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Two proteins with molecular weights of 61,000 and 73,000 were found to be induced by UV light in Escherichia coli mutants in which the SOS responses are constitutively expressed. The induction of these proteins by UV light and nalidixic acid was shown to be independent of the recA+ lexA + regulatory system. Analysis of these proteins by two-dimensional gel electrophoresis and comparison with the "heatshock" proteins of E. coli revealed that the Mr 61,000 protein comigrated with the groEL gene product, that the Mr 73,000 protein comigrated with the dnaK gene product, and that other heat-shock proteins were also induced. The induction of groEL and dnaK by UV light and nalidixic acid is controlled by the htpR locus. The results suggest that the regulatory response of E. coli to agents such as UV light and nalidixic acid is more complex than previously thought. MATERIALS AND METHODSBacterial strains used are listed in Table 1. Early log cultures grown at 30'C were labeled with [35S]methionine (5 ,uCi/ml, final concentration for one-dimensional gels; 30 ,uCi/ml, final concentration for two-dimensional gels; 1 Ci = 37 GBq) for 5 min and chased for 1 min after various treatments (6). Cell extracts were prepared and the proteins were separated on 10-12% NaDodSO4/polyacrylamide gels as described by Laemmli (10). Two-dimensional gel electrophoresis was carried out as described by O'Farrell (11). Extracts were prepared as for one-dimensional gels and were then diluted 1:3 with sample dilution buffer (12). Fluorographic exposures using Kodak XAR5 film were made of gels treated with EN3HANCE (New England Nuclear).Over the past several years there has been a considerable increase in our understanding of how Escherichia coli responds to DNA damage. Two independent regulatory networks have been identified that are induced by damage to the cell's DNA-the SOS response (1) and the adaptive response (2).Of the two, the adaptive response seems to be the simpler. It is induced by exposure to methylating or ethylating agents but not by agents such as UV irradiation or 4-nitroquinoline-1-oxide (3). Two proteins have been shown to be induced in this response, the 06-methylguanine-DNA methyltransferase and a 3-methyladenine glycosylase (4); the product of the ada gene regulates their induction (4).The SOS response seems to be more complex (1). It is induced by agents and conditions that either damage DNA or interfere with DNA replication. Typical inducing agents are UV irradiation, nalidixic acid, and mitomycin C. The expression of genes in the SOS network is controlled by two regulatory elements, the recA and lexA proteins. The lexA protein serves as the repressor of each of the din (damage-inducible) genes (5) that have been identified to date (1); lexA(Def) mutations that eliminate lexA function cause the high-level constitutive expression of din genes (1, 6). After SOS-inducing treatments, a protease activity of the recA protein is activated that then cleaves the lexA protein leading to the induction of the din genes (1). To...

A mutation of a cloned gene that has been made by introducing a transposon or some other selectable genetic determinant can be crossed into the gene's original replicon by linearizing the cloned DNA and transforming a recB recC sbcB mutant. A number of applications of this method are described with genes of either chromosomal or plasmid origin.

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The 51-kDa telomere protein from Euplotes crassus binds to the extreme terminus of macronuclear telomeres, generating a very salt-stable telomeric DNA-protein complex. The protein recognizes both the sequence and the structure of the telomeric DNA. To explore how the telomere protein recognizes and binds telomeric DNA, we have examined the DNA-binding specificity of the purified protein using oligonucleotides that mimic natural and mutant versions of Euplotes telomeres. The protein binds very specifically to the 3' terminus of single-stranded oligonucleotides with the sequence (T4G4) > or = 3 T4G2; even slight modifications to this sequence reduce binding dramatically. The protein does not bind oligonucleotides corresponding to the complementary C4A4 strand of the telomere or to double-stranded C4A4.T4G4-containing sequences. Digestion of the telomere protein with trypsin generates an N-terminal protease-resistant fragment of approximately 35 kDa. This 35-kDa peptide appears to comprise the DNA-binding domain of the telomere protein as it retains most of the DNA-binding characteristics of the native 51-kDa protein. For example, the 35-kDa peptide remains bound to telomeric DNA in 2 M KCl. Additionally, the peptide binds well to single-stranded oligonucleotides that have the same sequence as the T4G4 strand of native telomeres but binds very poorly to mutant telomeric DNA sequences and double-stranded telomeric DNA. Removal of the C-terminal 15 kDa from the telomere protein does diminish the ability of the protein to bind only to the terminus of a telomeric DNA molecule.