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An intronless gene encoding a protein of 674 amino acid residues with a molecular mass of 73,403 Da showing homology to the cytoplasmic form of the 70 kDa heat shock proteins has been cloned and sequenced from the intestinal pathogen Cryptosporidium parvum. Monospecific polyclonal antibodies obtained to recombinant protein recognized a single band with an approximate molecular mass of 70 kDa on a Western blot of C. parvum proteins, as well as the 70 kDa heat shock protein from bovine brain. Southern blot analysis suggested the gene was single copy in the C. parvum genome. Eleven perfect repeats of the sequence GGMP were found in the predicted protein near the carboxyl terminus.

Ultraviolet light is being considered as a disinfectant by the water industry because it appears to be very effective for inactivating pathogens, including Cryptosporidium parvum. However, many organisms have mechanisms for repairing ultraviolet light-induced DNA damage, which may limit the utility of this disinfection technology. Inactivation of C. parvum was assessed by measuring infectivity in cells of the human ileocecal adenocarcinoma HCT-8 cell line, with an assay targeting a heat shock protein gene and using a reverse transcriptase polymerase chain reaction to detect infections. Oocysts of five different isolates displayed similar sensitivity to ultraviolet light. An average dosage of 7.6 mJ/cm2 resulted in 99.9% inactivation, providing the first evidence that multiple isolates of C. parvum are equally sensitive to ultraviolet disinfection. Irradiated oocysts were unable to regain pre-irradiation levels of infectivity, following exposure to a broad array of potential repair conditions, such as prolonged incubation, pre-infection excystation triggers, and post-ultraviolet holding periods. A combination of data-mining and sequencing was used to identify genes for all of the major components of a nucleotide excision repair complex in C. parvum and Cryptosporidium hominis. The average similarity between the two organisms for the various genes was 96.4% (range, 92-98%). Thus, while Cryptosporidum spp. may have the potential to repair ultraviolet light-induced damage, oocyst reactivation will not occur under the standard conditions used for storage and distribution of treated drinking water.

The rad6-1 and rad6-3 mutants are highly UV sensitive and show an increase in spontaneous and UV induced mitotic heteroallelic recombination in diploids. Both rad6 mutants are proficient in spontaneous and UV induced unequal sister chromatid recombination in the reiterated ribosomal DNA sequence and are deficient in UV induced mutagenesis. In contrast to the above effects where both mutants appear similar, rad6-1 mutants are deficient in sporulation and meiotic recombination whereas rad6-3 mutants are proficient. The differential effects of these mutations indicate that the RAD6 gene is multifunctional. The possible role of the RAD6 gene in error prone excision repair of UV damage during the G1 phase of the cell cycle in addition to its role in postreplication repair is discussed.

A yeast strain was constructed that had a disruption of the chromosomal RAD3 gene and carried a series of centromeric plasmids with defined mutations in this gene. Using this isogenic collection, we examined sensitivity to UV radiation, spontaneous and UV radiation-induced mutagenesis, and mitotic recombination. Several alleles resulted in a marked increase in UV sensitivity. Most of these alleles were found to carry mutations located in consensus motifs for DNA helicases. Other alleles caused a modest or no increase in UV sensitivity and carried mutations in regions of the Rad3 polypeptide that are apparently not conserved. This correlation suggests that the DNA helicase activity of Rad3 protein is required for nucleotide excision repair of DNA. Some rad3 alleles conferred a marked increase in the frequency of spontaneous mutagenesis, including nonsuppressor reversion of the lys2-1 ochre mutation. These alleles also showed a good correlation with conserved DNA helicase domains, suggesting that the Rad3 DNA helicase also plays a role in the fidelity of DNA synthesis or postreplicative mismatch correction. Several rad3 mutator alleles also resulted in increased levels of mitotic recombination. Increased spontaneous mutagenesis and mitotic recombination are characteristic features of the Rem-phenotype. However, in contrast to the prototypic Rem-phenotype, the rad3 mutator alleles identified in this study did not confer inviability in the presence of mutations in the RAD50 or RAD52 gene required for strand break repair of DNA.Nucleotide excision repair in the yeast Saccharomyces cerevisiae is genetically complex, involving the products of multiple genes (7,11). Five of these genes (RADJ, RAD2, RAD3, RAD4, and RADIO) are required for damage-specific incision of UV-irradiated DNA and for excision of bulky photoproducts such as pyrimidine dimers (36,50). Among this group, the RAD3 gene is of particular interest. In addition to its requirement for nucleotide excision repair, this gene appears to have several other functions, as evidenced by the phenotypes of mutant alleles. Some rad3 mutations (specifically deletions and disruptions) are lethal to haploid yeast cells and cause recessive lethality in diploid cells (12,32). In addition, certain rad3 alleles (which result in only slight UV sensitivity) confer increased frequencies of mitotic recombination and spontaneous mutations, as well as lethality in the presence of the rad5O-I or rad52-1 mutation (14,24,29). The RAD5O and RAD52 genes belong to the RAD52 epistasis group required for recombinational repair of DNA strand breaks (11). This last group of characteristics is referred to as the Rem-(recombination-mutagenesis) phenotype of rad3 mutants (8).Rad3 protein has been purified from yeast cells overexpressing the cloned gene (9, 44) and has been shown to be a DNA-dependent ATPase/DNA helicase (9,43,44 Previous studies in one of our laboratories resulted in the isolation of a collection of rad3 mutant alleles following site-directed or random mutagenesis of the clone...

