Reduction of the number of mutant copies of mitochondrial DNA in tissues of irradiated mice in the postradiation period

Changes in the number of mutant copies of mitochondrial DNA (mtDNA) were studied in the brain and spleen tissues of mice after their X-irradiation at a dose of 5 Gy. For this purpose, heteroduplexes obtained via hybridization of the products of PCR amplification of mtDNA (ND3 gene and two D-loop regions) from irradiated and control mice were digested with the CelI nuclease capable of specific mismatch cleavage. Heteroduplexes obtained via hybridization of the products of PCR amplification of mtDNA from irrradiated and control mice were digested by the CelI nuclease to a greater degree than heteroduplexes of the PCR products of mtDNA of mice from the control group. This suggests the presence of mutations in mtDNA regions in irradiated mice. Digestion by the CelI nuclease of heteroduplexes obtained via hybridization of the PCR products of mtDNA (ND3 gene and D-loop regions) on day 8 after irradiation is essentially more efficient than digestion of heteroduplexes obtained via hybridization of the PCR products of mtDNA isolated from mouse tissues on days 14 and 28 of the postradiation period. These results indicate a reduction in the number of mtDNA copies with mutations in tissues of irradiated mice by day 28 of the postradiation period. The reduction in the level of mutant mtDNA copies by this term is especially significant in the spleen. The total number of mtDNA copies in the mouse brain and spleen tissues estimated by real-time PCR, relative to the nuclear β-actin gene, is also decreased by 30–50% as compared to the control on days 8 to 28 after irradiation. The results of the study suggest that mutant mtDNA copies are eliminated from tissues of irradiated animals in the postradiation period. This elimination can be regarded either as a result of selective degradation of mitochondria carrying mutant DNA copies or as a result of cell death being continued in tissues of irradiated animals.     

Mutant copies of mitochondrial DNA in tissues and plasma of mice subjected to X-ray irradiation

Impairments of mitochondrial genome are associated with a wide spectrum of degenerative diseases, development of tumors, aging, and cell death. We studied the content of mitochondrial DNA (mtDNA) with mutations and the total content of mutations in the brain and the spleen of mice subjected to X-ray irradiation at a dose of 1–5 Gy at 8–28 days after treatment. In these mice, we studied the number of mutant copies of extracellular mtDNA (ec-mtDNA) and its total content in blood plasma. We estimated mutations in control and irradiated mice using cleavage of heteroduplexes prepared by hybridization of PCR amplicons of mtDNA (D-loop region) mediated by CEL-I endonuclease, an enzyme that specifically cleaves unpaired bases. Changes in the total number of mtDNA copies relative to nuclear DNA were assessed by real time PCR using the ND-4 and GAPDH genes, respectively. We found that the number of mutant mtDNA copies was significantly increased in the brain and the spleen of irradiated mice and reached the maximum level at the eighth day after treatment; it then decreased by the 28th day after treatment. In nuclear genes similar to mutagenesis, mutagenesis of mtDNA in the brain and spleen tissues linearly depended on irradiation dose. In contrast to mutant nuclear genes, most mutant mtDNA copies were eliminated in the brain and spleen tissues, whereas the total content of mtDNA did not change within 28 days after irradiation. Our data show that, during this period, a high level of ec-mtDNA with mutations was observed in DNA circulating in blood plasma with the maximum level found at the 14th day. We suppose that mutant mtDNA copies are eliminated from cells of animals subjected to irradiation during the posttreatment period. Higher content of ec-mtDNA in blood plasma can be considered as a potential marker of radiation damage to the body.     

Extracellular mutant mitochondrial DNA content is dramatically elevated in the blood plasma of irradiated mice

Nucleic acids circulating in blood plasma and other biological fluids are of interest as potential markers for the diagnosis of various pathologies and the monitoring of stresses. Mitochondrial DNA (mtDNA) is a more vulnerable target for many genotoxic agents than nuclear DNA, and mutations in the mitochondrial genome can serve as markers for many diseases. In the present study, extracellular mtDNA with mutations was assayed in the blood plasma of mice exposed to X radiation at a dose of 5 Gy. For this purpose, heteroduplexes obtained by the hybridization of mtDNA PCR amplicons (ND3 gene and D loop region) from the blood plasma of irradiated and control mice were cleaved with CEL endonuclease, a mismatch-specific enzyme. The total amount of mtDNA (ND4 gene) copies vs. nuclear DNA (GAPDH gene) was measured by real-time PCR. The content of mtDNA with mutations in murine blood plasma remained high within one month after irradiation but varied with time. The measurements were performed on days 1, 4, 8, 14, and 28 after irradiation, and the maximum level was detected on day 14. The elevated content of extracellular mutant mtDNA in blood plasma of X-irradiated mice is a sensitive candidate biomarker for the assessment of radiation injury and effects of other genotoxic agents.     

