Application of mRNA Differential Display to the Identification of Genes Related to Embryonic Development

mRNA differential display was established by Liang and Pardee in 1992 for the purpose of displaying the mRNA differences between two tissues. The early embryonic development in animals is primarily controlled by the maternal RNAs stored in egg. These mRNAs are being degraded as the development proceeds. In some animals, such as fish and amphibian, new transcripts do not appear until the midblastula stage (midblastula transition, MBT). In other animals, for example in mouse, the zygotic genes are expressed during very early stages of development. The mRNA programmed synthesis and degradation during embryonic development controls the cell differentiation, germlayer formation and pattern formation. All these mRNA changes could be displayed side by side as cDNA band differences by mRNA differential display and the genes corresponding to these differential mRNAs could thus be obtained.

Progress in mRNA Differential Display

The mRNA Differential Display is a new molecular biological strategy for detecting and characterizing altered gene expression in eukaryotic cells which wad developed in 1992.Because of its simplicity,sensitivity and reproducibility,this method should find wide-ranging underpaid application developmental and molecular biology.Therecent successful applications of this method to gene hunting and the technological improvement promise great potential of mRNA Differential Display.

ESTABLISHMENT OF DIFFERENTIAL DISPLAY mRNA AND ITS APPLICATION IN THE STUDY ON THE MECHANISM OF LUNG CANCER INDUCED BY RADIATION

Objective: A method for separating mRNAs by means of the polymerase chain reaction (differential display mRNA), and identifying the genes related to radiation-induced lung cancer was introduced. Methods: The RNAs were isolated from two pairs of samples, SV40-immortalized human fetal tracheal fibroblast cell (SHTF) versus αSHTF cell (transformed SHTF cell induced by α particles) and lung cancer tissue versus normal lung tissue obtained from one miner, and amplified by RTPCR. The differential expressed gene fragments were displayed by autoradiograph or silver nitrate stain. Results: The differential display mRNA method was established using both cell and tissue samples. The bands stained by silver nitrate were clearer than those on X-ray film. The rate of reamplification of differentially expressed gene fragments stained by silver nitrate is 80%, higher than that by autoradiograph, 50%. Conclusion: Differential display mRNA method was established successfully on both cell and tissue samples. The modified method for staining band increased the rate of reamplification and established the basis for confirming relative genes.

Identification of Differentially Expressed Genes Induced by Ammonium Nitrogen in Rice Using mRNA Differential Display

RNAs isolated from ammonium- and nitrate-treated rice leaves were used to screen differentially expressed genes through mRNA differential display. A total of 72 bands appeared significant differences and some of them were further confirmed by reverse Northern and Northern blot. The results showed that two genes, A-02 （Oryza sativa drought stress related mRNA） and A-03 （Zea mays partial mRNA for TFIIB-related protein） were highly up-regulated in the ammonium-fed rice leaves. The enzyme assays showed that the activities of the two anti-oxidative enzymes, catalase and peroxidase, and the content of a non-enzymic antioxidant, glutathione, were significantly higher in the ammonium-fed rice leaves than those in the nitrate-fed ones, indicating that the ammonium nutrition might be beneficial for rice plants to improve the stress resistance during growth and development.

Isolation, Cloning, and Identification of Expressed Sequence Tags from the Backfat Tissue in Duroc and Tongcheng Pigs by mRNA Differential Display

mRNA differential display technique was performed to discuss the differential expression of genes in fat tissue between introduced European and Chinese indigenous pigs. Four anchor primers in combination with five arbitrary primers （20 sets in total） were used and nearly 300 bands were observed in polyacrylamide gel, among which 29 differential display bands were obtained. Twelve of 29 cDNA fragments were identified using reverse Northern dot blot, and subsequently cloned and sequenced. Eight of 12 cDNAs had no matches in GenBank and were submitted to GenBank, and the other 4 showed similarity to identified genes from GenBank. Three among 8 novel ESTs were selected to be further identified by semiquantitative RT-PCR. In our experiment, silver staining DDRT-PCR and DIG primer DNA labeling reverse Northern dot blot were used to avoid radioactive pollution. The result showed that the expressions of 5 among 8 novel ESTs were stronger in the backfat of Tongcheng pigs and the others were weaker than that in Duroc pigs. These novel ESTs were prepared for selecting genes related to adipose cells.

