Acceleration of Senescence induced in Wheat by Light

CELLS from patients with G-trisomy or E-trisomy and XXY cells from patients with XY/XXY mosaic Klinefelter's syndrome are more susceptible to transformation in vitro by SV40 than are cells from normal individuals^1–3. We have used triploid (69XXY) human cells to determine whether the presence of extra chromosomes per se increases susceptibility to transformation.     

Expression and DNA methylation pattern of reproduction-related genes in partially fertile triploid Pacific oysters <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

Partial or complete sterility is an obvious feature in triploid Pacific oyster (Crassostrea gigas) which contributes to improving rearing performances. Despite the significance of sterility, the molecular mechanism behind it remains elusive and related research was limited. This study focused on six reproduction-related genes and compared their different behavior in gene expression and DNA methylation pattern between triploid and diploid oysters in order to provide more molecular information. The gonadal development of triploid oyster was examined by histology before molecular analysis. Gametogenesis disturbance was observed in triploid oysters at different development stages (stage II and III) with more serious impairment in females. QPCR showed significant gene expression difference between diploid and triploid in two genes: putative Vg and cgER. Gene expression of putative Vg was delayed in triploids while for cgER triploid oyster showed higher expression and the difference was significant at stage III. DNA methylation pattern of these two genes were further investigated by bisulfite sequencing. Between diploid and triploid oysters, no difference was observed in total methylation level but some individual loci showed different patterns: significantly high methylation rate of loci 2284 in cgER was observed in triploid oyster which has a higher expression of this gene. This study indicated that putative Vg and cgER might play a role in partial sterile in triploid C. gigas. Gene expression could be regulated by the methylation pattern at specific individual locus, which deserves equivalent attention as well as total DNA methylation level.     

The <Emphasis Type="Italic">alm1</Emphasis><Superscript><Emphasis Type="Italic">+</Emphasis></Superscript> gene from <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> encodes a coiled-coil protein that associates with the medial region during mitosis

We have isolated a cDNA that encodes a 142-kDa protein by immunoscreening of a Schizosaccharomyces pombe expression library with a new antibody, mAb8, that reveals spindle poles and equatorial ring-like structures in several organisms. This cDNA encodes a putative protein which we termed Alm (for abnormal long morphology). The protein is predicted to be a coiled-coil protein, containing a central α-helical domain flanked by non-helical terminal domains. Immunofluorescence analysis showed that Alm1 is localized in the medial region of the cell from anaphase to the end of cytokinesis. Cells carrying an alm1::ura4 ⁺ disruption are viable and exhibit an elongated morphology. Homozygous alm1::ura4 ⁺ diploids sporulated normally but the spores did not germinate. Spores that have inherited the disruption allele from a heterozygous alm1 ⁺ / alm1::ura4 ⁺ diploid germinated but generated smaller colonies. We propose that Alm1 participates in the structural organization of the medial region in S. pombe.     

Embryological and genetic evidence of amphimixis and apomixis in <Emphasis Type="Italic">Boehmeria tricuspis</Emphasis>

Apomixis is a widespread alternative mode of sexual reproduction resulting in offspring that are genetically identical to the maternal plant. Boehmeria tricuspis (Hance) Makino is a perennial, wind-pollinated, herbaceous plant in the nettle family Urticaceae. The diploid B. tricuspis is monoecious but the triploid B. tricuspis is gynoecious, bearing female inflorescences only. Apomixis in B. tricuspis was first reported 50 years ago, but the mode of apomixis in the species has not been described yet. Here, we provide embryological observations of the embryo sac formation proving that triploid B. tricuspis reproduced apomictically following the Antennaria type of diplospory, and that the diploid individuals were the sexual genotype with the classical Polygonum-type maturation pattern of embryo sac development. A subsequent flow cytometry seed screen (FCSS) showed that the triploids were obligate apomicts with autonomous endosperm development, and the diploids reproduced sexually. In addition, a progeny test by molecular marker assays further demonstrated the above results.     

