ABSTRACT
Friedreich ataxia (FRDA) is a progressive neurodegenerative disease caused by deficiency of frataxin protein, with the primary sites of pathology being the large sensory neurons of the dorsal root ganglia and the cerebellum. FRDA is also often accompanied by severe cardiomyopathy and diabetes mellitus. Frataxin is important in mitochondrial iron-sulfur cluster (ISC) biogenesis and low-frataxin expression is due to a GAA repeat expansion in intron 1 of the FXN gene. FRDA cells are genomically unstable, with increased levels of reactive oxygen species and sensitivity to oxidative stress. Here we report the identification of elevated levels of DNA double strand breaks (DSBs) in FRDA patient and YG8sR FRDA mouse model fibroblasts compared to normal fibroblasts. Using lentivirus FXN gene delivery to FRDA patient and YG8sR cells, we obtained long-term overexpression of FXN mRNA and frataxin protein levels with reduced DSB levels towards normal. Furthermore, γ-irradiation of FRDA patient and YG8sR cells revealed impaired DSB repair that was recovered on FXN gene transfer. This suggests that frataxin may be involved in DSB repair, either directly by an unknown mechanism, or indirectly via ISC biogenesis for DNA repair enzymes, which may be essential for the prevention of neurodegeneration.
Subject(s)
Friedreich Ataxia/therapy , Genetic Therapy , Genomic Instability , Iron-Binding Proteins/genetics , Animals , Cells, Cultured , DNA Breaks, Double-Stranded , Fibroblasts/metabolism , Humans , Iron-Binding Proteins/metabolism , Lentivirus/genetics , Mice , Recombinational DNA Repair , FrataxinABSTRACT
Friedreich's ataxia (FRDA) is an inherited neurodegenerative disease. The mutation consists of a GAA repeat expansion within the FXN gene, which downregulates frataxin, leading to abnormal mitochondrial iron accumulation, which may in turn cause changes in mitochondrial function. Although, many studies of FRDA patients and mouse models have been conducted in the past two decades, the role of frataxin in mitochondrial pathophysiology remains elusive. Are the mitochondrial abnormalities only a side effect of the increased accumulation of reactive iron, generating oxidative stress? Or does the progressive lack of iron-sulphur clusters (ISCs), induced by reduced frataxin, cause an inhibition of the electron transport chain complexes (CI, II and III) leading to reactive oxygen species escaping from oxidative phosphorylation reactions? To answer these crucial questions, we have characterised the mitochondrial pathophysiology of a group of disease-relevant and readily accessible neurons, cerebellar granule cells, from a validated FRDA mouse model. By using live cell imaging and biochemical techniques we were able to demonstrate that mitochondria are deregulated in neurons from the YG8R FRDA mouse model, causing a decrease in mitochondrial membrane potential (âµΨm) due to an inhibition of Complex I, which is partially compensated by an overactivation of Complex II. This complex activity imbalance leads to ROS generation in both mitochondrial matrix and cytosol, which results in glutathione depletion and increased lipid peroxidation. Preventing this increase in lipid peroxidation, in neurons, protects against in cell death. This work describes the pathophysiological properties of the mitochondria in neurons from a FRDA mouse model and shows that lipid peroxidation could be an important target for novel therapeutic strategies in FRDA, which still lacks a cure.
