Morphology and molecular phylogeny of a new hypotrich ciliate,Anteholosticha songi nov. spec., and an American population of Holosticha pullaster (Müller, 1773) Foissner et al., 1991 (Ciliophora,Hypotrichia)

A new urostylid ciliate, Anteholosticha songi nov. spec., isolated from forest soil in Tibet, and an American population of Holosticha pullaster (Müller, 1773) Foissner et al., 1991, isolated from a freshwater pond in the USA, are investigated in terms of their morphology, ontogenesis, and molecular biology. Anteholosticha songi nov. spec. is characterized by a slender to ellipsoidal body measuring 160–205 × 40–55 μm in vivo; rod-shaped yellowish cortical granules arranged in irregular short rows; four dorsal kineties; adoral zone consisting of 35–40 membranelles; three frontal, one buccal, one parabuccal, two frontoterminal, two pretransverse, and four to six transverse cirri and 14–25 midventral pairs; 12–22 ellipsoidal macronuclear nodules longitudinally arranged in pairs left of cell mid-line. Supplemental information on morphogenesis in Holosticha pullaster is also presented. The phylogenetic relationship of Anteholosticha and Holosticha inferred from SSU rDNA sequence data are concordant with previous studies and showing that Holosticha is monophyletic whereas Anteholosticha is polyphyletic and should be split into two or more genera.     

Free energy of imperfect nucleic acid helices. 3. Small internal loops resulting from mismatches

Physical studies of enzymioally synthesized oligoribonucleotides of defined sequence are used to evaluate quantitatively the destabilizing influence of mismatched bases in a double helix. The series (A-)₄G(-C)_n(-U)₄, N = 1 to 6, exist as imperfect dimer helices when N is equal to or less than 4, and as monomolecular hairpin helices when N is 5 and 6. Internal loops become progressively more destabilizing as their size increases from 2 to 4 to 6 nucleotides resulting from 1, 2 and 3 consecutive mismatched base pairs. However, the stability of a helix will generally be greater if a given number of mismatched pairs occur consecutively rather than in isolation from one another.These data may be used for improved calculations of stability of RNA secondary structure, to estimate the frequency of structural fluctuations in a double helix and to assess the stability of modified polynucleotide helices. An unmodified double helix of one million randomly arranged base pairs should contain on the time average approximately 10 G.C and 500 A.U pairs in non-hydrogen bonded, unstacked conformations at 25 °C. Our estimate of the effect of mismatching on T_m values of high polymers is less precise because of the long temperature extrapolation required. However, we estimate that DNA or RNA treated with mutagens which interrupt up to 20% of the nucleotide pairs will show a drop of about 1.2 deg. C in melting temperature with each unit per cent of modification.     

Topography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication.

Investigation of the DNA binding properties of the simian virus 40 (SV40) A protein (large T antigen) and the hybrid adenovirus-SV40 D2 protein revealed that both viral proteins protect similar regions of SV40 DNA from digestion by DNase I or methylation by dimethyl sulfate. However, the interaction of D2 protein with DNA was more sensitive to increases of NaCl concentration than was the interaction of wild-type SV40 A protein. Dimethylsulfate footprinting identified 13 DNA pentanucleotide contact sites at the viral origin of replication. The sequences of these sites corresponded to the consensus family 5'-(G greater than T) (A greater than G)GGC-3'. The pentanucleotides were distributed in three regions of origin DNA. Region I contained three pentanucleotide contact sites arranged as direct repetitions encompassing a span of 23 base pairs. In region II, four pentanucleotides were oriented as inverted repetitions that also spanned a total of 23 base pairs. Region III had six recognition pentanucleotides arranged as direct repetitions in a space of 59 base pairs. These fundamental variations in DNA arrangement are likely to determine different patterns of protein binding in each region.     

