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PubMed2003

[DNA microarrays (DNA chips) used in molecular medical research].

Microchip technology and mapping of the human genome has led to the invention of DNA-micro-array technology. In that technology thousands of genes are analysed in parallel on a small glass surface. The very large data material that is generated requires bioinformatic analysis in order to be transformed to understandable biological results. There are different forms of DNA-micro-arrays. Some are used for measuring gene expression, i.e. which genes are turned on and which genes are turned off. Others are used for sequencing, and finally some are used for measuring single nucleotide polymorphisms (SNPs). Some application possibilities of the arrays within the area of cell biology and classification of diseases are presented. Especially within the area of classification of cancer diseases the technology is promising, and a scientific breakthrough could occur in the area of classification of very aggressive cancers.

Computational Biology↗

PubMed2002

[The construction of cDNA expression library from the tentacles of Sagartia rosea].

A cDNA expression library of the tentacles of Sagartia rosea was constructed. The cDNA was cloned into eukaryotical expression plasmid pcDNA3. SMART protocol was used for cDNA library construction and bioinformatics analysis was carried out. 71 novel EST clones were obtained from 130 sequences in the library, of which there were 21 full-length clones, including cytolysin genes, flourescent protein, ubiquinol-cytochrome C reductase gene, elongation factor, ferritin gene riboflavin kinase gene, ribosomal protein. This provides a base for further investigating their biological activity and application.

Animals↗

PubMed2003

Putative hAPN receptor binding sites in SARS_CoV spike protein.

AIM: To obtain the information of ligand-receptor binding between the S protein of SARS-CoV and CD13, identify the possible interacting domains or motifs related to binding sites, and provide clues for studying the functions of SARS proteins and designing anti-SARS drugs and vaccines. METHODS: On the basis of comparative genomics, the homology search, phylogenetic analyses, and multi-sequence alignment were used to predict CD13 related interacting domains and binding sites in the S protein of SARS-CoV. Molecular modeling and docking simulation methods were employed to address the interaction feature between CD13 and S protein of SARS-CoV in validating the bioinformatics predictions. RESULTS: Possible binding sites in the SARS-CoV S protein to CD13 have been mapped out by using bioinformatics analysis tools. The binding for one protein-protein interaction pair (D757-R761 motif of the SARS-CoV S protein to P585-A653 domain of CD13) has been simulated by molecular modeling and docking simulation methods. CONCLUSION: CD13 may be a possible receptor of the SARS-CoV S protein, which may be associated with the SARS infection. This study also provides a possible strategy for mapping the possible binding receptors of the proteins in a genome.

Amino Acid Sequence↗

PubMed2003

[Study of DNA microarray chip of associated genes of hydatidiform mole].

OBJECTIVE: To screen genes which may associate with the development and malignant transformation of hydatidiform mole. METHOD: The differentially expressed genes were analyzed between the tissues of two cases of hydatidiform mole and the normal placental tissues of two cases of pregnancy which had almost the same pregnant ages as hydatidiform mole, using cDNA chips containing 4,096 genes. RESULTS: There were total 89 genes significantly differently expressed in all hydatidiform moles, counting for 2.2% of total genes. Compared with normal villi, 24 genes in hydatidiform mole were up-regulated and 65 genes were down-regulated. Bioinformatical analysis of genes showed genes associated with cell proliferative inhibition, such as Ras GTPase activating protein, TGF-beta IIR alpha, BTG2 were significantly down-regulated, whereas genes associated with cell proliferation, malignant transformation and tumor metastasis as thymidine kinase, ribonucleotide reductase, glucose transport protein were highly up-regulated. CONCLUSION: cDNA chip technique is one kind of very effective methods in screening associated genes in hydatidiform mole.

Chorionic Villi↗

PubMed2000

[Alternative splicing of de novo methyltransferase gene 3b in adult and newborn mice].

