• BMB Reports
• /
• v.37 no.5
• /
• pp.527-532
• /
• 2004

A subtracted cDNA library specific to osmotic stress of Haloxylon ammodendron (Mey.) Bge seedlings was constructed by suppression subtractive hybridization (SSH) and T/A cloning. SSH was performed between two groups of H. ammodendron seedlings, one was cultivated in Hoagland (H) solution as a driver and the other group was treated with osmotic stress of the Hoagland solution by the addition of 400 mM mannitol (M), as a tester. The library consisted of about 400 recombinant clones, with the average size being of 500 bp, ranging from 300 bp to 1500 bp. Using a PCR-select differential screening kit, 100 recombinant clones were randomly chosen from the subtracted cDNA library and hybridized with forward,reverse subtracted and unsubtracted probes for two rounds. As a result, 21 positive clones specific to osmotic stress were obtained and some of them were verified by Northern blot analysis. The sequencing analysis of 6 positive clones and the following homology comparison to GenBank [blastx] non-redundant databases characterized that two sequences obtained in this experiment may contribute to novel drought-related genes.

• Journal of Photoscience
• /
• v.7 no.3
• /
• pp.103-108
• /
• 2000

We have evaluated a new mutagenesis strategy called random insertional mutagenesis with subtracted cDNA fragments. The cDNAs from long day Arabidopsis plants were subtracted by cDNAs from short day plants using PCR based cDNA subtraction. The subtracted cDNAs were inserted between 35S promoter and 3'-NOS terminator regardless of orientation. When the cDNA library was used for the random insertion into Arabidopsis genome by Agrobacterium-mediated transformation, approximately 15% of transformants showed abnormal development in leaf, floral organ, shoot apex. When 20 mutants were analyzed, 12 mutants showed single cDNA fragment insertion and 8 mutants showed more than 2 transgene insertions. Only two mutants among 12 mutants that have single cDNA insert showed consistent phenotype at T2 generation, suggesting the genetic instability of the mutants.

• Development and Reproduction
• /
• v.6 no.2
• /
• pp.89-96
• /
• 2002

The present study was conducted to elucidate genes involved in the primordial-primary follicular transition. By using suppression subtractive hybridization, day1- and day5-subtracted cDNA libraries were obtained with the forward and reverse subtraction method, respectively. In toto, 357 clones were sequenced and analyzed by BLAST and RIKEN program. Sequences of 330 clones significantly matched database entries while 27 clones were novel. Forty-two and 47 genes with known functions were different between day1 and day5 ovaries. Four genes, GDF8, lats2, septin2, and wee1, from the day1 subtracted cDNA library, and 6 genes, HSP84, laminin2, MATER, MTi7, PTP, and wrn, from day5-subtracted cDNA library were chosen, and their differential expression was evaluated using RNAs from whole ovaries as well as captured primordial and primary follicles by laser captured microdissection. Results from the present study would provide insight for the future study on the mechanisms involved in primordial-primary follicle transition in the mouse in addition to the human ovary.

• Radiation Oncology Journal
• /
• v.23 no.1
• /
• pp.43-50
• /
• 2005

Purpose : A number of genes and their products are Induced early or late following exposure of cells to ionizing radiation. These radiation-Induced genes have various effects on irradiated cells and tissues. Suppression subtractive hybridization (SSH) based on PCR was used to Identify the differentially expressed genes by radiation in cervix carcinoma cells. Materials and Methods : Total RNA and poly $(A)^+$ mRNA were Isolated from Irradiated and non-irradiated HeLa cells. Forward- and reverse-subtracted cDNA libraries were constructed using SSH. Eighty-eight clones of each were used to randomly select differentially expressed genes using reverse Northern blotting (dot blot analysis). Northern blotting was used to verify the screened genes. Results : Of the 17t clones, 10 genes in the forward-subtracted library and 9 genes In the reverse-subtracted library were identified as differentially expressed radiation-induced genes by PCR-select differential screening. Three clones from the forward-subtracted library were confirmed by Northern blotting, and showed increased expression in a dose-dependent manner, including a telomerase catalytic subunit and sodium channel-like protein gene, and an ESTs (expressed sequence tags) gene. Conclusion : We Identified differentially expressed radiation-induced genes with low-abundance genes with SSH, but further characterization of theses genes are necessary to clarify the biological functions of them.