Subject(s)
Iron-Binding Proteins/genetics , Lipid Peroxidation/genetics , Membrane Potential, Mitochondrial , Mitochondria/metabolism , Neurons/metabolism , Animals , Cerebellum/metabolism , Cerebellum/pathology , Disease Models, Animal , Electron Transport Complex I/genetics , Electron Transport Complex I/metabolism , Electron Transport Complex II/genetics , Electron Transport Complex II/metabolism , Electron Transport Complex III/genetics , Electron Transport Complex III/metabolism , Friedreich Ataxia/genetics , Friedreich Ataxia/metabolism , Friedreich Ataxia/pathology , Gene Expression Regulation , Glutathione/metabolism , Humans , Iron/metabolism , Iron-Binding Proteins/metabolism , Mice , Mitochondria/pathology , Mutation , Neurons/pathology , Oxidative Stress , Primary Cell Culture , Reactive Oxygen Species/metabolism , Signal Transduction , FrataxinABSTRACT
Many of the spinocerebellar ataxias (SCAs) are caused by expansions of CAG trinucleotide repeats encoding abnormal stretches of polyglutamine. SCA3 or Machado-Joseph disease (MJD) is the commonest dominant inherited ataxia disease, with pathological phenotypes apparent with a CAG triplet repeat length of 61-84. In this study a mouse model of SCA3 has been examined which was produced using a human yeast artificial chromosome containing the MJD gene with a CAG triplet expansion of 84 repeats. These mice have previously been shown to possess a mild progressive cerebellar deficit. NMR-based metabolomics/metabonomics in conjunction with multivariate pattern recognition identified a number of metabolic perturbations in SCA3 mice. These changes included a consistent increase in glutamine concentration in tissue extracts of the cerebellum and cerebrum and spectra obtained from intact tissue using magic angle spinning (1)H-NMR spectroscopy. Furthermore, these profiles demonstrated metabolic abnormalities were present in the cerebrum, a region not previously implicated in SCA3. As well as an increase in glutamine both brain regions demonstrated decreases in GABA, choline, phosphocholine and lactate (representing the summation of lactate in vivo, and postmortem glycolysis of glucose and glycogen). The metabolic changes are discussed in terms of the formation of neuronal intranuclear inclusions associated with SCA3. This study suggests high-resolution (1)H-NMR spectroscopy coupled with pattern recognition may provide a rapid method for assessing the phenotype of animal models of human disease.
Subject(s)
Brain/metabolism , Disease Models, Animal , Machado-Joseph Disease/genetics , Machado-Joseph Disease/metabolism , Nerve Tissue Proteins/genetics , Animals , Ataxin-3 , Brain/pathology , Cerebellum/metabolism , Cerebellum/pathology , Choline/metabolism , Glucose/metabolism , Glutamine/metabolism , Glycogen/metabolism , Humans , Lactic Acid/metabolism , Machado-Joseph Disease/pathology , Magnetic Resonance Spectroscopy , Mice , Mice, Transgenic , Nuclear Proteins , Phenotype , Phosphorylcholine/metabolism , Repressor Proteins , Telencephalon/metabolism , Telencephalon/pathology , Transcription Factors , Trinucleotide Repeat Expansion/genetics , gamma-Aminobutyric Acid/metabolismABSTRACT
We have generated and characterised transgenic mice that contain the entire Friedreich's ataxia gene (FRDA) within a human YAC clone of 370 kb. In an effort to overcome the embryonic lethality of homozygous Frda knockout mice and to study the behaviour of human frataxin in a mouse cellular environment, we bred the FRDA YAC transgene onto the null mouse background. Phenotypically normal offspring that express only YAC-derived human frataxin were identified. The human frataxin was expressed in the appropriate tissues at levels comparable to the endogenous mouse frataxin, and it was correctly processed and localised to mitochondria. Biochemical analysis of heart tissue demonstrated preservation of mitochondrial respiratory chain function, together with some increase in citrate synthase and aconitase activities. Thus, we have demonstrated that human frataxin can effectively substitute for endogenous murine frataxin in the null mutant. Our studies are of immediate consequence for the generation of Friedreich's ataxia transgenic mouse models, and further contribute to the accumulating knowledge of human-mouse functional gene replacement systems.
