cells in hair and iris. The introduction of cells into post-implantation embryos may provide the means of studying patterns of cell migration in mammalian development at a level of sophistication which so far has been restricted to the chicken system.

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Retrovirus insertion inactivates mouse alpha 1(I) collagen gene by blocking initiation of transcription.

Mov13 mice carry a single Moloney murine leukaemia virus (M-MuLV) proviral copy in the first intron of the alpha 1(I) collagen gene. Virus insertion interferes with the synthesis of stable alpha 1(I) collagen messenger RNA and causes a recessive lethal mutation. The virus insertion has induced changes of the methylation pattern as well as the chromatin conformation in the mutated gene. Specifically, a DNase-hypersensitive site which is associated with active transcription of the wild-type collagen gene is not present in the mutant allele. The block of collagen expression could be caused by virus-induced instability of collagen mRNA or by impaired initiation of transcription. To distinguish between these possibilities, we have compared the activity of the alpha 1(I) collagen gene promoter in cell lines derived from wild-type and Mov13 embryos by nuclear run-on transcription experiments and S1 mapping of nuclear RNA. We show here that initiation of transcription of the mutant gene is reduced 20-100-fold. This indicates that the virus-induced change of chromatin structure in the promoter region of the mutant gene prevents RNA polymerase from binding to its DNA template. Our results are consistent with the notion that the promoter-associated DNase-hypersensitive site is a prerequisite for rather than a consequence of gene activity.

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High frequency of unequal recombination in pseudoautosomal region shown by proviral insertion in transgenic mouse.

The mammalian X and Y chromosomes, in contrast to the autosomes, pair during male meiosis only near the telomeres. Alleles localized in this region can undergo reciprocal exchange during meiosis. Because such sequences do not show strict sex-linked inheritance, they have been termed pseudoautosomal. In man, several DNA sequences have been described which show pseudoautosomal transmission and which are localized in the pairing region at the ends of the short arms of both the X and Y chromosomes (refs 6-9, and D. Page, unpublished results). We now show that the transgenic mouse strain, Mov-15, contains a single Moloney murine leukaemia virus (M-MuLV) genome in its germline, and genetic evidence indicates that the provirus is integrated into the pseudoautosomal region of the sex chromosome. Proviral copies are lost or gained in 7% of male meioses in this strain, and mouse sequences flanking the provirus are tandemly repeated and highly variable. We conclude that unequal recombination events occur with high frequency in the pairing region, possibly because of the presence of repeated sequences.

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