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PubMed2026

FGF19 as a site-specific candidate biomarker in colorectal neuroendocrine carcinomas.

PURPOSE: Gastrointestinal neuroendocrine carcinomas (GI-NECs) are aggressive tumors with marked site-specific heterogeneity, yet molecular markers for colorectal origin are lacking. This study characterized genomic and protein expression profiles to identify origin-specific biomarkers. METHODS: Nineteen GI-NECs (7 esophageal, 6 gastric, 6 colorectal) were analyzed by targeted next-generation sequencing (NGS) of 425 genes and immunohistochemistry (IHC). Genetic variations across primary sites were compared, and associations between FGF19 expression, clinicopathological features, microsatellite (MS) status, and tumor mutational burden (TMB) were assessed. FGF19 transcriptional expression was further examined in The Cancer Genome Atlas (TCGA) colorectal cohort using the UALCAN platform. RESULTS: A total of 163 genomic alterations were identified. FGF19 was the only gene showing site-specific alterations, being exclusively mutated or amplified in colorectal NECs (50%, 95% CI: 11.8-88.2%) with significantly elevated protein expression (83.3%, 95% CI: 35.9-99.6%) compared with other sites. A microsatellite instability-high (MSI-H) subgroup (10.5%, 95% CI: 1.3-33.1%) exhibited markedly higher TMB. TCGA data confirmed upregulated FGF19 in colorectal tumors but showed no survival association, consistent with the prognostic neutrality in our cohort. CONCLUSIONS: FGF19 may act as a site-specific candidate biomarker for colorectal NECs, with 83.3% protein positivity and exclusive site-specific alterations in 50% of cases. Detection of MSI-H suggests that mismatch repair (MMR) testing may be considered in selected patients with suggestive clinical or family histories to inform immunotherapy decisions.

FGF19↗

PubMed2006

The porcine lymphotropic herpesvirus 1 encodes functional regulators of gene expression.

The porcine lymphotropic herpesviruses (PLHV) are discussed as possible risk factors in xenotransplantation because of the high prevalence of PLHV-1, PLHV-2 and PLHV-3 in pig populations world-wide and the fact that PLHV-1 has been found to be associated with porcine post-transplant lymphoproliferative disease. To provide structural and functional knowledge on the PLHV immediate-early (IE) transactivator genes, the central regions of the PLHV genomes were characterized by genome walking, sequence and splicing analysis. Three spliced genes were identified (ORF50, ORFA6/BZLF1(h), ORF57) encoding putative IE transactivators, homologous to (i) ORF50 and BRLF1/Rta, (ii) K8/K-bZIP and BZLF1/Zta and (iii) ORF57 and BMLF1 of HHV-8 and EBV, respectively. Expressed as myc-tag or HA-tag fusion proteins, they were located to the cellular nucleus. In reporter gene assays, several PLHV-promoters were mainly activated by PLHV-1 ORF50, to a lower level by PLHV-1 ORFA6/BZLF1(h) and not by PLHV-1 ORF57. However, the ORF57-encoded protein acted synergistically on ORF50-mediated activation.

Animals↗

PubMed1998

Two genes, COL4A3 and COL4A4 coding for the human alpha3(IV) and alpha4(IV) collagen chains are arranged head-to-head on chromosome 2q36.

We first isolated and characterized genomic DNA fragments that cover the 5' flanking sequences of COL4A3 and COL4A4 encoding the human basement membrane alpha3(IV) and alpha4(IV) collagen chains, respectively. Nucleotide sequence analysis indicated that the two genes are arranged head-to-head. To determine transcription start site for COL4A4 gene, we performed RACE and RNase protection assays, indicating that there are two alternative transcripts presumably derived from two different promoters. Interestingly, one transcription start site (from exon 1') of COL4A4 is only 5 bp away from the reported transcription start site of COL4A3, whereas the other transcript (from exon 1) starts 373 nucleotides downstream from the first one, generating the two kinds of transcripts that differ in the 5' UTR regions. Expression of these two transcripts appears tissue-specific; exon 1 transcript was expressed predominantly in epithelial cells, while exon 1' transcript showed rather ubiquitous and low expression. The nucleotide sequence of the promoter region is composed of dense CpG dinucleotides, GC boxes, CTC boxes and a CCAAT box but no TATA box. These results provide information to delineate the promoter activity for the tissue-specific expression of the six type IV collagen genes and basement membrane assembly in different tissues and organs.

Alternative Splicing↗

PubMed1999

Complete exon-intron organization of the mouse fibulin-1 gene and its comparison with the human fibulin-1 gene.

Fibulin-1 is a 90 kDa calcium-binding protein present in the extracellular matrix and in the blood. Two major variants, C and D, differ in their C-termini as well as the ability to bind the basement membrane protein nidogen. Here we characterized genomic clones encoding the mouse fibulin-1 gene, which contains 18 exons spanning at least 75 kb of DNA. The two variants are generated by alternative splicing of exons in the 3' end. By searching the database we identified most of the exons encoding the human fibulin-1 gene and showed that its exon-intron organization is similar to that of the mouse gene.

Alternative Splicing↗

PubMed2002

The human alphaE-catenin gene CTNNA1: mutational analysis and rare occurrence of a truncated splice variant.

Abnormal expression of the alphaE-catenin protein, a component of the E-cadherin/catenin cell adhesion complex, is frequently observed in human cancer cells. An inverse correlation between alphaE-catenin expression and tumor malignancy can be of prognostic value. Mutations of the alphaE-catenin gene, CTNNA1, were described in several human cancer cell lines and were found to result in aberrant cell adhesion. We have developed a polymerase chain reaction/single-strand conformation polymorphism-based method for mutation analysis of this gene in human tumor DNA. This approach enabled us to identify several polymorphisms in a set of desmoid tumors, demonstrating that this method is suitable for alphaE-catenin mutational analysis. On the basis of our genomic characterization data, we found that the previously reported alternative splicing of the alphaE-catenin gene actually generates a frame-shift, resulting in a truncated alphaE-catenin protein. This finding is unlike the other alpha-catenin family members alphaN-catenin and vinculin, which show in-frame alternative inserts. Furthermore, real-time quantitative reverse transcriptase-PCR analysis did not reveal relevant expression levels of this alternatively spliced alphaE-catenin variant neither in any human tissue or cell line tested, nor at any mouse developmental stage tested. Thus, contrary to previous notions, alternative splicing with in-frame insertion nearby the C-terminal end of the protein is not a general feature for all members of the alpha-catenin/vinculin family.

Alternative Splicing↗

PubMed2001

Regulation of human P2X1 promoter activity by beta helix-loop-helix factors in smooth muscle cells.

We isolated and characterized genomic clones of the human P2X1 receptor (hP2X1) gene in an effort to understand its tissue specific expression. The hP2X1 gene contains 12 exons spanning 20 kb, with exon sizes ranging from 59 to 143 bp. A 385 bp upstream fragment promoted hP2X1 gene expression in smooth muscle (A7R5 and primary trachealis) and fibroblast (NIH3T3) cell lines, and mutation of a consensus E box sequence (CACCTG) within this fragment (-340 to -345) did not alter basal promoter activity. However, co-transfected bHLH factors regulated activity of the 385 bp minimal P2X1 promoter in a tissue-specific manner. E12 expression inhibited and ITF2b augmented activity in A7R5 cells, but had no effect in NIH3T3 cells. ITF2a, Myo-D, and Id1 proteins had no effect on either cell line, but co-expression of ITF2a blocked E12 inhibition in A7R5 cells, while ITF2b failed to reverse the inhibition. Northern analysis of A7R5 RNA identified high levels of E12 and ITF2b transcripts, and gel shift assays using A7R5 and NIH3T3 nuclear extracts indicated the formation of a protein-DNA complex with an oligonucleotide corresponding to -330 and -348, which was abolished by base substitutions within the E box motif. Our results identify a critical E box response element in the hP2X1 promoter that binds bHLH factors and demonstrate smooth muscle specific transcriptional regulation by E proteins.