mRNA differential display on gene expression in settlement metamorphosis process of Ruditapes philippinarum larvae

The mRNA differential display (DDRT-PCR) technique was adopted to find out the genes related tosettlement metamorphosis development process of Ruditapes philippinarum larvae.In this study,we haveobtained three hundred and forty-six amplification bands in total from pediveliger larvae,veliger larvae,eye spot larvae and post-larvae.Sixty-five out of three hundred and forty-six bands are distinctly differen-tial display from band pattern,which can be put into four groups,standing for different expression char-acters.Sixteen differential display bands were cloned,sequenced and analyzed and nine different se-quences are obtained in the study.Three sequences have higher similarity to the cDNAs deposited indatabase and three are very similar to the rDNA of other species,considered as the rDNA of Ruditapesphilippinarum.The rest three sequences are found to be novel sequences after analyzed.Their accessionnumbers are AY916799,AY916798,and AY916797 respectively.We thought the novel sequences arepossibly relevant to the early embryo development of Ruditapes philippinarum larvae and can provide somefundamental understandings that are helpful for the improvement of scallop seed raising industry.

Identification of differentially expressed genes in dorsal root ganglion in early diabetic rats

Objective To screen and identify differentially expressed genes in the dorsal root ganglion （DRG） in early experimental diabetic rats. Methods Diabetic model rats were induced by single intraperitoneal injection of streptozotocin （STZ）. At the second week after STZ injection, the sensory nerve conduction velocities （SNCV） of sciatic nerve were measured as an indicator of neuropathy. The technique of silver-staining mRNA differential display polymerase chain reaction （DD-PCR） was used to detect the levels of differentially expressed genes in rat DRG. The cDNA fragments that displayed differentially were identified by reverse-hybridization, cloned and sequenced subsequently, and then confirmed by Northern blot. Results The SNCV in the diabetic model group [n = 9, （45.25±10.38） m/s] reduced obviously compared with the control group [n = 8, （60.10± 11.92） m/s] （P 〈 0.05）. Seven distinct cDNA clones, one was up-regulated gene and the others were downregulated ones, were isolated by silver-staining mRNA differential display method and confirmed by Northern blot. According to the results of sequence alignment with GenBank data, majority of the clones had no significant sequence similarity to previously reported genes except only one that showed high homology to 6-pyruvoyl-tetrahydropterin synthase mRNA （accession No., BC059140）, which had not been reported to relate to diabetic neuropathy. Conclusion These differentially expressed genes in the diabetic DRG may contribute to the pathogenesis of diabetic peripheral neuropathy.

Genes Differentially Expressed in Human Lung Fibroblast Cells Transformed by Glycidyl Methacrylate

Objective To define the differences in gene expression patterns between glycidyl methacrylate (GMA)-transformed human lung fibroblast cells (2BS cells) and controls. Methods The mRNA differential display polymerase chain reaction (DD-PCR) technique was used. cDNAs were synthesized by reverse transcription and amplified by PCR using 30 primer combinations. After being screened by dot blot analysis, differentially expressed cDNAs were cloned, sequenced and confirmed by Northern blot analysis. Results Eighteen differentially expressed cDNAs were cloned and sequenced, of which 17 were highly homologous to known genes (homology = 89%-100%) and one was an unknown gene. Northern blot analysis confirmed that eight genes encoding human zinc finger protein 217 (ZNF217), mixed-lineage kinase 3 (MLK-3), ribosomal protein (RP) L15, RPL41, RPS16, TBX3, stanniocalcin 2 (STC2) and mouse ubiquitin conjugating enzyme (UBC), respectively, were up-regulated, and three genes including human transforming growth factor b inducible gene (Betaig-h3), a-1,2-mannosidase 1A2 (MAN 1A2) gene and an unknown gene were down-regulated in the GMA-transformed cells. Conclusion Analysis of the potential function of these genes suggest that they may be possibly linked to a variety of cellular processes such as transcription, signal transduction, protein synthesis and growth, and that their differential expression could contribute to the GMA-induced neoplastic transformation.