OBJECTIVE: To unravel the biological significance of the alternative splicing of de novo methyltransferase 3b, expression of the Dnmt3b gene in various tissues and developmental stages was investigated in postnatal mice. METHODS: RT-PCR and capillary electrophoresis were employed to analyze the alternative splicing pattern of Dnmt3b in tissues of newborn and adult mice. The results had been further reaffirmed by repeating and statistics analysis. Bioinformatics tools were used to predict the structure and hydrophobicity of the Dnmt3b exon10 coding sequence. RESULTS: Isoform with Dnmt3b exon10 was the abundant form in lung of newborn mice and in liver of both newborn and adult mice, while in other tissues of newborn and adult mice, the spliced isoform was presented as the predominant one. Peptide encoded by Dnmt3b exon10 was mainly random coil on the surface of Dnmt3b protein. CONCLUSIONS: The data demonstrate that the specific expression of Dnmt3b exists in tissues and developmental stages of postnatal mice. The alternative splicing of the exon 10 of Dnmt3b is possibly involved in the regulation of Dnmt3b's catalytic function. These results provide an insight into the developmental regulation and physiological function of the alternative splicing of the Dnmt3b gene.

Alternative Splicing↗

PubMed2003

Characterization of a strong repression domain in the hinge region of orphan nuclear receptor hB1F/hLRH-1.

Human hepatitis B virus enhancer II B1 binding factor (hB1F also known as NR5A2, LRH-1, FTF or CPF) is an orphan nuclear receptor and belongs to the fushi tarazu factor I (FTZ-F1) subfamily. It plays important roles in the transcriptional regulation of a number of genes involved in bile acid biosynthesis pathway, hepatitis B virus (HBV) replication and liver specific regulatory network. Like other nuclear receptors, hB1F is composed of modular functional domains. We characterized a domain in its hinge region that imposes a strong repression on the transcriptional activity of hB1F, which is important for the function of hB1F on regulating the activity of HBV enhancer II/core promoter. Mutations of the core residues in this domain abrogate the repression. Bioinformatic analysis reveals that the amino acid sequence of this region is highly conserved only among members of the FTZ-F1 subfamily. The repression is observed in five cell lines tested, while the degree of the repression varies greatly, which does not parallel with the expression level of the DEAD box protein of 130 kD (DP103), a potential interacting protein of a homologous domain in the steroidogenic factor 1 (SF-1). Moreover, the repression is not affected by the silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT) and steroid receptor coactivator 1 (SRC-1). Collectively, these data suggest a novel regulatory mechanism for the transcriptional activity of hB1F.

Amino Acid Sequence↗

PubMed2003

Putative caveolin-binding sites in SARS-CoV proteins.

AIM: To obtain the information of protein-protein interaction between the SARS-CoV proteins and caveolin-1, identify the possible caveolin-binding sites in SARS-CoV proteins. METHODS: On the basis of three related caveolin-binding motifs, amino acid motif search was employed to predict the possible caveolin-1 related interaction domains in the SARS-CoV proteins. The molecular modeling and docking simulation methods were used to confirm the interaction between caveolin-1 and SARS-CoV proteins. RESULTS: Thirty six caveolin-binding motifs in the SARS-CoV proteins have been mapped out using bioinformatics analysis tools. Molecular modeling and simulation have confirmed 8 caveolin-binding sites. These caveolin-binding sites located in replicase 1AB, spike protein, orf3 protein, and M protein, respectively. CONCLUSION: Caveolin-1 may serve as a possible receptor of the SARS-CoV proteins, which may be associated with the SARS-CoV infection, replication, assembly, and budding.

Amino Acid Motifs↗

PubMed2004

[The cloning of human augmentor of liver regeneration-interacting proteins].

AIM: To screen human augmentor of liver regeneration(hALR)-interacting proteins and to explore the mechanisms of hALR in liver regeneration. METHODS: The open reading frame of hALR was used to construct the "bait" plasmid and the genes encoding hALR-interacting proteins were screened from the human liver cDNA library that was pretransformed into yeast Y187 by yeast two-hybrid system. Bioinformatic analysis of the sequences of the positive clones were performed. RESULTS: The positive clones encoding metallothionein, albumin, selenoprotein P,Na/K-ATPase, and 2 unknown proteins were screened out. CONCLUSION: The successful cloning of the genes encoding proteins interacting with hALR may pave the way for studying the interaction between the above proteins and hALR, and the molecular mechanisms of biological effect of hALR.

