Mirror Orientation Selection Research Articles

Identification of early-response genes involved in cadmium resistance in shiitake mushrooms (Lentinula edodes)

Basidiomycete species accumulate high levels of heavy metals, particularly cadmium (Cd). Heavy metal toxicity has been widely investigated, and different key factors affecting tolerance to heavy metals have been identified in plants and fungi. Nevertheless, molecular insights into different mechanisms of fungi to directly resist reactions with particular heavy metals remain insufficient. As such, early-response genes in basidiomycete fungi must be identified to elucidate the mechanisms that confer resistance to heavy metal stress, particularly for Cd. The basidiomycete fungus Lentinula edodes (Berk.) Pegler, an edible mushroom (shiitake), is the most economically important cultivated mushroom. Suppression subtractive hybridization (SSH) combined with mirror orientation selection (MOS) was performed to identify Cd-induced genes in L. edodes treated with 50 μM Cd for 24 h. A total of 72 L. edodes genes were identified from the forward subtractive library. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was then employed to confirm the induction of the identified genes upon Cd exposure. Bioinformatics analysis revealed that 53 of the identified genes encode proteins with known functions and may be involved in early response to Cd exposure, whereas 19 genes encode hypothetical proteins with unknown functions. The diversity of the putative functions of the identified genes indicates that Cd stress elicits complex responses in L. edodes, and complex metabolic, transport, and signaling pathways may be involved in Cd resistance. This study presents a group of potential early-response genes to Cd exposure and may be beneficial to elucidate heavy metal resistance in basidiomycete fungi.

The Trametes hirsuta 072 laccase multigene family: Genes identification and transcriptional analysis under copper ions induction.

Laccases, blue copper-containing oxidases, ≿ an play an important role in a variety of natural processes. The majority of fungal laccases are encoded by multigene families that express closely related proteins with distinct functions. Currently, only the properties of major gene products of the fungal laccase families have been described. Our study is focused on identification and characterization of laccase genes, which are transcribed in basidiomycete Trametes hirsuta 072, an efficient lignin degrader, in a liquid medium, both without and with induction of laccase transcription by copper ions. We carried out production of cDNA libraries from total fungal RNA, followed by suppression subtractive hybridization and mirror orientation selection procedures, and then used Next Generation Sequencing to identify low abundance and differentially expressed laccase transcripts. This approach resulted in description of five laccase genes of the fungal family, which, according to the phylogenetic analysis, belong to distinct clusters within the Trametes genus. Further analysis established similarity of physical, chemical, and catalytic properties between laccases inside each cluster. Structural modeling suggested importance of the sequence differences in the clusters for laccase substrate specificity and catalytic efficiency. The implications of the laccase variations for the fungal physiology are discussed.

Differential gene expression in the brain of Cuvier, Sebastiscus marmoratus, in response to exposure to tributyltin

Tributyltin (TBT), a ubiquitous marine environmental contaminant, has been reported to affect functioning of the central nervous system. However, the mechanism of its neurotoxicity remains unknown. The effects of TBT exposure on the Sebastiscus marmoratus were investigated using a suppression subtractive hybridisation (SSH) combined with mirror orientation selection (MOS) method. A total of 50 candidate clones from SSH libraries were picked for sequencing and homology searching. The results showed that 23 unique differentially expressed genes were obtained, including 11 up-regulated and 12 down-regulated fragments. The functions with which they are associated include energy-related metabolism, polypeptide translation and location, mitosis and cell proliferation. Expression of 11 candidate genes was analysed in brain by real-time fluorescent quantitative PCR. The present study provided a basis for investigating the response of fish to TBT exposure and allowed the characterisation of new potential neurotoxic biomarkers of TBT contamination in seawater.

Subtractive and differential hybridization molecular analyses of Ceratitis capitata XX/XY versus XX embryos to search for male-specific early transcribed genes.

