Structure of the Mutant Fibrillin-1 Gene in the Tight Skin (TSK) Mouse

Mice carrying the tight skin (TSK) mutation harbors a 3.0-kbgenomic duplication (exons 17–40) of the fibrillin-1 gene(Fbn-1) locatedon band F of chromosome 2 as TSK mutation. Wecloned and sequenced the mutated Fbn-1 gene, since it is believedto be responsible for TSK syndrome. Sequence analysis showednumerous amino aciddifferences in the 5' and 3' segments betweenthe TSK mutation and wild-type fbn-1 gene, but any amino aciddifference between the TSK mutation and C57BL/6 mice. (TSK andC57Bl/6 mice are genetically similar, differing only by TSKmutation.) Four amino acid differences were observed betweentwo copies of TSK's fbn-1 gene encoded by exons 17–40.Our results suggest that the majority of structural differencesoccurred in the N and C termini segments during strain divergenceand only a few after the duplication event.     

Effect of targeted mutation in collagen V alpha 2 gene on development of cutaneous hyperplasia in tight skin mice.

Collagen V plays a major regulatory role in the formation of heterotypic fibers of the dermis and cartilaginous tissues as well as in the assembly of extracellular matrix. The pN/pN mouse, which is defective in collagen V alpha 2 gene, exhibits skeletal abnormalities, skin fragility, and alterations in the collagen fiber organization, whereas the TSK/+ mouse, which is defective in fibrillin-1, the major component of microfibrils present in the extracellular matrix, develops cutaneous hyperplasia and autoimmunity. We have studied the role of collagen V in the formation of heterotypic collagen fibers in F1 mice, which are obtained by breeding pN/pN with TSK/+ mice. Our results show that F1 progeny neither develop cutaneous hyperplasia nor produce anti-topoisomerase I autoantibodies, unlike TSK/+ mice. The diameter of the collagen fibrils in the skin is also comparable to that found in control mice. Thus, the phenotypic changes observed in the TSK mouse could be reversed by genetic complementation with a collagen V-defective mouse.     

Intercellular adhesion molecule-1 deficiency attenuates the development of skin fibrosis in tight-skin mice

The tight-skin (TSK/+) mouse, a genetic model for systemic sclerosis, develops cutaneous fibrosis. Although a fibrillin 1 gene mutation and immunological abnormalities have been demonstrated, the roles of adhesion molecules have not been investigated. To directly assess roles of adhesion molecules in skin fibrosis, TSK/+ mice lacking L-selectin and/or ICAM-1 were generated. The deficiency of ICAM-1, but not L-selectin, significantly suppressed ( approximately 48%) the development of skin sclerosis in TSK/+ mice. Similarly, ICAM-1 antisense oligonucleotides inhibited skin fibrosis in TSK/+ mice. Although T cell infiltration was modest into the skin of TSK/+ mice, ICAM-1 deficiency down-regulated this migration, which is consistent with the established roles of endothelial ICAM-1 in leukocyte infiltration. In addition, altered phenotype or function of skin fibroblasts was remarkable and dependent on ICAM-1 expression in TSK/+ mice. ICAM-1 expression was augmented on TSK/+ dermal fibroblasts stimulated with IL-4. Although growth or collagen synthesis of TSK/+ fibroblasts cultured with IL-4 was up-regulated, it was suppressed by the loss or blocking of ICAM-1. Collagen expression was dependent on the strain of fibroblasts, but not on the strain of cocultured T cells. Thus, our findings indicate that ICAM-1 expression contributes to the development of skin fibrosis in TSK/+ mice, especially via ICAM-1 expressed on skin fibroblasts.     