Isolation, Identification and Tissue Expression Profile Analysis of One Novel Differentially Expressed Sequence Tag in the Longissimus dorsi Muscle from Meishan, Meishan × Large White Hybrid and Large White Pigs

In order to detect the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performed to investigate the differences of gene expression in the Longissimus dorsi tissue from Meishan, Meishan × Large White hybrid and Large White pigs with nine 3'-end anchored primers in combination with ten 5'-end arbitrary primers and nearly 3000 reproducible bands were examined. One novel expressed sequence tag (EST4, GenBank accession number: AY553914) that was differentially expressed in Meishan, Meishan× Large White hybrid and Large White pigs was isolated from the Longissimus dorsi muscle tissue and identified through semi-quantitative RT-PCR. BLAST analysis revealed that the 350 bp long EST (EST4) was not homologous to any of the known porcine genes. Tissue expression profile analyses showed that the EST4 was expressed in most of tissues.LIU Yong-gang, Ph D candidate

Differential Gene and Protein Expression in Soybean at Early Stages of Incompatible Interaction with Phytophthora sojae

Soybean root and stem rot caused by Phytophthora sojae is a destructive disease worldwide. Using genetic resistance is an important and major component in the integrated pest management of this disease. To understand molecular mechanisms of root and stem rot resistance in soybeans, the gene and protein expression in hypocotyls and stems of variety Suinong 10 carrying resistance genes Rps1a and Rps2 was investigated by using mRNA differential display reverse transcription PCR and two-dimensional electrophoresis at 0, 0.5, 1, 2, and 4 h after inoculation with P. sojae race 1. The results of the comparison of gene and protein expression showed that at least eight differential fragments at the transcriptional level were related to metabolic pathway, phytoalexin, and signal transduction in defense responses. Sequence analyses indicated that these fragments represented cinnamic acid 4-hydroxylase gene, ATP b gene coding ATP synthase b subunit and ubiquitin-conjugating enzyme gene which upregulated at 0.5 h post inoculation, blue copper protein gene and UDP-N-acetyl-a-D-galactosamine gene which upregulated at 2 h post inoculation, TGA-type basic leucine zipper protein TGA1.1 gene, cyclophilin gene, and 14-3-3 protein gene which upregulated at 4 h post inoculation. Three resistance-related proteins, a-subunit and b-subunit of ATP synthase, and cytochrome P450-like protein, were upregulated at 2 h post inoculation. The results suggested that resistance-related multiple proteins and genes were expressed in the recognition between soybean and P. sojae during zoospore germination, penetration and mycelium growth of P. sojae in soybean.

Isolation, Identification of Differentially Expressed Sequence Tags in the Backfat Tissue from Meishan, Large White and MeishanLarge White Cross Pigs

In order to detect the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performedto investigate the differences of gene expression in the backfat tissue from Meishan, Large White and MeishanLargeWhite cross pigs. Nine 3'-end anchored primers in combination with ten 5'-end arbitrary primers were used to perform thedifferential display PCR and nearly 3 000 reproducible bands were examined. Fifteen expressed sequence tags that weredifferentially expressed were isolated and then identified through semi-quantitative RT-PCR. BLAST analysis revealedthat the fifteen expressed sequence tags (ESTs) were not homologous to any of the known porcine genes or ESTs. Thesenovel ESTs were then submitted to GenBank.

Identification of tumor metastasis-related gene TMSG-1 by mRNA differential display

To investigate genes involved in cancer metastasis, mRNA differential display was used to compare the levels of gene expression of two cancer sublines derived from prostate carcinoma cell PC-3M that had different metastatic potentials. The differentially expressed genes were confirmed by Northern blot, and sequenced. The full-length cDNA of a tumor metastasis suppressor gene (TMSG-1) was obtained by using EST assembling and verified by RT-PCR and sequencing. The results showed that expression levels of TMSG-1 were lower in the highly metastatic cell line 1E8, compared with the non-metastatic cell line 2B4. The difference was significant. Full-length cDNA of TMSG-1 was about 2 kb, containing an open reading frame that encoded a protein of 230 amino acids. GenBank Blastn showed no marked homology with known genes. The functional prediction of amino acids sequence encoded by TMSG-1 gene indicated TMSG-1 protein was transmembrane protein, with 3 transmembrane domains, 3 putative protein kinase phosphorylation sites, 2 casein kinase II phosphorylation sites and 1 N-myristoylation site. The pattern of TMSG-1 expression in 6 types of human tumor tissues indicated levels of transcripts were the highest in prostate carcinoma. TMSG-1 had lower expression in metastases of lung carcinoma compared to primary lung carcinoma. Similarly the expression levels were higher in well-differentiated colon carcinoma than that in poorly differentiated colon carcinoma. TMSG-1 could also be detected in breast, ovarian, and pancreatic carcinoma. In 9 samples of primary gastric carcinoma tissues, RT-PCR and densitometric analysis demonstrated TMSG-1 expression levels in samples with lymph node metastases had a decreased tendency, compared to those without lymph node metastases. The difference was significant by student's t test (p<0.05). These results indicated TMSG-1 expression levels were inversely correlated with tumor metastatic potential.

mRNA differential display between sterile and fertile anther of rice and analysis of cDNA differential fragments

Rice anther and leaf mRNAs from two cytoplasmic male sterile lines (Maxie A and Congguong 41A ) were compared with those from their maintainers and F\-1 hybrids by using mRNA differential display to study gene expression pattern in sterile anther during pollen abortion. Anther cDNA differential bands between sterile and fertile plants were more than those of leaf. It was indicated that the expression of fertility gene(s) in the anther was more activable and sufficient than in the leaf. The gene transcript pattern in one of different types of anther was not only associated with its pollen sterility degree but also with its stage of pollen abortion. The anthers with full or partial sterility or with early abortion produced more cDNA differential bands than those fertile or late abortion anthers. In twelve recovered differential cDNA fragments, two were probably associated with male sterility, i.e. one is (AB\-4A\-5), which was specifically expressed in the sterile anther, and the other (AB\-3B\-2), which contained some sequence homologous to a mitochondrial gene (coxII) and whose expression was partially suppressed in the sterile anther.

Differential display of mRNA between hybrid F_1 and its parental inbred lines in maize (Zea mays L.)

Heterosis is a universal biological phenomenon in living things. It has been utilizedextensively in crop breeding, the improvement of fish and the breed of domestic animal fora long time. Great success has been achieved in practical production of the utilization ofheterosis, but in theory, we have known little about the mechanism of heterosis. At thebeginning of this century, Bruce et al. proposed the dominance hypothesis to explain

Screening for glutamate-induced and dexamethasone-downregulated epilepsy-related genes in rats by mRNA differential display

Background It is known that excessive release of glutamate can induce excitotoxicity in neurons and lead to seizure. Dexamethasone has anti-seizure function. The aim of this study was to investigate glutamatedexamethasone interaction in the pathogenesis of epilepsy, identify differentially expressed genes in the hippocampus of glutamate-induced epileptic rats by mRNA differential display, and observe the effects of dexamethasone on these genes expression. Methods Seizure models were established by injecting 5μl （250 μg/μl） monosodium glutamate （MSG） into the lateral cerebral ventricle in rats. Dexamethasone （5 mg/kg） was injected intraperitoneally at 30 minutes after MSG inducing convulsion. The rats＇ behavior and electroencephalogram （EEG） were then recorded for 1 hour. The effects of dexamethasone on gene expression were observed in MSG-induced epileptic rats at 1 hour and 6 hours after the onset of seizure by mRNA differential display. The differentially expressed genes were confirmed by Dot blot. Results EEG and behaviors showed that MSG did induce seizure, and dexamethasone could clearly alleviate the symptom, mRNA differential display showed that MSG increased the expression of some genes in epileptic rats and dexamethasone could downregulate their expression. From more than 10 differentially expressed eDNA fragments, we identified a 226 bp eDNA fragment that was expressed higher in the hippocampus of epileptic rats than that in the control group. Its expression was reduced after the administration of dexamethasone. Sequence analysis and protein alignment showed that the predicted amino acid sequence of this cDNA fragment kept 43% identity to agmatinase, a member of the ureohydrolase superfamily. Conclusions The results of the current study suggest that the product of the 226 bp eDNA has a function similar to agmatinase. Dexamethasone might relax alleviate seizure by inhibiting expression of the gene.

