Isolation and characterization of thermostable DNA polymerase of the hyperthermophilic archaeum <Emphasis Type="Italic">Thermococcus litoralis</Emphasis> Sh1AM

Overall, 30 strains of hyperthermophilic archaea, representing seven species of the genera Thermococcus, Desulfurococcus, Thermoproteus, and Acidilobus, were tested for the presence of thermostable DNA polymerases. Thermostabilities of the polymerases varied distinctly among the strains within one species. Polymerases of five strains retained 60–100% activity upon incubation of the preparations at 95°C for 120 min. A new DNA polymerase was isolated from the strain Thermococcus litoralis Sh1AM, possessing the enzyme with the most promising properties, and characterized. Molecular weight of the enzyme is 90–100% kDa. The purified DNA polymerase preserved 50% of the initial activity upon incubation at 95°C for 120 min. The polymerase isolated displayed an associated 3–5 exonuclease activity. The error rate when extending a DNA strand was at least twofold lower compared with Taq polymerase. The main physicochemical and enzymatic properties of the new polymerase are similar to the known DNA polymerases of family B.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 40–47.Original Russian Text Copyright © 2005 by Slobodkina, Chernykh, Lopatin, Ilina, Bannikova, Ankenbauer, Eldarov, Varlamov, Bonch-Osmolovskaya.     

The molecular genetic differentiation of cultured <Emphasis Type="Italic">Saccharomyces</Emphasis> strains

A comparative molecular genetic study of cultured Saccharomyces strains isolated from the surface of berries and various fermentation processes showed that bakers yeast and black-currant isolates contain not only Saccharomyces cerevisiae but also S. cerevisiae × S. bayanus var. uvarum hybrids. The molecular karyotyping of bakers, brewers, and wine yeasts showed their polyploidy. The restriction enzyme analysis of noncoding rDNA regions (5.8S-ITS and IGS2) makes it possible to differentiate species of the genus Saccharomyces and to identify interspecies hybrids. The microsatellite primer (GTG)₅ can be used to study the populations of cultured S. cerevisiae strains.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 215–223.Original Russian Text Copyright © 2005 by Naumova, Zholudeva, Martynenko, Naumov.     

Eggs and embryos in <Emphasis Type="Italic">Xenoturbella</Emphasis> (phylum uncertain) are not ingested prey

Xenoturbella is an enigmatic animal that has puzzled science for almost a century. The eggs and embryos found in Xenoturbella have recently been interpreted as ingested prey. However, PCR on individual eggs as well as in situ hybridisation and in situ PCR unambiguously show that they are Xenoturbellas own. The eggs and embryos are individually enclosed within follicles with the same ultrastructure. The cortical granules in oocytes and eggs from Xenoturbella but not Nucula stained positively with an antiserum against Reissners substance. The embryos incorporated 5-bromodeoxyuridine in vivo, i.e. they replicate their genome and are living.Electronic Supplementary Material Supplementary material is available for this article at .     

Sucrose in storage media and cultivar affects post-storage regrowth of <Emphasis Type="Italic">in vitro</Emphasis> Hosta propagules

The goal of this research was to investigate if culturing in high sucrose (5%) liquid media during multiplication phase (stage II) would enhance endogenous sugar levels and dry matter sufficiently to allow storage of in vitro plants in sugar free media without adversely affecting post-storage recovery. Hosta tokudama Newberry Gold (NBG) and Hosta Striptease were cultured in Murashige and Skoog (MS) media containing 5% sucrose during stage II and transferred to rooting phase (stage III) in MS medium without (0%) sucrose or with 3% sucrose for 4weeks. At the end of stage III, cultures were stored, with the remaining media, at 10°C with 5molm^–2s^–1 photosynthetic photon flux (PPF) from cool white fluorescent lamps for 7 or 14weeks with or without a 2-week dark period prior to removal from storage. In both cultivars, stage III plants cultured in 3% sucrose media had higher soluble sugar levels and greater shoot and root biomass than those cultured in 0% sucrose media. Shoot and root soluble sugars decreased during storage. Shoot growth ceased during storage in both media. Root dry matter continued to increase in plants stored in 3% sucrose media but did not change in 0% sucrose media. Plants cultured in 3% sucrose media had less leaf chlorosis and less mortality after 7 or 14weeks of low temperature storage than the plantlets from sugar free media. Extending the storage period from 7 to 14weeks or introduction of 2-week dark period at the end of storage did not affect leaf chlorosis or plant mortality during acclimatization. Post-storage growth varied with the cultivar. Benefit of having sucrose in storage media was to develop a strong root system that aided the acclimatization and post-storage growth following 7 or 14week storage. Sucrose loading by culturing plants in liquid media containing 5% sucrose did not allow storage in sugar free media without adversely affecting post-storage growth in both cultivars.     

Cross-effects of extracellular factors of adaptation to stress in <Emphasis Type="Italic">Luteococcus casei</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Saccharomyces cerevisiae yeasts (lower eukaryotes) were shown to produce a protein exometabolite with reactivation activity. We demonstrated cross-effects of extracellular protein factors of adaptation to stress (heat and UV irradiation) in yeasts and Luteococcus casei bacteria. The possibility for isolation and partial purification of protein exometabolites from the culture liquid of yeasts and bacteria by similar methods, as well as the similarity of elution profiles for the active proteins in high-performance liquid chromatography, suggests that the proteins (or fragments thereof) of the organisms studied are homologous.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 171–175.Original Russian Text Copyright © 2005 by Vorobeva, Khodzhaev, Ponomareva.     

Discovery,localization, and sequence characterization of molecular markers for the crown rust resistance genes <Emphasis Type="Italic">Pc38, Pc39</Emphasis>, and <Emphasis Type="Italic">Pc48</Emphasis> in cultivated oat (<Emphasis Type="Italic">Avena sativa</Emphasis> L.)

Molecular markers for the crown rust resistance genes Pc38, Pc39, and Pc48 in cultivated oat (Avena sativa L.) were identified using near-isogenic lines and bulked segregant analysis. Six markers for Pc48, the closest being 6 cM away, were found in a Pendek-39 × Pendek-48 (Pendek3948) population, but none was found in a Pendek-48 × Pendek-38 (Pendek4838) population. Three markers for Pc39 were found in the Pendek3948 population, one of which cosegregated with the gene. This same marker was found to be 6 cM away from the gene in an OT328 × Dumont (OT328Du) population. Nine markers for Pc38 were found in the Pendek4838 population, eight of which are within 2 cM of the gene. One other marker for Pc38 was found in the OT328Du population; however, comparative mapping suggests that the Pc38 region in OT328Du is in a different location than that in Pendek4838. A number of markers unlinked to the genes under study formed linkage groups in both the Pendek3948 and Pendek4838 populations. Four of these show homology or homoeology to each other and to the Pc39 region in Pendek3948. Two RFLP clones closely linked to Pc38 code for a putative leucine-rich repeat transmembrane protein kinase and a cre3 resistance gene analogue. This study provides information to support molecular breeding in oat, and contributes to ongoing research into genomic regions associated with fungal pathogen resistance.     

An evaluation of evolutionary theories based on genomic structures in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Encephalitozoon cuniculi</Emphasis>

Codon usage patterns in 16 chromosomes coincided with each other in Saccharomyces cerevisiae, and the same result was obtained from Encephalitozoon cuniculi consisting of 11 chromosomes, although each chromosome function differs. In addition, preferential codon usage in the regenerated coding systems for Leu and Lys differed between Saccharomyces cerevisiae and Encephalitozoon cuniculi. These results cannot be explained by Darwins natural selection theory or by the neutral theory proposed against Darwins. Furthermore, the codon usage patterns were examined in both prokaryotes and eukaryotes. The use of G or C at the third codon position was much lower than T or A in Ureaplasma urealyticum, whereas inversely the use of G or C at the third codon position was much higher than T or A in Mycobacterium tuberculosis. Additionally, Candida albicans and Plasmodium falciparum also showed a very low usage of G or C at the third codon position. It is a difficult leap to speculate that the inverse codon usage change occurred over the genome during biological evolution. Thus, the present results strongly suggest that organisms were derived from different origins, indicating that the origin of life was plural, based on genomic structures.     

Adaptogenic effects of furostanol glycosides of <Emphasis Type="Italic">Dioscorea deltoidea</Emphasis> wall on oxidative processes in tomato plants during biotic stress

The effect of furostanol glycosides of a cell culture of Dioscorea deltoidea Wall on oxidative processes in tomato plants subjected to invasion with the gall nematode Meloidogyne incognita Kofoid et White was studied. We showed that furostanol glycosides induce a nonspecific defensive response in plants. Exposure of cell membranes to furostanol glycosides causes rearrangements in fatty acids, resulting in the formation of conjugated dienes, which makes molecules thermodynamically more stable under stress conditions. The study of changes in the activity of peroxidases of intact plants and plants affected with the nematode, which were treated with furostanol glycosides, showed that the protective effect of the guaiacol-dependent peroxidase is more long-term than the effect of the benzidine-dependent peroxidase.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 3, 2005, pp. 347–353.Original Russian Text Copyright © 2005 by Vasileva, Vanyushkin, Zinoveva, Udalova, Volkova, Nosov, Paseshnichenko.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Bambusa balcooa</Emphasis> Roxb.: Factors affecting changes of morphogenetic competence in the axillary buds

Axillary buds from field-grown culms of Bambusa balcooa were used as explants to induce multiple shoots in liquid Murashige and Skoogs (MS) medium supplemented with 11.25 M of 6-benzylaminopurine (BAP) and 4.5 M kinetin (Kn). A clump of at least 3 shoots was used for root induction in half strength MS medium with 1.0 M of 3-indolebutyric acid (IBA). Morphogenetic competence of the axillary buds varied widely in different months of two consecutive calendar years. The highest morphogenetic potentials were observed in October. The major problem encountered was presence of systemic fungal contaminants. Perhaps, rainfall positively contributed to induce morphogenetic competence. A moderately high phenolic content of the nodal explant was also detrimental for in vitro morphogenesis. The morphogenetic competence of B. balcooa correlated with the season in which the explants were excised from the natural stands. To the best of our knowledge this is the first report on in vitro regeneration of B. balcooa from mature field-grown axillary buds.     

A novel family of mitochondrial proteins is represented by the <Emphasis Type="Italic">Drosophila</Emphasis> genes <Emphasis Type="Italic">slmo</Emphasis>, <Emphasis Type="Italic">preli-like</Emphasis> and <Emphasis Type="Italic">real-time</Emphasis>

Mitochondria play essential roles in development and disease. The characterisation of mitochondrial proteins is therefore of particular importance. The slowmo (slmo) gene of Drosophila melanogaster has been shown to encode a novel type of mitochondrial protein, and is essential in the developing central nervous system. The Slmo protein contains a conserved PRELI/MSF1p domain, found in proteins from a wide variety of eukaryotic organisms. However, the function of the proteins of this family is currently unknown. In this study, the evolutionary relationships between members of the PRELI/MSF1p family are described, and we present the first analysis of two novel Drosophila genes predicted to encode proteins of this type. The first of these, preli-like (prel), is expressed ubiquitously during embryonic development, whilst the second, real-time (retm), is expressed dynamically in the developing gut and central nervous system. retm encodes a member of a novel conserved subclass of larger PRELI/MSF1p domain proteins, which also contain the CRAL-TRIO motif thought to mediate the transport of small hydrophobic ligands. Here we provide evidence that, like Slmo, both the Prel and Retm proteins are localised to the mitochondria, indicating that the function of the PRELI/MSF1p domain is specific to this organelle.Edited by P. Simpson     

Comparative analysis of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> natural isolates

A comparative analysis was performed of the polymorphism of the oyster mushroom Pleurotus ostreatus (Fr.) Kumm naturally occurring strains isolated from the natural substrates from two geographically remote Russian natural preserves, the Central Forest Biosphere Tver State Preserve and the Moscow State University Zvenigorod Biological Station (Moscow oblast), and within the city of Moscow. The results of the frequency analysis for the allozyme loci alleles and for the sexual and somatic incompatibility groups are presented; the genetic structure and the interpopulation relations among 58 Pleurotus ostreatus dikaryotic strains are estimated. The natural samples from the Moscow and Tver oblasts are shown to have a high degree of polymorphism with a genetic differentiation of 0.743; in spite of their territorial remoteness, they are, however, actively exchanging genetic material. The natural fungal isolates form two reproductively isolated groups.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 231–238.Original Russian Text Copyright © 2005 by Shtaer, Yu. Belokon, M. Belokon, Shnyreva.     

First Record of Tropical Crabs <Emphasis Type="Italic">Portunus sanguinolentus</Emphasis> (Herbst, 1783) and <Emphasis Type="Italic">Plagusia depressa tuberculata</Emphasis> Lamarck, 1818 in Peter the Great Bay,Sea of Japan

Crabs Portunus sanguinolentus (Herbst, 1783) and Plagusia depressa tuberculata Lamarck, 1818 were found in Peter the Great Bay (Sea of Japan) on a drifting buoy. The occurrence of the members of tropical fauna along the coast of Primorye was due, in all likelihood, to the enlargement of the amount of anthropogenic garbage transported with sea currents that serves as a substrate for settlement of different marine organisms.Original Russian Text Copyright © 2005 by Biologiya Morya, Kepel, Tsareva.     

Silver nitrate and aminoethoxyvinylglycine affect <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated apple transformation

In Fuji, the production of ethylene was increased with the addition of AgNO₃ and inhibited with the addition of 10 M aminoethoxyvinylglycine (AVG). The addition of 80 M AgNO₃ to transformed explants of Fuji cultured on selection medium resulted in increased ethylene production (20 l l^–1) at 3 weeks. Under examining the effect of AgNO₃ in Fuji, the 40 M AgNO₃ showed with higher 33.8% and 6.5% in the efficiency of regeneration and transformation. However, ethylene production in Gala explants treated with 10M AgNO₃ (3 l l^–1) decreased after 2 weeks compared with the control (5 l l^–1). Although the regeneration efficiency of Gala with 10 M AgNO₃ was higher (41.1%) than the control (20.1%), there was no significant difference in the transformation efficiency at the same concentration. Shoot regeneration of Fuji and Gala was completely inhibited with 10 M AVG. These results suggest that the addition of AgNO₃ affects the efficiency of Agrobacterium-mediated gene transfer in Fuji.Eun Soo Seong, Ill Min Chung- These two Authors Contributed equally to this work     

Population analysis of electrophoretic variation in blood serum albumins of European (<Emphasis Type="Italic">Acipenser ruthenus</Emphasis> Linnaeus) and Siberian (<Emphasis Type="Italic">A. ruthenus marsiglii</Emphasis> Brandt) sterlet

We studied blood serum albumins in European (Acipenser ruthenus L.) and Siberian (A. ruthenus marsiglii Brandt) sterlet using disk electrophoresis in polyacrylamide gel. The albumins were shown to be controlled by three codominant alleles of a single locus (ALB* a, b, c). In European sterlet, all six theoretically possible genotypes were described, one of which (ALB*c/c) occurred extremely rarely (one individual). Siberian sterlet was found to be monomorphic for albumins: all fish examined had the ALB*a/a genotype. There was no correlation between albumin patterns and fish fatness. In a number of samples from the Volga River basin, spatial and temporal differentiation was found and analyzed. The results suggest that construction of hydroelectric stations may provoke massive and prolonged sterlet migrations.Translated from Genetika, Vol. 41, No. 2, 2005, pp. 246–253.Original Russian Text Copyright © 2005 by Kuzmin, Kuzmina.     