Albumins↗

PubMed2004

[Cloning full-length homologous cDNAs of pollen allergens in Humulus Scandens(Lour.) Merr by degenerate primer].

AIM: To establish a stable and reliable method for fast cloning homologous genes of pollen allergens in allergen-containing plants. METHODS: Degenerate primers were designed based on the bioinformatic analysis of numerous allergens available from the database. Subsequent amplification of the allergen genes was conducted in the weed pollen cDNA pool by a selective PCR profile. Following the truncated gene cloning, RACE method was used to isolate full-length cDNA. Gene function was deduced by sequence alignment in GenBank database. The degenerate ability of the primer was compared with the full-length cDNA sequences. RESULTS: Three full-length cDNAs were obtained. Sequence analysis showed that these new genes shared as high as 79%-85% homology with a large amount of known allergen profilins and were hence regarded as members of panallergen profilin family. Comparing these genes with the degenerate primers that were initially used in truncated gene cloning revealed that alternative nucleotide degeneracy occurred beyond the degenerate site predesigned, suggesting that further degeneracy was expanded by Touchdown-gradient PCR. CONCLUSION: Cloning of homologous genes or allergen genes can be efficiently achieved by using the combination of degenerate primer with Touchdown-gradient RT-PCR in the species such as Humulus scandens that has not yet been investigated.

Allergens↗

PubMed2004

Missense mutations in the BRCT domain of BRCA-1 from high-risk women frequently perturb strongly hydrophobic amino acids conserved among mammals.

Inherited missense mutations in the tumor suppressor gene, BRCA-1, may predispose to breast or ovarian cancer, but the exact effects on the protein are generally unknown. The COOH-terminal region of BRCA-1 encodes two BRCT repeats, which are partially conserved in mammalian species (human, dog, rat, and mouse; 60% amino acid identity). A bioinformatic analysis was conducted to evaluate 246 BRCT missense mutations from high-risk breast and/or ovarian cancer patients (reported in the NIH Breast Cancer Information Core database). It was hypothesized that amino acids conserved in evolution would be disproportionately targeted by the mutations and that conserved amino acids with strongly hydrophobic side chains would be disproportionately perturbed. A statistical model was developed, and chi(2) tests were used to determine whether missense mutations are randomly distributed throughout the BRCT repeats or whether they disproportionately target certain amino acids. The results showed that missense mutations disproportionately target amino acids that are identical in all four mammals (chi(2) = 46.01, P < 0.001). In addition, missense mutations disproportionately perturb conserved amino acids with strongly hydrophobic side chains (chi(2) = 68.57, P < 0.001) and alter the strongly hydrophobic property. The two most frequently observed known cancer-predisposing missense mutations in the BRCT repeats, M1775R and A1708E, conform to this pattern. These results suggest that missense mutations affecting highly conserved amino acids with strongly hydrophobic side chains can disturb important features of the BRCA-1 protein and may play a role in breast and ovarian cancer formation.

Amino Acid Sequence↗

PubMed2004

[Expression of cytochrome P450 enzymes in human nasopharyngeal carcinoma and non-cancerous nasopharynx tissue].

BACKGROUND & OBJECTIVE: The etiology of nasopharyngeal carcinoma (NPC) is associated with environmental and hereditary factors. Xenobiotics from environment must be activated to derive carcinogens. Several cytochrome P450 (CYP450) metabolic enzymes participate to the activation of pre-carcinogens, and the genetic polymorphism of those genes is associated with metabolic polymorphism and susceptibility to cancer. We performed a preliminary investigation on the xenobiotics metabolism of human nasopharynx by determining the expression of CYP450 genes in NPC and non-cancerous nasopharynx tissues. METHODS: The following two methods were used: (1)A cDNA library from the mixed RNA sample of seven non-cancerous nasopharynx tissues was generated, followed by clone sequencing and bioinformatics analysis. (2)RNA of 14 NPC and 8 non-cancerous nasopharynx tissues were reversely transcribed, and the expression of CYP450 genes in those samples was determined by PCR amplification. RESULTS: Eight ESTs of CYP450 genes including CYP1B1, CYP2F1, CYP2J2, CYP4B1, CYP4F12, CYP5A(TBXAS1), CYP20A1, and CYP51A1 were detected in non-cancerous nasopharynx cDNA library, among these CYP4B1 exhibited the highest expression level with 16 copies of ESTs. Positive expression of fourteen CYP450 genes including CYP1A1, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2F1, CYP3A4, CYP3A5, CYP3A7, and CYP4B1 were detected by RT-PCR, among these, CYP1B1, CYP2B6, and CYP4B1 had also been detected in the cDNA library. A total of 19 CYP450 genes expression were detected in NPC and non-cancerous nasopharynx tissues, and the expression levels of CYP1B1, CYP2C8, CYP2C9, CYP2D6, CYP3A5, and CYP4B1 were higher than those of the other genes. CONCLUSION: The expression of a great number of CYP450 genes was detected in human nasopharynx, some of which might participate to the activation of pre-carcinogen in human nasopharynx. Contribution of these genes to the risk of NPC needs further investigation.

Adult↗

PubMed2004

Transcriptional gene expression profile of human nasopharynx.

Inflammation in the nasopharynx is very common worldwide and nasopharyngeal carcinoma (NPC) results in significant morbidity and mortality in southeast Asia and north Africa. To facilitate the understanding of pathogenesis of these diseases as well as normal nasopharynx biology, transcriptional gene expression profile of normal human nasopharynx, from undissected biopsies, was established in this study by generating a large amount of ESTs, followed by bioinformatics analysis. A total of 27,209 ESTs generated from human nasopharynx cDNA library were integrated into 8,420 non-redundant clusters, of which, 6,070 (72.09%) corresponded to known genes, 156 (1.85%) to known ESTs, and 2,194 (26.06%) to novel sequences, respectively. Functional classification revealed that transcripts constituting enzymes (2,284, 28.30%) and those participating in cell growth/maintenance (3,306, 46.33%) were the largest population expressed in the nasopharynx, followed by genes coding for binding proteins (2,135, 26.45%) and involved in cell communication process (1,832, 25.68%). In addition, through comparison of the nasopharynx gene expression profile with those of 7 other human tissues, 59 transcripts preferentially expressed and 22 transcripts unique in the nasopharynx were identified. Our study acquired an initial assessment of qualitative diversity of gene expression in the nasopharynx, providing the first step toward comprehensive characterization of the molecular features of the nasopharyngeal disorders.

Base Sequence↗

PubMed2004

[The chromosomal location of AD7C-NTP gene and the prediction of transmembrane domains of its deduced protein].

AIM: To characterize the chromosomal location of AD7C-NTP gene and predict the transmembrane domains and sub-cellular location of its deduced protein. METHODS: The AD7C-NTP mRNA sequence was alignmented with human genomic DNA sequence by Blat server. The transmembrane domains and sub-cellular location of AD7C-NTP protein were predicted by using PHDhtm, TMHMM2.0, HMMTOP2.0, SMART and PSORT servers, etc. RESULTS: The AD7C-NTP gene located in minus strand of 1p36.11, without intron. The AD7C-NTP protein was predicted to have 3 potential transmembrane domains and locate on peroxisome's membrane. CONCLUSION: Bioinformatics analysis of the AD7C-NTP gene and its deduced protein provides valuable clues for further gene cloning and study of function.

Alzheimer Disease↗

PubMed2004

[Correction of five different types of errors of model REFSEQs appeared in NCBI human gene database only by using two novel human genes C17orf32 and ZNF362].

