Evidence for two chemosensory pathways in Rhodobacter sphaeroides

In contrast to enteric bacteria, chemotaxis in Rhodobacter sphaeroides requires transport and partial metabolism of chemoattractants. Although a chemotaxis operon has been identified containing homologues of the enteric cheA, cheW, cheR genes and two homologues of the cheY gene, deletion of the entire chemotaxis operon had only minor effects on chemotactic behaviour under the conditions tested. Responses to sugars were enhanced in tethered cells but in all other chemotaxis assays behaviour of the operon deletion mutant was wild type. The mutant also showed wild-type responses to weak organic acids such as acetate and propionate, the dominant chemoattractants for this organism, under all conditions. This is in direct contrast to the enterics in which CheA, CheW and CheY are absolutely essential for taxis to PTS sugars, oxygen and MCP-dependent chemoeffectors. The operon deletion mutant was subjected to Tn5 transposon mutagenesis and new mutants selected using a chemotaxis and phototaxis screen. One mutant, JPA203, was non-chemotactic on swarm plates and showed inverted responses when tethered or subjected to changes in light intensity. Characterization of the Tn5 insertion in JPA203 identified a second chemotaxis operon in R. sphaeroides that contains homologues of cheY, cheA and cheR, the first homologue of cheB and two homologues of cheW. The new genes were labelled orf10, cheY_III, cheA_II, cheW_II, cheW_III, cheR_II, cheB and tlpC. When introduced into a wild-type background, deletion of cheA_II produced a chemotaxis minus phenotype in R. sphaeroides, suggesting that cheA_II forms part of a dominant chemotactic pathway, whereas the earlier identified operon plays only a minor role under laboratory conditions. The data presented here support the existence of two chemosensory pathways in R. sphaeroides, a feature that so far is unique in bacterial chemotaxis.     

A family of Ca2+-dependent activator proteins for secretion: comparative analysis of structure, expression, localization, and function

Ca2+-dependent activator protein for secretion (CAPS) 1 is an essential cytosolic component of the protein machinery involved in large dense-core vesicle (LDCV) exocytosis and in the secretion of a subset of neurotransmitters. In the present study, we report the identification, cloning, and comparative characterization of a second mammalian CAPS isoform, CAPS2. The structure of CAPS2 and its function in LDCV exocytosis from PC12 cells are very similar to those of CAPS1. Both isoforms are strongly expressed in neuroendocrine cells and in the brain. In subcellular fractions of the brain, both CAPS isoforms are enriched in synaptic cytosol fractions and also present on vesicular fractions. In contrast to CAPS1, which is expressed almost exclusively in brain and neuroendocrine tissues, CAPS2 is also expressed in lung, liver, and testis. Within the brain, CAPS2 expression seems to be restricted to certain brain regions and cell populations, whereas CAPS1 expression is strong in all neurons. During development, CAPS2 expression is constant between embryonic day 10 and postnatal day 60, whereas CAPS1 expression is very low before birth and increases after postnatal day 0 to reach a plateau at postnatal day 21. Light microscopic data indicate that both CAPS isoforms are specifically enriched in synaptic terminals. Ultrastructural analyses show that CAPS1 is specifically localized to glutamatergic nerve terminals. We conclude that at the functional level, CAPS2 is largely redundant with CAPS1. Differences in the spatial and temporal expression patterns of the two CAPS isoforms most likely reflect as yet unidentified subtle functional differences required in particular cell types or during a particular developmental period. The abundance of CAPS proteins in synaptic terminals indicates that they may also be important for neuronal functions that are not exclusively related to LDCV exocytosis.     

