Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. The HTLV-1 transactivator, Tax, is implicated as the viral oncoprotein. Naïve cells expressing Tax for the first time develop severe cell cycle abnormalities that include increased DNA synthesis, mitotic arrest, appearance of convoluted nuclei with decondensed DNA, and formation of multinucleated cells. Here we report that Tax causes a drastic reduction in Pds1p/securin and Clb2p/cyclin B levels in yeast, rodent, and human cells and a loss of cell viability. With a temperature-sensitive mutant of the CDC23 subunit of the anaphase-promoting complex (APC), cdc23 ts ; a temperature-sensitive mutant of cdc20; and a cdh1-null mutant, we show that the diminution of Pds1p and Clb2p brought on by Tax is mediated via the Cdc20p-associated anaphase-promoting complex, APC Cdc20p . This loss of Pds1p/securin and Clb2p/cyclin B1 occurred before cellular entry into mitosis, caused a G 2 /M cell cycle block, and was accompanied by severe chromosome aneuploidy in both Saccharomyces cerevisiae cells and human diploid fibroblasts. Our results support the notion that Tax aberrantly targets and activates APC Cdc20p , leading to unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1, a delay or failure in mitotic entry and progression, and faulty chromosome transmission. The chromosomal instability resulting from a Tax-induced deficiency in securin and cyclin B1 provides an explanation for the highly aneuploid nature of adult T-cell leukemia cells.Human T-lymphotropic virus type 1 (HTLV-1) causes a malignancy of CD4 ϩ T lymphocytes called adult T-cell leukemia and a neurological disorder known as HTLV-1-associated myelopathy/tropical spastic paraparesis. Adult T-cell leukemia occurs in 2 to 6% of HTLV-1-infected individuals after a latency period of up to 20 to 40 years. The mechanism for progression from clinical latency to T-cell malignancy is not well understood but involves the unique viral transactivatoroncoprotein Tax, a regulatory protein critical for viral replication and T-cell transformation. Tax performs two major functions during the HTLV-1 life cycle: first, it mediates potent activation of viral transcription; second, it usurps regulatory mechanisms critical for cell growth and division to facilitate viral replication.Although there is general agreement on the mechanism of Tax-mediated HTLV-1 long terminal repeat transactivation, the exact mechanism through which Tax promotes oncogenesis is not fully resolved. The effects that Tax exerts on cells are pleiotropic and include potent 16,20,45,46), cell cycle perturbation (1,8,15,24,(30)(31)(32)38), and cell transformation (13,29,36,46). More recently, Jin et al. reported that the interaction between Tax and the human spindle checkpoint protein HsMAD-1 causes a spindle checkpoint defect that results in DNA aneuploidy, microsatellite instability, and the formation of multinucleated giant cells (18,21). In an earlier study, we showed that, in naïve mammalian cells, Tax expressi...

Background: Definitive molecular diagnosis of mitochondrial disorders has been greatly hindered by the tremendous clinical and genetic heterogeneity, the heteroplasmic condition of pathogenic mutations, and the presence of numerous homoplasmic mitochondrial DNA (mtDNA) variations with unknown significance. We used temporal temperature gradient gel electrophoresis (TTGE) to detect heteroplasmic mutations from homoplasmic variations in the whole mitochondrial genome. Methods: We screened 179 unrelated patients by TTGE with use of 32 overlapping primer pairs. Mutations were identified by direct sequencing of the PCR products and confirmed by PCR with allele-specific oligonucleotide or restriction fragment length polymorphism analysis. Results: We detected 71 heteroplasmic and 647 homoplasmic banding patterns. Sequencing of the heteroplasmic fragments identified 68 distinct novel mutations and 132 reported sequence variations and mutations; most of them occurred only once. The deleterious nature of some of the novel mutations was established by analyzing the asymptomatic family members and the biochemical and molecular characteristics of the mutation. When the number of mutations was normalized to the size of the region, the occurrence of mutations was 2.4 times more frequent in the tRNA genes than in the mRNA (protein coding) regions. Conclusions: Screening by TTGE detects low proportions of mutant mtDNA and distinguishes heteroplasmic from homoplasmic variations. Results from comprehensive molecular analysis should be followed up with clinical correlation to establish a guideline for complete mutational analysis of the entire mitochondrial genome and to facilitate the diagnosis of mitochondrial disorders.