Hydrogen-bonding and packing features of membrane proteins: functional implications

The recent structural elucidation of about one dozen channels (in which we include transporters) has provided further evidence that these membrane proteins typically undergo large movements during their function. However, it is still not well understood how these proteins achieve the necessary trade-off between stability and mobility. To identify specific structural properties of channels, we compared the helix-packing and hydrogen-bonding patterns of channels with those of membrane coils; the latter is a class of membrane proteins whose structures are expected to be more rigid. We describe in detail how in channels, helix pairs are usually arranged in packing motifs with large crossing angles (|τ| ≈ 40°), where the (small) side chains point away from the packing core and the backbones of the two helices are in close contact. We found that this contributes to a significant enrichment of Cα-H…O bonds and to a packing geometry where right-handed parallel (τ = −40° ± 10°) and antiparallel (τ = +140° ± 25°) arrangements are equally preferred. By sharp contrast, the interdigitation and hydrogen bonding of side chains in helix pairs of membrane coils results in narrowly distributed left-handed antiparallel arrangements with crossing angles τ = −160° ± 10° (|τ| ≈ 20°). In addition, we show that these different helix-packing modes of the two types of membrane proteins correspond to specific hydrogen-bonding patterns. In particular, in channels, three times as many of the hydrogen-bonded helix pairs are found in parallel right-handed motifs than are non-hydrogen-bonded helix pairs. Finally, we discuss how the presence of weak hydrogen bonds, water-containing cavities, and right-handed crossing angles may facilitate the required conformational flexibility between helix pairs of channels while maintaining sufficient structural stability.     

On molecular mechanisms of nucleic acid synthesis fidelity aspects. 1. Contribution of base interactions

This paper is concerned with the molecular mechanisms of spontaneous replacements of base pairs in the processes of template synthesis of nucleic acids. The method of atom-atom potential functions was used to calculate the energies of interaction in non-complementary base pairs arranged in a common plane so that the mutual position of the glycosidic bonds does not differ appreciably from their position for Watson-Crick pairs in the DNA double helix. A number of local minima of this energy have been found, which could occur in template synthesis and result in insertion of incorrect bases into the double helix. The calculation results are indicative of formation of purine-purine pairs with one of the purine nucleotides in syn-conformation, which can be regarded as a typical pathway of transversion, and that of wobble-pairs TG and AC, which can be regarded as a typical pathway of transition. The contribution of intramolecular interactions of nucleic acids as well as interactions of polynucleotide chains with an enzyme to the fidelity of template synthesis of nucleic acids is discussed. The calculation results are compared with the experimental data on the frequency of spontaneous mutations and the frequency of errors in template synthesis of nucleic acids in vitro.     

Crystalline sheets of tropomyosin

In the presence of spermine tropomyosin forms sheets having two-dimensional crystallinity and tactoids. The most common form of sheet has cmm symmetry with a = 80 nm and b = 5 nm. The structure of this sheet has been solved in projection to a nominal resolution of 1.5 nm by combining data from electron diffraction and electron microscopy. Analysis of this pattern and that of rarely observed sheets having p2 symmetry (a = 40 nm, b = 5 nm and γ = 80 °) indicated that the cmm structure was formed by superposition of two p2 sheets. The tropomyosin molecules in each p2 sheet were arranged in rows directed along the p2 (0, 1) lattice lines, with all the molecules in one row having the same polarity and lying antiparallel to the molecules in adjacent rows. These rows associated in pairs, possibly by the supercoiling of the molecules in one row about those in the neighbouring row.     

