Genetic mapping of chronic childhood-onset spinal muscular atrophy to chromosome 5q11.2-13.3

SPINAL muscular atrophy (SMA) describes a group of heritable degenerative diseases that selectively affect the alpha-motor neuron. Childhood-onset SMAs rank second in frequency to cystic fibrosis among autosomal recessive disorders, and are the leading cause of heritable infant mortality. Predictions that genetic heterogeneity underlies the differences between types of SMA, together with the aggressive nature of the most-severe infantile form, make linkage analysis of SMA potentially complex. We have now analysed 13 clinically heterogeneous SMA families. We find that 'chronic' childhood-onset SMA (including intermediate SMA or SMA type II, and Kugelberg-Welander or SMA type III) is genetically homogeneous, mapping to chromosomal region 5q11.2-13.3.     

Identification of a receptor for protein import into mitochondria

Anti-idiotypic antibodies, prepared using a chemically synthesized signal peptide of a mitochondrial precursor protein, recognized a mitochondrial integral membrane protein (p32). Fab fragments derived from both anti-idiotypic antibodies and monospecific antibodies against purified p32 inhibited protein import into mitochondria. Moreover, anti-p32 antibodies specifically immunoprecipitated a precursor-p32 complex after detergent solubilization of mitochondria. Immunoelectron microscopy and subfractionation of mitochondria indicate that p32 is located in contact sites between the outer and inner mitochondrial membranes.     

Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs

The aminoacyl-transfer RNA synthetases (aaRS) catalyse the attachment of an amino acid to its cognate transfer RNA molecule in a highly specific two-step reaction. These proteins differ widely in size and oligomeric state, and have limited sequence homology. Out of the 18 known aaRS, only 9 referred to as class I synthetases (GlnRS, TyrRS, MetRS, GluRS, ArgRS, ValRS, IleRS, LeuRS, TrpRS), display two short common consensus sequences ('HIGH' and 'KMSKS') which indicate, as observed in three crystal structures, the presence of a structural domain (the Rossman fold) that binds ATP. We report here the sequence of Escherichia coli ProRS, a dimer of relative molecular mass 127,402, which is homologous to both ThrRS and SerRS. These three latter aaRS share three new sequence motifs with AspRS, AsnRS, LysRS, HisRS and the beta subunit of PheRS. These three motifs (motifs 1, 2 and 3), in a search through the entire data bank, proved to be specific for this set of aaRS (referred to as class II). Class II may also contain AlaRS and GlyRS, because these sequences have a typical motif 3. Surprisingly, this partition of aaRS in two classes is found to be strongly correlated on the functional level with the acylation occurring either on the 2' OH (class I) or 3' OH (class II) of the ribose of the last nucleotide of tRNA.     

Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death

The t(14; 18) chromosomal translocation of human follicular B-cell lymphoma juxtaposes the bcl-2 gene with the immunoglobulin heavy chain locus. The bcl-2 immunoglobulin fusion gene is markedly deregulated resulting in inappropriately elevated levels of bcl-2 RNA and protein. Transgenic mice bearing a bcl-2 immunoglobulin minigene demonstrate a polyclonal expansion of resting yet responsive IgM-IgD B cells which display prolonged cell survival but no increase in cell cycling. Moreover, deregulated bcl-2 extends the survival of certain haematopoietic cell lines following growth-factor deprivation. By using immunolocalization studies we now demonstrate that Bcl-2 is an integral inner mitochondrial membrane protein of relative molecular mass 25,000 (25k). Overexpression of Bcl-2 blocks the apoptotic death of a pro-B-lymphocyte cell line. Thus, Bcl-2 is unique among proto-oncogenes, being localized to mitochondria and interfering with programmed cell death independent of promoting cell division.     

Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C

Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.     

Voltage-dependent InsP3-insensitive calcium channels in membranes of pancreatic endoplasmic reticulum vesicles.

Stimulus-secretion coupling in exocrine glands involves Ca2+ release from intracellular stores. In endoplasmic reticulum vesicle preparations from rat exocrine pancreas, an inositol 1,4,5-trisphosphate(InsP3)-sensitive, as well as an InsP3-insensitive, Ca2+ pool has been characterized. But Ca2+ channels in the endoplasmic reticulum of rat exocrine pancreas have not been demonstrated at the level of single-channel current. We have now used the patch-clamp technique on endoplasmic reticulum vesicles fused by means of the dehydration-rehydration method. In excised patches, single Ba2(+)- and Ca2(+)-selective channels were recorded. The channel activity was markedly voltage-dependent. Caffeine increased channel open-state probability, whereas ruthenium red and Cd2+ blocked single-channel currents. Ryanodine, nifedipine and heparin had no effect on channel activity. The channel activity was not dependent on the free Ca2+ concentration, the presence of InsP3, or pH. We conclude that this calcium channel mediates Ca2+ release from an intracellular store through an InsP3-insensitive mechanism.     

Localization of the human GM-CSF receptor gene to the X-Y pseudoautosomal region

Mammalian sex chromosomes share a small terminal region of homologous DNA sequences, which pair and recombine during male meiosis. Alleles in this region can be exchanged between X and Y chromosomes and are therefore inherited as if autosomal. Genes from this so-called pseudoautosomal region (PAR) are present in two doses in both males and females, and escape inactivation of the X chromosome in females. Indirect evidence suggests that there must be several pseudoautosomal genes, and several candidates have been proposed. Until now, the only gene that has been unequivocally located in the PAR is MIC2, which encodes a cell-surface antigen of unknown function. We now report the localization of a gene of known function to this region--the gene for the receptor of the haemopoietic regulator, granulocyte-macrophage colony stimulating factor. The chromosomal localization of this gene may be important in understanding the generation of M2 acute myeloid leukaemia.