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SUMMARY The green macroalgal genus Ulva (incl. Entemmorpha) contains economically valuable species, is of relevance for coastal management (green tides), and certain taxa serve as experimental organisms for fundamental research in green algae. The nuclear genome size of Ulva (Entemmorpha) compressa Linnaeus was measured in propidium iodide stained nuclei using laser scanning cytometry. Nuclei of fixed gametes yielded reproducible values, whereas nuclei extracted from multicellular gametophytes were unsuitable. With nuclei of Arabidopsis thaliana (L.) Heynh and Saccharomyces cerevisiae Hansen as references, the haploid nuclear genome size of U. compressa was calculated as 135 ± 7 Mbp. This is the smallest genome so far known from any species of Ulva.

Two FtsZ paralogues (NbFtsZ1 and NbFtsZ2) were isolated from the unicellular green alga Nannochloris bacillaris Naumann. These sequences encoded proteins of 435 and 439 amino acids with tubulin signature motifs (GGGTG[T/S]G), which are important for GTP binding activity. NbFtsZ1 and NbFtsZ2 had four and three introns, respectively, and two different putative core promoters; a TATA box (TATAAAA) and an initiator element (CCCAGG) were located 40 bp and 80 bp upstream of the coding regions of NbFtsZ1 and NbFtsZ2, respectively. Southern blot hybridization and contour-clamped homogeneous electric field electrophoresis showed that N. bacillaris contained at least one copy of each gene and that NbFtsZ1 was located on chromosome 5 and NbFtsZ2 on chromosome 3 or 4. Phylogenetically, NbFtsZ1 and NbFtsZ2 belong to the vascular plant protein families FtsZ1 and FtsZ2, respectively. The FtsZ1 proteins do not contain carboxy-terminal consensus sequences, whereas all FtsZ2 proteins possess the consensus sequence (I/V)PxFL(R/K)(K/R)(K/R). Our study has shown that NbFtsZ2 possesses a similar consensus sequence (VPDFLRRK), whereas NbFtsZ1 does not, further supporting their classification as FtsZ2 and FtsZ1. Escherichia coli ftsZ mutants transformed with cloned NbFtsZ1, and NbFtsZ2 cDNAs were restored for the capacity to divide by binary fission, suggesting that the proteins retained the ability to function in the bacterium. An anti-NbFtsZ2 antibody specifically recognized a single protein band of approximately 51 kDa on an immunoblot of N. bacillaris cellular proteins. Immunostaining of the algal cells with this antibody produced an intense fluorescent signal as a ring near the middle of the cell, which corresponded to the chloroplast division site.

In the haploid dioecious liverwort, Marchantia polymorpha, the X chromosome, but not the Y, carries a cluster of ribosomal RNA genes (rDNAs). Here we show that sequences of 5S, 17S, 5.8S and 26S rDNAs are highly conserved (>99% identity) between the X chromosomal and autosomal rDNA repeat units, but the intergenic spacer sequences differ considerably. The most prominent difference is the presence of a 615-bp DNA fragment in the intergenic spacer, X615, which has accumulated predominantly in the rDNA cluster of the X chromosome. These observations suggest that the rDNA repeat unit on the X chromosome evolved independently of that on autosomes, incorporating sex chromosome-specific sequences.

The molecular cytogenetic organization of 17S ribosomal RNA genes (17S rDNA), a part of the 45S rDNA repeat, was investigated on the chromosomes of the liverwort Marchantia polymorpha using fluorescence in-situ hybridization (FISH). The numbers of 17S rDNA loci visualized in female and male chromosomes were ten and nine, respectively. This heterogeneous localization was due to the presence of an additional 17S rDNA locus on the X chromosome and its absence on the Y chromosome. The signal on the X chromosome covered almost the entire region of its long arm. The other nine signals were observed on the same loci of respective autosomes in both sexes. Southern hybridization analysis revealed an additional band including 17S rDNA exclusively on EcoRI digested female genomic DNA supporting the existence of an additional 17S rDNA locus on the X chromosome.