The Molecular Cytogenetic Characterization of Pistachio (Pistacia vera L.) Suggests the Arrest of Recombination in the Largest Heteropycnotic Pair HC1

This paper represents the first molecular cytogenetic characterization of the strictly dioecious pistachio tree (Pistacia vera L.). The karyotype was characterized by fluorescent in situ hybridization (FISH) with probes for 5S and 45S rDNAs, and the pistachio specific satellite DNAs PIVE-40, and PIVE-180, together with DAPI-staining. PIVE-180 has a monomeric unit of 176–178 bp and high sequence homology between family members; PIVE-40 has a 43 bp consensus monomeric unit, and is most likely arranged in higher order repeats (HORs) of two units. The P. vera genome is highly heterochromatic, and prominent DAPI positive blocks are detected in most chromosomes. Despite the difficulty in classifying chromosomes according to morphology, 10 out of 15 pairs (2n = 30) could be distinguished by their unique banding patterns using a combination of FISH probes. Significantly, the largest pair, designated HC1, is strongly heteropycnotic, shows differential condensation, and has massive enrichment in PIVE-40 repeats. There are two types of HC1 chromosomes (type-I and type-II) with differing PIVE-40 hybridization signal. Only type-I/II heterozygotes and type-I homozygotes individuals were found. We speculate that the differentiation between the two HC1 chromosomes is due to suppression of homologous recombination at meiosis, reinforced by the presence of PIVE-40 HORs and differences in PIVE-40 abundance. This would be compatible with a ZW sex-determination system in the pistachio tree.     

THE ULTRASTRUCTURE OF PHALACROCLEPTES VERRUCIFORMIS, AN UNCILIATED CILIATE PARASITIZING THE POLYCHAETE SCHIZOBRANCHIA INSIGNIS

The organization of Phalacrocleptes verruciformis is, in general, less complex than that of other ciliates, and no kinetosomes have been observed. However, there are numerous suctorial tentacles at the surface of the body, and the pellicle is characterized by close-set villus-like projections. The tentacles are very small (about 430 mµ in length, and about the same in diameter), but show the essential features of tentacles of suctorians such as Tokophrya, Podophrya, and Ephelota. Each tentacle is reinforced by eight pairs of fibrils arranged concentrically just within its wall, and contains a single missile-like body (MLB). The tentacles become attached to the cilia of the host, and serve for feeding upon the plasmatic contents of the cilia as well as for maintaining contact with the host. The MLB's originate in the endoplasm, and then migrate toward the surface and become incorporated into the tentacles. When feeding is initiated, the membrane covering the outermost nozzle-like portion of the MLB becomes continuous with the membrane of the cilium, and there are other changes in the structure of the MLB which suggest enzymatic activity. Although it appears that Phalacrocleptes is a suctorian, the complete absence of kinetosomes sets this organism apart from other members of the group.     

Estinnophyton wahnbachense (Kräusel et Weyland) comb. nov., une plante remarquable du siegenien d'allemagne

Specimens collected from the type locality of Protolepidodendron wahnbachense Kräusel et Weyland 1932 (Germany) are studied. The narrow axes, with persistent leaves which are bifurcated and spirally arranged, resemble those of P. wahnbachense. However, the vegetative and fertile leaves bifurcate twice and the four resulting segments are spread in various planes; the fertile leaves support two pairs of stalked elongate sporangia which are directed towards the axis. Estinnophyton wahnbachense (Kräusel et Weyland) comb. nov. is thus proposed as a name for these Siegenian plant remains, the questionable affinities of which are discussed.     

Crystallization of purple membrane in three dimensions

Large aggregates of purple membrane have been produced by solubilization of purple membrane in Triton X100, followed by removal of the detergent by dialysis against 0.9 m or 1.0 m-phosphate (pH 5.0). Although the crystals are not yet large enough for X-ray analysis, they appear microscopically crystalline in three dimensions, with space group P321 or P312.Freeze-fracture electron microscopy and diffraction analysis show that each membrane forming the aggregate has the normal P3 packing ($\text{a = 63}\text{A}\text{?}$). Membranes are arranged in pairs, with their extracellular surfaces in close contact, and these pairs then stack up to form large aggregates. The large aggregates appear to be perfect three-dimensional crystals only over small regions, up to 12 membranes thick. There are occasional inclusions of solvent between membrane pairs, and “coincidence lattice” defects occur.     

