Molecular cloning, polymorphism, and functional activity of the bovine and water buffalo Mx2 gene promoter region.

BACKGROUND: Bovine Mx2 gene sequences were already reported, but further information about the gene properties is not yet available. The objective of the current study was to elucidate the structural properties of the bovine Mx2 gene mainly the promoter region and its possible functional role. If available, such information would help in assessing the functional properties of the gene, which was reported to confer antiviral action against recombinant VSV. RESULTS: Examinations on the bovine genomic BAC clone-confirmed to contain the Mx2 gene-revealed 883-bp sequences. A computer scan unequivocally identified a 788-bp promoter region containing a typical TATA box, three ISREs and other promoter-specific motifs. Comparative analysis of nine bovine genomic DNA samples showed 19 nucleotide substitutions suggesting the existence of five different genotypes in the promoter region. The water buffalo Mx2 promoter region was determined by using primers based on the bovine Mx2 promoter region disclosing 893-bp, with 56 substitutions, two insertions, 9 and 1 nt at two different sites. A functional analysis of the putative ISRE indicated that ISRE played a synergetic role in the activation of bovine Mx2 gene transcription. CONCLUSION: Bovine and water buffalo Mx2 promoter region was identified disclosing, the conserved ISRE, located in the proximal end of the promoter region like other members of the antiviral family, suggesting functional activity under interferon stimulation.

BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.

X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells.

A novel protein, MAPO1, that functions in apoptosis triggered by O6-methylguanine mispair in DNA.

O(6)-Methylguanine produced in DNA induces mutation due to its ambiguous base-pairing properties during DNA replication. To suppress such an outcome, organisms possess a mechanism to eliminate cells carrying O(6)-methylguanine by inducing apoptosis that requires the function of mismatch repair proteins. To identify other factors involved in this apoptotic process, we performed retrovirus-mediated gene-trap mutagenesis and isolated a mutant that acquired resistance to a simple alkylating agent, N-methyl-N-nitrosourea (MNU). However, it was still sensitive to methyl methanesulfonate, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea, etoposide and ultraviolet irradiation. Moreover, the mutant exhibited an increased mutant frequency after exposure to MNU. The gene responsible was identified and designated Mapo1 (O(6)-methylguanine-induced apoptosis 1). When the expression of the gene was inhibited by small interfering RNA, MNU-induced apoptosis was significantly suppressed. In the Mapo1-defective mutant cells treated with MNU, the mitochondrial membrane depolarization and caspase-3 activation were severely suppressed, although phosphorylation of p53, CHK1 and histone H2AX was observed. The orthologs of the Mapo1 gene are present in various organisms from nematode to humans. Both mouse and human MAPO1 proteins expressed in cells localize in the cytoplasm. We therefore propose that MAPO1 may play a role in the signal-transduction pathway of apoptosis induced by O(6)-methylguanine-mispaired lesions.

Long-term forskolin stimulation induces AMPK activation and thereby enhances tight junction formation in human placental trophoblast BeWo cells.

BeWo cells, derived from human choriocarcinoma, have been known to respond to forskolin or cAMP analogues by differentiating into multinucleated cells- like syncytiotrophoblasts on the surfaces of chorionic villi of the human placenta. In this study, we demonstrated that long-term treatment with forskolin enhances the tight junction (TJ) formation in human placental BeWo cells. Interestingly, AMPK activation and phosphorylation of acetyl-CoA carboxylase (ACC), a molecule downstream from AMPK, were induced by long-term incubation (>12h) with forskolin, despite not being induced by acute stimulation with forskolin. In addition, co-incubation with an AMPK inhibitor, compound C, as well as overexpression of an AMPK dominant negative mutant inhibited forskolin-induced TJ formation. Thus, although the molecular mechanism underlying AMPK activation via the forskolin stimulation is unclear, the TJ formation induced by forskolin is likely to be mediated by the AMPK pathway. Taking into consideration that TJs are present in the normal human placenta, this mechanism may be important for forming the placental barrier system between the fetal and maternal circulations.

Bovine and water buffalo Mx2 genes: polymorphism and antiviral activity.