Genetic architecture of the cryptic species complex of Acanthocyclops vernalis (Crustacea: Copepoda). II. Crossbreeding experiments, cytogenetics, and a model of chromosomal evolution

Collectively, populations of Acanthocyclops vernalis, a species complex of freshwater copepods, are remarkably similar as to morphology and DNA content, despite variability in chromosome number. Reproductive isolation had been reported among some populations, but with each new investigation the species boundaries and factors that may influence them appeared less clear. To clarify the pattern of biological species within this group of populations, we adopted a comprehensive approach and examined patterns of reproductive isolation in populations for which morphology, chromosome number, DNA content, and 18S rDNA sequences are known. In this study we established nine isofemale lines from four sites in Wisconsin and performed 266 crosses. Crosses within and among these lines were used to relate the degree of reproductive isolation to chromosome differences and to construct a model to explain the origin and maintenance of chromosome number variability. Different gametic and somatic chromosome numbers were observed among specimens within some isofemale lines. In a few cases, gametes with different haploid numbers were produced by a single female. Matings within isofemale lines always produced at least some reproductively successful replicate crosses (produced viable, fertile offspring). Crosses between lines from the same site showed reduced success relative to within-line crosses. Crosses between populations from distant sites showed limited genetic compatibility, producing viable, fertile F1 offspring but infertile F2 adults. One cross between lines with different chromosome numbers (one with 2n = 8 and one with 2n = 10) produced fertile viable offspring, which reproduced for at least 60 generations. These hybrids had either eight or nine chromosomes in the third generation of inbreeding, and eight chromosomes after 20 generations. These hybrids also had reduced nuclear DNA contents at the third generation, a level that persisted through the 20th generation. Successful backcrosses between some hybrids and their parental lines further demonstrated the potential for genetic compatibility among forms with different chromosome numbers. We propose a model in which alterations due to Robertsonian fusions, translocations, and/or loss of chromosomal fragments generate heritable variation, only some of which leads to reproductive isolation. Hence, some of the criteria traditionally used to recognize species boundaries in animals (morphology, DNA content, chromosome number) may not apply to this species complex.     

Involvement of protons in the ion transport cycle of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase

The effect of pH on electrogenic sodium transport by the Na⁺,K⁺-ATPase has been studied. Experiments were carried out by admittance recording in a model system consisting of a bilayer lipid membrane with adsorbed membrane fragments containing purified Na⁺,K⁺-ATPase. Changes in the membrane admittance (capacitance and conductance increments in response to photo-induced release of ATP from caged ATP) were measured as function of AC voltage frequency, sodium ion concentration, and pH. In solutions containing 150 mM Na⁺, the frequency dependence of capacitance increments was not significantly dependent on pH in the range between 6 and 8. At a low NaCl concentration (3 mM), the capacitance increments at low frequencies decreased with the increasing pH. In the absence of NaCl, the frequency-dependent capacitance increment at low frequencies was similar to that measured in the presence of 3 mM NaCl. These results may be explained by involvement of protons in the Na⁺,K⁺-ATPase pump cycle, i.e., electroneutral exchange of sodium ions for protons under physiological conditions, electrogenic transport of sodium ions at high pH, and electrogenic transport of protons at low concentrations (and in the absence) of sodium ions.     

Effects of auricular electrostimulation and naloxone on nociceptive sensitivity in rabbits

Auriculo-acupuncture electrostimulation (AES) (15 H2) decreased the amplitude of somatosensory evoked potential (EP) in response to tooth pulp electrostimulation in 64% of acupuncture-sensitive unanesthetized rabbits and didn't induce the changes of EP in 36% of animals (acupuncture-resistant rabbits). The systemic naloxone (0.2 mg/kg) injection reversed the AES analgetic effect and induced the hyperalgesic one in acupuncture-sensitive rabbits but induced the analgetic effect in acupuncture-resistant animals. It has been suggested that the differences of individual characteristics of endogenous opioid system determined different naloxone action in acupuncture-sensitive and acupuncture-resistant rabbits.     

