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PubMed2006

cDNA-AFLP reveals differentially expressed genes related to cell aggregation of Azospirillum brasilense.

The response regulatory gene flcA controls the differentiation process of Azospirillum brasilense from vegetative state to cyst-like forms, both in culture and in association with plants. In contrast to the wild type parental strain Sp7, strain Sp72002, a Tn5 induced flcA(-) mutant, does not aggregate, does not differentiate from motile, vibroid cells into nonmotile, cyst-like forms and lacks some of the exopolysaccharide material on its cell surface. Here we aimed to identify differentially expressed genes whose expression could be modulated by flcA. Using cDNA-amplified fragment length polymorphism, we detected 81 transcript-derived fragments showing differential expression during exponential growth phase in an aggregation-inducing medium containing high C : N ratio. The fragments were sequenced and analyzed, and expression of ten genes encoding known proteins was compared between the two strains by reverse transcriptase polymerase chain reaction-(RT-PCR). RT-PCR analyses confirmed the differential patterns observed by cDNA-AFLP in nine of those genes. Among them are genes homologous to nodQ, involved in sulfation; narK, involved in nitrite/nitrate transport, and flp, involved in autoaggregation; as well as genes encoding a biopolymer transport protein, and the signal recognition particle. This work demonstrates the usefulness of the cDNA-AFLP approach to reveal genes that are differentially expressed during aggregation in Azospirillum brasilense and provides insights into the aggregation process of this bacterium.

Amino Acid Sequence↗
PubMed2000

Optimization of cDNA representational difference analysis for the identification of differentially expressed mRNAs.

Representational difference analysis (RDA) is a powerful and sensitive tool for identification of differentially expressed genes (M. Hubank and D. G. Schatz, 1999, Methods Enzymol. 303, 325-349; 1994, Nucleic Acids Res. 22, 5640-5648) that will identify both up- and downregulated genes differentially expressed between two cDNA populations. This manuscript provides a thorough description of an optimized RDA method. This procedure while still based on the traditional RDA originally developed by Lisitsyn and co-workers(N. A. Lisitsyn, 1995, Trends Genet. 11, 303-307; N. A. Lisitsyn, F. S. Leach, B. Vogelstein, and M. H. Wigler, 1994, Cold Spring Harbor Symp. Quant. BioL 59, 585-587; N. Lisitsyn, N. Lisitsyn, and M. Wigler, 1993, 259, 946-951) and modified by Hubank and Schatz for RNA (1994, Nucleic Acids Res. 22, 5640-5648) is improved and requires less starting material than many existing methods. Several key modifications are included (1). Size-exclusion gel-filtration microspin columns are used throughout the procedure to remove the primers and low molecular weight cDNAs. This results in reducing the number of ethanol precipitations required and in improving the yield of desirable amplification products (2). Elimination of the mung bean nuclease treatment in favor of a simple dilution of PCR serves as a means of markedly reducing the single-stranded cDNAs that can interfere with the amplification of differentially expressed products (3). The use of up to six unique noninteracting primers ensures that no anomalous amplification occurs due to carryover of primers or incomplete digestion from the ends of the cDNAs (4). A set of cDNA standards was developed and various concentrations were used to better characterize the ability of representational difference analysis to identify rare messages in a complex cDNA population (5). Integral to this manuscript, a detailed laboratory protocol is available from the authors (craig.byus@ucr.edu) and provides a step-by-step description of the modified procedure.

Base Sequence↗
PubMed2006

[Effects of leukemic bone marrow stromal cells on gene differential expression in human leukemic Jurkat T-cell line exposed to daunorubicin].