Marine Biology and Ecology Research Program, Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology, Yokosuka, Kanagawa 236-0061, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8562, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, School of Marine Sciences and Technology, Tokai University, Shimizu-Orido, Shizuoka 424-8610, and Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan

Y chromosomes are different from other chromosomes because of a lack of recombination. Until now, complete sequence information of Y chromosomes has been available only for some primates, although considerable information is available for other organisms, e.g., several species of Drosophila . Here, we report the gene organization of the Y chromosome in the dioecious liverwort Marchantia polymorpha and provide a detailed view of a Y chromosome in a haploid organism. On the 10-Mb Y chromosome, 64 genes are identified, 14 of which are detected only in the male genome and are expressed in reproductive organs but not in vegetative thalli, suggesting their participation in male reproductive functions. Another 40 genes on the Y chromosome are expressed in thalli and male sexual organs. At least six of these genes have diverged X-linked counterparts that are in turn expressed in thalli and sexual organs in female plants, suggesting that these X- and Y-linked genes have essential cellular functions. These findings indicate that the Y and X chromosomes share the same ancestral autosome and support the prediction that in a haploid organism essential genes on sex chromosomes are more likely to persist than in a diploid organism.

Cell-wall synthesis in Chlorella vulgaris, an autospore-forming alga, was observed using the cell wall-specific fluorescent dye Fluostain I. The observation suggested two clearly distinguishable stages in cell-wall synthesis: moderate synthesis during the cell-growth process and rapid synthesis at the cell-division stage. We used electron microscopy to examine the structural changes that occurred with growth in the premature daughter cell wall during the cell-growth and cell-division phases. The cell began to synthesize a new daughter cell wall shortly after its release from the autosporangium. A very thin daughter cell wall, with a thickness of about 2 nm, was formed inside the mother cell wall and completely enveloped the outer surface of the plasma membrane of the cell. The daughter cell wall gradually increased in thickness from 2 to 3.8 nm. During the protoplast-division phase in the cell-division stage, the daughter cell wall expanded on the surface of the invaginating plasma membrane of the cleavage furrow, accompanied by active synthesis of the cell wall, which increased in thickness from 3.8 to 6.1 nm. The daughter cell matured into an autospore while completely enclosed by its own thickening (from 6.1 to 17 nm) wall. Finally, the released daughter cell was enclosed by its own cell wall after the mother cell wall burst. The daughter cell with mature wall thickness (17-21 nm) emerged as a small, but complete, autospore.

In the haploid dioecious liverwort, Marchantia polymorpha, the X chromosome, but not the Y, carries a cluster of ribosomal RNA genes (rDNAs). Here we show that sequences of 5S, 17S, 5.8S and 26S rDNAs are highly conserved (99% identity) between the X chromosomal and autosomal rDNA repeat units, but the intergenic spacer sequences differ considerably. The most prominent difference is the presence of a 615-bp DNA fragment in the intergenic spacer, X615, which has accumulated predominantly in the rDNA cluster of the X chromosome. These observations suggest that the rDNA repeat unit on the X chromosome evolved independently of that on autosomes, incorporating sex chromosome-specific sequences.

The temporal organization of secretion of the prothoracicotropic hormone (PTTH) and ecdysone during larval-pupal development of Samia cynthia ricini was studied by ligations, with particular attention to the circadian control of the timing of hormone release. PTTH and ecdysone are required first for the induction of prodromes of pupation and again later for pupal-cuticle formation. PTTH release in the first step occurs during the second or third photophase after the last-larval ecdysis under a photoperiod of 12 hr light and 12 hr darkness and is thought to be under the control of a circadian clock. Ecdysone release follows 1.5 days later, i.e. during the scotophase that precedes the gut purge. In the second secretory step, PTTH is released 2 days after purging the gut, and ecdysone release follows 6 hr later. The PTTH release at this time occurs at a fixed time after the gut purge irrespective of light conditions, accounting for light insensitivity of the timing for pupal ecdysis. Possible mechanisms relating to the inconsistent association of a circadian clock with PTTH release, and those underlying the determination of timing of the gut purge are discussed.