Subject(s)
Chromosomes, Artificial, Yeast , Friedreich Ataxia/genetics , Friedreich Ataxia/physiopathology , Iron-Binding Proteins , Mice, Knockout/genetics , Animals , Disease Models, Animal , Genes, Lethal , Homozygote , Humans , Mice , Phenotype , Phosphotransferases (Alcohol Group Acceptor)/genetics , Transgenes/genetics , FrataxinSubject(s)
Frameshift Mutation , Friedreich Ataxia/genetics , Iron-Binding Proteins , Adolescent , Age of Onset , Base Sequence , Child , DNA/chemistry , DNA/genetics , DNA Mutational Analysis , Family Health , Female , Humans , Male , Mutation , Pedigree , Phosphotransferases (Alcohol Group Acceptor)/genetics , Polymorphism, Single-Stranded Conformational , Sequence Deletion , FrataxinABSTRACT
The STM7 gene encodes a novel phosphatidylinositol-4-phosphate 5-kinase (PtdInsP 5-kinase) that is subject to alternative splicing and developmental control. We have recently presented data indicating that several splice variants of STM7 incorporate elements of the X25 sequence, previously implicated in the pathogenesis of Friedreich's ataxia by the detection of an intronic GAA repeat expansion as the predominant mutation in affected individuals. We now report the exon-intron structure of STM7.I and primer sequences designed to facilitate full characterization, including details relating to a novel exon (STM7; exon 17) derived from the 3'-UTR of the PRKACG gene. The detection of a mutation(s) within these exons would provide additional support for the hypothesis that a defect in phosphoinositide metabolism gives rise to the disease phenotype.
Subject(s)
Iron-Binding Proteins , Phosphotransferases (Alcohol Group Acceptor)/genetics , Alternative Splicing , Base Sequence , DNA Primers/genetics , Exons , Friedreich Ataxia/enzymology , Friedreich Ataxia/genetics , Humans , Introns , Minisatellite Repeats , Molecular Sequence Data , Mutation , Phenotype , Polymerase Chain Reaction , Trinucleotide Repeats , FrataxinABSTRACT
The STM7 gene on chromosome 9 was recently 'excluded' as a candidate for Friedreich's ataxia following the identification of an expanded intronic GAA triplet repeat in the adjacent gene, X25, in patients with the disease. Using RT-PCR, northern and sequence analyses, we now demonstrate that X25 comprises part of the STM7 gene, contributing to at least four splice variants, and report the identification of new coding sequences. Functional analysis of the STM7 recombinant protein corresponding to the reported 2.7-kilobase transcript has demonstrated PtdlnsP 5-kinase activity, supporting the idea that the disease is caused by a defect in the phosphoinositide pathway, possibly affecting vesicular trafficking or synaptic transmission.
Subject(s)
Friedreich Ataxia/genetics , Genes/genetics , Iron-Binding Proteins , Phosphotransferases (Alcohol Group Acceptor)/genetics , Proteins/genetics , Adult , Alternative Splicing/genetics , Amino Acid Sequence , Chromosome Mapping , Exons/genetics , Friedreich Ataxia/enzymology , Humans , Molecular Sequence Data , Organ Specificity , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Placenta , RNA, Messenger/analysis , RNA, Messenger/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , FrataxinABSTRACT
Forty loci (16 polymorphic and 24 non-polymorphic) together with 23 cosmids isolated from a chromosome 11-specific library were used to construct a detailed genetic map of 11p13-11q13. The map was constructed by using a panel of 13 somatic cell hybrids that sub-divided this region into 19 intervals, a meiotic mapping panel of 33 multiple endocrine neoplasia type 1 (MEN1) families (134 affected and 269 unaffected members) and a mitotic mapping panel that was used to identify loss of heterozygosity in 38 MEN1-associated tumours. The results defined the most likely order of the 16 loci as being: 11pter-D11S871-(D11S288, D11S149)-11cen-CNTF-PGA-ROM1-D11S480-PYGM- SEA-D11S913-D11S970-D11S97- D11S146-INT2-D11S971-D11S533-11qter. The meiotic mapping studies indicated that the most likely location of the MEN1 gene was in the interval flanked by PYGM and D11S97, and the results of mitotic mapping suggested a possible location of the MEN1 gene telomeric to SEA. Mapping studies of the gene encoding mu-calpain (CAPN1) located CAPN1 to 11q13 and in the vicinity of the MEN1 locus. However, mutational analysis studies did not detect any germ-line CAPN1 DNA sequence abnormalities in 47 unrelated MEN1 patients and the results therefore exclude CAPN1 as the MEN1 gene. The detailed genetic map that has been constructed of the 11p13-11q13 region should facilitate the construction of a physical map and the identification of candidate genes for disease loci mapped to this region.
