Identification of Phosphorus Starvation Inducible Genes in Rice by Suppression Subtractive Hybridization

Phosphorus is one of the three essential macroelements for plant growth. Plants respond to phosphorus starvation through adaptive mechanisms involved in morphological, biochemical and molecular changes. To investigate the molecular background of the adaptive mechanisms, the suppression subtractive hybridization (SSH) method was used to construct a rice phosphorus-starvation ( Pi-starvation) induced cDNA library. Through screening of the cDNA library and sequencing of the enriched cDNAs, 18 known genes and 47 novel genes were identified. The known genes are involved in different metabolic processes, including phosphate uptake and transport, signal transduction, protein synthesis and degradation, carbon metabolism and stress response. Northern analysis was performed to detect the expression patterns of some known genes and novel genes under different phosphorus levels. Different expression patterns of the selected genes were identified, which suggests that genes involved in different pathways may have different responses to Pi-starvation.

Screening for metronidazole-resistance associated gene fragments of H pylori by suppression subtractive hybridization

AIM： To screen for metronidazole （MTZ）-resistance associated gene fragments of H pylori by suppression subtractive hybridization （SSH）. METHODS： Five MTZ-resistant （tester, T） and 1 MTZ- susceptible （driver, D） clinical H pylori isolates were selected. Genomic DNAs were prepared and submitted to Rsa I digestion. Then two different adaptors were ligated respectively to the 5＇-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNAs. The specific inserts of tester strains were screened and MTZ-resistance related gene fragments were identified by dot blotting. RESULTS： Among the randomly selected 120 subtractive colonies, 37 DNA fragments had a different number of DNA copies （≥ 2 times） in resistant and susceptible strains and 17 of them had a significantly different number of DNA copies （≥ 3 times）. Among the sequences obtained from the 17 DNA fragments, new sequences were found in 10 DNA fragments and duplicated sequences in 7 DNA fragments, representing respectively the sequences of depeptide ABC transporter periplasmic dipeptide-binding protein （dppA）, permease protein （dppB）, ribosomal protein S4 （rps4）, ribonuclease Ⅲ （rnc）, protease （pqqE）, diaminopimelate epimerase （dapF）, acetatekinase （ackA）, H pylori plasmid pHP51 and Hpylori gene 1334. CONCLUSION： Gene fragments specific to MTZ-resistant H pylori strains can be screened by SSH and may be associated with MTZ-resistant Hpylori.

Identification and analysis of differentially expressed genes involved in dark-induced photoperiod response and senescence of soybean leaves by suppression subtractive hybridization

A cDNA subtractive library enriched for dark-induced up-regulated ESTs was constructed by suppression subtractive hybridization(SSH) from leaf tissues of soybean cultivar DongNong L13 treated with short-day(8-h light/16-h dark) and long-day(16-h light/8-h dark) conditions.A total of 148 clones were sequenced,representing 76 unique ESTs which corresponded to about 20% of 738 clones from the cDNA library and showed a significant up-regulation of at least three fold verified by dot blot hybridization.The putative functions of ESTs were predicted by Blastn and Blastx.The 43 differentially expressed genes identified by subtractions were classified according to their putative functions generated by Blast analysis.Genetic functional analysis indicated that putative proteins encoded by these genes were related to diverse functions during organism development,which include biological regulation pathways such as transcription,signal transduction and programmed cell death,protein,nucleic acid and carbohydrate macromolecule degradation,the cell wall modification,primary and secondary metabolism and stress response.Two soybean transcription factors enhanced in SD conditions,GAMYB-binding protein and DNA binding protein RAV cDNAs that may be involved in SD soybean photoperiod response,had been isolated using 5'-and 3'-rapid amplification of cDNA ends(RACE)(Genbank Accession numbers DQ112540 and DQ147914).

Lung Cancer Metastatic Related Genes by Suppression Subtractive Hybridization

Background and Objective The invasion and metastasis are not only the malignant markers and characteristics of lung cancer, but also the leading cause of failure to treatment and patient’

Identifcation of up-regulated genes in human uterine leiomyoma by sup-pression subtractive hybridization

In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression sub-tractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes werefirstly associated with UL. Three genes with notable difference were selected for Northern confirmationOur results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showedup-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obviousexpression in prostate, testis, liver, heart and skeletal muscle.

Identification of up-regulated genes in human uterine leiomyoma by sup-pression subtractive hybridization

In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression sub-tractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes werefirstly associated with UL. Three genes with notable difference were selected for Northern confirmationOur results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showedup-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obviousexpression in prostate, testis, liver, heart and skeletal muscle.

