ABSTRACT
Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has been mapped to human chromosome 2q33. Here we report the identification of two independent deletion mutations linked to ALS2 in the coding exons of the new gene ALS2. These deletion mutations result in frameshifts that generate premature stop codons. ALS2 is expressed in various tissues and cells, including neurons throughout the brain and spinal cord, and encodes a protein containing multiple domains that have homology to RanGEF as well as RhoGEF. Deletion mutations are predicted to cause a loss of protein function, providing strong evidence that ALS2 is the causative gene underlying this form of ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , GTP Phosphohydrolases/metabolism , Guanine Nucleotide Exchange Factors/genetics , Mutation , Amino Acid Sequence , Animals , Chromosome Mapping , Chromosomes, Human, Pair 2 , Female , Guanine Nucleotide Exchange Factors/chemistry , Humans , Male , Mice , Molecular Sequence Data , Polymorphism, Genetic , Sequence Homology, Amino AcidABSTRACT
Huntington's disease (HD) is associated with a significant expansion of a CAG trinucleotide repeat, which results in a lengthened polyglutamine tract in the single gene product, huntingtin, on human 4p16.3. We isolated cDNA clones that encompassed the entire coding sequence of the miniature pig HD gene (Sus HD) from two porcine testis cDNA libraries. The cDNA contig revealed a 12,749-nucleotide transcript coding for a 345-kDa protein (3139 amino acid residues), which exhibited 96% peptide sequence homology to human huntingtin. Northern blot analysis revealed that the Sus HD gene was ubiquitously expressed as two large transcripts of approximately 11 and 13 kb in size in all the tested tissues, much like the human HD gene. The CAG trinucleotide repeat was found to be interrupted by CAA triplets and to encode 17 or 18 consecutive glutamine residues. In our laboratory stock of miniature pig, three allotypes in the triplet repeat sequence were found. Thus, the Sus HD gene closely resembles its human counterpart in terms of sequence and expression pattern. In particular, human-miniature pig similarities in the normal length of the CAG triplet repeat as well as its repeat-number polymorphism may indicate that miniature pig would provide a good animal model for Huntington's disease.
Subject(s)
Huntington Disease/genetics , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , 3' Untranslated Regions/genetics , 5' Untranslated Regions/genetics , Alleles , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Conserved Sequence , DNA, Complementary/chemistry , DNA, Complementary/genetics , Female , Humans , Huntingtin Protein , Male , Molecular Sequence Data , Peptides/genetics , Polymorphism, Genetic , RNA/genetics , RNA/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Swine, Miniature , Tissue Distribution , Trinucleotide Repeats/geneticsABSTRACT
We have recently identified a tandem repetitive DNA sequence that we designated the RS447 megasatellite. In this study, we describe a functional novel deubiquitinating enzyme (USP17, 60 kDa) gene that is intronless and encoded by the RS447 repeating unit. Northern blot analysis in conjunction with 5' and 3' rapid amplification of cDNA ends confirmed the presence of poly(A)(+) containing RS447 RNA in normal cells. We also identified a functional promoter sequence as well as an open reading frame within every RS447 repeat. When USP17 was expressed in Escherichia coli, it exhibited deubiquitinating activity in vivo. An antibody against USP17 detected USP17 protein in human cells. Our results indicate that the RS447 repeating unit on this megasatellite repeat codes for and actively expresses a functional deubiquitinating enzyme. Although it is expressed ubiquitously in human tissues, USP17 exhibited a unique expression pattern in that its complementary strand is transcribed as an antisense transcript that may modulate the level of USP17 expression in the human brain.