HIGH-RESOLUTION ELECTRON MICROSCOPIC ANALYSIS OF THE AMYLOID FIBRIL

The ultrastructural organization of the fibrous component of amyloid has been analyzed by means of high resolution electron microscopy of negatively stained isolated amyloid fibrils and of positively stained amyloid fibrils in thin tissue sections. It was found that a number of subunits could be resolved according to their dimensions. The following structural organization is proposed. The amyloid fibril, the fibrous component of amyloid as seen in electron microscopy of thin tissue sections, consists of a number of filaments aggregated side-by-side. These amyloid filaments are approximately 75–80 A in diameter and consist of five (or less likely six) subunits (amyloid protofibrils) which are arranged parallel to each other, longitudinal or slightly oblique to the long axis of the filament. The filament has often seemed to disperse into several longitudinal rows. The amyloid protofibril is about 25–35 A wide and appears to consist of two or three subunit strands helically arranged with a 35–50-A repeat (or, less likely, is composed of globular subunits aggregated end-to-end). These amyloid subprotofibrillar strands measure approximately 10–15 A in diameter.     

Chromatin-Like Structures in Polyoma Virus and Simian Virus 40 Lytic Cycle

Nucleoprotein complexes containing viral DNA and cellular histones were extracted from nuclei of permissive cells infected with polyoma virus or simian virus 40 (SV40) and examined by electron microscopy. Polyoma and SV40 nucleoprotein complexes are almost identical. They appear as relaxed circular molecules consisting of 20 to 21 globular particles interconnected by thin filaments. Their contour length in 0.02 M salt is 2.7 times shorter than that of viral DNA form I obtained after dissociation of the proteins in 1 M NaCl. The nucleosomes have an average diameter of 12.5 nm. Each nucleosome contains 175 to 205 DNA base pairs condensed fivefold in length. The nucleosomes are regularly spaced on the circular molecule. The internucleosomal filaments are made of naked DNA, and each filament contains about 55 base pairs. The partial sensitivity of the nucleoprotein complex to cleavage by EcoR1 endonuclease suggests that the nucleosomes are not formed at specific sites on the viral genome. Faster sedimenting nucleoprotein complexes containing replicative intermediates were studied. Isopycnic centrifugation in metrizamide gradients in the absence of aldehyde fixation showed that these molecules conserved the same DNA-to-protein ratio as the form I DNA-containing complexes.     

The arthropodsmimetaster andvachonisia from the devonian hunsrück shale

A combined X-ray/surface study ofMimetaster andVachonisia has revealed several new features.Mimetaster has a head with stalked eyes and ocelli, a ventral organ, a large labrum, and two pairs of strong uniramous legs revealing muscle strands. The body carries up to more than 30 pairs of bifid appendages.Vachonisia, without visible eyes, appears to have three pairs of walking legs on the head. Two pairs of structures directed towards the mouth may be strong endites or discrete manipulating appendages. The body carries about 80 pairs of appendages, which are bifid and similar to those of Mimetaster. Both arthropods have one pair of antennae.Mimetaster seems to have been a deposit feeder,Vachonisia a carnivore or raptator. The former was related toMarrella, and these two probably toVachonisia, all of which may have been distantly related to the trilobite-chelicerate group.     

Differentiation of primordial germ cells during embryogenesis of Allacma fusca (L.) (Collembola: Symphypleona)

The youngest primordial germ cells (PGCs) of Allacma fusca (L.) (Collembola: Sminthuridae) can be identified in embryos at the blastoderm stage as scattered in the yolk mass. They are arranged in pairs connected via intercellular bridges and dispersed among the yolk granules over a relatively small area but they never form multicellular clusters. With progressing development, the mesoderm of the germ band differentiates, the PGCs migrate to the abdominal part of the germ band and enter among mesoderm cells making two clusters of cells in the left and right parts of the abdomen. The mesoderm cells neighbouring the PGC cluster differentiate into a one-layered gonad envelope and produce a thin basal lamina separating the gonad from the rest of the mesoderm. The PGCs are still connected in pairs. At the end of the embryonic development, the gonads have regular spherical shapes and are enclosed within the envelope built up by a layer of flat somatic cells. Now, the PGCs do not occur only in pairs, but chains of cells connected with a sequence of intercellular bridges can also be seen.     