Hepatocyte growth factor ameliorates dermal sclerosis in the tight-skin mouse model of scleroderma

The tight-skin (TSK/+) mouse, a genetic model of systemic sclerosis (SSc), develops cutaneous fibrosis and defects in pulmonary architecture. Because hepatocyte growth factor (HGF) is an important mitogen and morphogen that contributes to the repair process after tissue injury, we investigated the role of HGF in cutaneous fibrosis and pulmonary architecture defects in SSc using TSK/+ mice. TSK/+ mice were injected in the gluteal muscle with either hemagglutinating virus of Japan (HVJ) liposomes containing 8 mug of a human HGF expression vector (HGF-HVJ liposomes) or a mock vector (untreated control). Gene transfer was repeated once weekly for 8 weeks. The effects of HGF gene transfection on the histopathology and expression of tumor growth factor (TGF)-beta and IL-4 mRNA in TSK/+ mice were examined. The effect of recombinant HGF on IL-4 production by TSK/+ CD4+ T cells stimulated by allogeneic dendritic cells (DCs) in vitro was also examined. Histologic analysis revealed that HGF gene transfection in TSK/+ mice resulted in a marked reduction of hypodermal thickness, including the subcutaneous connective tissue layer. The hypodermal thickness of HGF-treated TSK/+ mice was decreased two-fold to three-fold compared with untreated TSK/+ mice. However, TSK/+ associated defects in pulmonary architecture were unaffected by HGF gene transfection. HGF gene transfection significantly inhibited the expression of IL-4 and TGF-beta1 mRNA in the spleen and skin but not in the lung. We also performed a mixed lymphocyte culture and examined the effect of recombinant HGF on the generation of IL-4. Recombinant HGF significantly inhibited IL-4 production in TSK/+ CD4+ T cells stimulated by allogeneic DCs. HGF gene transfection inhibited IL-4 and TGF-beta mRNA expression, which has been postulated to have a major role in fibrinogenesis and reduced hypodermal thickness, including the subcutaneous connective tissue layer of TSK/+ mice. HGF might represent a novel strategy for the treatment of SSc.     

Hepatocyte growth factor ameliorates dermal sclerosis in the tight-skin mouse model of scleroderma

The tight-skin (TSK/+) mouse, a genetic model of systemic sclerosis (SSc), develops cutaneous fibrosis and defects in pulmonary architecture. Because hepatocyte growth factor (HGF) is an important mitogen and morphogen that contributes to the repair process after tissue injury, we investigated the role of HGF in cutaneous fibrosis and pulmonary architecture defects in SSc using TSK/+ mice. TSK/+ mice were injected in the gluteal muscle with either hemagglutinating virus of Japan (HVJ) liposomes containing 8 μg of a human HGF expression vector (HGF-HVJ liposomes) or a mock vector (untreated control). Gene transfer was repeated once weekly for 8 weeks. The effects of HGF gene transfection on the histopathology and expression of tumor growth factor (TGF)-β and IL-4 mRNA in TSK/+ mice were examined. The effect of recombinant HGF on IL-4 production by TSK/+ CD4⁺ T cells stimulated by allogeneic dendritic cells (DCs) in vitro was also examined. Histologic analysis revealed that HGF gene transfection in TSK/+ mice resulted in a marked reduction of hypodermal thickness, including the subcutaneous connective tissue layer. The hypodermal thickness of HGF-treated TSK/+ mice was decreased two-fold to three-fold compared with untreated TSK/+ mice. However, TSK/+ associated defects in pulmonary architecture were unaffected by HGF gene transfection. HGF gene transfection significantly inhibited the expression of IL-4 and TGF-β1 mRNA in the spleen and skin but not in the lung. We also performed a mixed lymphocyte culture and examined the effect of recombinant HGF on the generation of IL-4. Recombinant HGF significantly inhibited IL-4 production in TSK/+ CD4⁺ T cells stimulated by allogeneic DCs. HGF gene transfection inhibited IL-4 and TGF-β mRNA expression, which has been postulated to have a major role in fibrinogenesis and reduced hypodermal thickness, including the subcutaneous connective tissue layer of TSK/+ mice. HGF might represent a novel strategy for the treatment of SSc.     

