Protein aggregation and inclusion body formation in Escherichia coli rpoH mutant defective in heat shock protein induction.

Mutations in the rpoH gene, encoding sigma 32, an alternative factor required for transcription of the heat shock genes, result in the extensive aggregation of virtually all cellular proteins and formation of inclusion bodies both under stress and non-stress conditions. Inhibitors of protein synthesis suppress this aggregation, suggesting that newly synthesized proteins preferentially aggregate in rpoH mutants. These data suggest that the heat shock proteins are involved in acquisition of the soluble state (i.e. correct conformation) of the bulk of intracellular proteins after their translation.

Heat shock response in Escherichia coli promotes assembly of plasmid encoded RNA polymerase beta-subunit into RNA polymerase.

Escherichia coli cells, carrying a rifampicin sensitive RNA polymerase beta-subunit gene in the chromosome and a rifampicin resistant beta-subunit gene placed under the control of a strong promoter in a multicopy plasmid, are unable to grow in the presence of rifampicin, despite the accumulation of large quantities of the resistant subunit. A major portion of the overproduced subunit is found in an insoluble form. Conditions known to induce the heat shock proteins (hsps), e.g. elevated temperature or the presence of ethanol in the growth medium, increase the amount of the plasmid-borne beta-subunit which apparently assembles into active RNA polymerase and makes the plasmid bearing cells rifampicin resistant. Alternatively, plasmid-borne subunits assemble into RNA polymerase with low efficiency in rpoH mutant cells known to have reduced level of hsps. We suggest that the plasmid-borne subunit is poorly assembled into RNA polymerase and that hsps promote the assembly by interfering with beta-subunit aggregation.

[Deletion-insertion mapping of the region non-essential for functioning of the beta-subunit of Escherichia coli RNA polymerase]. / Deletsionno-insertsionnoe kartirovanie oblasti, ne sushchestvennoi dlia funktsionirovaniia beta-sub"edinitsy RNK-polimerazy Escherichia coli.

A plasmid has been constructed containing the gene of beta-subunit of RNA polymerase of Escherichia coli under control of the PR promoter of bacteriophage lambda. PR promoter may be induced by heating up to 42 degrees C. In frame insertions of different sequences between 989 and 990 or 1010 and 1011 codons of the rpoB gene do not inactivate the beta-subunit function. Deletions in the region of 1011-1027 codons result in inactivation of beta-subunit. We localized antigene determinant of monoclonal anti-beta-antibodies which do not inactivate RNA polymerase in vitro. The borders of non-essential region of beta-subunit were accurately determined.

Stability of the pBR322 plasmid as affected by the promoter region of the tetracycline-resistance gene.

A region affecting the pBR322 plasmid maintenance has been located within the region of the TcR gene promoter. On the basis of stability analysis of pBR322 derivatives comprising the modified region of the TcR gene, we deduced that it is the nucleotide sequence localized in the region of the HindIII site that causes destabilization of the plasmid and not the TcR gene product or active transcription of this region. The destabilizing effect is manifested both in cis and in trans.

[Effect of a fragment of pBR322 plasmid in the region of tetracycline resistance gene on the stability of this plasmid]. / Vliianie fragmenta DNK plazmidy pBR322 v oblasti gena ustoichivosti k tetratsiklinu na stabil'nost' étoi plazmidy.

A fragment destabilizing the pBR322 plasmid has been localized in the region of pBR322 tet gene promoter. On the basis of stability analysis of pBR322 derivatives comprising the modified region of tet gene, we deduced that it is the DNA sequence localized at the beginning of tet gene in the region of HindIII splitting site that ensures the plasmid destabilization, and not tet gene product or the active transcription of this region. The destabilizing effect of this fragment of the plasmid is manifested both in cis and in trans. Possible molecular mechanisms of the phenomenon are discussed.

[Effect of the heat shock reaction on the phenotypic manifestation of mutations in rifampicin resistance affecting the gene of the RNA polymerase beta-subunit under the control of the lactose promotor]. / Vliianie reaktsii teplovogo shoka na fenotipicheskoe proiavlenie mutatsii ustoichivosti k rifampitsinu, zatragivaiushchikh gen beta-sub''edinitsy RNK-polimerazy, postavlennoi pod kontrol' laktoznogo promotora.

Plasmids bearing the rifampicin-resistant RNA polymerase beta-subunit gene under control of the lac promoter failed to provide Escherichia coli cells with resistance to rifampicin, despite the accumulation of large quantities of the resistant subunit. The effect proved to be connected with the aggregation of the plasmid-borne subunit. Conditions that induce synthesis of heat-shock proteins make the plasmid-containing cells resistant to rifampicin. This finding suggests that heat-shock proteins prevent the formation of protein aggregates.

[Effectiveness of expression of the chloramphenicol acetyltransferase gene controlled by foreign regulatory regions in Escherichia coli cells. I. Construction of vectors for the cloning of transcription regulatory elements]. / Issledovanie éffektivnosti ékspressii gena khloramfenikolatsetiltransferazy pod kontrolem chuzherodnykh reguliatornykh oblastei v kletkakh Escherichia coli. I. Konstruirovanie vektorov dlia klonirovaniia reguliatornykh élementov transkriptsii.

New plasmids pML2.1 and pML4 were constructed for cloning the transcription regulatory regions. In the pML2.1 the structural part of chloramphenicol acetyltransferase gene of the pBR325 is under control of the lacUV5-promotor. Because the unique BamH1 cleavage site is in the joint region, one may use it for cloning transcription termination regions and selecting recombinant clones with the AprCms phenotype. As for the pML4, the foreign fragment integration is carried directly before the structural part of cat-gene and it is expressed only if the promotor regions are present. The plasmids were sequenced and their restriction maps were established. Small molecular weight (about 2,0 MDa, AprCmr) or only Apr intact genes and convenient disposition of many unique cleavage sites by restriction endonucleases make these plasmids useful for different genetic engineering experiments.