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Biomedical subjects

J Kania

Publications and source records attributed to J Kania.

28 records · Page 2Linked to original sources
PubMed1978

beta-Galactosidase chimeras: primary structure of a lac repressor-beta-galactosidase protein.

A protein possessing both lac repressor and beta-galactosidase activities in a single polypeptide of about 155,000 daltons was purified from a deletion mutant of Escherichia coli in which the lacI and Z genes are fused. A 77-residue cyanogen bromide peptide containing the fusion joint was isolated. A radioimmunoassay with an antibody prepared against CNBr2 (residues 3-92) of beta-galactosidase was used to monitor its purification. The sequence of the joining peptide was determined by analysis of tryptic peptides and by automatic sequencer analysis. The site of joining is from residue 355 of lac repressor to residue 24 of beta-galactosidase (or 356 to 25), indicating that the last 4 residues at the carboxyl terminus of lac repressor and the first 23 residues at the amino terminus of beta-galactosidase are not essential for the activities of these two proteins. The exact site of the fusion is not known because lac repressor residue 356 and beta-galactosidase residue 24 are both leucine residues. Examination of the nucleotide sequences around the two end points of the deletion revealed a homology of 9 identities in a stretch of 11 base pairs.

Amino Acid Sequence↗
PubMed1977

Construction, isolation and implications of repressor-galactosidase - beta-galactosidase hybrid molecules.

Escherichia coli heterogenotes, which produce hybrid molecules between the chimaeric protein repressor-galactosidase and the enzyme beta-galactosidase, were constructed. Repressor-galactosidase in which fully active lac repressor is covalently linked to active beta-galactosidase, is an aggregate with a core structure of four beta-galactosidase parts and two peripheral lac repressor dimers. The lac repressor dimers, which are separated by tetrameric beta-galactosidase, retain all the biological activities of tetrameric lac repressor. Substitution of repressor-galactosidase subunits with beta-galactosidase subunits leads to hybrid molecules with y beta-galactosidase subunits aggregated with (4-y) repressor-galactosidase subunits (where y = 1, 2 or 3). A 2:2 hybrid, i.e. a tetrameric beta-galactosidase core with one lac repressor dimer grafted to it, binds at least 100 times less strongly to 32P-labelled lambdaplac DNA than pure lac repressor or repressor-galactosidase. The data suggest a model in which lac repressor binds with two subunits to lac operator and with the other two subunits elsewhere on the DNA, possibly on sequences like the lac operator.

Bacterial Proteins↗
PubMed1976

Use of a sequence-specific DNA-binding ligand to probe the environments of EcoRI restriction endonuclease cleavage sites.

The DNAs of bacteriophage lambda and adenovirus were incubated with the sequence-specific DNA-binding ligand 6,4'-diamidino-2-phenylindole. Digestion of the ligand-DNA complexes with EcoRI nuclease and subsequent agarose gel electrophoresis demonstrated that the ligand inhibited nuclease activity at some sites, but not at others. The results suggest that diamidino-2-phynylindole can be used to probe the immediate environments of the EcoRI cleavage sites.

Adenoviridae↗
PubMed1976

The functional repressor parts of a tetrameric lac repressor-beta-galactosidase chimaera are organized as dimers.

The chimaeric protein repressor-galactosidase, in which fully active lac repressor is covalently linked to the active enzyme beta-galactosidase, was used as a system for probing the quaternary structure of lac repressor. Electron micrographs revealed repressor-galactosidase to be a tetrameric aggregate. When lac repressor, alone, was crosslinked with dimethyl suberimidate, dimers, trimers, tetramers, and oligomers of the protein subunit were produced, whereas crosslinking of the tetrameric repressor-galactosidase resulted in the production of only dimers of the chimaera. Treatment of lac repressor with iodine resulted in the formation of protein dimers; the same result was obtained with repressor-galactosidase. After limited proteolysis of lac repressor, no crosslinking was obtained after treatment with dimethyl suberimidate, whereas iodine still produced a covalent linkage. These results are interpreted as evidence that the lac repressor parts of the tetrameric repressor-galactosidase-chimaera are organized as dimers on the tetrameric-beta-galactosidase core. Because this chimaera has been previously shown to have normal repressor activity [B. Müller-Hill and J. Kania (1974) Nature, 249,561-563], we conclude that lac repressor still is biologically active as a dimeric aggregate.

Bacterial Proteins↗