Mutation detection using Surveyor nuclease

We have developed a simple and flexible mutation detection technology for the discovery and mapping of both known and unknown mutations. This technology is based on a new mismatch-specific DNA endonuclease from celery, Surveyor nuclease, which is a member of the CEL nuclease family of plant DNA endonucleases. Surveyor nuclease cleaves with high specificity at the 3' side of any mismatch site in both DNA strands, including all base substitutions and insertion/deletions up to at least 12 nucleotides. Surveyor nuclease technology involves four steps: (i) PCR to amplify target DNA from both mutant and wild-type reference DNA; (ii) hybridization to form heteroduplexes between mutant and wild-type reference DNA; (iii) treatment of annealed DNA with Surveyor nuclease to cleave heteroduplexes; and (iv) analysis of digested DNA products using the detection/separation platform of choice. The technology is highly sensitive, detecting rare mutants present at as low as 1 in 32 copies. Unlabeled Surveyor nuclease digestion products can be analyzed using conventional gel electrophoresis or high-performance liquid chromatography (HPLC), while end labeled digestion products are suitable for analysis by automated gel or capillary electrophoresis. The entire protocol can be performed in less than a day and is suitable for automated and high-throughput procedures.     

Release of mtDNA from mitochondria and activation of its replication in tissues of irradiated mice

A nessessary condition for normal functioning of mitochondria is the maintenance of certain numbers of intact mtDNA molecules. In the present study, we investigasted changes in the number of mtDNA copies in brain and spleen cells of mice subjected to irradiation. For the first time, we observed the irradiation-induced output of mtDNA fragments into brain and spleen cell cytosol. In the cytosol of these cells, examined in mice 5 h after 5 Gy irradiation, 1841 h.p. mtDNA fragments were detected able to persist for at 3 weeks. In addition, larger fragments of mtDNA (10,090 b.p.) were detected in the cytosol of brain cells of irradiated mice. The occurrence of mtDNA fragments in the cytosol of brain cells is accompanied with an increase in the number of mtDNA copies in the mitochondrial matrix. The induction of mtDNA replication in brain cells of irradiated animals may be considered as a compensatory reaction in response to mtDNA damage. A sharp decrease in the amount of mtDNA copies in the mitochondrial matrix of spleen cells on the first day after irradiation may be considered as apoptosis development. However, the compensatory reaction in brain cells was also noticed but in later terms.     

CELⅠ酶的粗提取及其活性检测

CEL I酶是第一个从真核生物中提取的用于高效特异切割DNA双链碱基错配和DNA扭曲的内切酶, 因而也是TILLING技术中用到的一种关键酶。文章对CELI酶的粗提取及其活性检测进行了研究。错配切割实验表明, CELI酶在包含有G→A点突变的杂合双链中, 能有效地在错配位点进行切割, 并可以通过ABI377测序仪获得直观的检测结果, 从而可以用于TILLING分析。     

Regional variation in mitochondrial DNA copy number in mouse brain

Mitochondria have their own DNA (mitochondrial DNA [mtDNA]). Although mtDNA copy number is dependent on tissues and its decrease is associated with various neuromuscular diseases, detailed distribution of mtDNA copies in the brain remains uncertain. Using real-time quantitative PCR assay, we examined regional variation in mtDNA copy number in 39 brain regions of male mice. A significant regional difference in mtDNA copy number was observed (P<4.8×10(-35)). High levels of mtDNA copies were found in the ventral tegmental area and substantia nigra, two major nuclei containing dopaminergic neurons. In contrast, cerebellar vermis and lobes had significantly lower copy numbers than other regions. Hippocampal dentate gyrus also had a relatively low mtDNA copy number. This study is the first quantitative analysis of regional variation in mtDNA copy number in mouse brain. Our findings are important for the physiological and pathophysiological studies of mtDNA in the brain.     

