Phyletic and evolutionary relationships ofBrachyscome lineariloba (Compositae)

A comparison of karyotypes ofBrachyscome breviscapis (2n = 8),B. lineariloba cytodemes E (2n = 10), B (2n = 12) and C (2n = 16) suggests that these species have a homoelogous basic set of four chromosome pairs, two large pairs and two small, and that theB. lineariloba cytodemes E, B and C are related toB. breviscapis by successive additions of small chromosomes. A pronounced asynchrony of chromosome condensation between these large and small chromosomes has been observed. In the artificial hybrids betweenB. dichromosomatica (2n = 4) ×B. breviscapis, and theB. lineariloba cytodemes, theB. dichromosomatica chromosomes are similar in size and condensation behaviour to the small chromosomes ofB. breviscapis and ofB. lineariloba cytodemes E, B and C. Meiotic pairing in these hybrids also demonstrates the strong affinities between these chromosomes. It is suggested thatB. breviscapis may be of amphidiploid origin between a species with two large early condensing chromosome pairs and another,B. dichromosomatica-like species with two small late condensing pairs. It seems most likely that the additional small and late condensing chromosomes inB. lineariloba cytodemes E, B and C are derived from theB. dichromosomatica-like parent, and that each addition increases vigour, fecundity and drought tolerance, allowing these cytodemes to colonize more open and arid environments. Transmission of the univalents in the quasidiploidB. lineariloba cytodeme E was verified as being via the pollen, and not via the embryo sacs.The cytology ofBrachyscome lineariloba (Compositae, Asteroidae), 10.     

Different types of plant chromatin associated with modified histones H3 and H4 and methylated DNA

Eukaryotic chromosomes are organized into two large and distinct domains, euchromatin and heterochromatin, which are cytologically characterized by different degrees of chromatin compaction during interphase/prophase and by post-synthesis modifications of histones and DNA methylation. Typically, heterochromatin remains condensed during the entire cell cycle whereas euchromatin is decondensed at interphase. However, a fraction of the euchromatin can also remain condensed during interphase and appears as early condensing prophase chromatin. 5S and 45S rDNA sites and telomere DNA were used to characterize these regions in metaphase and interphase nuclei. We investigated the chromosomal distribution of modified histones and methylated DNA in the early and late condensing prophase chromatin of two species with clear differentiation between these domains. Both species, Costus spiralis and Eleutherine bulbosa, additionally have a small amount of classical heterochromatin detected by CMA/DAPI staining. The distribution of H4 acetylated at lysine 5 (H4K5ac), H3 phosphorylated at serine 10 (H3S10ph), H3 dimethylated at lysine 4 or 9 (H3K4me2, H3K9me2), and 5-methylcytosine was compared in metaphase, prophase, and interphase cells by immunostaining with specific antibodies. In both species, the late condensing prophase chromatin was highly enriched in H4K5ac and H3K4me2 whereas the early condensing chromatin was very poor in these marks. H3K9me2 was apparently uniformly distributed along the chromosomes whereas the early condensing chromatin was slightly enriched in 5-methylcytosine. Signals of H3S10ph were restricted to the pericentromeric region of all chromosomes. Notably, none of these marks distinguished classical heterochromatin from the early condensing euchromatin. It is suggested that the early condensing chromatin is an intermediate type between classical heterochromatin and euchromatin.     

RRP20, a component of the 90S preribosome,is required for pre-18S rRNA processing in Saccharomyces cerevisiae

A strain of Saccharomyces cerevisiae, defective in small subunit ribosomal RNA processing, has a mutation in YOR145c ORF that converts Gly235 to Asp. Yor145c is a nucleolar protein required for cell viability and has been reported recently to be present in 90S pre-ribosomal particles. The Gly235Asp mutation in YOR145c is found in a KH-type RNA-binding domain and causes a marked deficiency in 18S rRNA production. Detailed studies by northern blotting and primer extension analyses show that the mutant strain impairs the early pre-rRNA processing cleavage essentially at sites A₁ and A₂, leading to accumulation of a 22S dead-end processing product that is found in only a few rRNA processing mutants. Furthermore, U3, U14, snR10 and snR30 snoRNAs, involved in early pre-rRNA cleavages, are not destabilized by the YOR145c mutation. As the protein encoded by YOR145c is found in pre-ribosomal particles and the mutant strain is defective in ribosomal RNA processing, we have renamed it as RRP20.     