A missense mutant myostatin causes hyperplasia without hypertrophy in the mouse muscle

Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia.     

Transgenic small interfering RNA halts amyotrophic lateral sclerosis in a mouse model

Many autosomal dominant diseases such as familial amyotrophic lateral sclerosis (ALS) with copper/zinc superoxide dismutase (SOD1) mutation may be induced by missense point mutations that result in the production of proteins with toxic properties. Reduction in the encoding of proteins from such mutated genes can therefore be expected to improve the disease phenotype. The duplex of 21-nucleotide RNA, known as small interfering RNA (siRNA), has recently emerged as a powerful gene silencing tool. We made transgenic (Tg) mice with modified siRNA, which had multiple mismatch alternations within the sense strand, to prevent the "shutdown phenomenon" of transgenic siRNA. Consequently, the in vivo knockdown effect of siRNA on SOD1 expression did not diminish over four generations. When we crossed these anti-SOD1 siRNA Tg mice with SOD1G93A Tg mice, a model for ALS, siRNA prevented the development of disease by inhibiting mutant G93A SOD1 production in the central nervous system. Our findings clearly proved the principle that siRNA-mediated gene silencing can stop the development of familial ALS with SOD1 mutation.     

Introduction of the human recombinant angiogenin at the early stages of postnatal development inhibits the growth of mice

We studied the influence of recombinant DNA containing the cloned angiogenin gene, plasmid DNA without angiogenin gene, and purified recombinant angiogenin injected to Tg8 mice at the age of two days on the body mass of 28- and 40-day old mice. The body mass of mice that were injected with the cloned angiogenin gene or purified angiogenin was less than in the control mice. The body mice of Tg8 mice injected with recombinant DNA containing the cloned angiogenin gene did not increase from day 2 to day 40, while in the mice with purified recombinant angiogenin and control mice it increased by 24 and 57%, respectively. These data suggest that the elevated level of angiogenin at the early developmental stages inhibits the increase of body mass. The effect we described as related, in al likelihood, to the known inhibitory effect of angiogenin on protein synthesis.     

Introduction of Human Recombinant Angiogenin at the Early Stages of Postnatal Development Inhibits the Growth of Mice

We studied the influence of recombinant DNA containing the cloned angiogenin gene, plasmid DNA without angiogenin gene, and purified recombinant angiogenin injected to Tg8 mice at the age of two days on the body mass of 28- and 40-day old mice. The body mass of mice that were injected with the cloned angiogenin gene or purified angiogenin was less than in the control mice. The body mice of Tg8 mice injected with recombinant DNA containing the cloned angiogenin gene did not increase from day 28 to day 40, while in the mice with purified recombinant angiogenin and control mice it increased by 24 and 57%, respectively. These data suggest that the elevated level of angiogenin at the early developmental stages inhibits the increase of body mass. The effect we described is related, in al likelihood, to the known inhibitory effect of angiogenin on protein synthesis.     

Detected microsatellite polymorphisms in genetically altered inbred mouse strains