The use of temporal temperature gradient gel electrophoresis to reveal mutations in peripheral blood mitochondrial DNA

The mutations in mitochondrial DNA (mtDNA) arise at a higher frequency than in nuclear DNA, and their appearance in peripheral blood can be considered as a sensitive marker to estimate the level of genotoxic load. For revealing the presence of mutations in mtDNA of peripheral blood, we used the method of temporal temperature gradient gel electrophoresis (TTGE). The samples of whole blood DNA from four donor groups were used. Group I contained 10 young (23-26 years) donors and Group II 12 elderly (65-74 years) donors. Group III was formed from patients with breast cancer (12 women) past sessions of radio-chemotherapies (RCHT). Group IV was made of professionals of a nucleus plant occupationally exposed to chronic gamma-irradiation. PCR was carried out on four coding sequences and on one hypervariable sequence of the D-loop (DloopI) of mtDNA. PCR products were tested with TTGE. Most mutations were revealed in the DloopI. Heteroplasmy in the region of DloopI was registered in the blood of each donor of Group III 7 days after the RCHT session. Also, mutations in mtDNA Dloop1 were found in 6 of 13 individuals of Group IV. The blood of this donor group was taken 16 to 28 years after prolonged irradiations in a dose range of 250-350 cGy. In the elderly donor group, the same results were observed in 3 of 12 individuals. The results show that the method of TTGE can be used in mass analyses to assess the effects of radiation and other genotoxic agents in man by detection of unknown mutations in peripheral blood mtDNA.     

The increase in mitochondrial DNA copy number in the tissues of gamma-irradiated mice

Changes in the number of mitochondrial DNA (mtDNA) copies in the brain and spleen tissues of gamma-irradiated (3 Gy) mice were studied by comparative analysis of the long-extension PCR products of mtDNA (15.9 kb) and a fragment of the cluster nuclear beta-globin gene (8.7 kb) amplified simultaneously in one and the same test-tube within total DNA. The analysis showed that, compared to the nuclear beta-globin gene, an increase in mtDNA copy number (polyploidization) took place in the brain and spleen cells of mice exposed to gamma-radiation. This data led to the suggestion that the major mechanism for maintenance of the mitochondrial genome, which is constantly damaged by endogenous ROS and easily affected by ionizing radiation or other exogenous factors, is the induction of synthesis of new mtDNA copies on intact or little affected mtDNA templates because the repair systems in the mitochondria function at a low level of efficiency.     

Incision at nucleotide insertions/deletions and base pair mismatches by the SP nuclease of spinach

Spinach leaves contain a highly active nuclease called SP. The purified enzyme incises single-stranded DNA, RNA, and double-stranded DNA that has been destabilized by A-T-rich regions and DNA lesions [Strickland et al. (1991) Biochemistry 30, 9749-9756]. This broad range of activity has suggested that SP may be similar to a family of nucleases represented by S1, P1, and the mung bean nuclease. However, unlike these single-stranded nucleases that require acidic pH and low ionic strength conditions, SP has a neutral pH optimum and is active over a wide range of salt concentrations. We have extended these findings and showed that an outstanding substrate for SP is a mismatched DNA duplex. For base-substitution mismatches, SP incises at all mismatches except those containing a guanine residue. SP also cuts at insertion/deletions of one or more nucleotides. Where the extrahelical DNA loop contains one nucleotide, the preference of extrahelical nucleotide is A > T approximately C but undetectable at G. The inability of SP to cut at guanine residues and the favoring of A-T-rich regions distinguish SP from the CEL I family of neutral pH mismatch endonucleases recently discovered in celery and other plants [Oleykowski et al. (1998) Nucleic Acids Res. 26, 4597-4602]. SP, like CEL I, does not turn over after incision at a mismatched site in vitro. Similar to CEL I, the presence of a DNA polymerase or a DNA ligase allows SP to turn over and stimulate its activity in vitro by about 20-fold. The possibility that the SP nuclease may be a natural variant of the CEL I family of mismatch endonucleases is discussed.     