Down-regulation of DPH2L gene during cellular differentiation/apoptosis:Use of mRNA differential display

To characterize the genes associated with differentiation/apoptosis induced by all-trans retinoic acid (ATRA) in human lung cancer cells, mRNA differential display was employed. Six cDNA fragments have been isolated, and one of them corresponds to a sequence-known gene DPH2L with unknown function, which was first isolated from the critical region of deletion on chromosome 17p13.3 in human ovarian carcinoma, and regarded as a candidate tumor suppressor gene. Results show that DPH2L is a wide expressed gene, and has another major transcript besides the previously reported 2.3 kb transcript. It is proved that the DPH2L gene is dawn-regulated during differentiation or apoptosis in several kinds of cancer cells induced by all-trans retinoic acid and N-(4-hydroxyphenyl) retinamide (4HPR). This may suggest that DPH2L does not play a role as a tumor suppressor gene, on the contrary, its down-regulation may be functionally involved in the transition from cell growth to differentiation or apoptosis.

Preliminary study on search for genes for early embryonic development in goldfish by mRNA differential display

MATERNAL RNAs stored in eggs play an essential role during the initial stage of embryonic de-velopment in animals and they are degraded as the development proceeds. In some animals (e.g. fish and amphibian), zygotic genes begin to transcribe new mRNA only when the embryosdevelop to about midblastula stage. This degradation of maternal RNA and synthesis of

SCREENING OF DRUG RESISTANCE-RELATED GENES FROM HUMAN OVARIAN CANCER CELL LINE OC3/ADR BY DD-PCR

Objective: To screen novel genes related to adriamycin (Adr) resistance from human ovarian cancer resistance cell line OC3/Adr. Methods: Multidrug resistant ovarian cancer cell line OC3/Adr was induced by intermittent treatment of the human parent cell line OC3 with high concentration Adr. The difference of gene expression was screened by using different display analysis to the acquired Adr-resistance subline OC3/Adr and its parent cell line OC3. Results: OC3/Adr cell line was obtained which was more resistance to Adr than the parent cell line OC3 with the resistance index (RI) of 15.4. The OC3/Adr cell line also showed cross-resistance to other anti-cancer drugs (VP16, CDDP,5FU). It grew slowly and exhibited changes of cell cycle. A number of differentially expressed ESTs (Expressed Sequence Tags, ESTs) were identified at mRNA level between the OC3/Adr and OC3. Four of 18 different ESTs were sequenced. The 431/432 base pair S1 was homologous to human sperm zona pellucida binding protein, while the other two ESTs, S3 and S4, were new gene segments, which were registered to GenBank with the number of AF 117656 and AF 126507 respectively. Particularly, the expression of S2 sequence increased in all the drug-resistance cell lines and S3 sequence overexpressed in human ovarian cancer tissues as compared with benign ovarian tumors. Conclusion: Drug resistance induced by Adr in ovarian cancer OC3/Adr is involved with changes of multiple gene expressions.

Stage-specific expression genes of the Spirometra erinaceieuropaei plerocercoid screened by mRNA differential display technique

Background The stage-specific expression of genes is o ne of the most characteristics of parasites. It has been found that a lot of gen es of Spriometra erinaceieuropaei are specifically expressed in pleroceroid in large amount, but not expressed when the plerocercoid development into adult worm. The study is to screen other stage-specific ecpression genes of plerocerc oid of Spirtmetra erinceieuropaei.Methods RNA was separately extracted by acid guanidinium thiocyanate-phenol-chloroform from plerocercoids and adult worms of Spirometra erinaceieuropaei, DN A contaminated in the RNA was digested by RNase-free DNase. After the RNA was reverse transcripted to cDNA using T 12 MA, T 12 MC, T 12 MG and T 12 MT anchor-primers, PCR was done using the same T 12 MN and one random primer with α 35 S-dATP in the system. The PCR products were fractionated on an 8% denatured polyacrylamide gel. Differential bands of the plerocercoid found in the gel were cut out, amplified by PCR and sequenced. Northern hybridization was used to identify the stage-specific expression genes.Results Eleven differential bands were selected from the gel and classified into 3 kinds of gene fragments by hybridization, after they were amplified by PCR. Fragments 1 (238 bp) and 2 (383 bp), were confirmed by Northern hybridization, as being expressed in the plerocercoid. However, fragment 3 (433 bp), was expressed in both the plerocercoid and the adult worm. Data from the 3 gene fragments underwent homological analysis in GenBank. The sequence which was homologous with fragments 1 and 2 was not found, but fragment 3 had high homology with many kinds of 28S rRNA. Conclusions The gene expressions of plerocercoids are different from adult worms because they live in different hosts. Two types of different gene fragments from the plerocercoid were found by mRNA differential display technique.