Subject(s)
Calpain/genetics , Chromosome Mapping/methods , Chromosomes, Human, Pair 11/genetics , Cosmids/genetics , Multiple Endocrine Neoplasia Type 1/genetics , Animals , Base Sequence , Female , Gastrinoma/genetics , Genetic Linkage , Germ-Line Mutation/genetics , Humans , Hybrid Cells , Insulinoma/genetics , Male , Meiosis , Mitosis , Molecular Sequence Data , Pancreatic Neoplasms/genetics , Parathyroid Neoplasms/genetics , Pedigree , Pituitary Neoplasms/genetics , Polymorphism, Genetic , Sequence Deletion/geneticsABSTRACT
EagI and NotI linking libraries were prepared in the lambda vector, EMBL5, from the mouse-human somatic cell hybrid 1W1LA4.9, which contains human chromosomes 11 and Xp as the only human component. Individual clones containing human DNA were isolated by their ability to hybridise with total human DNA and digested with SalI and EcoRI to identify the human insert size and single-copy fragments. The mean (+/- SD) insert sizes of the EagI and NotI clones were 18.3 +/- 3.2 kb and 16.6 +/- 3.6 kb, respectively. Regional localisation of 66 clones (52 EagI, 14 NotI) was achieved using a panel of 20 somatic cell hybrids that contained different overlapping deletions of chromosomes 11 or Xp. Thirty-nine clones (36 EagI, 3 NotI) were localised to chromosome 11; 17 of these were clustered in 11q13 and another nine were clustered in 11q14-q23.1. Twenty-seven clones (16 EagI, 11 NotI) were localised to Xp and 10 of these were clustered in Xp11. The 66 clones were assessed for seven different microsatellite repetitive sequences; restriction fragment length polymorphisms for five clones from 11q13 were also identified. These EagI and NotI clones, which supplement those previously mapped to chromosome 11 and Xp, should facilitate the generation of more detailed maps and the identification of genes that are associated with CpG-rich islands.
Subject(s)
Chromosomes, Human, Pair 11/genetics , Cloning, Molecular/methods , Deoxyribonucleases, Type II Site-Specific , X Chromosome/genetics , Animals , Base Sequence , Chromosome Mapping , Genomic Library , Humans , Hybrid Cells , Mice , Microsatellite Repeats , Molecular Sequence Data , Polymorphism, Restriction Fragment LengthABSTRACT
We have previously assigned the mutation causing Friedreich's ataxia (FRDA) to 9q13 by genetic linkage and fluorescent in situ hybridization analysis, and identified recombination events which position the gene centromeric to D9S5. We report here the extension of a yeast artificial chromosome contig to span the 860 kb interval immediately proximal to this marker, which includes the D9S886 and D9S887/888 loci reported to flank the FRDA locus, and the construction of a high resolution cosmid contig initiated from the D9S888 locus. Exon trapping and cDNA library screening strategies have resulted in the isolation of a candidate gene which traverses the centromeric boundary of the FRDA critical region. The gene spans a genomic interval greater than 220 kb with at least two of the coding exons located proximal to the D9S887/888 loci. Expression is complex, with multiple transcripts detected in a variety of tissues and evidence of alternative splicing and developmental control. The predicted amino acid sequence for the 2.7 kb transcript reported here shows a marked homology to the deduced amino acid sequence of the Saccharomyces cerevisiae MSS4 protein, proposed to function within the phosphoinositide cycle, suggesting a potential role for the human homologue in signal transduction. Whilst no evidence for mutation has been detected in this transcript, the sequence represents only one of the shorter alternatively spliced species identified by Northern analysis and direct sequencing. This gene remains a strong candidate for FRDA.
