Selection of reliable reference genes for gene expression study in nasopharyngeal carcinoma.

AIM: To construct a system for selecting reference genes (RGs) and to select the most optimal RGs for gene expression studies in nasopharyngeal carcinoma (NPC). METHODS: The total RNAs from 20 NPC samples were each labeled with Cy5-dUTP. To create a common control, the total RNA from 15 nasopharyngeal phlogistic (NP) tissues was mixed and labeled via reverse transcription with Cy3-dUTP. cDNA microarrays containing 14 112 genes were then performed. A mathematical approach was constructed to screen stably expressed genes from the microarray data. Using this method, three genes (YARS, EIF3S7, and PFDN1) were selected as candidate RGs. Furthermore, 7 commonly used RGs (HPRT1, GAPDH, TBP, ACTB, B2M, G6PDH, and HBB) were selected as additional potential RGs. Real-time PCR was used to detect these 10 candidate genes' expression levels and the geNorm program was used to find the optimal RGs for NPC studies. RESULTS: On the basis of the 10 candidate genes' expression stability level, geNorm analysis identified the optimal single RG (YARS or HPRT1) and the most suitable set of RGs (HPRT1, YARS, and EIF3S7) for NPC gene expression studies. In addition, this analysis determined that B2M, G6PDH, and HBB were not appropriate for use as RGs. Interestingly, ACTB was the least stable RG in our study, even though previous studies had indicated that it was one of the most stable RGs. Three novel candidate genes (YARS, EIF3S7, and PFDN1), which were selected from microarray data, were all identified as suitable RGs for NPC research. A RG-selecting system was then constructed, which combines microarray data analysis, a literature screen, real-time PCR, and bioinformatic analysis. CONCLUSION: We construct a RG-selecting system that helps find the optimal RGs. This process, applied to NPC research, determined the single RG (YARS or HPRT1) and the set of RGs (HPRT1, YARS, and EIF3S7) that are the most suitable internal controls.

Grb10 interacts with Bim L and inhibits apoptosis.

Bim is a proapoptotic member of the Bcl-2 family and is primarily involved in the regulation of the intrinsic apoptotic pathway. However, the detail of regulation of Bim's proapoptotic activity has not been clarified yet. Using Bim L as bait, we screened a human fetal cDNA library for interacting proteins and identified Grb10 as an interactor. This interaction was verified by co-immunoprecipitation and intracellular co-localization studies. The potential segment of Bim L that binds Grb10 was identified via a yeast mating test. Grb10 interacted with the DBD (dynein binding domain) of Bim and inhibited apoptosis triggered by overexpression of DBD containing Bim isoforms. The putative phosphorylation sites on DBD of Bim play a role for the anti-proapoptotic activity of Grb10. Our results suggest that Grb10 interacts with Bim L and inhibits its proapoptotic activity in a phosphorylation-dependant manner.

A stochastic model for identifying differential gene pair co-expression patterns in prostate cancer progression.

BACKGROUND: The identification of gene differential co-expression patterns between cancer stages is a newly developing method to reveal the underlying molecular mechanisms of carcinogenesis. Most researches of this subject lack an algorithm useful for performing a statistical significance assessment involving cancer progression. Lacking this specific algorithm is apparently absent in identifying precise gene pairs correlating to cancer progression. RESULTS: In this investigation we studied gene pair co-expression change by using a stochastic process model for approximating the underlying dynamic procedure of the co-expression change during cancer progression. Also, we presented a novel analytical method named 'Stochastic process model for Identifying differentially co-expressed Gene pair' (SIG method). This method has been applied to two well known prostate cancer data sets: hormone sensitive versus hormone resistant, and healthy versus cancerous. From these data sets, 428,582 gene pairs and 303,992 gene pairs were identified respectively. Afterwards, we used two different current statistical methods to the same data sets, which were developed to identify gene pair differential co-expression and did not consider cancer progression in algorithm. We then compared these results from three different perspectives: progression analysis, gene pair identification effectiveness analysis, and pathway enrichment analysis. Statistical methods were used to quantify the quality and performance of these different perspectives. They included: Re-identification Scale (RS) and Progression Score (PS) in progression analysis, True Positive Rate (TPR) in gene pair analysis, and Pathway Enrichment Score (PES) in pathway analysis. Our results show small values of RS and large values of PS, TPR, and PES; thus, suggesting that gene pairs identified by the SIG method are highly correlated with cancer progression, and highly enriched in disease-specific pathways. From this research, several gene interaction networks inferred could provide clues for the mechanism of prostate cancer progression. CONCLUSION: The SIG method reliably identifies cancer progression correlated gene pairs, and performs well both in gene pair ontology analysis and in pathway enrichment analysis. This method provides an effective means of understanding the molecular mechanism of carcinogenesis by appropriately tracking down the process of cancer progression.

