The isolation and characterization of a calmodulin-encoding gene (CMD1) from the dimorphic fungus Candida albicans.

Candida albicans is a dimorphic, opportunistic pathogen of humans, and calcium and calmodulin have been implicated in its morphogenic transition. The C. albicans calmodulin-encoding gene, CMD1, was isolated from cDNA and genomic phage lambda libraries using the Saccharomyces cerevisiae CMD gene as a hybridization probe. Southern-blot hybridization analysis of genomic DNA suggests the existence of only one type of calmodulin gene in C. albicans. Comparison of cDNA and genomic sequences identified a 222-bp intron located immediately after the Met start codon. The predicted amino acid sequence was 60% identical with yeast CMD and 70% identical with CMDs of filamentous fungi and vertebrates. We have localized the CMD1 gene to chromosome 3 using the contour-clamped homogeneous electric field gel electrophoresis. The CMD1 gene hybridized to a single 650-nucleotide transcript which was present in equivalent amounts in both the yeast and hyphal forms of the organism.

Role of exonuclease III and endonuclease IV in repair of pyrimidine dimers initiated by bacteriophage T4 pyrimidine dimer-DNA glycosylase.

The role of exonuclease III and endonuclease IV in the repair of pyrimidine dimers in bacteriophage T4-infected Escherichia coli was examined. UV-irradiated T4 showed reduced survival when plated on an xth nfo double mutant but showed wild-type survival on either single mutant. T4 denV phage were equally sensitive when plated on wild-type E. coli or an xth nfo double mutant, suggesting that these endonucleases function in the same repair pathway as T4 pyrimidine dimer-DNA glycosylase. A uvrA mutant of E. coli in which the repair of pyrimidine dimers was dependent on the T4 denV gene carried on a plasmid was constructed. Neither an xth nor an nfo derivative of this strain was more sensitive than the parental strain to UV irradiation. We were unable to construct a uvrA xth nfo triple mutant. In addition, T4, which turns off the host UvrABC excision nuclease, showed reduced plating efficiency on an xth nfo double mutant.

Nucleotide sequence of the nfo gene of Escherichia coli K-12.

The nfo gene of Escherichia coli K-12 which encodes endonuclease IV has been sequenced. The predicted gene product has a molecular weight of 31,562, in good agreement with the size of the gene product estimated by maxicell analysis. The nfo promoter was mapped by primer extension of in vivo transcripts. Inspection of the nucleotide sequence revealed no regions of potential secondary structure corresponding to a transcriptional terminator downstream from the structural gene; however, there was a potential open reading frame immediately downstream from the nfo structural gene.

Nucleotide sequence of the xth gene of Escherichia coli K-12.

The xth gene of Escherichia coli K-12, which encodes exonuclease III, has been sequenced. Exonuclease III from a cloned copy of the E. coli K-12 gene has been purified and characterized. The molecular weight (30,921), the amino-terminal amino acid sequence, and the amino acid composition of the polypeptide predicted from the nucleotide sequence are in excellent agreement with those properties determined for the purified enzyme. The xth promoter was mapped by primer extension of in vivo transcripts. Inspection of the nucleotide sequence reveals that a region of dyad symmetry which could form a hairpin stem-loop structure in RNA characteristic of a rho-dependent terminator lies immediately downstream from the xth gene.

Endonuclease IV (nfo) mutant of Escherichia coli.

A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested.