Analysis Of Expressed Sequence Tag Database Research Articles

Expression pattern of human SERPINE2 in a variety of human tumors.

Serine proteinase inhibitor, clade E member 2 (SERPINE2), also known as protease nexin-1 (PN-1), is a member of the serpin family. Despite several reported roles of SERPINE2 in tumor development the histological distribution of SERPINE2 and its expression levels in a large variety of tumors remains unclear. Through expressed sequence tag database analysis, immunohistochemical staining of tissue microarrays and a literature review, it was revealed that SERPINE2 expression varied according to growth stages and tissue types. SERPINE2 is differentially expressed in a number of tumors and their normal tissue counterparts. SERPINE2 is identified most abundantly in adenocarcinomas. SERPINE2 serves diverse roles in a variety of tumors and therefore may serve as a promising biomarker for tumor diagnosis and prognosis.

Molecular characterization of DnaJ 5 homologs in silkworm Bombyx mori and its expression during egg diapause

A comparison of the cDNA sequences (1 056 bp) of Bombyx mori DnaJ 5 homolog with B. mori genome revealed that unlike in other Hsps, it has an intron of 234 bp. The DnaJ 5 homolog contains 351 amino acids, of which 70 contain the conserved DnaJ domain at the N-terminal end. This homolog of B. mori has all desirable functional domains similar to other insects, and the 13 different DnaJ homologs identified in B. mori genome were distributed on different chromosomes. The expressed sequence tag database analysis of Hsp40 gene expression revealed higher expression in wing disc followed by diapause-induced eggs. Microarray analysis revealed higher expression of DnaJ 5 homolog at 18th h after oviposition in diapause-induced eggs. Further validation of DnaJ 5 expression through qPCR in diapause-induced and nondiapause eggs at different time intervals revealed higher expression in diapause eggs at 18 and 24 h after oviposition, which coincided with the expression of Hsp70 as the Hsp 40 is its co-chaperone. This study thus provides an outline of the genome organization of Hsp40 gene, and its role in egg diapause induction in B. mori.

Putative Vitis vinifera Rop- and Rab-GAP-, GEF-, and GDI-interacting proteins uncovered with novel methods for public genomic and EST database analysis

To understand how grapevine Rop and Rab proteins achieve their functional versatility in signalling, identification of the putative VvRop- and VvRab-interacting proteins was performed using newly designed tools. In this study, sequences encoding eight full-length proteins for VvRop GTPase-activating proteins (GAPs), five for VvRabGAPs, six for VvRop guanine nucleotide exchange factors (GEFs), one for VvRabGEF, five for VvRop GDP dissociation inhibitors (GDIs), and three for VvRabGDIs were identified. These proteins had a CRIB motif or PH domain, a TBC domain, a PRONE domain, a DENN domain, or GDI signatures, respectively. By bootstrap analysis, an unrooted consensus phylogenetic tree was constructed which indicated that VvRopGDIs and VvRopGEFs--but not VvRopGAP--belonged to the same clade, and that VvRabGEF1 protein was more closely related to VvRopGAPs than to the other putative VvRab-interacting proteins. Twenty-two genes out of 28 encoding putative VvRop- and VvRab-interacting proteins could be located on identified grapevine chromosomes. Generally one gene was anchored on one chromosome, but in some cases up to four genes were located on the same chromosome. Expression patterns of the genes encoding putative VvRop- and VvRab-interacting proteins were also examined using a newly developed tool based on public expressed sequence tag (EST) database analysis. Expression patterns were sometimes found to be specific to an organ or a developmental stage. Although some limitations exist, the use of EST database analysis is stressed, in particular in the case of species where expression data are obtained at high costs in terms of time and effort.