Gene expression profiling related to powdery mildew resistance in wheat with the method of suppression subtractive hybridization

'Bainong 3217 × Mardler' BC5F4 wheat line at the initial stage of inoculation with powdery mildew pathogen (Erysiphe graminis DC) was used to construct a suppression subtractive hybridization (SSH) cDNA library. Totally 760 ESTs were obtained through sequencing. Similarity analysis of ESTs based on BLASTn and BLASTx with the sequences in GenBank, in combination with macroarray differential screening, revealed that 199 ESTs of 65 kinds were known to be functionally disease resistance related. Based on the gene expression profiling in the present study, it is postulated that salicylic acid (SA) and MAP-related signal transduction pathways were involved in powdery mildew resistance in wheat. System acquired resistance genes were predominant in terms of kinds and quantity. With the initiation of cell defense reaction, the genes conferring anti-oxidation substances were largely expressed and thus cell protection mechanism was activated. Much evidence revealed that phenylpropanes metabolic pathway was

Identification of differentially expressed genes in anagen dermal pap illa by suppression subtractive hybridization

Background We constructed a cDNA subtractive library of dermal papilla cells (DPCs) in anagen with suppression subtractive hybridization (SSH) technique and clone differentially expressed genes related to DPCs in anagen. Methods Total mRNA was isolated from DPCs of anagen and telogen follicles. Moreover, single-strand (ss) and double-strand (ds) cDNAs were synthesized in turn using SMART PCR cDNA synthesis technology. ds cDNAs then were digested with Rsa I and divided into two groups, and ligated to the specific adaptor 1 and adaptor 2R, respect ively. After cDNAs were hybridized with each other twice and underwent two rounds of nested PCR. PCR products were ligated with arms of T/A plasmid vectors to set up the subtractive library. Selected clones were demonstrated by reverse Northern blot and sequenced. The acquired sequence data were aligned against the Genbank nucleotide database. Results cDNA subtractive library of DPCs in anagen follicles was set up successfully with high subtractive efficiency. Thirty-five genes were identified in this study with 22 known functional genes and 13 unknown functional genes. Conclusions All results confirm the effectiveness and sensitivity of SSH in detecting differentially expressed genes from a small amount of clinical samples. Information about such alterations in gene expression could be useful for elucidating the genetic events in hair follicle growth regulation.

Suppression subtractive hybridization for identifying differentially expressed genes in renal cell carcinoma

Objective To construct a renal cell carcinoma (RCC) cDNA subtractive library using suppression subtractive hybridization.Methods Polyadenylated RNA ［Poly (A)+ RNA］ was isolated from tissues of RCC and normal kidney, and single-strand cDNAs and double-strand cDNAs were synthesized in turn. RCC cDNAs were divided into two groups and ligated to the specific adaptors l and 2, and then hybridized with normal kidney cDNA twice with two rounds of suppression PCR. Second round PCR products were cloned to T/A plasmid vectors to set up the subtractive library. One hundred clones were randomly picked to perform enzyme digest analysis, and some underwent sequence analysis and Northern blot to identify RCC specifically expressed genes. SMART RACE procedure was operated to clone full length novel RCC specifically expressed genes.Results A human RCC subtractive library with high subtractive efficiency was successfully set up. The amplified library contains 350 positive clones. Random analysis of 100 clones with enzyme restriction showed that 85 plasmids in the clones contained 50-400?bp inserts. Sequence analysis was performed for 10 clones. All the 10 sequences were unknown before and derived from 6 unique, novel genes among which the cDNA insert RCC18 had five copies. Northern blot analysis showed that RCC18 cDNA was highly expressed in RCC, but no signal could be detected in normal kidney. Using SMART RACE technique, we obtained the full length of the novel gene RCC18.Conclusions The constructed cDNA subtractive library of human RCC is a highly efficient one and lays a solid foundation for large scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel specifically expressed genes provided an important clue for studying the mechanisms of occurrence and development of RCC.

Use of suppression subtractive hybridization strategy for cloning and identifying specifically expressed genes of renal cell carcinoma

Using suppression subtractive hybridization, a renal cell carcinoma (RCC) cDNA subtractive library which only contains differently expressed cDNAs between human RCC and normal kidney has been constructed. 200 clones were picked out randomly to perform enzyme digest analysis, a part of them underwent sequence analysis and Northern blot to identify RCC specially expressed genes. Results showed that 190 clones contain 50-400 bp inserts respectively. Sequence analysis was performed in 10 clones. All the 10 sequences were unknown before and derived from 6 unique novel genes among which the cDNA insert RCC18 has five copies. Northern blot analysis showed that RCC18 cDNA expressed highly in RCC, but there was no signal detected in normal kidney, and the full length of RCC18 was about 2.5 kb. The constructed cDNA subtractive library of human RCC is a highly efficient one and lays the solid foundation for large-scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel

Identification of the Rice Vacuolar ATPase B Subunit Gene and Its Expression Pattern Analysis Under Phosphorus Deficiency

A vacuolar ATPase (V-ATPase.) B subunit gene has been cloned and characterized front a phosphorus starvation induced rice root subtractive cDNA library by suppression subtractive hybridization (SSH) method and RT-PCR amplification. This gene encodes a polypeptide of 487 amino acid residues, containing a conservative ATP binding site and with a molecular weight of 54.06 kD and an isoelectric point of 4.99, southern analysis of the. genomic DNA indicates that V-ATPase B subunit is encoded by a single gene in rice genome. The amino acid homologies of V-ATPase B subunits among different organisms range from 76% to 97% and reveals that the evolution of V-ATPase B subunit is accompanied with the biological evolution. Expression pattern analysis indicated that the maximal expression of V-ATPase B subunit gene occurred at an early stage (6 - 12 h) after phosphorus starvation in roots, and lately stage (24 - 48 It) in leaves. Under phosphorus deficiency, the up-regulated expression of V-ATPase gene was presumed to strengthen the proton transport and provide the required energy to maintain an electrochemical gradient across the tonoplast to facilitate Phosphorus transport.

Identification of differently expressed genes in human colorectal adenocarcinoma

AIM： To investigate the differently expressed genes in human colorectal adenocarcinorna.METHODS： The integrated approach for gene expression profiling that couples suppression subtractive hybridization, high-throughput cDNA array, sequencing, bioinformatics analysis, and reverse transcriptase real- time quantitative polymerase chain reaction （PCR） was carried out. A set of cDNA clones including 1260 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with florescent-labeled probes prepared from RNA of human colorectal adenocarcinoma （HCRAC） and normal colorectal tissues.RESULTS： A total of 86 genes were identified, 16 unknown genes and 70 known genes. The transcription factor Sox9 influencing cell differentiation was downregulated. At the same time, Heat shock protein 10 KDis downregulated and Calmoulin is up-regulated.CONCLUSION： Downregulation of heat shock protein 10 KD lost its inhibition of Ras, and men attenuated the Ras GTPase signaling pathway, increased cell proliferation and inhibited cell apoptosis. Down-regulated transcription factor Sox9 influences cell differentiation and cell-specific gene expression. Down-regulated Sox9 also decreases its binding to calmodulin, accumulates calmodulin as receptor-activated kinase and phosphorylase kinase due to the activation of PhK.

Cloning and Identification of Porcine HSPC117 Gene Differentially Expressed in F_(1)Crossbreds and Their Parents

To investigate the molecular basis of porcine heterosis, suppression subtractive hybridization （SSH） was performed to detect the differences in gene expression between porcine longissimus dorsi of Meishan X Large White （MS × LW） F1 hybrids and their parents Meishan pigs. An expression sequence tag （EST） differentially expressed was found, designated as ML556, which was homologous to a hypothetical protein HSPC117, from human hematopoietic stem/progenitor cells （HSPCs）, and the full-length cDNA of porcine HSPC117 was cloned using rapid amplification of cDNA ends （RACE） method. Translation of the mRNA transcript revealed an open reading frame （ORF） of 505 amino acid residues encoding a peroxisomal targeting signal （PTS） with theoretical molecular weight of 55 kDa. Alignment analysis revealed that the deduced protein sequence exhibit 98, 98, 98, 97, and 97% identity with that of cattle, human, dog, rat, and mouse, respectively. The tissue expression analysis indicated that the porcine HSPC117 gene is highly expressed in muscle, spleen, lung, kidney, uterus, ovary and testis, moderately expressed in fat, heart, and liver, and not expressed in stomach and small intestine. The possible role of porcine HSPC117 and its relationship with porcine heterosis were discussed.

