Phenotypic changes associated with DYNACTIN-2 (DCTN2) over expression characterise SJSA-1 osteosarcoma cells.

DYNACTIN-2 (DCTN2) localises to chromosome 12q13-q15, a region prone to stable amplification in several cancers. Transient DCTN2 overexpression has a significant impact on cellular phenotype primarily due to disruption of the DYNEIN-dynactin motor. Changes reported include alterations of microtubule-directed movement of molecular (e.g. TP53) and organelle (e.g. Golgi) cargoes towards the nucleus, centrosome biology, cellular movement and mitosis with a potential predisposition to mitotic block and polyploidy. These changes would be expected to be of relevance to carcinogenesis. To investigate this, we report the first study of DCTN2 genomic amplification and sustained DCTN2 overexpression in cancer cells. QFMPCR was employed to characterise the extent of chromosome 12q13-q15 amplicons in SJSA-1, SJRH30, U373MG and CCF-STTG1 cancer cells. DCTN2 amplification was present in SJSA-1, U373MG and SJRH30 cells, yet was incomplete at the 5'-end in SJRH30 cells. Only SJSA-1 cells were characterised by DCTN2 overexpression on Western blot analyses. Microscopy studies distinguished SJSA-1 cells by greater DCTN2 immunofluorescence and diminished centrosome and 58K protein Golgi-marker focus compared to SJRH30 cells. Indirect evidence derived from the published work of others indicated that TP53 transport into the nucleus was unimpaired. Furthermore, we observed that SJSA-1 cells were easy to propagate. In conclusion, persistent DCTN2 overexpression can be tolerated in SJSA-1 cancer cells despite phenotypic abnormalities predicted from transient overexpression studies. This preliminary study does not support a major role for DCTN2 overexpression in carcinogenesis, although further studies would be necessary to confirm this.

Homozygosity for a missense mutation in the 67 kDa isoform of glutamate decarboxylase in a family with autosomal recessive spastic cerebral palsy: parallels with Stiff-Person Syndrome and other movement disorders.

BACKGROUND: Cerebral palsy (CP) is an heterogeneous group of neurological disorders of movement and/or posture, with an estimated incidence of 1 in 1000 live births. Non-progressive forms of symmetrical, spastic CP have been identified, which show a Mendelian autosomal recessive pattern of inheritance. We recently described the mapping of a recessive spastic CP locus to a 5 cM chromosomal region located at 2q24-31.1, in rare consanguineous families. METHODS: Here we present data that refine this locus to a 0.5 cM region, flanked by the microsatellite markers D2S2345 and D2S326. The minimal region contains the candidate gene GAD1, which encodes a glutamate decarboxylase isoform (GAD67), involved in conversion of the amino acid and excitatory neurotransmitter glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). RESULTS: A novel amino acid mis-sense mutation in GAD67 was detected, which segregated with CP in affected individuals. CONCLUSIONS: This result is interesting because auto-antibodies to GAD67 and the more widely studied GAD65 homologue encoded by the GAD2 gene, are described in patients with Stiff-Person Syndrome (SPS), epilepsy, cerebellar ataxia and Batten disease. Further investigation seems merited of the possibility that variation in the GAD1 sequence, potentially affecting glutamate/GABA ratios, may underlie this form of spastic CP, given the presence of anti-GAD antibodies in SPS and the recognised excitotoxicity of glutamate in various contexts.

PRELI (protein of relevant evolutionary and lymphoid interest) is located within an evolutionarily conserved gene cluster on chromosome 5q34-q35 and encodes a novel mitochondrial protein.

The characterization of mitochondrial proteins is important for the understanding of both normal cellular function and mitochondrial disease. In the present study we identify a novel mitochondrial protein, PRELI (protein of relevant evolutionary and lymphoid interest), that is encoded within the evolutionarily conserved MAD3/PRELI/RAB24 gene cluster located at chromosome 5q34-q35. Mouse Preli is expressed at high levels in all settings analysed; it is co-expressed with Rab24 from a strong bi-directional promoter, and is regulated independently from the S-phase-specific Mad3 gene located at its 3' end. PRELI contains a stand-alone 170 amino acid PRELI/MSF1p' motif at its N-terminus. This domain is found in a variety of proteins from diverse eukaryotes including yeast, Drosophila and mammals, but its function is unknown, and the subcellular location of higher eukaryotic PRELI/MSF1P' proteins has not been determined previously. We show here that PRELI is located in the mitochondria, and by using green-fluorescent-protein fusion proteins we identify a mitochondrial targeting signal at its N-terminus.

Identification of SATB2 as the cleft palate gene on 2q32-q33.

