Ambiguities in results obtained with 2D gel replicon mapping techniques.

Recently, two 2-dimensional (2D) gel techniques, termed neutral/neutral and neutral/alkaline, have been developed and employed to map replication origins in eukaryotic plasmids and chromosomal DNA (1-11). The neutral/neutral technique, which requires less DNA for analysis, has been preferentially used in recent studies. We show here that the signal predicted for an origin is not detected using the neutral/neutral technique if the origin is located near the end of the analyzed restriction fragment. We also demonstrate that analysis of the same batch of DNA by the two different mapping techniques can generate apparently contradictory results: in some situations where neutral/alkaline 2D analysis indicates that a certain origin is always used, neutral/neutral 2D analysis suggests that the origin is not always used. Several possible explanations for this type of disagreement between the two techniques are discussed, and we conclude that it is important to use both techniques in combination in order to minimize possible misinterpretations.     

Cell-wall proteins in pollen and roots of Lilium longiflorum: extraction and partial characterization

Cell-wall proteins of pollen grains, in-vitro-germinated pollen and young roots of Lilium longiflorum were studied by gel electrophoresis and amino-acid analysis. The proteins were removed from extensively purified walls by successive saline and alkali extractions. The major part including the hydroxyproline-containing proteins is covalently bound to the wall. Clear differences were observed between the proteins, especially the glycoproteins, of the pollen grain and the pollen tube. During elongation of the tubes some proteins decrease in quantity and many new proteins appear. The amount of protein in the cell walls is much lower in roots than in pollen and the root cell walls also contain fewer glycoproteins.     

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Distribution of unesterified and esterified pectins in cell walls of pollen tubes of flowering plants

Immunocytochemical localization of polygalacturonic acid (pectin) and methyl-esterified pectin in the walls of pollen tubes of 20 species of flowering plants grown in vitro was investigated by using monoclonal antibodies (MAbs) JIM5 and JIM7 and by means of confocal laser scanning microscopy (CLSM). In general, periodic annular deposits of pectins were found coating the tube wall in species possessing solid styles, and a more uniform pectin sheath in tube walls in species having hollow styles or no styles. We hypothesize that the periodic ring-like structure of the pectin sheath reinforces pollen tubes for passing through the transmitting tract in the style. Esterified pectin which prevents Ca²⁺-induced gelification of pectate is located predominantly at the apex. This implies that pectin esterification is related to tip wall loosening that is required for cell wall expansion during tip growth of pollen tubes. The occurrence of unesterified pectins in other areas of pollen tube walls suggests that de-esterification of pectin following tip expansion leads to a more rigid form of pectin that contributes to the construction of the pollen tube wall.