New use of BCG for recombinant vaccines

BCG, a live attenuated tubercle bacillus, is the most widely used vaccine in the world and is also a useful vaccine vehicle for delivering protective antigens of multiple pathogens. Extrachromosomal and integrative expression vectors carrying the regulatory sequences for major BCG heat-shock proteins have been developed to allow expression of foreign antigens in BCG. These recombinant BCG strains can elicit long-lasting humoral and cellular immune responses to foreign antigens in mice.     

A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA

Hepatitis delta virus genomic and antigenomic RNAs contain a self-cleavage site hypothesized to function in processing the viral RNA during replication. Self-cleavage requires only a divalent cation and is mediated at the genomic site by a sequence of less than 85 nucleotides. We propose that the genomic self-cleaving sequence element and a corresponding sequence from the anti-genomic RNA could generate related secondary structures. The region of the antigenomic sequence, predicted from the proposed structure, was synthesized and shown to be sufficient for self-cleavage. Evidence for two stems which form a tertiary interaction was obtained by site-specific mutagenesis of the antigenomic sequence. Efficient self-cleavage in 10 M formamide or 5 M urea, also a property of the genomic sequence, was dependent on base-pairing in both stems. But in the absence of denaturants, the stem distal to the site of cleavage was not required, suggesting that the tertiary interaction stabilizes the structure required for self-cleavage.     

A novel cyclin encoded by a bcl1-linked candidate oncogene

We have previously identified a candidate oncogene (PRAD1 or D11S287E) on chromosome 11q13 which is clonally rearranged with the parathyroid hormone locus in a subset of benign parathyroid tumours. We now report that a cloned human placental PRAD1 complementary DNA encodes a protein of 295 amino acids with sequence similarities to the cyclins. Cyclins can form a complex with and activate p34cdc2 protein kinase, thereby regulating progress through the cell cycle. PRAD 1 messenger RNA levels vary dramatically across the cell cycle in HeLa cells. Addition of the PRAD1 protein to interphase clam embryo lysates containing inactive p34cdc2 kinase and lacking endogenous cyclins allows it to be isolated using beads bearing p13suc1, a yeast protein that binds cdc2 and related kinases with high affinity and coprecipitates kinase-associated proteins. Addition of PRAD1 also induces phosphorylation of histone H1, a preferred substrate of cdc2. These data suggest that PRAD1 encodes a novel cyclin whose overexpression may play an important part in the development of various tumours with abnormalities in 11q13.     

Mutational analysis of a protein-folding pathway

The effects of amino-acid replacements on the disulphide-coupled folding pathway of bovine pancreatic trypsin inhibitor have been examined. Replacements at three sites destabilize the native protein relative to the unfolded state, but have different effects on the relative stabilities of the disulphide-bonded folding intermediates, thus allowing the roles of the altered residues during folding to be distinguished.     

Inhibition of endocytic vesicle fusion in vitro by the cell-cycle control protein kinase cdc2

Membrane transport between the endoplasmic reticulum and the plasma membrane, which involves the budding and fusion of carrier vesicles, is inhibited during mitosis in animal cells. At the same time, the Golgi complex and the nuclear envelope, as well as the endoplasmic reticulum in some cell types, become fragmented. Fragmentation of the Golgi is believed to facilitate its equal partitioning between daughter cells. In fact, it has been postulated that both the inhibition of membrane traffic and Golgi fragmentation during mitosis are due to an inhibition of vesicle fusion, while vesicle budding continues. Although less is known about the endocytic pathway, internalization and receptor recycling are also arrested during mitosis. We have now used a cell-free assay to show that the fusion of endocytic vesicles from baby hamster kidney cells is reduced in Xenopus mitotic cytosol when compared with interphase cytosol. We reconstituted this inhibition in interphase cytosol by adding a preparation enriched in the starfish homologue of the cdc2 protein kinase. Inhibition was greater than or equal to 90% when the added cdc2 activity was in the range estimated for that in mitotic Xenopus eggs, which indicates that during mitosis the cdc2 kinase mediates an inhibition of endocytic vesicle fusion, and possibly other fusion events in membrane traffic.     

Phenotypic differences between alpha beta versus beta T-cell receptor transgenic mice undergoing negative selection

T-cell differentiation in the thymus is thought to involve a progression from the CD4-CD8- phenotype through CD4+CD8+ intermediates to mature CD4+ or CD8+ cells. There is evidence that during this process T cells bearing receptors potentially reactive to 'self' are deleted by a process termed 'negative selection' One example of this process occurs in mice carrying polymorphic Mls antigens, against which a detectable proportion of T cells are autoreactive. These mice show clonal deletion of thymic and peripheral T-cell subsets that express the autoreactive V beta 3 segment of the T-cell antigen receptor, but at most a two-fold depletion of thymic cells at the CD4+CD8+ stage. By contrast, transgenic mice bearing both alpha and beta chain genes encoding autoreactive receptors recognizing other ligands, show severe depletion of CD4+CD8+ thymocytes as well, suggesting that negative selection occurs much earlier. We report here the Mls 2a/3a mediated elimination of T cells expressing a transgene encoded V beta 3-segment, in T-cell receptor alpha/beta and beta-transgenic mice. Severe depletion of CD4+CD8+ thymocytes is seen only in the alpha/beta chain transgenic mice, whereas both strains delete mature V beta 3 bearing CD4+ and CD8+ T cells efficiently. We conclude that severe CD4+CD8+ thymocyte deletion in alpha/beta transgenic mice results from the premature expression of both receptor chains, and does not reflect a difference in the timing or mechanism of negative selection for Mls antigens as against the allo- and MHC class 1-restricted antigens used in the other studies.     

Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology

Neurotransmission effected by GABA (gamma-aminobutyric acid) is predominantly mediated by a gated chloride channel intrinsic to the GABAA receptor. This heterooligomeric receptor exists in most inhibitory synapses in the vertebrate central nervous system (CNS) and can be regulated by clinically important compounds such as benzodiazepines and barbiturates. The primary structures of GABAA receptor alpha- and beta-subunits have been deduced from cloned complementary DNAs. Co-expression of these subunits in heterologous systems generates receptors which display much of the pharmacology of their neural counterparts, including potentiation by barbiturates. Conspicuously, however, they lack binding sites for, and consistent electrophysiological responses to, benzodiazepines. We now report the isolation of a cloned cDNA encoding a new GABAA receptor subunit, termed gamma 2, which shares approximately 40% sequence identity with alpha- and beta-subunits and whose messenger RNA is prominently localized in neuronal subpopulations throughout the CNS. Importantly, coexpression of the gamma 2 subunit with alpha 1 and beta 1 subunits produces GABAA receptors displaying high-affinity binding for central benzodiazepine receptor ligands.