[Application of oligonucleotide array in single nucleotide polymorphism typing of cytokines and cytokine receptors].

OBJECTIVE: To develop an oligonucleotide array for single nucleotide polymorphism (SNP) typing of cytokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-10, tumor growth factor (TGF)-betal, IL-4, and IL-6, and their receptors and evaluate its function by direct sequencing. METHODS: According to relevant literature, SNP database of NCBI and SNP500 Cancer database of NCI, SNP loci and sequences of cytokines of clinical importance, TNF-a, IL-10, TGF-bl, IL-4 and IL-6, and matched cytokine receptors were chosen and 59 synthesized oligonucleotide probes were immobilized on a glass support, then the primer and Cy5-dCTP were used in multi-PCR, thus the products were labeled with Cy5. The labeled PCR products were hybridized with the probes in the array, and the signals were scanned by Scanner and then analyzed by Image software. Peripheral blood samples were collected from 80 healthy donors and 50 patients with uremia to isolate lymphocytes and DNA so as to undergo typing by this array, and the results were validated with direct sequencing. RESULTS: All the samples with PCR products except for 10 from uremia patients had been genotyped by cytokine array successfully. No diversity was found in genotyping for three times. Incorrect locus was not found with direct sequencing. CONCLUSION: With high specificity, sensitivity, repetitiveness, and throughout, and easy to manipulate, oligonucleotide array technique is an ideal molecular method for SNP genotyping of cytokine and cytokine receptor.

Discovery and analysis of pancreatic adenocarcinoma genes using cDNA microarrays.

AIM: To study the pathogenetic processes and the role of gene expression by microarray analyses in expediting our understanding of the molecular pathophysiology of pancreatic adenocarcinoma, and to identify the novel cancer-associated genes. METHODS: Nine histologically defined pancreatic head adenocarcinoma specimens associated with clinical data were studied. Total RNA and mRNA were isolated and labeled by reverse transcription reaction with Cy5 and Cy3 for cDNA probe. The cDNA microarrays that represent a set of 4 096 human genes were hybridized with labeled cDNA probe and screened for molecular profiling analyses. RESULTS: Using this methodology, 184 genes were screened out for differences in gene expression level after nine couples of hybridizations. Of the 184 genes, 87 were upregulated and 97 downregulated, including 11 novel human genes. In pancreatic adenocarcinoma tissue, several invasion and metastasis related genes showed their high expression levels, suggesting that poor prognosis of pancreatic adenocarcinoma might have a solid molecular biological basis. CONCLUSION: The application of cDNA microarray technique for analysis of gene expression patterns is a powerful strategy to identify novel cancer-associated genes, and to rapidly explore their role in clinical pancreatic adenocarcinoma. Microarray profiles provide us new insights into the carcinogenesis and invasive process of pancreatic adenocarcinoma. Our results suggest that a highly organized and structured process of tumor invasion exists in the pancreas.

[Impacts of donor and recipient's SNP of cytokine and cytokine receptor on early acute renal allograft rejection].

OBJECTIVE: To investigate the impact of donor and recipient's SNP of cytokine and cytokine receptor on early acute rejection after renal transplantation. METHODS: (1) 129 cases of cadaveric renal allograft recipients were divided into two groups according to the presence or absence of acute graft rejection. The distribution of 21 single nucleotide polymorphisms in cytokines and cytokine receptors gene were compared between two groups as well as latent factors affecting the development of acute rejection. (2) Based on the result of HLA-DR matching between donor and recipient, the recipients with AR were stratified into two conditions, 0-1 locus mismatched (0-1MM) and completely mismatched (2MM). By aids of SPSS 11.5 software, association analysis was assessed using Kruskal Wallis test, 2 x 2 or 2 x n contingency table, the Chi-square test. RESULTS: (1) Of 129 recipients of renal transplantation, 39 developed acute graft rejection (30.2%). (2) Compared with recipients without acute rejection, the number of HLA-DR mismatching was significantly higher in rejection group. (3) In rejection group and non-rejection group, the gene polymorphism distribution was significantly different. (4) 0-1MM group and 2MM group were various in the gene polymorphism distribution. CONCLUSIONS: In the whole, the susceptibility of acute rejection after renal transplantation may be predicted by the donor and recipient's SNP of cytokine and cytokine receptor.