Two new species of mazocraeid monogeneans from Clupanodon punctatus (Temminck & Schlegel) (Clupeiformes: Clupeidae) found in Daya Bay, South China Sea, with the proposal of Chelimazocraes n. g.

Chelimazocraes liaoi n. g., n. sp. and Chelimazocraes ascidiformis n. sp. (Monogenea: Mazocraeidae) are described from the gills of Clupanodon punctatus (Temminck & Schlegel) in Daya Bay (South China Sea). The new genus is characterised by the following features: (i) the haptor is distinctly separated from the body proper, and the arrangement of the clamps is bilaterally symmetrical but longitudinally heteromorphic; (ii) the anterior three pairs of clamps are of the mazocraeid-type, whereas the fourth pair is of a non-mazocraeid type with three sclerites; (iii) all three pairs of clamps are similar in shape but their size gradually becomes smaller from the anterior to the posterior; (iv) the inner spines of the copulatory organ have a similar shape; and (v) the testes are numerous and arranged longitudinally posterior to the ovary. The two new species are easily distinguished from other members of the Mazocraeidae by the unique structure of the fourth pair of clamps; however, there are some noticeable differences between the two species. The major differences are as follows: (i) the body of C. liaoi n. sp. is significantly larger than that of C. ascidiformis n. sp.; (ii) the anterior three pairs of clamps consist of different sclerites in the two species; and (iii) the copulatory organ has one pair of outer spines and 15–16 pieces of inner spines in C. liaoi n. sp. (vs two pairs of outer spines and 22–26 pieces of inner spines in C. ascidiformis n. sp.).     

Hexagonal surface array in a protein-secreting bacterium, Bacillus brevis 47

Bacillus brevis 47, a protein-secreting bacterium, contained two major proteins with approximate molecular weights of 150 000 and 130 000 in the cell wall. The cell surface was covered with a hexagonally arranged array of six structural units about 4 nm in diameter with a lattice constant of 14.5 nm. The regular array structure as well as the chemical composition of cell envelopes remained the same regardless of the growth conditions. A mutant, strain 47–57, which was isolated as a phage resistant colony, contained only the 150 000 protein as a major cell wall protein. Although the mutant had hexagonally arranged arrays with the same lattice constant as that of wild-type cells, the distribution of mass in the unit cell differed considerably from that of the wild-type cells. The number of structural units in the unit cell of the mutant was reduced from six to three. Taking these results together with filtered images of the wild-type and mutant envelopes, two possible models for the surface array of B. brevis 47 are discussed.     

An electron microscope study of the DNA sequence organization of the human genome

The arrangements of inverted-repeated and repeated DNA sequences in the human genome have been investigated by an electron microscope method. The arrangement of the interspersed repeated DNA sequences is found to be similar to the corresponding arrangement found in Xenopus. This arrangement consists of 300-nucleotide-long repeated DNA sequences interspersed with roughly gene-size single-copy DNA sequences. The inverted-repeated sequences are also 300 nucleotides in length and are interspersed with the other DNA sequence classes.Most inverted-repeated sequences (64%) are spaced by another sequence which is recognized by electron microscopy as a single-stranded loop in a hairpin structure. The average length of this spacer loop is 1.6 kilobases. Although some pairs of inverted-repeated sequences are clustered, most seem to be randomly distributed throughout the genome. The average distance separating two pairs of inverted-repeated sequences is 10 to 20 kilobases. The interspersed repeated sequences and inverted-repeated sequences are arranged simultaneously in a portion of the human genome resulting in an interspersion of all three sequence classes.