Microsatellites are 50–200 repetitive DNA sequences composed of 1- to 6-base-pair-long reiterative motifs within the genome. They are vulnerable to DNA modifications, such as recombination and/or integration, and are recognized as “sentinel” DNA. Our previous report indicated that the genotypes of the microsatellite loci could change from mono- to poly-morphisms (CMP) in gene knockout (KO) mice, implying that genetic modification induces microsatellite mutation. However, it is still unclear whether the random insertion of DNA fragments into mice genomes produced via transgene (Tg) or N-ethyl-N-nitrosourea (ENU) would also result in microsatellite mutations or microsatellite loci genotypes changes. This study was designed to find possible clues to answer this question. In brief, 198 microsatellite loci that were distributed among almost all of the chromosomes (except for the Y) were examined through polymerase chain reaction to screen possible CMPs in six Tg strains. First, for each strain, the microsatellite sequences of all loci were compared between Tg and the corresponding background strain to exclude genetic interference. Simultaneously, to exclude spontaneous mutation-related CMPs that might exist in the examined six strains, mice from five spontaneously mutated inbred strains were used as the negative controls. Additionally, the sequences of all loci in these spontaneous mutated mice were compared to corresponding genetic background controls. The results showed that 40 of the 198 (20.2 %) loci were identified as having CMPs in the examined Tg mice strains. The CMP genotypes were either homozygous or heterozygous compared to the background controls. Next, we applied the 40 CMP positive loci in ENU-mutated mice and their corresponding background controls. After that, a general comparison of CMPs that exist among Tg, ENU-treated and KO mouse strains was performed. The results indicated that four (D11mit258, D13mit3, D14mit102 and DXmit172) of the 40 (10 %) CMP loci were shared by Tg and KO mice, two (D15mit5 and D14mit102) (5 %) by Tg and ENU-treated mice, and one (D14mit102) (2.5 %) by all three genetic modifications. Collectively, our study implies that genetic modifications by KO, Tg or chemical mutant can trigger microsatellite CMPs in inbred mouse strains. These shared microsatellite loci could be regarded as “hot spots” of microsatellite mutation for genetic monitoring in genetic modified mice.     

Genome structure of Halobacterium halobium: plasmid dynamics in gas vacuole deficient mutants

Halobacterium halobium contains two gas vacuole protein genes that are located in plasmid pHH1 (p-vac) and in the chromosomal DNA (c-vac). The mutation frequency for these genes is different: the constitutively expressed p-vac gene is mutated with a frequency of 10(-2), while the chromosomal gene expressed in the stationary phase of growth is mutated with a frequency of 10(-5). The difference in the mutation susceptibility is due to the dynamics of plasmid pHH1. p-vac gene mutations are caused (i) by the integration of an insertion element or (ii) by a deletion event encompassing the p-vac gene region. In contrast, c-vac mutants analyzed to date incurred neither insertion elements nor deletions. Deletion events within pHH1 occur at high frequencies during the development of a H. halobium culture. The investigation of the fusion regions resulting from deletion events indicates that insertion elements are involved. The analysis of pHH1 deletion variants led to a 4 kilobase pair DNA region containing the origin of replication of the pHH1 plasmid.     

Physiological levels of Pik3ca(H1047R) mutation in the mouse mammary gland results in ductal hyperplasia and formation of ERα-positive tumors

PIK3CA, the gene coding for the p110α subunit of phosphoinositide 3-kinase, is frequently mutated in a variety of human tumors including breast cancers. To better understand the role of mutant PIK3CA in the initiation and/or progression of breast cancer, we have generated mice with a conditional knock-in of the common activating mutation, Pik3ca(H1047R), into one allele of the endogenous gene in the mammary gland. These mice developed a ductal anaplasia and hyperplasia by 6 weeks of age characterized by multi-layering of the epithelial lining of the mammary ducts and expansion of the luminal progenitor (Lin(-); CD29(lo); CD24(+); CD61(+)) cell population. The Pik3ca(H1047R) expressing mice eventually develop mammary tumors with 100% penetrance but with a long latency (>12 months). This is significantly longer than has been reported for transgenic models where expression of the mutant Pik3ca is driven by an exogenous promoter. Histological analysis of the tumors formed revealed predominantly ERα-positive fibroadenomas, carcinosarcomas and sarcomas. In vitro induction of Pik3ca(H1047R) in immortalized mammary epithelial cells also resulted in tumor formation when injected into the mammary fat pad of immunodeficient recipient mice. This novel model, which reproduces the scenario of a heterozygous somatic mutation occurring in the endogenous PIK3CA gene, will thus be a valuable tool for investigating the role of Pik3ca(H1047R) mutation in mammary tumorigenesis both in vivo and in vitro.     

Mutations in PVRL4, Encoding Cell Adhesion Molecule Nectin-4, Cause Ectodermal Dysplasia-Syndactyly Syndrome

Ectodermal dysplasias form a large disease family with more than 200 members. The combination of hair and tooth abnormalities, alopecia, and cutaneous syndactyly is characteristic of ectodermal dysplasia-syndactyly syndrome (EDSS). We used a homozygosity mapping approach to map the EDSS locus to 1q23 in a consanguineous Algerian family. By candidate gene analysis, we identified a homozygous mutation in the PVRL4 gene that not only evoked an amino acid change but also led to exon skipping. In an Italian family with two siblings affected by EDSS, we further detected a missense and a frameshift mutation. PVRL4 encodes for nectin-4, a cell adhesion molecule mainly implicated in the formation of cadherin-based adherens junctions. We demonstrated high nectin-4 expression in hair follicle structures, as well as in the separating digits of murine embryos, the tissues mainly affected by the EDSS phenotype. In patient keratinocytes, mutated nectin-4 lost its capability to bind nectin-1. Additionally, in discrete structures of the hair follicle, we found alterations of the membrane localization of nectin-afadin and cadherin-catenin complexes, which are essential for adherens junction formation, and we found reorganization of actin cytoskeleton. Together with cleft lip and/or palate ectodermal dysplasia (CLPED1, or Zlotogora-Ogur syndrome) due to an impaired function of nectin-1, EDSS is the second known “nectinopathy” caused by mutations in a nectin adhesion molecule.     

Analysis of streptomycin-resistance of Escherichia coli mutants.

We previously reported about Escherichia coli transformation experiments yielding streptomycin-resistant cells carrying a C912 to T transition in a plasmid-born 16S rRNA gene. These experiments were based on results obtained with streptomycin-resistant Euglena chloroplasts bearing an equivalent mutation in the single chloroplast 16S rRNA gene. We extended this study and transformed E. coli with plasmid constructs having a mutated 16S rRNA gene at position 914 (A to C) or a double mutation at positions 912 and 888 (C to T:G to A) or a mutation in the S12 gene (Lys-42 to Thr). We tested the transformed cells before and after a screening procedure in the presence of streptomycin. We find that the plasmid-born mutations protect colonies against a short streptomycin exposure, but ribosomes carrying mutated 16S rRNA do not significantly reduce codon misreading in vitro. However, ribosomes isolated from transformed cells after the screening procedure resist misreading. These ribosomes have acquired a second mutation in the S12 protein as shown in one case by sequencing and by transformation experiments. Furthermore, we show that the A914 to C mutation prevents (strongly reduces) base methylation in the central domain of 16S rRNA.     

Self-cloning yeast strains containing novel FAS2 mutations produce a higher amount of ethyl caproate in Japanese sake

Point mutation of Gly1250Ser (1250S) of the yeast fatty acid synthase gene FAS2 confers cerulenin resistance. This mutation also results in a higher production of the apple-like flavor component ethyl caproate in Japanese sake. We mutated the 1250th codon by in vitro site-directed mutagenesis to encode Ala (1250A) or Cys (1250C) and examined cerulenin resistance and ethyl caproate production. The mutated FAS2 genes were inserted into a binary plasmid vector containing a drug-resistance marker and a counter-selectable marker, GALp-GIN11M86. The plasmids were integrated into the wild-type FAS2 locus of a sake yeast strain, and the loss of the plasmid sequences from the integrants was done by growth on galactose plates, which is permissive for loss of GALp-GIN11M86. These counter-selected strains contained either the wild type or the mutated FAS2 allele but not the plasmid sequences, from which FAS2 mutant strains were selected by allele-specific PCR. The FAS2-1250C mutant produced a higher amount of ethyl caproate in sake than FAS2-1250S, while FAS2-1250A produced an ethyl caproate level intermediate between FAS2-1250S and the parental Kyokai no. 7 strain. Interestingly, these mutants only showed detectable cerulenin resistance. These 'self-cloning' yeast strains should be acceptable to the public because they can improve sake quality without the presence of extraneous DNA sequences.     

