A PCR-RFLP method on faecal samples to distinguish <Emphasis Type="Italic">Martes martes</Emphasis>, <Emphasis Type="Italic">Martes foina</Emphasis>, <Emphasis Type="Italic">Mustela putorius</Emphasis> and <Emphasis Type="Italic">Vulpes vulpes</Emphasis>

A non-invasive genetic approach has been recently employed in the population genetics of wild species, using faeces that could be easily collected, particularly of elusive or endangered species. However, faeces of pine (Martes martes) and beech marten (M. foina) can be morphologically similar and could be confused with those of polecat (Mustela putorius) and red fox (Vulpes vulpes). On this basis, a rapid, simple and inexpensive RFLP protocol using a cytochrome b (Cyt b) gene fragment of mtDNA, isolated from faecal samples, was performed to distinguish the above-mentioned species.     

Hair density in the Eurasian otter <Emphasis Type="Italic">Lutra lutra</Emphasis> and the Sea otter <Emphasis Type="Italic">Enhydra lutris</Emphasis>

The hair density of adult Eurasian otters Lutra lutra (Linnaeus, 1758) and sea otters Enhydra lutris (Linnaeus, 1758) was analysed using skin samples taken from frozen carcasses. Lutra lutra exhibited a mean hair density of about 70 000 hairs/cm² (whole body, appendages excepted), the mean individual density ranging from about 60 000 to 80 000 hairs/cm². The dominant hair type were secondary hairs (wool hairs), the hair coat comprising only 1.26% of primary hairs (PH). Secondary hair (SH) density remained constant over the body (appendages excepted), whereas a few variations in PH density were observed. Neither an influence of the sex, nor a seasonal variation of the hair coat was found, moulting seems to be continuous. Enhydra lutris had a hair density between 120 000 and 140 000 hairs/cm², the primary hairs representing less than 1% within the hair coat. Hair density remained quite constant over the regions of the trunk but was lower at the head (about 60 000 hairs/cm² on the cheek). The hair follicles were arranged in specific groups with different bundles of varying size, normally comprising dominant numbers of wool hair (SH) follicles. Invariably there was always a large central primary hair follicle and numerous sebaceous glands between the bundles and principally around the PH follicles. The results are discussed related to possible ecological influences on hair coat density.     

<Emphasis Type="Italic">FORMOSA</Emphasis> controls cell division and expansion during floral development in <Emphasis Type="Italic">Antirrhinum</Emphasis><Emphasis Type="Italic">majus</Emphasis>

Control of organ size is the product of coordinated cell division and expansion. In plants where one of these pathways is perturbed, organ size is often unaffected as compensation mechanisms are brought into play. The number of founder cells in organ primordia, dividing cells, and the period of cell proliferation determine cell number in lateral organs. We have identified the Antirrhinum FORMOSA (FO) gene as a specific regulator of floral size. Analysis of cell size and number in the fo mutant, which has increased flower size, indicates that FO is an organ-specific inhibitor of cell division and activator of cell expansion. Increased cell number in fo floral organs correlated with upregulation of genes involved in the cell cycle. In Arabidopsis the AINTEGUMENTA (ANT) gene promotes cell division. In the fo mutant increased cell number also correlates with upregulation of an Antirrhinum ANT-like gene (Am-ANT) in inflorescences that is very closely related to ANT and shares a similar expression pattern, suggesting that they may be functional equivalents. Increased cell proliferation is thought to be compensated for by reduced cell expansion to maintain organ size. In Arabidopsis petal cell expansion is inhibited by the BIGPETAL (BPE) gene, and in the fo mutant reduced cell size corresponded to upregulation of an Antirrhinum BPE-like gene (Am-BPE). Our data suggest that FO inhibits cell proliferation by negatively regulating Am-ANT, and acts upstream of Am-BPE to coordinate floral organ size. This demonstrates that organ size is modulated by the organ-specific control of both general and local gene networks. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Impact of <Emphasis Type="Italic">cry1AC</Emphasis>-carrying <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> on leaf bacterial community

