Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally.

Homozygous targeted disruption of the mouse Caspase 8 (Casp8) gene was found to be lethal in utero. The Caspase 8 null embryos exhibited impaired heart muscle development and congested accumulation of erythrocytes. Recovery of hematopoietic colony-forming cells from the embryos was very low. In fibroblast strains derived from these embryos, the TNF receptors, Fas/Apo1, and DR3 were able to activate the Jun N-terminal kinase and to trigger IkappaB alpha phosphorylation and degradation. They failed, however, to induce cell death, while doing so effectively in wild-type fibroblasts. These findings indicate that Caspase 8 plays a necessary and nonredundant role in death induction by several receptors of the TNF/NGF family and serves a vital role in embryonal development.

The yeast two-hybrid screening technique and its use in the study of protein-protein interactions in apoptosis.

The yeast two-hybrid technique provides a general approach for cloning cDNAs merely by exploiting the ability of their encoded proteins to bind to a protein of interest. The technique therefore offered a useful access to the analysis of the mechanisms of cell death at the initial stage of their study, when only a few of the proteins involved and very little about their mode of action were known. Conversely, the knowledge of cell death mechanisms gained by this technique provided a useful insight into both the potential and the limitations of this technique.

A protein related to a proteasomal subunit binds to the intracellular domain of the p55 TNF receptor upstream to its 'death domain'.

A novel protein that binds specifically to the intracellular domain of the p55 tumor necrosis factor (TNF) receptor was cloned by two-hybrid screening of a HeLa cell cDNA library. Data bank searches revealed high sequence similarity of the protein (55.11) to yeast, nematode and plant proteins, whose functions are yet unknown. Significant similarity was also found between 55.11 and SEN3, the yeast equivalent of the p112 subunit of the 26S proteasome. Deletion analysis showed that the protein binds to the p55 receptor upstream to the region involved in induction of cell death.

A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain.

Signaling for cell death by Fas/APO1 occurs via a distinct region in its intracellular domain. This region contains a conserved sequence motif, the death domain motif, that is also found in the intracellular domains of the p55 tumor necrosis factor receptor and the low affinity nerve growth factor receptor, as well as in the regulatory domain of the ankyrins. A novel protein that specifically binds to the death domain of Fas/APO1 but not to Fas/APO1 molecules with a loss of function point mutation occurring in lprcg mice was cloned by a two-hybrid screen of a HeLa cells' cDNA library. The cloned protein itself contains a death domain motif, and this region binds to the death domain of Fas/APO1, while the region upstream to the death domain prompts self-association of the protein. Induced expression of the protein results in ligand-independent triggering of cytotoxicity, suggesting that it is involved in cell death induction by Fas/APO1.

Self-association of the "death domains" of the p55 tumor necrosis factor (TNF) receptor and Fas/APO1 prompts signaling for TNF and Fas/APO1 effects.

Signaling by the p55 tumor necrosis factor (TNF) receptor and by the structurally related receptor Fas/APO1 is initiated by receptor clustering. Data presented here and in other recent studies (Wallach, D., Boldin, M., Varfolomeev, E. E., Bigda, Y., Camonis, H.J. and Mett, I. (1994) Cytokine 6, 556; Song, H.Y., Dunbar, J.D., and Bonner, D.B. (1994) J. Biol. Chem. 269, 22492-22495) indicate that part of that region within the intracellular domains of the two receptors that is involved in signaling for cell death, as well as for some other effects (the "death domain", specifically self-associates. We demonstrate also the expected functional consequence of this association; a mere increase in p55 TNF receptor expression, or the expression just of its intracellular domain, is shown to trigger signaling for cytotoxicity as well as for interleukin 8 gene induction, while expression of the intracellular domain of Fas/APO1 potentiates the cytotoxicity of co-expressed p55 TNF receptor. These findings indicate that the p55 TNF and Fas/APO1 receptors play active roles in their own clustering and suggest the existence of cellular mechanisms that restrict the self-association of these receptors, thus preventing constitutive signaling.

[Molecular cloning and structural-functional analysis of the arginine biosynthesis genes of the thermophilic bacterium Bacillus stearothermophilus]. / Molekuliarnoe klonirovanie i strukturno-funktsional'nyi analiz genov biosinteza arginina termofil'noi bakterii Bacillus stearothermophilus.

Genes encoding arginine biosynthesis of Bacillus stearothermophilus strain 718 were cloned in the mutant argA strain of the Escherichia coli K-12. The arg genes were shown to be located on the 3.7 kb DNA fragment in the following order: argA--argE--argB. The expression of the argA gene of B. stearothermophilus on the multicopy vehicle is twofold higher in argR- strain of E. coli K-12 than is isogenic argR+ strain. According to hybridization analysis argA genes of B. stearothermophilus and B. subtilis have low level of homology, which is the evidence of their evolutional divergence.

[Restriction mapping of recombinant plasmids carrying the genes for arginine biosynthesis in Escherichia coli K-12]. / Restriktsionnoe kartirovanie rekombinantnykh plazmid, nesushchikh geny biosinteza arginina Escherichia coli K-12.

ArgA and argECBH genes of Escherichia coli K-12 were cloned on the pBR322 vector. Restriction maps of the recombinant plasmids were constructed. Deletion mutants of these recombinant plasmids, retaining the functional argA and argE genes, were obtained using different restriction enzymes. All of the recombinant derivatives have the replication properties of the pBR322 vector.

[Presence of genes of ribosomal and transfer RNAs in kinetoplast DNA from two Crithidia species]. / O nalichii genov ribosomnykh i transportnykh RNK v kinetoplastnoi DNK dvukh vidov kritidii.

The coding properties of kinetoplast DNA from two respresentatives of the order Kinetoplastidae--Crithidia oncopelti and C. fasciculata--were studied. Experiments on hybridization of the whole network and fraction of minicircles with labelled 23S and 16S rRNA and with tRNA isolated from kinetoplasts of C. oncopelti clearly demonstrated the presence of the genes for these RNAs in the whole network and their absence in the minicircles. It may be thus concluded that the genes of ribosomal and transfer RNAs are localized in the maxicircular molecules. Similar efficiency of hybridization of rRNAs from C. oncopelti with kDNA from C. fasciculata and C. oncopelti revealed significant conservativity of ribosomal genes in the protozoa under study.

[DNA-dependent RNA-polymerase activity of isolated kinetoplasts of crithidia oncopelti]. / DNK-zavisimaia RNK-polimeraznaia aktivnost' izolirovannykh kinetoplastov Crithidia oncopelti

DNA-dependent RNA-polymerase activity was found in the kinetoplasts of Crithidia oncopelti. Kinetic patterns of 14C-UTP incorporation into the acid-insoluble fraction of isolated kinetoplasts at 25 degrees, 30 degrees and 35 degrees C were estimated. The effects of different antibiotics and intercalating agents on RNA synthesis in kinetoplasts were studied. alpha-amanitin, a specific inhibitor of the nuclear enzyme, does not affect the RNA-polymerase activity of the kinetoplasts. Streptolidigin and rifampicin, inhibitors of bacterial RNA-polymerase, have little effect on RNA synthesis in the kinetoplasts even at high concentrations. Kinetoplasts preincubation in the phosphate buffer increases the permeability of their membranes for rifampicin. Intercalating agents, acriflavin and ethidium bromide, strongly inhibit the kinetoplast RNA synthesis. Similar specific effects of some antibiotics and intercalating agents on RNA synthesis in kinetoplasts and typical mitochondria may be indicative of similarity of functional properties of RNA-polymerases in those organelles.