The multiplex polymerase chain reaction-allele specific oligonucleotides (PCR/ASO) dot blot hybridization method was used to detect 44 mitochondrial DNA point mutations in 2,000 patients suspected as having mitochondrial DNA disorders. These point mutations are classified into four categories. Category I consists of primary disease-causing, heteroplasmic point mutations. Homoplasmic nucleotide substitutions that have been reported to be possibly disease associated are in Category II. Homoplasmic nucleotide substitutions that are thought to be benign polymorphism are included in category III. The novel nucleotide substitutions recently discovered in our laboratory by single strand conformation polymorphism analysis are in category IV. Frequencies of these 44 nucleotide substitutions in 2,000 patients and 262 control individuals were studied. The results indicated that analysis of 12 recurrent disease-causing point mutations in category I identified 5.4% of the patients suspected as having mitochondrial DNA disorders. Since the mitochondrial disorders are a group of complex, heterogeneous, and multisystemic diseases, it is often difficult to confirm clinical diagnosis without molecular studies. Thus, the multiplex PCR/ASO method is an effective approach for initial screening of mtDNA mutations in patients suspected as having mitochondrial DNA disorders.

A 21-year-old woman who has been suspected of mitochondrial cytopathy, but negative for common mitochondrial DNA (mtDNA) point mutations and deletions, was screened for unknown mutations in the entire mitochondrial genome by temporal temperature gradient gel electrophoresis (TTGE). Her asymptomatic mother's blood DNA was also analyzed and used as a reference. Two tRNA regions showing different TTGE patterns between the proband and her mother were sequenced. Two novel mutations, G15995A in tRNA(pro) and A8326G in tRNA(lys), were revealed. These mutations are present in heteroplasmic states. They both occurred at a nucleotide position that is highly conserved throughout evolution. This patient is also a compound heterozygote for the cystic fibrosis (CF) mutations, DeltaF508 and R347P. The phenotype for R347P has been associated with mild disease. Due to the mild features of the R347P mutation in the CF transmembrane conductance regulator (CFTR) gene and the heterogeneous clinical presentation of the mtDNA disease, the patient was not definitively diagnosed until age 21. This case underscores the importance of a complete mutational analysis of the entire mitochondrial genome when a patient suspected of mitochondrial disorder is negative for common mtDNA mutations.

The multiplex polymerase chain reaction-allele specific oligonucleotides (PCR/ASO) dot blot hybridization method was used to detect 44 mitochondrial DNA point mutations in 2,000 patients suspected as having mitochondrial DNA disorders. These point mutations are classified into four categories. Category I consists of primary disease-causing, heteroplasmic point mutations. Homoplasmic nucleotide substitutions that have been reported to be possibly disease associated are in Category II. Homoplasmic nucleotide substitutions that are thought to be benign polymorphism are included in category III. The novel nucleotide substitutions recently discovered in our laboratory by single strand conformation polymorphism analysis are in category IV. Frequencies of these 44 nucleotide substitutions in 2,000 patients and 262 control individuals were studied. The results indicated that analysis of 12 recurrent disease-causing point mutations in category I identified 5.4% of the patients suspected as having mitochondrial DNA disorders. Since the mitochondrial disorders are a group of complex, heterogeneous, and multisystemic diseases, it is often difficult to confirm clinical diagnosis without molecular studies. Thus, the multiplex PCR/ASO method is an effective approach for initial screening of mtDNA mutations in patients suspected as having mitochondrial DNA disorders.