Found that there exist many mistakes in the REFSEQ issued in the genome annotation project of NCBI, the result of which indicates that people be cautious in using REFSEQ database in NCBI. By adopting the technical route combining bioinformatics analysis and experimental verification, through the comparison of the cloned genes in the non-redundant database, we found that there were many mistakes in the computer annotation human genome coding sequences that were issued on the internet. First we quoted nine wrong types of novel human genes anticipated by NCBI GENOME Annotation Project. Here we give one example in detail: (1) Comparison of the sequences between novel human gene C17orf32 and hypothetical human gene LOC124919. LOC123722 is a modified sequence of C17orf32 cDNA with an inserted G between 406 -407 nucleotides. The base G in the 401 position of LOC123722 cDNA is a redundant insert, which causes a reading frame shift in the translation of an alternative protein. This inserted G has not been found in our experimental clone, and is fully rejected by human EST alignment, and is shown as a redundance by genomic GT/AG organization analysis. (2) Comparison of the sequences between novel human gene C17orf32 and hypothetical human gene LOC147007. C17orf32 gene (ORF from 31 to 657 nucleotides) is located on human chromosome 17(Accession No. NT_010808.7), and is only linked with a hypothetical human gene LOC147007 (ORF from 55 to 435 nucleotides) at present. This hypothetical human gene sequence has not been verified by experiment, and is a wrong form of our verified C17orf32 gene. The full-length 1 679 bp cDNA sequence of C17orf32 exhibits overall homology to that of LOC147007 of 625 bp mRNA, with matching percentage of 37% in 36% of total window over the full-length nucleotide, especially 121 approximately 366 bp of LOC147007 is just the same as 316 approximately 561 bp of C17orf32. Thus, the 126 aa protein encoded by XP_097165 of LOC147007 exhibits overall homology to the 208 aa protein encoded by C17orf32, with matching percentage of 50% in 48% of total window over the full-length protein, especially 23 approximately 104 aa of XP_097165 is just the same as 96 approximately 177 aa of C17orf32 protein. Both flanking regions of LOC147007 outside the same ORF central part are wrong assembly of non-relative cDNA. In addition, we have in silico cloned a novel mouse gene, ORF32 (open reading frame 32) with TPA accession number of BK000258, which is the mouse ortholog of human C17orf32. Our strategy is helpful in both finding out more novel human genes and correcting the mistakes in the REFSEQs issued by NCBI genome annnotation project. For example, we adopted the gene anticipating method, through automatic calculation and analysis, anticipated two modes reference sequences (LOC124919 and LOC147007) from NCBI contig NT_ 010808. Both of them should be C17orf32, but the fact is that both of them are various wrong forms of C17orf32, respectively are the first type and second type of mistakes. Another example, we adopted gene anticipation method, through automatic calculation and analysis, anticipated three modes reference sequences (LOC14907, LOC200084 and LOC91126) from NCBI contig NT_004511 which really are one type of gene of ZNF362, but submitted three different wrong forms of ZNF362, respectively are: the fourth, fifth, and seventh type of mistakes. We can correct or avoid the currently wrong human genome coding sequence by using in silico clone and combining experimental verification. People should be cautious in treating the computer's annotation which may exist all type of wrong human genome coding sequences. The correct identification and annotation of the novel human genes still remain to be a long and arduous task.

Amino Acid Sequence↗

PubMed2004

[Cloning and expression of a homologue of human macrophage migration inhibitory factor from P. falciparum 3D7].

OBJECTIVE: To clone and express a homologue of human macrophage migration inhibitory factor (MIF) from P. falciparum 3D7--PfMIF. METHODS: The nucleotide sequence of PfMIF was found through blast P. falciparum genomic sequence databases with the amino acid sequence of human MIF (HuMIF). RT-PCR, DNA sequencing, and bioinformatics analysis were used for the cloning of Pfmif gene. The recombinant protein was expressed in E. coli and purified through the affinity column. RESULTS: The full length of Pfmif gene was cloned and sequenced. It was composed of 351 nucleotides and encoded 116 amino acids with the typical characteristic of MIF family. The recombinant protein was successfully expressed and purified. CONCLUSIONS: The Pfmif gene and recombinant protein were successfully isolated and PfMIF was preliminarily identified as a novel member of MIF family.