3T3 Cells↗

PubMed2003

Pentamer vocabularies characterizing introns and intron-like intergenic tracts from Caenorhabditis elegans and Drosophila melanogaster.

Overall compositional properties at the level of bases, dinucleotides and longer oligos characterize genomes of different species. In Caenorhabditis elegans, using recurrence analysis, we recognized the existence of a long-range correlation in the oligonucleotide usage of introns and intergenic regions. Through correlation analysis, this is confirmed here to be a genome-wide property of C. elegans non-coding portions. We then investigate the possibility of extracting a typical vocabulary through statistical analysis of experimentally confirmed introns of sufficient length (>1 kb), deprived of known splice signals, the focus being on distributed lexical features rather than on localized motifs. Lexical preferences typical of introns could be exposed using principal component analysis of pentanucleotide frequency distributions, both in C. elegans and in Drosophila melanogaster. In either species, the introns' pentamer preferences are largely shared by intergenic tracts. The pentamer vocabularies extracted for the two species exhibit interesting symmetry properties and overlap in part. A more extensive investigation of the interspecies relationship at the level of oligonucleotide preferences in non-coding regions, not related by sequence similarity, might form the basis of new approaches for the study of the evolutionary behaviour of these regions.

Animals↗

PubMed1997

The wacA gene of Dictyostelium discoideum is a developmentally regulated member of the MIP family.

We isolated a cDNA from Dictyostelium discoideum that encodes a 30 kDa protein with significant similarity to members of the major intrinsic protein (MIP) family of membrane transporters. The most closely related protein in the public data bases is an aquaporin from Cicadella viridis which shows 34% identity. The cDNA was used to isolate and characterize genomic fragments carrying the Dictyostelium gene which we named wacA. Genomic probes were used to recognize wacA mRNA isolated at various stages of development. The results showed that the gene is developmentally regulated such that the mRNA first appears at 12 h of development and is retained throughout the remainder of development. In situ hybridization of whole mounts prepared at 15 h of development showed that wacA mRNA accumulates exclusively in prespore cells and is absent from prestalk cells. Although wacA expression is prespore specific, disruption of the gene by homologous recombination did not result in observable alterations in the formation of spores or their resistance to osmotic challenges.

Amino Acid Sequence↗

PubMed1997

Cloning, structural analysis and expression of the gene encoding Hsp32 from Dictyostelium discoideum.

We have isolated and characterized genomic clones encoding a novel small heat shock (HS) protein (Hsp32) from Dictyostelium discoideum that is not homologous to the alpha-crystallin family Hsps. Besides its induction by HS, this gene is also regulated during the life cycle of the organism. At physiologic temperatures hsp32 is expressed at high levels in growing cells and at low levels in cells starved to initiate their developmental programme. However, in both cases the gene can be induced by HS. A DNA fragment containing the upstream region of hsp32 was shown to confer HS induction to a cat reporter gene, indicating a transcriptional regulation for this gene. A single transcription start site, located at position -152 relative to the initiator Met, 17 nucleotides downstream from a putative TATA box, was determined both in vegetative cells and cells starved for 6 h. This site was unchanged when either vegetative or starved cells were submitted to HS at 30 degrees C for 30 min. Despite HS induction, a perfect HSE element was not found in the 5' regulatory region of the gene. The hsp32 coding region is interrupted by a single intron located near its 5' end, which is properly spliced even under HS conditions.

Amino Acid Sequence↗

PubMed2000

Molecular organization of the mouse gastrin-releasing peptide receptor gene and its promoter.

