Meiotic role of SWI6 in Saccharomyces cerevisiae.

The transcript levels of DNA replication genes and some recombination genes in Saccharomyces cerevisiae fluctuate and peak at the G1/S boundary in the mitotic cell cycle. This fluctuation is regulated by MCB (Mlu I cell cycle box) elements which are bound by the DSC1/MBF1 complex consisting of Swi6 and Mbp1. It is also known that some of the MCB-regulated genes are induced by treatment with DNA damaging agents and in meiosis. In this report, the function of SWI6 in meiosis was investigated. Delta swi6 cells underwent sporulation as did wild-type cells. However, the deletion mutant cells showed reduced spore viability and lower frequency of recombination. The transcript levels of the recombination genes RAD51 and RAD54 , which have MCB elements, were reduced in Delta swi6 cells. The transcript levels of SWI6 itself were also induced and declined in meiosis. Furthermore, an increased dosage of SWI6 enhanced the transcript level of the RAD51 gene and also the recombination frequency in meiosis. These results suggest that SWI6 enhances the expression level of the recombination genes in meiosis in a dosage-dependent manner, which results in an effect on the frequency of meiotic recombination.

Matrix-assisted laser desorption/ionization for sequencing single-stranded and double-stranded DNA.

The DNA sequence of a single-stranded and double-stranded template was determined. The templates were sequenced using the chain termination method and cycle sequencing method and detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The sequencing products were analyzed successfully without the laborious and expensive methods for removal of the template. Direct sequencing of the double-stranded template was achieved with minimal post-reaction purifications, which could be extremely important for mutation analysis and clinical diagnosis. A systematic study of the mechanisms and kinetics of sequencing reactions was also performed. The details of this analysis and directions for future improvements of the quality of sequencing are presented.

Laser desorption mass spectrometry for point mutation detection.

A point mutation can be associated with the pathogenesis of inherited or acquired diseases. Laser desorption mass spectrometry coupled with allele specific polymerase chain reaction (PCR) was first used for point mutation detection. G551D is one of several mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene present in 1-3% of the mutant CFTR alleles in most European populations. In this work, two different approaches were pursued to detect G551D point mutation in the cystic fibrosis gene. The strategy is to amplify the desired region of DNA template by PCR using two primers that overlap one base at the site of the point mutation and which vary in size. If the two primers based on the normal sequence match the target DNA sequence, a normal PCR product will be produced. However, if the alternately sized primers that match the mutant sequence recognize the target DNA, an abnormal PCR product will be produced. Thus, the mass spectrometer can be used to identify patients that are homozygous normal, heterozygous for a mutation or homozygous abnormal at a mutation site. Another approach to identify similar mutations is the use of sequence specific restriction enzymes which respond to changes in the DNA sequence. Mass spectrometry is used to detect the length of the restriction fragments generated by digestion of a PCR generated target fragment.

The study of 2,3,4-trihydroxyacetophenone and 2,4,6-trihydroxyacetophenone as matrices for DNA detection in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometric study of DNA detection using 2,3,4-trihydroxyacetophenone, 2,4,6-trihydroxyacetophenone, and their combination has been carried out systematically. The results show that a mixture of 2,4,6-trihydroxyacetophenone, 2,3,4-trihydroxyacetophenone and ammonium citrate with molar ratios of 2:1:1 serves as a good matrix for the detection of DNA, especially for samples containing a small quantity of DNA such as polymerase chain reaction product. The resolution and shot-to-shot reproducibility using this matrix are better than, and the MALDI sensitivity comparable to, that obtained when using 3-hydroxy picotinic acid (3-HPA), PA and ammonium citrate matrix (9:1:1). The mechanism of desorption/ionization using this matrix is discussed.

Site-directed mutagenesis study on DNA binding regions of the mouse homologue of Suppressor of Hairless, RBP-J kappa.

To map regions important for DNA binding of the mouse homologue of Suppressor of Hairless or RBP-J kappa protein, mutated mouse RBP-J kappa cDNAs were made by insertion of oligonucleotide linkers or base replacement. DNA binding assays using the mutated proteins expressed in COS cells showed that various mutations between 218 Arg and 227 Arg decreased the DNA binding activity drastically. The DNA binding activity was not affected by amino acid replacements within the integrase motif of the RBP-J kappa protein (230His-269His). Replacements between 291Arg and 323Tyr affected the DNA binding activity slightly but reproducibly. These results indicate that the region encompassing 218Arg-227Arg is critical for the DNA binding activity of RBP-J kappa. This region did not show any significant homology to motifs or domains of the previously described DNA binding proteins. Using a truncation mutant protein RBP-J kappa was shown to associate with DNA as a monomer.