[Detecting genetic polymorphisms of CYP1 A1 and GSTM1 simultaneously with oligonucleotide microarray].

Cytochrome P4501A1 plays a major role in the bioactivation of a number of tobacco procarcinogens. Glutathione S-transferase( GSTM1), a member of the class of GST gene family, has been shown to be polymorphic because of gene deletion resulting in a failure to express the GSTM1 gene in 50% approximately 60% of individuals. Some CYP1 A1/GSTM1 null genotype combinations seem to predispose the lung, esophagus, and oral cavity of smokers to an even higher risk for cancer or DNA damage, requiring, however, confirmation. An easy and reliable oligonuleotide microarray approach validated through direct sequencing method is developed in order to accurately detect single nucleotide polymorphisms of CYP1 A1 gene and discriminate the presence and absence of GSTM1 gene. The m1 (Msp I) and m2 (Ile462Val) polymorphisms of CYP1 A1 gene and GSTM1 null genotype were also determined in a random population of 84 healthy, unrelated volunteers with developed microarray-based method. Of 84 cases, 47.6% were calssified as GSTM1 null, close to the published data. It's interesting that there lack three genotypes of m1 -m2 locus in the population: TT-AG, TT-GG and TC-GG. However, according to the data of the genotype frequencies independently happened at both m1 and m2 site, the combination frequencies of above three genotypes are 11.4%, 2.6%, and 3.1% respectively. Therefore we assume that the haplotypes of m1 -m2 are only T-A, C-A and C-G, but not T-G, as it were,there is no recombination happened between m1 site and m2 site. The frequencies of three haplotypes of T-A, C-A and C-G, calculated through corresponding genotypes, are 69.6%, 7.7% and 22.6% respectively.

Over-expression of Bim alpha3, a novel isoform of human Bim, result in cell apoptosis.

Bim proteins are essential factors of apoptosis. Nine isoforms of Bim have been submitted to GenBank database. In order to improve the understanding of the regulation of Bims' proapoptotic activity, we screened a multiple tissue cDNA panels for Bim isoforms and tested their proapoptotic activity by over-expression. Two novel cDNA isoforms of Bim family are generated by alternative splicing. One isoform encodes a 79 residue putative protein with a BH3 domain, named Bim alpha3. There is not any significant ORF found in another isoform, named Bim beta5. Subcellular localized analysis of EGFP-Bim fusion protein suggests Bim alpha3 distributed to both plasma and nucleus of HEK 293 cell. HEK 293 cells transfected with pcDNA-Bim alpha3 presented a similar proapoptotic activity (32.05 +/- 6.42%) with Bim alpha2 (30.14 +/- 2.66%). The proapoptotic activity of Bim alpha3 was obviously weaker than that of Bim S (46.52 +/- 5.07%) and Bim L (55.53 +/- 1.99%). Anti-sense over-expression of Bim in HEK 293 cells results in a weak down-regulated proapoptotic level. Expression pattern analysis reveals that both the novel cDNAs are expressed widely in normal tissue just like the other reported isoforms. The expression pattern of Bim isoforms shows tissue specific obviously. The results suggest that BH3 domain is sufficiency for proapoptotic activity of Bim proteins. The functional state of Bims might be regulated both in the transcript and post transcript process.

Cloning and expression of human GTDC1 gene (glycosyltransferase-like domain containing 1) from human fetal library.

The glycosyltransferases (GTs) catalyze the synthesis of the carbohydrate portions of glycoproteins, glycolipids, and proteoglycans. Here we report the cloning and characterization of a novel human GTDC1 (glycosyltransferase-like domain containing 1) gene, which locates on human chromosome 2q22. The GTDC1 cDNA is 2954 bp in length, encoding a putative protein of 458 amino acids. At protein level human GTDC1 has 75 and 37% identity with its homologous counterparts in the mouse and fruitfly, respectively. RT-PCR analysis revealed its relatively high expression level in the adult lung, spleen, testis, and peripheral blood leukocyte.

Analysis of gene expression profiles in human HL-60 cell exposed to cantharidin using cDNA microarray.