The murine gastrin-releasing peptide receptor (mGRP-R) is a member of the G protein-coupled receptor family and mediates important physiological actions of its specific ligand, the gastrointestinal hormone/neurotransmitter GRP, including mitogenic properties in the mouse Swiss 3T3 fibroblasts. Glucocorticoids and increases in intracellular cAMP are reported to alter GRP-R gene transcription, but the molecular basis for these effects is unknown. To begin to identify possible gene regulatory mechanisms that are responsible for modifying mGRP-R expression, we determined its structure and investigated its basal promoter activity. We isolated and characterized genomic bacteriophage P1 clones encoding the mouse gastrin-releasing peptide receptor (mGRP-R). By DNA sequencing and Southern blot analyses, we determined the protein coding region to be contained in three exons interrupted by two introns 20 and 2kb in length. The open reading frame of the putative GRP-R gene encodes for a 384-amino-acid protein which demonstrates 48% identity with the mouse BRS-3 protein and 53% identity with the mouse NMB-R protein. The mGRP-R gene locus extends over 29kb and was mapped to the X-chromosome (DXMit20) utilizing a minisatellite polymorphism in the 5' UTR and by fluorescent in-situ hybridization (FISH). In Swiss 3T3 cells, which natively express mGRP-R, two gene-specific mRNA species of 3 and 7kb can be detected by Northern blot analysis. With RNase protection assays, and independently with inverse PCR of 5' RACE clones, common mRNA initiation sites were identified clustered between 21 and 61bp downstream of a TTTAAA motif, which is located 450bp upstream of the ATG translation start site. However, different polyadenylation sites are utilized. A 2kb genomic DNA fragment extending from 2147 to 141 bases 5' to the ATG translation start was cloned into a luciferase reporter plasmid and shown to contain promoter activity in Swiss 3T3 and COS-7 cells. Progressive promoter truncations and mutations of a cyclic AMP response element (CRE) located 83bp upstream of the TTTAAA motif demonstrate that transcriptional mGRP-R activation in Swiss 3T3 cells only occurs when both the TTTAAA motif and the intact CRE site are retained. With the availability of the full structure of the mGRP-R gene and the minimal promoter sequences reported in this study, it will be possible in future studies to investigate the molecular basis for transcriptional regulation of the mGRP-R gene by glucocorticoids, cAMP and other factors.

3T3 Cells↗

PubMed1993

Structure and organization of the human alpha class glutathione S-transferase genes and related pseudogenes.

We have isolated and characterized genomic DNA encoding several human Alpha class glutathione S-transferase genes and pseudogenes. All the genes are composed of seven exons with boundaries identical to those of the Alpha class genes in rats. The GSTA1 gene is approximately 12 kb in length and is closely flanked by other Alpha class gene sequences. The complete sequence of the 1.7-kb intergenic region between exon 7 of an upstream pseudogene and exon 1 of the GSTA1 gene has been determined. An additional gene that encodes an uncharacterized Alpha class glutathione S-transferase has been identified. The protein derived from this gene would have 19 amino acid substitutions compared with the GSTA1 isoenzyme. Several pseudogenes with single-base and/or complete exon deletions have been identified, but no reverse-transcribed pseudogenes have been detected. The occurrence of multiple genes and pseudogenes on a single fragment of cloned genomic DNA and the prior identification of a single chromosomal region (6p12) of hybridization (Board and Webb, 1987, Proc. Natl. Acad. Sci. USA 84:2377-2381) suggest that all the Alpha class genes are members of a closely linked gene family that has evolved by duplication and gene conversion events.

Amino Acid Sequence↗

PubMed2003

Antibiotic safety assessment.

Antibiotics usually have positive risk-benefit ratios, their adverse effects being generally mild and reversible on treatment cessation. However, severe adverse drug reactions (ADR), associated with significant mortality and morbidity have resulted in the withdrawal of several active antibiotics, including new fluoroquinolones. Adverse reactions to antibiotics are often poorly documented. The purpose of this article is to examine current tools for investigating and preventing antibiotic toxicity and to suggest future lines of investigation. Structure/ADR relationships have been investigated with various antibiotics (beta-lactams, macrolides, quinolones, etc.) in an attempt to reduce the risk of adverse reactions. Some reactions can be linked to the drug's stereochemical composition. In the case of quinolones for instance, particularly ofloxacin and its derivatives, experimental data show that individual enantiomers have different toxicities. Another major factor that influences the risk of ADRs in a given population is metabolic variability, due to genetic differences in the relevant drug-metabolizing enzymes. Idiosyncratic antibiotic toxicity can be caused by a chemically reactive metabolite. Recent advances in molecular biology, and especially in individual genomic characterization (DNA chip technology, etc.), could in future be useful for identifying patients who are at a special risk of ADR. Finally, certain pharmacokinetic parameters (AUC, Cmax, etc.) can be used to predict adverse effects.

