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Rearrangement and reactivation of the myelin basic protein locus in myelin-deficient (mld) mouse brain
Y Sun1, J Xu, E Barbarese
1Neuroscience Program, University of Connecticut Health Center, Farmington 06030, USA.
Abstract:
Myelin-deficient (mld) is a complex mutation affecting the myelin basic protein (MBP) locus of the mouse. It consists of duplication and partial inversion of the MBP gene and results in a dysfunctional MBP locus. The mutant phenotype is reversed, both in vivo and in vitro, in approximately 5% of mld oligodendrocytes. One possible mechanism for the somatic reversion is recombination between homologous sequences of the duplicated gene copies to reconstitute a functional MBP locus. There are several possible recombination events that could reconstitute a functional MBP locus by DNA rearrangement. Two of these would result in reinversion and circularization of specific MBP gene sequences, respectively. In this work polymerase chain reaction analysis was used to detect both reinverted and circularized MBP gene sequences in mld mouse tissues, indicating that DNA rearrangement at the MBP locus does occur. Analysis of individually harvested cells showed that in revertant MBP-positive mld oligodendrocytes DNA rearrangement at the MBP locus was correlated with reactivation of the MBP gene. Fluctuation analysis showed that reactivation of the MBP locus is a stochastic event occurring with a frequency of approximately 1.4 x 10(-6) per cell per cell cycle during oligodendrocyte development. The frequency of rearrangement and reactivation of the MBP locus was comparable in double mutant (mld/mld, scid/scid) and single mutant (mld/mld, +scld/+scld) mice, indicating that the scid factor is not required for MBP gene reactivation in mld. The significance of DNA rearrangement in mammalian development is discussed.
Insights
Somatic DNA rearrangement can restore myelin basic protein (MBP) gene function in myelin-deficient (mld) mice. This DNA repair mechanism occurs spontaneously in some oligodendrocytes, reactivating the MBP gene during development.
Area of Science:
- Neuroscience
- Genetics
- Molecular Biology
Background:
- Myelin-deficient (mld) is a mouse mutation impacting the myelin basic protein (MBP) gene locus.
- The mutation involves duplication and inversion, leading to a non-functional MBP locus and myelin deficiency.
- A small percentage of mld oligodendrocytes exhibit spontaneous phenotypic reversion.
Purpose of the Study:
- To investigate the mechanism of somatic reversion in mld oligodendrocytes.
- To determine if DNA rearrangement at the MBP locus underlies MBP gene reactivation.
- To quantify the frequency and developmental timing of MBP gene reactivation.
Main Methods:
- Polymerase chain reaction (PCR) analysis to detect specific DNA rearrangements (reinversion, circularization) of the MBP gene.
- Individual cell analysis to correlate DNA rearrangement with MBP gene expression.
- Fluctuation analysis to determine the frequency of MBP locus reactivation.
Main Results:
- PCR detected both reinverted and circularized MBP gene sequences in mld mouse tissues, confirming DNA rearrangement.
- In revertant cells, MBP gene rearrangement was directly correlated with MBP gene reactivation.
- MBP locus reactivation is a stochastic event with a frequency of approximately 1.4 x 10^-6 per cell per cell cycle.
- The scid mutation did not affect MBP gene reactivation frequency, indicating it's independent of the scid factor.
Conclusions:
- DNA rearrangement at the MBP locus is a mechanism for somatic reversion in mld mice.
- MBP gene reactivation is a stochastic process during oligodendrocyte development.
- The findings highlight the role of DNA rearrangement in mammalian development and gene repair.