Hybridization success is largely limited to homoploid <Emphasis Type="Italic">Prunus</Emphasis> hybrids: a multidisciplinary approach

Prunus fruticosa is a rare shrub occurring in Eurasian thermophilous forest-steppe alliances. The species frequently hybridizes with cultivated Prunus species in Europe (allochthonous tetraploid P. cerasus and partly indigenous diploid P. avium). Propidium iodide flow cytometry, distance-based morphometrics, elliptic Fourier analysis and embryology were employed to evaluate the extent of hybridization in six Slovak populations. Flow cytometric analyses revealed three ploidy levels: diploid (P. avium), triploid (P. × mohacsyana) and tetraploid (P. fruticosa, P. × eminens and P. cerasus). In addition, P. fruticosa and P. cerasus, at the tetraploid level, were found to differ in absolute genome size. An embryological evaluation suggested the existence of a triploid block in P. × mohacsyana and significant potential for hybridization among tetraploid taxa (indicated also by a continuous distribution of genome size data and further mirrored by morphometrics). Although hybrids significantly differ in ploidy level and embryological characteristics, they are almost indistinguishable using morphological characters. Hybridization with P. cerasus thus turns out to be a significant threat to wild populations of P. fruticosa compared to the relatively weak influence of P. avium.     

Thermotolerance and place memory in adult <Emphasis Type="Italic">Drosophila</Emphasis> are independent of natural variation at the <Emphasis Type="Italic">foraging</Emphasis> locus

Natural polymorphisms at the foraging (for) gene influence several behaviors. However, it is seldom clear how different for alleles could be selected. In one case, Drosophila with the rover allele (for ^r ) have higher locomotor activity in the presence of food than animals with the sitter allele (for ^s ), suggesting a complementary feeding strategy. There are, in addition, differences between for ^r and for ^s Drosophila in some tests of short-term memory (for ^r animals generally perform at higher levels) and thermotolerance (for ^s larvae are more resistant to the effects of high-temperature). We asked whether there could be a direct compensating advantages in adult for ^s flies that could maintain the natural for variants. First, are adult for ^s flies more thermotolerant? Second, do for ^r flies have a higher short-term place memory? Third, as an alternative, might for ^s flies have higher place memory? Our results do not confirm these possibilities. Thus, a thermotolerance advantage of for ^s flies does not compensate for a potential for ^r short-term memory advantage; for ^r flies do not have a universal advantage in short-term memory; and for ^s flies do not have an advantage in place memory that could compensate for for ^r advantages in other learning contexts.     

Distinct mechanisms of phenotypic effects of inactivation and prionization of Swi1 protein in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Prions are proteins that under the same conditions can exist in two or more conformations, and at least one of the conformations has infectious properties. The prionization of a protein is typically accompanied by its functional inactivation due to sequestration of monomers by the prion aggregates. The most of prions has been identified in the yeast Saccharomyces cerevisiae. One of them is [SWI ⁺], a prion isoform of the Swi1 protein, which is a component of the evolutionarily conserved chromatin remodeling complex SWI/SNF. Earlier, it was shown that the prionization of [SWI ⁺] induces a nonsense suppression, which leads to weak growth of the [SWI ⁺] strains containing mutant variants of the SUP35 gene and the nonsense allele ade1-14 _UGA on selective medium without adenine. This effect occurs because of [SWI ⁺] induction that causes a decrease in the amount of the SUP45 mRNA. Strains carrying the SWI1 deletion exhibit significantly higher suppression of the ade1-14 _UGA nonsense mutation than the [SWI ⁺] strains. In the present study, we identified genes whose expression is altered in the background of the SWI1 deletion using RNA sequencing. We found that the ade1-14 _UGA suppression in the swi1Δ strains is caused by an increase in the expression of this mutant allele of the ADE1 gene. At the same time, the SUP45 expression level in the swi1Δ strains does not significantly differ from the expression level of this gene in the [swi ^–] strains. Thus, we have shown that the phenotypic effects of Swi1 prionization and deletion are mediated by different molecular mechanisms. Based on these data, we have concluded that the prionization of proteins is not only unequal to their inactivation, but also can lead to the acquisition of novel phenotypic effects and functions.     

Inactivation of bone morphogenetic protein 1 (<Emphasis Type="Italic">Bmp1</Emphasis>) and tolloid-like 1 (<Emphasis Type="Italic">Tll1</Emphasis>) in cells expressing type I collagen leads to dental and periodontal defects in mice

