Caffeine inhibition of cytokinesis: Ultrastructure of cell plate formation/degradation

Summary Caffeine is a potent inhibitor of cell plate formation in dividing plant cells. Previous studies living cells reveal that the drug always permits the cell plate to arise and grow normally until about 80% complete, but then causes it to break down. In the present investigation we examine this formation/degradation cycle at the ultrastructure level. Our results show that during the formation phase the caffeine treated plate is indistinguishable from untreated controls. Phragmoplast microtubules arise and align in the interzone, Golgi vesicles are produced and aggregate in a line that defines the young cell plate, and considerable fusion of these vesicles occurs to form islands of plate material. However, under the influence of caffeine these islands do not fuse to form the enlarged lamellar expanses characteristic of maturing cell plates. Instead, the partially fused material reverts to small vesicles which appear to become resorbed by the cellular membrane systems. The resorption process continues leaving no evidence of the previously developing plate, although occasionally we observe a stub of fused vesicles attached to the parent wall. Following cell plate disintegration the reformed nuclei move close together and occupy the central region of the cell. These observations focus attention on the consolidation phase of cell plate formation as the one being maximally affected by caffeine.Dedicated to the memory of Professor Oswald Kiermayer     

Mutation analysis of the cystic fibrosis transmembrane regulator gene in native American populations of the southwest

We report DNA and clinical analyses of cystic fibrosis (CF) in two previously unstudied, genetically isolated populations: Pueblo and Navajo Native Americans. Direct mutation analysis of six mutations of the CFTR gene--namely, delta F508, G542X, G551D, R553X, N1303K, and W1282X--was performed on PCR-amplified genomic DNA extracted from blood samples. Haplotype analyses with marker/enzyme pairs XV2c/TaqI and KM19/PstI were performed as well. Of the 12 affected individuals studied, no delta F508 mutation was detected; only one G542X mutation was found. None of the other mutations was detected. All affected individuals have either an AA, AC, or CC haplotype, except for the one carrying the G542X mutation, who has the haplotype AB. Clinically, six of the affected individuals examined exhibit growth deficiency, and five (all from the Zuni Pueblo) have a severe CF phenotype. Four of the six Zunis with CF are also microcephalic, a finding not previously noted in CF patients. Our DNA data have serious implications for risk assessment of CF carrier status for these people.     

The cumulative effect of wetlands on stream water quality and quantity. A landscape approach

A method was developed to evaluate the cumulative effect of wetland mosaics in the landscape on stream water quality and quantity in the nine-county region surrounding Minneapolis—St. Paul, Minnesota. A Geographic Information System (GIS) was used to record and measure 33 watershed variables derived from historical aerial photos. These watershed variables were then reduced to eight principal components which explained 86% of the variance. Relationships between stream water quality variables and the three wetland-related principal components were explored through stepwise multiple regression analysis. The proximity of wetlands to the sampling station was related to principal component two, which was associated with decreased annual concentrations of inorganic suspended solids, fecal coliform, nitrates, specific conductivity, flow-weighted NH₄ flow-weighted total P, and a decreased proportion of phosphorus in dissolved form(p < 0.05). Wetland extent was related to decreased specific conductivity, chloride, and lead concentrations. The wetland-related principal components were also associated with the seasonal export of organic matter, organic nitrogen, and orthophosphate. Relationships between water quality and wetlands components were different for time-weighted averages as compared to flow-weighted averages. This suggests that wetlands were more effective in removing suspended solids, total phosphorus, and ammonia during high flow periods but were more effective in removing nitrates during low flow periods.     