Multi-step Loading of Human Minichromosome Maintenance Proteins in Live Human Cells

Once-per-cell cycle replication is regulated through the assembly onto chromatin of multisubunit protein complexes that license DNA for a further round of replication. Licensing consists of the loading of the hexameric MCM2–7 complex onto chromatin during G₁ phase and is dependent on the licensing factor Cdt1. In vitro experiments have suggested a two-step binding mode for minichromosome maintenance (MCM) proteins, with transient initial interactions converted to stable chromatin loading. Here, we assess MCM loading in live human cells using an in vivo licensing assay on the basis of fluorescence recovery after photobleaching of GFP-tagged MCM protein subunits through the cell cycle. We show that, in telophase, MCM2 and MCM4 maintain transient interactions with chromatin, exhibiting kinetics similar to Cdt1. These are converted to stable interactions from early G₁ phase. The immobile fraction of MCM2 and MCM4 increases during G₁ phase, suggestive of reiterative licensing. In late G₁ phase, a large fraction of MCM proteins are loaded onto chromatin, with maximal licensing observed just prior to S phase onset. Fluorescence loss in photobleaching experiments show subnuclear concentrations of MCM-chromatin interactions that differ as G₁ phase progresses and do not colocalize with sites of DNA synthesis in S phase.     

Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens

During early embryogenesis of the nematode Parascaris univalens (2n=2) the processes of chromatin diminution and segregation of the germ and somatic cell lineages take place simultaneously. In this study we analyzed the nucleolar cycle in early embryos, both in germinal and somatic blastomeres, by means of silver staining and antibodies against the nucleolar protein fibrillarin. We observed an identical nucleolar cycle in both types of blastomeres, hence, the chromatin diminution process has no effect on the nucleolar cycle of somatic blastomeres. We report the existence of outstanding differences between this cycle and those previously reported during early embryogenesis of other species. There is a true nucleolar cycle in early embryos that shows a peculiar nucleolar disorganization at prophase, and a preferential localization of prenucleolar bodies only on the euchromatic regions during nucleologenesis. Moreover, fibrillarin does not form a perichromosomal sheath in metaphase or anaphase holocentric chromosomes, probably owing to their special centromeric organization. The number and location of nucleolus organizer regions (NORs) in the chromosomal complement have been determined using silver impregnation, chromomycin A₃/distamycin A staining, and fluorescent in situ hybridization using an rDNA probe. There are only two NORs, one per chromosome, and these are lost in blastomeres after chromatin diminution. Moreover, the constant presence of two nucleoli in somatic blastomeres suggests that NORs are not affected during the fragmentation of euchromatic regions when this process occurs.     

Ribonuclease activities in bean roots

Vicia faba meristematic and elongating root cells (zones 0–4 and 10–20 mm) contained one nuclease (A₁) and four ribonucleases (A₂, A₃, C₁, C₂). When the overall activity of each enzyme was expressed per cell, the elongating cells contained 4-, 4-, 4-, 10- and 17-fold more activity than meristematic cells for A₁, C₁, C₂, A₂ and A₃, respectively.     

Effect of outer sphere anions on the structure and color of nitrosylpentaamminechromium complex

Three kinds of crystalline compounds containing the nitrosylpentaamminechromium complexes [Cr(NO)(NH₃)₅]²⁺(A) were obtained: chloride ACl₂ (red-orange), chloride perchlorate ACl(ClO₄) (brown), and perchlorate A(ClO₄)₂ (green). The cause of the color change of the complex A with the change of outer sphere anions was sought using X-ray structural data of ACl₂, ACl(ClO₄), and A(ClO₄)₂. Crystal data: ACl₂, orthorhombic, space group Cmcm, a=10.0236 (9) Å, b=9.098 (3) Å, c=10.357(1) Å, V=944.5 (5) ų, Z=4; ACl(ClO₄), tetragonal, space group P4/nmm, a=7.6986 (8) Å, c=9.9566(8) Å, V=590.1 (1) Å^3,Z=2; A(ClO₄)₂, orthorhombic, space group Pnma, a=15.760 (2) Å, b=11.480(2) Å, c=7.920 (2) Å, V=1432.9 (4) ų, Z=4. The complex cation in ACl₂ has a distorted octahedral structure with a linear CrNO moiety. The short CrN (nitrosyl) distance of 1.692 (7) Å indicates the presence of multiple bonding between the chromium atom and the nitrogen atom in the nitrosyl group. The interatomic distances and angles within the complex cations hardly change with the change of the counter anions, while the distances between the complex cations in each crystal increase in the order ACl₂<ACl(ClO₄)<A(ClO₄)₂. The bulky perchlorate anions seems to separate the complex cations, while smaller chloride anions are not large enough to separate them. The distance (3.213(5) Å) between O(NO) and N(NH₃ in the adjacent complex cation) is rather short in the crystal of ACl₂, and there are six hydrogen bonds, where the NO group is surrounded by four NH₃ ligands. The distance (4.002(5) Å) between O(NO) and N(NH₃) is much longer in the crystal of A(ClO₄)₂, indicating the presence of no hydrogen bonding. In the crystal of ACl(ClO₄) the distance (3.452(4) Å) between O(NO) and N(NH₃) is in between those of ACl₂ and A(ClO₄)₂. The presence of hydrogen bonding between O(NO) and N(NH₃ in the adjacent complex cation) seems to cause the color change with the change of outer sphere anions.     

Gibberellins and the Legume-Rhizobium Symbiosis : I. Endogenous Gibberellins of Lima Bean (Phaseolus lunatus L.) Stems and Nodules

The content of gibberellin-like substances in nodules formed by Bradyrhizobium species strain 127E14 on roots of lima bean (Phaseolus lunatus L.) has been previously found to be relatively high. The objectives of the present study were to purify and identify the endogenous gibberellins from the stems and nodules of lima bean. By sequential silica gel partition column chromatography, C₁₈ reverse-phase high performance liquid chromatography, and combined gas chromatography-mass spectrometry, the gibberellins A₁, A₃, A₁₉, A₂₀, A₂₉, and A₄₄ were identified from root nodules. Gibberellins A₁, A₃, A₁₉, A₂₀, and A₄₄ were also identified from lima bean stem tissue. These data provide the first mass spectral-based evidence that gibberellins are present in leguminous root nodules. The presence of the gibberellins identified indicates that the early 13-hydroxylation gibberellin biosynthetic pathway predominates in stem and nodule tissue. However, it is not known if the gibberellins within the nodules are produced in situ, or if they are imported from some remote host plant tissue.     

Nonredundant Requirement for Multiple Histone Modifications for the Early Anaphase Release of the Mitotic Exit Regulator Cdc14 from Nucleolar Chromatin

In Saccharomyces cerevisiae, the conserved phosphatase Cdc14 is required for the exit from mitosis. It is anchored on nucleolar chromatin by the Cfi1/Net1 protein until early anaphase, at which time it is released into the nucleoplasm. Two poorly understood, redundant pathways promote Cdc14 release, the FEAR (Cdc fourteen early release) network and the MEN (mitotic exit network). Through the analysis of genetic interactions, we report here a novel requirement for the ubiquitination of histone H2B by the Bre1 ubiquitin ligase in the cell cycle–dependent release of Cdc14 from nucleolar chromatin when the MEN is inactivated. This function for H2B ubiquitination is mediated by its activation of histone H3 methylation on lysines 4 and 79 (meH3K4 and meH3K79) but, surprisingly, is not dependent on the histone deacetylase (HDAC) Sir2, which associates with Cdc14 on nucleolar chromatin as part of the RENT complex. We also observed a defect in Cdc14 release in cells lacking H3 lysine 36 methylation (meH3K36) and in cells lacking an HDAC recruited by this modification. These histone modifications represent previously unappreciated factors required for the accessibility to and/or action on nucleolar chromatin of FEAR network components. The nonredundant role for these modifications in this context contrasts with the notion of a highly combinatorial code by which histone marks act to control biological processes.     