Anti-Bacterial Agents↗

PubMed2003

Vaccines for parasitic and bacterial diseases.

The first decade of the millennium should mark the beginning of a new era in vaccine development, reaping the rewards of advances in genome characterization, antigen identification, understanding the molecular bases of protective immune responses, and adjuvant design and development. Advances in all of these areas have culminated in vaccine candidates entering clinical testing. These include vaccines against two of humankind's oldest and deadliest diseases, tuberculosis and malaria. Several vaccine candidates for each of these diseases will be tested in humans during the next few years. A candidate vaccine for leishmaniasis, an infection that has taught us much about T-cell regulation of protection and disease in animal models, has been developed and is now in the clinic. There are indications both in animal models and in patients that vaccines may be used not only to protect but also to treat leishmania infections.

Humans↗

PubMed2004

Immune responses against adenoviral vectors and their transgene products: a review of strategies for evasion.

Human adenoviruses have been adopted as attractive vectors for in vivo gene therapy since they have a well-characterized genomic organization, can be grown to high titres and efficiently transduce a wide spectrum of dividing and non-dividing cells. However, the first-generation of adenoviral (Ad) vectors yielded only transient expression of the transgene in most immunocompetent mice. This constituted a major limitation of this early vector type. In contrast, persistent transgene expression can be established in immunodeficient mice. This suggests that the immunogenicity of adenoviral vectors limits the effective period of adenovirus-based gene therapy. Much effort has been put in devising strategies to circumvent the limitations imposed onto gene therapy by the immune system. Improvements in vector design have significantly improved the performance of the adenovirus vectors. Based on these results it is reasonable to anticipate that new modifications of the vectors will overcome some of the immunological barriers and will further expand the applicability of adenovirus-derived vectors.

Adenoviridae↗

PubMed2003

A comparative study and phage typing of silage-making Lactobacillus bacteriophages.

To investigate basic characteristics of 10 virulent phages active on silage-making lactobacilli, morphological properties, host ranges, protein composition and genome characterization were separated into five groups based on host ranges and basic properties. The seven phages of groups I, II and V were active on Lactobacillus plantarum and Lactobacillus pentosus. Phage phiPY4 (group III) infected both L. casei and Lactobacillus rhamnosus. Phage phiPY5 (group IV) specifically infected Lactobacillus casei. Morphologically, three phages of groups I belonged to the Myoviridae family, while seven other phages of groups II, III and V belonged to the Siphoviridae family. SDS-PAGE profiles, restriction analysis, G + C contents of DNA and Dot blot hybridization revealed a high degree of homology in each group. Clustering derived from host range analysis was closely related to results of DNA and protein analyses. These phages may be applicable to phage typing for silage-making lactobacilli.

Journal Article↗

PubMed1994

Structural characterization and alternate splicing of the gene encoding the preadipocyte EGF-like protein pref-1.