Glomerulus-specific mRNA transcripts and proteins identified through kidney expressed sequence tag database analysis

The kidney glomerulus plays a crucial role in blood filtration but the molecular composition and physiology of the glomerulus is not well understood. We previously constructed and large-scale sequenced four mouse glomerular expressed sequence tag (EST) libraries from newborn and adult mouse glomeruli. Here, we compared glomerular EST profiles with whole kidney EST profiles, thereby identifying 497 transcripts corresponding to UniGene clusters that were glomerulus-enriched, that is expressed more abundantly in glomeruli than in whole kidney. These include several known protein-coding glomerulus-specific transcripts critical for glomerulus development and function, but also a large number of gene transcripts, which have not previously been shown to be expressed in the glomerulus, or implicated in glomerular functions. We used in situ hybridization to demonstrate glomerulus-specific RNA expression for six novel glomerular genes and the public Human Protein Atlas to verify glomerular protein expression for another two. The higher mRNA abundance for the eight genes in glomeruli compared with whole kidney was also verified by Taqman quantitative polymerase chain reaction. We surmise that the further characterization of these genes and proteins will increase our understanding of glomerular development and physiology.

The Kallikrein Gene 5 Splice Variant 2 Is a New Biomarker for Breast and Ovarian Cancer

The presence of more than one mRNA form for the same gene is common among kallikreins, and many of the kallikrein splice variants may hold significant clinical value. The human kallikrein gene 5 (KLK5) is a member of the human kallikrein gene family of serine proteases on chromosome 19q13.4. KLK5 has been shown to be differentially expressed in a variety of endocrine tumors including ovarian, breast and prostate cancer. Utilizing Expressed Sequence Tag database analysis and reverse transcriptase polymerase chain reaction, we identified a new alternatively spliced form of KLK5(KLK5-splice variant 2, KLK5-SV2). This variant mRNA is 1,438 bp in length; formed of 195 bp of 5′ untranslated region, 882 bp of protein coding sequence and a 3′ untranslated region of 326 nucleotides. KLK5-SV2 has 7 exons, the first 2 of which are untranslated, and 6 intervening introns. KLK5-SV2 is different from the classic form of the KLK5 mRNA in its 5′ untranslated region, where the first 5′ untranslated exon of the classic form is split into 2 exons with an intervening intron of 135 nucleotides. KLK5-SV2 is expressed in a variety of tissues, with higher expression levels in the mammary gland, cervix, salivary gland and trachea. The steroid hormone receptor-positive breast cancer cell line BT-474 was used to examine the effect of different steroids on the expression levels of KLK5-SV2. Expression levels were significantly higher after stimulation with androgens, but not estrogens, progestins, aldosterone or corticosteroids. While relatively high levels of expression were found in all 10 normal breast tissues examined, no expression was detected in 16 breast cancer tissues, and expression was significantly lower than normal in the remaining 4 cancers. Expression levels comparable to normal were found in only 1 breast cancer cell line. Weak to no expression was detected in 3 other breast cancer cell lines. KLK5-SV2 was not detectable in any of the 10 normal ovarian tissues examined. It was, however, expressed at relatively high levels in 10 out of 20 ovarian cancer tissues, and lower levels were found in 4 other cancers. No expression was detected in the remaining 6 cancers. High expression levels were also detected in the CAOV-3 ovarian cancer cell line. KLK5-SV2 is a potential biomarker for breast and ovarian cancers.

Molecular screens for inner ear genes.

Identification of the genes that encode proteins that are important for proper function of specific inner ear cell types is central to our understanding of the molecular basis of hearing and balance. Whereas the combination of electrophysiology and biophysics has resulted in an exquisite understanding of inner ear function, little is known about the proteins that confer these properties at the cellular level. Furthermore, the genes that control inner ear development, susceptibility to wear and tear, regeneration from damage, and age-related degeneration, are largely unknown. This review discusses tools that have been developed during the past few years to address this imbalance between a thorough physiologic characterization of inner ear function and a detailed understanding at a molecular level of the proteins involved in these functions. Creation of inner ear cDNA libraries has laid the foundation for the discovery of genes that are specifically expressed by cell types of the inner ear and that encode proteins that are important for molecular processes in these cells. In conjunction with expressed sequence tag database analysis, cDNA subtraction, and DNA arrays, functionally important genes, whose specific expression patterns are usually verified by gene expression analysis, can be identified. Discussion of these techniques takes into account the specific characteristics of the inner ear in relation to its study using molecular biological approaches.

The discovery and confirmation of single nucleotide polymorphisms in the human p53R2 gene by EST database analysis.

