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PubMed2004

Exploratory differential gene expression analysis in microarray experiments with no or limited replication.

We describe an exploratory, data-oriented approach for identifying candidates for differential gene expression in cDNA microarray experiments in terms of alpha-outliers and outlier regions, using simultaneous tolerance intervals relative to the line of equivalence (Cy5 = Cy3). We demonstrate the improved performance of our approach over existing single-slide methods using public datasets and simulation studies.

Analysis of Variance↗
PubMed2005

Mutational and expression analysis of tbnat and its response to isoniazid.

A gene (nat) encoding arylamine N-acetyltransferase (NAT) has been found in Mycobacterium tuberculosis. The gene is expressed and the enzyme is active in growing M. tuberculosis cells. N-Acetyltransferase acetylates and inactivates isoniazid (INH), which is a front-line drug used in tuberculosis (TB) therapy. In this study, it was shown that a previously reported G619A single nucleotide polymorphism (SNP) was conserved in two M. tuberculosis strain families found in the Western Cape Province of South Africa (strain families 3 and 28). Further sequence analysis of isolates in strain family 3 identified a new T529C SNP in NAT resulting in a histidine instead of a tyrosine at position 177. This SNP was found only in isolates from strain family 3, and this mutation affects the highly conserved tyrosine residue close to the active site. Using real-time PCR, the expression of M. tuberculosis nat (tbnat) was determined over a 28 day growth cycle of the M. tuberculosis reference strain (H37Rv). The expression of tbnat occurs early in growth and reaches maximum levels at mid-exponential phase. The exposure of INH-susceptible isolates to low levels of INH resulted in an increase of tbnat expression (reference strain H37Rv, which is wild-type for tbnat, and isolate 1430, containing both SNPs). An INH-resistant isolate (816) exposed to INH showed no change in tbnat expression. The increased expression in the susceptible isolates suggests that INH affects tbnat expression. tbnat may contribute to INH susceptibility, but in combination with other factors.

Antitubercular Agents↗
PubMed2006

Sequence and expression analysis of an interferon stimulated gene (ISG15) from Atlantic cod (Gadus morhua L.).

Suppression subtractive hybridization was used to examine gene expression in Atlantic cod head kidney cells treated with polyinosinic polycytidylic acid (poly I:C). One of the most abundant genes was ISG15, showing 24-53% amino acid similarity to ISG15 from both mammals and teleosts. The promoter was cloned by genome walking and three potential interferon-stimulated response elements (ISREs) were identified. Analysis of the gene structure revealed a single intron in the 5' untranslated region (UTR) of cod ISG15, which also seems to be present in zebrafish and pufferfish ISG15. A quantitative real time PCR assay was established to monitor the gene expression of cod ISG15. Injection of cod with poly I:C strongly induced the expression of ISG15 in all organs investigated. Stimulation was most pronounced the first day with a gradual decline the following days. The expression of ISG15 in head kidney cells was also induced in vitro by treatment with poly I:C, but not significantly with LPS. However, injection of formalin killed Vibrio anguillarum-induced ISG15 expression in head kidney.

Amino Acid Sequence↗
PubMed2005

Molecular characterization of a novel quinolizidine alkaloid O-tigloyltransferase: cDNA cloning, catalytic activity of recombinant protein and expression analysis in Lupinus plants.

A novel acyltransferase committed to the final step of quinolizidine alkaloid biosynthesis, tigloyl-CoA:(-)-13alpha-hydroxymultiflorine/(+)-13alpha-hydroxylupanine O-tigloyltransferase, has been purified from Lupinus albus. The internal amino acid sequences were determined with protease-digested fragments of 25 and 30 kDa bands, allowing design of primers for amplification of cDNA fragments by polymerase chain reaction. Using an amplified fragment as the probe, a full-length cDNA clone was isolated. Sequence analysis revealed that the cDNA encodes a protein of 453 amino acids with a molecular mass of 51.2 kDa. Phylogenetic analysis of the deduced amino acid sequences indicated that this alkaloid acyltransferase belongs to a unique subfamily of a plant acyl-CoA-dependent acyltransferase gene family. The cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity purified and cleaved to yield the recombinant enzyme for the study of catalytic properties. The recombinant enzyme catalyzed the acyltransfer reaction from tigloyl-CoA to (-)-13alpha-hydroxymultiflorine and (+)-13alpha-hydroxylupanine. Benzoyl-CoA could also serve efficiently as an acyl donor for these hydroxylated alkaloids. RNA blot analysis suggested that the gene was expressed in roots and hypocotyls but not in cotyledons and leaves. These results indicated that this specialized acyltransferase, isolated for the first time as tigloyltransferase from nature, is committed to control the quinolizidine alkaloid patterns in a tissue-specific manner.