Cytogenetic evidence, in the form of deletions and balanced translocations, points to the existence of a locus on 2q32-q33, for which haploinsufficiency results in isolated cleft palate (CPO). Here we show by high-resolution FISH mapping of two de novo CPO-associated translocations involving 2q32-q33 that one breakpoint interrupts the transcription unit of the gene encoding the DNA-binding protein SATB2 (formerly KIAA1034). The breakpoint in the other translocation is located 130 kb 3' to the SATB2 polyadenylation signal, within a conserved region of non-coding DNA. The SATB2 gene is transcribed in a telomeric to centromeric direction and lies in a gene-poor region of 2q32-q33; the nearest confirmed gene is 1.26 Mb centromeric to the SATB2 polyadenylation signal. SATB2-encoding transcripts are assembled from 11 exons that span 191 kb of genomic DNA. They encode a protein of 733 amino acids that has two CUT domains and a homeodomain and shows a remarkable degree of evolutionary conservation, with only three amino acid substitutions between mouse and human. This protein belongs to the same family as SATB1, a nuclear matrix-attachment region binding protein implicated in transcriptional control and control of chromatin remodelling. There are also sequence similarities to the Drosophila protein DVE. Whole mount in situ hybridization to mouse embryos shows site- and stage-specific expression of SATB2 in the developing palate. Despite the strong evidence supporting an important role for SATB2 in palate development, mutation analysis of 70 unrelated patients with CPO did not reveal any coding region variants.

Detection by fluorescence in situ hybridization of microdeletions at 1p36 in lymphomas, unidentified on cytogenetic analysis.

The chromosomal band 1p36 exhibits frequent loss of heterozygosity in a variety of human malignancies, suggesting the presence of an as yet unidentified tumor suppressor gene. The faint terminal subbands often make cytogenetic analysis of 1p36 particularly difficult. Small deletions at this locus may therefore escape detection on analysis by conventional cytogenetics, a hypothesis that we have explored using fluorescence in situ hybridization (FISH) in malignant lymphoma. The study cohort consisted of 20 cases of lymphoma of various subtypes without any 1p abnormality on G-banded karyotyping. FISH was performed using a human chromosome 1 paint and a bacterial artificial chromosome probe RP4-755G5 localizing to 1p36.33, the most telomeric subband of 1p36. Tumors demonstrating 1p36.33 deletions were additionally analyzed by FISH using a second probe from the proximal 1p36.1 subband, to further define the breakpoint. Eight cases of follicular lymphoma (FL), 5 diffuse large B-cell lymphomas (DLBCL), 2 Hodgkin disease, 2 B-cell small lymphocytic lymphomas, 2 T-cell lymphomas, and 1 marginal zone lymphoma were analyzed. FISH identified deletions at 1p36.33 in 5 of the 20 cases: 3 DLBCL and 2 FL. FISH is considerably more sensitive for identifying lymphoma genetic alterations than conventional cytogenetics. Deletion of the distal part of the 1p36 may be a much more common aberration than previously recognized in lymphoma.

Expression of mOb1, a novel atypical 73 amino acid K50-homeodomain protein, during mouse development.

We report the initial characterization of mOb1 (Odd homeoBox 1), which encodes an atypical 73 amino acid K50-homeodomain protein localised in the cytoplasm and absent from nuclei during mouse development. Conserved orthologues were present in man, rat, cow, pig and chicken, but not in fish, amphibians or invertebrates. Temporo-spatial patterns of mOb1 transcript and mOb1 protein expression were coincident in developing mouse embryos. Cardiac expression was first observed at E8.25 in linear heart tube myocardium and briefly in both horns of the sinus venosus. Myocardial expression peaked at E13.5, where after it diminished and was not detectable above background by adulthood. At no stage was expression observed in endocardium, endocardial cushion tissue, the coronary arteries or great vessels. At E13.5 and E15.5, mOb1 expression broadened to include skeletal muscle, stratified epithelium (upper aerodigestive tract and skin), epithelium of developing airways, vibrissae, midbrain/hindbrain junction, meninges, mesenchymal cellular condensations that preceded cartilage formation and chondrocytes.

Expression of mOb1, a novel atypical 73 amino acid K50-homeodomain protein, during mouse development.

We report the initial characterization of mOb1 (Odd homeoBox 1), which encodes an atypical 73 amino acid K50-homeodomain protein localised in the cytoplasm and absent from nuclei during mouse development. Conserved orthologues were present in man, rat, cow, pig and chicken, but not in fish, amphibians or invertebrates. Temporo-spatial patterns of mOb1 transcript and mOb1 protein expression were coincident in developing mouse embryos. Cardiac expression was first observed at E8.25 in linear heart tube myocardium and briefly in both horns of the sinus venosus. Myocardial expression peaked at E13.5, where after it diminished and was not detectable above background by adulthood. At no stage was expression observed in endocardium, endocardial cushion tissue, the coronary arteries or great vessels. At E13.5 and E15.5, mOb1 expression broadened to include skeletal muscle, stratified epithelium (upper aerodigestive tract and skin), epithelium of developing airways, vibrissae, midbrain/hindbrain junction, meninges, mesenchymal cellular condensations that preceded cartilage formation and chondrocytes.