Participation of hup gene product in ori2-dependent replication of fertility plasmid F

The fertility plasmid F'gal was not stably maintained in a hupA-hupB double mutant of Escherichia coli. Moreover, mini-F plasmids pFZY1, pFTC1 and pFTC2 were unable to transform the double mutant, though these plasmids efficiently transformed cells harboring a hupA or hupB single mutation. The composite plasmid pFHS1, which consists of the f5 DNA fragment of F plasmid and the whole DNA of a pSC101 derivative that carries a temperature-sensitive mutation for DNA replication, was not stably maintained in the hup double mutant at 42°C. These findings strongly suggest that HU protein is required for ori2-dependent replication of the F plasmid.     

Gonadotropin-releasing hormone in third ventricular cerebrospinal fluid of the heifer during the estrous cycle

The release profile of GnRH in cerebrospinal fluid (CSF) and its correlation with LH in peripheral blood of ovary-intact heifers during the estrous cycle were investigated. A silicon catheter was placed into the third ventricle of six heifers using ultrasonography. During the mid-luteal phase, the heifers were injected with prostaglandin F(2alpha) to induce luteolysis. Surges of CSF GnRH (66.7 h after prostaglandin F(2alpha) administration) and peripheral LH (66.3 h) occurred simultaneously and were coincident with the onset of estrus (67.0 h). Duration of elevated GnRH concentration considerably overlapped with the estrous phase in each of the heifers. Mean pulse frequencies of both GnRH and LH were significantly higher during the proestrous and early luteal phases than during the mid-luteal phase, while mean concentration and pulse amplitude of both GnRH and LH were not different between these three phases. Of all the GnRH pulses identified, more than 80% were accompanied by an LH pulse during the proestrous and early luteal phases. However, the proportion of GnRH pulses that were coincident with an LH pulse during the mid-luteal phase decreased to 60%. The results clearly demonstrate that a dynamic (pulse) and longer-term (surge) changes of GnRH release into CSF are physiologically expressed during the estrous cycle in heifers, and the pattern of pulsatile GnRH secretion in heifers depends upon their estrous cycle.     

Molecular cloning and characterization of a cDNA for a novel ethylene receptor, NT-ERS1, of tobacco (Nicotiana tabacum L.)

The cDNA encoding a novel member (NT-ERS1) of ethylene receptor family of tobacco (Nicotiana tabacum L.) was obtained by a combination of RT-PCR and 5'-/3'-RACE cloning. The cDNA was 2,092 nucleotides long and had an open reading frame of 1,911 bp encoding 637 amino acids. The deduced polypeptide lacked a response regulator domain, indicating that the ethylene receptor belongs to an ERS-group. The amino acid sequence was similar to respective members of the tobacco ethylene receptor family: 67.8% to NT-ETR1, 39.1% to NTHK1 and 31.1% to NTHK2. Comparison of amino acid sequence suggested that NT-ERS1 is the counterpart of Nr in the ethylene receptor family of tomato, which belongs to Solanaceae as does tobacco. Northern blot analysis showed that mRNA of NT-ERS1 was present in leaf, shoot and root tissues, and accumulated in leaves treated with exogenous ethylene. A mutated NT-ERS1 cDNA transgene, obtained by introducing one nucleotide substitution into NT-ETR1 cDNA, conferred ethylene insensitivity in tobacco plants, indicating that the translation product of the cDNA actually functioned in the plants.     

Indispensability of transmembrane domains of Golgi UDP-galactose transporter as revealed by analysis of genetic defects in UDP-galactose transporter-deficient murine had-1 mutant cell lines and construction of deletion mutants

UDP-galactose transporter is a membrane protein localized in the Golgi apparatus. It translocates UDP-galactose from the cytosol into the Golgi lumen, thus providing galactosyltransferases with their substrate. We characterized murine UDP-galactose transporter through molecular cloning for the following purposes: (i) to elucidate the molecular bases underlying the genetic defects of murine Had-1 mutants, which are deficient in UDP-galactose transporting activity, and (ii) to obtain information that would help us in planning rational approaches to identify functionally essential regions, based on comparison of primary structures between human and murine UDP-galactose transporters. We identified five nonsense mutations, one missense Gly178Asp mutation, and two aberrant splicing mutations. Although glycine178 is highly conserved among nucleotide-sugar transporters, a Gly178Ala variant was functional. The species-differences between human and murine UDP-galactose transporters were largely confined to the N- and C-terminal regions of the transporters. Substantial deletions in the N- and C-terminal regions did not lead to loss of UDP-galactose transporting activity, indicating that these cytosolic regions are dispensable for the transporting activity. The transporter was fused with green-fluorescent protein at the C-terminal cytosolic tail without impairing the functions of either protein. Our results demonstrate the importance of the transmembrane core region of the UDP-galactose transporter protein.     

Subunit Interacting Surfaces of Human Hemoglobin in Solution: Localization of the α–β Subunit Interacting Surfaces on the α-Chain by a Comprehensive Synthetic Strategy

By using synthetic overlapping peptides encompassing the entire -chain of adult human hemoglobin (HbA), we have mapped on the -chain the regions responsible for its binding to the -chain in solution. These binding surfaces were, in general, in good agreement with those expected from the crystal structure (peptides 81–95, 101–115, 111–125, and 131–141). However, we observed some significant differences in the levels of binding found here in solution and those expected from the crystal structure. Peptide 31–45, which in the crystal had the highest number of contact residues of all the -chain peptides, did not bind the -chain in solution. Similarly, peptide 91–105, with seven contact residues in the crystal, showed low binding with the -chain in solution. On the other hand, peptides 41–55 and 121–135 possessed much higher binding activity in solution than would be expected from their contribution to subunit association in the crystal. In fact, peptide 121–135 had the highest binding activity of the -chain peptides. These studies and our previous findings, which localized on the -chain the regions that bind to the -chain in solution, have shown that the regions of subunit association in solution are close to, but not identical with, those in the crystal. The approach should be quite useful for mapping subunit association in oligomeric proteins and could even be applied to proteins that are isolated only in traces or whose three-dimensional structure is not yet known.     