Bone morphogenetic protein 1 (BMP1) and tolloid-like 1 (TLL1) belong to the BMP1/tolloid-like proteinase family, which cleaves secretory proteins. The constitutive deletion of the Bmp1 or Tll1 genes causes perinatal or embryonic lethality in mice. In this study, we first studied the β-galactosidase activity in mice in which an IRES-lacZ-Neo cassette was inserted in the intron of either the Bmp1 or the Tll1 gene; the β-galactosidase activities were used to reflect the expression of endogenous Bmp1 and Tll1, respectively. Our X-gal staining results showed that the odontoblasts in the tooth and cells in the periodontal ligament express both Bmp1 and Tll1. We then created Bmp1 ^flox/flox and Tll1 ^flox/flox mice by removing the IRES-lacZ-Neo cassette. By breeding 2.3 kb Col1a1-Cre mice with the Bmp1 ^flox/flox and Tll1 ^flox/flox mice, we further generated Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice in which both Bmp1 and Tll1 were inactivated in the Type I collagen-expressing cells. We employed X-ray radiography, histology and immunohistochemistry approaches to characterize the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice. Our results showed that the molars of the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice had wider predentin, thinner dentin and larger pulp chambers than those of the normal controls. The dentinal tubules of the molars in the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice appeared disorganized. The level of dentin sialophosphoprotein in the molars of the 6-week-old Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice was lower than in the normal controls. The periodontal ligaments of the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice were disorganized and had less fibrillin-1. Our findings indicate that the proteinases encoded by Bmp1 and Tll1 genes play essential roles in the development and maintenance of mouse dentin and periodontal ligaments.     

Search for genes influencing the maintenance of the [<Emphasis Type="Italic">ISP</Emphasis><Superscript>+</Superscript>] prion-like antisuppressor determinant in yeast with the use of an insertion gene library

The prion-like determinant [ISP ⁺] manifests itself as an antisuppressor of certain sup35 mutations. To establish that [ISP ⁺] is indeed a new yeast prion, it is necessary to identify the gene that codes for the protein whose prion form is [ISP ⁺]. Analysis of the transformants obtained by transformation of an [ISP ⁺] strain with an insertion gene library revealed three genes controlling the [ISP ⁺] maintenance: UPF1, UPF2, and SFP1. SFP1 codes for a potentially prionogenic protein, which is enriched in Asn and Gln residues, and is thereby the most likely candidate for the [ISP ⁺] structural gene. UPF1 and UPF2 code for components of nonsense-mediated mRNA decay. The [ISP ⁺] elimination caused by UPF1 and UPF2 inactivation was reversible, and Upf1p and Upf2p were not functionally related to phosphatase Ppz1p, which influences the [ISP ⁺] manifestation. Possible mechanisms sustaining the influence of UPF1 and UPF2 on [ISP ⁺] maintenance are discussed.     

Growth and wax ester production of an <Emphasis Type="Italic">Acinetobacter baylyi</Emphasis> ADP1 mutant deficient in exopolysaccharide capsule synthesis

Acinetobacter baylyi ADP1 naturally produces wax esters that could be used as a raw material in industrial applications. We attempted to improve wax ester yield of A. baylyi ADP1 by removing rmlA, a gene involved in exopolysaccharide production. Growth rate, biomass formation and wax ester yield on 4-hydroxybenzoate were not affected, but the rmlA ^? strain grew slower on acetate, while reaching similar biomass and wax ester yield. The rmlA ^? cells had malformed shape and large size and grew poorly on glucose without expression of the gene for pyruvate kinase (pykF) from Escherichia coli. The pykF-expressing rmlA ^? strain had similar growth rate, lowered biomass formation and improved wax ester production on glucose as compared to the wild-type strain expressing pykF. Cultivation of the pykF-expressing rmlA ^? strain on an elevated glucose concentration in a medium supplemented with amino acids resulted in doubled molar wax ester yield and acetate production.     

Modulation of proline metabolic gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> under water-stressed conditions by a drought-mitigating <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strain

Although amelioration of drought stress in plants by plant growth promoting rhizobacteria (PGPR) is a well reported phenomenon, the molecular mechanisms governing it are not well understood. We have investigated the role of a drought ameliorating PGPR strain, Pseudomonas putida GAP-P45 on the regulation of proline metabolic gene expression in Arabidopsis thaliana under water-stressed conditions. Indeed, we found that Pseudomonas putida GAP-P45 alleviates the effects of water-stress in A. thaliana by drastic changes in proline metabolic gene expression profile at different time points post stress induction. Quantitative real-time expression analysis of proline metabolic genes in inoculated plants under water-stressed conditions showed a delayed but prolonged up-regulation of the expression of genes involved in proline biosynthesis, i.e., ornithine-Δ-aminotransferase (OAT), Δ ¹ -pyrroline-5-carboxylate synthetase1 (P5CS1), Δ ¹ -pyrroline-5-carboxylate reductase (P5CR), as well as proline catabolism, i.e., proline dehydrogenase1 (PDH1) and Δ ¹ -pyrroline-5-carboxylate dehydrogenase (P5CDH). These observations were positively correlated with morpho-physiological evidences of water-stress mitigation in the plants inoculated with Pseudomonas putida GAP-P45 that showed better growth, increased fresh weight, enhanced plant water content, reduction in primary root length, enhanced chlorophyll content in leaves, and increased accumulation of endogenous proline. Our observations point towards PGPR-mediated enhanced proline turnover rate in A. thaliana under dehydration conditions.     

