Accumulation of interspersed and sex-specific repeats in the non-recombining region of papaya sex chromosomes

Background

The papaya Y chromosome has undergone a degenerative expansion from its ancestral autosome, as a consequence of recombination suppression in the sex determining region of the sex chromosomes. The non-recombining feature led to the accumulation of repetitive sequences in the male- or hermaphrodite-specific regions of the Y or the Y^h chromosome (MSY or HSY). Therefore, repeat composition and distribution in the sex determining region of papaya sex chromosomes would be informative to understand how these repetitive sequences might be involved in the early stages of sex chromosome evolution.

Results

Detailed composition of interspersed, sex-specific, and tandem repeats was analyzed from 8.1 megabases (Mb) HSY and 5.3 Mb corresponding X chromosomal regions. Approximately 77% of the HSY and 64% of the corresponding X region were occupied by repetitive sequences. Ty3-gypsy retrotransposons were the most abundant interspersed repeats in both regions. Comparative analysis of repetitive sequences between the sex determining region of papaya X chromosome and orthologous autosomal sequences of Vasconcellea monoica, a close relative of papaya lacking sex chromosomes, revealed distinctive differences in the accumulation of Ty3-Gypsy, suggesting that the evolution of the papaya sex determining region may accompany Ty3-Gypsy element accumulation. In total, 21 sex-specific repeats were identified from the sex determining region; 20 from the HSY and one from the X. Interestingly, most HSY-specific repeats were detected in two regions where the HSY expansion occurred, suggesting that the HSY expansion may result in the accumulation of sex-specific repeats or that HSY-specific repeats might play an important role in the HSY expansion. The analysis of simple sequence repeats (SSRs) revealed that longer SSRs were less abundant in the papaya sex determining region than the other chromosomal regions.

Conclusion

Major repetitive elements were Ty3-gypsy retrotransposons in both the HSY and the corresponding X. Accumulation of Ty3-Gypsy retrotransposons in the sex determining region of papaya X chromosome was significantly higher than that in the corresponding region of V. monoica, suggesting that Ty3-Gypsy could be crucial for the expansion and evolution of the sex determining region in papaya. Most sex-specific repeats were located in the two HSY expansion regions.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-335) contains supplementary material, which is available to authorized users.     

Development of a novel telomerase assay using the PPDK–luciferin–luciferase detection system

Telomerase participates in malignant transformation or immortalization of cells, and has attracted attention as an anticancer drug screening and diagnostic tumor marker. We developed a novel telomerase assay called the PPDK–luciferin–luciferase system bioluminescence assay (PLLBA) using pyruvate phosphate dikinase (PPDK). In this assay, pyrophosphate produced by the telomerase reaction and polymerase chain reaction (PCR) is converted to ATP by PPDK, and ATP is detected by the firefly luciferin–luciferase reaction. In this work, telomerase substrate was obtained in accordance with the telomeric repeat amplification protocol (TRAP). Telomerase‐positive (500 cells/assay), ‐inactive (heated for 10 min at 85 °C) and ‐negative (only Chaps lysis buffer) samples were used. As a result, the findings clearly showed that the signal‐to‐noise (S/N) ratio of the positive cells was 39.5. After the telomerase reaction and PCR, PLLBA was completed ~ 120 s later. A high level of reproducibility was obtained with ‐ coefficient of variation (CV) of 4.1% (positive cells). The detection limit for cells using telomerase was one cell per assay. This assay for telomerase activity was also shown to be adaptable to human cancer‐derived cell lines. Copyright © 2013 John Wiley & Sons, Ltd.     

Stimulation of Akt poly-ubiquitination and proteasomal degradation in P388D1 cells by 7-ketocholesterol and 25-hydroxycholesterol

Akt plays a role in protecting macrophages from apoptosis induced by some oxysterols. Previously we observed enhanced degradation of Akt in P388D1 moncocyte/macrophages following treatment with 25-hydroxycholesterol (25-OH) or 7-ketocholesterol (7-KC). In the present report we examine the role of the ubiquitin proteasomal pathway in this process. We show that treatment with 25-OH or 7-KC results in the accumulation of poly-ubiquitinated Akt, an effect that is enhanced by co-treatment with the proteasome inhibitor MG-132. Modification of Akt by the addition of a Gly-Ala repeat (GAr), a domain known to block ubiquitin-dependent targeting of proteins to the proteasome, resulted in a chimeric protein that is resistant to turn-over induced by 25-OH or 7-KC and provides protection from apoptosis induced by these oxysterols. These results uncover a new aspect of oxysterol regulation of Akt in macrophages; oxysterol-stimulated poly-ubiquitination of Akt and degradation by the proteasomal pathway.     