OBJECTIVE: To investigate the inhibition effect of leukemic bone marrow stromal cells (BMSCs) on daunorubicin (DNR) induced apoptosis of human Jurkat cell line, and analyze the differentially expressed genes between Jurkat cells cocultured with leukemic BMSCs or without. METHODS: Suppression subtractive hybridization (SSH) was employed to establish subtracted cDNA library of differentially expressed genes in Jurkat cells cocultured with leukemic BMSCs and DNR. The cDNA fragments were sequenced and analyzed. RESULTS: The differentially expressed gene cDNA library was successfully developed. Primary screening was done by reverse Northern hybridization. Thirty up-regulated and 22 down-regulated cDNA fragments were isolated and sequenced. Analysis and comparison were performed in GenBank using BLAST. These genes are related to cell cycle regulation, cell apoptosis and energy metabolism. CONCLUSION: Leukemic BMSCs influence gene expression of Jurkat cells. The resulting differentially expressed genes might be associated with the protection of leukemic cells by BMSCs from injury.

Apoptosis↗
PubMed2003

Reg IV, a differentially expressed gene in colorectal adenoma.

OBJECTIVE: To discover and identify differentially expressed genes associated with colorectal adenoma formation and the role of RegIV in colorectal adenoma differentiation. METHODS: A subtracted cDNA library was constructed with cDNAs that were isolated from either the normal mucosa or adenoma tissue of a single patient. Suppressive subtractive hybridization (SSH) combined with virtual northern blotting was used to characterize differentially expressed genes and contigs were assembled by electronic cloning (in silico cloning) with the EST database. Semi-quantitative RT-PCR was performed in 9 colorectal adenomas. RESULTS: The amino acid sequence was determined with open reading frame (ORF) prediction software and was found to be 100% homologous to the protein product of RegIV (a novel gene isolated from a large inflammatory bowel disease library). RegIV was found to be highly expressed in all of the adenoma samples (9/9) compared with the normal mucosa samples, while 5/6 cases showed RegIV to be more strongly expressed in adenocarcinoma. CONCLUSION: RegIV may play an important role in the initiation of colorectal adenoma differentiation, and its detection may be useful in the early diagnosis of colorectal adenoma formation.

Adenoma↗
PubMed2002

[Screening of novel genes differentially expressed in human renal cell carcinoma by suppression subtractive hybridization].

BACKGROUND AND OBJECTIVE: Identifying the differentially expressed genes in renal cell carcinoma (RCC) contributes to the elucidation of its genetic basis. However, the above knowledge has not yet been fully understood. The aims of this experiment were to screen novel genes differentially expressed in RCC tissues by suppression subtractive hybridization (SSH) and clone RCC-specific related genes. METHODS: To construct SSH library of RCC by using the mRNA from RCC tissues and matched normal kidney tissues as tester and driver, respectively. Partial positive clones in the library were selected randomly and sequenced, then analyze the sequences with the BLAST software. To confirm the location of the fragments of interest in human chromosome through comparing their sequences with the human genome draft. mRNA levels of the novel genes in RCC and matched normal kidney tissues were determined by Northern blot and semi-quantitative RT-PCR analysis. RESULTS: The SSH library contained 414 positive clones. Random analysis of 280 clones with enzyme restriction showed that 265 clones contained cDNA fragments distributed mainly between 300-900bp. Among 80 arbitrary clones with were derived from above 265 clones and sequenced, No. 28, 158, 170, and 249 clones are previously unknown genes and located in human chromosome 21q22, 4p15.3, 9q34, and 22q11.2 by electronic mapping, respectively. The consequence of semi-quantitative RT-PCR demonstrated that mRNA levels of the two novel genes were overexpressed in RCC compared to matched normal tissues by more than 2-6 folds. Northern blot analysis confirmed the above results. CONCLUSIONS: SSH is a reliable strategy for screening novel genes differentially expressed in RCC. The novel gene fragments can be used to clone their full length and further to study their functions.

Blotting, Northern↗
PubMed2002

[Differential expression of the genes in leukemia cell apoptosis induced by TRAIL].