A FRET-based analysis of SNPs without fluorescent probes

Fluorescence resonance energy transfer (FRET) is a simple procedure for detecting specific DNA sequences, and is therefore used in many fields. However, the cost is relatively high, because FRET-based methods usually require fluorescent probes. We have designed a cost-effective way of using FRET, and developed a novel approach for the genotyping of single nucleotide polymorphisms (SNPs) and allele frequency estimation. The key feature of this method is that it uses a DNA-binding fluorogenic molecule, SYBR Green I, as an energy donor for FRET. In this method, single base extension is performed with dideoxynucleotides labeled with an orange dye and a red dye in the presence of SYBR Green I. The dyes incorporated into the extended products accept energy from SYBR Green I and emit fluorescence. We have validated the method with ten SNPs, which were successfully discriminated by end-point measurements of orange and red fluorescence intensity in a microplate fluorescence reader. Using a mixture of homozygous samples, we also confirmed the potential of this method for estimation of allele frequency. Application of this strategy to large-scale studies will reduce the time and cost of genotyping a vast number of SNPs.     

Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery

Proper patterns of genome-wide DNA methylation, mediated by DNA methyltransferases DNMT1, -3A and -3B, are essential for embryonic development and genomic stability in mammalian cells. The de novo DNA methyltransferase DNMT3B is of particular interest because it is frequently overexpressed in tumor cells and is mutated in immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. In order to gain a better understanding of DNMT3B, in terms of the targeting of its methylation activity and its role in genome stability, we biochemically purified endogenous DNMT3B from HeLa cells. DNMT3B co-purifies and interacts, both in vivo and in vitro, with several components of the condensin complex (hCAP-C, hCAP-E and hCAP-G) and KIF4A. Condensin mediates genome-wide chromosome condensation at the onset of mitosis and is critical for proper segregation of sister chromatids. KIF4A is proposed to be a motor protein carrying DNA as cargo. DNMT3B also interacts with histone deacetylase 1 (HDAC1), the co-repressor SIN3A and the ATP-dependent chromatin remodeling enzyme hSNF2H. Further more, DNMT3B co-localizes with condensin and KIF4A on condensed chromosomes throughout mitosis. These studies therefore reveal the first direct link between the machineries regulating DNA methylation and mitotic chromosome condensation in mammalian cells.     

Phylogeography of the component species of broad-leaved evergreen forests in Japan,based on chloroplast DNA variation

In order to elucidate the past distribution and colonization routes of broad-leaved evergreen (lucidophyllous) forests, we investigated the intraspecific phylogeographic patterns of lucidophyllous forests in Japan and surrounding areas. We selected 6 component species with a similar geographic distributions growing in Castanopsis-dominant forests. We defined possible important refugia during the glacial periods as the regions rich in rare haplotypes (with a frequency of 5% or less), or as regions rich in the number of common haplotypes (with a frequency of more than 5%). We then located the sites of refuge by comparing the intraspecific phylogeographic patterns among 6 component species of lucidophyllous forests with respect to these two parameters (i.e., haplotype uniqueness and the number of haplotypes). The following results were obtained during the course of this study: (1) rare haplotypes were distributed among islands around the main islands of Japan; (2) rare subtypes and the greatest numbers of common haplotypes were observed in Kyushu, a finding which agreed with fossilized pollen data demonstrative of the past existence of refugia in southern Kyushu; and (3) rare haplotypes were found on the Muroto Peninsula, and the second greatest numbers of common haplotypes were observed on the Kii Peninsula, a finding which suggested the existence of additional important refugia along the Pacific coast of Japan during the glacial ages.     

Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germ cell number in mouse embryos

Estrogen related receptor beta (ERR-beta) is an orphan nuclear receptor specifically expressed in a subset of extra-embryonic ectoderm of post-implantation embryos. ERR-beta is essential for placental development since the ERR-beta null mutants die at 10.5dpc due to the placenta abnormality. Here, we show that the ERR-beta is specifically expressed in primordial germ cells (PGC), obviously another important cell type for reproduction. Expression of the ERR-beta mRNA in embryonic germ cells started at E11.5 as soon as PGC reached genital ridges, and persisted until E15-E16 in both sexes. Immunostaining with anti-ERR-beta antibody revealed that the ERR-beta protein is exclusively expressed in germ cells in both male and female gonads from E11.5 to E16. 5. To study function of the ERR-beta in PGC, we complemented placental defects of the ERR-beta null mutants with wild-type tetraploid embryos, and analyzed germ cell development in the rescued embryos. It was found that development of gonad and PGC was not apparently affected, but number of germ cells was significantly reduced in male and female gonads, suggesting that the ERR-beta appears to be involved in proliferation of gonadal germ cells. The rescued embryos could develop to term and grow up to adulthood. The rescued ERR-beta null male were found to be fertile, but both male and female null mutants exhibited behavioural abnormalities, implying that the ERR-beta plays important roles in wider biological processes than previously thought.