Embryonic Lethals and T-DNA Insertional Mutagenesis in Arabidopsis

T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development. We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens. Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis. The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers. Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families. Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene. Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process. DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion. A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis.     

A cytokinesis-defective mutant of Arabidopsis ( cyt1 ) characterized by embryonic lethality, incomplete cell walls, and excessive callose accumulation

The genetic control of cell division in eukaryotes has been addressed in part through the analysis of cytokinesis-defective mutants. Two allelic mutants of Arabidopsis ( cyt1–1 and cyt1–2 ) altered in cytokinesis and cell-wall architecture during embryogenesis are described in this report. Mutant embryos appear slightly abnormal at the heart stage and then expand to form a somewhat disorganized mass of enlarged cells with occasional incomplete walls. In contrast to the keule and knolle mutants of Arabidopsis and the cyd mutant of pea, which also exhibit defects in cytokinesis during embryogenesis, cyt1 embryos cannot be rescued in culture, are desiccation-intolerant at maturity, and produce cell walls with excessive callose as revealed through staining with the aniline blue fluorochrome, Sirofluor. Some cyt1 defects can be partially phenocopied by treatment with the herbicide dichlobenil, which is thought to interfere with cellulose biosynthesis. The distribution of unesterified pectins in cyt1 cell walls is also disrupted as revealed through immunocytochemical localization of JIM 5 antibodies. These features indicate that CYT1 plays an essential and unique role in plant growth and development and the establishment of normal cell-wall architecture.     

The tertiary structure of endo-beta-1,4-glucanase B (CenB), a multidomain cellulase from the bacterium Cellulomonas fimi.

Endo-beta-1,4-glucanase B (CenB) is a large (110 kDa) extracellular enzyme from the cellulolytic bacterium Cellulomonas fimi. CenB contains five domains, including a typical C.fimi cellulose-binding domain, separated by distinctive linker polypeptides (Meinke et al., 1991b). X-ray scattering analyses show that CenB has a highly elongated shape resembling beads on a string. The sizes of the polypeptides produced by treatment of CenB with proteases, together with their N-terminal amino acid sequences, show that at least two of the four linkers connecting the five domains of CenB are more sensitive to proteolysis than the domains themselves. It is concluded that the beads represent the domains of CenB, the string represents the linkers.     

Physical properties of cytoplasmic membrane-associated DNA

Some of the physical properties of a cytoplasmic membrane-associated DNA isolated from a diploid human lymphocyte cell line have been examined. Cytoplasmic membrane-associated DNA extracted from lymphocytes labeled with either [³H]or [¹⁴C]thymidine had a specific activity lower than nuclear DNA extracted from the same cells. Analysis of cytoplasmic membrane-associated DNA in the electron microscope shows that the molecules are linear and have a mean length of 1·75 μm; the average sedimentation coefficient of this DNA is 16·6 S, which corresponds to a molecular weight of 4·2×10⁶. Cytoplasmic membrane-associated and nuclear DNA band at identical positions in both neutral and alkaline CsCl gradients with buoyant densities of 1·699 g/ml and 1·752 g/ml, respectively. Native cytoplasmic membrane-associated DNA is double-stranded and has a mole fraction of guanine plus cytosine of 40± l %. Sheared, denatured cytoplasmic membrane-associated DNA reassociates as two distinct fractions whose rates of reassociation differ by about four decades: the complexity of the reassociation of this DNA tends to rule out the possibility that it arises from either mycoplasmal or viral contamination of our cell cultures. The slowly reassociating fraction of cytoplasmic membrane-associated DNA reassociates about ten times faster than the unique sequences of nuclear DNA. This could represent potential genetic information for about 100,000 diverse genes of 1000 nucleotide pairs each. At present the function of cytoplasmic membrane-associated DNA in these cells is unknown.     

Design and synthesis of potent and subtype-selective PPARalpha agonists

Beginning with a moderately potent PPARgamma agonist 9, a series of potent and highly subtype-selective PPARalpha agonists was identified through a systematic SAR study. Based on the results of the efficacy studies in the hamster and dog models of dyslipidemia and the desired pharmacokinetic data, the optimized compound 39 was selected for further profiling.     

Nodulisporic acid side-chain modifications: access to the 2", 3", 4", and 6" registers

Efficient routes to access the 2", 3", 4", and 6" registers of the nodulisporic acid (NsA) side chain are disclosed. A mild one-carbon, Ph(2)CdoublebondNCH(2)CtriplebondN mediated homologation of NsA's 3"-aldehyde permitted access to the 4"-register. Curtius reaction of NsA's 3"-acid yielded the corresponding 2"-aldehyde 4 from which the unnatural Delta(2",3")-olefin isomer 2b was obtained. In addition, Arndt-Eistert reactions of the parent NsA permitted a one-carbon homologation to the 6" register. These efforts identified new analogues with significant flea activity and illustrated the biological significance of unsaturation at the 1",2" register.     

Characterization of general esterases from methyl parathion-resistant and -susceptible populations of western corn rootworm (Coleoptera: Chrysomelidae)

A consistent correlation between elevated esterase activity and methyl parathion resistance among Nebraska western corn rootworm, Diabrotica virgifera virgifera LeConte, populations has previously been documented. Characterization of general esterase activity using naphtholic esters as model substrates indicated that differences between resistant and susceptible strains could be maximized by optimizing assay conditions. The optimal conditions identified here were similar to those reported for other insect species. The majority of general esterase activity was found in the cytosolic fractions of resistant populations, whereas the activity was more evenly distributed between cytosolic and mitochondrial/nuclear fractions in the susceptible population. General esterase activity was predominately located in the adult thorax and abdomen. Although there were significant differences in general esterase activities between resistant and susceptible populations, the differences exhibited in single beetle activity assays did not provide sufficient discrimination to identify resistant individuals. In contrast, single larva activity assays provided greater discrimination and could be considered as an alternative to traditional bioassay techniques.