The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, is a fruit crop pest of very high economic relevance in different continents. The strategy to separate Ceratitis males from females (sexing) in mass rearing facilities is a useful step before the sterilization and release of male-only flies in Sterile Insect Technique control programs (SIT). The identification of genes having early embryonic male-specific expression, including Y-linked genes, such as the Maleness factor, could help to design novel and improved methods of sexing in combination with transgenesis, aiming to confer conditional female-specific lethality or female-to-male sexual reversal.We used a combination of Suppression Subtractive Hybrydization (SSH), Mirror Orientation Selection (MOS) and differential screening hybridization (DSH) techniques to approach the problem of isolating corresponding mRNAs expressed in XX/XY embryos versus XX-only embryos during a narrow developmental window (8-10 hours after egg laying, AEL ). Here we describe a novel strategy we have conceived to obtain relatively large amounts of XX-only embryos staged at 8-10 h AEL and so to extract few micrograms of polyA+ required to apply the complex technical procedure. The combination of these 3 techniques led to the identification of a Y-linked putative gene, CcGm2, sharing high sequence identity to a paralogous gene, CcGm1, localized either on an autosome or on the X chromosome.We propose that CcGm2 is a first interesting putative Y-linked gene which could play a role in sex determination. The function exterted by this gene should be investigated by novel genetic tools, such as CRISPR-CAS9, which will permit to target only the Y-linked paralogue, avoiding to interfere with the autosomal or X-linked paralogue function.

Identification of cadmium-induced Agaricus blazei genes through suppression subtractive hybridization

Cadmium (Cd) is one of the most serious environmental pollutants. Filamentous fungi are very promising organisms for controlling and reducing the amount of heavy metals released by human and industrial activities. However, the molecular mechanisms involved in Cd accumulation and tolerance of filamentous fungi are not fully understood. Agaricus blazei Murrill, an edible mushroom with medicinal properties, demonstrates high tolerance for heavy metals, especially Cd. To investigate the molecular mechanisms underlying the response of A. blazei after Cd exposure, we constructed a forward subtractive library that represents cadmium-induced genes in A. blazei under 4ppm Cd stress for 14days using suppression subtractive hybridization combined with mirror orientation selection. Differential screening allowed us to identify 39 upregulated genes, 26 of which are involved in metabolism, protein fate, cellular transport, transport facilitation and transport routes, cell rescue, defense and virulence, transcription, and the action of proteins with a binding function, and 13 are encoding hypothetical proteins with unknown functions. Induction of six A. blazei genes after Cd exposure was further confirmed by RT-qPCR. The cDNAs isolated in this study contribute to our understanding of genes involved in the biochemical pathways that participate in the response of filamentous fungi to Cd exposure.

Identification of fungal genes involved in the preinfection events between ectomycorrhizal association (Pisolithus tinctorius and Pinus massoniana)

The preinfection stage of establishment of the mycorrhiza before physical contact between symbionts is crucial, as changes that occur throughout mycorrhiza formation are set in motion at this time. To improve the understanding of the molecular mechanisms involved in the fungus Pisolithus tinctorius during its interaction with its symbiotic partner Pinus massoniana, we set up a cellophane-separated culture system avoiding physical contact between the symbionts. A cDNA subtraction library representing the differentially expressed genes of P.tinctorius during the preinfection stage was constructed using suppression subtractive hybridization combined with mirror orientation selection (SSH/MOS). Differential screening of the SSH library allowed us to identify 133 unique cDNA clones. Sequences analysis of the expressed sequence tags (ESTs) showed that they represent 23 differentially expressed genes, of which 10 are involved in metabolism and energy, transcription and protein fate, membrane proteins and signaling, and cell rescue and defense, and 13 encoding hypothetical proteins with unknown function. Some fungal genes have not been previously identified in other ectomycorrhizal associations. Expression of the genes that code for ubiquitin-conjugating enzyme E2, 40S ribosomal protein, maternal g10 transcript, ADP ribosylation factor 6, and cytochrome P450 were evaluated by RT-qPCR, confirming the activation of these genes during the preinfection stage.

Differential Expression in Hibiscus cannabinus (Mesta) Plants After Infection with Mesta Yellow Vein Mosaic Virus as Revealed by Suppression Subtractive Hybridization

AbstractYellow vein mosaic disease induced by a whitefly transmitted monopartite begomovirus causes a devastating foliar disease of Hibiscus cannabinus (mesta) crops across India. Characterization of the causal virus at molecular level and different epidemiological factors associated with the disease have already been investigated to understand the role of driving components behind continued spread of the disease. We have investigated the global gene expression profiling to increase knowledge of transcriptional changes taken place in a compatible interaction between Mesta yellow vein mosaic virus (MeYVMV) and H. cannabinus plants by PCR‐based suppression subtractive hybridization supplemented with mirror orientation selection. Dot‐blot analysis of forward and reverse subtracted libraries with respective cDNA probes confirmed the differential regulation of 100 clones of forward subtracted library and 70 clones of reverse‐subtracted library of 220 positive colonies (proved by colony PCR and restriction release) picked for analysis (from both reactions), and these clones were sequenced. Sequence analysis and virtual Northern blot at varying time points of the infection process finally confirmed the consistent up‐regulation of 11 and down‐regulation of seven gene fragments (ESTs) in infected plant. The up‐regulated transcripts could be functionally categorized in three different groups: (i) members of signal transduction cascades, (ii) host defence‐responsive elements and (iii) factors involved in metabolism and transport. Down‐regulation of the gene encoding SGT1 protein in infected plants suggested the possible modulation by the virus to overcome host defence responses. Other down‐regulated ESTs were found to be members of photosynthetic pathways, electron transport chain and metabolism. Differential regulations of a few genes from both libraries were subsequently confirmed by Northern analysis. Our results present the first evidence of genes that might be involved in recognition and signalling routes in the mesta plant after infection with MeYVMV and facilitate the design of new crop protection strategies.

Transcriptional profiling in cadmium-treated rice seedling roots using suppressive subtractive hybridization

Cadmium (Cd), a non-essential metal, is a kind of toxic heavy metal to life, which can accumulate in rice tissues including seeds, thus posing a risk to human health through food chain. To investigate the molecular mechanisms of rice response to Cd exposure, suppression subtractive hybridization and mirror orientation selection were used to compare gene expression profiles in seedling roots of Cd-exposed and control (unexposed) rice plants (Oryza sativa L., Nipponbare). Approximately 1700 positive clones, with insertions ranging from 250 to 1300bp, were identified through reverse cDNA microarray analysis. Gene expression was further confirmed by real time RT-PCR. A number of differentially expressed genes were found in Cd-exposed rice roots, including 28 up-regulated genes and 19 down-regulated genes. They were found to be involved in diverse biological processes, such as metabolism, stress response, ion transport and binding, protein structure and synthesis, as well as signal transduction. Notably a number of known functional genes were identified encoding membrane proteins and stress-related proteins such as heat shock proteins, monosaccharide transporters, CBL-interacting serine/threonine-protein kinases and metal tolerance proteins. The cDNAs isolated in this study contribute to our understanding of genes and the biochemical pathways that may play a key role in the response of plants to metal exposure in the environment.

Barley DNA-binding methionine aminopeptidase, which changes the localization from the nucleus to the cytoplasm by low temperature, is involved in freezing tolerance

The polymerase chain reaction-based Mirror Orientation Selection (MOS) method was used to isolate low temperature-induced genes from cold-treated winter barley (Hordeum vulgare L. cv. Dongbori). MOS screening identified a novel methionine (Met) aminopeptidase (MAP) designated as HvMAP. The deduced HvMAP protein was determined to possess an aminopeptidase domain and a nuclear localization signal. An in vitro enzyme assay using recombinant HvMAP protein demonstrated MAP activity. The expression of this gene was induced by low temperature and abscisic acid treatment, and overexpression of this gene conferred stronger freezing tolerance to Arabidopsis transgenic plants as compared to wild-type plants. Interestingly, low temperature treatment changed the localization of HvMAP from the nucleus to the cytoplasm. These findings suggest that HvMAP is a novel MAP that functions in freezing tolerance by facilitating protein maturation.

Role of normalization in the elimination of abundant myelin sequences in spinal cord cDNA libraries produced by suppression subtractive hybridization.

Spinal cord libraries subtracted against visual cortex using suppression subtractive hybridization SSH are dominated by abundant gene sequences derived from myelin elements. We compared our subtracted library results of three of these abundant sequences to published expressed sequence tag libraries that are not normalized and not subtracted and presumed representatives of murine spinal cord mRNA abundance. We show that: all three abundant sequences, myelin basic protein (Mbp), proteolipid protein (Plp1) and Ferretin heavy chain (Fth1) are highly expressed in spinal cord when this structure is compared to visual cortex; myelin basic protein is represented in our subtracted libraries but at a low frequency, whereas Plp1 and Fth1 represent nearly one-third of all sequences in these libraries; mirror orientation selection, a procedure designed to reduce background sequences, generates libraries very similar in abundance to SSH; proteolipid protein can be reduced in these libraries by adding Plp1 sequences to the driver in the SSH procedure and also by subtracting Plp1 directly from tester and driver. We conclude that adequate normalization is essential to reduce the presence of abundant sequences in SSH libraries.

Chloroplast DNA Copy Number May Link to Sex Determination in Leucadendron (Proteaceae)

Leucadendron (Proteaceae) is a South African genus, the flowers of which have become a popular item in the Australian cut-flower industry. All species are dioecious. In general the female flowers are the more desirable as cut flowers. The availability of a molecular marker linked to sex determination is therefore needed both to maximize the efficiency of breeding programs and to supply markets with flowers from the preferred sex. The polymerase chain reaction-based method of suppression subtractive hybridization (SSH) combined with mirror orientation selection (MOS) were applied in an attempt to identify genome differences between male and female plants of Leucadendron discolor . Screening of 416 clones from a male-subtracted genomic DNA library and 282 clones from a female-subtracted library identified 13 candidates for male-specific genomic fragments. Sequence analyses of the 13 candidate DNA fragments showed that they were fragments of the chloroplast DNA, raising the possibility that chloroplast DNA copy number is linked to sex determination in Leucadendron .

Differential gene expression in White Spot Syndrome Virus (WSSV)-infected naïve and previously challenged Pacific white shrimp Penaeus ( Litopenaeus) vannamei

White Spot Syndrome Virus (WSSV) is the most devastating viral disease affecting cultivated shrimp around the world. The CENIACUA breeding programme included host resistance as strategy to control WSSV. Since 1999, all families were subjected to WSSV challenge tests and up to 5% of the animals survived. We suspect that a unique genetic profile could be responsible for survival. By Subtractive Suppression Hybridization improved with Mirror Orientation Selection we compared gene expression in haemocytes of WSSV-infected shrimp; seven were previously challenged (pre-challenged) and 10 naïve. Out of 1167 clones, 993 (85%) were obtained from the WSSV-infected pre-challenged library, while 174 (15%) were from the WSSV-infected naïve library. Differential expression was confirmed by hybridization using PCR DIG-labelled probes from unsubtracted cDNA. The 216 (18.5%) clones differentially expressed in the WSSV-infected pre-challenged library and 23 (2%) in the WSSV-infected naïve animals were sequenced. Relative expression of relevant gene products from both libraries was confirmed by reverse transcription real time PCR. Genes related to shrimp immune response, such as the antimicrobial peptide penaeidin, crustin, C-type lectin, protease inhibitor and chitin binding domain-containing protein were found in the library of pre-challenged shrimp. On the other hand, genes from the naïve library coded for viral structural and functional proteins. To our knowledge, this is the first report demonstrating that penaeidin-3 isoforms and crustin are over expressed in haemocytes of WSSV-infected pre-challenged P. vannamei.