Cantharidin is a natural toxin that has antitumor properties and causes leukocytosis as well as increasing sensitivity of tumor cells resistant to other chemotherapeutic agents. There is limited information, however, on the molecular pharmacological mechanisms of cantharidin on human cancer cells. We have used cDNA microarrays to identify gene expression changes in HL-60 promyeloid leukemia cells exposed to cantharidin. Cantharidin-treated cells not only decreased expression of genes coding for proteins involved in DNA replication (e.g., DNA polymerase delta), DNA repair (e.g., FANCG, ERCC), energy metabolism (e.g., isocitrate dehydrogenase alpha, ADP/ATP translocase), but also decreased expression of genes coding for proteins that have oncogenic activity (e.g., c-myc, GTPase) or show tumor-specific expression (e.g., phosphatidylinositol 3-kinase). In contrast, these treated cells overexpressed several genes that encode intracellular and secreted growth-inhibitory proteins (e.g., BTG2, MCP-3) as well as proapoptotic genes (e.g., ATL-derived PMA-responsive peptide). Our findings suggest that alterations in specific genes functionally related to cell proliferation or apoptosis may be responsible for cantharidin-mediated cytotoxicity. We also found that exposure of HL-60 cells to cantharidin resulted in the decreased expression of multidrug resistance-associated protein genes (e.g., ABCA3, MOAT-B), suggesting that cantharidin may be used as an oncotherapy sensitizer, and the increased expression of genes in modulating cytokine production and inflammatory response (e.g., NFIL-3, N-formylpeptide receptor), which may partly explain the stimulating effects on leukocytosis. Our data provide new insight into the molecular mechanisms of cantharidin.

Assignment of human sprouty 4 gene to chromosome segment 5q32 approximately 33 and analysis of its pattern of expression.

The human sprouty 4 (SPRY4) gene was localized to chromosome band 5q32 approximately 33 by screening the Stanford radiation hybrid G3 panel using a SPRY4-specific primer pair for PCR. Northern blot analysis revealed two different mRNAs (5 kb and 2 kb) in liver, skeletal muscle, heart, lung, kidney, spleen, placenta and small intestine. Reverse transcriptase-PCR analysis showed that SPRY4 was expressed in all tested tissues to different levels.

Cloning and identification of a novel cDNA which encodes a putative protein with a DnaJ domain and a thioredoxin active motif, human macrothioredoxin.

A 3345 bp cDNA was isolated from the fetal brain cDNA library by high throughput cDNA sequencing. The cDNA with an open reading fragment (ORF) of 2241 bp encodes a 747 amino acids putative protein with a DnaJ N-terminus domain and four thioredoxin active sets. So it is named human macrothioredoxin (hMTHr). We used Northern blot to detect a band with a length of about 5 kb, which was ubiquitously expressed in human adult tissues with different intensities. The expression pattern was verified by RT-PCR, revealing that the transcripts were ubiquitously expressed in fetal tissues and human tumor tissues also. The transcripts in fetal tissues were more numerous than in adult tissues. The transcripts were high in adult testis, adult pancreas, fetal thymus, and fetal kidney. We have found three splice isoforms with lengths of 3483, 3345, and 2982 bp in the sequencing analysis of the RT-PCR products. They encode three putative proteins with 793, 747, and 275 amino acids, respectively. The first two putative proteins contain a DnaJ domain and four thioredoxin domains. The third putative protein only contains an N-terminus DnaJ domain and one thioredoxin active set. Blast analysis against the NCBI database revealed that the gene spans a genome sequence of about 75 kb that contains at least 25 exons and is located in human chromosome 2q32.1. The organization of the functional motifs of hMTHr suggests that the protein might be a member of a molecular chaperone family.

Purification, crystallization and preliminary X-ray studies of a p-nitrophenylphosphatase from Bacillus stearothermophilus.

Thermostable p-nitrophenylphosphatase from Bacillus stearothermophilus has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group C(2), with unit-cell parameters a = 67.17 A, b = 57.84 A, c = 62.49 A and alpha = 90.0 degrees, beta = 95.4 degrees, gamma = 90.0 degrees. Diffraction data were collected to 1.40 A resolution with a completeness of 94.7% (96.6% for the last shell), an R(fac) value of 0.074 (0.341) and an I/sigma (I) value of 30.1 (2.67).

[Validation of cDNA microarray technology].

cDNA microarray is a technological approach that has the potential to globally measure changes in mRNA expression levels. Self-comparison experiments with the same kind of tissue and differential expression experiments with the different kinds of tissue have been done to verify the reproducibility and the accuracy of this technique. The parameter of the reliability and the reproducibility of the microarray data were analyzed by correlation coefficient (R), coefficient of variation (CV) and false positive rate (FPR) etc. Meanwhile, the error resource also has been inspected. These results showed that generally the correlation coefficient of data from this cDNA microarray system was more than 0.9, the coefficient of variation was about 15%, and the false positive rate was below 3%. The result proves the accuracy of the cDNA microarray data. Consistence rate (CR) was advanced here as a new parameter to evaluate the reproducibility of two replicate experiments. It has some advantages over correlation coefficient and coefficient of variation. The influence of some important factors in the experiments, such as different concentration of spotted DNA, mRNA and total RNA, different batches of slides and different processes of labeling, have been investigated by comparing the results. It was shown that most of the false position produced by the experiment system could be reduced by replicate experiments.