Millennia-long selective pressure of single-strand RNA viruses on the bovine Mx locus has increased the advantages of using the bovine Mx protein to evaluate the ultimate significance of the antiviral role of Mx proteins. The conclusions of research based only on the bovine Mx1 protein showed the need for comprehensive studies that demonstrate the role of all isoforms, individually or together, especially in the presence of a second isoform, the bovine Mx2 gene. This study provides information about bovine and water buffalo Mx2 genes, as well as their allelic polymorphism and basic antiviral potential. Observation of an Mx2 cDNA sequence (2,381 bp) obtained from 15 animals from 11 breeds using primers based on a previous sequence (NCBI accession no. AF335147) revealed several nucleotide substitutions, with eight different alleles and two amino acid exchanges: Gly to Ser at position 302 and Ile to Val at position 354, though the latter was found only in the NCBI database. A water buffalo Mx2 cDNA sequence was identified for the first time, revealing 46 nucleotide substitutions with 12 amino acid variations, in addition to a 9-bp insertion in the 5' untranslated region UTR, compared with the bovine Mx2 cDNA. Transfected 3T3 cells expressing bovine Mx2 mRNAs coding Gly or Ser at position 302, water buffalo Mx2 mRNA, positive control bovine Mx1 mRNA-expressing cells, and negative control parental 3T3 were subjected to infection with recombinant vesicular stomatitis virus (VSVDeltaG*-G), as were empty pCI-neo vector-transfected cells. The positive control and all cells expressing Mx2 mRNAs displayed significantly higher levels of antiviral activity against VSVDeltaG*-G (P < 0.01) than did the negative controls.

Genetic polymorphisms and antiviral activity in the bovine MX1 gene.

Bovine MX1 cDNAs consisting of 2280 bp from 11 animals of five breeds and from a cultured cell line were sequenced and compared with previously reported data. Ten nucleotide substitutions were synonymous mutations, and a single nucleotide substitution at 458 resulted in an amino acid exchange of Ile (ATT) and Met (ATG). A 13-bp deletion-insertion mutation was also found in the 3'-UTR. Based on the nucleotide substitutions found in this study, bovine MX1 cDNA was classified into 11 genotypes. A phylogenetic tree of the 11 genotypes suggested that the genotypes observed in Brahman were a great genetic distance from other genotypes. An 18-bp deletion-insertion variation at position 171 was found to be the result of alternative splicing. The 18-bp deletion-insertion is located at the boundary between exon 3 and intron 3. Permanently transfected 3T3 cell lines expressing bovine MX1 mRNA were established to analyse the antiviral potential against VSVDeltaG*-G infection. Transfected cell clones expressing bovine MX1 mRNA showed a significantly smaller number of cells infected with VSVDeltaG*-G compared with the control cells. These results indicate that the bovine MX1 protein has potent antiviral activity.

Early postnatal ataxia and abnormal cerebellar development in mice lacking Xeroderma pigmentosum Group A and Cockayne syndrome Group B DNA repair genes.

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are rare autosomal recessive disorders associated with a defect in the nucleotide excision repair (NER) pathway required for the removal of DNA damage induced by UV light and distorting chemical adducts. Although progressive neurological dysfunction is one of the hallmarks of CS and of some groups of XP patients, the causative mechanisms are largely unknown. Here we show that mice lacking both the XPA (XP-group A) and CSB (CS-group B) genes in contrast to the single mutants display severe growth retardation, ataxia, and motor dysfunction during early postnatal development. Their cerebella are hypoplastic and showed impaired foliation and stunted Purkinje cell dendrites. Reduced neurogenesis and increased apoptotic cell death occur in the cerebellar external granular layer. These findings suggest that XPA and CSB have additive roles in the mouse nervous system and support a crucial role for these genes in normal brain development.

UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency.

Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (-/-) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP.

A novel cytoplasmic GTPase XAB1 interacts with DNA repair protein XPA.

