Mutation accumulation in <Emphasis Type="Italic">Tetrahymena</Emphasis>

Background  

The rate and fitness effects of mutations are key in understanding the evolution of every species. Traditionally, these parameters are estimated in mutation accumulation experiments where replicate lines are propagated in conditions that allow mutations to randomly accumulate without the purging effect of natural selection. These experiments have been performed with many model organisms but we still lack empirical estimates of the rate and effects of mutation in the protists.     

Random segment deletion based on IS<Emphasis Type="Italic">31831</Emphasis> and Cre/<Emphasis Type="Italic">loxP</Emphasis> excision system in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

A simple and random genome deletion method combining insertion sequence (IS) element IS31831 and the Cre/loxP excision system generated 42 Corynebacterium glutamicum mutants (0.2–186 kb). A total of 393.6 kb (11.9% of C. glutamicum R genome) coding for 331 genes was confirmed to be nonessential under standard laboratory conditions. The deletion strains, generated using only two vectors, varied not only in their lengths but also the location of the deletion along the C. glutamicum R genome. By comparing and analyzing the generated deletion strains, identification of nonessential genes, the roles of genes of hitherto unknown function, and gene–gene interactions can be easily and efficiently determined. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Diversity and recombination in <Emphasis Type="Italic">Wolbachia</Emphasis> and <Emphasis Type="Italic">Cardinium</Emphasis> from <Emphasis Type="Italic">Bryobia</Emphasis>spider mites

BACKGROUND: Wolbachia and Cardinium are endosymbiotic bacteria infecting many arthropods and manipulating host reproduction. Although these bacteria are maternally transmitted, incongruencies between phylogenies of host and parasite suggest an additional role for occasional horizontal transmission. Consistent with this view is the strong evidence for recombination in Wolbachia, although it is less clear to what extent recombination drives diversification within single host species and genera. Furthermore, little is known concerning the population structures of other insect endosymbionts which co-infect with Wolbachia, such as Cardinium. Here, we explore Wolbachia and Cardinium strain diversity within nine spider mite species (Tetranychidae) from 38 populations, and quantify the contribution of recombination compared to point mutation in generating Wolbachia diversity. RESULTS: We found a high level of genetic diversity for Wolbachia, with 36 unique strains detected (64 investigated mite individuals). Sequence data from four Wolbachia genes suggest that new alleles are 7.5 to 11 times more likely to be generated by recombination than point mutation. Consistent with previous reports on more diverse host samples, our data did not reveal evidence for co-evolution of Wolbachia with its host. Cardinium was less frequently found in the mites, but also showed a high level of diversity, with eight unique strains detected in 15 individuals on the basis of only two genes. A lack of congruence among host and Cardinium phylogenies was observed. CONCLUSIONS: We found a high rate of recombination for Wolbachia strains obtained from host species of the spider mite family Tetranychidae, comparable to rates found for horizontally transmitted bacteria. This suggests frequent horizontal transmission of Wolbachia and/or frequent horizontal transfer of single genes. Our findings strengthens earlier reports of recombination for Wolbachia, and shows that high recombination rates are also present on strains from a restrictive host range. Cardinium was found co-infecting several spider mite species, and phylogenetic comparisons suggest also horizontal transmission of Cardinium among hosts.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Revealing the anti-HRP epitope in <Emphasis Type="Italic">Drosophila</Emphasis> and <Emphasis Type="Italic">Caenorhabditis</Emphasis>

Antibodies are very often used as specific cell and/or tissue markers. An example of this is anti-horseradish peroxidase (HRP), an antibody raised against a plant glycoprotein, which was shown some twenty-five years ago to specifically stain neural tissue in an animal, Drosophila melanogaster. This peculiar finding was later expanded to other invertebrate species including Caenorhabditis elegans, which were also shown to bear anti-HRP epitopes. Initial experiments indicated that the epitopes recognised by anti-HRP in invertebrates are of carbohydrate nature. Indeed, more recent experiments have characterised relevant core α1-3-fucosylated N-glycan structures that act as epitopes in various model and parasitic organisms. Moreover, a number of enzymes required for the synthesis of such structures have been identified. Over the years, medically-relevant roles of these structures have become apparent as regards allergenicity and immunoregulation. Although major advances have been made in understanding of the underlying mechanisms and structures related to the anti-HRP epitope, the in vivo role of the relevant epitopes in neural and other tissues is yet to be resolved. Current understanding of the anti-HRP epitopes synthesis and their relevance is discussed and elaborated.