INFERENCES ABOUT QUANTITATIVE INHERITANCE BASED ON NATURAL POPULATION STRUCTURE IN THE YELLOW MONKEYFLOWER,MIMULUS GUTTATUS

We used a nonmanipulative, marker-based method to study quantitative genetic inheritance in two habitats of a common monkeyflower population. The method involved regressing quantitative trait similarity on marker-estimated relatedness between individuals sampled in the field. We sampled 300 adult plants from each of two transects, one along a stream habitat and another through a meadow habitat. For each plant we measured 10 quantitative characters and assayed 10 polymorphic isozyme loci. In the meadow habitat, relatedness of plants within 1 m was moderate (r = 0.125, corresponding to half-sibs) as was actual variance of relatedness (V_r = 0.044). Significant heritabilities of 50–70% were found for corolla width and the fitness characters of flower number and plant weight. Genetic correlations were strongly positive, but sharing of environmental effects within 1 m was weak. In the stream habitat, levels of relatedness were lower and similar heritabilities were indicated. To detect dominance variance and the correlation of phenotypes due to shared inbreeding, we also estimated higher-order coefficients of relationship and inbreeding, but these did not significantly differ from zero. Laboratory-based estimates of heritability in the field were lower than the marker-based estimates, indicating that natural heritabilities and genetic correlations may be stronger than indicated by controlled studies.     

Sperm motility initiation factor is a minor component of the Pacific herring egg chorion

Polyclonal antibodies were generated to the 105 kDa herring sperm motility initiation factor (SMIF) and used to explore the role of SMIF in sperm-egg interaction. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting with SMIF antibodies, it was demonstrated that SMIF is present as a minor (4–7% of total chorion protein) component of the chorion. The major polypeptides in the chorion migrated at 117 kDa and in a grouping between 48–54 kDa, with other minor bands above and below. The only detectable glycosylated component was the 105 kDa band, which was resolved at two isoelectric points (8.22 and 8.31) after isoelectric focusing gel electrophoresis. Using antibodies to SMIF, fertilization was blocked, sperm motility was inhibited in vitro in the presence of solubilized SMIF and SMIF binding sites on sperm were localized. Lastly, SMIF was localized to the region of the herring egg that encircles the micropyle.     

Eph Family Receptors and Their Ligands Distribute in Opposing Gradients in the Developing Mouse Retina

The Eph family of receptor tyrosine kinases and their ligands can be divided into two specificity subclasses: the Eck-related receptors and their GPI-anchored ligands, and the Elk-related receptors and their transmembrane ligands. Previous reports demonstrated that Eck- and Elk-related receptors in the retina distribute in high temporal–low nasal and high ventral–low dorsal gradients, respectively. While others have focused on complementary ligand gradients in the retinal axon target, the tectum, we report that ligands from each subclass also distribute in gradients opposing those of their corresponding receptors within the retina itself. Moreover, ligand gradients in the retina precede ganglion cell genesis. These results support an intraretinal role for Eph family members in addition to their previously proposed role in the development of retinotectal topography. The distinct distributions of Eph family members suggest that each subclass specifies positional information along independent retinal axes.     

Construction of hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and non-transformableNeisseria mutants

A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, orori _fd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the genetic manipulation of pathogenicNeisseriae are described.     

EagI andNotI linking clones from human chromosomes 11 and Xp

EagI and NotI linking libraries were prepared in the lambda vector, EMBL5, from the mouse-human somatic cell hybrid 1W1LA4.9, which contains human chromosomes 11 and Xp as the only human component. Individual clones containing human DNA were isolated by their ability to hybridise with total human DNA and digested with SalI and EcoRI to identify the human insert size and single-copy fragments. The mean (± SD) insert sizes of the EagI and NotI clones were 18.3 ± 3.2 kb and 16.6 ± 3.6 kb, respectively. Regional localisation of 66 clones (52 EagI, 14 NotI) was achieved using a panel of 20 somatic cell hybrids that contained different overlapping deletions of chromosomes 11 or Xp. Thirty-nine clones (36 EagI, 3 NotI) were localised to chromosome 11; 17 of these were clustered in 11q13 and another nine were clustered in 11q14–q23.1. Twenty-seven clones (16 EagI, 11 NotI) were localised to Xp and 10 of these were clustered in Xp11. The 66 clones were assessed for seven different microsatellite repetitive sequences; restriction fragment length polymorphisms for five clones from 11q13 were also identified. These EagI and NotI clones, which supplement those previously mapped to chromosome 11 and Xp, should facilitate the generation of more detailed maps and the identification of genes that are associated with CpG-rich islands. Received: 27 December 1995 / Revised: 30 January 1996