Increase of mitochondrial DNA copies with low level of DNA repair in tissue cells of gamma-irradiated mice

The damage and the change in the number of mitochondrial DNA (mtDNA) copies in brain and spleen tissues of gamma-irradiated mice were studied. The changes in the number of mitochondrial DNA (mtDNA) copies were assayed by the comparative analysis of the density values of long-extension PCR products of the mtDNA fragments (16 kb) and the cluster nuclear gene of beta-globin (8.7 kb). PCRs of mtDNA fragments and the nuclear gene of beta-globin were carried out simultaneously in one test-tube within total DNA. Our results showed that in brain and in spleen cells of mice exposed to gamma-radiation an increase in copy number (polyploidization) of mtDNA with regard to the nuclear gene beta-globin took place. The induction of polyploidization of mtDNA observed in cells of gamma-irradiated animals is regarded as the development of a compensatory reaction because of the energy deficiency due to the increased ATP consumption and structural alteration of genes controlling OXPHOS. The data enabled the assumption that because of the low efficiency of repair systems in mitochondria the induction of synthesis of new mtDNA copies on intact or little affected mtDNA templates may be the major mechanism for the retention of the mitochondrial genome which is constantly damaged by the endogenous ROS and is affected by ionizing radiation and/or other exogenous factors.     

Reactive oxygen species stimulate mitochondrial allele segregation toward homoplasmy in human cells

Mitochondria that contain a mixture of mutant and wild-type mitochondrial (mt) DNA copies are heteroplasmic. In humans, homoplasmy is restored during early oogenesis and reprogramming of somatic cells, but the mechanism of mt-allele segregation remains unknown. In budding yeast, homoplasmy is restored by head-to-tail concatemer formation in mother cells by reactive oxygen species (ROS)–induced rolling-circle replication and selective transmission of concatemers to daughter cells, but this mechanism is not obvious in higher eukaryotes. Here, using heteroplasmic m.3243A > G primary fibroblast cells derived from MELAS patients treated with hydrogen peroxide (H₂O₂), we show that an optimal ROS level promotes mt-allele segregation toward wild-type and mutant mtDNA homoplasmy. Enhanced ROS level reduced the amount of intact mtDNA replication templates but increased linear tandem multimers linked by head-to-tail unit-sized mtDNA (mtDNA concatemers). ROS-triggered mt-allele segregation correlated with mtDNA-concatemer production and enabled transmission of multiple identical mt-genome copies as a single unit. Our results support a mechanism by which mt-allele segregation toward mt-homoplasmy is mediated by concatemers.     

Bile salt-stimulated carboxyl ester lipase influences lipoprotein assembly and secretion in intestine: a process mediated via ceramide hydrolysis.

Bile salt-stimulated carboxyl ester lipase (CEL), also called cholesterol esterase, is one of the major proteins secreted by the pancreas. The physiological role of CEL was originally thought to be its mediation of dietary cholesterol absorption. However, recent studies showed no difference between wild type and CEL knockout mice in the total amount of cholesterol absorbed in a single meal. The current study tests the hypothesis that CEL in the intestinal lumen may influence the type of lipoproteins produced. A lipid emulsion containing 4 mm phospholipid, 13.33 mm [(3)H]triolein, and 2.6 mm [(14)C]cholesterol in 19 mm taurocholate was infused into the duodenum of lymph fistula CEL(+/+) and CEL(-/-) mice at a rate of 0.3 ml/h. Results showed no difference between CEL(+/+) and CEL(-/-) mice in the rate of cholesterol and triglyceride transport from the intestinal lumen to the lymph. However, CEL(-/-) mice produced predominantly smaller lipoproteins, whereas the CEL(+/+) mice produced primarily large chylomicrons and very low density lipoprotein. The proximal intestine of CEL(-/-) mice was also found to possess significantly less ceramide hydrolytic activity than that present in CEL(+/+) mice. By using Caco2 cells grown on Transwell membranes as a model, sphingomyelinase treatment inhibited the secretion of larger chylomicron-like lipoproteins without affecting total cholesterol secretion. In contrast, the addition of CEL to the apical medium increased the amount of large lipoproteins produced and alleviated the inhibition induced by sphingomyelinase. Taken together, this study identified a novel and physiologically significant role for CEL, namely the promotion of large chylomicron production in the intestine. The mechanism appears to be mediated through CEL hydrolysis of ceramide generated during the lipid absorption process.     

Purification, cloning, and characterization of the CEL I nuclease

CEL I, isolated from celery, is the first eukaryotic nuclease known that cleaves DNA with high specificity at sites of base-substitution mismatch and DNA distortion. The enzyme requires Mg(2+) and Zn(2+) for activity, with a pH optimum at neutral pH. We have purified CEL I 33 000-fold to apparent homogeneity. A key improvement is the use of alpha-methyl-mannoside in the purification buffers to overcome the aggregation of glycoproteins with endogenous lectins. The SDS gel electrophoresis band for the homogeneous CEL I, with and without the removal of its carbohydrate moieties, was extracted, renatured, and shown to have mismatch cutting specificity. After determination of the amino acid sequence of 28% of the CEL I polypeptide, we cloned the CEL I cDNA. Potential orthologs are nucleases putatively encoded by the genes BFN1 of Arabidopsis, ZEN1 of Zinnia, and DSA6 of daylily. Homologies of CEL I with S1 and P1 nucleases are much lower. We propose that CEL I exemplifies a new family of neutral pH optimum, magnesium-stimulated, mismatch duplex-recognizing nucleases, within the S1 superfamily.     

Assaying the probabilities of obtaining maternally inherited heteroplasmy as the basis for modeling OXPHOS diseases in animals

Gross alterations in cell energy metabolism underlie manifestations of hereditary OXPHOS (oxidative phosphorylation) diseases, many of which depend on proportion of mutant mitochondrial DNA (mtDNA) in tissues. An animal model of OXPHOS disease with maternal inheritance of mitochondrial heteroplasmy might help understanding the peculiarities of abnormal mtDNA distribution and its effect on pre- and postnatal development. Previously we obtained mice that carry human mtDNA in some tissues. It co-existed with murine mtDNA (heteroplasmy) and was transmitted maternally to the progeny of animals developed from zygotes injected with human mitochondria. To analyze the probability of obtaining heteroplasmic mice we increased the number of experiments with early embryos and obtained more specimens from F1. About 33% of zygotes injected with human mtDNA developed into post-implantation embryos (7th-13th days). Lower amount of such developed into neonate mice (ca. 21%). Among post-implantation embryos and in generations F0 and F1 percentages of human mtDNA-carriers were ca. 14-16%. Such percentages are sufficient for modeling maternally inherited heteroplasmy in small animal groups. More data are needed to understand the regularities of anomalous mtDNA distribution among cells and tissues and whether heart and muscles frequently carrying human mtDNA in our experiments are particularly susceptible to heteroplasmy.     

Mitochondrial Nucleases ENDOG and EXOG Participate in Mitochondrial DNA Depletion Initiated by Herpes Simplex Virus 1 UL12.5

Herpes simplex virus 1 (HSV-1) rapidly eliminates mitochondrial DNA (mtDNA) from infected cells, an effect that is mediated by UL12.5, a mitochondrial isoform of the viral alkaline nuclease UL12. Our initial hypothesis was that UL12.5 directly degrades mtDNA via its nuclease activity. However, we show here that the nuclease activities of UL12.5 are not required for mtDNA loss. This observation led us to examine whether cellular nucleases mediate the mtDNA loss provoked by UL12.5. We provide evidence that the mitochondrial nucleases endonuclease G (ENDOG) and endonuclease G-like 1 (EXOG) play key redundant roles in UL12.5-mediated mtDNA depletion. Overall, our data indicate that UL12.5 deploys cellular proteins, including ENDOG and EXOG, to destroy mtDNA and contribute to a growing body of literature highlighting roles for ENDOG and EXOG in mtDNA maintenance.     

The control of mtDNA replication during differentiation and development

Background

Mitochondrial DNA (mtDNA) is important for energy production as it encodes some of the key genes of electron transfer chain, where the majority of cellular energy is generated through oxidative phosphorylation (OXPHOS). MtDNA replication is mediated by nuclear DNA-encoded proteins or enzymes, which translocate to the mitochondria, and is strictly regulated throughout development. It starts with approximately 200 copies in each primordial germ cell and these copies undergo expansion and restriction events at various stages of development.

Scope of review

I describe the patterns of mtDNA replication at key stages of development. I explain that it is essential to regulate mtDNA copy number and to establish the mtDNA set point in order that the mature, specialised cell acquires the appropriate numbers of mtDNA copy to generate sufficient adenosine triphosphate (ATP) through OXPHOS to undertake its specialised function. I discuss how these processes are dependent on the controlled expression of the nuclear-encoded mtDNA-specific replication factors and that this can be modulated by mtDNA haplotypes. I discuss how these events are altered by certain assisted reproductive technologies, some of which have been proposed to prevent the transmission of mutant mtDNA and others to overcome infertility. Furthermore, some of these technologies are predisposed to transmitting two or more populations of mtDNA, which can be extremely harmful.

