Identification of survival motor neuron as a transcriptional activator-binding protein.

Spinal muscular atrophy (SMA) is an inherited neuro-muscular disease characterized by specific degeneration of spinal cord anterior horn cells and subsequent muscle atrophy. Survival motor neuron ( SMN ), located on chromosome 5q13, is the SMA-determining gene. In the nucleus, SMN is present in large foci called gems, the function of which is not yet known, while cytoplasmic SMN has been implicated in snRNP biogenesis. In SMA patients, SMN protein levels and the number of gems generally correlate with disease severity, suggesting a critical nuclear function for SMN. In a screen for proteins associated with the nuclear transcription activator 'E2' of papillomavirus, two independent SMN cDNAs were isolated. The E2 and SMN proteins were found to associate specifically in vitro and in vivo. Expression of SMN enhanced E2-dependent transcriptional activation, and patient-derived SMN missense mutations reduced E2 gene expression. Our results demonstrate that SMN interacts with a nuclear transcription factor and imply that SMN may serve a role in regulating gene expression. These observations suggest that SMA may in part result from abnormal gene expression and that E2 may influence viral gene expression through SMN interaction.

SMN oligomerization defect correlates with spinal muscular atrophy severity.

Spinal muscular atrophy (SMA) is a motor-neuron disorder resulting from anterior-horn-cell death. The autosomal recessive form has a carrier frequency of 1 in 50 and is the most common genetic cause of infant death. SMA is categorized as types I-III, ranging from severe to mild, based upon age of onset and clinical course. Two closely flanking copies of the survival motor neuron (SMN) gene are on chromosome 5q13 (ref. 1). The telomeric SMN (SMN1) copy is homozygously deleted or converted in >95% of SMA patients, while a small number of SMA disease alleles contain missense mutations within the carboxy terminus. We have identified a modular oligomerization domain within exon 6 of SMN1. All previously identified missense mutations map within or immediately adjacent to this domain. Comparison of wild-type to mutant SMN proteins of type I, II and III SMA patients showed a direct correlation between oligomerization and clinical type. Moreover, the most abundant centromeric SMN product, which encodes exons 1-6 but not 7, demonstrated reduced self-association. These findings identify decreased SMN self-association as a biochemical defect in SMA, and imply that disease severity is proportional to the intracellular concentration of oligomerization-competent SMN proteins.

The survival motor neuron protein in spinal muscular atrophy.

The 38 kDa survival motor neuron (SMN) protein is encoded by two ubiquitously expressed genes: telomeric SMN (SMN(T)) and centromeric SMN (SMN(C)). Mutations in SMN(T), but not SMN(C), cause proximal spinal muscular atrophy (SMA), an autosomal recessive disorder that results in loss of motor neurons. SMN is found in the cytoplasm and nucleus. The nuclear form is located in structures termed gems. Using a panel of anti-SMN antibodies, we demonstrate that the SMN protein is expressed from both the SMN(T) and SMN(C) genes. Western blot analysis of fibroblasts from SMA patients with various clinical severities of SMA showed a moderate reduction in the amount of SMN protein, particularly in type I (most severe) patients. Immunocytochemical analysis of SMA patient fibroblasts indicates a significant reduction in the number of gems in type I SMA patients and a correlation of the number of gems with clinical severity. This correlation to phenotype using primary fibroblasts may serve as a useful diagnostic tool in an easily accessible tissue. SMN is expressed at high levels in brain, kidney and liver, moderate levels in skeletal and cardiac muscle, and low levels in fibroblasts and lymphocytes. In SMA patients, the SMN level was moderately reduced in muscle and lymphoblasts. In contrast, SMN was expressed at high levels in spinal cord from normals and non-SMA disease controls, but was reduced 100-fold in spinal cord from type I patients. The marked reduction of SMN in type I SMA spinal cords is consistent with the features of this motor neuron disease. We suggest that disruption of SMN(T) in type I patients results in loss of SMN from motor neurons, resulting in the degeneration of these neurons.

Longitudinal study of human herpesvirus 6 infection in organ transplant recipients.

Human herpesvirus 6 (HHV-6) has been frequently isolated from immunocompromised patients. To determine if a routine survey of HHV-6 infection is needed after organ transplantation, as is the case for human cytomegalovirus infection, we observed patients who had received kidney, liver, and kidney-liver transplants; these patients were followed up for the first 3 months after transplantation. HHV-6 infection was diagnosed by isolation of the virus and by the results of serological tests. Antibodies to HHV-6 were detected in 28 (87.5%) of the 32 recipients, before the transplant, whereas only 4 (12.5%) of the 32 recipients were seronegative for HHV-6. After engraftment, HHV-6 infection occurred in 10 (31%) of the 32 recipients; infection was diagnosed by isolation of the virus (6 of 32 recipients) or by the results of serological tests (4 of 32 recipients). Regardless of whether they had HHV-6 primary infection or reactivation, severe clinical manifestations were observed only in patients who had concomitant cytomegalovirus infection, and no correlation could be found between graft rejection and HHV-6 infection. These results suggest that HHV-6 infection occurs frequently in organ transplant recipients and that it is usually not associated with severe clinical manifestations unless accompanied by a concomitant CMV infection.

A new luciferase promoter insertion vector for the analysis of weak transcriptional activities.

A new luciferase-encoding expression vector was generated by inserting the strong transcription termination signal from the mouse c-mos oncogene upstream from a multiple cloning site. This construct significantly reduced background transcription in NIH3T3 cells and has proven useful in the study of a weak promoter from the murine growth-arrest-specific gene gas-1.