Acyltransferases↗
PubMed2001

Molecular cloning and expression analysis of feline melanoma antigen (MAGE) obtained from a lymphoma cell line.

Melanoma antigens (MAGE) are regarded as inducing tumor-specific immune response and thought to be potential therapeutical agents for cancer immunotherapy. We hereby report the cloning of feline MAGE cDNA obtained from a lymphoma cell line derived from cat malignant lymphoma, and its expression pattern in tumor and normal tissues. The cDNA encoding the MAGE is 1668 base pairs (bp) in length, and contains an open reading frame (ORF) of 936 bp encoding a protein of 311 amino acids. The predicted amino acid sequence has 29-46% of homology with other MAGE proteins from human and mouse. mRNA transcripts for the feline MAGE were detected in certain tumors, but not in adult cat normal tissues except in testis, by reverse transcription polymerase chain reaction (RT-PCR) analysis. This indicates that the expression pattern of feline MAGE mRNA is similar to those of other MAGE family genes in tumors and normal tissues.

Amino Acid Sequence↗
PubMed2006

Cloning and expression analysis of SKn-type dehydrin gene from bean in response to heavy metals.

A heavy metal responsive gene PvSR3 (GenBank accession number U54703) encoding an acid dehydrin was isolated from a mercuric chloride-treated bean (Phaseolus vulgaris L.) leaf cDNA library by differential screening using cDNAs derived from treated and untreated plants. The PvSR3 cDNA is 981-bp long and has a 606-bp open-reading frame with a 202-residue-deduced amino acid sequence. The PvSR3 sequence contains two conserved repeats of the characteristic lysine-rich K segment (EKKGIMDKIKEKLPG) preceded by an 8-serine residue stretch, whereas the Y segment (DEYGNP) conserved motif is absent. The deduced protein has a calculated molecular weight of 23 kDa and an isoelectric point of 5.2. Sequence similarity and comparative analysis showed that PvSR3 shares 70 and 73% similarity with the dehydrin of poplar and pepper, respectively. Southern hybridizations indicated that PvSR3 was a low copy-number gene. Northern blot analysis revealed that PvSR3 mRNA was weakly detected in seedling leaves. However, the gene expression was strongly stimulated by heavy metals, such as mercury, cadmium, arsenic, and copper, whereas virus infection and salt had little effect on it. In contrast, PvSR3 was not responsive to drought or abscisic acid (ABA), and was downregulated by UV radiation. Furthermore, PvSR3 was upregulated by the exogenous signaling molecules, including salicylic acid (SA) and hydrogen peroxide (H2O2). It is suggested that PvSR3 is extremely related to heavy metal stress, and might play an important role in metal detoxification and resistance to the damage caused by heavy metals.

Abscisic Acid↗
PubMed2003

Genomic organization and expression analysis of B7-H4, an immune inhibitory molecule of the B7 family.

B7-H4 is a recently identified B7 family member that negatively regulates T cell immunity by the inhibition of T cell proliferation, cytokine production, and cell cycle progression. In this study, we report that the genomic DNA of human B7-H4 is mapped on chromosome 1 comprised of six exons and five introns spanning 66 kb, of which exon 6 is used for alternative splicing to generate two different transcripts. Similar B7-H4 structure is also found in mouse genomic DNA in chromosome 3. A human B7-H4 pseudogene is identified in chromosome 20p11.1 with a single exon and two stop codons in the coding region. Immunohistochemistry analysis using B7-H4-specific mAb demonstrates that B7-H4 is not expressed on the majority of normal human tissues. In contrast, up to 85% (22 of 26) of ovarian cancer and 31% (5 of 16) of lung cancer tissues constitutively express B7-H4. Our results indicate a tight regulation of B7-H4 expression in the translational level in normal peripheral tissues and a potential role of B7-H4 in the evasion of tumor immunity.

Animals↗
PubMed2005

Full genome gene expression analysis of the heat stress response in Drosophila melanogaster.

