Effects of donor cell type and genotype on the efficiency of mouse somatic cell cloning

Although it is widely assumed that the cell type and genotype of the donor cell affect the efficiency of somatic cell cloning, little systematic analysis has been done to verify this assumption. The present study was undertaken to examine whether donor cell type, donor genotype, or a combination thereof increased the efficiency of mouse cloning. Initially we assessed the developmental ability of embryos that were cloned from cumulus or immature Sertoli cells with six different genotypes (i.e., 2 x 6 factorial). Significantly better cleavage rates were obtained with cumulus cells than with Sertoli cells (P < 0.005, two-way ANOVA), which probably was due to the superior cell-cycle synchrony of cumulus cells at G0/G1. After embryo transfer, there was a significant effect of cell type on the birth rate, with Sertoli cells giving the better result (P < 0.005). Furthermore, there was a significant interaction (P < 0.05) between the cell type and genotype, which indicates that cloning efficiency is determined by a combination of these two factors. The highest mean birth rate (10.8 +/- 2.1%) was obtained with (B6 x 129)F1 Sertoli cells. In the second series of experiments, we examined whether the developmental ability of clones with the wild-type genotype (JF1) was improved when combined with the 129 genotype. Normal pups were cloned from cumulus and immature Sertoli cells of the (129 x JF1)F1 and (JF1 x 129)F1 genotypes, whereas no pups were born from cells with the (B6 x JF1)F1 genotype. The present study clearly demonstrates that the efficiency of somatic cell cloning, and in particular fetal survival after embryo transfer, may be improved significantly by choosing the appropriate combinations of cell type and genotype.     

The Commelina yellow mottle virus promoter drives companion-cell-specific gene expression in multiple organs of transgenic tobacco

Summary.  Previous work has demonstrated that some endogenous plant gene promoters are active in selective companion cells of the phloem, depending on organ types and developmental stages. Here we report that the Commelina yellow mottle virus (CoYMV) promoter is active in the companion cells of leaves, stems and roots of transgenic Nicotiana tabacum cv. Xanthi NN, using β-glucuronidase (GUS) as a reporter. Thus, the CoYMV promoter has a broad organ specificity. This promoter can be useful in molecular studies on the functions of companion cells in many aspects of phloem biology, such as regulation of long-distance transport, macromolecular traffic, plant development and interaction with pathogens. It may also be useful in engineering crops that produce specific gene products in the companion cells to block long-distance movement of pathogens. Received February 5, 2002; accepted March 27, 2002; published online July 4, 2002 RID="*" ID="*" Correspondence and reprints: Department of Plant Biology and Plant Biotechnology Center, 207 Rightmire Hall, Ohio State University, 1060 Carmack Road, Columbus, OH 43210, U.S.A.     

3-Chloro-DL-alanine resistance by L-methionine-alpha-deamino-gamma-mercaptomethane-lyase activity

The antibacterial agent 3-chloro-DL-alanine (3CA) is an inhibitor of peptidoglycan synthesis. Fusobacterium nucleatum and Porphyromonas gingivalis, the bacteria responsible for oral malodor, are shown to be resistant to 1 mM 3CA, whereas Streptococcus mutans and Escherichia coli are sensitive to this antibacterial agent at the same concentration. We isolated the 3CA resistance gene from F. nucleatum and showed that the gene encodes an L-methionine-alpha-deamino-gamma-mercaptomethane-lyase that catalyzes the alpha,gamma-elimination of L-methionine to produce methyl mercaptan. The enzyme also exhibits 3CA chloride-lyase (deaminating) activity. This antibacterial agent is expected to be useful for specific selection of malodorous oral bacteria producing high amounts of methyl mercaptan.     

