Binding of a nuclear factor to a consensus sequence in the 5' flanking region of zein genes from maize

The genomic organization of the zein structural genes and of regulatory loci influencing their expression suggests that control of zein gene expression will involve interactions between cis elements in the flanking DNA sequences and products from trans-acting genes. The interaction between fragments from the 5' flanking region of a zein gene and specific, double-stranded oligonucleotides with crude nuclear extracts from maize endosperm have been studied by nitrocellulose filter binding, gel retention and DNase I footprinting assays. Specific binding of a nuclear factor was observed and the exact position of the protein binding site was determined. The 22-nt binding site included 14 bp of a 15-bp sequence conserved in all zein genes.     

Interactions of 14N:15N stearic acid spin-label pairs: effects of host lipid alkyl chain length and unsaturation

Electron-electron double resonance (ELDOR) and saturation recovery electron paramagnetic resonance (EPR) spectroscopy have been employed to examine the interactions of 14N:15N stearic acid spin-label pairs in fluid-phase model membrane bilayers composed of a variety of phospholipids. The [14N]-16-doxylstearate:[15N]-16-doxylstearate (16:16) pair was utilized to measure lateral diffusion of the spin-labels, while the [14N]-16-doxylstearate:[15N]-5-doxylstearate (16:5) pair provided information on vertical fluctuations of the 16-doxylstearate nitroxide moiety toward the membrane surface. Three saturated host lipids of varying alkyl chain length [dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC)], an alpha-saturated, beta-unsaturated lipid [1-palmitoyl-2-oleoylphosphatidylcholine (POPC)], and phosphatidylcholine from a natural source [egg yolk phosphatidylcholine (egg PC)] were utilized as host lipids. Lateral diffusion of the stearic acid spin-labels was only slightly affected by alkyl chain length at a given reduced temperature (Tr) in the saturated host lipids but was significantly decreased in POPC at the same Tr. Lateral diffusion in DMPC, POPC, and egg PC was quite similar at 37 degrees C. A strong correlation was noted between lateral diffusion constants and rotational mobility of [14N]-16-doxylstearate. Vertical fluctuations were likewise only slightly influenced by alkyl chain length but were strongly diminished in POPC and egg PC relative to the saturated systems. This diminution of the 16:5 interaction was observed even under conditions where no differences were discernible by conventional EPR. These studies indicate that vertical fluctuation of 16-doxylstearate is quite sensitive to host lipid unsaturation and that ELDOR studies of interactions between 14N:15N spin-label pairs can provide information on spin-label motion beyond that given by conventional EPR.     

A functional splice site in the 5'' untranslated region of a zein gene.

Zein genes, the genes coding for the zein storage proteins of maize, have a unique gene structure where at least two promoters lie upstream of the coding region. Between the P1 promoter (900 base pairs upstream of the coding region) and the translation initiation AUG codon are 18 short reading frames. A discrepancy between the signals obtained by S1-mapping and primer extension and the DNA sequence in the region of one of these signals suggests the presence of a 3' splice site lying 40 nucleotides upstream of the coding region. A splicing event removing all of the short reading frames from the mRNA transcribed from the P1 promoter would bring this mRNA into a translatable form. Further evidence for a functional 3' splice site has been obtained from sequencing of primer extension products and in vitro splicing of a hybrid intron in the HeLa cell in vitro splicing system.     

Isolation and characterization of high-mobility-group proteins from maize

Chromosomal nonhistone high-mobility-group (HMG) proteins were purified from nuclei of maize (Zea mays L. cv. A619) endosperm and leaf tissue. Tissuespecific differences were observed in their polypeptide patterns, in in-vitro phosphorylation experiments with a casein-kinase type II, and by Western blot analysis with antisera against different HMG proteins. Gelfiltration chromatography demonstrated that maize HMG proteins occur as monomers. By measuring the capacity of the HMG proteins to bind to the 5 flanking region of a zein gene, the sensitivity of the proteins to different temperatures, salt concentrations and pH values was determined.Abbreviations EMSA electrophoretic-mobility-shift assay - FPLC fast protein liquid chromatography - HMG high-mobility group - kDa kilodaltons - PVDF polyvinylidenedifluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We would like to thank Mrs. E. Brutzer for excellent technical assistance. We are indebted to Mrs. M. Strecker and Dr. W. Bessler of the Institut für Immunbiologie, Freiburg, FRG, for the preparation of antisera and we gratefully acknowledge helpful discussions with Drs. T. Quayle, R. Grimm and U. Müller of this institute. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fond der Chemischen Industrie.     

