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PubMed1994

Mutational analysis of the tobacco vein mottling virus genome.

We have used a cDNA clone of the potyvirus, tobacco vein mottling virus, to construct 19 mutants bearing 12-nt insertions in the viral genome. These mutants display a variety of phenotypes in inoculated tobacco plants or protoplasts. All mutants with insertions in P3, CI, 6K, NIa, or NIb failed to produce detectable amounts of progeny viral RNA in protoplasts or plants which suggests that they all may be directly involved in replication. Mutants (one in P1 and one in HCpro) presumably affected in polyprotein processing also did not replicate in plants or protoplasts. Seven mutants, with insertions in the 5' noncoding region, P1, HCpro, or CP regions of the genome, were able to infect protoplasts. Three of the 7 mutants (1 in the 5' noncoding region and 2 in HCpro) were able to infect protoplasts but not plants. The remaining 4 mutants replicated in protoplasts and were able to cause systemic infection in plants. The mutation in the CP had no effect on virus accumulation or symptom development in inoculated plants, whereas the other 3 (1 in P1 and 2 in HCpro) induced cyclical patterns of symptom expression. These symptoms ranged from very mild to wild-type-like as new leaves emerged and, as the plants continued to grow, this pattern was repeated. These results support the assignment of roles in replication to five coding regions in the genome and demonstrate that sequence alterations in many parts of other regions of the viral genome may have pronounced effects on replication and the expression of disease symptoms.

Amino Acid Sequence↗

PubMed2004

Evidence for coxsackievirus infection in primary Sjögren's syndrome.

OBJECTIVE: Primary Sjögren's syndrome (SS) is an autoimmune disease characterized by activation of minor salivary gland (MSG) epithelial cells and B and T lymphocytic infiltrates. These findings have long encouraged the hypothesis that a persistent viral infection of the MSG epithelial cells may drive the autoimmune response; however, the identity of that virus has remained elusive. The aim of this study was to test this hypothesis. METHODS: We applied the differential display protocol to MSG RNA samples from patients with primary SS and healthy controls. We then used seminested reverse transcriptase-polymerase chain reaction to amplify the 5'-noncoding region (5'-NCR) of the enteroviral genome in 8 patients with primary SS, 9 patients with secondary SS, and 8 control subjects. Immunohistochemistry was performed to study the expression of the VP1 enteroviral capsid protein in MSG biopsy samples from 12 patients with primary SS, 8 patients with secondary SS, and 16 controls. RESULTS: Differential display analysis yielded a 94-bp fragment of coxsackievirus B4 (CVB4) P2A gene in the primary SS samples. The 5'-NCR was amplified in 7 samples from patients with primary SS and in no samples from patients with secondary SS or controls. The 7 amplified products were sequenced; 4 of the sequences were found to be 98-99% identical to the 5'- NCR of CVB4, and 3 were found to be 97-98% identical to the 5'-NCR of CVA13. Immunohistochemistry for the enteroviral capsid protein VP1 revealed positive staining in epithelial cells and lymphocytic infiltrates in 11 primary SS samples, 1 secondary SS sample, and no control samples. CONCLUSION: We provide evidence that primary SS may be associated with coxsackievirus infection of the MSG epithelial cells and focal lymphocytic infiltrates. Our findings are formulated in a hypothesis concerning the possible role of coxsackieviruses in the induction and maintenance of autoimmunity in primary SS.

Coxsackievirus Infections↗

PubMed2002

Drift in the hypervariable region of the hepatitis C virus during 27 years in two patients.

Serial serum samples were obtained over a 27-year period from a hepatitis C virus (HCV)-infected patient and from a nurse who appeared to become infected by this patient. The hypervariable region 1 (HVR1) and 5'noncoding region (5'NCR) of the HCV genome were amplified from each serum sample by polymerase chain reaction (PCR) and cloned. In the first serum specimen from the patient and the first two serum specimens from the nurse, most of the 20 clones from each serum sample had one common sequence in the HVR1 gene. All later serum samples contained a heterogeneous mixture of HCV quasispecies. The uniformity of the HVR1 sequence in the early samples and the emergence of greater diversity in later serum samples is consistent with the apparent transmission of HCV between the patient and nurse and the eventual emergence of other quasispecies as the virus replicated in the new host. In addition, the immune globulin given to the nurse may have been responsible for some of the HCV quasispecies changes observed in her serum.

5' Untranslated Regions↗

PubMed2003

Mitochondrial DNA alterations in thyroid cancer.