Analysis of compound heterozygotes reveals that the mouse floxed <Emphasis Type="Italic">Pax6</Emphasis><Superscript><Emphasis Type="Italic">tm1Ued</Emphasis></Superscript> allele produces abnormal eye phenotypes

Analysis of abnormal phenotypes produced by different types of mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are not equivalent to wild-type alleles and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6 ^tm1Ued (Pax6 ^fl ) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6 ^fl/fl and heterozygous Pax6 ^fl/+ mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6 ^fl/fl corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6 ^Sey-Neu (Pax6 ^?) null allele. Pax6 ^fl/? compound heterozygotes had more severe eye abnormalities than Pax6 ^+/? heterozygotes, implying that Pax6 ^fl differs from the wild-type Pax6 ⁺ allele. Immunohistochemistry showed that the Pax6 ^fl/? corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6 ^fl allele provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic effects of floxed alleles.     

Allelic differentiation at the <Emphasis Type="Italic">Sg-1</Emphasis> locus for the terminal sugar of the C-22 position of group A saponin in Chinese wild soybean (<Emphasis Type="Italic">Glycine soja</Emphasis> Sieb. &amp; Zucc.)

Group A saponins are thought to be the cause of bitter and astringent tastes in processed foods of soybean (Glycine max), and the elimination of group A saponins is an important breeding objective. The group A saponins include two main Aa and Ab types, controlled by codominant alleles at the Sg-1 locus that is one of several key loci responsible for saponin biosynthesis in the subgenus Glycine soja. However, A0 mutant lacking group A saponin is a useful gene resource for soybean quality breeding. Here, eight Chinese wild soybean A0 accessions were sequenced to reveal the mutational mechanisms, and the results showed that these mutants were caused by at least three kinds of mechanisms involving four allelic variants (sg-1^0-b2, sg-1^0-b3, Sg-1^b-0, and Sg-1^b-01). The sg-1^0-b2 had two nucleotide deletions at positions +?72 and +?73 involving in the 24th and 25th amino acids. The sg-1^0-b3 contained a stop codon (TGA) at the 254th residue. The Sg-1^b-0 and Sg-1^b-01 were two novel A0-type mutants, which likely carried normal structural alleles, and nevertheless did not encode group A saponin due to unknown mutations beyond the normal coding regions. In addition, to reveal the structural features, allelic polymorphism, and mechanisms of the abiogenetic absence of group A (i.e., A0 phenotype), nucleotide sequence analysis was performed for the Sg-1 locus in wild soybean (Glycine soja). The results showed that Sg-1 alleles had a lower conservatism in the coding region; as high as 18 sequences were found in Chinese wild soybeans in addition to the Sg-1^a (Aa) and Sg-1^b (Ab) alleles. Sg-1^a and Sg-1^b alleles were characterized by eight synonymous codons and nine amino acid substitutions. Two evolutionarily transitional allelic sequences (Sg-1^a7 and Sg-1^b2) from Sg-1^a toward Sg-1^b were detected.     

Structural organization characteristics of the <Emphasis Type="Italic">DIP1</Emphasis> gene in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> strains mutant for the <Emphasis Type="Italic">flamenco</Emphasis> gene