No abstract

We report improved separation of chromosome-sized DNA molecules of the coccidian parasite Cryptosporidium parvum with contour-clamped homogeneous electric fields (CHEF). We used scanning densitometry to determine that the most likely number of chromosomes is eight. Molecular probes consisting of cloned genes were used to distinguish each of five bands visible on CHEF gels. We have also identified a low-molecular-size DNA molecule possibly related to the 35-kb circular DNAs found in other Apicomplexa.

A nuclease-sensitive region forms in chromatin containing a 273-base-pair (bp) segment of simian virus 40 DNA encompassing the viral origin of replication and early and late promoters. We have saturated this region with short deletion mutations and compared the nuclease sensitivity of each mutated segment to that of an unaltered segment elsewhere in the partially duplicated mutant. Although no single DNA segment is required for the formation of a nuclease-sensitive region, a deletion mutation (d145) which disrupted both exact copies of the 21-bp repeats substantially reduced nuclease sensitivity. Deletion mutations limited to only one copy of the 21-bp repeats had little, if any, effect. A mutant (d1135) lacking all copies of the 21-and 72-bp repeats, while retaining the origin of replication and the TATA box, did not exhibit a nuclease-sensitive region. Mutants which showed reduced nuclease sensitivity had this effect throughout the nuclease-sensitive region, not just at the site of the deletion, indicating that although multiple determinants must be responsible for the nuclease-sensitive chromatin structure they do not function with complete independence. Mutant d19, which lacks the late portion of the 72-bp segment, showed reduced accessibility to BglI, even though the Bgll site is 146 bp away from the site of the deletion.Simian virus 40 (SV40)-infected cells contain SV40 DNA in a nucleoprotein structure with many similarities to cellular chromatin. The availability of an amplified, homogeneous DNA species in a chromatin-like structure has provided convenient material for the study of specific features of chromatin. Of particular interest is the short region adjacent to the viral origin of replication (ori site) which is hypersensitive to cleavage by endonucleases (23,26,27,29) and which appears as a gap in the nucleosome pattern when visualized in the electron microscope (16,22). This region contains the early and late gene promoters, the transcriptional enhancer sequences, and the viral origin of replication. The distinctive chromatin structure may be relevant to any or all of these functions.To identify which sequences are responsible for the nuclease-sensitive region, partially duplicated SV40 mutants have been studied. All essential information required for the hypersensitive site is contained within a 273-base-pair (bp) segment of DNA spanning the origin of replication (11,30 We have investigated the genetic elements responsible for the chromatin structure in this region by analyzing a number of additional mutants. No single segment is essential for nuclease sensitivity. However, overall accessibility to nuclease was dramatically decreased by a mutant affecting only the two exact copies of the 21-bp repeated sequences. Insertion of a foreign DNA segment into this region also substantially diminished nuclease sensitivity. By contrast, deletion of only one of the two exact copies of the 21-bp repeats had little effect on the pattern of nuclease cleavage.These results imply that the region of the genome e...