BACKGROUND: Alterations in mitochondrial DNA have been identified in a number of solid tumor types, including gastric, head and neck, breast, colorectal, lung, and bladder carcinomas. Recently, a homopolymeric C stretch (D310) located within the noncoding D-loop of the mitochondrial genome was identified and described as a mutational hotspot. The objective of the present study was to examine a series of thyroid cancers for genetic alterations in this region. METHODS: Seventy-two (72) thyroid cancers were examined for alterations in D310 using PCR-based methods. The primary tumors tested included 35 papillary carcinomas, 18 medullary carcinomas, 9 anaplastic carcinomas, 9 follicular carcinomas, and 1 insular carcinoma. RESULTS: Alterations in D310 were observed in 2/35 papillary carcinomas (5.7%), 1/18 medullary carcinomas (5.6%), 1/9 anaplastic carcinomas (11.1%), and 1/9 follicular carcinomas (11.1%). Overall, the rate of alterations was 5/72 (6.9%). CONCLUSIONS: Mutations in the D310 region of the D-loop of mitochondrial DNA are found in thyroid tumors of varying histologic types and grades. This mutation rate is lower than the reported rate of alteration in tumors of epithelial origin, and shows no relationship to histologic grade.

Adenocarcinoma↗

PubMed1997

Human gamma-aminobutyric acid-type A receptor alpha5 subunit gene (GABRA5): characterization and structural organization of the 5' flanking region.

The gamma-aminobutyric acid-type A receptor alpha5 subunit gene (GABRA5) is widely expressed in brain and localized to the imprinted human chromosome 15q11-q13. A combination of cDNA library screening and 5' RACE analysis led to identification of three distinct mRNA isoforms of GABRA5 in human adult and fetal brain tissues, each of which differs only in the noncoding 5' UTR sequence. Alignment of the genomic and cDNA sequences of GABRA5 revealed that the mRNA isoforms resulted from three alternative first exons 1A, 1B, and 1C. Northern blot analysis showed that the expression of GABRA5 was not only tissue specific but region specific in brain. CAT reporter assays revealed promoter elements in the 5' proximity of each first exon. The GABRA5 promoter regions lacked TATA and CCAAT boxes but contained several other consensus transcriptional factor recognition sequences. These findings suggest that the differential exon 1 usage of GABRA5 arises as a consequence of alternative promoter activation.

Adult↗

PubMed2002

Quasispecies heterogeneity and constraints on the evolution of the 5' noncoding region of hepatitis C virus (HCV): relationship with HCV resistance to interferon-alpha therapy.

Hepatitis C virus (HCV) polyprotein translation depends on direct internal entry of the 40S ribosomal subunit mediated by an internal ribosome entry segment (IRES) located in the 5' noncoding (5'NC) region of the viral genome. HCV is genetically heterogeneous and is characterized by the existence of a quasispecies distribution of the virus population within a single infected individual. Cloning and sequencing strategies were used to characterize 5'NC quasispecies genetically. Similar to coding regions, the HCV 5'NC region was distributed as a quasispecies, but it appeared to be subjected to stronger conservatory constraints than other regions of the HCV genome, probably due to the need for structural (and functional) conservation of the IRES. Indeed, most of the quasispecies substitutions were in unpaired regions of the IRES or clustered such that base-pairing was maintained, whereas only 21% were expected to result in a loss of base-pairing. Quasispecies-related structural changes could be predicted in the core cruciform of IRES domain III composed of the RNA helices which extend from the four-way junction JIIIabc, mostly in minor variants, but sometimes in major ones. The results presented here suggest the simultaneous presence in infected patients of a mixture of genetically distinct but closely related IRES sequences that may have different structures. No significant genetic changes of 5'NC quasispecies were observed after interferon-alpha treatment, except in patients with mixed genotype infection who cleared one of the infecting strains during therapy, suggesting that the quasispecies distribution of IRES sequences does not play a role in HCV resistance to interferon-alpha therapy. In contrast, the overall quasispecies distribution of HCV genomes (including IRES sequences) might participate in regulation of hepatic and extrahepatic HCV replication.

5' Untranslated Regions↗

PubMed1991

Structure and expression of mouse germline immunoglobulin gamma 3 heavy chain transcripts induced by the mitogen lipopolysaccharide.

Germline immunoglobulin heavy chain gene transcription is though to direct isotype switching by modulating the accessibility of specific switch regions to a recombinase. In this study, cloned cDNA copies of mouse germline Igh-8 RNAs have been used to characterize the Igh-8 transcription unit. The 5' end of these transcripts are derived from an exon denoted Ig3, located 1 kilobase 5' of the Igh-8 switch region. Sequence analysis of cDNA and genomic clones reveals that these RNAs are noncoding. In splenic B cell cultures treated with lipopolysaccharide (LPS), germline Igh-8 transcript levels are upregulated after 8 h due to increased transcription. This induction is consistent with the identification of a putative binding site for the LPS inducible transcription factor NF-kappa B approximately 150 nucleotides upstream of the sites of transcript initiation. Furthermore, nucleotide sequence comparisons reveal that the region encompassing the site of germline Igh-8 transcription initiation is highly homologous to part of the Ig2b exon, and is also conserved upstream of the Igh-1 switch region. The implications of these findings for the control of germline Igh-8 transcription is discussed.

Animals↗

PubMed2003

Strand compositional asymmetries of nuclear DNA in eukaryotes.

Both DNA replication and transcription are structurally asymmetric processes. An asymmetric nucleotide substitution pattern has been observed between the leading and the lagging strand, and between the coding and the noncoding strand, in eubacterial, viral, and organelle genomes. Similar studies in eukaryotes have been rare, because the origins of replication in nuclear genomes are mostly unknown and the replicons are much shorter than those of prokaryotes. To circumvent these predicaments, all possible pairs of neighboring genes that are located on different strands of nuclear DNA were selected from the complete genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Plasmodium falciparum, Encephalitozoon cuniculi, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Anopheles gambiae, Mus musculus, and Homo sapiens. For such a pair of genes, one is likely coded from the leading strand and the other from the lagging strand. By examining the introns and the fourfold degenerate sites of codons in the genes of each pair, we found that the relative frequencies of T vs. A and of G vs. C are significantly skewed in most eukaryotes studied. In a gene pair, the potential effects of replication- and transcription-associated mutation bias on strand asymmetry are in the same direction for one gene where leading strand synthesis shares the same template with transcription, while they tend to be canceled out in the other gene. Our study demonstrates that DNA replication-associated and transcription-associated mutation bias and/or selective codon usage bias may affect the strand nucleotide composition asymmetrically in eukaryotic genomes.

Animals↗

PubMed2006

Real-time quantitative assay of HCV RNA using the duplex scorpion primer.

A novel real-time quantitative method for detecting HCV in serum was established in which the duplex scorpion primer was used to provide a unimolecular probing mechanism for hybridizing the highly conserved 5' noncoding region (5' NCR) of the HCV genome specifically. Through methodological evaluation, we found this new method had a wide linearity, high sensitivity, repeatability and specificity. Compared to the commercial TaqMan method, this method was found to be more sensitive and less costly, and the final results were obtained more quickly. Therefore, it could be applied to diagnose and monitor HCV infection in clinical practice.

DNA Primers↗

PubMed1999

Molecular cloning of two bovine aquaporin-4 cDNA isoforms and their expression in brain endothelial cells.

Two cDNA isoforms of bovine aquaporin-4 (bAQP4-A and bAQP4-B) were newly isolated. Sequence analysis of both cDNAs revealed open reading frames of 972 (bAQP4-A) and 906 nucleotides (bAQP4-B) with deduced proteins of 323 (bAQP4-A) and 301 amino acid residues (bAQP4-B). Partial 5'-genomic sequence analysis showed that the 5'-noncoding sequences specific to bAQP4-A and -B transcripts were contained in distinct exons, exon 0 for bAQP4-A and new exon X for bAQP4-B. RNase protection assay demonstrated the definite expression of both isoforms in bovine brain. The deduced amino acid sequence of bAQP4-A was highly homologous to the human (97%), rat (95%), and mouse (93%) AQP4. Reverse transcription-PCR detected the expression of AQP4 mRNAs in bovine brain endothelial cells as well as in a variety of bovine organs such as brain, lung, spleen, and kidney. Northern blot analysis indicated that a 6.0 kb message is predominantly expressed in bovine brain and lung.

Amino Acid Sequence↗

PubMed2001

Detection and genotyping of GBV-C/HGV variants in China.

We detected GBV-C/HGV sequences in the sera from 64 out of a total of 324 subjects in the south of China. In agreement with findings of others, we noted an especially high rate of infection among intravenous drug addicts and patients with chronic hepatitis C virus infection. The detection was achieved by nested PCR to amplify the 5' noncoding region (5'NCR) of the viral genome. Sequence analysis of the resulting 234 bp product revealed a total of 26 different sequences of which 25 were found to belong to the genotype G3, which is the most prevalent genotypes among Asian isolates, and one belonged to genotype G1, common among African isolates. The sequence divergence between the genotypes was largely clustered in a short variable region (V2) within the 5'NCR, and we showed that genotyping may be achieved equally well by analysis of this variable region as by the more detail analysis of the entire 5'NCR or of the entire viral genome.