Dual functions of fractalkine/CX3C ligand 1 in trafficking of perforin+/granzyme B+ cytotoxic effector lymphocytes that are defined by CX3CR1 expression

Fractalkine/CX3C ligand 1 and its receptor CX3CR1 are known to mediate both cell adhesion and cell migration. Here we show that CX3CR1 defines peripheral blood cytotoxic effector lymphocytes commonly armed with intracellular perforin and granzyme B, which include NK cells, gammadelta T cells, and terminally differentiated CD8(+) T cells. In addition, soluble fractalkine preferentially induced migration of cytotoxic effector lymphocytes. Furthermore, interaction of cytotoxic effector lymphocytes with membrane-bound fractalkine promoted subsequent migration to the secondary chemokines, such as macrophage inflammatory protein-1beta/CC ligand 4 or IL-8/CXC ligand 8. Thus, fractalkine expressed on inflamed endothelium may function as a vascular regulator for cytotoxic effector lymphocytes, regardless of their lineage and mode of target cell recognition, through its ability to capture them from blood flow and to promote their emigration in response to other chemokines.     

Functional expression of Acetabularia acetabulum vacuolar H(+)-pyrophosphatase in a yeast VMA3-deficient strain

The function of the translation product of cDNA for Acetabularia vacuolar H(+)-pyrophosphatase was examined using the Saccharomyces cerevisiae VMA3-deficient strain. The open reading frame of Acetabularia H(+)-pyrophosphatase was revealed to encode 751 amino acids (721 or 751 amino acids in a previous paper). The acidification of the vacuole was observed by fluorescence microscopy when the cDNA was constructed in pYES2. Immunoblot analysis also supported the localization of the translation product in the vacuolar-membrane-enriched fraction.     

Human B cells immortalized with Epstein-Barr virus upregulate CCR6 and CCR10 and downregulate CXCR4 and CXCR5

Compared to peripheral blood resting B cells, Epstein-Barr virus (EBV)-immortalized B cells consistently express CCR6 and CCR10 at high levels and CXCR4 and CXCR5 at low levels. Accordingly, these cells vigorously responded to the ligands of CCR6 and CCR10 but not to those of CXCR4 and CXCR5. In a human EBV-negative B-cell line, BJAB, stable expression of EBNA2 upregulated CCR6, while stable expression of EBNA2 as well as LMP1 downregulated CXCR4. On the other hand, upregulation of CCR10 or downregulation of CXCR5 was not induced in BJAB by stable expression of EBNA2 or LMP1. Thus, these changes may be due to a plasmablast-like stage of B-cell differentiation fixed by EBV immortalization. EBV-infected B cells in infectious mononucleosis are known to avoid germinal centers and accumulate under the mucosal surfaces. EBV-associated opportunistic lymphomas also tend to occur in extranodal sites. These preferred sites of in vivo localization are consistent with the unique profile of chemokine receptor expression exhibited by EBV-immortalized B cells.     

Cloning of the maltose phosphorylase gene from Bacillus sp. strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis

The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.     

Highly sensitive time-resolved fluorometric determination of estrogens by high-performance liquid chromatography using a beta-diketonate europium chelate

A new fluorescent europium chelate labeling reagent, 5-(4"-chlorosulfo-1',1"-diphenyl-4'-yl)-1,1,1,2,2-pentafluoro-3,5-pentanedione (CDPP), was synthesized for the time-resolved fluorometric detection of HPLC. The label can be directly bound to amino or phenolic hydroxyl groups of analytes with its chlorosulfonyl group, and the labeled analytes are separated on a HPLC column. After separation, EuCl(3), TOPO (tri-n-octylphosphine oxide), and Triton X-100 were added by post-column introduction to the eluent, and the fluorescence of the europium chelate was measured with the time-resolved fluorometric detector. Estrone (E1), 17beta-estradiol (E2), ethynylestradiol (EE2) and estriol (E3) were measured with the detection limits of 0.65, 0.65, 0.65 and 0.60 ng/ml, respectively. The recovery for river water samples was in the range of 86.0-105.1% with the RSD of 1.9-5.8%. The method was applied to the analysis of a river water sample and estrone (E1) was determined to be 2.1 ng/l. The results and processing have been compared with those of a GC-MS method and a high degree of correlation (r> or =0.98) was observed.     

Crystal structure of the RuvA-RuvB complex: a structural basis for the Holliday junction migrating motor machinery

We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration. Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring. The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode.