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PubMed1990

Polymerase chain-reaction-mediated cloning and expression of the coat protein gene of potato virus Y in Escherichia coli.

Complementary DNAs (cDNAs) to the RNA genome of a necrotic strain of potato virus Y in Japan (Hokkaido Univ. isolate of PVY-T:PVY-TH) were synthesized and cloned into a plasmid pBR322. About 4.3 kbp of the cDNA sequence containing the 3'-poly(A) tract of PVY-TH was inserted into a recombinant plasmid pBRYT88. The coat protein coding region (CP gene) in pBRYT88 was amplified using the polymerase chain reaction (PCR) and subcloned into a plasmid pUC119. The nucleotide sequence of the CP gene was determined from both the PCR-mediated clones and pBRYT88. The CP gene of PVY-TH consisted of 801 nucleotides, corresponding to 267 amino acids of Mr 29,811. The predicted amino acid sequence of the PVY-TH CP gene was different from that of PVYN (1) in only five amino acids and displayed 98.1% sequence homology. This result indicates that PVY-TH is closely related to PVYN (1). The cDNAs of the PVY-TH CP gene containing an additional initiation codon (ATG) at the 5' end and a stop codon at the 3' end were constructed by PCR amplification and subcloned into an E. coli expression vector, pKK223-3. Five transformed E. coli colonies expressing the PVY-TH CP were identified by immunoscreening using both polyclonal rabbit antiserum against PVY-TH and mouse monoclonal antibody (MoAb) specific to PVY-T. The CP of PVY-TH produced in the E. coli colonies had an electrophoretic mobility identical to that of native PVY-TH CP and reacted strongly to a specific MoAb to PVY-T, but did not react to a specific MoAb to an ordinary strain of PVY (PVY-O). The maximum expression of the CP in E. coli was approximately 7% of the total soluble proteins. The result indicates that the CP gene cloned by PCR was functional and the PCR procedure was useful for producing biologically active cDNA clones from a single, long positive-sense RNA genome encoding a single, large polyprotein precursor, such as potyviruses.

Amino Acid Sequence↗
PubMed1993

Molecular cloning, sequencing, and expression in Escherichia coli of the potato virus Y cytoplasmic inclusion gene.

Complete nucleotide sequences of cytoplasmic inclusion (CI) genes of two strains of potato virus Y (PVY) were determined from six polymerase chain reaction (PCR)-amplified cDNA clones. The size of the CI genes of both ordinary (PVY-O) and necrotic strains (PVY-T13) was 1902 nucleotides, with a sequence homology of 83.4%. Comparison of the predicted amino acid sequences showed more than 90% homology. When these were compared with those of other potyviruses, the homology ranged from 53 to 61%. cDNAs of all or a part of the PVY-O CI gene containing an additional initiation codon (ATG) at the 5' end and a stop codon at the 3' end were constructed by PCR amplification and cloned into an Escherichia coli expression vector, pKK 223-3. Complete and truncated PVY-O CI proteins were successfully produced in E. coli as judged by reactivities with PVY-O CI protein-specific antiserum. To our knowledge, this is the first report on expression of PVY CI proteins in E. coli.

Amino Acid Sequence↗
PubMed2005

Mutations of the APC, beta-catenin, and axin 1 genes and cytoplasmic accumulation of beta-catenin in oral squamous cell carcinoma.

PURPOSE: The Wnt pathway is involved in carcinogenesis and three regulatory genes of the Wnt pathway, APC, beta-catenin and Axin are mutated in some primary human cancers. Mutations in these genes can impair the down regulation of beta-catenin, which results in the stabilization of beta-catenin, accumulation of free beta-catenin and subsequent activation of the Wnt pathway. To clarify the genetic alterations of components of the Wnt pathway in oral squamous cell carcinoma (SCC), we examined mutations in the APC, beta-catenin and Axin genes and subcellular localization of beta-catenin. METHODS: 20 oral SCC tissues and four cell lines derived from oral SCC were used. Mutational analysis was performed by a single-strand conformation polymorphism (SSCP) method and direct sequencing analysis. The samples were also examined by immunohistochemical staining and immunoblot analysis. RESULTS: In 3 of 4 cell lines, mutations were observed in the APC and Axin1 genes without amino acid substitutions. In a clinical sample, a mutation in the Axin1 gene was detected; a T insertion at codon 250 resulted in the formation of a stop codon at codon 259. In addition, cytoplasmic accumulation of beta-catenin was observed in 3 (75%) of 4 cell lines and 18 (90%) of 20 cancer tissue samples. CONCLUSION: The Axin1 gene may be one of the mutational target in oral SCC. In addition, the cytoplasmic accumulation of beta-catenin is a common characteristic of oral SCC, but is not closely associated with mutational alterations in the APC, beta-catenin and Axin1 genes.

