Purification and Properties of 5-Enolpyruvylshikimate-3-Phosphate Synthase from Dark-Grown Seedlings of Sorghum bicolor

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phospho-shikimate 1-carboxyvinyltransferase; EC 2.5.1.19) was purified 1300-fold from etiolated shoots of Sorghum bicolor (L.) Moench. Native polyacrylamide gel electrophoresis revealed three barely separated protein bands staining positive for EPSP synthase activity. The native molecular weight was determined to be 51,000. Enzyme activity was found to be sensitive to metal ions and salts. Apparent K_m values of 7 and 8 micromolar were determined for the substrates shikimate-3-phosphate and phosphoenolpyruvate (PEP), respectively. The herbicide glyphosate was found to inhibit the enzyme competitively with respect to PEP (K_i = 0.16 micromolar). Characterization studies support the conclusion of a high degree of similarity between EPSP synthase from S. bicolor, a monocot, and the enzyme from dicots. A similarity to bacterial EPSP synthase is also discussed. Three EPSP synthase isozymes (I, II, III) were elucidated in crude homogenates of S. bicolor shoots by high performance liquid chromatography. The major isozymes, II and III, were separated and partially characterized. No significant differences in pH activity profiles and glyphosate sensitivity were found. This report of isozymes of EPSP synthase from S. bicolor is consistent with other reports for shikimate pathway enzymes, including EPSP synthase.     

VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells.

Agrobacterium tumefaciens transfers single-stranded DNAs (T strands) into plant cells. VirE1 and VirE2, which is a single-stranded DNA binding protein, are important for tumorigenesis. We show that T strands and VirE2 can enter plant cells independently and that export of VirE2, but not of T strands, depends on VirE1.     

Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex

Two regions of the genome, a 1-kbp portion of the zeste locus and a 1.1- kbp portion of the yolk protein 2 locus, were sequenced in six individuals from each of four species: Drosophila melanogaster, D. simulans, D. mauritiana, and D. sechellia. The species and strains were the same as those of a previous study of a 1.9-kbp region of the period locus. No evidence was found for recent balancing or directional selection or for the accumulation of selected differences between species. Yolk protein 2 has a high level of amino acid replacement variation and a low level of synonymous variation, while zeste has the opposite pattern. This contrast is consistent with information on gene function and patterns of codon bias. Polymorphism levels are consistent with a ranking of effective population sizes, from low to high, in the following order: D. sechellia, D. melanogaster, D.mauritiana, and D. simulans. The apparent species relationships are very similar to those suggested by the period locus study. In particular, D. simulans appears to be a large population that is still segregating variation that arose before the separation of D. mauritiana and D. sechellia. It is estimated that the separation of ancestral D. melanogaster from the other species occurred 2.5-3.4 Mya. The separations of D. sechellia and D. mauritiana from ancestral D. simulans appear to have occurred 0.58- 0.86 Mya, with D. mauritiana having diverged from ancestral D. simulans 0.1 Myr more recently than D. sechellia.      

Intersection of Small RNA Pathways in Arabidopsis thaliana Sub-Nuclear Domains

In Arabidopsis thaliana, functionally diverse small RNA (smRNA) pathways bring about decreased RNA accumulation of target genes via several different mechanisms. Cytological experiments have suggested that the processing of microRNAs (miRNAs) and heterochromatic small interfering RNAs (hc-siRNAs) occurs within a specific nuclear domain that can present Cajal Body (CB) characteristics. It is unclear whether single or multiple smRNA-related domains are found within the same CB and how specialization of the smRNA pathways is determined within this specific sub-compartment. To ascertain whether nuclear smRNA centers are spatially related, we localized key proteins required for siRNA or miRNA biogenesis by immunofluorescence analysis. The intranuclear distribution of the proteins revealed that hc-siRNA, miRNA and trans-acting siRNA (ta-siRNA) pathway proteins accumulate and colocalize within a sub-nuclear structure in the nucleolar periphery. Furthermore, colocalization of miRNA- and siRNA-pathway members with CB markers, and reduced wild-type localization patterns in CB mutants indicates that proper nuclear localization of these proteins requires CB integrity. We hypothesize that these nuclear domains could be important for RNA silencing and may partially explain the functional redundancies and interactions among components of the same protein family. The CB may be the place in the nucleus where Dicer-generated smRNA precursors are processed and assigned to a specific pathway, and where storage, recycling or assembly of RNA interference components takes place.     