The effects of Chinese cabbage (Brassica rapa subsp. pekinensis) carrying cry1AC derived from Bacillus thuringiensis (Bt) on leaf bacterial community were examined by analyzing the horizontal transfer of trans-gene fragments from plants to bacteria. The effect of plant pathogenic bacteria on the gene transfer was also examined using Pseudomonas syringae pathovar. maculicola. The frequency of hygromycin-resistant bacteria did not alter in Bt leaves, though slight increase was observed in Pseudomonas-infected Bt leaves with no statistical significance. The analysis of bacterial community profiles using the denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated that there were slight differences between Bt and control Chinese cabbage, and also that infected tissues were dominated by P. syringae pv. maculicola. However, the cultured bacterial pools were not found to contain any transgene fragments. Thus, no direct evidence of immediate gene transfer from plant to bacteria or acquisition of hygromycin resistance could be observed. Still, long-term monitoring on the possibility of gene transfer is necessary to correctly assess the environmental effects of the Bt crop on bacteria.     

Dynamics of <Emphasis Type="Italic">Vulmar/VulMITE</Emphasis> group of transposable elements in <Emphasis Type="Italic">Chenopodiaceae</Emphasis> subfamily <Emphasis Type="Italic">Betoideae</Emphasis>

Transposable elements are important factors driving plant genome evolution. Upon their mobilization, novel insertion polymorphisms are being created. We investigated differences in copy number and insertion polymorphism of a group of Mariner-like transposable elements Vulmar and related VulMITE miniature inverted-repeat transposable elements (MITEs) in species representing subfamily Betoideae. Insertion sites of these elements were identified using a modified transposon display protocol, allowing amplification of longer fragments representing regions flanking insertion sites. Subsequently, a subset of TD fragments was converted into insertion site-based polymorphism (ISBP) markers. The investigated group of transposable elements was the most abundant in accessions representing the section Beta, showing intraspecific insertion polymorphisms likely resulting from their recent activity. In contrast, no unique insertions were observed for species of the genus Beta section Corollinae, while a set of section-specific insertions was observed in the genus Patellifolia, however, only two of them were polymorphic between P. procumbens and P. webbiana. We hypothesize that Vulmar and VulMITE elements were inactivated in the section Corollinae, while they remained active in the section Beta and the genus Patellifolia. The ISBP markers generally confirmed the insertion patterns observed with TD markers, including presence of distinct subsets of TE insertions specific to Beta and Patellifolia.     

A reliable genetic technique for sex determination of giant panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>) from non-invasively collected hair samples

Extractions from non-invasive hair samples usually yield low amounts of highly degraded DNA. Previously developed mammal molecular sexing methods were not designed with such sub-optimal conditions in mind. We developed a simple and reliable PCR-based sexing method aimed at degraded, low yield DNA extractions from the giant panda (Ailuropoda melanoleuca). Comparisons of this new primer set with others showed that the reliability of sex determination from low-yield, degraded DNA extractions was improved if; amplification products were short (<170 bp); and the Y-chromosome amplification product was shorter than the X-chromosome amplification product. The primers developed in this study appear useful for sex determination in other bear species.     

Targeted knockout and <Emphasis Type="Italic">lacZ</Emphasis> reporter expression of the mouse <Emphasis Type="Italic">Tmhs</Emphasis> deafness gene and characterization of the <Emphasis Type="Italic">hscy-2J</Emphasis> mutation

The Tmhs gene codes for a tetraspan transmembrane protein that is expressed in hair cell stereocilia. We previously showed that a spontaneous missense mutation of Tmhs underlies deafness and vestibular dysfunction in the hurry-scurry (hscy) mouse. Subsequently, mutations in the human TMHS gene were shown to be responsible for DFNB67, an autosomal recessive nonsyndromic deafness locus. Here we describe a genetically engineered null mutation of the mouse Tmhs gene (Tmhs ^tm1Kjn ) and show that its phenotype is identical to that of the hscy missense mutation, confirming the deleterious nature of the hscy cysteine-to-phenylalanine substitution. In the targeted null allele, the Tmhs promoter drives expression of a lacZ reporter gene. Visualization of β-galactosidase activity in Tmhs ^tm1Kjn heterozygous mice indicates that Tmhs is highly expressed in the cochlear and vestibular hair cells of the inner ear. Expression is first detectable at E15.5, peaks around P0, decreases slightly at P6, and is absent by P15, a duration that supports the involvement of Tmhs in stereocilia development. Tmhs reporter gene expression also was detected in several cranial and cervical sensory ganglia, but not in the vestibular or spiral ganglia. We also describe a new nontargeted mutation of the Tmhs gene, hscy-2J, that causes abnormal splicing from a cryptic splice site within exon 2 and is predicted to produce a functionally null protein lacking 51 amino acids of the wild-type sequence.     