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Establishment of an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation system for <Emphasis Type="Italic">Fortunella crassifolia</Emphasis>

Epicotyl segments of kumquat (Fortunella crassifolia Swingle cv. Jindan) were transformed with Agrobacterium tumefaciens GV3101 harboring neomycin phosphotransferase gene (npt II) containing plant expression vectors. Firstly, the explants were cultured in darkness at 25 °C on kanamycin free shoot regeneration medium (SRM) for 3 d, and then on SRM supplemented with 25 mg dm⁻³ kanamycin and 300 mg dm⁻³ cefotaxime for 20 d. Finally, they were subcultured to fresh SRM containing 50 mg dm⁻³ kanamycin monthly and grown under 16-h photoperiod. Sixty five kanamycin resistant shoots were regenerated from 500 epicotyl explants after four-month selection. Shoot tips of 20 strong shoots were grafted to 50-day-old kumquat seedlings and survival rate was 55 %. Among the 11 whole plants, 3 were transgenic as confirmed by Southern blotting. This is the first report on transgenic kumquat plants, and a transformation efficiency of 3.6 % was achieved.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Regulation of stipule development by <Emphasis Type="Italic">COCHLEATA</Emphasis> and <Emphasis Type="Italic">STIPULE</Emphasis>-<Emphasis Type="Italic">REDUCED</Emphasis> genes in pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.     

Responses of the ciliates <Emphasis Type="Italic">Tetrahymena</Emphasis> and <Emphasis Type="Italic">Paramecium</Emphasis> to external ATP and GTP

The unicellular ciliates Paramecium and Tetrahymena are the simplest eukaryotic cells to show reliable depolarizing responses to micromolar concentrations of external ATP and GTP. Their simplicity allows for combined analysis of swimming behavior, electrophysiology, receptor binding, behavioral mutant and drug screens as well as molecular genetic approaches such as RNAi and gene knockouts experiments. ATP and GTP are depolarizing chemorepellents in both ciliates, producing measurable receptor potentials and Ca²⁺-based action potentials that are correlated with jerking behaviors called avoiding reactions (AR). GTP also causes repetitive continuous ciliary reversals (CCR) and oscillating plateau depolarizations in Paramecium. Both ciliates show high affinity, saturable external binding of ³²P-GTP and ³²P-ATP but GTP does not compete for ATP binding and vice versa. Chemosensory adaptation occurs after continued exposure (15 min) to these ligands, producing a loss of external binding and forward swimming. However, cells adapted to ATP still bind and respond to GTP and GTP-adapted cells still bind and respond to ATP. This, combined with pharmacological analyses, suggests that there are two separate receptor systems: A metabotropic ATP receptor pathway and a different, novel GTP receptor pathway. A Paramecium mutant (ginA) lacks the GTP-induced oscillating depolarizations but does show AR in GTP, unveiling isolated GTP-receptor potentials for study. An ecto-ATPase is also present that may be involved in inactivation of ATP and GTP signals. Gene knockout experiments are currently underway to determine the roles of the ecto-ATPase and a putative 7-transmembrane spanning receptor in these responses.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Effect of <Emphasis Type="Italic">cra</Emphasis> gene knockout together with <Emphasis Type="Italic">edd</Emphasis> and <Emphasis Type="Italic">iclR</Emphasis> genes knockout on the metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved. Dayanidhi Sarkar and Khandaker Al Zaid Siddiquee have contributed equally.     

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.     

Small phosphatidate phosphatase (<Emphasis Type="Italic">TtPAH2</Emphasis>) of <Emphasis Type="Italic">Tetrahymena</Emphasis> complements respiratory function and not membrane biogenesis function of yeast <Emphasis Type="Italic">PAH1</Emphasis>