The xeroderma pigmentosum group A protein (XPA) plays a central role in nucleotide excision repair (NER). To identify proteins that bind to XPA, we screened a HeLa cDNA library using the yeast two-hybrid system. Here we report a novel cytoplasmic GTP-binding protein, designated XPA binding protein 1 (XAB1). The deduced amino acid sequence of XAB1 consisted of 374 residues with a molecular weight of 41 kDa and an isoelectric point of 4.65. Sequence analysis revealed that XAB1 has four sequence motifs G1-G4 of the GTP-binding protein family in the N-terminal half. XAB1 also contains an acidic region in the C-terminal portion. Northern blot analysis showed that XAB1 mRNA is expressed ubiquitously, and immunofluorescence analysis revealed that XAB1 is localized mainly in the cytoplasm. Consistent with the GTP-binding motif, purified recombinant XAB1 protein has intrinsic GTPase activity. Using the yeast two-hybrid system, we elucidated that XAB1 binds to the N-terminal region of XPA. The deletion of five amino acids, residues 30-34 of XPA, required for nuclear localization of XPA abolished the interaction with XAB1. These results suggest that XAB1 is a novel cytoplasmic GTPase involved in nuclear localization of XPA.

XAB2, a novel tetratricopeptide repeat protein involved in transcription-coupled DNA repair and transcription.

Nucleotide excision repair is a highly versatile DNA repair system responsible for elimination of a wide variety of lesions from the genome. It is comprised of two subpathways: transcription-coupled repair that accomplishes efficient removal of damage blocking transcription and global genome repair. Recently, the basic mechanism of global genome repair has emerged from biochemical studies. However, little is known about transcription-coupled repair in eukaryotes. Here we report the identification of a novel protein designated XAB2 (XPA-binding protein 2) that was identified by virtue of its ability to interact with XPA, a factor central to both nucleotide excision repair subpathways. The XAB2 protein of 855 amino acids consists mainly of 15 tetratricopeptide repeats. In addition to interacting with XPA, immunoprecipitation experiments demonstrated that a fraction of XAB2 is able to interact with the transcription-coupled repair-specific proteins CSA and CSB as well as RNA polymerase II. Furthermore, antibodies against XAB2 inhibited both transcription-coupled repair and transcription in vivo but not global genome repair when microinjected into living fibroblasts. These results indicate that XAB2 is a novel component involved in transcription-coupled repair and transcription.

Decreased UV sensitivity, mismatch repair activity and abnormal cell cycle checkpoints in skin cancer cell lines derived from UVB-irradiated XPA-deficient mice.

Xeroderma pigmentosum group A gene (XPA)-deficient mice are defective in nucleotide excision repair (NER) and are therefore highly sensitive to ultraviolet (UV)-induced skin carcinogenesis. We established cell lines from skin cancers of UVB-irradiated XPA-deficient mice to investigate the phenotypic changes occurring during skin carcinogenesis. As anticipated, the skin cancer cell lines were devoid of NER activity but were less sensitive to killing by UV-irradiation than the XPA(-/-) fibroblast cell line. The lines were also more resistant to 6-thioguanine (6-TG) than XPA(-/-) and XPA(+/+) fibroblasts, which was suggestive of a mismatch repair (MMR) defect. Indeed, in vitro mismatch binding and MMR activity were impaired in several of these cell lines. Moreover, these cell lines displayed cell cycle checkpoint derangements following UV-irradiation and 6-TG exposure. The above findings suggest that MMR downregulation may help cells escape killing by UVB, as was seen previously for methylating agents and cisplatin, and thus that MMR deficient clones are selected for during the tumorigenic transformation of XPA(-/-) cells.

Studies of in vivo mutations in rpsL transgene in UVB-irradiated epidermis of XPA-deficient mice.

We have established xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair (NER) deficiency, which rapidly developed skin tumors when exposed to a low dose of chronic UV like XP-A patients, confirming that the NER process plays an important role in preventing UVB-induced skin cancer. To examine the in vivo mutation in the UVB-irradiated epidermis, we established XPA (-/-), (+/-) and (+/+) mice carrying the Escherichia coli rpsL transgene with which the mutation frequencies and spectra in the UVB-irradiated epidermal tissue can be examined conveniently. The XPA (-/-) mice showed a higher frequency of UVB-induced mutation in the rpsL transgene with a low dose (150 J/m(2)) of UVB-irradiation than the XPA (+/-) and (+/+) mice, while, at a high dose (900 J/m(2)) they showed almost the same frequency of mutation as the XPA (+/-) and (+/+) mice, probably because of cell death in the epidermis of the XPA (-/-) mice. However, CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the XPA (-/-) mice than the XPA (+/-) and (+/+) mice at both doses of UVB. This rpsL/XPA mouse system will be useful for further analyzing the role of NER in the mutagenesis and carcinogenesis induced by various carcinogens.

