Meiotic recombination requires the meiosis-specific RecA homolog Dmc1 as well as the mitotic RecA homolog Rad51. Here, we show that the two meiosis-specific proteins Mei5 and Sae3 are necessary for the assembly of Dmc1, but not for Rad51, on chromosomes including the association of Dmc1 with a recombination hot spot. Mei5, Sae3, and Dmc1 form a ternary and evolutionary conserved complex that requires Rad51 for recruitment to chromosomes. Mei5, Sae3, and Dmc1 are mutually dependent for their chromosome association, and their absence prevents the disassembly of Rad51 filaments. Our results suggest that Mei5 and Sae3 are loading factors for the Dmc1 recombinase and that the Dmc1-Mei5-Sae3 complex is integrated onto Rad51 ensembles and, together with Rad51, plays both catalytic and structural roles in interhomolog recombination during meiosis.

Assembly and disassembly of Rad51 and Rad52 complexes were monitored by immunofluorescence during homologous recombination initiated by an HO endonuclease-induced double-strand break (DSB) at the MAT locus. DSB-induced Rad51 and Rad52 foci colocalize with a TetR-GFP focus at tetO sequences adjacent to MAT. In strains in which HO cleaves three sites on chromosome III, we observe three distinct foci that colocalize with adjacent GFP chromosome marks. We compared the kinetics of focus formation with recombination intermediates and products when HO-cleaved MATalpha recombines with the donor, MATa. Rad51 assembly occurs 1 h after HO cleavage. Rad51 disassembly occurs at the same time that new DNA synthesis is initiated after single-stranded (ss) MAT DNA invades MATa. We present evidence for three distinct roles for Rad52 in recombination: a presynaptic role necessary for Rad51 assembly, a synaptic role with Rad51 filaments, and a postsynaptic role after Rad51 dissociates. Additional biochemical studies suggest the presence of an ssDNA complex containing both Rad51 and Rad52.

The functions of acetylpolyamines were examined with respect to stimulation of protein synthesis and cell growth. Unlike polyamines, acetylpolyamines could not lower the optimal Mg2+ concentration of protein synthesis, and the degree of stimulation of protein synthesis by acetylpolyamines was small. The addition of N1-acetylspermine did not stimulate cell growth of a polyamine-requiring mutant of Escherichia coli MA261, although acetylspermine was accumulated in the cells. Acetylspermine did not interfere with polyamine stimulation of protein synthesis and cell growth of E. coli MA261. The binding of acetylpolyamines to RNA was very weak, and the binding of polyamines to RNA was not disturbed significantly by the presence of acetylpolyamines. When the growth of E. coli MA261 was stimulated by addition of polyamines, significant amounts of acetylpolyamines were also formed in the cells. These results suggest that acetylation of polyamines, together with polyamine excretion, may regulate the intracellular level of the parent polyamines when excess amounts of polyamines accumulate intracellularly.

-In The Toxicogenomics Project, about 150 chemicals are administered to rats, and gene expression in the liver analyzed by Affymetrix GeneChip and stored in the database. As the quality of RNA greatly influences the accuracy of gene expression data, conditions of the storage of the sample are very important. Recently, an RNA stabilization solution, RNAlater ® , has become commercially available. In this study, the new storage method was compared with the traditional storage method (stored in freezer or liquid nitrogen) under various conditions by looking at the degradation of RNA assessed by its total yield, OD260/280 ratio, 28S/18S ratio, and quantity of β-actin. It was confirmed that RNAlater ® preserved the liver tissue sample by maintaining the quality of RNA for one year (in liquid N 2 or −80°C), for 3 days (4°C), or for 2 hr (room temperature) without degradation of RNA. Quality of RNA samples dissolved in buffer RLT and stored at −20°C tended to decrease, but samples stored at −80°C were almost equivalent to those stored in liquid nitrogen. In conclusion, we recommend the following procedure for preservation of liver tissue for extraction of RNA: 1) tissues removed should be put into chilled RNAlater ® as soon as possible; 2) samples in RNAlater ® must be stored overnight or longer at 4°C and can be left for as long as 2 weeks without freezing; 3) samples in RNAlater ® can be stored for at least one year under less than −20°C and 4) samples dissolved in buffer RLT can be preserved at least for one year under −80°C.

Histopathological examination of formalin-fixed paraffin-embedded (FFPE) tissues that had been stored for 30 years was conducted, and reconstructivity of the results was verified. These FFPE tissues, which were from all organs of male and female rats, were re-sectioned and histopathologically examined using hematoxylin and eosin (HE) staining. In particular, the stainability and morphology of HE sections and reproducibility of microscopic findings in the liver and kidney demonstrated in the original final reports were evaluated. Although the stainability of hematoxylin was slightly weaker and some morphological artifacts were observed in tissues in re-prepared slides, these deteriorations in the quality of HE sections were considered to be permissible for histopathological examination so long as control sections were also prepared. Most microscopic findings recorded in the original final reports were confirmed using re-prepared HE sections in the present study. While some focal findings, which were judged to be either incidental or spontaneous in nature, were not observed in the sections as expected, this was not considered to be a problem in reconstructing the results of the original histopathological examination because most findings related to the test articles were generally observed diffusely or multifocally in each organ. We concluded that results of the original histopathological examinations could be reconstructed using paraffin blocks that had been stored for up to 30 years. Appropriate archiving of records and materials generated in good laboratory practice (GLP) studies is required to comply with the principles of GLP 1 . This is because the maintenance of raw data associated with a specific study and the specimens generated from that study are the only means that can be used to reconstruct the study. In several countries, the retention period for specimens of a GLP study is set such that it expires when they can no longer be evaluated. Consequently, formalin-fixed organs, paraffin-embedded blocks, and hematoxylin and eosin (HE)-stained slides have been stored for long periods in each test facility.The quality of long-term stored formalin-fixed wet tissues has been histopathologically evaluated by producing HE sections 2-4 . To the best of our knowledge, no studies have reported the quality evaluation of long-term stored formalin-fixed paraffin-embedded (FFPE) tissues. Here we re-prepared HE sections from FFPE tissues that had been previously stored for approximately 30 years and histopathologically evaluated the quality of these tissues and reproducibility of microscopic findings. We then investigated the possibility of reconstructing the results of the original histopathological examination using re-prepared HE sections from long-term stored paraffin blocks.This study evaluated FFPE tissues that had been stored for approximately 30 years. FFPE blocks evaluated in this study contained all the organs of 4 males and 4 females, i.e., 1 or 2 males and females each from three GLP toxicity studie...