Triplex configuration in the nick-free DNAs that constitute the chromosomal scaffolds in grasshopper spermatids.

After applying proper deoxyribonucleic acid (DNA) probes, fluorescence in situ hybridization (FISH) showed that the 8/9 centromeres-one per chromatid of the male haploid complement (X0) of Pyrgomorpha conica grasshopper-colocalized at the spermatid blunt end, where the spermatozoa flagellum inserts. A bundle of aligned 4',6-diamidino-2-phenylindole-positive chromatid scaffolds, which formed the central spermatid core, was observed after DNA breakage detection followed by FISH. Modular nature of scaffold DNA was occasionally evident. The technique also showed that in the early spermatid, the chromatid scaffolds lacked any DNA nick, whereas abundant breaks accumulated in the surrounding loops. Moreover, immunodetection showed that scaffold DNA participated in the formation of triplex DNA, while this configuration was absent from the loops. During spermatid maturation, triplex DNA disappeared from the scaffold in parallel with loop retraction, while protamines replace histones. Thus, the presence of triplex DNA in the chromatid scaffold correlates with the anchoring of expanded DNA loops to it. After loop retraction, the scaffolds of all chromatids coiled as a single unit in the spermatid head. This cooperative coiling produced enlargement and tilting of the distal telomeric signals, which were distributed along the spermatid head according to the length of each chromosome. We propose that specific DNA sequences dispersed throughout the whole chromatid fold forward and backward coaxially to chromatid length, forming individual scaffold modules whose linear assembly accounts for the minimum length of each individual chromatid. Finally, the core of the grasshopper male spermatid should be considered as a single chromosome in which the DNA scaffolds of the whole set of the nonhomologous chromosomes of the haploid complement are interconnected. This pattern of chromatin organization applies probably to other elongated spermatids.

Do phytotropins inhibit auxin efflux by impairing vesicle traffic?

Phytotropins such as 1-N-naphthylphthalamic acid (NPA) strongly inhibit auxin efflux, but the mechanism of this inhibition remains unknown. Auxin efflux is also strongly decreased by the vesicle trafficking inhibitor brefeldin A (BFA). Using suspension-cultured interphase cells of the BY-2 tobacco (Nicotiana tabacum L. cv Bright-Yellow 2) cell line, we compared the effects of NPA and BFA on auxin accumulation and on the arrangement of the cytoskeleton and endoplasmic reticulum (ER). The inhibition of auxin efflux (stimulation of net accumulation) by both NPA and BFA occurred rapidly with no measurable lag. NPA had no observable effect on the arrangement of microtubules, actin filaments, or ER. Thus, its inhibitory effect on auxin efflux was not mediated by perturbation of the cytoskeletal system and ER. BFA, however, caused substantial alterations to the arrangement of actin filaments and ER, including a characteristic accumulation of actin in the perinuclear cytoplasm. Even at saturating concentrations, NPA inhibited net auxin efflux far more effectively than did BFA. Therefore, a proportion of the NPA-sensitive auxin efflux carriers may be protected from the action of BFA. Maximum inhibition of auxin efflux occurred at concentrations of NPA substantially below those previously reported to be necessary to perturb vesicle trafficking. We found no evidence to support recent suggestions that the action of auxin transport inhibitors is mediated by a general inhibition of vesicle-mediated protein traffic to the plasma membrane.