Satoshi Tabata

General recombination mechanisms in extracts of meiotic cells.

RecA-like proteins have been purified from somatic and meiotic cells of mouse and lily. The rec proteins have been designated “s-rec” and “m-rec” to indicate their respective tissues of origin. The two proteins differ in molecular weight and in their response to temperature, the latter being consistent with the optimal temperature for physiological function of their tissues of origin. There is a major increase in m-rec protein with the entry of cells into meiosis, the peak of activity being early pachytene. Extracts of the cells and also those of yeast (Saccharomyces cerevisiae) have been prepared that have the capacity to catalyze homologous recombination. These extracts behave similarly to the m-rec proteins upon entry of cells into meiosis. Yeast transferred to sporulation medium displays a 100-fold increase in the recombination activity of the extract at about the time of entry into meiosis. The occurrence of peak levels of m-rec and recombination activity in extracts from cells in early pachytene points strongly to that stage as the time at which the enzymatic phase of recombination occurs.

Identification and characterization of genes induced during sexual differentiation in Schizosaccharomyces pombe

Five cDNA clones, harboring genetic messages preferentially expressed during the sexual differentiation process, were isolated from a cDNA library of Schizosaccharomyces pombe by subtractive screening. Transcription of the corresponding genes, termed isp3, 4, 5, 6, and 7, was dependent on nitrogen starvation and their induction occurred at several stages of spore formation. Analysis of the cDNA prmary structures revealed a capacity for the coding of polypeptides of 19.2 kDa, 88.3 kDa, 60.1 kDa, 49.7 kDa, and 43.8 kDa, respectively. The translated amino-acid sequences of isp5 and isp6 were found to show significant similarities to those of amino-acid permeases and proteinase B of Saccharomyces cerevisiae, respectively. Disruption of isp6 arrested the cell cycle prior to conjugation and caused a drastic blocking effect on spore formation.

Isolation and characterization of a yeast gene that is homologous with a meiosis-specific cDNA from a plant

SummaryBy using as probe a meiosis-specific cDNA clone LIM15 from the monocotyledonous plant, Lilium longiflorum, a clone containing a 2.8 kb DNA fragment was isolated from a genomic library of Saccharomyces cerevisiae. Primary structure analysis revealed that the clone includes two complete open reading frames, designated ISC2 and ISC10, capable of coding for a 36.6 kDa and a 31.6 kDa polypeptide, respectively, with the former frame being interrupted by a 92 by intron. The predicted amino acid sequence of Isc2 was 56% identical with the putative gene product of lily cDNA clone LIM15, and showed limited sequence similarity with the yeast RAD57 gene product. Transcripts of the two genes begin accumulating 2.5 h and 7.5 h after induction of meiosis, respectively, according to a Northern hybridization analysis. Since disruption of either one of these genes had a drastic effect on the ability to form spores, ISC2 and ISC10 are expected to play significant roles in the formation of reproductive cells.

Anion-mediated Fe3+ release mechanism in ovotransferrin C-lobe: a structurally identified SO4(2-) binding site and its implications for the kinetic pathway.

The differential properties of anion-mediated Fe3+ release between the N- and C-lobes of transferrins have been a focus in transferrin biochemistry. The structural and kinetic characteristics for isolated lobe have, however, been documented with the N-lobe only. Here we demonstrate for the first time the quantitative Fe3+ release kinetics and the anion-binding structure for the isolated C-lobe of ovotransferrin. In the presence of pyrophosphate, sulfate, and nitrilotriacetate anions, the C-lobe released Fe3+ with a decelerated rate in a single exponential progress curve, and the observed first order rate constants displayed a hyperbolic profile as a function of the anion concentration. The profile was consistent with a newly derived single-pathway Fe3+ release model in which the holo form is converted depending on the anion concentration into a “mixed ligand” intermediate that releases Fe3+. The apo C-lobe was crystallized in ammonium sulfate solution, and the structure determined at 2.3 Å resolution demonstrated the existence of a single bound SO 4 2 − in the interdomain cleft, which interacts directly with Thr461-OG1, Tyr431-OH, and His592-NE2 and indirectly with Tyr524-OH. The latter three groups are Fe3+-coordinating ligands, strongly suggesting the facilitated Fe3+ release upon the anion occupation at this site. The SO 4 2 − binding structure supported the single-pathway kinetic model.

The effect of temperature on recombination activity in testes of rodents

An extractable enzyme system capable of catalyzing recombination in vitro was described in murine spermatocytes [Hotta et al. (1985) Chromosoma 93, 140-151]. The system is specific to meiosis, its activity increasing 400-fold between the premeiotic S-phase and mid-pachytene. The present study examines the effect of temperature on this system since the elevation of testicular temperature is one of the major factors causing impairment of testicular function. A strong depression of in vitro recombination activity occurred immediately after raising the testicular temperature in vivo by translocating the testes into the abdominal cavity (cryptorchid). The in vitro study also showed that the extract from spermatocytes preferred lower temperatures (30-32 degrees C) than somatic cells (37 degrees C) for maximal activity of recombination. These results suggest that the strong depression of recombination activity may be an important factor which causes degeneration of testes by heat.