Preadipocyte factor 1 (pref-1), a member of the EGF-like protein family, is a transmembrane protein with six tandem EGF-like repeats in the putative extracellular domain. Expression of pref-1 is abolished during the in vitro differentiation of 3T3-L1 preadipocytes to adipocytes, and constitutive expression of pref-1 in preadipocytes inhibits their differentiation [Smas, C.M., & Sul, H.S. (1993) Cell 73, 725-734]. In the present studies, we have isolated and characterized genomic clones for pref-1 and have identified multiple pref-1 transcripts generated by alternate splicing. The pref-1 gene consists of five exons and four introns spanning approximately 7.3 kb. By primer extension analysis, the transcription start site was determined to be 169 bp upstream from the translation initiation codon. We have identified functional promoter sequences by transient transfection using a 2.1 kb fragment of the pref-1 5' flanking region linked to a luciferase gene; the pref-1-luciferase fusion gene construct gave 20-fold higher promoter activity as compared to the promoterless vector. Analysis of exon-intron junctions reveals that unlike the majority of the mammalian EGF-like genes, EGF-like repeats of pref-1 are not encoded by discrete exons. Through RT-PCR and the isolation and analysis of multiple pref-1 cDNA clones, we have identified, in addition to full-length pref-1, five alternately spliced forms with various in-frame deletions of all or a part of the sixth EGF-like repeat, juxta-membrane, and predicted transmembrane domains.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1999

Characterization of the gene encoding human TAFI (thrombin-activable fibrinolysis inhibitor; plasma procarboxypeptidase B).

Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described human plasma zymogen that is related to pancreatic carboxypeptidase B. The active form of TAFI (TAFIa), which is formed by thrombin cleavage of the zymogen, likely inhibits fibrinolysis by removal from partially degraded fibrin of the carboxyl-terminal lysine residues which act to stimulate plasminogen activation. We have isolated and characterized genomic clones which encompass the entire human TAFI gene from lambda phage and bacterial artificial chromosome genomic libraries. The complete TAFI gene contains 11 exons and spans approximately 48 kb of genomic DNA. The positions of intron/exon boundaries are conserved between the TAFI gene and the rat pancreatic carboxypeptidase A1, A2, and B and the human mast cell carboxypeptidase A genes, indicating that these carboxypeptidases arose from a common ancestral gene. However, the intron lengths diverge significantly among all of these genes. The TAFI promoter lacks a consensus TATA sequence, and transcription is initiated from multiple sites. Transient transfection of reporter plasmids containing portions of the TAFI 5'-flanking region into mammalian cells allowed localization of the promoter and identified a approximately 70 bp region crucial for liver-specific transcription. Sequence analysis of cDNA clones obtained from human liver RNA indicated that the TAFI transcript is polyadenylated at three different sites. Our findings will facilitate the assessment of the regulation of TAFI expression by transcriptional and/or posttranscriptional mechanisms. Furthermore, knowledge of the genomic structure of the TAFI gene will aid in the identification of mutations that may be associated with the tendency to either bleed or thrombose.

3' Untranslated Regions↗

PubMed2000

Structural study of the porcine Na+/H+ exchanger NHE1 gene and its 5'-flanking region.

The Na+/H+ exchanger is an ubiquitous mammalian plasma membrane protein that is important for the regulation of intracellular pH and cell volume. In order to provide some insight into the molecular basis of NHE1 expression we have isolated and characterized genomic DNA clones containing the coding region and 5'-flanking region of the porcine NHE1 gene. The gene spans more than 30 kb in length and consists of twelve exons that are flanked by typical splice donor and acceptor sequences at the exon-intron boundaries. The positions of the splicing sites are conserved in relation to the human NHE1 gene. The 5' distal transcription initiation site, identified by primer extension analysis, is positioned 766 bp upstream of the translation initiation codon and 36 bp downstream of a TATA box. A 5'-flanking region of 1.62 kb in length contains a number of potential regulatory elements, and exhibits several features that distinguish the pig gene from those of rabbit, mouse and human. The NHE1 gene is located in a CpG island. The promoter sequence of 500 bp is compared with that for NHE1 genes from different species. The homology between the porcine and the human, rabbit and mouse genes is 78, 76 and 75%, respectively. Several consensus elements for transcription factors, including AP-1, C/EBP, and Sp1 are phylogenetically conserved between pig and human, while AP3 and PEA3 are found only in pig. Some conserved elements are found in the pig in multiple copies. These results suggest broadly similar regulatory mechanisms for NHE1 transcription among the different mammalian species but show some species or tissue-specific differences.

5' Untranslated Regions↗