The availability of full genome sequences has allowed the construction of microarrays, with which screening of the full genome for changes in gene expression is possible. This method can provide a wealth of information about biology at the level of gene expression and is a powerful method to identify genes and pathways involved in various processes. In this study, we report a detailed analysis of the full heat stress response in Drosophila melanogaster females, using whole genome gene expression arrays (Affymetrix Inc, Santa Clara, CA, USA). The study focuses on up- as well as downregulation of genes from just before and at 8 time points after an application of short heat hardening (36 degrees C for 1 hour). The expression changes were followed up to 64 hours after the heat stress, using 4 biological replicates. This study describes in detail the dramatic change in gene expression over time induced by a short-term heat treatment. We found both known stress responding genes and new candidate genes, and processes to be involved in the stress response. We identified 3 main groups of stress responsive genes that were early-upregulated, early-downregulated, and late-upregulated, respectively, among 1222 differentially expressed genes in the data set. Comparisons with stress sensitive genes identified by studies of responses to other types of stress allow the discussion of heat-specific and general stress responses in Drosophila. Several unexpected features were revealed by this analysis, which suggests that novel pathways and mechanisms are involved in the responses to heat stress and to stress in general. The majority of stress responsive genes identified in this and other studies were downregulated, and the degree of overlap among downregulated genes was relatively high, whereas genes responding by upregulation to heat and other stress factors were more specific to the stress applied or to the conditions of the particular study. As an expected exception, heat shock genes were generally found to be upregulated by stress in general.

Animals↗
PubMed2000

cDNA cloning, chromosomal localization, and expression analysis of human BEHAB/brevican, a brain specific proteoglycan regulated during cortical development and in glioma.

BEHAB (Brain Enriched HyAluronan Binding)/brevican, a brain-specific member of the lectican family of chondroitin sulfate proteoglycans (CSPGs), may play a role in both brain development and human glioma. BEHAB/brevican has been cloned from bovine, mouse and rat. Two isoforms have been reported: a full-length isoform that is secreted into the extracellular matrix (ECM) and a shorter isoform with a sequence that predicts a glycophosphatidylinositol (GPI) anchor. Here, we report the characterization of BEHAB/brevican isoforms in human brain. First, BEHAB/brevican maps to human chromosome 1q31. Second, we report the sequence of both isoforms of human BEHAB/brevican. The deduced protein sequence of full-length, secreted human BEHAB/brevican is 89.7, 83.3 and 83.2% identical to bovine, mouse and rat homologues, respectively. Third, by RNase protection analysis (RPA) we show the developmental regulation of BEHAB/brevican isoforms in normal human cortex. The secreted isoform is highly expressed from birth through 8years of age and is downregulated by 20years of age to low levels that are maintained in the normal adult cortex. The GPI isoform is expressed at uniformly low levels throughout development. Fourth, we confirm and extend previous studies from our laboratory, here demonstrating the upregulation of BEHAB/brevican mRNA in human glioma quantitatively. RPA analysis shows that both isoforms are upregulated in glioma, showing an approximately sevenfold increase in expression over normal levels. In contrast to the developmental regulation of BEHAB/brevican, where only the secreted isoform is regulated, both isoforms are increased in parallel in human glioma. The distinct patterns of regulation of expression of the two isoforms suggest distinct mechanisms of regulation of BEHAB/brevican during development and in glioma.

Amino Acid Sequence↗
PubMedDate not supplied

Epidermal growth factor receptor (EGFR): mutational and protein expression analysis in gastric cancer.

BACKGROUND: Since specific epidermal growth factor receptor (EGFR) somatic mutations have been demonstrated to influence the response to anti-EGFR therapy in non-small cell lung cancer, EGFR gene mutational analysis of different types of neoplasm is under investigation. PATIENTS AND METHODS: EGFR protein expression and gene mutations in exons 18, 19 and 21 were investigated in 49 gastric adenocarcinomas. RESULTS: no specific EGFR gene mutations were detected, while EGFR positive staining was detected in 6% of the cases. CONCLUSION: Our findings indicate that in gastric adenocarcinoma, specific EGFR gene mutations are very rare or absent and the rate of EGFR protein expression is low.

Adenocarcinoma↗
PubMed2001

Cloning and Expression Analysis of a Novel Gene, UBAP1, Possibly Involved in Ubiquitin Pathway.