Axin Protein↗
PubMed2003

Efficient disruption of a polyketide synthase gene ( pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis.

Glarea lozoyensis produces pneumocandin B(0), a potent inhibitor of fungal glucan synthesis. This industrially important filamentous fungus is slow-growing, is very darkly pigmented, and has not been easy to manipulate genetically. Using a PCR strategy to survey the G. lozoyensis genome for polyketide synthase (PKS) genes, we have identified pks1, a gene that consists of five exons interrupted by four introns of 56, 400, 50 and 341 bp. It encodes a 2124-amino acid protein with five catalytic modules: ketosynthase, acyltransferase, two acyl carrier sites, and thioesterase/Claisen cyclase. The transcriptional initiation and termination sites were found 43 bp upstream of the translational start codon and 295 bp downstream of the translational stop codon, respectively. Cluster analysis of 37 fungal ketosynthase modules grouped the Pks1p with PKSs involved in the biosynthesis of 1,8-dihydroxynaphthalene melanin. Disruption of pks1 yielded knockout mutants that displayed an albino phenotype, suggesting that pks1 encodes a tetrahydroxynaphthalene synthase. Gene replacement was achieved by Agrobacterium-mediated transformation, which proved to be simple and efficient. Loss of pigmentation occurred in more than half the transformants, and examination of six non-pigmented transformants showed that the functional genomic copy of the pks1 gene had been replaced by the disruption cassette in each case. A putative 1215-bp ORF (dsg) devoid of introns was present downstream from pks1. BLAST analysis of the 405-amino acid sequence of its predicted product showed a high degree of similarity with Zn(II)(2)Cys(6) binuclear cluster DNA-binding proteins, a class of fungal transcription factors involved in the regulation of polyketide production and other pathways.

Amino Acid Sequence↗
PubMed1999

A novel nonsense mutation at Glu-631 in a Spanish family with complement component 7 deficiency.

Deficiency of the seventh component of complement (C7D) is frequently associated with recurrent neisserial infections. We report in the present study the genetic basis for C7D in a Spanish family. We used exon-specific polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) analysis as a screening step for mutations, followed by direct sequencing of the target exon. The mutation in the proband was a homozygous G-to-T transversion at nucleotide 1957, the first nucleotide of the codon GAG for Glu-631, leading to a stop codon TAG (E631X). Our result provides further evidence that the molecular pathogenesis of C7D is heterogeneous.

Adult↗
PubMed1992

Hormonal and nutritional stimuli modulate apolipoprotein B mRNA editing in mouse liver.

Human livers produce apoB-100, a major protein of VLDL, while intestines produce apoB-48, the major protein of chylomicrons. ApoB-48 is translated from apoB-100 mRNAs that are post-transcriptionally edited at codon 2153, converting CAA (glutamine) to TAA, a stop codon. In contrast to humans, mouse and rat livers contain the apoB-100 mRNA editing mechanism. Because hormones and nutrients affect the metabolism of apoB containing lipoproteins, we studied the effects of sex hormones and diets on apoB mRNA editing. Groups of male and female C3H/HeJ mice were castrated and treated with 17 beta-estradiol at 0.16 (E2L) or at 5 micrograms (E2H), or with testosterone propionate at 1 microgram/g body weight/day for 14 days. Plasma apoB levels and ratios of apoB-100/apoB-48 both increased 2-fold, but only in the E2H group. To determine if the increased apoB-100/apoB-48 ratios were associated with altered levels of apoB-100 and apoB-48 mRNA, both forms of apoB mRNA were quantified. We found that indeed ApoB-100 mRNA increased 1.8-fold (p < 0.025) compared to apoB-48 mRNA only in the E2H group. Next, we studied the individual effects of dietary fatty acids and dietary cholesterol on the relative abundance of apoB-100 and apoB-48 mRNA. Contrary to the estrogen effect, the high fat-combination diet increased apoB-48 mRNA relative to apoB-100 mRNA. Total plasma apoB as well as apoB-48 synthesis in liver also increased. Our studies demonstrate that estrogens and high fat diet both modulate apoB editing in mouse liver, but that estrogens and fat diet affected apoB mRNA editing in opposite directions.

Animals↗
PubMed1990

Cloning and sequence analysis of the Erythrina corallodendron lectin cDNA.

Examination of the hemagglutinating activity of extracts from seeds of Erythrina corallodendron at various maturation stages revealed that the level of lectin increases markedly past mid-maturation. Seeds at this stage of maturation served as a source of mRNA for the construction of an expression cDNA library in the vector lambda Zap, which generates fusion proteins with an N-terminal portion of beta-galactosidase. The library was screened with rabbit polyclonal anti-ECorL antiserum. Four immunopositive clones were isolated. Western blot analysis of cell extracts from one of the clones (pIEcL-B) showed a 36 kDa protein that reacted with the antiserum, as well as with a mouse monoclonal antibody raised against the lectin. DNA sequence analysis by the chain termination method revealed that clone pIEcl-C has an insert of 1017 bp with the entire coding sequence of ECorL, beginning with an initiation codon ATG at position 26 and ending with stop codon TAA at position 868. This fragment encodes a polypeptide of 281 amino acids consisting of a signal leader sequence of 25 amino acids and a mature protein of 256 amino acids. The deduced amino acid sequence from this fragment is identical to the sequence of the first 244 amino acids of ECorL, as determined at the protein level, except at 7 positions.

Amino Acid Sequence↗
PubMed1986

Structure of the gene encoding the exoglucanase of Cellulomonas fimi.

In Cellulomonas fimi the cex gene encodes an exoglucanase (Exg) involved in the degradation of cellulose. The gene now has been sequenced as part of a 2.58-kb fragment of C. fimi DNA. The cex coding region of 1452 bp (484 codons) was identified by comparison of the DNA sequence to the N-terminal amino acid (aa) sequence of the Exg purified from C. fimi. The Exg sequence is preceded by a putative signal peptide of 41 aa, a translational initiation codon, and a sequence resembling a ribosome-binding site five nucleotides (nt) before the initiation codon. The nt sequence immediately following the translational stop codon contains four inverted repeats, two of which overlap, and which can be arranged in stable secondary structures. The codon usage in C. fimi appears to be quite different from that of Escherichia coli. A dramatic (98.5%) bias occurs for G or C in the third position for the 35 codons utilized in the cex gene.

Amino Acid Sequence↗
PubMed1991

Analysis of the cbhE' plasmid gene from acute disease-causing isolates of Coxiella burnetii.

A gene termed cbhE' was cloned from the QpH1 plasmid of Coxiella burnetii. Expression of recombinants containing cbhE' in vitro and in Escherichia coli maxicells, produced an insert-encoded polypeptide of approx. 42 kDa. The CbhE protein was not cleaved when intact maxicells were treated with trypsin. Hybridizations of total DNA isolated from the six strains of C. burnetii indicate that this gene is unique to C. burnetii strains associated with acute disease, i.e., Hamilton[I], Vacca[II], and Rasche[III]. The cbhE' gene was not detected in strains associated with chronic disease (Biotzere[IV] and Corazon[V]) or the Dod[VI] strain. The cbhE' open reading frame (ORF) is 1022 bp in length and is preceded by a predicted promoter/Shine-Dalgarno (SD) region of TCAACT(-35)-N16-TAAAAT(-10)-N14-AGAAGGA (SD) located 10 nucleotides (nt) before the presumed AUG start codon. The ORF ends with a single UAA stop codon and has no apparent Rho-factor-independent terminator following it. The cbhE' gene codes for the CbhE protein of 341 amino acid (aa) residues with a deduced Mr of 39,442. CbhE is predominantly hydrophilic with a predicted pI of 4.43. The function of CbhE is unknown. No nt or aa sequences with homology to cbhE' or CbhE, respectively, were found in searches of a number of data bases.