Evidence of meiosis-specific regulation of gene expression in lily microsporocytes

Abstract Transfection experiments were performed with DNA constructs bearing the GUS reporter gene and various promoter elements to assess meiosis specificity of expression. Plasmid DNA, pBI221, including the CaM 35S promoter fused to the GUS gene, was expressed in protoplasts derived from lily calli and young anthers but not microsporocytes. In contrast, plasmid DNAs bearing the meiotic promoter mei2Pro (δ12) and mei2Pro (δ12-s) isolated from Schizosaccharomyces pombe, fused to the GUS gene, were expressed in protoplasts from lily microsporocytes but not from somatic cells. Furthermore, DNAs with a mutated mei2 promoter fused to the GUS gene showed reduced activity. Transfection of the plasmid DNA, pUC19-recA-mei2, bearing mei2Pro (δ12) in front of the E. coli recA gene, was associated with higher recombinogenic activities assayable in vitro. The induction of GUS in meiocytes by transfection of plasmid DNA was dependent on the presence of mei2, as shown by the reduction of activity when the promoter was mutated and the stage of meiosis. The developmentally regulated expression of a yeast promoter in plant cells demonstrated in this paper may be the first to be reported.

Meiosis specific transcription and functional proteins

We have discussed and/or demonstrated the following: 1. Many enzymes and structural proteins have been identified as meiosis-specific proteins. These can be classified according to their metabolic behavior. 2. We obtained and analyzed 18 cDNA clones from lily meiocytes. One of them, LIM15, was similar to known genes like RecA, RAD57, and DMC1/ISC2, and might function in pairing and recombination. 3. Transcription of these genes is regulated by their regulator region(s). When such a regulator, mei2 promoter sequence isolated from S. pombe, was ligated with the proper vector and transfected, it functioned specifically in meiotic cells but not in the somatic cells tested. 4. Presence of a new lamin, lamin B3 was identified in mammalian spermatocytes and the transfection of lamin B3 gene (inserted into vector) into somatic cells alters the nuclear shape, possibly expressing a characteristic shape of meiotic nuclei. Lamin B3 was synthesized after meiosis-specific processing of lamin B2 mRNA. Other protein specific to meiotic nuclear-skeleton (MNS1) were found and characterized. All these events were studied basically focussing on homologous pairing and recombination which take place in meiosis I. We recognize the necessity of further studies on these and other events like the structure and segregation of chromosomes and the suppression of somatic gene expression during meiosis.

Isolation of synaptonemal complexes from lily microsporocytes

Abstract Synaptonemal complexes (SCs) were isolated from microsporocytes of Lilium. Nuclei prepared from microsporocytes at pachytene in meiotic prophase were treated with DNase II and SCs released from the nuclei were purified by sedimentation through a density gradient of Nycodenz. The SC fraction thus obtained contained fibrous structures consisting of two parallel filaments as major components. They were ∼ 10 μm in length and 200 nm apart from each other and were considered to be the segments of the lateral elements. According to sodium dodecyl sulfate (SDS)-gel electrophoretic analyses, twenty or more proteins were identified as components of the SC fraction. Among these, five proteins having Mr values of 42, 50, 52 116 and 140 kDa were consistently present in various preparations. Antiserum raised against the isolated SCs recognized several kinds of antigens including the proteins of 42, 50 and 52 kDa, which were likely to be candidates for constituents of SCs.

Genome Structure of Jatropha curcas L.

The recent progress in DNA sequencing technology has allowed us to acquire information on the structures of whole genomes of various agronomically important plants in a relatively short period of time. In order to understand the genetic systems carried by Jatropha curcas and to accelerate the process of molecular breeding, comprehensive analyses of genes and the genome of this plant have been conducted using both conventional and advanced technologies, and a large quantity of sequence data has been accumulated. The latest draft sequence of the genome of J. curcas is 297 Mb long, and is presumed to cover 99 % of the gene space, with an average GC content of 33.8 %. By combining with the transcriptome information, a total of 30,203 protein-encoding genes, in addition to the 17,575 transposon-related genes and 2,124 putative pseudogenes, were assigned to the genome. Information on the genomic sequences and genes is available at http://www.kazusa.or.jp/jatropha/.

Proanthocyanidin Biosynthesis in Forage Legumes with Especial Reference to the Regulatory Role of R2R3MYB Transcription Factors and Their Analysis in Lotus japonicus

Proanthocyanidins (condensed tannins) can play an important part in ruminant nutrition both by increasing ruminal efficiency and preventing pasture bloat. In this chapter we discuss the control of this pathway by transcription factors and focus particularly upon genes of the bHLH and R2R3MYB classes. Results from studies using transgenic approaches, tilling and similar techniques are discussed here.