5' Untranslated Regions↗

PubMed1997

GB virus C/hepatitis G virus infection among Korean patients with liver diseases and general population.

GB virus C and hepatitis G virus (GBV-C/HGV) have been identified from the patients with acute or chronic liver diseases as possible agents of non-B, non-C hepatitis by two different groups, independently. To investigate whether GBV-C/HGV plays a role among Korean patients with liver diseases, GBV-C/HGV RNA were evaluated in 337 sera by the reverse transcription polymerase chain reaction (RT-PCR) using specific primers derived from 5'-noncoding region of GBV-C/HGV genome. GBV-C/HGV RNA was identified in 11/337 (3.3%). They consisted of 1/160 (0.6%) and 10/177 (3.3%) among the general population and patients with liver diseases, respectively (P < 0.01). Nucleotide sequences of all PCR amplicons were determined by the dideoxy chain termination method and analyzed by molecular evolutionary methods. The phylogenetic tree showed all sequences could be divided into three genotypes. These results indicate that: (1) GBV-C/HGV already exist in Korea; (2) GBV-C/HGV may play some role as an etiologic factor among the Korean patients with liver diseases; (3) GBV-C/HGV infection is rare among Korean general population; and (4) there are at least three different types of GBV-C/HGV in Korea.

Adolescent↗

PubMed1998

High prevalence of infection with hepatitis G virus in patients with hepatic and extrahepatic malignancies.

BACKGROUND/AIMS: The pathogenic role of hepatitis G virus, the recently discovered blood-borne agent, is controversial. Our aim was to ascertain the prevalence of hepatitis G virus infection in hepatic and in extrahepatic malignancies. METHODS: We studied 166 Italian patients (112 male, 54 female, mean age 61.8+/-9.3, mean+/-SD, range 34-85). One hundred and eighteen had cirrhosis, which was complicated by hepatocellular carcinoma in 66 cases. Forty-eight patients had extra-hepatic malignancies. Circulating HGV RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) of both the nonstructural-3 and 5'noncoding regions of the hepatitis G virus genome. Antibodies to the E2 protein of hepatitis G virus were detected by means of an enzyme-linked immunosorbent assay. RESULTS: Ongoing HGV infection was detected in 30/66 (46%) patients with hepatocellular carcinoma, 12/52 (23%) patients with cirrhosis, and 14/48 (29%) patients with extrahepatic malignancies (p<0.05). Evidence of exposure to hepatitis G virus (detection of either HGV RNA or anti-E2 antibodies) was found in 46% of patients with cirrhosis, 66% of patients with hepatocellular carcinoma, and 39% of patients with extrahepatic malignancies. Serum HGV RNA positivity was associated with a hematocrit value < or = 0.35 and with history of exposure to blood products (p<0.005). CONCLUSIONS: Ongoing hepatitis G virus infection is detected at a very high rate in patients with hepatocellular carcinoma, but is also fairly common in extrahepatic malignancies. Hepatitis G virus infection in these patients is likely to originate from exposure to blood products, and to persist because of deficient immune surveillance.

Adult↗

PubMedDate not supplied

Dissociation of transforming and trans-activation functions for bovine papillomavirus type 1.

It has been shown that genetic information encoded by the 3' open reading frames (ORFs), E2, E3, E4 and E5, of bovine papillomavirus type 1 (BPV-1), is sufficient to induce cellular transformation of certain mouse cells. The product of the E2 ORF has further been shown to be responsible for the trans-activation of a transcriptional regulatory element located in the noncoding region (NCR) of the BPV-1 genome. To examine whether or not the E2 trans-activation function is encoded by the same gene that encodes the 3' ORF viral transformation function, we have now analysed the expression of the trans-activation function in series of mouse C127 cells transformed by BPV-1 deletion mutants. In addition, using mutated complementary DNA clones generated by the insertion of a premature translational termination linker into different sites of a BPV-1 cDNA clone containing the 3' ORFs intact, we demonstrate that transformation and transcriptional trans-activation functions can be dissociated and that they map respectively to the E5 and E2 ORFs.

Acetyltransferases↗

PubMed1986

Structure and sequence of the human c-sis/platelet-derived growth factor 2 (SIS/PDGF2) transcriptional unit.

