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. 2002 Jul;22(14):5212-21.
doi: 10.1128/MCB.22.14.5212-5221.2002.

Targeted deletion of both thymidine phosphorylase and uridine phosphorylase and consequent disorders in mice

Misako Haraguchi  1 , Hiroaki Tsujimoto, Masakazu Fukushima, Itsuro Higuchi, Hideto Kuribayashi, Hideo Utsumi, Atsuo Nakayama, Yoshio Hashizume, Junko Hirato, Hiroki Yoshida, Hiromitsu Hara, Shinjiro Hamano, Hiroaki Kawaguchi, Tatsuhiko Furukawa, Kohei Miyazono, Fuyuki Ishikawa, Hideo Toyoshima, Tadashi Kaname, Masaharu Komatsu, Zhe-Sheng Chen, Takenari Gotanda, Tokushi Tachiwada, Tomoyuki Sumizawa, Kazutaka Miyadera, Mitsuhiro Osame, Hiroki Yoshida, Tetsuo Noda, Yuji Yamada, Shin-ichi Akiyama
Affiliations

Targeted deletion of both thymidine phosphorylase and uridine phosphorylase and consequent disorders in mice

Misako Haraguchi et al. Mol Cell Biol. 2002 Jul.

Abstract

Thymidine phosphorylase (TP) regulates intracellular and plasma thymidine levels. TP deficiency is hypothesized to (i) increase levels of thymidine in plasma, (ii) lead to mitochondrial DNA alterations, and (iii) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In order to elucidate the physiological roles of TP, we generated mice deficient in the TP gene. Although TP activity in the liver was inhibited in these mice, it was fully maintained in the small intestine. Murine uridine phosphorylase (UP), unlike human UP, cleaves thymidine, as well as uridine. We therefore generated TP-UP double-knockout (TP(-/-) UP(-/-)) mice. TP activities were inhibited in TP(-/-) UP(-/-) mice, and the level of thymidine in the plasma of TP(-/-) UP(-/-) mice was higher than for TP(-/-) mice. Unexpectedly, we could not observe alterations of mitochondrial DNA or pathological changes in the muscles of the TP(-/-) UP(-/-) mice, even when these mice were fed thymidine for 7 months. However, we did find hyperintense lesions on magnetic resonance T(2) maps in the brain and axonal edema by electron microscopic study of the brain in TP(-/-) UP(-/-) mice. These findings suggested that the inhibition of TP activity caused the elevation of pyrimidine levels in plasma and consequent axonal swelling in the brains of mice. Since lesions in the brain do not appear to be due to mitochondrial alterations and pathological changes in the muscle were not found, this model will provide further insights into the causes of MNGIE.