The 9p21-22 region shows loss of heterozygosity in up to 60% of human nasopharyngeal carcinomas (NPC), indicating the presence of a tumor suppressor gene in this region. We have identified a novel minimal common deletion region at 9p21-22. Twenty-two epithelial-derived expressed sequence tags (ESTs) in this critical region were systematically screened by differential RT-PCR to investigate the expression patterns in NPC cell line HNE1 and primary cultures of normal nasopharyngeal epithelial cells. One of these ESTs was found down-expressed in HNE1, whose differential expression was confirmed by Northern blot. Subsequently the corresponding gene sequence for this EST was established by cDNA cloning and RACE procedures (GenBank Accession No.AF222043). Furthermore, a mouse homologueof this gene was identified (GenBank Accession No.AF275549). This gene is 2.7 kb long and contains two UBA domains. It is a new member of UBA domain protein family, encoding a putative protein of 502 amino acids with a theoretical molecular mass of 55 kD, so we have named this gene UBAP1 for ubiquitin associated protein 1 (HUGO Gene Nomenclature Committee-approved symbol). Northern blot and RT-PCR analysis demonstrated a ubiquitous pattern of gene expression in human and mouse tissues. Direct sequencing analysis of the coding region of hUBAP1 following RT-PCR failed to reveal any mutations in a preliminary screening of NPC cell line HNE1 and primary nasopharyngeal carcinomas samples. However, more detailed analysis is to be performed to reveal if fine mutations of this gene are present in NPC.

Journal Article↗
PubMed2004

Expression analysis of the human testis-specific serine/threonine kinase (TSSK) homologues. A TSSK member is present in the equatorial segment of human sperm.

Two members of the human testis-specific serine/threonine (Ser/Thr) kinase family, TSSK 1 and TSSK 2, were cloned and sequenced from a human testis adaptor-ligated cDNA library using a PCR strategy. Within the cDNA, open reading frames (ORF) were defined encoding proteins of 367 and 358 amino acids respectively, as well as conserved kinase domains typical of the superfamily of Ser/Thr kinases. Both genes were intronless and mapped to chromosomes 5 and 22 respectively. The human and mouse homologues of TSSK 1 and TSSK 2, together with TSSK 3 and SSTK/FKSG82, constitute a kinase subfamily closely related to the calmodulin kinases and SNF/nim 1 kinase subfamilies. Similar to the mouse, tissue expression by northern and dot blot analysis revealed that human TSSK 1 and 2 messages are expressed exclusively in the testis. However, mRNA for these kinases can be detected in other tissues using real-time PCR. In addition, TSKS, the human homologue of a putative substrate of TSSK 1 and 2, was cloned. TSKS had an ORF of 592 amino acids and was also expressed exclusively in the testis as demonstrated by northern and dot blot analyses; however, lower levels of expression in other tissues were detected using real-time PCR. Human TSSK 2 and TSKS interacted in a yeast two-hybrid system and also co-immunoprecipitated after in vitro translation. TSSK 2 expressed in yeast and bacteria was able to autophosphorylate and also phosphorylated recombinant TSKS in vitro. Antibodies against recombinant TSSK 2 demonstrated that a member of the TSSK family was present in human testis and localized to the equatorial segment of ejaculated human sperm. In contrast, TSKS was only found in the testis. The finding of a TSSK family member in mature sperm suggests that this family of kinases might play a role in sperm function.

Amino Acid Sequence↗
PubMed2000

Complete sequencing and mRNA expression analysis of the MEN-I gene in adrenal myelolipoma.

The molecular pathogenesis of adrenal myelolipoma is unclear. Endocrine activity of these tumors and association with other endocrine tumors have stimulated the hypothesis that it may belong to the group of sporadic tumors caused by defects of the gene responsible for multiple endocrine neoplasia type I (MEN-I). DNA of blood and tumoral sections from two patients with adrenal myelolipoma were analyzed by examination of variable number of tandem repeats (VNTR) loci PYGM, D11S987, D11S480, and D11S449 on chromosome 11q13 and by complete direct DNA sequencing of all coding exons and splice junctions of the MEN-I gene. Menin expression was examined by RT-PCR. RT-PCR did not detect menin expression in one adrenal myelolipoma. No loss of heterozygozity on chromosome 11q13 was identified. Intragenic heterozygozity was retained in codon 418 of the menin gene in both patients. No mutation was identified in the coding exons of the menin gene. Complete DNA sequencing yielded no hint that defects of the MEN-I gene are responsible for the formation of adrenal myelolipomas. Adrenal myelolipomas do not share the loss of heterozygozity on chromosome 11q13 observed in some benign adenomatous and many malignant adrenocortical tumors.

Adrenal Gland Neoplasms↗
PubMed2003

Molecular cloning and expression analysis of a novel gene DGCR8 located in the DiGeorge syndrome chromosomal region.