OBJECTIVE: To identify the genes differentially expressed in leukemia cell apoptosis induced by recombinant soluble tumor necrosis factor-related apoptosis inducing ligand (rsTRAIL). METHODS: Suppression subtractive hybridization (SSH) and polymerase chain reaction (PCR) were used for the cloning and identification of the genes differentially expressed in the apoptotic Jurkat cells induced by TRAIL. Slot blot and Northern blot were used for the expression pattern analysis of the genes. Automatic DNA sequencing was used for DNA sequence analysis. RESULTS: Six cDNA fragments differentially expressed in the Jurkat leukemia cells treated with TRAIL were found, in which four were inhibited and two were activated during the Jurkat cell apoptosis treated with TRAIL. Among which the five genes of A14, X1, D1, A23 and C5 were found at the first time by DNA sequencing and GeneBank database searching. So that they were registered in GeneBank as AW731601, AW731602, AW731603, AW731604 and BE239235, respectively. It was found that the gene D1 was expressed higher in Jurkat leukemia cells and MCF-7 breast cancer cells than that in K562 leukemia, 825 gastric cancer and 7721 liver cancer cells. CONCLUSIONS: Five novel cDNA fragments were found, and among which D1 might be a tumor specific gene.

Antineoplastic Agents↗
PubMed2004

[Construction of a reverse-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state].

OBJECTIVE: To inquire into the mechanism of prothrombotic state (PTS) and the roles of liver therein by constructing a reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS. METHODS: The reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS was constructed by suppression subtractive hybridization. The rat model of PTS was induced by a high-carbohydrate diet. Poly A+ mRNAs were isolated from PTS and control rats, and cDNAs were synthesized from the mRNAs. After digestion by means of Ras I, cDNAs 400-600 bp in size were obtained. For suppression subtractive hybridization, cDNAs from PTS rat were used as Driver and the cDNAs from control rat as Tester. The Tester was divided into two parts and ligated to adaptor 1 and adaptor 2R respectively. After two times of subtractive hybridization and two times of nested PCR, the products of the last PCR amplification were inserted into T/A plasmid vectors to transform the Escherichia coli JM109 cells. The transformed cells were incubated at 37 degrees C overnight on a LB agar plate containing ampicillin (50 micrograms/ml), IPTG and X-gal. The colonies were counted. RESULTS: 78% of the colonies were white and the reverse-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state was successfully constructed. CONCLUSION: Prothrombotic state caused by malfunction of homeostasis and fibrinolysis is an important risk factor of cardiovascular disease. Liver plays important roles in the development of PTS, for the majority of the factors in the coagulation as well as fibrinolytic cascades are generated by the liver and secreted into the bloodstream. The reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS, successfully constructed in the present study, provides an efficient way to further investigate the mechanism of PTS and the relevant liver functions.

Animals↗
PubMed1999

Specific gene expression in pancreatic beta-cells: cloning and characterization of differentially expressed genes.

Identification and characterization of genes expressed preferentially in pancreatic beta-cells will clarify the mechanisms involved in the specialized properties of these cells, as well as providing new markers of the development of type 1 diabetes. Despite major efforts, relatively few beta-cell-specific genes have been characterized. We applied representational difference analysis to identify genes expressed selectively in the pancreatic beta-cell line betaTC1 compared with the pancreatic alpha-cell line alphaTC1 and isolated 26 clones expressed at higher levels in the beta-cells than in the alpha-cells. DNA sequencing revealed that 14 corresponded to known genes (that is, present in GenBank). Only four of those genes had been shown previously to be expressed at higher levels in beta-cells (insulin, islet amyloid polypeptide, neuronatin, and protein kinase A regulatory subunit [RIalpha]). The known genes include transcription factors (STAT6) and mediators of signal transduction (guanylate cyclase). The remaining 12 genes are absent from the GenBank database or are present as expressed sequence tag (EST) sequences (4 clones). Some of the genes are expressed in a highly specific pattern-expression in betaTC1 and islet cells and in relatively few of the non-beta-cell types examined; others are expressed in most cell types tested. The identification of these differentially expressed genes may aid in attaining a clearer understanding of the mechanisms involved in beta-cell function and of the possible immunogens involved in development of type 1 diabetes.

Amyloid↗
PubMed2000

Identification of genes differentially expressed in C6 glioma cells transfected with connexin43.

