Isolation of root hairs from seedlings of Pisum sativum. Identification of root hair specific proteins by in situ labeling

A procedure was developed which allows the large-scale isolation of root hairs from seedlings of Pisum sativum . L. cvs. Kleine Rheinländerin and Rosa Krone. The method may yield up to 50 g fresh weight of root hairs per 3.10⁴ seedlings. In a modified form considerable amounts of root hair material may be harvested, even after incubation of the roots in aqueous solutions. Thus, detailed biochemical studies on the root hair system have become feasible.
The occurrence of specific proteins in membrane fractions of P. sativum root hairs was demonstrated as follows: Incubation of root hairs in situ with 3-azidonaphthalene-2,7-disulfonate – a strongly anionic, photoactivated fluorescent marker – followed by gel electrophoresis of membrane fractions showed the presence of root-hair specific proteins which, since the system was intact, suggests that they are on the outer surface of the cells.     

The involvement of biological quinones in the formation of hydroxyl radicals via the Haber-Weiss reaction

The present investigation was made to evaluate biologically relevant quinones as possible catalysts in the generation of hydroxyl radicals from hydrogen peroxide and superoxide radicals. ESR spectra demonstrated that menadione, plastoquinone, and ubiquinone derivatives could all be reduced to their semiquinone forms by electron transfer from superoxide radicals. Reductive homolytic cleavage of H₂O₂ was observed to be dependent upon the presence of semiquinones in the reaction medium. Our data strongly support the concept that quinones normally involved in physiological processes may also play a role as catalysts of the Haber-Weiss reaction in the biological generation of hydroxyl radicals.     

Ribosomal gene amplification does not occur in the oocytes of Locusta migratoria

It has been suggested that Locusta migratoria amplifies its ribosomal RNA genes in the growing oocytes (Kunz (1967) Chromosoma20, 332–370). Cloned ribosomal DNA of L. migratoria was used to analyze rDNA structure and number. The rDNA is localized on three chromosome pairs in six nucleolus organizers. It was found that all structural variants of the rRNA genes which have been described previously are represented in the same relative amounts in DNA from isolated oocytes as in somatic cells. Furthermore, the rRNA gene number is not increased in oocyte DNA, i.e., amplification does not occur. Therefore, the large number of multiple nucleoli seen in the growing oocytes has to be interpreted as the fully extended and fully active set of chromosomal rRNA genes. The total rRNA gene number was determined by dot blot hybridization to be about 3300 genes/haploid genome.     

N-Terminal Sequence of Pig Brain Choline Acetyltransferase Purified by a Rapid Procedure

A procedure is reported that allows the purification and amino terminal sequencing of pig brain choline acetyltransferase. The enzyme (present in extremely low amounts in this tissue) is eluted together with its antibody from an affinity column by a mild pH shift and the resulting enzyme-antibody complex separated by gel electrophoresis. The band corresponding to the enzyme is electroeluted from the gel using volatile solutions allowing the direct determination of the amino acid composition and partial sequence. The first 11 residues are: Pro-Ile-Leu-Glu-Lys-Thr-Pro-Pro-Lys-Met-Ala.     

Brain mitochondrial cytochrome P-450scc: spectral and catalytic properties

The cytochrome P-450-dependent cholesterol side chain cleavage system of the brain has been studied using nonsynaptic mitochondria as the source of enzymatic activity. The system has been found to bind cholesterol and 11-deoxycorticosterone, producing type I difference spectra, whereas the binding of pregnenolone induced a reverse type I difference spectrum. Inhibitors of cytochrome P-450-linked monooxygenase activities produced type II spectra. The formation of labeled pregnenolone after incubation of brain mitochondria with [4-14C]cholesterol has been obtained, and this formation was inhibited by glutethimide, a specific inhibitor of cytochrome P-450scc. The functional significance of this enzymatic activity is discussed.     

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.     

Construction and properties of a chimeric bacteriophage lysis gene

Abstract The genomes of DNA phage ΦX174 and of RNA phage MS2 each encode a single lysis protein, E protein and L protein, respectively. Based on the known DNA and protein sequences, and with the aid of structural predictions of the proteins, a chimeric gene was constructed. The resulting protein was composed of the N-terminal sequence of E protein and the C-terminal sequence of L protein. The truncated E and L polypeptides used in this construction were functionally inactive. However, the chimeric gene product had very high lysis-inducing activity. This construction is an example which could be extended to the engineering of other lysis proteins designed with specific biotechnological processes in mind.     

Liver glutathione S-transferase polymorphism in Japanese and its pharmacogenetic importance

Summary A total of 168 autopsy liver extracts from Japanese individuals were examined for the glutathione S-transferase (GST) isozymes by means of starch gel electrophoresis. The gene frequencies of GST1^*1, GST1^*2, and GST1^*0 in Japanese were 0.252, 0.057, and 0.691, respectively. GST1^*3 was detected as a rare variant allele. The incidence of GST1 0 in 41 liver biopsy samples from patients suffering from various liver diseases was investigated using polyacrylamide gel isoelectric focusing. The GST1 0 phenotype was found more frequently in livers with hepatitis and carcinoma than in control livers. The isozymes coded by different GST loci were partially purified and characterized to study their biochemical properties. The apparent Km values with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate for the isozymes at the GST1, GST2, GST3, and GST4 loci were 604, 1345, 776 and 591 M, respectively.     

Performance of recombinant inbred lines in Brussels sprouts (Brassica oleracea var. gemmifera)

Summary Performance of a random array of recombinant inbred lines derived by single seed descent from five different source populations of Brussels sprouts (Brassica oleracea var. gemmifera) is presented. A total of 2,356 lines were tested in trials during 1985 and 1986. Three of the source populations were derived from double crosses between F₁ hybrids. These hybrids show a considerable heterotic advantage over their inbred parents for the most important agronomic traits. The recombinant inbred lines performed, on average, less well than the parental inbred material, indicating that additive x additive genie interactions may make a significant contribution to the performance of current inbred material. Nevertheless, the very large variation among the recombinant inbred lines permitted many lines to be identified which outperformed the best parental inbred for all traits. Two lines outperformed the reference F₁ hybrid, Gower, for an index that included marketable yield and quality. Consideration was also given to the dangers of misinterpreting phenotypically based proportions. Accordingly, response equations were used to ascertain the real genetic progress that was made. Advance seemed small when compared with the large heterotic effect, which is consistent with the segregation of a large number of loci. The distribution of the recombinant inbred lines was compared to predictions made from early generation trials. There was broad agreement but significant discrepancies existed which, it is suggested, may arise from the effects of genotype-environment interactions.     

Membranous tubes in pseudoscorpion spermiogenesis

The highly complicated differentiation of the spermatid in the pseudoscorpion Diplotemnus sp. is accomplished without the presence of microtubules. Instead membranous tubes measuring approximately 50 nm in diameter and closely associated with endoplasmic reticulum are found from early to mid spermatids. The lumen of the tube is devoid of electron dense contents but a fluffy material is attached to the cytoplasmic side. They run straight or slightly bent and are in open connection with the cell membrane. First appearing near the cell bridge of the interconnected spermatids they form a bundle in the longitudinal axis during a transitory phase of elongation. When the cell rounds off again the tubules together with the endoplasmic reticulum disappear. The arrangement of the tubes and their presence during abortive elongation of the spermatid suggest a supportive function commonly attributed to microtubules. Moreover, the open connection with the cell membrane and their close association with the endoplasmic reticulum may indicate their participation also in transport.