In situ hybridization to metaphase chromosomes in six species ofPhaseolus andVigna using ribosomal DNA as the probe

In situ hybridization with a biotin-labeled rice ribosomal DNA (rDNA) probe to the somatic metaphase chromosomes of six species ofPhaseolus andVigna (P. angularis, P. calcaratus, P. coccineus, P. vulgaris, V. sesquipedalis andV. sinensis) was done to determine the sites of rDNA. Hybridization signals were present in the terminal and subterminal chromosome regions of each of the six species. The number of rDNA sites was two inP. angularis andP. calcaratus, four inP. coccineus andP. vulgaris, and six inV. sesquipedalis andV. sinensis.     

Expression of the [beta]-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica,and Peony (Paeonia lactiflora) by Particle Bombardment

A [beta]-glucuronidase (GUS) gene that is under the control of the anther-specific LAT52 promoter of tomato (Lycopersicon esculentum) and the nopaline synthetase polyadenylation terminator was successfully expressed in pollen of Lilium longiflorum, Nicotiana tabacum, Nicotiana rustica, and Paeonia lactiflora using a pneumatic particle gun. The GUS gene in plasmid pBI221 was also expressed, to a lesser extent, in pollen of all of these species. The presence of methanol in the substrate solution for histochemical GUS assay and the incubation time in this solution influenced successful detection of GUS expression in bombarded pollen. Cytological analysis of GUS-expressing pollen of lily showed that introduced gold particles were seen in intracellular compartments of pollen, including the vegetative cytoplasm, vegetative nucleus, and generative cytoplasm.     

Stable transformation of cultured cells of the liverwortMarchantia polymorpha by particle bombardment

Suspension-cultured cells (A-18 line) of the liverwortMarchanta polymorpha were bombarded by a pneumatic particle gun with plasmid pCH harbouring the hygromycin phosphotransferase (HPT) gene (hpt) under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and the nopaline synthase polyadenylation region. Nine weeks after bombardments, 128 hygromycin-resistant calluses were obtained from an approximate total of 7×10⁶ cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain thehpt gene in the genome, demonstrating that these lines are transformants. An HPT enzyme activity assay confirmed the expression of the gene in all of the transformant lines.     

Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment

Gold particles coated with -glucuronidase (GUS) mRNA with a 5 cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.     

Genetic Variation in VP7 Gene of Human Rotavirus Serotype 2 (G2 Type) Isolated in Japan,China, and Pakistan

Sequence analysis of the gene encoding the major neutralization glycoprotein (VP7) was performed on sixteen human isolates of serotype 2 of rotavirus in Japan, China, and Pakistan and their genetic variations were examined. Comparative studies of their nucleotide and deduced amino acid sequences between the sixteen isolates and the HU5 strain revealed an overall homology of more than 94%. A higher degree of homology in nucleotides was observed among the sixteen isolates than between HU5 and the isolates. A total of thirteen amino acid residues frequently converted to another amino acid. Out of the thirteen, five amino acid residues belonging to the major neutralizing epitope regions (C, E, and F in this communication) converted frequently. From the amino acid sequences three subtypes, subtype 1, subtype 2, and intermediate, were suggested to be classified as previously reported for serotype 1 (Xin et al, Virology, 1993, 197: 813-816).     

Determination of the NMR solution structure of a specific DNA complex of the Myb DNA-binding domain