Astrocytes are characterized by extensive gap junctional intercellular communication (GJIC) mediated primarily by channels composed of connexin43. In contrast, C6 glioma cells are deficient in connexin expression and gap junctional communication. Transfection of these glioma cells with connexin cDNAs results in changes in cellular phenotype following increased GJIC. Specifically, connexin expression correlates with reduced cellular proliferation and tumorigenicity. To characterize the role of gap junctions in this growth control, we have screened for changes in gene expression by differential display. We have observed that these changes in GJIC are associated with changes in expression of several genes, including those coding for a number of secreted factors which may play a role in modulating the tumor phenotype of these cells. These include the immediate early gene cyr61, ostoepontin and the KC gene (murine homologue of the human gro gene).

Animals↗
PubMed2006

[Screening and cloning the genes differentially expressed in human embryonic AGM-derived stromal cells].

To screen and separate the genes differentially expressed in human embryonic aorta-gonad-mesonephros (AGM)-derived stromal cells, a subtracted library was generated through the suppression subtractive hybridization using the cDNA of human embryonic AGM-derived stromal cells as target and human fetal liver (FL)-derived stromal cells as drivers. Then a high though screening technique, gene chip, was used to screen the differentially expressed genes in the established subtractive library. Approximately 18 of the resulting subtracted cDNA clones were partially sequenced and analyzed by blastn in the GenBank database. The results showed that 211 Clones were selected and identified from the established subtractive library, the positive ratio was amount to 76.4%. 18 over-expressed genes were screened by gene chip with more than a 5-fold difference expression levels between AGM and FL-derived stromal cells, and were selected to sequence, results of sequencing indicated that the 18 sequences was compared to known sequences in the GenBank database, and among the sequenced clones, 14 sequences were considered as part of the known genes, and 4 sequences representing previously unknown genes. The known genes were reported to involve the regulation of cell migration, cell differentiation, cell proliferation, cell cycle, signal transduction, and angiogenesis. Most of these genes have not been reported to relate to the haematogenesis in ontogeny. It is concluded that many genes both known and unknown are differentially expressed in human embryonic aorta-gonad-mesonephros-derived stromal cells. Discovery of these genes provides a solid foundation to elucidate the mechanism of haematogenesis in ontogeny.

Aorta↗
PubMed2005

Proteomic analysis of differentially expressed proteins between the male and female worm of Schistosoma japonicum after pairing.

Identification of differentially expressed proteins between the male and female worm of Schistosoma japonicum may provide new insights into the development of schistosomes, especially the molecular mechanism of female worm maturation induced by the male worm after pairing. Comparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry were employed to separate and identify differentially expressed proteins between the male and female worm after pairing. Soluble and hydrophobic proteins from egg, schistosomulum (14 days), and female and male worms at adult stage (42 days) were separated by a sequential extraction method followed by 2-DE and 2-DE images were constructed. There were 1016 +/- 67, 1808 +/- 89, 1142 +/- 45 and 1288 +/- 32 spots detected for soluble proteins and 1425 +/- 108, 952 +/- 59, 847 +/- 75 and 965 +/- 69 spots for hydrophobic proteins from egg, schistosomulum, and adult stage female and male worms, respectively. The differentially and uniquely expressed proteins from male and female worms after pairing (42 days) include 41 +/- 4 and 23 +/- 2 unique spots for soluble proteins, and 11 +/- 1 and 26 +/- 3 unique spots for hydrophobic proteins, respectively. Matrix-assisted laser desorption/ionization-time of flight and electrospray ionization-tandem mass spectrometry were employed to analyze 12 unique spots from the female worm and 16 unique spots from the male worm for peptide mass fingerprinting and sequencing. The results showed that the main functions of these differentially expressed proteins were in signal transduction, metabolism and transcriptional regulation etc. Comparison of the schistosomes proteome between male and female worms may permit the identification of protein candidates for the development of vaccines or new targets for drug development against schistosomiasis.

Animals↗
PubMed2001

Identifying differentially expressed genes in cDNA microarray experiments.

