Biomedical subjects
Publications and source records attributed to P Herrlich.
Chimaeric plasmids carrying EcoRI fragments of the F sex factor have been used to identify proteins involved in conjugation and to assign them to tra cistrons. Most of these proteins are incorporated into the cell envelope and are individually regulated at the post-transcriptional level.
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Mitochondria and bacteria possess protein synthesizing machineries which are similar in many respects; The regulation of gene expression in mitochondria is unknown. We, therefore, tried to use a well-established prokaryotic regulatory system for the exploration of mitochondrial gene regulation. DNA of the bacterial virus can be used as a template for gene expression in a mitochondrial in vitro system. The gene directed enzyme synthesis in the mitochondrial system is the basis for a study of regulation in mitochondrial protein synthesis.
Co- and superinfection of cells with T3/T7 result in exclusion (mutual or superinfection exclusion). The exclusion mechanism is also directed against homologous (or identical) virus. Exclusion is established after the adsorption but before the genome becomes available for gene expression or replication, that is only one virus per cell develops. The exclusion is triggered by a constituant of the viral particle. An early T7 gene (M gene) (Schweiger et al., 1975) is essential for the formation of exclusion competent virions.
Infection of E. coli with the viruses T7 or T3 leads to a dramatic efflux of potassium ions. This ion efflux is caused by the virus particle since no concomitant protein synthesis is required. T7 mutants carrying deletions in the M-gene (Schweiger et al., 1975), however, yield virus particles disturbed in the ion release.
Nitrofurans, a class of antibacterial drugs in extensive use, interferes with gene expression in a highly specific manner. While in the low dose range (0.5-25 mug/ml), 5-nitro-2-furfurylidene-1-aminohydantoin has no effect on transcription, it inhibits specifically the expression of one class of genes in translation. The specific inhibition concerns the inducible genes. The inhibition of messenger RNA expression occurs at the initiation step. The action of nitrofurans, thus, indicates heterogeneity in the population of mRNA molecules and in the translational machinery and suggests the possibility of selective translational control.
The cause of T7 exclusion by the F episome was investigated. Extracts from neither normal nor infected F+ cells contained an inhibitor of gene expression in vitro. The protein synthesizing systems prepared in vitro from these cells supported T7 early and late protein synthesis with normal efficiency. The content of translational initiation factors in F- and F+ cells, both noninfected and infected, was almost identical. The episome-dependent block of T7 gene expression was observed only in intact cells and detailed kinetics of gene expression in vivo revealed a stop of all transcription and translation at or just before 11 min after T7 infection. The mechanism of F+-dependent T7 exclusion involves both episomal and viral gene products. The data indicate that a T7-induced membrane alteration of the F+ cell membrane leads to cessation of T7 development as well as to the death of the host cell ('suicide').
A protein kinase, ATP:protein phosphotransferase (EC 2.7.1.37) was detected in Escherichia coli after infection with bacteriophage T7. The enzyme was purified from the ribosomal wash fraction by conventional methods, affinity chromatography on Cibacron blue and on lysozyme coupled to Sepharose, and by cellogel electrophoresis. An approximately 5000-fold purification was achieved.
Protein kinase, which was isolated from cells infected with T7, is indeed a viral gene product. This is shown by DNA-dependent synthesis in vitro. The protein kinase transfers phosphate from ATP to seryl or threonyl residues in protein. The enzyme has only a relative requirement for magnesium ions, but is only active at low ionic strength. The best substrate is lysozyme. T7 protein kinase activity is not stimulated by cyclic 3':5'-AMP and/or cyclic 3':5'-GMP. The T7 protein kinase carries -- SH groups essential for activity. There is indication that the enzyme phosphorylates itself and causes self inactivation, which may explain the fast disappearance of enzyme activity in vivo. Bacteriophage T3 also induces a protein kinase which is similar to the T7-induced enzyme in all respects tested.
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Transcriptional units for ribosomal proteins in Escherichia coli were measured using the ultraviolet sensitivities of the rates of synthesis of individual ribosomal proteins. The ultraviolet sensitivities of gene transcriptions are proportional to the distances from the promoters. The longest transcriptional units for ribosomal proteins are 3.6 x 10(6) of DNA molecular weight corresponding to 1.8 x 10(6) of RNA or to 180 000 of protein. The length would cover 10--12 genes of ribosomal proteins (of an average Mr of 15000-18000).
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After bacteriophage T7 infection, a protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) activity can be demonstrated in E. coli in vivo by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Cell-free extracts catalyzed the transfer of the terminal phosphoryl group of [(gamma)-(32)P]ATP to endogenous protein acceptor or to added histone. The bond between phosphate and protein shows the characteristics of serine phosphate: it is stable in 1 N HCl (100 degrees ) and cleaved by 1 N KOH (37 degrees ) and by alkaline phosphatase treatment. Moreover, after partial acid hydrolysis, radiophosphate migrates with marker O-phosphoserine on polyethyleneimine-cellulose thin-layer chromatograms. Enzyme activity in uninfected cells is negligible. Ultraviolet irradiation of the phage genome prevents the appearance of the protein kinase; irradiation of the host genome does not. The enzyme activity occurs 4 min after infection and its gene maps in the early region (promoter proximal to gene 1). Ribosomal proteins are phosphorylated in vivo and are substrates in vitro. Enzyme activity in vitro is not changed by addition of cyclic AMP or cyclic GMP.
Phage T7 discontinues host gene expression by translational and transcriptional control mechanisms. Translational control is exerted by the T7 translational-repressor. This protein inhibits the synthesis of beta-galactosidase (EC 3.2.1.23) in vivo and in vitro and the synthesis of the T3 enzyme S-adenosylmethioninehydrolase (EC 3.3.1.-). The translational-repressor does not interfere with T7-specific enzyme synthesis. The T7 translational-repressor purifies with the initiation factors. The repressor interacts with the initiation of translation of host enzymes. The translational-repressor gene is close to the promotor for RNA polymerase of Escherichia coli.
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