Subject(s)
Friedreich Ataxia/genetics , Signal Transduction/genetics , Alternative Splicing , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Chromosomes, Artificial, Yeast , Chromosomes, Human, Pair 9/genetics , Cloning, Molecular , Cosmids , DNA, Complementary/genetics , Exons , Genetic Markers , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Proteins/genetics , Sequence Homology, Amino AcidABSTRACT
Dent's disease is a familial proximal renal tubular disorder which is associated with low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, kidney stones and renal failure. The mode of inheritance and the primary defect for this disorder are unknown. An analysis of 5 unrelated British families revealed a greater disease severity in males and an absence of male to male transmission. This suggested an X-linked inheritance and we investigated this further by linkage studies in 33 members (12 affected, 21 unaffected) from two 3-generation families. Twenty X-linked polymorphic markers were used and linkage was established with the Xp11 loci ARAFI, DXS426, DXS255 and DXS988 with peak LOD scores and recombination fractions (theta) of 5.42 (theta = 0.000), 3.61 (theta = 0.000), 5.48 (theta = 0.000) and 4.25 (theta = 0.045) respectively. In addition, DXS255 revealed a microdeletion in the affected members of one family, thereby further localising Dent's disease to Xp11.22. Combined multilocus linkage analysis and deletion mapping studies defined the locus order Xpter-MAOB-(ARAFI, DXS426)-SYP-TFE3-(DXS255, DENT'S)-DXS988-Xcen, thereby mapping the microdeletion associated with Dent's disease to a 4 centiMorgan interval flanked by TFE3 and DXS988. Thus, Dent's disease is an X-linked disorder which is associated with a microdeletion of Xp11.22, and a further characterisation of this gene will help to elucidate the factors controlling proximal renal tubular function and the development of kidney stones.
Subject(s)
Fanconi Syndrome/genetics , Polymorphism, Genetic , Sequence Deletion , X Chromosome , Calcinosis/genetics , Chromosome Mapping , DNA, Satellite/genetics , Female , Genetic Linkage , Genetic Markers , Humans , Kidney Calculi/genetics , Lymphocytes/pathology , Male , Pedigree , SoftwareABSTRACT
Mapping studies using a panel of 22 rodent-human somatic cell hybrids have helped to localize the Tamm-Horsfall glycoprotein (uromodulin) gene (UMOD), which has previously been reported to map to 16p13.11, to the region 16p12.3-qter. The combined results indicate that UMOD is located distal to D16S295 and proximal to D16S287 and in the region 16p12.3-16p13.11. Uromodulin is known to affect the formation of calcium-containing kidney stones, and this localization of UMOD will help in studies of families with autosomal forms of nephrolithiasis.
Subject(s)
Chromosomes, Human, Pair 16 , Mucoproteins/genetics , Animals , Base Sequence , Chromosome Mapping , Humans , Hybrid Cells , Kidney Calculi/genetics , Molecular Sequence Data , Osteopontin , Proteins/genetics , Rodentia , UromodulinABSTRACT
X-linked recessive nephrolithiasis is associated with kidney stones and renal tubular dysfunction in childhood progressing to renal failure in adulthood. The primary defect causing this renal tubular disorder is unknown and determining the chromosomal location of the mutant gene would represent an important step toward defining the biochemical basis. We have performed linkage studies in 102 members (10 affected males, 47 unaffected males, 15 obligate heterozygote females, and 30 unaffected females) from five generations of one family. As genetic markers we used 10 cloned human X chromosome fragments identifying restriction fragment length polymorphisms and seven pairs of oligonucleotide primers identifying microsatellite polymorphisms. Linkage with the locus DXS255 was established with a peak LOD score = 5.91 at 3.6% recombination, thereby localizing the X-linked recessive nephrolithiasis gene to the pericentromeric region of the short arm of the X chromosome (Xp11.22). Multilocus analysis indicated that the mutant gene was distal to DXS255 but proximal to the Duchenne muscular dystrophy locus on Xp. Thus, the gene that causes X-linked recessive nephrolithiasis maps to the pericentromeric region of the short arm of the X chromosome (Xp11.22), and further characterization of this gene will help to elucidate the factors controlling renal tubular function and mineral homeostasis.