Molecular cloning of the DIP1 gene located in the 20A4-5 region has been performed from the following strains with the flamenco phenotype: flam ^SS (SS) and flam ^MS (MS) characterized by a high transposition rate of retrotransposon gypsy (mdg4), flam ^{py + (P)} carrying the insertion of a construction based on the P element into the region of the flamenco gene, and flamenco ⁺. The results of restriction analysis and sequencing cloned DNA fragments has shown that strains flam ^SS , flam ^MS considerably differ from flam ^{py + (P)}, and flamenco ⁺ in the structure of DIP1. Strains flam ^SS and flam ^MS have no DraI restriction site at position 1765 in the coding region of the gene, specifically, in the domain determining the signal of the nuclear localization of the DIP1 protein. This mutation has been found to consist in a nucleotide substitution in the recognition site of DraI restriction endonuclease, which is transformed from TTTAAA into TTTAAG and, hence, is not recognized by the enzyme. This substitution changes codon AAA into AAG and is translationally insignificant, because both triplets encode the same amino acid, lysine. The DIP1 gene of strains flam ^SS and flam ^MS has been found to contain a 182-bp insertion denoted IdSS (insertion in DIP1 strain SS); it is located in the second intron of the gene. The IdSS sequence is part of the open reading frame encoding the putative transposase of the mobile genetic element HB1 belonging to the Tc1/mariner family. This insertion is presumed to disturb the conformations of DNA and the chromosome, in particular, by forming loops, which alters the expression of DIP1 and, probably, neighboring genes. In strains flamenco ⁺ and flam ^{py + (P)}, the IdSS insertion within the HB1 sequence is deleted. The deletion encompasses five C-terminal amino acid residues of the conserved domain and the entire C-terminal region of the putative HB1 transposase. The obtained data suggest that DIP1 is involved in the control of gypsy transpositions either directly or through interaction with other elements of the genome.     

Features of the genetic structure of the earthworms <Emphasis Type="Italic">Aporrectodea</Emphasis> (Superspecies) <Emphasis Type="Italic">Caliginosa</Emphasis> (Oligochaeta: Lumbricidae) complex in Ukraine

An investigation of the genetic structure of the Aporrectodea caliginosa-A. trapezoids diploidpolyploid complex of earthworms found in the Ukraine is performed by means of biochemical genetic marking with respect to six loci (Aat, Es-1,-2,-3, and-4, and Mdh) and karyotyping. All 646 individuals from 21 samples are analyzed at the biochemical gene level and karyotype samples are obtained from 70 specimens. As a result, diploid amphimictic A. caliginosa individuals (2n = 36), which form panmictic populations, and triploid (2n = 54) A. trapezoides individuals, represented by 19 hypothetical clones discovered in the course of an analysis of 157 specimens, are clearly differentiated. A clear trend towards dominance of the triploid forms in the steppe zone of Ukraine is discovered. Here they represent roughly 70% as against 12% of all A. (superspecies) caliginosa individuals in the northern forest regions. Based on the stated nature of the heterozygosity of the loci studied and the behavior of the chromosomes in meiosis, it may be claimed that the cloned forms are allotriploids, formed as a result of hybridization of a series of related forms, which, judging from the allelic pools, does not include amphimictic species that now inhabit the territory of Ukraine. That is, either the apomictic clones of A. trapezoides lack a local origin and their appearance among the fauna of Ukraine is a consequence of settlement in arid steppe regions that are unfavorable for earthworms or, on the other hand, amphimictic A. caliginosa is an invasive species.     

Defects in meiotic recombination delay progression through pachytene in <Emphasis Type="Italic">Tex19.1</Emphasis><Superscript><Emphasis Type="Italic">−/−</Emphasis></Superscript> mouse spermatocytes

Recombination, synapsis, chromosome segregation and gene expression are co-ordinately regulated during meiosis to ensure successful execution of this specialised cell division. Studies with multiple mutant mouse lines have shown that mouse spermatocytes possess quality control checkpoints that eliminate cells with persistent defects in chromosome synapsis. In addition, studies on Trip13^mod/mod mice suggest that pachytene spermatocytes that successfully complete chromosome synapsis can undergo meiotic arrest in response to defects in recombination. Here, we present additional support for a meiotic recombination-dependent checkpoint using a different mutant mouse line, Tex19.1^?/?. The appearance of early recombination foci is delayed in Tex19.1^?/? spermatocytes during leptotene/zygotene, but some Tex19.1^?/? spermatocytes still successfully synapse their chromosomes and we show that these spermatocytes are enriched for early recombination foci. Furthermore, we show that patterns of axis elongation, chromatin modifications and histone H1t expression are also all co-ordinately skewed towards earlier substages of pachytene in these autosomally synapsed Tex19.1^?/? spermatocytes. We also show that this skew towards earlier pachytene substages occurs in the absence of elevated spermatocyte death in the population, that spermatocytes with features of early pachytene are present in late stage Tex19.1^?/? testis tubules and that the delay in histone H1t expression in response to loss of Tex19.1 does not occur in a Spo11 mutant background. Taken together, these data suggest that a recombination-dependent checkpoint may be able to modulate pachytene progression in mouse spermatocytes to accommodate some types of recombination defect.