Social mutilation in the Ponerine ant <Emphasis Type="Italic">Diacamma</Emphasis>: cues originate in the victims

Summary. In the queenless ponerine ant genus Diacamma, all workers eclose with a pair of innervated thoracic appendages termed gemmae. The gamergate (= mated egg laying worker) maintains reproductive monopoly by mutilating the gemmae of all eclosing individuals. Such mutilation leads to irreversible behavioural and neurological changes such that the individual lacking gemmae becomes incapable of appropriate sexual calling and mating. In one population related to Diacamma ceylonense from India, Diacamma sp. from Nilgiri (hereafter referred to as nilgiri), gamergates do not mutilate their nestmates and yet maintain reproductive monopoly. To understand what triggers mutilation, we exchanged cocoons between the mutilating D. ceylonense colonies and the non mutilating nilgiri colonies. nilgiri callows were not mutilated even in D. ceylonense colonies while D. ceylonense callows were mutilated even in nilgiri colonies, suggesting that the cues for mutilation originate in the victims (callows), presumably in the gemmae themselves. This finding should facilitate understanding the proximate mechanism and evolutionary significance of mutilation of gemmae as a method of resolution of reproductive conflicts in the genus Diacamma.Received 27 August 2003; revised 26 April 2004; accepted 27 April 2004.     

Survey of gorillas (<Emphasis Type="Italic">Gorilla gorilla gorilla</Emphasis>) and chimpanzees (<Emphasis Type="Italic">Pan troglodytes troglodytes</Emphasis>) in Southwestern Cameroon

A study on the distribution and population density of the western lowland gorilla (Gorilla g. gorilla) and the central chimpanzee (Pan t. troglodytes) was undertaken between December 1997 and August 2000 in the Campo and Maan Forests in southwestern Cameroon. The aim of this survey was to estimate the densities of the apes in different parts of the area, to assess the importance of the region for the conservation of these endangered species and to determine the influence of human activities such as logging and hunting. The survey was based on night nest counts on a total of 665.5 km of line transects. The overall density in the Campo Forest was estimated at 0.2 gorillas/km² and at 0.63–0.78 chimpanzees/km². The overall density of chimpanzees in the Maan Forest was estimated at 0.8–1 individuals/km². Gorilla density in this area was too low to allow an estimation. The highest gorilla nest density was found in secondary forest. The gorilla density in unlogged forest was significantly lower. Chimpanzees showed a clear preference for less disturbed areas. In unlogged forest, old secondary forests (logging more than 23 years ago) and areas of recent logging with large remaining patches of primary forest, significantly higher densities were calculated than inside the more heavily exploited logging concession. In areas with both logging and high hunting pressure both species were rare or even absent. The Campo Maan area is considered a very important area for the conservation of gorillas and chimpanzees. Conservation measures are urgently required to reduce the impact of logging and hunting. The creation of the Campo Maan National Park in January 2000 is an important measure to preserve the unique biodiversity in this so far hardly protected area.     