Subject(s)
Chromosome Aberrations , Kidney Calculi/genetics , X Chromosome , Adolescent , Aged , Animals , Base Sequence , Child , Child, Preschool , Chromosome Mapping , DNA, Satellite/genetics , Female , Genes, Recessive/genetics , Genetic Linkage , Genetic Markers , Humans , Hybrid Cells , Male , Molecular Sequence Data , New York , Nucleic Acid Hybridization , Pedigree , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Rodentia , Sex CharacteristicsABSTRACT
The molecular pathology of somatotrophinomas has been investigated by a combined search for dominant mutations of the gene encoding the Gs alpha protein and for recessive mutations involving chromosome 11q13, which contains the gene causing multiple endocrine neoplasia type 1 (MEN1). Somatotrophinomas and peripheral leukocytes were obtained from thirteen patients with acromegaly; one patient also suffered from MEN1. Five DNA probes identifying restriction fragment length polymorphisms from 11q revealed allele loss in pituitary tumors from five (four non-MEN1 and one MEN1) patients. Deletion mapping revealed that the region of allele loss common to the somatotrophinomas involved 11q13. An analysis for similar allelic deletions at 12 other loci from chromosomes 1-5, 7-9, 12-14, and 17 did not reveal generalized allele loss in the somatotrophinomas. These results, which represent the first report of chromosome 11 allele loss occurring in non-MEN1 somatotrophinomas, indicate that a recessive oncogene on 11q13 is specifically involved in the monoclonal development of somatotrophinomas. In addition Gs alpha mutations were detected in two non-MEN1 somatotrophinomas, one of which also revealed allele loss of chromosome 11. Thus, our results reveal that the development of somatotrophinomas is associated with alterations in both dominant and recessive oncogenes and further characterization of these genetic abnormalities will help to elucidate the multistep etiology and progression of somatotrophinomas.
Subject(s)
Alleles , Chromosome Deletion , Chromosomes, Human, Pair 11 , GTP-Binding Proteins/genetics , Growth Hormone/metabolism , Pituitary Neoplasms/genetics , Acromegaly/genetics , Adult , Aged , Base Sequence , Chromosome Mapping , Female , Genes, Dominant/genetics , Genes, Recessive/genetics , Genes, Suppressor/genetics , Humans , Male , Middle Aged , Molecular Sequence Data , Oncogenes/geneticsABSTRACT
Familial multiple endocrine neoplasia type 1 (MEN 1) is an autosomal dominant disorder characterized by the combined occurrence of tumors of the parathyroid glands, the endocrine pancreas, and the pituitary gland. MEN 1 tumors have previously been shown to be associated with the loss of alleles on chromosome 11, and deletion mapping studies together with family linkage studies have localized the MEN 1 gene to 11q13. A detailed genetic map around the MEN 1 locus is required to facilitate further characterization and cloning of the gene (MEN1). We have characterized a panel of seven rodent-human somatic cell hybrids which contain fragments of human chromosome 11 with breakpoints in the pericentromeric region by using eight DNA sequences (D11S149, PGA, PYGM, D11S97, INT2, D11S37, D11S533, and D11S147) to define the region containing MEN1. This will facilitate the rapid localization of additional DNA sequences in this region. In addition, we have used a highly polymorphic repetitive degenerate hexanucleotide sequence, designated D11S533, for segregation studies in one family with MEN 1. These molecular genetic approaches will help to define a precise 1 to 2 centiMorgan map around MEN1.