Application of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">parE</Emphasis> sequence similarity analyses for differentiation of species within the genus <Emphasis Type="Italic">Geobacillus</Emphasis>

The primary structures of the genes encoding the β-subunits of a type II topoisomerase (gyrase, gyrB) and a type IV topoisomerase (parE) were determined for 15 strains of thermophilic bacteria of the genus Geobacillus. The obtained sequences were used for analysis of the phylogenetic similarity between members of this genus. Comparison of the phylogenetic trees of geobacilli constructed on the basis of the 16S rRNA, gyrB, and parE gene sequences demonstrated that the level of genetic distance between the sequences of the genes encoding the β-subunits of type II topoisomerases significantly exceeded the values obtained by comparative analysis of the 16S rRNA gene sequences of Geobacillus strains. It was shown that, unlike the 16S rRNA gene analysis, comparative analysis of the gyrB and parE gene sequences provided a more precise determination of the phylogenetic position of bacteria at the species level. The data obtained suggest the possibility of using the genes encoding the β-subunits of type II topoisomerases as phylogenetic markers for determination of the species structure of geobacilli.     

Multiplex polymerase chain reaction method discriminating <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Shigella</Emphasis> sp.

To distinguish between Escherichia coli and other bacteria that have similar biochemical characteristics, 3 polymerase chain reaction techniques were combined. The primer sets cydA-F2-A2 and cydA-R2-A2 were designed to amplify 605 base pairs of nucleotide sequence specific for the cydA gene of Escherichia coli; primer sets lacZ-F-A and lacZ-R-A to amplify 1,023 bp of nucleotide sequence specific for the lacZ gene of Escherichia coli; and primers lacA-F2-A2 and lacA-R2-A2 to amplify 325 bp of nucleotide sequence specific for the lacA gene of Escherichia coli. As a result, 3 nucleotide fragments were generated when 3 samples DNA from Escherichia coli were used as template. On the other hand, 1,023- and 605-bp products were obtained when DNA of Shigella sonnei was used, and a 605-bp product was obtained when DNA of Shigella flexneri was used. The specificity of the technique was confirmed by comparing it with the conventional culture test; the consistency rate of both tests was 0.749. These results suggest that the technique described in the present study will be useful for distinguishing Escherichia coli from Shigella species with accuracy and specificity.     

Molecular analysis shows differential expression of <Emphasis Type="Italic">R</Emphasis>-<Emphasis Type="Italic">spondin1</Emphasis> in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) gonads

R-spondin1 (RSPO1) is a potential female-determining gene in human (Homo sapiens) and mouse (Mus musculus). Its differential expression in these mammals is correlated with signaling for sex determination. As a way of studying sex determination in fish we cloned and analyzed a RSPO1 gene in zebrafish (Danio rerio). Using real-time PCR, we observed that RSPO1 is expressed more strongly in ovaries than in testes, suggesting that RSPO1 may have a role in gonad differentiation. High RSPO1 expression was detected in some non-gonadal organs like muscle and kidneys. In situ hybridization results demonstrate that RSPO1 is expressed in premature germ cells, in oogonia and primary oocytes in ovaries and in spermatogonia and spermatocytes in testes. It is also expressed in gonad somatic cells during gonadal development: in granulosa cells and theca cells of early and late cortical-alveolar stage follicles in ovaries, and in Leydig cells in testes. This differential expression may indicate that RSPO1 has a role(s) in zebrafish gonad development and differentiation. By fusing zebrafish RSPO1 with a green fluorescent protein gene, we found that RSPO1 is located in the cytosol and Golgi apparatus but not the nucleus of fish epithelioma papulosum cyprinid (EPC) cells. These preliminary findings suggest some aspects of RSPO1 like differential expression linked to sex determination may be conserved in fish while other aspects like subcellular localization differ from the mammalian RSPO1.     