Characterization and expression of genes from the RubisCO gene cluster of the chemoautotrophic symbiont of <Emphasis Type="Italic">Solemya velum</Emphasis>: <Emphasis Type="Italic">cbbLSQO</Emphasis>

Chemoautotrophic endosymbionts residing in Solemya velum gills provide this shallow water clam with most of its nutritional requirements. The cbb gene cluster of the S. velum symbiont, including cbbL and cbbS, which encode the large and small subunits of the carbon-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), was cloned and expressed in Escherichia coli. The recombinant RubisCO had a high specific activity, 3 mol min^–1 mg protein ^–1, and a K _CO2 of 40.3 M. Based on sequence identity and phylogenetic analyses, these genes encode a form IA RubisCO, both subunits of which are closely related to those of the symbiont of the deep-sea hydrothermal vent gastropod Alviniconcha hessleri and the photosynthetic bacterium Allochromatium vinosum. In the cbb gene cluster of the S. velum symbiont, the cbbLS genes were followed by cbbQ and cbbO, which are found in some but not all cbb gene clusters and whose products are implicated in enhancing RubisCO activity post-translationally. cbbQ shares sequence similarity with nirQ and norQ, found in denitrification clusters of Pseudomonas stutzeri and Paracoccus denitrificans. The 3 region of cbbO from the S. velum symbiont, like that of the three other known cbbO genes, shares similarity to the 3 region of norD in the denitrification cluster. This is the first study to explore the cbb gene structure for a chemoautotrophic endosymbiont, which is critical both as an initial step in evaluating cbb operon structure in chemoautotrophic endosymbionts and in understanding the patterns and forces governing RubisCO evolution and physiology.     

Dynamics of byssal thread production in <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> and <Emphasis Type="Italic">Modiolus modiolus</Emphasis> (Bivalvia) upon reattachment to substrate

It was established that the process of repeated attachment to a substrate in bivalve mollusks Crenomytilus grayanus (Grays mussel) and Modiolus modiolus (the northern horse mussel) involved several successive stages which took in vitro about one month at a water temperature of 19 ± 2°C. Comparison with Grays mussel revealed that the northern horse mussel had a higher rate of byssal thread production and a greater thread number by the end of the complete formation of the byssus complex. The observed differences are explained by the adaptation of mollusks to habitation in different biotopes.Original Russian Text Copyright © 2004 by Biologiya Morya, Selin, Vekhova.     

Characterization of Na,K-ATPase genes in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>) and comparative genomic organization with rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

A combination of molecular and in silico approaches was employed to assemble a survey of Na, K-ATPase genes contained in the ancestrally tetraploid genome of the Atlantic salmon (Salmo salar). Molecular characterization of genomic clones coding for the subunit revealed two single genes (1a and 2) and two pairs of presumably homeologous genes (1b/i-ii and 1c/i-ii). Each of the six genes showed high sequence similarity to isoforms previously isolated from rainbow trout and extensive structural differences relative to putative orthologs in the human genome. In silico analysis of expressed sequence tag (EST) collections indicated that at least five (1a, 1b, 1c, 2, and 3) and four (1a, 1b, 2, and 3b) subunit isoforms are expressed in Atlantic salmon. Meiotic linkage analysis further showed that Na, K-ATPase genes are dispersed throughout the salmon genome, with the exception of two multigene clusters on linkage groups AS-22 and AS-28. Duplicate gene copies for the isoform 1b were assigned to linkage groups with multiple homeologous anchors (AS-22 and AS-23), while 2 duplicates suggested a new homeologous affinity between AS-05 and AS-28. In addition, the comparison of linkage arrangements with rainbow trout also showed that the genomic organization of Na, K-ATPase genes is consistent with the evolutionary conservation of syntenic chromosome regions between these species.Electronic Supplementary Material Supplementary material is available for this article at .     

Structural diversity of O-polysaccharides and serological classification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">garcae</Emphasis> and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I:3)--L Rha (12)-- L Rha (12)--L-Rha-(13)--L Rha (1;II: 2)--L-Rha-(13) -L-Rha-(12)--L-Rha-(13)--L Rha (1.The branched O-polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588^T have the same L-rhamnan backbone with repeating units I and II and a lateral chain of 14)- or 13)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral -D-Fuc3NAc residues were characterized.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 777–789.Original Russian Text Copyright © 2004 by Ovod, Zdorovenko, Shashkov, Kocharova, Knirel.