Suppression Subtractive Hybridization and Differential Screening Reveals Endodormancy-associated Expression of an SVP/AGL24-type MADS-box Gene in Lateral Vegetative Buds of Japanese Apricot

To understand the molecular basis of the endodormancy of buds of perennial plants, we searched for the genes that are expressed preferentially in endodormant lateral buds of the deciduous fruit tree japanese apricot (Prunus mume Sieb. et Zucc.) using suppression subtractive hybridization with mirror orientation selection (SSH/MOS). We generated two SSH/MOS libraries containing gene pools that are expressed preferentially in endodormant buds in comparison with paradormant or ecodormant buds to search for the genes that are upregulated by endodormancy induction or down-regulated by endodormancy release, respectively. Differential screening and sequencing indicated that genes involved in gibberellin metabolism, stress resistance, cell wall modification, and signal transduction, such as transcription factors, are upregulated in endodormant buds. After a further expression survey and full-length cDNA cloning, we found that a gene similar to the SVP/AGL24-type MADS-box transcription factor showed endodormancy-associated expression. Seasonal expression analysis suggested that the SVP/AGL24 homolog in japanese apricot might be involved in endodormancy regulation of its lateral buds.

Gene expression analysis of germinating rice seeds responding to high hydrostatic pressure

High hydrostatic pressure (HHP) is an extreme thermal-physical stress affecting multiple cellular activities. Recently, we found that HHP treatment caused various physiological changes in rice. To investigate the molecular mechanisms of plant response to HHP, we constructed forward and reverse subtracted cDNA libraries of rice seeds treated with 75MPa hydrostatic pressure for 12h by suppression subtractive hybridization in combination with mirror orientation selection. Of 97 clones isolated through microarray dot-blot and sequenced, 45 were unique genes. Among these 45 unique cDNAs, 29 clones showed significant sequence similarity to known genes, 12 were homologous to genes with unknown function, and the remaining 4 clones did not match any known sequences. Most of the genes with known function were involved in metabolism, defense response, transcriptional regulation, transportation regulation, and signal transduction. To our knowledge, this is the first gene expression analysis of rice in response to HHP. The expression profiles of the genes identified in this study provide useful information regarding molecular processes, including alteration of metabolism and adaptation response caused by HHP.

Identification of Differential Genes by Suppression Subtractive Hybridization: IV. Mirror Orientation Selection (MOS)

INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. The major drawback of SSH is the presence of background clones that represent nondifferentially expressed DNA species in the subtracted libraries. In some cases, the number of background clones may considerably exceed the number of target clones. This protocol describes mirror orientation selection (MOS)--a simple procedure that substantially decreases the number of background clones in libraries generated by SSH. The MOS technique is based on the rationale that after PCR amplification, during SSH, background molecules will be present in one orientation only, relative to the adapter sequences. Genuine SSH clones will be present in both sequence orientations.

Identification of an osteopontin‐like protein in fish associated with mineral formation

Fish has been recently recognized as a suitable vertebrate model and represents a promising alternative to mammals for studying mechanisms of tissue mineralization and unravelling specific questions related to vertebrate bone formation. The recently developed Sparus aurata (gilthead seabream) osteoblast-like cell line VSa16 was used to construct a cDNA subtractive library aimed at the identification of genes associated with fish tissue mineralization. Suppression subtractive hybridization, combined with mirror orientation selection, identified 194 cDNA clones representing 20 different genes up-regulated during the mineralization of the VSa16 extracellular matrix. One of these genes accounted for 69% of the total number of clones obtained and was later identified as theS. aurata osteopontin-like gene. The 2138-bp full-length S. aurata osteopontin-like cDNA was shown to encode a 374 amino-acid protein containing domains and motifs characteristic of osteopontins, such as an integrin receptor-binding RGD motif, a negatively charged domain and numerous post-translational modifications (e.g. phosphorylations and glycosylations). The common origin of mammalian osteopontin and fish osteopontin-like proteins was indicated through an in silico analysis of available sequences showing similar gene and protein structures and was further demonstrated by their specific expression in mineralized tissues and cell cultures. Accordingly, and given its proven association with mineral formation and its characteristic protein domains, we propose that the fish osteopontin-like protein may play a role in hard tissue mineralization, in a manner similar to osteopontin in higher vertebrates.