Identification and Expression Analysis of Salinity-induced Genes in Rangpur lime(Citrus limonia)

Among the economically important horticultural crops,citrus is one of the most vulnerable crops to soil salinity.Rangpur lime is more tolerant to high soil salinity than other commonly used citrus rootstocks.However,the molecular mechanism involved in salinity tolerance has not been explored in Rangpur lime.In this study,a cDNA library was constructed from leaves of Rangpur lime watered for 30 days with 100mmol·L^−1 NaCl in tap water or tap water using suppression subtractive hybridization(SSH).Two hundred cDNA clones randomly selected from this library were sequenced,and an average of 569 bp was obtained from the majority of clones.Fifty-six salinity-induced genes,showing 2-to 6-fold increases in their expression levels,were identified by macroarray hybridizations.Salinity-induced genes were associated with transcription(5.36%),stress response and signaling(21.43%),metabolism(16.07%),transport facilitation(10.71%),photosynthesis(10.71%),protein synthesis and fate(19.64%)and cellular biogenesis(3.57%).Stress response-and signaling-related genes constituted the largest functional group,associated with the production of compatible solutes,regulation of stomatal movement,lipid modification,oxidative stress,antioxidant defense,and stress signaling.Expression levels of 13 identified genes were induced 1.8-to 3.1-fold,which were validated in salt-treated and untreated Rangpur lime.The functions of salinity-induced,stress-related genes and their potential roles in salinity tolerance in Rangpur lime were discussed.

Identification of differentially expressed genes in two new human bladder carcinoma cell lines

Objective To screen and identify differentially expressed genes in two new human urothelial carcinoma cell lines, BLS-211 and BLX. Methods Suppression subtractive hybridization (SSH) was used to createa subtracted library, and clones were sequenced. Results Totally 13 over-expressed genes in BLX and 9 in BLS-211 cells were obtained, respectively. Among them, 18 were known genes and 4 were new ESTs (Expressed Sequence Tag), and were collected by GenBank dbEST database (The access number was EB390424-7). Conclusion SSH is a powerful method for the identification of differentially expressed genes. The differential expression of some BCG-associated genes in different cells may be related to the different responses to clinical BCG therapy. The identified new ESTs can be cloned for full length to further study their functions.

Identification and Characterization of Putative Virulent Genes in Streptococcus equi ssp. zooepidemicus

Suppression subtractive hybridization （SSH） was performed with virulent strain ATCC35246 and avirulent strain ST171 to identify novel genes associated with virulence in Streptococcus equi ssp. zooepidemicus （SEZ）. There were fourteen genomic regions that only presented in virulent strain ATCC35246. These regions encoded 14 proteins, some of them were homologous to proteins associated with cellular surface structure, molecular synthesis, energy metabolism, regulation, transport systems, and other unknown functions. Primers for 6 particular regions were designed from the already published SEZ sequence. Then, we used PCR to evaluate the distribution and conservation of these 6 DNA fragments in various SEZ strains collected from different sources, regions, groups, and times. The results showed that these 6 DNA fragments were widely distributed in SEZ strains, yet they were not existence in the avirulent strain ST171. Moreover, these fragments could not be detected in other Streptococcus groups.

Identification of differentially expressed genes in mouse hepatocarcinoma ascites cell line with low potential of lymphogenous metastasis

AIM: To identify genes differentially expressed in mouse hepatocarcinoma ascites cell line with low potential of lymphogenous metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with low potential of lympho- genous metastasis Hca-P and its synogenetic cell line Hca-F with high metastatic potential was constructed by suppression subtracted hybridization (SSH) method. The screened clones of the subtracted library were sequenced and GenBank homology search was performed. RESULTS: Fifteen differentially expressed cDNA fragments of Hca-P were obtained which revealed 8 known genes, 4 expressed sequence tags (ESTs) and 3 cDNAs showed no homology. CONCLUSION: Tumor metastasis is an incident involving multiple genes. SSH is a useful technique to detect differentially expressed genes and an effective method to clone novel genes.

Identification and Expression Analysis of Regulatory Genes Induced by Near-Ultraviolet Irradiation in <i>Bipolaris oryzae</i>

Bipolaris oryzae is the causal agent of brown leaf spot disease in rice, and its asexual spore (conidium) formation is known to be induced by near-ultraviolet (NUV) irradiation. In order to reveal the photomorphogenic response and to identify new genes upregulated by NUV irradiation, suppression subtractive hybridization (SSH) was carried out in B. oryzae. To confirm the differential gene expression in NUV-irradiated mycelia, quantitative real-time PCR (qRT-PCR) analysis was performed among 301 genes arbitrarily chosen from 1170 cDNA clones. The expression of 46 genes (named NUV01 to NUV46) was found to be significantly enhanced (>4-fold) by NUV irradiation. Sequence analysis revealed that 23 out of the 46 sequences (50%) showed significant matches to known fungal genes. The 46 genes were categorized as either BLR1-dependent or BLR1-independent expression groups using the BLR1-deficient mutant, which presumably lacks the blue/UVA-absorbing photoreceptor. This finding demonstrates that NUV irradiation can induce gene regulation, and that this response may be mediated by both a blue/UVA-absorbing photoreceptor and an as-yet-unidentified photoreceptor in B. oryzae.