Biochemical and genetic evidence for a macromolecular -glucuronidase complex in microsomal membranes

In the mouse β-glucuronidase is present in both microsomes and lysosomes and the enzyme at both sites is coded by the same structural gene. Electrophoresis on polyacrylamide gels showed that liver, kidney and lung from normal strains contained five enzyme forms designated L, M1, M2, M3 and M4 in order of decreasing mobility toward the anode. Band L is found primarily in lysosomes and is a tetramer of 260,000 molecular weight. Bands M1 to M4 are found exclusively in microsomes and range in molecular weight from 310,000 to 470,000. The increase in molecular weight is due to sequential addition of an accessory protein chain. When glucuronidase is highly induced in kidneys of female mice by injection of dihydrotestosterone, a sixth electrophoretic form of glucuronidase, designated X, appears. Form X appears early in induction, is localized in microsomes, and has a molecular weight (260,000) equal to that of the tetramer form L.Mice homozygous for the eg ° mutation, and thus deficient in microsomal glucuronidase, completely lack the microsomal forms M1 to M4. They do contain form X, and this increases after testosterone induction in kidney. The form X present in eg ° mice is indistinguishable from the form X seen in normal induced kidney.It appears that mice synthesize two different tetrameric forms of glucuronidase from the same structural gene. One, form L, is lysosomal; the other, form X, gives rise to microsomal enzyme forms M1 to M4 by the successive addition of up to four accessory protein chains. The eg ° mutant is blocked in the conversion of X to M1.     

Purification of hepatitis B virus gene X product synthesized in Escherichia coli and its detection in a human hepatoblastoma cell line producing hepatitis B virus

A fused gene containing 94% of the hepatitis B virus (HBV) open reading frame X was expressed in Escherichia coli, and its 17-kDa product was purified by ion-exchange chromatography. Antibody elicited against the X-gene product reacted with materials proximal to the nuclear membrane of a human hepatoblastoma cell line producing HBV particles. No such reaction was observed with the same cell line that did not produce HBV particles.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

De novo organogenesis and plant regeneration in <Emphasis Type="Italic">Pongamia pinnata</Emphasis>, oil producing tree legume

Role of Thidiazuron (TDZ) in inducing adventitious organogenesis in Pongamia was studied. TDZ at different concentrations (0, 0.45, 2.27, 4.54, 6.71, 9.08, 11.35, 13.12 and 22.71 μM) were used for induction of caulogenic bud formation in deembryonated cotyledon explants. Each cotyledon was cut into three segments and identified as proximal, middle and distal. Duration of TDZ exposure, influence of the segment and orientation of the explant were studied. TDZ at 11.35 μM concentration was optimum for the induction of shoots and rapid elongation. Shoots induced at higher concentration elongated after several passages in growth regulator free medium, thereby extending the period of differentiation. Exposure of the explant for 20 days yielded more number of buds than 10 days. Proximal segment of the cotyledon was more responsive. Contact of abaxial surface in the medium was more effective and generated more buds than the adaxial side. Buds differentiated and elongated on transfer to MS basal medium for 8–12 passages of 15 days each. Rooting and elongation of shoots was achieved in charcoal supplemented half-strength MS medium. Rooted plantlets survived on transfer to sand soil mixture. The plants were hardened and transferred to green house. This is the first report on in vitro regeneration of Pongamia pinnata via adventitious organogenesis using TDZ. This protocol may find application in studies in genetic transformation, isolation of somaclonal variants and in induction of mutants. It also provides a system to study the inhibitory role of TDZ on shoot differentiation.     

Early events in the life of apple roots: variation in root growth rate is linked to mycorrhizal and nonmycorrhizal fungal colonization

Early events of mycorrhizal and nonmycorrhizal fungal colonization in newly-emerging roots of mature apple (Malus domestica Borkh) trees were characterized to determine the relationship of these events to fine root growth rate and development. New roots were traced on root windows to measure growth and then collected and stained to quantify microscopically the presence of mycorrhizal and nonmycorrhizal fungal structures. Most new roots were colonized by either mycorrhizal or nonmycorrhizal fungi but none less 25 days old were ever internally colonized by both. Compared to nonmycorrhizal colonization, mycorrhizal colonization was associated with faster growing roots and roots that grew for a longer duration, leading to longer roots. While either type of fungi was observed in roots as soon as 3 days after root emergence, intraradical colonization by mycorrhizal fungi was generally faster (peaking at 7 to 15 days) than that by nonmycorrhizal fungi and often occurred more frequently in younger roots. Only 15 to 35% of the roots had no fungal colonization by 30 days after emergence. This study provides the first detailed examination of the early daily events of mycorrhizal and nonmycorrhizal fungal colonization in newly emerging roots under field conditions. We observed marked discrimination of roots between mycorrhizal and nonmycorrhizal fungi and provide evidence that mycorrhizal fungi may select for faster growing roots and possibly influence the duration of root growth by non-nutritional means.     

Accumulation of the labdane diterpene Marrubiin in glandular trichome cells along the ontogeny of Marrubium vulgare plants

The content of the diterpene Marrubiin was assessed by GC-FID in leaves of Marrubium vulgare plants along their ontogeny. Maximum accumulation occurred just before flowering time and in fully expanded leaves. After feeding the plants with radio labeled [³H]-geranyl geranyl diphosphate, up to 70% of the radioactivity was recovered in HPLC-Rt coincidental with authentic Marrubiin, which was also characterized by GC-EIMS, thus confirming that the biosynthesis of Marrubiin proceeds through the 1-deoxy-D-xylulose-5-phosphate pathway. The major accumulation of radioactivity occurred in glandular trichome cells, and the product remained stable throughout.     

Pigments and ultrastructures of pigment cells in xanthic sailfin mollies (Poecilia latipinna).

Electron micrographs of skin from xanthic (gold) sailfin mollies revealed numerous xanthophores, as well as scattered melanophores. The melanophores were seen to contain premelanosomes in various stages of development. This is consistent with the fact that xanthic mollies have been shown to be tyrosinase positive. Melanosomes in xanthic mollies appear to develop by one of two pathways: 1) from an endoplasmic reticulum-derived vesicle which develops an internal lamellar framework, and 2) by fusion of multiple Golgi-derived vesicles which lack an internal lamellar framework. Analysis of the pigments in the skin of the xanthic mollies identified four colorless pteridine pigments (xanthopterin, isoxanthopterin, neopterin, and pterin) and a carotenoid with an absorbance spectrum similar to beta-carotene. It appears that, unlike some other poeciliid fishes, sailfin mollies do not use pteridine pigments for orange coloration. Rather, they appear to rely primarily on carotenoids.