Acute Disease↗
PubMed1997

Characterization of a full-length cDNA for rabbit factor X.

A 300 bp probe generated by the PCR was derived from rabbit genomic DNA using primers from a highly conserved region of the DNA for human factor X (HFX). The probe was used in northern blot analysis of liver RNA to demonstrate an mRNA species of 1.6 kb for the rabbit factor X and subsequently for isolation and characterization of the cDNA for rabbit factor X (RFX) from a lambda Zap II cDNA library generated from rabbit liver mRNA. The cDNA contains 22 bases upstream from the 5'-translation initiation codon, 1470 nucleotides of open reading frame, a stop codon and a 3' poly (A) tail. The cDNA codes for a 40-residue signal/propeptide region, followed by a 447-residue mature protein. The deduced amino acid sequence shows a high degree of homology with the sequence of HFX. Inhibitory peptides derived from interactive sites of HFX for activators, cofactor and substrate exerted degrees of inhibition of RFX activation which showed a dependence on extent of homology with the corresponding regions of RFX.

Amino Acid Sequence↗
PubMed2003

Pseudogenization of the tumor-growth promoter angiogenin in a leaf-eating monkey.

Physiological functions of human genes may be studied by gene-knockout experiments in model organisms such as the mouse. This strategy relies on the existence of one-to-one gene orthology between the human and mouse. When lineage-specific gene duplication occurs and paralogous genes share a certain degree of functional redundancy, knockout mice may not provide accurate functional information on human genes. Angiogenin is a small protein that stimulates blood-vessel growth and promotes tumor development. Humans and related primates only have one angiogenin gene, while mice have three paralogous genes. This makes it difficult to generate angiogenin-knockout mice and even more difficult to interpret the genotype-phenotype relation from such animals should they be generated. We here show that in the douc langur (Pygathrix nemaeus), an Asian leaf-eating colobine monkey, the single-copy angiogenin gene has a one-nucleotide deletion in the sixth codon of the mature peptide, generating a premature stop codon. This nucleotide deletion is found in five unrelated individuals sequenced, and therefore is likely to have been fixed in the species. Five colobine species that are closely related to the douc langur have intact angiogenin genes, suggesting that the pseudogenization event was recent and unique to the douc langur lineage. This natural knockout experiment suggests that primate angiogenin is dispensable even in the wild. Further physiological studies of douc langurs may offer additional information on the role of this cancer-related gene in normal physiology of primates, including humans. Our findings also provide a strong case for the importance of evolutionary analysis in biomedical studies of gene functions.

Amino Acid Sequence↗
PubMed1997

The Staphylococcus aureus collagen adhesin-encoding gene (cna) is within a discrete genetic element.

Although the gene (cna) encoding the Staphylococcus aureus (Sa) collagen adhesin is not present in all strains, the DNA both upstream and downstream of cna is present in all Sa strains. Using oligo primers corresponding to the conserved nt flanking cna and template DNA from Sa strains that do not encode cna, we amplified a 372-bp fragment. These results illustrate that the conserved regions upstream and downstream of cna are contiguous in strains that do not encode cna. Using primers corresponding to the conserved flanking DNA together with primers corresponding to the 5' and 3' ends of cna, we also amplified DNA fragments containing the junctions between the cna genetic element and the conserved flanking sequences. Sequence comparisons of the amplification products from four cna negative and four cna positive strains revealed that cna is within a discrete genetic element that extends 202 bp upstream from the cna start codon and 100 bp downstream of the cna stop codon. Sequence analysis of the ends of the cna element did not reveal any of the repeats characteristic of transposable elements. These results suggest that cna may be part of a larger element (e.g., a phage) that may or may not contain cna. Alternatively, cna may be a subject to a precise excision event resulting in its deletion from the chromosome. Based on sequence analysis of the flanking DNA amplified from strains that do not encode cna, the presence of a cna genetic element does not disrupt an ORF.