The human expressed sequence tag (EST) database provides a wealth of resources, which can be used to rapidly screen for potential polymorphisms in proteins of physiological interest. The human p53R2 gene, a recently identified ribonucleotide reductase, plays an important role in DNA repair and is involved in the pathway of p53 activity in response to the presence of DNA damage. On the basis of the alignment of human EST sequences, we identified three candidate polymorphisms at nt 2752, 2759 and 4696 in the 3'-untranslated region of the p53R2 gene. The presence of these polymorphisms was confirmed in a Caucasian population (n = 82) by allele-specific PCR and PCR/restriction fragment length polymorphism analyses. The rare allele frequency at position 4696 (15.5%) is higher than either rare allele frequency at position 2752 or 2759 (6 and 6%). Our results suggest that the human EST data may serve as a valuable source for the rapid identification of genetic variation.

Discovery of germ cell–specific transcripts by expressed sequence tag database analysis

Objective: To identify transcripts whose expression is restricted to germ cells. Design: Expressed sequence tags (ESTs) from unfertilized egg libraries were utilized to perform in silico subtraction and identify germ cell–specific transcripts. Setting: Baylor College of Medicine, Houston, Texas. Animal(s): C57BL/6J/129SvEv hybrid. Intervention(s): Tissue harvesting from mice. Main Outcome Measure(s): Identification of germ cell–specific transcripts. Result(s): We have used the Unigene collection of mouse cDNA libraries to identify ESTs derived from unfertilized egg libraries. A total of 3,499 ESTs were identified from Knowles Solter and Ko unfertilized egg cDNA libraries. In silico subtraction identified 258 ESTs, which were found in these unfertilized egg libraries, but not in adult mouse tissue cDNA libraries. We performed reverse transcription polymerase chain reaction (RT-PCR) on multiple adult tissues with 43 selected ESTs and found 5 of them where expression was absent in heart, lung, liver, brain, spleen, stomach, intestines, kidneys, and uterus, but restricted to ovaries and testes. Three ESTs were further analyzed, and they were exclusively localized to the oocytes by in situ hybridization. Conclusion: We have shown that utilization of publicly available ESTs from murine EST libraries is a simple and rapid in silico approach to the identification of transcripts preferentially expressed in germ cells.

Hevin is down-regulated in many cancers and is a negative regulator of cell growth and proliferation.

We have cloned a human Hevin cDNA from omental adipose tissue of different patients by reverse transcription polymerase chain reaction and shown a sequence variation due to a possible polymorphism at amino acid position 161 (E/G). Hevin protein expressed in vitro showed molecular weights of approximately 75 kDa and 150 kDa, suggesting that Hevin may form a homodimer in vitro. Using Northern blots and a human expressed sequence tAg database analysis, Hevin was shown to be widely expressed in human normal or non-neoplastic diseased tissues with various levels. In contrast to this, its expression was strongly down-regulated in most neoplastic cells or tissues tested. However, neither the mechanism nor the physiological meaning of this down-regulation is known. As an initial step towards investigating the functional role of Hevin in cell growth and differentiation, we transiently or stably expressed this gene in cancer cells (HeLa 3S) that are devoid of endogenous Hevin and measured DNA synthesis (cell proliferation) by 5-bromo-2′-deoxyuridine incorporation. Hevin was shown to be a negative regulator of cell proliferation. Furthermore, we have shown that Hevin can inhibit progression of cells from G1 to S phase or prolong G1 phase. This is the first report which describes the function of Hevin in cell growth and proliferation. Through database analysis, Hevin was found to be located on chromosome 4 which contains loss of heterozygosity of many tumour suppressor genes. Taken together, these results suggest that Hevin may be a candidate for a tumour suppressor gene and a potential target for cancer diagnosis/therapy. © 2000 Cancer Research Campaign

Discovery of a functional polymorphism in human glutathione transferase zeta by expressed sequence tag database analysis