The ankyrin repeat gene family in rice: genome-wide identification,classification and expression profiling

Ankyrin repeat (ANK) containing proteins comprise a large protein family. Although many members of this family have been implicated in plant growth, development and signal transduction, only a few ANK genes have been reported in rice. In this study, we analyzed the structures, phylogenetic relationship, genome localizations and expression profiles of 175 ankyrin repeat genes identified in rice (OsANK). Domain composition analysis suggested OsANK proteins can be classified into ten subfamilies. Chromosomal localizations of OsANK genes indicated nine segmental duplication events involving 17 genes and 65 OsANK genes were involved in tandem duplications. The expression profiles of 158 OsANK genes were analyzed in 24 tissues covering the whole life cycle of two rice genotypes, Minghui 63 and Zhenshan 97. Sixteen genes showed preferential expression in given tissues compared to all the other tissues in Minghui 63 and Zhenshan 97. Nine genes were preferentially expressed in stamen of 1 day before flowering, suggesting that these genes may play important roles in pollination and fertilization. Expression data of OsANK genes were also obtained with tissues of seedlings subjected to three phytohormone (NAA, GA3 and KT) and light/dark treatments. Eighteen genes showed differential expression with at least one phytohormone treatment while under light/dark treatments, 13 OsANK genes showed differential expression. Our data provided a very useful reference for cloning and functional analysis of members of this gene family in rice. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development and interspecific transferability of genic microsatellite markers in Spartina spp. with different genome size

Flow cytometry analysis showed variation of nuclear DNA content among different species of Spartina. Spartina alterniflora had the biggest genome (1763.9 Mbp) and S. cynosuroides had the smallest genome (756.35 Mbp), whereas the genomes of S. patens (969.36 Mbp) and S. spartinae (979.78 Mbp) were comparable. Mining simple sequence repeats (SSR) from 1227 expressed sequence tags (EST) generated from salt stressed S. alterniflora showed an abundance of di- and tri-nucleotide repeats. Of 100 ESSR (EST-derived SSR) loci with five or more repeats, 81 loci were successfully amplified in eight S. alterniflora genotypes and 15 (22.2%) ESSR markers were polymorphic. Eleven of the 15 polymorphic ESSRs showed amplification across six different species of Spartina while 100% cross transferability was observed with at least one species of Spartina. The average number of alleles per marker was 3.9 and 5.8 within S. alterniflora and among Spartina species, respectively. The ESSR markers discriminated different members within and between species of Spartina genus.     

FMR1: A gene with three faces

The FMR1 gene is involved in three different syndromes, the fragile X syndrome (FXS), premature ovarian insufficiency (POI) and the fragile X-associated tremor/ataxia syndrome (FXTAS) at older age. Fragile X syndrome is caused by an expansion of a CGG repeat above 200 units in the FMR1 gene resulting in the absence of the FMR1 mRNA and protein. The FMR1 protein is proposed to act as a regulator of mRNA transport and of translation of target mRNAs at the synapse. FXS is seen as a loss of function disorder. POI and FXTAS are found in individuals with an expanded repeat between 50 and 200 CGGs and are associated with increased FMR1 mRNA levels. The presence of elevated FMR1 mRNA in FXTAS suggests that FXTAS may represent a toxic RNA gain-of-function effect. The molecular basis of POI is yet unknown. The role of the FMR1 gene in these disorders is discussed.     

Functional analysis of HvSPY,a negative regulator of GA response,in barley aleurone cells and <Emphasis Type="Italic">Arabidopsis</Emphasis>

SPINDLY (SPY) is an important regulator of plant development, and consists of an N-half tetratricopeptide repeat (TPR) domain containing 10 TPR motifs and a C-half catalytic domain, similar to O-GlcNAc transferase (OGT) of animals. The best characterised role of SPY is a negative regulator of GA signalling, and all known spy alleles have been isolated based on increased GA response. Of the eight alleles that directly affect the TPR domain, all alter TPRs 6, 8 and/or 9. To test the hypothesis that a subset of TPRs, including 6, 8 and 9, are both essential and sufficient for the regulation of GA response, we overexpressed the full-length barley (Hordeum vulgare L.) SPY protein (HvSPY) and several deletion mutants in barley aleurone cells and in Arabidopsis wild type (WT) and spy-4 plants. Transient assays in barley aleurone cells, that also express endogenous HvSPY, demonstrated that introduced HvSPY and HvTPR inhibited GA₃-induced α-amylase expression. With the exception of HvSPYΔ1–5, the other deletion proteins were partially active in the barley assay, including HvSPYΔ6–9 which lacks TPRs 6, 8 and 9. In Arabidopsis, analysis of seed germination under a range of conditions revealed that 35S:HvSPY increased seed dormancy. Hvspy-2, which lacks parts of the eighth and ninth TPRs, was able to partially complement all aspects of the spy-4 phenotype. In the presence of AtSPY, 35S:HvTPR caused some phenotypes consistent with a decrease in GA signalling, including increased seed sensitivity to paclobutrazol and delayed flowering. These plants also possessed distorted leaf morphology and altered epidermal cell shape. Thus, despite genetic analysis demonstrating that TPRs 6, 8 and 9 are required for regulation of GA signalling, our results suggest that these TPRs are neither absolutely essential nor sufficient for SPY activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.