The rem mutations in the ATP-binding groove of the Rad3/XPD helicase lead to Xeroderma pigmentosum-Cockayne syndrome-like phenotypes.

The eukaryotic TFIIH complex is involved in Nucleotide Excision Repair and transcription initiation. We analyzed three yeast mutations of the Rad3/XPD helicase of TFIIH known as rem (recombination and mutation phenotypes). We found that, in these mutants, incomplete NER reactions lead to replication fork breaking and the subsequent engagement of the homologous recombination machinery to restore them.

Assessment of the effectiveness of the studio format in introductory undergraduate biology.

Kansas State University converted its introductory biology course, previously taught as an audio-tutorial (A-T), to a studio format in 1997. We share with others information about the process involved and present assessment data for the studio format course that address 1) student exam performance in A-T and studio; 2) student course grades in A-T and studio; 3) student and instructor perceptions and attitudes for A-T and studio; 4) student performance in subsequent biology courses for A-T and studio; and 5) gains in student learning for the studio course and other traditional lecture/lab courses. Collectively, these measures demonstrate that the studio format is as effective as or more effective (for some measures) than the A-T approach and traditional approaches in providing an effective learning environment.

The response of Cryptosporidium parvum to UV light.

Ultraviolet (UV) light is being considered as a disinfectant by the water industry because it appears to be very effective for controlling potential waterborne pathogens, including Cryptosporidium parvum. However, many organisms have mechanisms such as nucleotide excision repair and photolyase enzymes for repairing UV-induced DNA damage and regaining preirradiation levels of infectivity or population density. Genes encoding UV repair proteins exist in C.

Irreversible UV inactivation of Cryptosporidium spp. despite the presence of UV repair genes.

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.

Improving baculovirus resistance to UV inactivation: increased virulence resulting from expression of a DNA repair enzyme.

The use of baculoviruses as biological control agents is hampered by their susceptibility to inactivation by ultraviolet (UV) light. In an attempt to reduce UV inactivation, an algal virus pyrimidine dimer-specific glycosylase, cv-PDG, was expressed in the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV), and the infectivity of recombinant viruses expressing cv-PDG was measured after exposure to UV light. Expression of cv-PDG resulted in a 3-fold decrease in inactivation of budded virus by UV as measured by plaque assay in Spodoptera frugiperda Sf21 cells.

Sequence of the gene encoding hsp90e from Cryptosporidium parvum.

A composite 2364 nt DNA sequence with an open reading frame (ORF) encoding an endoplasmic reticulum-associated heat shock protein 90 (CpHsp90e) was determined from clones isolated from genomic libraries constructed from the KSU-1 isolate of Cryptosporidium parvum. Transcription was verified by isolation of a clone from a cDNA library with a similar restriction map to that observed with genomic DNA. The predicted protein consists of 787 amino acids, has a predicted molecular size of 89.

Sequencing, analysis and expression in Escherichia coli of a gene encoding a 15 kDa Cryptosporidium parvum protein.

A previous paper presented data on a cDNA sequence encoding a protein associated with the AIDS related pathogen, Cryptosporidium parvum. However, the position of the start codon was uncertain, and the 5' end was continuous, lending doubt about the size and complete sequence of the final protein product. Herein we present the complete gene sequence and conclude the predicted size of the putative protein to be 16.

Molecular karyotype analysis of Cryptosporidium parvum: evidence for eight chromosomes and a low-molecular-size molecule.

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.

Sequence of the parasitic protozoan, Cryptosporidium parvum, putative protein disulfide isomerase-encoding DNA.

A composite 1876-bp DNA encoding a putative protein disulfide isomerase (PDI) has been constructed from clones isolated from Cryptosporidium parvum (C. parvum) genomic and cDNA libraries and the nucleotide sequence determined. As predicted from the open reading frame (ORF), the protein product has a predicted molecular size of 54 kDa and a high degree of homology to PDIs from other species.

The putative acetyl-CoA synthetase gene of Cryptosporidium parvum and a new conserved protein motif in acetyl-CoA synthetases.

We determined the nucleotide (nt) sequence of the putative gene encoding acetyl-coenzyme A synthetase (ACS) from the parasitic protozoan Cryptosporidium parvum. The gene is single copy, located on a chromosome of approximately 1.08 mb, and has no introns.

Cloning and analysis of a Cryptosporidium parvum gene encoding a protein with homology to cytoplasmic form Hsp70.

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.

Interactions among mutations affecting spontaneous mutation, mitotic recombination, and DNA repair in yeast.

The mutant alleles mms9-1, mms13-1, or mms21-1 of Saccharomyces cerevisiae confer pleiotropic effects, including sensitivity to the alkylating agent methyl methanesulfonate, elevations in spontaneous mutation and mitotic recombination, defects in meiosis, and cross-sensitivity to radiation. We constructed double-mutant strains containing an mms mutation and a defect in either excision repair, mutagenic repair, or recombinational repair and measured the levels of spontaneous mutation and mitotic recombination. Double mutants lacking excision repair show elevations in spontaneous mutation but with predominantly unchanged levels of mitotic recombination.

Analysis of the rad3-101 and rad3-102 mutations of Saccharomyces cerevisiae: implications for structure/function of Rad3 protein.

The mutations rad3-101 and rad3-102 (formerly rem1-1 and rem1-2) of the essential RAD3 gene of Saccharomyces cerevisiae confer a phenotype of semidominant enhancement of spontaneous mitotic recombination and mutation frequencies, but not extreme sensitivity to ultraviolet (UV) light. These properties differ from the previously published observations of other rad3 mutations, which are very UV-sensitive but do not alter recombination frequencies significantly. We have located the position of DNA sequence changes from wild-type RAD3 to the rad3-101 and rad3-102 mutations and have demonstrated that these sequence changes are necessary and sufficient to confer the (Rem-) mutant phenotype when transferred into otherwise wild-type RAD3 plasmids.

Interaction of excision repair gene products and mitotic recombination functions in yeast.

We have tested the ability of mutants of three additional genes in the excision repair pathway of Saccharomyces cerevisiae to suppress the hyper-recombination and rad52 double-mutant lethality phenotypes of the rad3-102 (formerly rem1-2) mutation. Such suppression has previously been observed with mutant alleles of RAD1 and RAD4. We had hypothesized that the rad3-102 mutation created elevated levels of DNA lesions which could be processed by the products of the RAD1 and RAD4 genes into recombinogenic double-strand breaks requiring the RAD52 product for repair.

Analysis of the spectrum of mutations induced by the rad3-102 mutator allele of yeast.

The product of the RAD3 gene of Saccharomyces cerevisiae is required for mitotic cell viability and excision repair of UV-induced pyrimidine dimers. Certain rad3 mutant alleles (originally called rem1) increase the rates of both spontaneous mitotic recombination and mutation. The increase in mutation rates is not dependent upon the presence of the RAD6 error-prone pathway.

Effects of multiple yeast rad3 mutant alleles on UV sensitivity, mutability, and mitotic recombination.

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.

A reexamination of the role of the RAD52 gene in spontaneous mitotic recombination.

The RAD52 gene is required for much of the recombination that occurs in Saccharomyces cerevisiae. One of the two commonly utilized mutant alleles, rad52-2, increases rather than reduces mitotic recombination, yet in other respects appears to be a typical rad52 mutant allele. This raises the question as to whether RAD52 is really necessary for mitotic recombination.

Spontaneous mitotic recombination in yeast: the hyper-recombinational rem1 mutations are alleles of the RAD3 gene.

The RAD3 gene of Saccharomyces cerevisiae is required for UV excision-repair and is essential for cell viability. We have identified the rem1 mutations (enhanced spontaneous mitotic recombination and mutation) of Saccharomyces cerevisiae as alleles of RAD3 by genetic mapping, complementation with the cloned wild-type gene, and DNA hybridization. The high levels of spontaneous mitotic gene conversion, crossing over, and mutation conferred upon cells by the rem1 mutations are distinct from the effects of all other alleles of RAD3.

Role of specific simian virus 40 sequences in the nuclease-sensitive structure in viral chromatin.

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 (dl45) which disrupted both exact copies of the 21-bp repeats substantially reduced nuclease sensitivity.

Hyper-recombination and mutator effects of the mms9-1, mms13-1, and mms21-1 mutations in Saccharomyces cerevisiae.

Spontaneous mitotic intragenic and intergenic recombination at various sites is enhanced 10 to 100 fold in the methyl methanesulfonate (MMS)-sensitive mutants mms9-1, mms13-1, and mms21-1 of Saccharomyces cerevisiae. All three mutants show elevated rates of spontaneous mutation. Sporulation is reduced in diploids homozygous for any of the three mutations, and a deficiency in meiotic recombination and meiotic chromosome segregation is observed.

Spontaneous mitotic recombination in mms8-1, an allele of the CDC9 gene of Saccharomyces cerevisiae.

The methyl methane sulfonate (MMS)-sensitive mutation mms8-1 increases the rate of spontaneous mitotic intragenic recombination at five heteroallelic loci on three chromosomes. Complementation, segregation, and mapping studies indicate that mms8-1 is allelic to cdc9, known to be defective in deoxyribonucleic acid ligase. Both mms8-1 and cdc9 mutants are lethal in combination with the recombination-defective mutant rad52-1.

Recombination and mutagenesis in rad6 mutants of Saccharomyces cerevisiae: evidence for multiple functions of the RAD6 gene.

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.

Effects of the RAD52 Gene on Recombination in SACCHAROMYCES CEREVISIAE.

Effects of the rad52 mutation in Saccharomyces cerevisiae on meiotic, gamma-ray-induced, UV-induced and spontaneous mitotic recombination were studied. The rad52/rad52 diploids undergo premeiotic DNA synthesis; sporulation occurs but inviable spores are produced. Both intra and intergenic recombination during meiosis were examined in cells transferred from sporulation medium to vegetative medium at different time intervals.