Under a 12-hr light and 12-hr dark photoperiod, haemolymph ecdysteroid titre of the last(5th)-instar larva of Samia cynthia ricini begins to rise in the early scotophase preceding gut purge, which marks the larval-prepupal transition, to reach a peak titre of 7.6 ng/ml ca. 4.5 hr before gut purge. This profile of ecdysteroid increment is phase-shifted in response to phase shifts of the scotophase immediately before gut purge, in parallel with gut-purge phase shifts, suggesting that ecdysone release is under the control of a circadian clock dictating gut-purge timing. Ecdysone and 20-hydroxyecdysone (10–20 μg), injected no later than 18 hr before the normal gate of gut purge induced well-defined peaks of precocious gut purge ca. 8 hr after injection. Earlier injections caused acceleration of gut purge but the degree of acceleration was unpredictable. These results suggest that the timed surge of ecdysteroids is responsible for the gated occurrence of gut purge and that 18 hr before gut purge larvae acquire the competence to undergo gut purge in a gated fashion provided that they are exposed to a sufficient surge of ecdysteroids.

Crude extracts of Bombyx mori brains can provoke adult development when injected into brain-removed dormant pupae of Bombyx mori and Samia Cynthia ricini. From this fact the prothoracicotropic hormone (PTTH) of Bombyx has long been thought to be species-nonspecifically active on Samia. Chemical fractionation of Bombyx brain or head extracts by fractional precipitation with acetone, Sephadex G-50 gel-filtration, and DEAE-Sepharose CL-6B chromatography, however, separated the fractions which activated Bombyx brainless pupae from those which activated Samia. Those results reveal the existence of two species-specific PTTHs.

Observations on the timing of ecdysis and neck ligation experiments on larvae of Samia cynthia ricini under various light-dark conditions show that an endogenous circadian clock controls the timing of larval ecdysis and prothoracicotropic hormone (PTTH) secretion preceding it. The clock, upon reaching a specific phase point, causes the brain to secrete PTTH provided that the brain has acquired the secretory competence. This time may vary, in relation to a previous ecdysis, according to the light-dark conditions by which the clock phase is specifically determined, but is fixed relative to a subsequent ecdysis. Thus, in the case of the ecdysis to the 5th instar, PTTH is secreted [15+nτ] hr (τ: free-running period, slightly less than 24 hr) after the clock has started when the rhythm is free-running, and in the second and third nights of the 4th instar under a photoperiod of 12 hr light and 12 hr dark. Full secretion of ecdysone occurs 6 hr after PTTH secretion and ecdysis ensues 34 hr thereafter to complete the ultimate sequence of ecdysis.

Blockage of adult development by brain removal and its resumption by application of the prothoracicotropic hormone (PTTH) were studied using pupae of a racial hybrid J-122 × C-115 of Bombyx mori. A log-linear dose-response relationship was obrained after injection of a PTTH solution. The Bombyx-unit of PTTH has been defined from this dose-response curve.

The prothoracicotropic hormone (PTTH) was isolated from adult heads of Bombyx mori . Fifty micrograms of pure PTTH was obtained from 648,000 heads through a 15-step purification procedure with a 2 × 10 6 -fold purification and an 8% recovery. Chemical analyses of this PTTH have shown that it is a single-chain peptide consisting of 40–43 amino acid residues (MW, 4330–4740), the N-terminus of which is glycine. As little as 0.1 ng of PTTH elicited adult development in a debrained pupa of Samia cynthia ricini . Five picograms of PTTH directly stimulated the prothoracic glands in vitro so as to enhance ecdysone release. The hemolymph ecdysteroids of brainless Samia pupae that were developed by PTTH injection increased with essentially the same pattern as in developing normal pupae.