The structure of the normal human c-sis/platelet-derived growth factor 2 (SIS/PDGF2) transcript was determined by a combination of cDNA cloning, nuclease S1 mapping, and primer extension. Nucleotide sequence analysis revealed that the 3373-nucleotide SIS/PDGF2 mRNA contained only a 723-base-pair (bp) coding sequence for the PDGF2 precursor polypeptide. The coding sequence was flanked by long 5' (1022 bp) and 3' (1625 bp) untranslated regions. The 5' noncoding region, as well as upstream flanking genomic sequences, contained clusters of specific short repeat sequences. A consensus transcriptional promoter sequence, TATAAA, was identified 24 bp upstream of the mRNA start site and an enhancer-like "TG element" was detected about 180 bp downstream from the site of polyadenylylation. These findings identify putative regulatory elements of the SIS/PDGF2 gene.

Amino Acid Sequence↗

PubMed2004

Long-range control of gene expression: emerging mechanisms and disruption in disease.

Transcriptional control is a major mechanism for regulating gene expression. The complex machinery required to effect this control is still emerging from functional and evolutionary analysis of genomic architecture. In addition to the promoter, many other regulatory elements are required for spatiotemporally and quantitatively correct gene expression. Enhancer and repressor elements may reside in introns or up- and downstream of the transcription unit. For some genes with highly complex expression patterns--often those that function as key developmental control genes--the cis-regulatory domain can extend long distances outside the transcription unit. Some of the earliest hints of this came from disease-associated chromosomal breaks positioned well outside the relevant gene. With the availability of wide-ranging genome sequence comparisons, strong conservation of many noncoding regions became obvious. Functional studies have shown many of these conserved sites to be transcriptional regulatory elements that sometimes reside inside unrelated neighboring genes. Such sequence-conserved elements generally harbor sites for tissue-specific DNA-binding proteins. Developmentally variable chromatin conformation can control protein access to these sites and can regulate transcription. Disruption of these finely tuned mechanisms can cause disease. Some regulatory element mutations will be associated with phenotypes distinct from any identified for coding-region mutations.

Animals↗

PubMed2001

DNA variation in the 5' upstream region of the Adh locus of the wild plants Arabidopsis thaliana and Arabis gemmifera.

To investigate the level and pattern of DNA polymorphism in the noncoding regulatory region in the plant nuclear genome, 2.4 kb of nucleotide sequence of the 5' upstream region of ADH: was determined for 14 ecotypes of Arabidopsis thaliana and five accessions of Arabis gemmifera. Using this data set and previously determined ADH: sequence data, DNA variation was analyzed in a 4.4-kb region of the locus. Two divergent sequence types detected in the transcriptional unit of ADH: were not present in the 5' region of the ADH: gene in A. thaliana. Nucleotide diversity of the entire 5' region was estimated to be 0.0040, which is lower than that in the transcriptional unit. The level of variation was not uniform. There were peaks of variations in a approximately 400-bp region where cis-regulatory elements for ADH: expression were clustered and in exon 4. In interspecific comparison with A. gemmifera, lower divergence was observed in the 5' flanking region than in the exons. High peaks of divergence in the 400-bp regulatory region and exon 4 were also detected, although there were many other peaks. These results indicate that regions of functional importance have a high level of polymorphism and divergence in the ADH: locus of these genera. The possibility of balancing selection in the ADH: gene of these plants is discussed.

5' Untranslated Regions↗

PubMed2019

Discovering human transcription factor physical interactions with genetic variants, novel DNA motifs, and repetitive elements using enhanced yeast one-hybrid assays.

Identifying transcription factor (TF) binding to noncoding variants, uncharacterized DNA motifs, and repetitive genomic elements has been technically and computationally challenging. Current experimental methods, such as chromatin immunoprecipitation, generally test one TF at a time, and computational motif algorithms often lead to false-positive and -negative predictions. To address these limitations, we developed an experimental approach based on enhanced yeast one-hybrid assays. The first variation of this approach interrogates the binding of >1000 human TFs to repetitive DNA elements, while the second evaluates TF binding to single nucleotide variants, short insertions and deletions (indels), and novel DNA motifs. Using this approach, we detected the binding of 75 TFs, including several nuclear hormone receptors and ETS factors, to the highly repetitive Alu elements. Further, we identified cancer-associated changes in TF binding, including gain of interactions involving ETS TFs and loss of interactions involving KLF TFs to different mutations in the TERT promoter, and gain of a MYB interaction with an 18-bp indel in the TAL1 superenhancer. Additionally, we identified TFs that bind to three uncharacterized DNA motifs identified in DNase footprinting assays. We anticipate that these enhanced yeast one-hybrid approaches will expand our capabilities to study genetic variation and undercharacterized genomic regions.

Algorithms↗