Summary The solution structure of a specific DNA complex of the minimum DNA-binding domain of the mouse c-Myb protein was determined by distance geometry calculations using a set of 1732 nuclear Overhauser enhancement (NOE) distance restraints. In order to determine the complex structure independent of the initial guess, we have developed two different procedures for the docking calculation using simulated annealing in four-dimensional space (4D-SA). One is a multiple-step procedure, where the protein and the DNA were first constructed independently by 4D-SA using only the individual intramolecular NOE distance restraints. Here, the initial structure of the protein was a random coil and that of the DNA was a typical B-form duplex. Then, as the starting structure for the next docking procedure, the converged protein and DNA structures were placed in random molecular orientations, separated by 50 Å. The two molecules were docked by 4D-SA utilizing all the restraints, including the additional 66 intermolecular distance restraints. The second procedure comprised a single step, in which a random-coil protein and a typical B-form DNA duplex were first placed 70 Å from each other. Then, using all the intramolecular and intermolecular NOE distance restraints, the complex structure was constructed by 4D-SA. Both procedures yielded the converged complex structures with similar quality and structural divergence, but the multiple-step procedure has much better convergence power than the single-step procedure. A model study of the two procedures was performed to confirm the structural quality, depending upon the number of intermolecular distance restraints, using the X-ray structure of the engrailed homeodomain-DNA complex.Abbreviations rmsd root-mean-square deviation - NOE nuclear Overhauser enhancement - 4D-SA simulated annealing in four-dimensional space - Myb-R2R3 repeats 2 and 3 of the DNA-binding domain of the c-Myb protein - DNA 16 Myb-specific binding DNA duplex with 16 base pairs - IHDD-C residues 3 to 59 of the C-chain of the engrailed homeodomain-DNA complex - DNA11 DNA duplex with base pairs 9 to 19 of the engrailed homeodomain-DNA complex     

Enhancement of penicillin acylase activity by cultivating immobilized Kluyvera citrophila

Summary Whole cells of Kluyvera citrophila were immobilized in polyacrylamide gel. The penicillin acylase activity of immobilized whole cells was 60%–70% of native cells. When the immobilized cells were continuously cultivated for 40 h in an aerated fermentor containing peptone medium and were treated with alkali in order to remove -lactamase activity, the immobilized cells produced ampicillin up to 4.4 times faster than noncultivated cells.Ampicillin production was investigated in a column system using these cultivated immobilized whole cells. The cultivated immobilized cells showed excellent performance in continuous ampicillin production.     

Infrared analysis of pea stem cell walls and oriented structure of matrix polysaccharides in them

Infrared absorption spectra of film specimens of the epidermaland parenchyma cell walls of the third internode of pea stem,before and after protease treatment and after treatment forremoval of lipid materials, pectic substances and hemicellulose,were recorded, and characteristic bands in the spectrum of thewall were assigned. Polarization spectrum measurements of thewall provided evidence indicating that the non-cellulosic polysaccharidematrix as well as cellulose microfibrils has an oriented structurein the wall which changes during extension growth as well asupon mechanical extension of the walls. (Received March 9, 1978; )     

Effect of auxin on changes in the oriented structure of wall polysaccharides in response to mechanical extension in oat coleoptile cell walls

The increase in the dichroism of the ester-C=O and COO^–bands in response to mechanical extension in oat coleoptilecell walls was much enhanced by IAA pretreatment while the decreasein that of the C-O-C band was not enhanced or even suppressed.The results indicate the importance of the ultrastructure ofnon-cellulosic polysaccharides in controlling auxin-inducedcell elongation. ¹ Present adress: Department of Agricultural Chemistry, KyotoPrefectural University, Shimogamo, Kyoto 606, Japan. (Received May 16, 1978; )     

Glutamyl-tRNA synthetase from Thermus thermophilus HB8. Molecular cloning of the gltX gene and crystallization of the overproduced protein.

The gene for the Glu-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8, was isolated using a synthetic oligonucleotide probe coding for the N-terminal amino acid sequence of Glu-tRNA synthetase. Nucleotide-sequence analysis revealed an open reading frame coding for a protein composed of 468 amino acid residues (Mr 53,901). Codon usage in the T. thermophilus Glu-tRNA synthetase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus: the G + C content in the third position of the codons was as high as 94%. In contrast, the amino acid sequence of T. thermophilus Glu-tRNA synthetase showed high similarity with bacterial Glu-tRNA synthetases (35-45% identity); the sequences of the binding sites for ATP and for the 3' terminus of tRNA(Glu) are highly conserved. The Glu-tRNA synthetase gene was efficiently expressed in Escherichia coli under the control of the tac promoter. The recombinant T. thermophilus Glu-tRNA synthetase was extremely thermostable and was purified to homogeneity by heat treatment and three-step column chromatography. Single crystals of T. thermophilus Glu-tRNA synthetase were obtained from poly(ethylene glycol) 6000 solution by a vapor-diffusion technique. The crystals diffract X-rays beyond 0.35 nm. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters of a = 8.64 nm, b = 8.86 nm and c = 8.49 nm.