Phosphatidate phosphatases (PAH) play a central role in lipid metabolism and intracellular signaling. Herein, we report the presence of a low-molecular-weight PAH homolog in the single-celled ciliate Tetrahymena thermophila. In vitro phosphatase assay showed that TtPAH2 belongs to the magnesium-dependent phosphatidate phosphatase (PAP1) family. Loss of function of TtPAH2 did not affect the growth of Tetrahymena. Unlike other known PAH homologs, TtPAH2 did not regulate lipid droplet number and ER morphology. TtPAH2 did not rescue growth and ER/nuclear membrane defects of the pah1? yeast cells, suggesting that the phosphatidate phosphatase activity of the protein is not sufficient to perform these cellular functions. Surprisingly, TtPAH2 complemented the respiratory defect in the pah1? yeast cells indicating a specific role of TtPAH2 in respiration. Overall, our results indicate that TtPAH2 possesses the minimal function of PAH protein family in respiration. We suggest that the amino acid sequences absent from TtPAH2 but present in all other known PAH homologs are critical for lipid homeostasis and membrane biogenesis.     

Horizontal transfer of the C-termini of <Emphasis Type="Italic">tccC</Emphasis> genes in <Emphasis Type="Italic">Photorhabdus</Emphasis> and <Emphasis Type="Italic">Xenorhabdus</Emphasis>

The high molecular weight insecticidal toxin complexes (Tcs), including four toxin-complex loci (tca, tcb, tcc and tcd), were first identified in Photorhabdus luminescens W14. Each member of tca, tcb or tcc is required for oral toxicity of Tcs. However, the sequence sources of the C-termini of tccC3, tccC4, tccC6 and tccC7 are unknown. Here, we performed a whole genome survey to identify the orthologs of Tc genes, and found 165 such genes in 14 bacterial genomes, including 40 genes homologous to tccC1-7 in P. luminescens TT01. The sequence sources of the C-termini of tccC2-6 were determined by sequence analysis. Further phylogenetic investigations suggested that the C-termini of 6 tccC genes experienced horizontal gene transfer events.     

<Emphasis Type="Italic">IGHV1</Emphasis>,<Emphasis Type="Italic"> IGHV5</Emphasis> and<Emphasis Type="Italic"> IGHV7</Emphasis> subgroup genes in the Rhesus macaque

The diversity of the antibody response is achieved, in part, by rearrangement of different immunoglobulin (Ig) genes. The Ig heavy chain is made up of a variable region (IGHV), a diversity region (IGHD) and a joining region (IGHJ). Human germline IGHV genes have been grouped into seven multigene subgroups. Size and usage of these subgroups is not equal, the IGHV3 subgroup is the most commonly used (36%), followed by IGHV1/7 (26%), then IGHV4, IGHV5, IGHV2, IGHV6 (15%, 12%, 4%, 3% respectively). The rhesus macaque (Macaca mulatta) is a useful non-human primate model for studies of infection and the database of germline Ig genes for the macaque is gradually growing to become a useful tool in the study of B-cell responses. The proportions of IGHV subgroup usage in the macaque are similar to those in man. Representatives from IGHV3 and IGHV4 subgroups for the macaque have been published, as have germline sequences of the IGHD and IGHJ genes. However, to date there have been no sequences published from the second largest IGHV subgroup, IGHV1. We report the isolation and sequencing of a genomic fragment containing an IGHV1 gene from the macaque. Polymerase chain reaction (PCR) primers designed from this sequence enabled us to amplify and sequence 25 new IGHV1 germline genes. We also isolated two IGHV7 genes, using the same primers, and two IGHV5 genes, using human IGHV5 primers.     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

Genomewide analysis of <Emphasis Type="Italic">ABCB</Emphasis>s with a focus on <Emphasis Type="Italic">ABCB1</Emphasis> and <Emphasis Type="Italic">ABCB19</Emphasis> in <Emphasis Type="Italic">Malus domestica</Emphasis>

The B subfamily of ATP-binding cassette (ABC) proteins (ABCB) plays a vital role in auxin efflux. However, no systematic study has been done in apple. In this study, we performed genomewide identification and expression analyses of the ABCB family in Malus domestica for the first time. We identified a total of 25 apple ABCBs that were divided into three clusters based on the phylogenetic analysis. Most ABCBs within the same cluster demonstrated a similar exon–intron organization. Additionally, the digital expression profiles of ABCB genes shed light on their functional divergence. ABCB1 and ABCB19 are two well-studied auxin efflux carrier genes, and we found that their expression levels are higher in young shoots of M106 than in young shoots of M9. Since young shoots are the main source of auxin synthesis and auxin efflux involves in tree height control. This suggests that ABCB1 and ABCB19 may also take a part in the auxin efflux and tree height control in apple.