Some conclusions on the role of redundant DNA and the mechanisms of eukaryotic genome evolution inferred from studies of chromatin diminution in Cyclopoida

The absence of progress in understanding the problem of redundant eukaryotic DNA is stated. This is caused primarily by the attempts to solve this problem either in terms of the traditional approaches (the general phenotypic parameters such as developmental rate, body size, etc. depend on the genome size) or by introducing such vague terms as egoistic, parasitic, or junk DNA. Studying chromatin diminution (CD) in copepods yielded two important conclusions. First, part of the genome of a certain size (94% in Cyclops kolensis first described by the authors) is not needed for somatic functions as it is eliminated during the early (fourth to seventh) cleavage divisions from the presumptive somatic cells. Second, this DNA is not redundant, let alone selfish or junk, relative to the germline cells. In this sense, it can be regarded as invariant (monomorphic) trait that characterizes the species. Analysis of cloned and sequenced DNA regions eliminated from the somatic cell genome by CD (i.e., confined to the germline), which was first carried out for C. kolensis, showed that the molecular structure of this DNA has at least two features of regular organization: a mosaic structure of repetitive sequences and high (sometimes up to 100%) homology between different repeats and subrepeats. We have suggested that the germline-restricted DNA forms a unique molecular portrait of the species genome, thus acting as a significant factor of genetic isolation. Yet, the phenomenon of CD proper as it occurs in Cyclopoida (without disintegration of the chromosome structure) may be regarded as a model of reductional genome evolution, which has repeatedly occurred in the history of eukaryotes.__________Translated from Genetika, Vol. 41, No. 4, 2005, pp. 466–479.Original Russian Text Copyright © 2005 by Akifyev, Grishanin.     

Thermal resistance,preferred and avoidance temperatures of <Emphasis Type="Italic">Cyclops strenuus</Emphasis> Fischer, 1851, and their relation to optimal,pessimal, and tolerant temperatures

Ranges of preferred and avoidance temperatures in Cyclops strenuus Fischer, 1851 were determined based on the results of its experimental testing in the thermal gradient device. It is established that the process of temperature selection occurs with an overshoot. It is noted that copepods started to select the final preferred temperatures on the 8th day (temperatures above 26°C were avoided; the avoidance of low temperatures was not recorded). The average value of the critical thermal maximum for the season was determined. It is found that optimal, pessimal, and tolerant temperatures can be calculated on the scale of the species tolerance according to values of preferred and avoidance temperatures as well as according to values of the temperature range of regulation of the critical thermal maximum.     

Chromosomal Radiosensitivity as Associated with Chromatin Diminution in Cyclops (Crustacea, Copepoda)

Chromosomal radiosensitivity inferred from the yield of chromosome aberrations (CAs) was for the first time studied in Cyclops (Crustacea, Copepoda) before and after chromatin diminution (CD). A comparison was made for C. kolensis, in which CD denudes somatic embryo cells of the greatest (94%) DNA amount known for multicellular organisms, and C. insignis, which lacks CD. The two species have similar genome sizes, 4.6 and 4.3 pg, respectively. Radiosensitivity of C. kolensis chromosomes proved to be extremely high during prediminution cleavage divisions. This was attributed to membrane damage in granules that contain enzymes (topoisomerases) normally involved in cleavage and ligation of chromosomal DNA during CD.     

The peculiarities of the radiation mutagenesis of Cyclops kolensis and Cyclops insignis (Crustacea, Copepoda)

The frequency of the chromosomes aberrations (FA) of Cyclops kolensis before chromatin diminution (CD), which were inducted by gamma-irradiation in 50 times exceeds the FA of Cyclops kolensis after CD, during the CD, reducing the genome of C. kolensis in 15 times. During the embryogenesis the FA of Cyclops insignis doesn't change. The obtained results show us a low level of the spontaneous mutagenesis of C. kolensis and C. insignis embrios. The FA of Cyclops kolensis is correlated with the doses 1, 2, 3, 5 Gy gamma-irradiation. The similarity of the CD mechanism and of the mechanism of the chromosome aberrations is supposed.