Digital image analysis of AgNORs in cervical smears of women with premalignant and malignant lesions of the uterine cervix

We performed a hospital-based, unmatched case-control study to investigate the association between progressive stages of cervical neoplasia and digital analysis of cell proliferation by silver stained nucleolus organizer region associated proteins (AgNORs). We measured cell proliferation levels in the cervical epithelial cells of 10 women with low grade squamous intraepithelial lesions (LG-SIL), eight with high grade squamous intraepithelial lesions (HG-SIL), 11 with cervical cancer (CC) and eight with no cervical lesions (controls) using the AgNORs technique. Cell proliferation was measured by digital image analysis (DIA). DIA revealed increased total areas of AgNORs in HG-SIL and CC compared to LG-SIL and control patients. AgNORs with a kidney or cluster shape exhibited greater areas than those with a spherical or long shape. We propose a cut-off of 118 pixels to differentiate benign (control and LG-SIL) from malignant (HG-SIL and CC) lesions. DIA of AgNORs is a simple and inexpensive method for studying proliferation. The increased total area of AgNORs in malignant lesions provides information regarding cell behavior and may be related to cervical carcinogenesis; however, further validation studies are required to establish its usefulness in cytological analysis.     

A test for a metastable epigenetic component of heterosis using haploid induction in maize

We conducted a test to detect if there is a heritable epigenetic component to hybrid vigor and/or inbreeding depression. The impetus for this work was a classical study of the effect of homozygosis on the expression of the maize red color (r1) locus. It had been shown that maintaining R1 mottling alleles in the homozygous state over several generations produces a progressive decrease of their paternally imprinted expression. This effect is reversed by R1/r1 allele heterozygosity. If this behavior were characteristic of many regulatory genes, then such a phenomenon could contribute to inbreeding depression and heterosis. To examine this question, inbreds of Mo17 and B73 and the two reciprocally produced hybrids were crossed by Stock 6 to generate four classes of maternal haploids. The mature haploid plants were measured for several quantitative traits. If inbreeding depression results from an accumulating heritable effect that is reversed by the hybrid state, one would expect the haploids derived from the hybrids to perform better than those derived from the inbred lines. The hybrid-derived haploids did not exhibit greater average performance than the inbred-derived haploids. These data fail to support the hypothesis that inbreeding depression and heterosis have a metastable epigenetic component.Communicated by D. Hoisington     

1-Thio-beta-D-galactofuranosides: synthesis and evaluation as beta-D- galactofuranosidase inhibitors

Beta-D-galactofuranosidase is a good chemotherapeutic target for the design of inhibitors, since beta-D-galactofuranose is a constituent of important parasite glycoconjugates but is not present in the host mammals. With this aim, we have synthesized for the first time alkyl, benzyl and aryl 1-thio-beta-D-galactofuranosides by condensation of penta-O-benzoyl-alpha,beta-D-galactofuranose with the corresponding thiols, in the presence of SnCl4as catalyst. The complete chemical and spectroscopical characterization of these compounds showed that the reaction was stereoselective. Debenzoylation with sodium methoxide afforded the beta-S-galactofuranosides in high yield. The thioglycosides were tested as inhibitors of the beta-D- galactofuranosidase of Penicillium fellutanum, using for the first time 4-nitrophenyl-beta-D-galactofuranoside as chromogenic substrate. The 4- aminophenyl-1-thio-beta-D-galactofuranoside, obtained by catalytic hydrogenation of the nitrophenyl derivative, was the best inhibitor being then an adequate ligand for the preparation of an affinity phase aimed at the isolation of beta-d-galactofuranosidases from different sources. Also the inhibitory activity of d-galactono-1, 4-lactone was shown.