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Figures

FIG. 1.
FIG. 1.
Targeted disruption of the TP gene. (a) Schematic representation of the murine TP locus (top), the targeting vector (middle), and the mutated allele (bottom). The closed boxes denote the 10 exons of the TP gene. The targeting vector was designed to replace an exon encoding a portion of the phosphate- or deoxyribose-binding site (top [K or L]) with a neomycin resistance gene. The position of the 3′-flanking probe used for Southern blot analysis is indicated. Positions of the PCR primers for the wild-type allele (TPf and TPr) and the mutant allele (TPf and neo) are also shown. S, n, N, A, and C represent the SpeI, NheI, NdeI, AflII, and ClaI sites, respectively. (b) Southern blot analysis of wild-type and mutant ES cell DNA. Genomic DNA from wild-type (+/+) and TP+/− (+/−) ES cell clones were digested with NheI (left) or NdeI (right) and hybridized with the 3′-flanking probe. The 10-kb wild-type fragment (WT) and the 15-kb mutant fragment (MT) obtained after NheI digestion are indicated on the left. The wild-type allele produced an 8.5-kb NdeI digestion product, whereas the mutant allele gave rise to an 11-kb hybridizing product (right). (c) PCR analysis of mouse tail DNA from the wild-type allele (+/+), heterozygous TP-null allele (+/−), and homozygous TP-null allele (−/−). Oligonucleotides TPf and TPr (see panel a) were used as primers for the upstream and downstream disruption sites, respectively. Oligonucleotides TPf and Neo were used as primers for the knockout allele. The amplified PCR products were analyzed on 1% agarose gels to separate the 640-bp wild-type (WT) and 400-bp TP-null (Null) targeted allele fragments. (d) Reverse transcription-PCR analysis of TP mRNA in the livers of wild-type mice (+/+), TP+/− (+/−) mice, and TP−/− (−/−) mice. The total RNA was isolated from the liver and reverse transcribed. The resulting cDNA was used for PCR analysis with specific primers for TP and for the murine glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene. A 300-bp band is amplified by the TP-specific primers (TP), and a 950-bp band represents mRNA for G3PDH.
FIG. 2.
FIG. 2.
Generation of mice with targeted deletions of both TP and UP. (a) Levels of F3dThd in plasma in mice after administration of F3dThd. F3dThd (100 mg/kg) was administered orally to TP+/+ and TP−/− mice. The concentration of F3dThd in plasma was determined as described previously (7). Statistical significance was determined by using the Student's t test (mean ± the standard error; n = 3). (b) TP activity in various tissues from wild-type, TP−/−, UP−/−, and TP−/− UP−/− mice. The rate of conversion of thymidine to thymine was measured radiometrically as described previously (6). TP activity is expressed as nanomoles of thymidine catalyzed to thymine per minute per milligram of protein. Tissues were collected from three 2-month-old mice. (c) PCR analysis of mouse tail DNA from animals with the following six phenotypes: wild-type allele (TP+/+ UP+/+), heterozygous TP-null allele (TP+/− UP+/+), homozygous TP-null allele (TP−/− UP+/+), heterozygous UP-null allele (TP+/+ UP+/−), homozygous UP-null allele (TP+/+UP−/−), heterozygous TP or UP allele (TP+/− UP+/−), and homozygous TP- and UP-null allele (TP−/− UP−/−). The sizes of the amplified PCR products were 210 bp for UP wild type, 640 bp for TP wild type, 313 bp for the UP targeted allele, and 400 bp for the TP targeted allele fragments. (d) Five female mice at 5 months of age were injected intraperitoneally with pentobarbital. The time during which postural reflex was lost was measured.
FIG. 3.
FIG. 3.
Elevated concentrations of thymidine in the plasma and brains of TP−/− UP−/− mice. (a) Concentrations of pyrimidine nucleosides in the plasma of wild-type, TP−/−, and TP−/− UP−/− mice. Blood was collected from five mice at the age of 2 months and centrifuged to obtain the plasma. An aliquot (0.2 ml) of mouse plasma was applied to a high-pressure liquid chromatography column. The concentrations of thymidine, uridine, and cytidine in plasma were then measured. (b) Concentrations of thymidine in the plasma and brains of wild-type mice, TP−/− UP−/− mice, and thymidine-fed mice (indicated as “wild-type+” and “TP−/− UP−/−+”).
FIG. 4.
FIG. 4.
Lack of significant features of MNGIE in the muscles of TP−/− UP−/− mice. The morphology of serial sections of muscles from 12-month-old wild-type (TP+/+) mice (a and b) and TP−/− mice (c and d) and 10-month-old TP−/− UP−/− mice (e and f) and thymidine-fed TP−/− UP−/− mice (g and h) was examined. Histochemical staining of soleus muscle or EDL sections was done with modified Gomori trichrome (GT; shown in panels b, d, f, and h), and histochemical staining was done to detect COX (shown in panels a, c, e, and g). (i) COX activities in the lysates of leg muscles and livers from three 2-month-old wild-type and TP−/− mice were measured. (j and k) Southern blot (j) and PCR (k) analyses of mtDNA. The total DNAs from the muscles of wild-type mice (lanes 1 and 2), TP−/− UP−/− mice (lanes 3 and 4), and thymidine-fed (dThd+) mice (wild type, lanes 5 and 6; TP−/− UP−/−, lanes 7 and 8) were tested to measure the amount of mtDNA.
FIG. 5.
FIG. 5.
Abnormalities of the brains as shown by MRI and electron micrographic studies. In the magnetic resonance images, the T2 maps at 9.4 T at the level of lateral ventricles in wild-type (a) and TP−/− UP−/− (b and c) mice are shown. The gray scale for T2 maps ranges from 0 to 80 ms. (d to f) Light microscopic sections of the brains from the wild-type and TP−/− UP−/− mice brain were examined by toluidine blue (TB) staining. (g to i) Ultrastructures of myelinated fibers as viewed by electron microscopy (EM). The enlarged myelinated fibers were conspicuous in TP−/− UP−/− mouse brains (see panels e, f, h, and i). Magnification, ×2,000. (j and k) Southern blot (j) and PCR (k) analyses of mtDNA. The total DNAs from the muscles of wild-type mice (lanes 1 and 2), TP−/− UP −/− mice (lanes 3 and 4), and thymidine-fed (dThd+) mice (wild type, lanes 5 and 6; TP−/− UP−/−, lanes 7 and 8) were tested to measure the amount of mtDNA.
FIG. 6.
FIG. 6.
The TP gene overlaps with the SCO2 gene. (a) Chromosomal positions of the human TP (NM_001953) and human SCO2 (XM_017992) genes. The best match of these genes determined by a BLAST search against the human genome sequence was a chromosome 22 working draft sequence segment (HS22_11683). The TP gene lies between positions 45508430 and 45504150, and the SCO2 gene lies between positions 45503950 and 45501950 within chromosome 22q13.33. (b) Schematic diagram of the mouse TP and SCO2 gene structures. Schematic representation of the murine TP gene (top), TP cDNA (middle), and murine SCO2 cDNA (bottom). The coding regions of both cDNAs are shown as black boxes, and noncoding regions are shown as white boxes. The cDNA sequence of SCO2 shares exons 9 and 10 of TP, indicated as white boxes (noncoding region) and a black box (coding region). In addition to these sequences, SCO2 cDNA has a unique sequence in the 5′-noncoding region as indicated by a gray box.
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