Purkinje cell degeneration in mice lacking the xeroderma pigmentosum group G gene.

Laboratory mice carrying the nonfunctional xeroderma pigmentosum group G gene (the mouse counterpart of the human XPG gene) alleles have been generated by using gene-targeting and embryonic stem cell technology. Homozygote animals of this autosomal recessive disease exhibited signs and symptoms, such as postnatal growth retardation, reduced levels of activity, progressive ataxia and premature death, similar to the clinical manifestations of Cockayne syndrome (CS). Histological analysis of the cerebellum revealed multiple pyknotic cells in the Purkinje cell layer of the xpg homozygotes, which had atrophic cell bodies and shrunken nuclei. Further examination by an immunohistochemistry for calbindin-D 28k (CaBP) showed that a large number of immunoreactive Purkinje cells were atrophic and their dendritic trees were smaller and shorter than in wild-type littermates. These results indicated a marked degeneration of Purkinje cells in the xpg mutant cerebellum. Study by in situ detection of DNA fragmentation in the cerebellar cortex demonstrated that some deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin in situ nick labeling (TUNEL)-positive cells appeared in the granule layer of the mutant mice, but few cell deaths were confirmed in the Purkinje layer. These results suggested Purkinje cell degeneration in the mutant cerebellum was underway, in which much Purkinje cell death had not appeared, and the appearance of some abnormal cerebellar symptoms in the xpg-deficient mice was not only due to a marked Purkinje cell degeneration, but also to damage of other cells.

Postnatal growth failure, short life span, and early onset of cellular senescence and subsequent immortalization in mice lacking the xeroderma pigmentosum group G gene.

The xeroderma pigmentosum group G (XP-G) gene (XPG) encodes a structure-specific DNA endonuclease that functions in nucleotide excision repair (NER). XP-G patients show various symptoms, ranging from mild cutaneous abnormalities to severe dermatological impairments. In some cases, patients exhibit growth failure and life-shortening and neurological dysfunctions, which are characteristics of Cockayne syndrome (CS). The known XPG protein function as the 3' nuclease in NER, however, cannot explain the development of CS in certain XP-G patients. To gain an insight into the functions of the XPG protein, we have generated and examined mice lacking xpg (the mouse counterpart of the human XPG gene) alleles. The xpg-deficient mice exhibited postnatal growth failure and underwent premature death. Since XPA-deficient mice, which are totally defective in NER, do not show such symptoms, our data indicate that XPG performs an additional function(s) besides its role in NER. Our in vitro studies showed that primary embryonic fibroblasts isolated from the xpg-deficient mice underwent premature senescence and exhibited the early onset of immortalization and accumulation of p53.

Chromosomal localization of the mouse and rat DNA double-strand break repair genes Ku p70 and Ku p80/XRCC5 and their mRNA expression in various mouse tissues.

The Ku p70 and Ku p80/XRCC5 genes are involved in DNA double-strand break repair and V(D)J recombination, and their gene products are the components of the DNA-dependent protein kinase. We have determined the chromosomal locations of the mouse Ku p70 and Ku p80/XRCC5 genes by both in situ hybridization and molecular linkage analysis: the Ku p70 gene was localized to mouse chromosome 15 and rat chromosome 7, and the Ku p80/XRCC5 gene was localized to mouse chromosome 1 and rat chromosome 9. Both genes were mapped to a region of conserved linkage homology among three species, i.e., the mouse, rat, and human. Molecular linkage analysis using interspecific backcross mice revealed that the murine Ku p70 locus was localized 0.7 cM terminal to D15Mit1 and that the murine Ku p80/XRCC5 locus was 0.7 cM proximal to D1Mit46. To determine the size and tissue transcription specificity of the mouse Ku p70 and Ku p80/XRCC5 mRNA, Northern blot analysis was carried out with six mouse tissues. Each tissue expressed one species of the Ku p70 gene transcript with 2.4 kb and one species of the Ku p80/XRCC5 gene transcript with 2.6 kb. In the latter case, however, the brain showed two sizes of transcript, 2.6 and 2.9 kb.

Complementary DNA sequence and chromosomal localization of xpg, the mouse counterpart of human repair gene XPG/ERCC5.

We have molecularly cloned and sequenced the mouse counterpart of the human repair gene XPG/ERCC5 cDNA. The mouse xpg cDNA had a single long open reading frame predicted to encode 1170 amino acid residues (predicted M(r) of 130,753). Northern blot analysis has been carried out to determine the size and tissue transcription specificity of the mouse xpg mRNA. The xpg gene expressed one species of transcript with 4.3 kb at similar levels in five mouse tissues examined. We have determined the chromosomal location of the xpg gene by both in situ hybridization and molecular linkage analysis. The xpg gene was localized at 2.3 cM proximal to the microsatellite locus D1Mit18 on the R-positive B band of mouse chromosome 1. By in situ hybridization with the mouse xpg probe, the rat homolog of the mouse xpg was localized on q22.3 band of rat chromosome 9, which has been known to have a conserved linkage homology to mouse chromosome 1. In the case of human, the XPG/ERCC5 gene has been reported to be assigned to human chromosome 13q32.3-q33.1, where any conserved linkage homology to mouse chromosome 1 has not been found so far. Thus, these results show new regions of conserved linkage homology among mouse chromosome 1, rat chromosome 9, and human chromosome 13q.

Location of the mouse complement factor H gene (cfh) by FISH analysis and replication R-banding.

A technique for replication R- and G-banding of mouse lymphocyte chromosomes was developed, and the replication R-banding pattern was analyzed. Optimal banding patterns were obtained with thymidine- and BrdU-treatment of lymphocytes in the same cell cycle. This produced replication R-band patterns that were the complete reverse of the G-band patterns on all chromosomes. Replication R-banding methods can be used in conjunction with nonisotopic, fluorescence in situ hybridization (FISH) to localize cloned probes to specific chromosomal bands on mouse chromosomes. with these methods the mouse complement factor H gene (cfh) was localized to the terminal portion of the F region of Chromosome 1. Q-banding patterns were also obtained by the replication R-banding method and may be useful for rapid identification of each chromosome.

A gene locus controlling a serum protein migrating electrophoretically in the beta region of mice and detected by using a strain derived from the Japanese wild mouse (Mus musculus molossinus).

Antigenic specificities of serum proteins from the MOL-ANJ strain of mice (a strain derived from Japanese wild mice, Mus musculus molossinus) were studied by gel precipitation with alloantisera produced by reciprocal alloimmunization between MOL-ANJ and BALB/c mice. An alloantigen which migrates immunoelectrophoretically in the beta region of serum proteins has been identified. Evidence indicates that this antigenic specificity is controlled by a co-dominant autosomal gene locus designated by the symbol Sas-2. The evidence also suggests that Sas-2 is genetically different from the previously described Sas-1 which controls a serum protein in the mouse. Sas-2 was located by linkage analysis between Idh-1 locus and Akp-1 locus on chromosome 1.