Premature termination of telomeric extension-PCR for detection of telomerase activity.

In this article, we report a simple, rapid, and efficient method to detect telomerase activity: the premature termination of telomeric extension-PCR (PTEP). Similar to the telomeric repeat amplification protocol (TRAP), this method is based on PCR amplification following the in vitro telomerase reaction, while the in vitro telomerase reaction here is prematurely, rather than randomly, terminated. Apart from this, the telomeric extension products are used as initial primers, instead of as templates, to trigger the amplification with a specially constructed plasmid DNA as the template that cannot be directly amplified with the telomerase primer. The end product is a specific 159-bp DNA fragment that reflects telomerase activity. Because its product can be clearly identified with routine agarose gel electrophoresis and ethidium bromide staining, PTEP allows even lesser-equipped laboratories to easily detect telomerase activity.

Purification, crystallization and preliminary X-ray studies of GMP reductase 2 from human.

GMP reductase 2 from human has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P3(2)21, with unit-cell parameters a = b = 110.6, c = 209.8 A, alpha = beta = 90, gamma = 120 degrees. Diffraction data were collected to 3.0 A with a completeness of 100% (100% for the last shell), an R(merge) value of 0.089 (0.189) and an I/sigma(I) value of 7.3 (3.2).

[Optimization of T7-based RNA amplification system for cDNA microarray].

cDNA microarrays are powerful parallel tools for gene expression profiling analysis, which help us to understand the molecular mechanism of diseases and to identify potential targets for therapeutic intervention. However, their broader application are hampered by the large amount of RNA required: up to 200 microg of total RNA or 5 microg of mRNA for one chip, making analysis of small samples difficult. In this work, combined with a template switching effect, the T7 RNA linear amplification procedure was optimized, providing multiple copies of anti-sense RNA with reduced sample inputs: no more than 3 microg total RNA for one chip. Using the same RNA sample in all cases, the anti-sense RNA labeling method was compared with standard total RNA and mRNA methods by two sets of self-comparison experiments. Furthermore, the three methods in profiling analysis were compared with the same pair RNA samples. All the results indicated that the fidelity, reproducibility, and reliability showed no significant difference with conventional total RNA or mRNA microarrays.

A novel cDNA encodes a putative hRALY-like protein, hRALYL.

High throughput cDNA sequencing and 5'-rapid amplification of cDNA ends (5'RACE) isolated two cDNAs that shared the same open reading fragment (ORF). Northern blot analysis with the fetal brain mRNA blots detected two transcripts with the length of 3.2 kb and 2.2 kb respectively. The ORF encodes a 291 residues putative protein that shares great homology with hRALY and hnRNPC. So it was named hRALY like protein, hRALYL. Expression pattern was detected by multiple-issue cDNA (MTC) panel based RT-PCR. It revealed that the transcripts of hRALYL were expressed ubiquitously in human tissues with different intensities. The transcript is highest in brain. Blast analysis found the cDNA corresponding to a contig NT_008292, which revealed the gene containing at least 9 exons and located the gene on human chromosome 8q21.13-8q21.2. hRALYL might be a member of hnRNPC subfamily.

Cloning, expression, and genomic structure of a novel human Rap2 interacting gene (RPIP9).

During a large-scale screen of a human fetal brain cDNA library, a full-length cDNA encoding a novel Rap2 interacting protein was isolated and sequenced. The cDNA is 3397bp long and has a predicted open reading frame encoding a protein of 329 aa. The predicted protein shows high homology to mouse and human RPIP8, and has a RUN domain near its C-terminus. The gene was mapped to human chromosome 7q21-7q22 and has 9 exons and 8 introns. The expression pattern was also detected by cycle-limited reverse polymerase chain reaction (RT-PCR).

[RAPD study of populations of Glycine tabacina].

The ecological genetic research on Glycine tabacina populations was based on RAPD technique, which revealed 100% polymorphisms, with minimum value of 0.41 in population MM, and maximum value of 0.82 in population PT. Cluster analysis showed that the populations' genetic variation was correlation to the environment gradient when the geographic distance among populations was big. In small geographic range,however, no correlation exists between genetic structure and ecological factors because of random genetic drift.