Occurrence of 9 homologous repeat units in the external spacer region of a nuclear maize rRNA gene unit.

A 14 kb maize DNA fragment carrying nuclear rRNA genes and spacer regions was isolated and characterised by restriction enzyme mapping. A complete 3020 bp long external spacer region was sequenced and revealed 9 tandemly arranged 200 bp long repeat units with high homology. The repeat units lie upstream from two prominent S1 mapping signals. The sequence of a typical repeat unit is compared to a corresponding 130 bp long wheat repeat unit. The possible functional relevance of the repeat units is discussed.     

Multiple proteins bind to the P2 promoter region of the zein gene pMS1 of maize

Summary A 216 by promoter fragment of the 19 kDa protein zein gene pMS1, containing the CCAAT and TATA boxes, was analysed by a variety of techniques for in vitro interactions with nuclear proteins from endosperm tissue. HMG proteins were found to form stable complexes with these A/T-rich promoter sequences and several specific DNA-binding proteins appear to be involved in the formation of DNA-protein complexes with this fragment. A 29 bp region spanning the two CCAAT boxes was protected from DNase I digestion in footprinting experiments.     

Membrane fluidity and myotonia: Effects of cholesterol and desmosterol on erythrocyte membrane fluidity in rats with 20,25-diazacholesterol-induced myotonia and on phospholipid liposomes

Previous spin-label and electromyographic experiments with rats fed 20,25-diazacholesterol, an inhibitor of the biosynthetic conversion of desmostero[ to cholesterol, demonstrated an increased erythrocyte membrane fluidity and myotonia, a prolonged muscle contraction upon stimulation. The current studies with rats showed normal erythrocyte fluidity in animals fed 20,25-diazacholesterol but maintained on a high-cholesterol diet and no myotonia. Studies of model membrane systems composed of phospholipid vesicles containing desmosterol, cholesterol, or both demonstrated that desmosterol increased membrane lipid fluidity relative to cholesterol, suggesting that in 20,25-diazacholesterol-induced myotonia, in which desmosterot accounts for 85% of the plasma sterol, the increased membrane fluidity previously observed in erythrocytes and sarcolemma m this animal model of human congenital myotonia may be due to desmosterol.     

Isolation and characterization of a genomic sequence of maize coding for a zein gene

Summary An EcoRI restriction endonuclease fragment of maize DNA coding for the 19,000 dalton zein protein was cloned in phage gt WES. The zein gene was identified by the electron microscopic analysis of RNA-DNA hybrids (R-loops) and DNA-DNA hybrids (D-loops). The R-loops were formed with poly(rA)-containing RNA isolated from 18 days post-pollination maize endosperm and showed no intervening non-hybridizing sequences (introns) within their 800 base length. A cDNA clone specific for the 19,000 dalton zein protein formed D-loops in the same position and orientation as the R-loops. The cloned fragment measured 4.4 kilobases (kb), the same size as an EcoRI fragment of maize DNA revealed by Southern analysis.     

RNA nicking activity associated with DNA ligase of T4 infected E. coli: properties and influence on in vitro reactions of ligase.

Highly purified DNA ligase from T4 infected E. coli displays an RNA nicking activity which cleaves endonucleolytically the RNA of ribo-desoxy-and ribo-ribo type doublestranded structures to oligonucleotides with 5'phosphoryl-and 3'hydroxy termini. In the presence of ATP the generated nicks are repaired by the ligase except at the ends of the doublestranded regions where some short oligonucleotides are released before ligation can occur. As judged from its behaviour during the various purification steps and from some of its properties, the nicking activity seems to be different from known nicking enzymes.