Mismatch Repair Genes on Chromosomes 2p and 3p Account for a Major Share of Hereditary Nonpolyposis Colorectal Cancer Families Evaluable by Linkage

Two susceptibility loci for hereditary nonpolyposis colo-rectal cancer (HNPCC) have been identified, and each contains a mismatch repair gene: MSH2 on chromosome 2p and MLH1 on chromosome 3p. We studied the involvement of these loci in 13 large HNPCC kindreds originating from three different continents. Six families showed close linkage to the 2p locus, and a heritable mutation of the MSH2 gene was subsequently found in four. The 2p-linked kindreds included a family characterized by the lack of extracolonic manifestations (Lynch I syndrome), as well as two families with cutaneous manifestations typical of the Muir-Torre syndrome. Four families showed evidence for linkage to the 3p locus, and a heritable mutation of the MLH1 gene was later detected in three. One 3p-linked kindred was of Amerindian origin. Of the remaining three families studied for linkage, one showed lod scores compatible with exclusion of both MSH2 and MLH1, while lod scores obtained in the other two families suggested exclusion of one HNPCC locus (MSH2 or MLH1) but were uninformative for markers flanking the other locus. Our results suggest that mismatch repair genes on 2p and 3p account for a major share of HNPCC in kindreds that can be evaluated by linkage analysis.     

Young transgenic hMTH1 mice are protected against dietary fat‐induced metabolic stress—implications for enhanced longevity

hMTH1 protects against mutation during oxidative stress. It degrades 8‐oxodGTP to exclude potentially mutagenic oxidized guanine from DNA. hMTH1 expression is linked to ageing. Its downregulation in cultured cells accelerates RAS‐induced senescence, and its overexpression in hMTH1‐Tg mice extends lifespan. In this study, we analysed the effects of a brief (5 weeks) high‐fat diet challenge (HFD) in young (2 months old) and adult (7 months old) wild‐type (WT) and hMTH1‐Tg mice. We report that at 2 months, hMTH1 overexpression ameliorated HFD‐induced weight gain, changes in liver metabolism related to mitochondrial dysfunction and oxidative stress. It prevented DNA damage as quantified by a comet assay. At 7 months old, these HFD‐induced effects were less severe and hMTH1‐Tg and WT mice responded similarly. hMTH1 overexpression conferred lifelong protection against micronucleus induction, however. Since the canonical activity of hMTH1 is mutation prevention, we conclude that hMTH1 protects young mice against HFD by reducing genome instability during the early period of rapid growth and maximal gene expression. hMTH1 protection is redundant in the largely non‐growing, differentiated tissues of adult mice. In hMTH1‐Tg mice, expression of a less heavily mutated genome throughout life provides a plausible explanation for their extended longevity.     

Novel nuclear and mitochondrial glycosylases revealed by disruption of the mouse Nth1 gene encoding an endonuclease III homolog for repair of thymine glycols

Endonuclease III, encoded by nth in Escherichia coli, removes thymine glycols (Tg), a toxic oxidative DNA lesion. To determine the biological significance of this repair in mammals, we established a mouse model with mutated mNth1, a homolog of nth, by gene targeting. The homozygous mNth1 mutant mice showed no detectable phenotypical abnormality. Embryonic cells with or without wild-type mNth1 showed no difference in sensitivity to menadione or hydrogen peroxide. Tg produced in the mutant mouse liver DNA by X-ray irradiation disappeared with time, though more slowly than in the wild-type mouse. In extracts from mutant mouse liver, we found, instead of mNTH1 activity, at least two novel DNA glycosylase activities against Tg. One activity is significantly higher in the mutant than in wild-type mouse in mitochondria, while the other is another nuclear glycosylase for Tg. These results underscore the importance of base excision repair of Tg both in the nuclei and mitochondria in mammals.