Major conclusions

The failure to regulate mtDNA replication and mtDNA transmission during development is disadvantageous.

General significance

Manipulation of oocytes and embryos can lead to significant implications for the maternal-only transmission of mtDNA.This article is part of a Special Issue entitled Frontiers of mitochondrial research.     

Metabolically induced heteroplasmy shifting and l-arginine treatment reduce the energetic defect in a neuronal-like model of MELAS

The m.3243A>G variant in the mitochondrial tRNA(Leu(UUR)) gene is a common mitochondrial DNA (mtDNA) mutation. Phenotypic manifestations depend mainly on the heteroplasmy, i.e. the ratio of mutant to normal mtDNA copies. A high percentage of mutant mtDNA is associated with a severe, life-threatening neurological syndrome known as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). MELAS is described as a neurovascular disorder primarily affecting the brain and blood vessels, but the pathophysiology of the disease is poorly understood. We developed a series of cybrid cell lines at two different mutant loads: 70% and 100% in the nuclear background of a neuroblastoma cell line (SH-SY5Y). We investigated the impact of the mutation on the metabolism and mitochondrial respiratory chain activity of the cybrids. The m.3243A>G mitochondrial mutation induced a metabolic switch towards glycolysis in the neuronal cells and produced severe defects in respiratory chain assembly and activity. We used two strategies to compensate for the biochemical defects in the mutant cells: one consisted of lowering the glucose content in the culture medium, and the other involved the addition of l-arginine. The reduction of glucose significantly shifted the 100% mutant cells towards the wild-type, reaching a 90% mutant level and restoring respiratory chain complex assembly. The addition of l-arginine, a nitric oxide (NO) donor, improved complex I activity in the mutant cells in which the defective NO metabolism had led to a relative shortage of NO. Thus, metabolically induced heteroplasmy shifting and l-arginine therapy may constitute promising therapeutic strategies against MELAS.     

Mutation detection using a novel plant endonuclease.

We have discovered a useful new reagent for mutation detection, a novel nuclease CEL I from celery. It is specific for DNA distortions and mismatches from pH 6 to 9. Incision is on the 3'-side of the mismatch site in one of the two DNA strands in a heteroduplex. CEL I-like nucleases are found in many plants. We report here that a simple method of enzyme mutation detection using CEL I can efficiently identify mutations and polymorphisms. To illustrate the efficacy of this approach, the exons of the BRCA1 gene were amplified by PCR using primers 5'-labeled with fluorescent dyes of two colors. The PCR products were annealed to form heteroduplexes and subjected to CEL I incision. In GeneScan analyses with a PE Applied Biosystems automated DNA sequencer, two independent incision events, one in each strand, produce truncated fragments of two colors that complement each other to confirm the position of the mismatch. CEL I can detect 100% of the sequence variants present, including deletions, insertions and missense alterations. Our results indicate that CEL I mutation detection is a highly sensitive method for detecting both polymorphisms and disease-causing mutations in DNA fragments as long as 1120 bp in length.     

Myelin Proteolipid Protein Gene Expression in Jimpy and Jimpymsd Mice

Proteolipid protein (PLP) gene expression was studied in the dysmyelinating mouse mutant jimpy(msd) (jpmsd; myelin synthesis deficient) and compared with that in wild-type mice and the allelic mutant, jimpy (jp). Southern analyses of genomic DNA from jpmsd mice revealed no major rearrangements of the PLP gene relative to the wild-type mouse PLP gene. PLP-specific mRNA levels were significantly reduced in these mutant mice, although both the 3.2- and 2.4-kilobase PLP-specific mRNAs were seen. Also, no size differences in either PLP or DM20 mRNAs were found by S1 nuclease assays of brain RNA from either jpmsd or wild-type mice. Both PLP and DM20 protein were detectable at low levels in jpmsd brain homogenates, and these proteins comigrated with PLP and DM20 protein from normal mice. Western analyses showed an altered PLP:DM20 ratio in jpmsd mice relative to wild-type mice; DM20 levels exceeded PLP levels. It is surprising that a similar pattern of expression was seen in normal mice at less than 10 days of age: DM20 protein expression preceding PLP expression. Thus, jpmsd mice are capable of synthesizing normal PLP and DM20 protein; however, the PLP gene defect has affected the normal developmental pattern of expression for these two proteins.