Mutational analysis of a function of xeroderma pigmentosum group A (XPA) protein in strand-specific DNA repair.

To analyze the function of the xeroderma pigmentosum group A (XPA) protein in strand-specific DNA repair, we examined repair of UV-induced cyclobutane pyrimidine dimer (CPD) in transcribed and non-transcribed strands of the dihydrofolate reductase gene of xeroderma pigmentosum group A (XP-A) cell line (XP12ROSV) which was transfected with various types of mutant XPA cDNA. The transfectant overexpressing mutant XPA with a defect in the interaction with either ERCC1, replication protein A (RPA), or general transcription factor TFIIH, showed more or less decreased repair of CPD in each strand in parallel, while in the transfectant overexpressing R207G (Arg207to Gly) mutant XPA derived from XP129, a UV-resistant XP12ROSV revertant, the rate of CPD repair was almost normal in each strand. We also examined the dose responses of the XPA protein on CPD repair in each strand by the modulation of the expression levels of wild-type or R207G mutant XPA using an inducible expression system, LacSwitchtrade mark promoter. There were good correlations between the rate of CPD repair in each strand and the amount of XPA protein produced in these Lac cells. Our results indicate that the XPA protein is equally important for the CPD repair in both transcribed and non-transcribed strands and that the R207G mutation found in XP129 may not be responsible for a selective defect in CPD repair in the non-transcribed strand in XP129.

A very large protein with diverse functional motifs is deficient in rjs (runty, jerky, sterile) mice.

Three radiation-induced alleles of the mouse p locus, p6H, p25H, and pbs, cause defects in growth, coordination, fertility, and maternal behavior in addition to p gene-related hypopigmentation. These alleles are associated with disruption of the p gene plus an adjacent gene involved in the disorders listed. We have identified this adjacent gene, previously named rjs (runty jerky sterile), by positional cloning. The rjs cDNA is very large, covering 15,264 nucleotides. The predicted rjs-encoded protein (4,836 amino acids) contains several sequence motifs, including three RCC1 repeats, a structural motif in common with cytochrome b5, and a HECT domain in common with E6-AP ubiquitin ligase. On the basis of sequence homology and conserved synteny, the rjs gene is the single mouse homolog of a previously described five- or six-member human gene family. This family is represented by at least two genes, HSC7541 and KIAA0393, from human chromosome 15q11-q13. HSC7541 and KIAA0393 lie close to, or within, a region commonly deleted in most Prader-Willi syndrome patients. Previous work has suggested that the multiple phenotypes in rjs mice might be due to a common neuroendocrine defect. In addition to this proposed mode of action, alternative functions of the rjs gene are evaluated in light of its known protein homologies.

RPA2, a gene for the 32 kDa subunit of replication protein A on chromosome 1p35-36, is not mutated in patients with familial melanoma linked to chromosome 1p36.

Although some cases of dysplastic naevi (DN) and familial melanoma have been linked to anonymous markers on chromosome 1p36, the gene has not been identified. A candidate gene, RPA2, which codes for the 32 kDa subunit of replication protein A, is located in the 1p35-36 region. We examined the RPA2 gene in seven lymphoblastoid cell lines from members of melanoma-prone families linked to chromosome 1p36. Southern and Northern blot analyses showed the DNA and RNA bands were of normal size and intensity. DNA sequencing demonstrated no nucleotide alterations in the RPA2 cDNA. Western blot analysis exhibited normal electrophoretic migration and intensity of the RPA2 protein. These results indicate that alterations do not occur in the RPA2 gene in these DN/familial melanoma families linked to chromosome 1p36.

Strand specificity and absence of hot spots for p53 mutations in ultraviolet B-induced skin tumors of XPA-deficient mice.

