Isolation and characterization of eceriferum (cer) mutants induced by T-DNA insertions in Arabidopsis thaliana

Thirteen Arabidopsis thaliana mutants with deviating epicuticular wax layers (i.e., cer mutants) were isolated by screening 13 000 transformed lines produced by the seed transformation method. After crossing the 13 mutants to some of the previously known cer mutant lines, 12 of our mutants mapped to 6 of the 21 known complementation groups (cer1 through cer4 as well as cer6 and cer10), while the other mutant corresponded to a previously unknown locus, cer21. Mutant phenotypes of 6 of the 13 mutant lines were caused by T-DNA insertions within cer genes. We also analyzed the chemical composition of the epicuticular wax layers of the cer mutants isolated in this study relative to that of Arabidopsis wild-type plants. Our results suggest that the five genes we tagged regulate different steps in wax biosynthesis, i.e., the decarbonylation of fatty aldehydes to alkanes, the elongation of hexacosanoic acid to octacosanoic acid, the reduction of fatty aldehydes to primary alcohols and the production of free aldehydes, while an insertion in the fifth gene causes an alteration in the chain length distribution of the different classes of wax compounds.     

Detection of CaMV gene I and gene VI protein products in vivo using antisera raised to COOH-terminal β-galactosidase fusion proteins

Specific antisera were prepared to the inclusion body protein (gene VI product) and the gene I product of cauliflower mosaic virus (CaMV). Translational fusions between the lacZ gene and gene VI or gene I were constructed by cloning the relevant DNA fragments into the expression vectors pUR290, pUR291 or pUR292. Large amounts of fusion protein were synthesized when the inserted DNA fragment was in frame with the lacZ gene of the expression vector. These fusion proteins were used to raise specific antisera to gene VI and gene I proteins of CaMV. Antiserum to the gene VI product detected a range of proteins in crude extracts and in a subcellular fraction enriched for virus inclusion bodies. This range of proteins was further shown to be related to gene VI by Staphylococcus aureus V8 partial proteolysis. Antiserum to the gene I product detected viral specific proteins of 46, 42 and 38 K in preparations of CaMV replication complexes from infected plants but not in any other subcellular fraction.     

Compartmentation of glucose 6-phosphate in hepatocytes.

Rat hepatocytes were incubated with 14C-labelled hexoses, and the specific radioactivities of glucose 6-phosphate, glucose 1-phosphate and fructose 6-phosphate were determined. (1) When suspensions of freshly isolated hepatocytes were incubated with [14C]glucose, the specific radioactivities of glucose 1-phosphate and fructose 6-phosphate were severalfold higher than that of glucose 6-phosphate. The ratios of the specific radioactivities decreased with time of incubation. These relationships were also found when incubations were carried out with primary cultures of rat hepatocytes or with crude homogenates of hepatocytes, but not with isolated nuclei. (2) When cells were incubated with [14C]fructose, the ratios of the specific radioactivities were higher than with [14C]glucose, and also decreased with time. (3) Paired incubations were carried out with a mixture of galactose and fructose, with one or other sugar being labelled with 14C. The specific radioactivity of glucose released into the medium was greater than that of glucose 6-phosphate when fructose was labelled, but not when galactose was labelled. Furthermore, glucose 6-phosphate and glucose in the medium differed with regard to the distribution of 14C between C-1 and C-6. These results are interpreted as evidence that glucose 6-phosphate in hepatocytes does not exist as a homogeneous pool, but that subcompartments exist which are associated with glucose phosphorylation, gluconeogenesis and glycogenolysis.     

Regression of blood vessels precedes cartilage differentiation during chick limb development

We have previously investigated distinct areas of vascular regression in the developing vascular system of the chick limb bud. Avascular areas appear in a characteristic spatial and temporal pattern, and are correlated with the position of developing cartilage. In the present study, we examined limb-bud sections which had been double labeled for endothelial cells and developing cartilage in order to determine the relationship between the appearance of cartilage and the disappearance of capillaries. Endothelial cells, which specifically take up acetylated low-density lipoprotein (acLDL), were labeled by intravenously injecting fluorescent acLDL (DiIacLDL) into chick embryos at Hamburger and Hamilton stages 26-30. Avascular zones, which correspond to the developing digits, were clearly visible within the fluorescently labeled distal vasculature. The same sections were labeled with monoclonal antibodies specific for cartilage. We found that progressing avascularity in the digital regions was followed by increased staining for cartilage antigens in the same areas. Zones of avascularity always developed earlier than morphologically and immunologically detectable cartilage in all planes of section and were always larger than the areas of cartilage. These results demonstrate that blood vessels disappear in predictable areas prior to the overt differentiation of cartilage.     

High-resolution dynamic and morphological G-bandings (GBG and GTG): a comparative study

Summary A high-resolution replication banding technique, dynamic GBG banding (G-bands after 5-bromodeoxyuridine [BrdUrd] and Giemsa), showed that, at a resolution of 850 bands/genome, GBG banding and GTG banding (G-bands after trypsin and Giemsa) produce almost identical patterns. RBG band (R-bands after BrdUrd and Giemsa) and RHG band (R-bands after heat denaturation and Giemsa) patterns were previously shown to be only 75%–85% coincident; thus GTG banding more accurately reflects replication patterns than does RHG banding. BrdUrd synchronization uses high concentrations of BrdUrd both to substitute early replicating DNA and to arrest cells before the late bands replicate. Release from the block is via a low thymidine concentration. The banding is revealed by the fluorochrome-photolysis-Giemsa (FPG) technique and produces the GBG banding that includes concomitant staining of constitutive heterochromatin. As opposed to other replication G-banding procedures, BrdUrd synchronization and GBG banding produces a reproducible replication band pattern. The discordance between homologs after GBG banding is similar to that after GTG banding and no lateral asymmetry of the constitutive heterochromatin has been observed. Also, BrdUrd synchronization neither significantly depresses the mitotic index, nor induces chromosome breaks. Thus, GBG banding seems as clinically useful as GTG banding and provides important information regarding replication time.