A major goal of microarray experiments is to determine which genes are differentially expressed between samples. Differential expression has been assessed by taking ratios of expression levels of different samples at a spot on the array and flagging spots (genes) where the magnitude of the fold difference exceeds some threshold. More recent work has attempted to incorporate the fact that the variability of these ratios is not constant. Most methods are variants of Student's t-test. These variants standardize the ratios by dividing by an estimate of the standard deviation of that ratio; spots with large standardized values are flagged. Estimating these standard deviations requires replication of the measurements, either within a slide or between slides, or the use of a model describing what the standard deviation should be. Starting from considerations of the kinetics driving microarray hybridization, we derive models for the intensity of a replicated spot, when replication is performed within and between arrays. Replication within slides leads to a beta-binomial model, and replication between slides leads to a gamma-Poisson model. These models predict how the variance of a log ratio changes with the total intensity of the signal at the spot, independent of the identity of the gene. Ratios for genes with a small amount of total signal are highly variable, whereas ratios for genes with a large amount of total signal are fairly stable. Log ratios are scaled by the standard deviations given by these functions, giving model-based versions of Studentization. An example is given.

Analysis of Variance↗
PubMed2005

[Differentially expressed genes of cell signal transduction associated with benzene poisoning by cDNA microarray].

OBJECTIVE: To detect the differential expression of cell signal transduction genes associated with benzene poisoning, and to explore the pathogenic mechanisms of blood system damage induced by benzene. METHODS: Peripheral white blood cell gene expression profile of 7 benzene poisoning patients, including one aplastic anemia, was determined by cDNA microarray. Seven chips from normal workers were served as controls. Cluster analysis of gene expression profile was performed. RESULTS: Among the 4265 target genes, 176 genes associated with cell signal transduction were differentially expressed. 35 up-regulated genes including PTPRC, STAT4, IFITM1 etc were found in at least 6 pieces of microarray; 45 down-regulated genes including ARHB, PPP3CB, CDC37 etc were found in at least 5 pieces of microarray. CONCLUSION: cDNA microarray technology is an effective technique for screening the differentially expressed genes of cell signal transduction. Disorder in cell signal transduction may play certain role in the pathogenic mechanism of benzene poisoning.

Apoptosis↗
PubMed2006

Genes differentially expressed in titanium implant healing.

Bone generation occurs around titanium implants; however, its underlying mechanisms are unknown. We hypothesized that molecular determinants distinct from those undertaking normal bone healing regulate osseointegration. Using differential display-polymerase chain-reaction in the male rat model, we isolated 3 genes that are differentially expressed in bone healing with implants, but not in osteotomy healing. A homology search indicated that these 3 genes are apolipoprotein E, prolyl 4-hydroxylase alpha-subunit, and an unknown transcript. Differential expression of these genes was remarkable during early healing stages up to week 2, and accelerated with rough acid-etched surfaces compared with machined surfaces. The differential expression was confirmed in the female rats, with enhanced expression for the acid-etched surfaces. The osseointegration-unfavorable condition created by gonadal estrogen deficiency reduced the level of differential expression. This study provides evidence that selected gene transcripts are induced by titanium implants under regulatory control strongly associated with the nature of osseointegration.

Acid Etching, Dental↗
PubMed2006

[Analysis of differential express gene between keloid and normal skin by suppression subtractive hybridization].

OBJECTIVE: To compare gene express difference of keloid and normal skin tissues by using the suppression subtractive hybridization (SSH) so as to find the differential express gene in keloid. METHODS: mRNA extracted from keloid and normal skin tissues was used as the template to synthesis cDNA of keloid and normal skin. The cDNA of keloid served as a tester, the cDNA of normal skin as a driver. cDNA was digested with Rsa I. Adaptor-ligated tester cDNA was prepared. Then first hybridization, second hybridization and PCR amplification were done. Differentially expressed cDNA was selectively amplified during these reactions. After SSH, the PCR mixture was ligated with T-vector. The positive clones were selected and the insert gene fragments were analyzed. Southern hybridization identified the keloid differential express genes. The positive clones of Southern hybridization were selected, and these sequences were analyzed. The results were compared with that of GeneBank. RESULTS: Thirteen differential genes were found in keloid, of which 11 gene clones have been known their function, and 2 clones have not known their function. CONCLUSION: Keloid differentially expressed gene was screened successfully by SSH.