Adhesins, Bacterial↗
PubMed2000

Identification and characterization of the Trichoplusia ni single capsid nuclear polyhedrosis virus p10 gene.

The p10 gene was identified and characterized from the Trichoplusia ni single capsid nuclear polyhedrosis virus (TniSNPV). The p10 open reading frame (ORF) sequence was identified following sequencing of the ends of the EcoRI-G clone. Subsequent sequencing of an EcoRI-SmaI subclone identified the entire p10 and a portion of a p26 homologue. The p10 ORF of 264 basepairs (bps), encoded a predicted protein of 88 amino acids (aas) with Mr 9527 Da. The putative late transcription initiation motif (TAAG) was found upstream of the translation initiation codon at position -46. Downstream of the translation stop codon, a putative poly(A) signal was identified. The p10 amino acid sequence contained the three conserved domains reported for all other p10 genes. The p10 amino acid sequence was most homologous (85% similarity and 67% identity) to that of Buzura suppresaria NPV p10 sequence.

Amino Acid Sequence↗
PubMed1994

Dimeric structure of a human apolipoprotein B mRNA editing protein and cloning and chromosomal localization of its gene.

Apolipoprotein B (apoB) mRNA editing consists of a posttranscriptional C-->U conversion involving the first base of the codon CAA encoding glutamine-2153 to UAA, a stop codon, in apoB mRNA. Using a cloned rat cDNA as a probe, we cloned the cDNA and genomic sequences of the gene for a human apoB mRNA editing protein. Expression of the cDNA in HepG2 cells results in editing of the intracellular apoB mRNA. By fluorescence in situ hybridization, we localized the gene for the editing protein to chromosome band 12p13.1-p13.2. By Northern blot analysis, it was shown that the human editing protein mRNA is expressed exclusively in the small intestine. The cDNA sequence predicts a translation product of 236-aa residues. By attaching an epitope tag sequence to the C terminus of the editing protein, we examined the polymerization state of the editing protein synthesized in vitro. We found that the editing protein undergoes spontaneous polymerization. The migration of the human apoB mRNA editing protein on an HPLC column and the stoichiometry of polymeric epitope-tagged to untagged protein indicate that the protein exists as a dimer. Dimerization does not require glycosylation of a consensus N-linked glycosylation sequence present in the protein and is not mediated by disulfide bridge formation. The human apoB mRNA editing protein is a cytidine deaminase showing structural homology to some known mammalian and bacteriophage deoxycytidylate deaminases. The latter enzymes exist as homopolymers. The fact that the apoB mRNA editing protein also exists as a homodimer has important implications for the mechanism of apoB mRNA editing in humans.

APOBEC-1 Deaminase↗
PubMed1999

An experimental solution for the Luria-Delbrück fluctuation problem in measuring hypermutation rates.

A cell line harboring all trans-acting elements necessary for hypermutation was transfected with a plasmid harboring the major cis-acting elements plus a green fluorescent protein gene containing a premature chain-termination codon. Transfected cells do not fluoresce unless the stop codon reverts. When a sizable cell population is purged of revertants by sorting, the frequency of mutants increases linearly with time, and there is no Luria-Delbrück fluctuation effect. Moreover, as mutant frequencies seemed to vary less than cell numbers in replicate cultures, it is suggested that hypermutation might not be coupled closely to cell division.

Cell Line, Transformed↗
PubMed1995

Alternative mRNA splicing and differential promoter utilization determine tissue-specific expression of the apolipoprotein B mRNA-editing protein (Apobec1) gene in mice. Structure and evolution of Apobec1 and related nucleoside/nucleotide deaminases.