We examined the spectrum of p53 mutations found in 40 UV-induced skin tumors of xeroderma pigmentosum group A gene (XPA)-deficient mice. p53 mutations were detected in 48% of the tumors. Nearly all of the mutations were induced at dipyrimidine sites. Ninety-three % of the mutations were G.C-->A.T transitions at dipyrimidine sites, including tandem transitions (CC-->TT), which are the hallmark of the UVB-induced mutation. Seventy-two % of the mutations at dipyrimidine sites could be ascribed to damage on the transcribed strand. In addition, no evident mutational hot spots were detected. This is in contrast to the UVB-induced skin tumors of normal mice, in which 92% of p53 mutations occurred as a result of DNA damage on the nontranscribed strand, and clear hot spots were observed. Thus, XPA-deficient mice showed significant mutation features that might be characteristic of the absence of nucleotide excision repair and may provide a good animal model for the analysis of the high incidence of skin cancer in xeroderma pigmentosum group A patients.

Loss of the xeroderma pigmentosum group A gene (XPA) enhances apoptosis of cultured cerebellar neurons induced by UV but not by low-K+ medium.

To study the involvement of the xeroderma pigmentosum group A gene (XPA) in neuronal apoptosis, we cultured cerebellar neurons from mice lacking XPA gene (XPA-/-) and induced apoptosis by exposure to UV irradiation or medium containing a low concentration of potassium (low-K+ medium). When cerebellar neurons from postnatal days 15-16 wild-type mice were treated with UV irradiation, apoptotic neuronal death was observed after 24-48 h. About 60% of neurons survived 48 h after UV irradiation at a dose of 5 J/m2. On the other hand, neurons from XPA-/- mice showed a significantly increased vulnerability to UV irradiation, and >90% of neurons died 48 h after UV irradiation at a dose of 5 J/m2. In contrast, low-K+ medium induced apoptosis of neurons from mice of each genotype with the same kinetics. These results suggest that the XPA gene is involved in neuronal DNA repair and that it thereby influences apoptosis induced by DNA damage in cultured cerebellar neurons.

Mutations in the XPD gene leading to xeroderma pigmentosum symptoms.

XP is a sun-sensitive and cancer-prone genetic disorder, consisting of eight (group A-G) genetically distinct complementation groups. Some XP group D patients exhibit clinical symptoms of other genetic disorders, CS, and TTD. The XP group D gene (XPD gene) product is required for nucleotide excision repair and is one of the components of basal transcription factor TFIIH as well. Therefore, different mutations in the XPD gene may result in a variety of clinical manifestations. Here we report on two causative mutations of the XPD gene in XP61OS, a Japanese XP group D patient who has only mild skin symptoms of XP without CS, TTD, or other neurological complications. One of the mutations was the 4-bp deletion at nucleotides 668-671, resulting in frameshift and truncation of the protein. The other was a nucleotide substitution leading to Ser-541 to Arg (S541R) in helicase domain IV of the XPD protein. The patient's father was heterozygous for the 4-bp deletion, while the mother was heterozygous for the S541R mutation. Thus, the parents were obligate carriers of the XP-D trait. The expression study showed that the XPD cDNA containing the deletion or the S541R missense mutation failed to restore the UV sensitivity of XP6BE, group DaXP cells, while the wild-type XPD cDNA restored it to the normal level. However, the transfectant expressing the XPD cDNA with the missense mutation was slightly more resistant than the parental XP6BE cells. These findings are consistent with the mild symptoms of the XP61OS patient.

High incidence of ultraviolet-B-or chemical-carcinogen-induced skin tumours in mice lacking the xeroderma pigmentosum group A gene.

Xeroderma pigmentosum (XP) is an autosomal recessive disorder characterized by a high frequency of skin cancer on sun-exposed areas, and neurological complications. XP has a defect in the early step(s) of nucleotide-excision repair (NER) and consists of eight different genetic complementation groups (groups A-G and a variant). We established XPA (group-A XP) gene-deficient mice by gene targeting of mouse embryonic stem (ES) cells. The XPA-deficient mice showed neither obvious physical abnormalities nor pathological alterations, but were defective in NER and highly susceptible to ultraviolet-B- or 9,10-dimethyl-1,2-benz[a]anthracene-induced skin carcinogenesis. These findings provide in vivo evidence that the XPA protein protects mice from carcinogenesis initiated by ultraviolet or chemical carcinogen. The XPA-deficient mice may provide a good in vivo model to study the high incidence of skin carcinogenesis in group A XP patients.