Subject(s)
Chromosome Mapping , Chromosomes, Human, Pair 11 , Multiple Endocrine Neoplasia/genetics , Humans , Hybrid Cells , Nucleic Acid Hybridization , Polymerase Chain Reaction , Polymorphism, Genetic , Repetitive Sequences, Nucleic Acid/geneticsABSTRACT
In order to study transcriptional patterns of expression of individual class I HLA genes we have constructed a series of cDNA libraries from human cell lines including normal lymphoblastoid cell lines MANN and HOM2, two colorectal carcinoma cell lines, WiDr and SW480, and a fetal lung fibroblast cell line, MRC-5. Between 0.5 and 1 x 10(6) independent clones were screened in each library using a class I HLA-specific DNA probe and the frequency of class I HLA cDNA clones was found to vary between 0.23% (WiDr) and 0.76% (HOM2). Polymerase chain reaction (PCR)-based analyses of possible alternative splicing events showed that each of 161 class I HLA cDNA clones which had insert sizes exceeding 0.6 kb exhibited normal splicing patterns for exons 5 and 6. Similar PCR-based analyses in clones with appropriately large inserts revealed no exceptions to the normal splicing patterns for each of exons 2, 3, 4, and 7. Sixty of the class I HLA cDNA clones selected from the WiDr, MRC-5, and MANN cDNA libraries were assigned to individual loci following identification of locus-specific DNA sequences by PCR sequencing across exon 5. The sequences obtained from the 60 clones were each interpreted to correspond to one of the classical loci, HLA-A, HLA-B, and HLA-C. While representatives of the HLA-A locus predominated in the MANN library, HLA-B-specific clones were the most abundant in the WiDr and MRC-5 libraries.
Subject(s)
Gene Expression Regulation/genetics , HLA Antigens/genetics , Polymerase Chain Reaction/methods , Base Sequence , Cell Line , Consensus Sequence , DNA/analysis , DNA Probes , Electrophoresis, Agar Gel , Exons/genetics , Gene Library , Humans , Molecular Sequence Data , Sequence Homology, Nucleic Acid , Tumor Cells, CulturedABSTRACT
Screening of a human cosmid library representing genomic DNA from an individual homozygous for the HLA-DR2 B7 A2 haplotype yielded 109 class I HLA-specific clones. One cosmid clone, Ice 6.23, had a full-length nonclassical class I gene within a 5.4-kb HindIII fragment. The Ice 6.23-5.4H gene was cloned into the unique NotI site of an expression vector pSV2.Not, a derivative of pSV2neo, which was constructed to contain a second SV40 early region promoter adjacent to an introduced NotI site. The resulting construct was transfected into the P815-B2M cell line, a derivative of the mouse mastocytoma P815 (HTR) line which expressed human beta2-microglobulin following stable transfection with a cloned human beta2-microglobulin gene. Following transfection the Ice 6.23-5.4 H gene was found to be expressed at both the mRNA and cell surface product levels. DNA sequencing of this gene suggests that it is allelic to the HLA-6.0 gene clone (HLA-G) of Geraghty et al. (Proceedings of the National Academy of Sciences USA, 84:9145, 1987); thereby revealing a HindIII restriction fragment length polymorphism at the HLA-G locus. An extraordinarily high degree of sequence similarity (99.92%) between these two genes, which derive from unrelated HLA haplotypes, suggests strong conservative selection pressure at the HLA-G locus. A flanking single copy sequence probe 4 kb distant from the Ice 6.23-5.4H gene was used to generate long-range restriction mapping at the HLA-G locus.