Amino Acid Sequence↗

PubMed2004

[Cloning and characterization of a novel human ceg1 gene cDNA].

ceg1 gene was a novel human gene, which was cloned by bioinformatics research and RT-PCR. It was a single exon gene and located on human chromosome 14. The length of the cDNA was 2050 bp. Bioinformatics analysis predicted a 1340 bp complete open reading frame (ORF) which encoded a 446 amino acid protein, containing an EGF-like and CLECT domain. We found that the homologous genes of ceg1 in mouse embryo and chicken embryo were specifically expressed in the brain by in-situ hybridization. The result of RT-PCR of the mature mouse organs showed it was widely expressed in many organs. The result indicates that ceg1 gene may have an essential role in the development of brain and the maintenance of the organs' normal function. The analysis of expression and function profile of ceg1 gene may provide valuable insights into the functions of ceg1 in the development and function of human body.

Amino Acid Sequence↗

PubMed2005

Identification of protein pattern in kidney cancer using ProteinChip arrays and bioinformatics.

Tumor biology of renal cell carcinoma (RCC) is not very well understood, although many studies on molecular and cellular biology have been performed. It is accepted now that cancer research has to be performed also with proteomic tools, because proteins are the real actors in the genesis and progression of cancer. Therefore, we used a ProteinChip System(R) (SELDI) which is able to detect minute amounts of protein and moreover to analyze a complex protein pattern. We analyzed 37 cases of clear cell RCC as a training set including corresponding normal tissue. From all samples protein lysates were made and spotted directly on different chip surfaces (SAX2, WCX). After a washing procedure the arrays were analyzed in the ProteinChip Reader. All profiles were subjected to a bioinformatical analysis including normalization, clustering, rule extraction and rating. Defined rules (markers) were evaluated using a test set of 24 samples (13 tumor tissues and 11 normal kidney tissues). The generated rule base for the SAX2 surface showed a sensitivity of 100% and a specificity of 97.3%. For the WCX arrays the optimal rule base showed worse results. A combined rule base for SAX2 and WCX did not result in a higher sensitivity or specificity. Using the optimal rule base for the SAX2 chip in the test set, sensitivity and specificity reached 76.9% and 100%, respectively. The ProteinChip System represents a key technology for the rapid detection of cancer specific proteomic patterns. It is possible to identify clear cell renal cancer with high sensitivity and specificity from minimal amounts of cells.

Cell Line, Tumor↗

PubMed2005

[Cloning and subcellular localization of apr-1--a new gene of tumor specific antigen family].

BACKGROUND & OBJECTIVE: apr-1 was cloned by improved polymerase chain reaction (PCR)-based subtractive hybridization from all-trans retinoic acid (ATRA)-induced apoptotic leukemia HL-60 cells in 1999. Preliminary results showed that apr-1 might be an apoptosis-related gene (GenBank ID: NM_014061). This study was to explore the background of apr-1 through gene cloning, bioinformatic analysis, and subcellular locating. METHODS: The cDNA encoding Apr-1 was amplified by reverse transcription-PCR (RT-PCR), and sequenced. Open reading frame (ORF) of apr-1 was analyzed with ORF finder software. Chromosome locus was defined by genome blast software. Conserved domains of amino acids were analyzed by protein blast software. Align (Cluster W) software in Vector NTI software package was used to analyze homogeneous genes (or proteins), and to draw the Phylogenetic Tree. Subcellular localization of apr-1 was performed. RESULTS: apr-1 was mapped to chromosome Xp11.22 with the ORF locating in 1 exon. Two MAGE conserved domains were found in Apr-1. Apr-1 shared homology with MAGE-A1, MAGE-B1, MAGE-C1, MAGE-D1, and Necdin. Phylogenetic analysis showed that Apr-1 was more closely related to MAGE-D1 and Necdin. Gene products of apr-1 were located in the nuclei of eukaryocytes. CONCLUSIONS: apr-1 is a member of MAGE family, and might belong to type II MAGE genes.

Amino Acid Sequence↗