DNA, Complementary↗
PubMed2003

3' Splicing variants of ret receptor tyrosine kinase are differentially expressed in mouse embryos and in adult mice.

The RET protooncogene encodes for a transmembrane receptor tyrosine kinase and plays a crucial role in nephrogenesis and the enteric nervous system (ENS) development. Alternative splicing at the 3' end of the RET gene generates 3' splicing variants that encode RET 9, RET 51 and RET 43 isoforms. It has been hypothesized that these isoforms perform distinct functions and that their expressions are differentially regulated during mammalian development. To gain an insight into the expression patterns of various ret isoforms during embryogenesis, we investigate the temporal and spatial expressions of ret gene in mouse embryos and in adult mice. We characterized the 3' end of the mouse ret gene and localized the alternatively spliced exons. Using 3' rapid amplification of cDNA ends (3' RACE) and reverse transcription-polymerase chain reaction (RT-PCR), ret 9 and ret 51 transcripts were identified in both mouse embryos and adult mouse tissues. However, the ret 43 transcript was not. Using in situ hybridization, we showed that ret 9 was the dominant ret encoding transcript in mouse embryos. Transcripts of ret 9 were detected in all cranial ganglia; in the sensory and autonomic ganglia of the trunk; in a subset of neurons of the dorsal root ganglion (DRG); in the motor neurons of the spinal cord; in the developing lung and excretory systems; in the enteric neuroblasts of the ENS; and in the thyroid lobes. In contrast, ret 51 expression was weak and restricted to the motor column of the spinal cord, the DRG, the enteric neuroblasts, the lung bud and the kidney. In adult mice, ret 9 expression was relatively widespread in many organs while that of ret 51 was rather restricted. Our data indicated that ret isoforms are temporally and spatially regulated in mouse embryos and adult mouse.

Alternative Splicing↗
PubMed2000

Use of RNA arbitrarily primed-PCR fingerprinting to identify Vibrio cholerae genes differentially expressed in the host following infection.

Evidence suggests that a repertoire of Vibrio cholerae genes are differentially expressed in vivo, and regulation of virulence factors in vivo may follow a different pathway. Our work was aimed at characterization of in vivo-grown bacteria and identification of genes that are differentially expressed following infection by RNA arbitrarily primed (RAP)-PCR fingerprinting. The ligated rabbit ileal loop model was used. The motility of in vivo-grown bacteria increased by 350% over that of in vitro-grown bacteria. Also, the in vivo-grown cells were more resistant to killing by human serum. By using the RAP-PCR strategy, five differentially expressed transcripts were identified. Two in vitro-induced transcripts encoded polypeptides for the leucine tRNA synthatase and the 50S ribosomal protein, and the three in vivo-induced transcripts encoded the SucA and MurE proteins and a polypeptide of unknown function. MurE is a protein involved in the peptidoglycan biosynthetic pathway. The lytic profiles of in vivo- and in vitro-grown cells suspended in distilled water were compared; the former was found to be slightly less sensitive to lysis. Ultrathin sections of both cells observed under the transmission electron microscope revealed that in contrast to the usual wavy discontinuous membrane structure of the in vitro-grown cells, in vivo-grown cells had a more rigid, clearly visible double-layered structure. The V. cholerae murE gene was cloned and sequenced. The sequence contained an open reading frame of 1,488 nucleotides with its own ribosome-binding site. A plasmid containing the murE gene of V. cholerae was transformed into V. cholerae 569B, and a transformed strain, 569BME, containing the plasmid was obtained. Ultrathin sections of 569BME viewed under a transmission electron microscope revealed a slightly more rigid cell wall than that of wild-type 569B. When V. cholerae 569B and 569BME cells were injected separately into ligated rabbit ileal loops, the transformed cells had a preference for growth in the ileal loops versus laboratory conditions.

Amino Acid Sequence↗