Optimizing the production of transformed pea (Pisum sativum L.) callus using disarmed Agrobacterium tumefaciens strains

For optimization of the transformation procedure with Pisum sativum L. stern explant callus was used to test the effect of disarmed Agrobacterium tumefaciens strains, cocultivation procedures (preconditioning of explants; use of Nicotiana tabacum L. nurse cultures), duration of cocultivation (2, 3 or 4 days), and agents for selection (kanamycin or hygromycin). The succinamopine strain EHA101(pBI1042) produced the highest percentage of transformed calli (77%) when used in conjunction with tobacco nurse culture during four days of cocultivation. Using this strain, kanamycin (76%) and hygromycin (77%) were equally effective selective agents, but for strain LBA4404(pBI1042) percentage of transformed calli was higher for hygromycin (63%) than for kanamycin (17%). The procedures and strains shown to be optimal for transformation of pea callus will now be complemented by a pea regeneration system.     

The sequence of the 5.8 S ribosomal RNA of the crustacean Artemia salina. With a proposal for a general secondary structure model for 5.8 S ribosomal RNA.

We report the primary structure of 5.8 S rRNA from the crustacean Artemia salina. The preparation shows length heterogeneity at the 5'-terminus, but consists of uninterrupted RNA chains, in contrast to some insect 5.8 S rRNAs, which consist of two chains of unequal length separated in the gene by a short spacer. The sequence was aligned with those of 11 other 5.8 S rRNAs and a general secondary structure model derived. It has four helical regions in common with the model of Nazar et al. (J. Biol. Chem. 250, 8591-8597 (1975)), but for a fifth helix a different base pairing scheme was found preferable, and the terminal sequences are presumed to bind to 28 S rRNA instead of binding to each other. In the case of yeast, where both the 5.8 S and 26 S rRNA sequences are known, the existence of five helices in 5.8 S rRNA is shown to be compatible with a 5.8 S - 26 S rRNA interaction model.     

Amino acid sequence determination of the ADP,ATP carrier from beef heart mitochondria. The sequence of the C-terminal acidolytic fragment

The primary structure of the C-terminal region (94 residues) of the ADP,ATP carrier of beef heart mitochondria is described. CNBr cleavage results in a large peptide (CB1) with Mr 22 000 and several small peptides (CB2 to CB8). Peptide separation was achieved by gel chromatography with 80% formic acid or with an ethanol/formic acid mixture. The amino acid sequence of the small CNBr peptides was determined by solid-phase techniques. Hydrolysis in formic acid cleaves the carrier protein into an Mr 23 000 fragment (A1) with the blocked N-terminus and an Mr 10 000 fragment (A2) starting with proline. The alignment of two CNBr fragments was possible by degradation of A2 by solid-phase methods for 34 steps. The remaining CNBr fragments were arranged by sequencing the tryptic peptides of citraconylated A2.     

Amino acid sequence of the N-terminal non-collagenous segment of dermatosparactic sheep procollagen type I.

The non-collagenous N-terminal segment of type I procollagen from dermatosparactic sheep skin was isolated in the form of the peptide Col 1 from a collagenase digest of the protein. The peptide has a blocked N-terminus, which was identified as pyrrolid-2-one-5-carboxylic acid. Appropriate overlapping fragments were prepared from reduced and alkylated peptide Col 1 by cleavage with trypsin at lysine, arginine and S-aminoethyl-cysteine residues and by cleavage with staphylococcal proteinase at glutamate residues. Amino acid sequence analysis of these fragments by Edman degradation and mass spectrometry established the whole sequence of peptide Col 1 except for a peptide junction (7--8) and a single Asx residue (44), and demonstrated that peptide Col 1 consists of 98 amino acid residues. The N-terminal portion of peptide Col 1 (86 residues) shows an irregular distribution of glycine, whereas the C-terminal portion (12 residues) possesses the triplet structure Gly-Xy and is apparently derived from the precursor-specific collagenous domain of procollagen. The central region of the peptide contains ten cysteine residues located between positions 18 and 73 and shows alternating polar and hydrophobic sequence elements. The regions adjacent to the cysteine-rich portion have a hydrophilic nature and are abundant in glutamic acid. The data are consistent with previous physicochemical and immunological evidence that distinct regions at the N- and C-termini of the non-collagenous domain possess a less rigid conformation than does the central portion of the molecule.     

Lumi-mestranol and epi-lumi-mestranol.

Treatment of lumi-estrone 3-methyl ether (I) with acetylene gave the C-17-epimeric compounds lumi-mestranol (3-methoxy-17 alpha-ethynyl-13 alpha-estra-1,3,5(10)-trien-17 beta-ol, III ) and epi-lumi-mestranol (3-methoxy-17 beta-ethynyl-13 alpha-estra-1,3,5(10)-trien-17 alpha-ol, IV). The structures of the two isomers were assigned on the basis of their molecular rotations and shift-reagent experiments in the NMR. The irradiation of estrone 3-methyl ether (II) to provide compound I was investigated in two solvent systems. Minor products of these reactions were the seco-steroids VII, VIII and X.     

The MRN-CtIP Pathway Is Required for Metaphase Chromosome Alignment

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Highlights? The MRN-CtIP pathway is required for metaphase chromosome alignment in egg extracts ? MRN inhibition interferes with spindle assembly around DNA-coated beads ? MRN inhibition in cells causes a metaphase delay and disrupts the RanGTP gradient ? The MRN complex regulates the stable association of RCC1 to chromatin