Analysis of the expressed sequence tag (EST) database by sequence alignment allows a rapid screen for polymorphisms in proteins of physiological interest. The human zeta class glutathione transferase GSTZ1 has recently been characterized and analysis of expressed sequence tag clones suggested that this gene may be polymorphic. This report identifies three GSTZ1 alleles resulting from A to G transitions at nucleotides 94 and 124 of the coding region, GSTZ1*A-A94A124; GSTZ1*B-A94G124; GSTZ1*C-G94G124. Polymerase chain reaction/restriction fragment length polymorphism analysis of a control Caucasian population (n = 141) showed that all three alleles were present, with frequencies of 0.09, 0.28 and 0.63 for Z1*A, Z1*B and Z1*C, respectively. These nucleotide substitutions are non-synonymous, with A to G at positions 94 and 124 encoding Lys32 to Glu and Arg42 to Gly substitutions, respectively. The variant proteins were expressed in Escherichia coli as 6X His-tagged proteins and purified by Ni-agarose column chromatography. Examination of the activities of recombinant proteins revealed that GSTZ1a-1a displayed differences in activity towards several substrates compared with GSTZ1b-1b and GSTZ1c-1c, including 3.6-fold higher activity towards dichloroacetate. This report demonstrates the discovery of a functional polymorphism by analysis of the EST database.

Molecular Cloning and Expression of a Mouse Thiamin Pyrophosphokinase cDNA

Thiamin pyrophosphokinase (EC 2.7.6.2) catalyzes the pyrophosphorylation of thiamin with adenosine 5'-triphosphate to form thiamin pyrophosphate. A mouse thiamin pyrophosphokinase cDNA clone (mTPK1) was isolated using a combination of mouse expressed sequence tag database analysis, a two-step polymerase chain reaction procedure, and functional complementation screening with a Saccharomyces cerevisiae thiamin pyrophosphokinase-deficient mutant (thi80). The predicted protein contained 243 amino acid residues with a calculated molecular weight of 27,068. When the intact mTPK1 open reading frame was expressed as a glutathione S-transferase fusion protein in Escherichia coli lacking thiamin pyrophosphokinase, marked enzyme activity was detected in the bacterial cells. The corresponding 2.5-kilobase pair mRNA was expressed in a tissue-dependent manner and was found at relatively high levels in the kidney and liver, indicating that the mode of expression of mTPK1 genes differs with cell type. The expression of mTPK1 genes in cultured mouse neuroblastoma and normal liver cells was unaffected by the thiamin concentration in the medium (10 microM versus 3.0 nM). This is the first report on identification of the primary sequence for mammalian thiamin pyrophosphokinase.

Structure and splice products of the human gene encoding sds22, a putative mitotic regulator of protein phosphatase-1.

sds22 is a regulatory subunit of protein phosphatase-1 that is required for the completion of mitosis in yeast. It consists largely of 11 tandem leucine-rich repeats of 22 residues that are expected to mediate interactions with other polypeptides, including protein phosphatase-1. In this paper, we report on the structure of the human gene encoding sds22, designated PPP1R7. This gene (33 kb) comprises 11 exons, but these do not coincide with the sequences encoding the leucine-rich repeats. Up to six splice variants can be generated by exon skipping and alternative polyadenylation, as revealed by expressed sequence tag database analysis, RT-PCR and Northern blot analysis. The sds22 transcripts are expected to encode four different polypeptides. sds22alpha1 corresponds to the variant cloned previously from human brain [Renouf et al. (1995) FEBS Lett. 375, 75-78]. Sds22beta1 is truncated within the ninth repeat and has a short and different C-terminus. Both variants also exist without the sequence corresponding to exon 2, and these are termed sds22alpha2 and sds22beta2. The 5'-flanking region of PPP1R7 contains two NF-Y-binding CCAAT boxes near the transcription start site and potential binding sites for the transcription factors c-Myb, Ik-2 and NF-1, which are conserved in the mouse gene.

Discovery of three genes specifically expressed in human prostate by expressed sequence tag database analysis.

A procedure is described to discover genes that are specifically expressed in human prostate. The procedure involves searching the expressed sequence tag (EST) database for genes that have many related EST sequences from human prostate cDNA libraries but none or few from nonprostate human libraries. The selected candidate EST clones were tested by RNA dot blots to examine tissue specificity and by Northern blots to examine the transcript size of the corresponding mRNA. The computer analysis identified 15 promising genes that were previously unidentified. When seven of these were examined in an RNA hybridization experiment, three were found to be prostate specific. The genes identified could be useful in the targeted therapy of prostate cancer. The procedure can easily be applied to discover genes specifically expressed in other organs or tumors.