We have identified and cloned a novel gene (DGCR8) from the human chromosome 22q11.2. This gene is located in the DiGeorge syndrome chromosomal region (DGCR). It consists of 14 exons spanning over 35kb and produces transcripts with ORF of 2322bp, encoding a protein of 773 amino acids. We also isolated a mouse ortholog Dgcr8 and found it has 95.3% identity with human DGCR8 at the amino acid sequence level. Northern blot analysis of human and mouse tissues from adult and fetus showed rather ubiquitous expression. However, the in situ hybridization of mouse embryos revealed that mouse Dgcr8 transcripts are localized in neuroepithelium of primary brain, limb bud, vessels, thymus, and around the palate during the developmental stages of embryos. The expression profile of Dgcr8 in developing mouse embryos is consistent with the clinical phenotypes including congenital heart defects and palate clefts associated with DiGeorge syndrome (DGS)/conotruncal anomaly face syndrome (CAFS)/velocardiofacial syndrome (VCFS), which are caused by monoallelic microdeletion of chromosome 22q11.2.

Amino Acid Motifs↗
PubMedDate not supplied

[Expression analysis of different collagens and cytokines in cartilage cells derived from arthrotic hip and knee joints].

AIM: Osteoarthritis (OA) is characterized by an irreversible destruction of articular cartilage. This is associated with a multiplicity of factors, causing an increased catabolic metabolism in cartilage. However, the prevalence of the OA is very variable in different joints. Therefore , we conducted a comparative analysis of chondrocytes derived from knee and hip joints with respect to their expression of inflammatory factors, such as IL-1beta, IL-1beta-receptorantagonist, iNOS, components of cartilage matrix (collagen I, II, and VI) as well as vimentin. METHODS: Different cytokines and proteins were detected by immune-histochemical staining of cartilage samples ex vivo. Further, chondrocytes were isolated from OA knee and hip joints, expanded in vitro and gene expression patterns were investigated by quantitative RT-PCR. RESULTS: Chondrocytes from knee and hip joints of OA patients express collagenes I, II and VI, IL-1beta and IL-1beta-RA, iNOS as well as Vimentin. A significant difference in gene expression patterns was not found in chondrocytes from the hip joints versus the knee joint ex vivo or in primary culture cells in vitro. However, in vitro the expression of type I collagen exceeded the expression of type II collagen. The IL-1beta-expression was high ex vivo, remained low during primary culture but was significantly elevated after primary culture in hip chondrocytes. CONCLUSION: Osteoarthritic gene expression patterns in cells derived from hip or knee joints ex vivo and in primary culture were not significantly different. We conclude that the rather frequent occurrence of OA in these joints in comparison to the ankle joint may be associated with a close physiological relation of cells in these joints. However, future studies which will include ankle cartilage must be investigated in further detail.

Aged↗
PubMed2004

Identification of the human ubiquitin specific protease 31 (USP31) gene: structure, sequence and expression analysis.

Parkinson's disease (PD) is a common neurodegenerative disorder with clinical features of bradykinesia, rigidity and resting tremor resulting from the deficiency of dopamine in the nigrostriatal system. Recently, PARK6 was identified as a novel locus associated with autosomal recessive PD. Here we report the identification and characterization of a novel human deubiquitylating gene (USP31), which maps to the critical PARK6 region. Database analysis and 5' RACE identified a 4070bp cDNA, encoded by 27 exons spanning approximately 105kbp of genomic sequence. The predicted protein of 1035 amino acids included a conserved ubiquitin hydrolase region (Prosite profile PS50235), a DUSP (domain in ubiquitin specific proteases-Smart00695) and a ubiquitin-like domain (Prosite pattern PS00299). Northern blot analysis revealed a single USP31 transcript of approximately 4 kb, which was primarily expressed in the testis and lung.

Amino Acid Sequence↗
PubMed2004

Cloning and expression analysis of a bZIP cDNA in Gossypium hirsutum L.

A new full-length cDNA clone was isolated from the fiber cDNA library of Gossypium hirsutum L. The cDNA, designated GhbZIP,encoded a polypeptide of 645 amino acids. GhbZIP protein had the structure characteristics of plant bZIP proteins, including two conserved domains, DUF630 and DUF632 with unknown functions, a proline-rich domain and a phenylalanine-rich domain. Meanwhile, the protein contained a leucine zipper-like motif in DUF632. The hydropathy analysis showed that the GhbZIP was a membrane protein. GhbZIP gene was preferentially expressed in ovule and fiber cells since three days post-anthesis, as indicated it might be involved in the transcription regulation of genes during cotton fiber elongation.

Amino Acid Sequence↗