Functional organization of the tympanal organ of the flour moth, Ephestia kuehniella

Data about electrical recordings from the tympanic organ of the flour moth, Ephestia kuehniella, to acoustic stimuli is presented. The stimuli had a frequency that ranged from 5 to 100 kHz, with minimal intensities of 40 to 50 db (Odb = 0.0002 dynes/cm²) and maximal up to 110 db. The tympanic organ of E. kuehniella responded in the whole range of frequencies used and showed two sensitivity maxima, one at 20 kHz and the other at 60 kHz. It responded from 45 to 110 db. The electrical activity of the tympanic nerve consisted of a spontaneous discharge of a type B cell and a tonic discharge in response to acoustic stimulation, produced by four acoustic sense cells, called A₁, A₂, A₃, and A₄. All these acoustic sense cells respond in the whole frequency range used and they differ in the heights of their action potentials and in their sensitivity to acoustic stimuli. The possible biological significance of hearing in pyralid moths is discussed.     

Segregation of Replicative DNA Polymerases during S Phase: DNA POLYMERASE ?, BUT NOT DNA POLYMERASES α/δ, ARE ASSOCIATED WITH LAMINS THROUGHOUT S PHASE IN HUMAN CELLS*

DNA polymerases (Pol) α, δ, and ϵ replicate the bulk of chromosomal DNA in eukaryotic cells, Pol ϵ being the main leading strand and Pol δ the lagging strand DNA polymerase. By applying chromatin immunoprecipitation (ChIP) and quantitative PCR we found that at G₁/S arrest, all three DNA polymerases were enriched with DNA containing the early firing lamin B2 origin of replication and, 2 h after release from the block, with DNA containing the origin at the upstream promoter region of the MCM4 gene. Pol α, δ, and ϵ were released from these origins upon firing. All three DNA polymerases, Mcm3 and Cdc45, but not Orc2, still formed complexes in late S phase. Reciprocal ChIP of the three DNA polymerases revealed that at G₁/S arrest and early in S phase, Pol α, δ, and ϵ were associated with the same nucleoprotein complexes, whereas in late S phase Pol ϵ and Pol α/δ were largely associated with distinct complexes. At G₁/S arrest, the replicative DNA polymerases were associated with lamins, but in late S phase only Pol ϵ, not Pol α/δ, remained associated with lamins. Consistently, Pol ϵ, but not Pol δ, was found in nuclear matrix fraction throughout the cell cycle. Therefore, Pol ϵ and Pol α/δ seem to pursue their functions at least in part independently in late S phase, either by physical uncoupling of lagging strand maturation from the fork progression, or by recruitment of Pol δ, but not Pol ϵ, to post-replicative processes such as translesion synthesis or post-replicative repair.     

One predominant 5′-undecanucleotide in adenovirus 2 late messenger RNAs

Oligonucleotides containing the 5′ termini of adenovirus 2 mRNA are selectively retained on columns of dihydroxyboryl cellulose. When total late adenovirus 2 mRNA was treated with RNAase T1, a single 5′ terminal oligonucleotide was isolated, although in several states of methylation. This oligonucleotide has the general structure m⁷GS^5′ppp^5′$\text{A}$mCmU(C₄,U₃)G. Since at least twelve individual species of mRNA must be present late after infection, this finding was unexpected and its significance is discussed.     

Synthesis of hybrid analogues of caffeine and eudistomin D and its affinity for adenosine receptors

Four bis-N-n-propyl analogues (3–6) in the uracil ring of two hybrid molecules (1 and 2) of caffeine and eudistomin D, a β-carboline alkaloid from a marine tunicate, were synthesized, and their affinity and selectivity for adenosine receptors A₁, A_2A, and A₃ were examined. All the compounds (3–6) showed better potency as adenosine receptor ligands than caffeine. Bis-N-n-propylation (3 and 4, respectively) of the uracil ring in 1 and 2 resulted in higher affinity for A₁ and A_2A adenosine receptors. Furthermore, it was found that a compound (5) possessing a n-propyloxy group at C-7 in compound 3 with a nitrogen at the β-position of the pyridine ring (β-N type) enhanced remarkably affinity for adenosine receptor A₃ subtype, while n-propyloxy substitution (compound 6) at C-5 in compound 4 with a nitrogen at the δ-position of the pyridine ring (δ-N type) reduced affinity for all the adenosine receptor, A₁, A_2A, and A₃. Among all the compounds (1–6) examined, compound 5 showed the most potent affinity for adenosine receptor A₃ subtype (K_i value, 0.00382 μM).