Isolation and enrichment of human genomic CpG islands by methylation-sensitive mirror orientation selection

CpG islands (CGIs) in human genomic DNA are GC-rich fragments whose aberrant methylation is associated with human disease development. In the current study, methylation-sensitive mirror orientation selection (MS–MOS) was developed to efficiently isolate and enrich unmethylated CGIs from human genomic DNA. The unmethylated CGIs prepared by the MS–MOS procedure subsequently were used to construct a CGI library. Then the sequence characteristics of cloned inserts of the library were analyzed by bioinformatics tools, and the methylation status of CGI clones was analyzed by HpaII PCR. The results showed that the MS-MOS method could be used to isolate up to 0.001% of differentially existed unmethylated DNA fragments in two complex genomic DNA. In the CGI library, 34.1% of clones had insert sequences satisfying the minimal criteria for CGIs. Excluding duplicates, 22.0% of the 80,000 clones were unique CGI clones, representing 60% of all the predicted CGIs (about 29,000) in human genomic DNA, and most or all of the CGI clones were unmethylated in human normal cell DNA based on the HpaII PCR analysis results of randomly selected CGI clones. In conclusion, MS–MOS was an efficient way to isolate and enrich human genomic CGIs. The method has powerful potential application in the comprehensive identification of aberrantly methylated CGIs associated with human tumorigenesis to improve understanding of the epigenetic mechanisms involved.

Differential gene expression in egg cells and zygotes suggests that the transcriptome is restructed before the first zygotic division in tobacco

We applied suppression subtractive hybridization and mirror orientation selection to compare gene expression profiles of isolated Nicotiana tabacum cv SR1 zygotes and egg cells. Our results revealed that many differentially expressed genes in zygotes were transcribed de novo after fertilization. Some of these genes are critical to zygote polarity and pattern formation during early embryogenesis. This suggests that the transcriptome is restructed in zygote and that the maternal-to-zygotic transition happens before the first zygotic division, which is much earlier in higher plants than in animals. The expressed sequence tags used in this study provide a valuable resource for future research on fertilization and early embryogenesis.

Differentially expressed genes during seed development in soybean

Little reliable information exists about the genetic events that control the onset and timing of seed‐fill in soybean cotyledons. To identify the genes involved in this process, cDNA libraries were prepared from mRNAs isolated from seeds at 7 and 21 days after flowering (DAF), which represent times just before and after the initiation of seed‐fill. For the soybean variety Resnik, which was used for this study, seed‐fill and the establishment of an endoreduplicative cell cycle occurred 12–14 DAF. Suppression subtractive hybridization was then applied to identify sequences that were differentially expressed at each of these two developmental stages. False positives in the libraries were reduced by using mirror orientation selection (MOS). The libraries of differentially expressed genes that resulted were analysed and the nucleotide sequences obtained were compared with those in existing databases. Several genes from each library were chosen and their expression profile during seed development was analysed by quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) using RNA preparations originating from different seed developmental stages. Candidate genes for control of the stage shift from dividing cells to endoreduplication were identified.

Analysis of differentially expressed transcripts from planthopper-infested wild rice (Oryza minuta)

A subtracted library was constructed from planthopper-infested wild rice (Oryza minuta) by suppression subtractive hybridization in combination with mirror orientation selection. To screen the differentially expressed transcripts in the library, we applied a cDNA microarray containing 960 random clones in a reverse Northern blot analysis using cDNA probes prepared from the mRNAs of control and planthopper-infested samples. On the basis of the signal intensities and expression ratios obtained from experiments performed in triplicate, we selected 383 clones. The elevated expression levels and overall profiles over time were verified by Northern blot analysis. Although Southern blot analysis showed similar copy numbers of the screened genes in O. minuta and O. sativa, it also revealed that the expression profiles had a different pattern. Functional categorization placed the identified transcripts in the categories of subcellular localization, metabolism, and protein fate. The presence of these expressed sequence tags implies that resistance of O. minuta to insect infestation can be achieved not only by an elevated expression of defense-related genes but also by enhanced metabolic activities.