Obtaining carrot (Daucus carota L.) plants in isolated microspore cultures.

Microspores were cultured on the modified B5 liquid medium containing 2.4D (0.1 mg L(-1)), NAA (0.1 mg L(-1)), L-glutamine (500 mg L(-1), L-serine (100 mg L(-1)), and sucrose (100 g L(-1)). The developmental stages of microspores and divisions were observed. Initially, the formation of binuclear and multicellular structures was noticed. Plants regenerated in the cultures in which the tetrad stage of microsporogenesis had predominated. Embryoids were still forming 24 weeks after the cultures were set up. Six weeks after the transfer of androgenetic embryos onto the B5 regeneration medium, they were converted into complete plants. Out of 90 androgenetic plants planted in a growth chamber, 42 plants adapted to the new conditions. All of those plants proved to be diploids in cytometric analysis.

Juvenile hormone binding protein and transferrin from Galleria mellonella share a similar structural motif.

It has been previously suggested that juvenile hormone binding protein(s) (JHBP) belongs to a new class of proteins. In the search for other protein(s) that may contain structural motifs similar to those found in JHBP, hemolymph from Galleria mellonella (Lepidoptera) was chromatographed over a Sephadex G-200 column and resulting fractions were subjected to SDS-PAGE, transferred onto nitrocellulose membrane and scanned with a monoclonal antibody, mAb 104, against hemolymph JHBP. Two proteins yielded a positive reaction with mAb 104, one corresponding to JHBP and the second corresponding to a transferrin, as judged from N-terminal amino acid sequencing staining. Transferrin was purified to about 80% homogeneity using a two-step procedure including Sephadex G-200 gel filtration and HPLC MonoQ column chromatography. Panning of a random peptide display library and analysis with immobilized synthetic peptides were applied for finding a common epitope present in JHBP and the transferrin molecule. The postulated epitope motif recognized by mAb 104 in the JHBP sequence is RDTKAVN, and is localized at position 82-88.

UV-difference and CD spectroscopy studies on juvenile hormone binding to its carrier protein.

Juvenile hormone binding protein (JHBP) from hemolymph of Galleria mellonella is a single-chain glycoprotein of molecular mass near 25,880 containing no Trp residues. The fourth derivative of the protein absorption spectrum shows the characteristic vibrational components of the phenylalanine spectrum within the range 240-270 nm. At longer wavelengths two main bands 0-0 and 0+800 cm(-1) appear, caused by vibrational levels of electronic transition, pi-->pi*, in the tyrosine residues, with maxima at 279 nm and 286 nm, respectively. Two intersection points of the second derivative absorption band with abscissa at about 288.8 nm and 283 nm were analysed for estimation of the environment polarity of Tyr residues in the JHBP molecule. The results obtained suggest that JHBP contains at least two classes of Tyr residues with very apolar environment, similar to that found in azurine. In the JHBP-JH complex only one class of Tyr residues located in a very apolar environment was found, and a small perturbation of disulphide bridges was deduced from the UV-difference spectrum. Ligand perturbation appears as a minimum at 243 nm of the UV-difference spectrum. Comparison of the circular dichroism (CD) spectra for free JHBP with the CD spectra for the JHBP-JH complex monitored in the far-UV (190-240 nm) region indicates rather small differences in the secondary structure of the protein. Although ligand binding induces distinct changes in the near-UV (250-300 nm) region of the CD spectrum of JHBP, it is apparent where both Tyr and Phe residues contribute.