Apolipoprotein (apo) B mRNA editing consists of a C-->U conversion involving the first base of the codon CAA, encoding Gln 2153, to UAA, a stop codon. Editing occurs in the intestine only in most mammals, and in both the liver and intestine in a few mammalian species including mouse. We have cloned the cDNA for the mouse apoB mRNA editing protein, apobec1. Expression of mouse apobec1 cDNA in HepG2 cells results in the editing of the intracellular apoB mRNA. The cDNA predicts a 229-amino acid protein showing 92, 66, and 70% identity to the rat, rabbit, and human proteins, respectively. Based on the estimated values of divergence of apobec1 sequences in terms of the numbers of synonymous and non-synonymous suhstitutions per site, we found that apobec1 is a fairly rapidly evolving protein. Sequence comparison among mammalian apobec1 sequences has permitted the identification of seven conserved regions that may be functionally important for editing activity. We present a phylogenetic tree relating apobec1 sequences to double-stranded RNA adenosine deaminase and other nucleotide/nucleoside deaminases. Northern blot analysis indicates that apobec1 mRNA exists in two different sizes, a approximately 2.2-kilobase (kb) form in small intestine and a approximately 2.4-kb form in liver, spleen, kidney, lung, muscle, and heart. To study the molecular basis for the different sized apobec1 mRNAs, we cloned the apobec1 gene and characterized its exon-intron organization together with the sequences expressed in the hepatic and intestinal mRNA. The mouse apobec1 gene contains 8 exons and spans approximately 25 kb, and is located in chromosome 6. The major hepatic mRNA contains all 8 exons, whereas the major small intestinal mRNA misses the first 3 exons and its transcription is initiated in exon 4. The intestinal mRNA also contains at its 5' end a unique 102-nucleotide piece that is absent in the liver mRNA. We also identified two alternatively spliced hepatic apobec1 mRNAs with different acceptor sites in exon 4. Transient expression studies using promoter-reporter gene constructs in HeLa, Hepa, and Caco-2 cells indicate that the 5'-flanking sequences of the liver mRNA (i.e. upstream of exon 1) have predominantly hepatic promoter activity and the 5'-flanking sequences of the major small intestine mRNA (i.e. upstream of exon 4) have preferential intestinal promoter activity.(ABSTRACT TRUNCATED AT 400 WORDS)

APOBEC-1 Deaminase↗
PubMed1999

A novel MDMX transcript expressed in a variety of transformed cell lines encodes a truncated protein with potent p53 repressive activity.

The MDMX gene product is related to the MDM2 oncoprotein, both of which interact with the p53 tumor suppressor. We have identified a novel transcript of the MDMX gene that is expressed in a variety of cell lines, and in particular, in growing and transformed cells. This transcript is identical to the published sequence yet it has a short internal deletion of 68 base pairs. This deletion produces a shift in the reading frame after codon 114, resulting in the inclusion of a stop codon at amino acid residue 127 (full-length MDMX is 489 residues). This truncated MDMX protein is termed MDMX-S ("short form"), represents only the p53-binding domain, and appears to bind p53 better than full-length MDMX. The MDMX-S protein can be detected in cell extracts and when overexpressed is much more effective than MDMX at inhibiting p53-mediated transcriptional activation and induction of apoptosis. Since MDMX-S lacks the central and carboxyl-terminal regions contained within full-length MDMX, it is likely to play a key role in the regulation of cell proliferation and apoptosis in a way distinct from MDMX.

3T3 Cells↗
PubMed1995

Precore mutant chronic hepatitis B in an autochthonous Belgian population: a study of ten patients.

The mode of contamination, presentation, clinical evolution and liver histology of 10 native Belgian patients with chronic hepatitis B likely due to a peculiar viral strain, the so called precore mutant hepatitis B virus were reviewed. This viral strain first described in Southern European regions is characterized by a mutation of a single base change at nucleotide 1896 in the preterminal codon of the precore region which creates a stop codon that prevents the production of HBeAg. In this setting, abnormal liver biochemistry and active viral replication as evidenced by HBV DNA positivity often coincides with a serological profile similar to that observed in healthy HBsAg carriers. Our data indicate that chronic hepatitis related to such a viral strain may be observed in a native Belgian population. This unusual presentation of chronic hepatitis B should thus be kept in mind when interpreting atypical sero-biochemical features, evaluating the risk of infectivity and the potential benefit of antiviral therapy.

Adolescent↗