• Proceedings of the KSAR Conference
• /
• 2004.06a
• /
• pp.195-195
• /
• 2004

To discover germ cell-specific transcripts, we prepared a cDNA library from adult testes of 35-day old mice and subtracted it with mRNA from the testes of juvenile mice. Real-time RT-PCR analysis indicated that 42 cDNA clones in the subtracted library were expressed more intensely in the adult testes than in the juvenile testes. One clone identified by subtraction is expressed preferentially in the late spermatid and is located on chromosome 17E3 in mouse and 2p22 in human. (omitted)

• The Journal of the Korean dental association
• /
• v.39 no.12 s.391
• /
• pp.1068-1080
• /
• 2001

• Journal of Animal Science and Technology
• /
• v.44 no.1
• /
• pp.13-22
• /
• 2002

To identify the differentially expressed genes at growth stage of Hanwoo, we constructed the subtractive cDNA library from loin mRNA of 12- and 24-month old Hanwoo by PCR-based subtraction. The fourteen genes were confirmed by sequencing and reverse northern blot analysis, and they were selected as candidate of putative genes differentially expressed at the growth stage of Hanwoo. Three subtracted cDNA fragments that expressed specific signal to cDNA probe for 6-month-old loin of Hanwoo were highly homologous to those of the genes encoding EPV 20, Ca2＋ATPase, and TCTP, respectively. The nine cDNA clones showed intense signal to cDNA probe from 12-month-old loin of Hanwoo, and highly homologus to those of genes encoding VCP, HSP 70, aldolase A, MSSK1, GM-2 activator protein, ryanodine receptor, acidic ribosomal phosphoprotein p1, ADP/ATP translocase, and UCP 2, respectively. Two subtracted cDNA clones that expressed specific signal to cDNA probes for 12- and 24-month-old loin of Hanwoo were detected. One of them was highly homologus to the gene encoding ferrochelatase and the other was highly homologus to the gene encoding ADRP.

• Proceedings of the Korean Society of Grassland Science Conference
• /
• 2001.06a
• /
• pp.88-88
• /
• 2001

• Korean Journal of Ichthyology
• /
• v.23 no.1
• /
• pp.21-29
• /
• 2011

Expression analysis of development-related genes was conducted using differential screening of 6-month-old [18M(-), 6M-18M] specific and 18-month-old [6M(-), 18M-6M] specific subtracted cDNA libraries constructed by subtractive hybridization using skeletal muscle of 6- and 18-month-old dark-banded rockfish Sebastes inermis. A total 202 cDNA clones displaying different expression levels in each stage were obtained; among them, 32 clones showing up-regulation were finally selected for further expression analysis. We sequenced the clones and analyzed individual sequences. Genes expressed specifically in 6-month-old skeletal muscle were identified as myosin, adenylate kinase, calsequestrin, dystrobrevin beta, and diphosphate kinase-Z1. Genes showing strong expression in 18-month-old rockfish were identified as desmin, TGFBR2 (transforming growth factor-beta receptor), muscle-type creatine kinase, and cathepsin D. Expression of these genes was checked further in 6-18-30-42 month-old dark-banded rock fish. Rapid reduction of expression was observed in dystrobrevin beta and diphosphate kinase. However, expression of creatine kinase (muscle type) and cathepsin D increased as dark-banded rockfish grew, and remained even after 18 months. The results reported here demonstrate that genes related to muscles contract are expressed at an early stage of development, and genes controlling energy in muscles are predominantly expressed at a late developmental stage.

• Development and Reproduction
• /
• v.6 no.1
• /
• pp.7-16
• /
• 2002

In order to obtain the specific genes of snailfish a subtracted cDNA library was constructed, and analysed by sequencing and GenBank search. Among them C90-171 clone was turned out to be genes showing low homology and nonredundant genes. This novel clone was named Gomsin(C90-171). Gomsin was shown to be intensely expressed in the epithelial cells, some mesenchymal cells, and sheaths of muscle bundles in the result of immunohistochemistry. In the cross reaction assay of Gomsin antibody against various human tissues, the Gomsin was strongly expressed in the ductal and acinar cells of salivary glands, which was similar to the expression patterns of proline-rich proteins(PRPs) of human. The antibody raised against the Gomsin was clearly cross-reacted with human salivary PRPs and also recombinant proteins of human PRPs in the Western blot and immunoprecipitation analysis. Contrast to the salivary PRPs, the Gomsin was not easily degraded in the mixed saliva, but rapidly attacked on the cultured keratocytes in vitro. The simulated protein structure of Gomsin was similar to the whorled pattern of PRPs, even though the amino acid sequence of Gomsin was quite different from those of PRPs. These data suggest that the Gomsin is a characteristic matrix protein in the skin and body of snailfish, which is also utilized for the tissue protection in the similar way to the PRPs of human muco-secretory organs.