Functional conservation of the human <Emphasis Type="Italic">EXT1</Emphasis> tumor suppressor gene and its <Emphasis Type="Italic">Drosophila</Emphasis> homolog <Emphasis Type="Italic">tout</Emphasis><Emphasis Type="Italic">velu</Emphasis>

Heparan sulfate proteoglycans play a vital role in signaling of various growth factors in both Drosophila and vertebrates. In Drosophila, mutations in the tout velu (ttv) gene, a homolog of the mammalian EXT1 tumor suppressor gene, leads to abrogation of glycosaminoglycan (GAG) biosynthesis. This impairs distribution and signaling activities of various morphogens such as Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp). Mutations in members of the exostosin (EXT) gene family lead to hereditary multiple exostosis in humans leading to bone outgrowths and tumors. In this study, we provide genetic and biochemical evidence that the human EXT1 (hEXT1) gene is conserved through species and can functionally complement the ttv mutation in Drosophila. The hEXT1 gene was able to rescue a ttv null mutant to adulthood and restore GAG biosynthesis.     

Polymorphism of the <Emphasis Type="Italic">CONSTANS</Emphasis> gene in <Emphasis Type="Italic">Brassica</Emphasis> plants

Using a direct amplification of genomic DNA from two Brassica rapa forms, we obtained two homologs of the CONSTANS gene, which controls the photoperiodic induction of flowering in Arabidopsis plants. The cloned fragments of B. rapa genome were identified as members of the CONSTANS-LIKE1 class. By aligning the nucleotide sequences of the CONSTANS gene and its homologs, three classes, CONSTANS, CONSTANS-LIKE1, and CONSTANS-LIKE2, were distinctly discerned by their primary structure. The pattern of restriction fragment length polymorphisms (RFLP) of the CONSTANS homologs in B. carinata, B. juncea, B. napus, B. nigra, B. oleracea, and B. rapa were genome-specific; in addition, the CONSTANS homologs were classified by plant geographic origin, and we assume that such classification is related to plant photoperiodic response.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 274–281.Original Russian Text Copyright © 2005 by Martynov, Khavkin.This revised version was published online in April 2005 with a corrected cover date.     

Screening of population genetic SCAR markers for mykiss <Emphasis Type="Italic">Parasalmo</Emphasis> (<Emphasis Type="Italic">Oncorhynchus</Emphasis>) <Emphasis Type="Italic">mykiss</Emphasis> from Kamchatka

Using AP-PCR, the genome of Kamchatka mykiss (Parasalmo (O.) mykiss) was examined. Polymorphic fragments, implying geographic differences among the samples, were selected, cloned, and sequenced. Based on these sequences, longer, specific SCAR primers were selected and constructed. Using the BLAST software program, the sequences were analyzed for analogy to those from the GenBank database. It seemed likely that all sequences obtained belonged to earlier unexamined repeated sequences, variable in the populations of the species of interest. A total of seven SCAR markers, characterized by population-significant variability of the DNA products in Kamchatka geographic group of rainbow trout were constructed. These markers can be used for further investigation of the species Parasalmo (O.) mykiss. The SCAR marker sequences were deposited in GenBank under the accession numbers EU805500 to EU805506.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Polyhydroxybutyrate in <Emphasis Type="Italic">Rhizobium</Emphasis> and <Emphasis Type="Italic">Bradyrhizobium</Emphasis>: quantification and <Emphasis Type="Italic">phbC</Emphasis> gene expression

Polyhydroxyalkanoates (PHAs) are hydroxyalkanoate polymers that are produced and accumulate by many kinds of bacteria. These polymers act as an energy store for bacteria. Polyhydroxybutyrate (PHB) is the most studied polymer in the PHA family. These polymers have awakened interest in the environmental and industrial research areas because they are biodegradable and have thermoplastic qualities, like polypropylene. In this work, we analyzed the PHB production in Bradyrhizobium sp., Rhizobium leguminosarum bv. phaseoli, and Rhizobium huautlense cultured with two different carbon sources. We did biochemical quantification of PHB production during the three phases of growth. Moreover, these samples were used for RNA extraction and phbC gene expression analysis via real-time PCR. The bacteria showed different manner of growth, PHB accumulation and phbC gene expression when different quantity and quality of carbon sources were used. These results showed that under different growth media conditions, the growth and metabolism of different species of bacteria were influenced. These differences reflect the increase or decrease in PHB accumulation.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies.