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At least 1,693 records · Page 94Linked to original sources
PubMed1994

Sense antisense DNA strand?

Recent evidence indicates that alphaherpesviruses express latency associated transcripts (LATs) from the antisense strand of immediate-early (IE) genes of the viral genome. It has been suggested that LATs containing extended open reading frames (ORFs), might be translated into (a) protein product(s). We found that a salient feature of some herpesvirus DNAs is a high GC preference at the third codon positions. The consequence of this feature is that the probability of a stop-codon appearing at two of the six reading frames of the DNA strand is very low. Therefore, the presence of an extended ORF does not necessarily mean that it is relevant to real translation.

Base Sequence↗
PubMed1996

Characterization of a cDNA encoding murine coagulation factor VII.

The cDNA encoding murine coagulation factor VII (mfVII) was isolated and reconstructed from a lambda Zap cDNA library generated from murine liver mRNA. The cDNA contains 1903 nucleotides spanning 15 bases upstream of the 5'-translation initiation codon, an open reading frame of 1338 nucleotides, 550 nucleotides downstream of the first stop codon and a 3' poly(A) tail. The translation product is composed of a 41-amino acid signal/propeptide region followed by a 405-residue mature protein. The latter is highly homologous to that of human, rabbit, bovine, Rhesus monkey, and canine fVII. All protein domains of hfVII are strictly conserved in mfVII.

Amino Acid Sequence↗
PubMed1997

P53 oncoprotein expression and gene mutations in some keratoacanthomas.

OBJECTIVE: To analyze the relationship of p53 oncoprotein overexpression in most keratoacanthomas (KAs) with gene mutations. DESIGN: Expression of p53 oncoprotein in immunohistochemical staining and its correlation to gene mutations in DNA extracted from KAs and tested in single-strand conformational polymorphism (SSCP) analysis and direct sequencing. SETTING: A micrographic surgery unit and a dermatopathology unit at a university medical center. PATIENTS: Sixteen formalin-fixed, paraffin-embedded skin biopsy specimens were retrieved from dermatopathology archives. Biopsies were performed to establish the diagnosis of KA before surgical treatment. MAIN OUTCOME MEASURES: Intensity of staining in immunohistochemical testing for p53 oncoprotein expression and sequencing of gene mutations. RESULTS: Immunohistochemical staining of 16 KA specimens detected p53 oncoprotein in 15 (94%), distributed as strong in 4 (25%), moderate in 2 (12%) mild in 9 (56%), and negative in 1 (6%), compared with control specimens. Specimens were screened by SSCP for mutations in the p53 gene, and 1 specimen showed a potential mutation in exon 7. Direct sequencing of the samples revealed 2 point mutations. One specimen showed a change of G:A for A:G in codon 146 of exon 5, predicting an amino acid substitution of tryptophan for a stop codon. Another specimen revealed a change of T:A for A:T in codon 234 of exon 7, predicting an amino acid substitution of tyrosine for asparagine. CONCLUSIONS: Ninety-four percent of KA specimens evaluated had detectable p53 oncoprotein. This protein was associated with a point mutation in the p53 gene in 2 of 16 KAs evaluated. In a small fraction of KAs, overexpression of p53 oncoprotein may be associated with point mutations in the p53 gene.

Aged↗
PubMed2000

Altered splicing of the ATDC message in ataxia telangiectasia group D cells results in the absence of a functional protein.

The ATDC gene was cloned using functional complementation and complements the radiosensitivity of ataxia telangiectasia (AT) group D cells. Although a number of transcripts have been detected, only a 3.0 kb cDNA found in a HeLa cell cDNA library has been cloned. Since AT group D cells express only a 2.4 kb transcript, efforts were made to clone and sequence this transcript. Using a biotinylated oligonucleotide probe, mRNA preparations were enriched in ATDC-related sequences. After this enrichment, 2.4 kb clones were obtained from the resulting library. The 2.4 kb transcript appears to be untranslated, since no protein from this transcript has been detected in AT group D cells, and this transcript is probably non-functional, since a splicing variation has positioned part of intron 1 near the first methionine codon in exon 1, eliminating most of exon 1 and important functional regions from this transcript. This transcript now has a stop codon located 33 bp in front of the first methionine, which would stop translation after the eleventh amino acid. As a result of these changes, the AT group D cell line (AT5BI) expresses no functional ATDC protein.

Alleles↗
PubMed1997

Novel insertion mutation in a non-Jewish Caucasian type 1 Gaucher disease patient.

Gaucher disease is the most prevalent lysosomal storage disorder. It is autosomalrecessive, resulting in lysosomal glucocerebrosidase deficiency. Three clinical forms of Gaucher disease have been described: type 1 (nonneuronopathic), type 2 (acute neuronopathic), and type 3 (subacute neuronopathic). We performed PCR-thermal cycle sequence analysis of glucocerebrosidase genomic DNA and identified a novel mutation in a non-Jewish type 1 Gaucher disease patient. It is a C insertion in exon 3 at cDNA nucleotide position 122 and genomic nucleotide position 1626. This mutation causes a frameshift and, subsequently, four of the five codons immediately downstream of the insertion were changed while the sixth was converted to a stop codon, resulting in premature termination of protein translation. The 122CC insertion abolishes a Cac81 restriction endonuclease cleavage site, allowing a convenient and reliable method for detection using RFLP analysis of PCR-amplified glucocerebrosidase genomic DNA. The mutation in the other Gaucher allele was found to be an A-->G substitution at glucocerebrosidase cDNA nucleotide position 1226 that so far has only been reported among type 1 Gaucher disease patients. Since mutation 122CC causes a frameshift and early termination of protein translation, it most likely results in a meaningless transcript and subsequently no residual glucocerebrosidase enzyme activity. We speculate that mutation 122CC may result in a worse prognosis than mutations associated with partial activity. When present in the homozygous form, it could be a lethal allele similar to what has been postulated for the other known insertion mutation, 84GG. Our patient, who is a compound heterozygote 122CC/1226G, has moderately severe type 1 Gaucher disease. Her clinical response to Ceredase therapy that began 31 months ago has been favorable, though incomplete.

Adult↗
PubMed1985

Cloning and characterization of the multifunctional his-3 gene of Neurospora crassa.

We have cloned the his-3 gene of Neurospora crassa and determined its nucleotide sequence. The gene specifies a protein of 863 amino acids (aa) and contains a 59-bp intron which interrupts aa 800, a proline residue. The 5' end of the his-3 transcript is heterogeneous with major starts 122 and 124 bp upstream from the start codon. There are three possible polyadenylation sites, 119, 120 and 121 bp after the UAA stop codon. The protein shows two regions of homology to the yeast HIS4 gene which correspond to the hisIE and hisD genes of Escherichia coli and Salmonella typhimurium. Northern analysis shows that the level of his-3 mRNA increases severalfold in cultures subjected to histidine starvation.

Base Sequence↗
PubMed1986

First position wobble in codon-anticodon pairing: amber suppression by a yeast glutamine tRNA.

A 2.4-kb fragment of DNA isolated from the Saccharomyces cerevisiae genome was found to suppress amber mutations when its carrier plasmid was present in high copy number. A 1.2-kb subclone of this fragment was sufficient to confer suppressor activity. Sequencing has established that this fragment carries a normal glutamine tRNA gene. Deletion of this tRNA gene from the subclone resulted in the loss of suppressor activity. The tRNAGln has the anticodon CUG that normally recognizes the glutamine codon CAG. We propose that suppression occurs via an inefficient readthrough of the UAG amber stop codons during translation. Such readthrough requires wobble in the first position of the codon.

Anticodon↗
PubMed2004

Transient idling of posttermination ribosomes ready to reinitiate protein synthesis.

The fate of ribosomes between termination and initiation during protein synthesis is very basic, yet poorly understood. Here we found that translational reinitiation of the alkaline phosphatase gene occurs in Escherichia coli from an internal methionine codon when the authentic translation is prematurely terminated at a nonsense codon that is within seven codons upstream of the reinitiation codon (which we refer to as "reinitiation window"). Changing the reading frame downstream of the stop codon did not abolish the reinitiation, while inactivating the upstream initiation codon abolished the reinitiation. Moreover, depletion of the ribosome recycling factor (RRF), which disassembles posttermination ribosomes in conjunction with elongation factor G, did not influence the observed reinitiation. These findings suggest that posttermination ribosomes can undergo a transient idling state ready to reinitiate protein synthesis even in the absence of the Shine-Dalgarno (SD) sequence within the reinitiation window by evading disengagement from the mRNA.

Alkaline Phosphatase↗
PubMed2005

Retroviral vectors to monitor somatic hypermutation.

The recent expansion of studies on hypermutation may benefit from a fast and uncomplicated way to measure mutation rates. In this paper we compare different retroviral vector designs for monitoring hypermutation in vivo. Retroviral vectors combine a high transduction rate with integration at random sites within the host cell genome, thus equalizing positional effects on the reporter gene. The vectors contain a reporter gene with a premature TAG termination codon; upon reversion, a full-length fluorescent protein is expressed. Any single point mutation at the amber codon activates the reporter--except the transition from G to A, which only creates the stop codon TAA. In the construct, the reporter gene is followed by an internal ribosome entry site and a second marker that allows selection of stably transduced cells. As a reporter gene, we tested the green and yellow fluorescence proteins (GFP and YFP); and various proteins with red fluorescence (dsRed). The second marker was either a drug resistance gene, or a second fluorescent protein. We also introduced various cis-acting enhancer elements into the reporter construct, to study the simultaneous activity of enhancers on transcription and hypermutation. We found that GFP as a reporter, combined with a drug selection marker, gave the most consistent and convenient mutation rate measurements. DsRed is a good alternative to GFP, but variants with greater fluorescence intensity are needed when combined with green fluorescence measurements. We also confirm that no immunoglobulin specific sequence is needed to target hypermutation. Depending on their position in these ectopically expressed constructs, enhancers can have positive or negative effects on hypermutation.

Animals↗
PubMed2005

The first position of a codon placed in the A site of the human 80S ribosome contacts nucleotide C1696 of the 18S rRNA as well as proteins S2, S3, S3a, S30, and S15.

Messenger RNA analogues (42-mers) containing a GAC codon (aspartic acid) in the middle of their sequence followed by a s(4)UGA stop codon were used to identify the components of the human ribosomal A site in direct contact with the photoactivatable 4-thiouridine (s(4)U) residue. We compared the behavior of the nonphased ribosome-mRNA complex, (-)tRNA(Asp), to the one of the phased complex, (+)tRNA(Asp), in the absence and in the presence of eRF1, the eukaryotic class 1 translation termination factor of human origin. The patterns of cross-links obtained for the three complexes were similar to those previously reported for rabbit ribosomes [Chavatte, L., et al. (2001) Eur. J. Biochem. 268, 2896-2904]. Cross-links involving proteins S2, S3, S3a, and S30 were poorly dependent on the presence of tRNA(Asp) and eRF1. Cross-linking to nucleotide C1696 of 18S rRNA occurred in all complexes, but its yield was at least two times higher in the phased complex with an empty A site than in the nonphased complex or when the A site was occupied by eRF1. In contrast, protein S15 cross-linked only in the phased complex in the absence of eRF1. The data obtained point to notable differences in organization of the decoding site between mammalian and prokaryotic ribosomes and to large internal mobility of the components of the tRNA (eRF1)-free A site.

Base Sequence↗
PubMed1996

Two-domain haemoglobin of the blood clam Barbatia lima resulted from the recent gene duplication of the single-domain delta chain.

The blood clam Barbatia lima subsp. from Amami-Oshima, Japan, expresses three types of haemoglobins in erythrocytes: a tetramer (alpha 2 beta 2), a homodimer (delta 2) and a polymer consisting of two kinds of chains, a 34 kDa two-domain (2D) globin and a delta chain. This is in sharp contrast to the congeneric clams B. reeveana (a North American species) and B. lima from Kochi, Japan, each containing a tetramer and a polymer consisting of the 2D globin, but not the delta chain. We have determined the cDNA-derived amino acid sequences of all four chains, alpha (163 residues), beta (155 residues), delta (152 residues) and 2D (308 residues) of B. lima (Amami-Oshima). The alpha chain has an extremely long N-terminal extension of 20 residues that may form a 'pre-A helix', and this makes the alpha chain the longest known globin. B. lima alpha and beta chains show about 50% sequence identity with the alpha and beta chains, respectively, of tetrameric haemoglobin from a related clam, Anadara trapezia. The B. lima homodimeric delta chain shows 71-74% identity with each of the two domains of the 2D chain, but only 39% identity with the homodimeric gamma chain of Anadara. In addition, the alignment of amino acid sequences of the delta chain and the two domains of the 2D chain revealed that the delta chain lacks one amino acid (Lys) at the C-terminus, suggesting that the C-terminal Lys (codon AAA or AAG) of the two domains of 2D chain could result from the stop codon TAA in the delta chain by nucleotide substitutions. These results, together with the fact that the delta and 2D chains form a polymeric haemoglobin, indicates that the delta chain is the ancestral single-domain globin for the 2D globin. The delta chain is expressed only in B. lima (Amami-Oshima), and appears to be a relic of molecular evolution.

Amino Acid Sequence↗
PubMed1983

Prenatal diagnosis of beta-thalassemia. Detection of a single nucleotide mutation in DNA.

We investigated a method employing synthetic oligonucleotides for the prenatal diagnosis of beta-thalassemia due to a single nucleotide mutation. The beta 0 thalassemia we tested is produced by a nonsense mutation and is commonly found in Sardinia and other parts of the Mediterranean. In this DNA lesion, the glutamine codon CAG at the beta 39 position is mutated to TAG, which results in a stop codon and premature termination of the beta-globin chain. We synthesized two oligonucleotides: one homologous to the normal beta A gene and the other to the beta 0 thalassemia gene at the beta 39 location. The oligonucleotides were labeled with 32P and used as hybridization probes for normal and thalassemic DNA. The beta A probe hybridized only to the normal DNA, and the beta-thalassemia probe only to thalassemic DNA, thus providing a technique for direct demonstration of the mutation. The method is sensitive enough to be applied directly to DNA that is isolated from uncultured cells obtained from only 20 ml of amniotic fluid as early as the 16th gestational week.

Amniocentesis↗
PubMed1994

Transient transfection of the enteric parasite Entamoeba histolytica and expression of firefly luciferase.

Development of DNA-mediated transfection in Entamoeba histolytica will facilitate basic research toward the control of this protozoan parasite. A transient transfection system was established by using the firefly luciferase gene ligated to the 5' and 3' flanking regions of the amebic hgl1 gene. The optimal construct tested encoded an hgl1-luciferase fusion protein and contained 1 kb of 5' flanking sequence with 16 bases of coding sequence from the hgl1 gene ligated in-frame to the luciferase start codon and 2.3 kb of 3' flanking sequence from hgl1 ligated 3' to the luciferase stop codon. Optimal electroporation conditions in strain HM-1:IMSS trophozoites when using this construct were 500 microF and 500 V/cm, which resulted in luciferase activity up to 5000-fold above background 9-12 hr after electroporation. Constructs that contained the luciferase gene without amebic flanking sequences or that contained a simian virus 40 promoter, enhancer, and polyadenylylation signal produced only background levels of luciferase activity. The ability to introduce and express genes in amebae will now permit a genetic analysis of the virulence of this organism, which remains a serious threat to world health.

Animals↗
PubMed1999

Phylogenetic analysis of the apolipoprotein B mRNA-editing region. Evidence for a secondary structure between the mooring sequence and the 3' efficiency element.

Apolipoprotein (apo) B mRNA editing is the deamination of C(6666) to uridine, which changes the codon at position 2153 from a genomically encoded glutamine (CAA) to an in-frame stop codon (UAA). The apoB mRNA-editing enzyme complex recognizes the editing region of the apoB pre-mRNA with exquisite precision. Four sequence elements spanning 139 nucleotides (nt) on the apoB mRNA have been identified that specify this precision. In cooperation with the indispensable mooring sequence and spacer element, a 5' efficiency element and a 3' efficiency element enhance editing in vitro. A phylogenetic comparison of 32 species showed minor differences in the apoB mRNA sequence, and the apoB mRNA from 31 species was robustly edited in vitro. However, guinea pig mRNA was poorly edited. Compared with the consensus sequences of these 31 species, guinea pig apoB mRNA has three variations in the 3' efficiency element, and the conversion of these to the consensus sequence increased editing to the levels in the other species. From this information, a model for the secondary structure was formulated in which the mooring sequence and the 3' efficiency element form a double-stranded stem. Thirty-one mammalian apoB mRNA sequences are predicted to form this stem positioning C(6666) two nucleotides upstream of the stem. However, the guinea pig apoB mRNA has a mutation in the 3' efficiency element (C(6743) to U) that predicts an extension of the stem and hence the lower editing efficiency. A test of this model demonstrated that a single substitution at 6743 (U to C) in the guinea pig apoB mRNA, that should reduce the stem, enhanced editing, and mutations in the 3' efficiency element that extended the stem for three base pairs dramatically reduced editing. Furthermore, the addition of a 20-nucleotide 3' efficiency element RNA, to a 58-nucleotide guinea pig apoB mRNA lacking the 3' efficiency element more than doubled the in vitro editing activity. Based on these results, a model is proposed in which the mooring sequence and the 3' efficiency element form a double-stranded stem, thus suggesting a mechanism of how the 3' efficiency element enhances editing.

Animals↗
PubMed2005

Identification of a novel alpha1-antitrypsin null variant (Q0Cairo).

Alpha1-antitrypsin deficiency (AATD) is a common hereditary disorder associated with high risk of developing pulmonary emphysema early in life and, to a lesser extent, chronic liver disease and cirrhosis. Among Northern Europeans and Northern Americans, more than 95% of individuals with emphysema associated with AATD carry the most frequent AAT deficient gene variants, PI*Z and PI*S. Rare AAT deficient variants account for 2-4% of AATD individuals. We extend the sequence data on AAT by characterizing a novel Null allele detected in 3 subjects: a carrier belonging to an Italian/Egyptian family and 2 members of a family originating from Southern Italy. The mutation raised on a M1 (Ala213) base allele and it is characterized by an A-->T transversion at exon III, nt 218, codon 259 (AAA-->TAA) (GeneBank accession number AY 256958). The transversion results in a premature stop codon (Lys259AAA-->Stop259TAA). The proposed nomenclature of Q0cairo is from the birthplace of the father of first recognized subject. Serum levels and isoelectric focusing of AAT were consistent with the presence of the Null variant.

Adolescent↗
PubMed2005

Peroxisomal monodehydroascorbate reductase. Genomic clone characterization and functional analysis under environmental stress conditions.

In plant cells, ascorbate is a major antioxidant that is involved in the ascorbate-glutathione cycle. Monodehydroascorbate reductase (MDAR) is the enzymatic component of this cycle involved in the regeneration of reduced ascorbate. The identification of the intron-exon organization and the promoter region of the pea (Pisum sativum) MDAR 1 gene was achieved in pea leaves using the method of walking polymerase chain reaction on genomic DNA. The nuclear gene of MDAR 1 comprises nine exons and eight introns, giving a total length of 3,770 bp. The sequence of 544 bp upstream of the initiation codon, which contains the promoter and 5' untranslated region, and 190 bp downstream of the stop codon were also determined. The presence of different regulatory motifs in the promoter region of the gene might indicate distinct responses to various conditions. The expression analysis in different plant organs by northern blots showed that fruits had the highest level of MDAR. Confocal laser scanning microscopy analysis of pea leaves transformed with Agrobacterium tumefaciens having the binary vectors pGD, which contain the autofluorescent proteins enhanced green fluorescent protein and enhanced yellow fluorescent protein with the full-length cDNA for MDAR 1 and catalase, indicated that the MDAR 1 encoded the peroxisomal isoform. The functional analysis of MDAR by activity and protein expression was studied in pea plants grown under eight stress conditions, including continuous light, high light intensity, continuous dark, mechanical wounding, low and high temperature, cadmium, and the herbicide 2,4-dichlorophenoxyacetic acid. This functional analysis is representative of all the MDAR isoforms present in the different cell compartments. Results obtained showed a significant induction by high light intensity and cadmium. On the other hand, expression studies, performed by semiquantitative reverse transcription-polymerase chain reaction demonstrated differential expression patterns of peroxisomal MDAR 1 transcripts in pea plants grown under the mentioned stress conditions. These findings show that the peroxisomal MDAR 1 has a differential regulation that could be indicative of its specific function in peroxisomes. All these biochemical and molecular data represent a significant step to understand the specific physiological role of each MDAR isoenzyme and its participation in the antioxidant mechanisms of plant cells.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1988

Identification of a CpG mutation in the coagulation factor-IX gene by analysis of amplified DNA sequences.

In a family with no previous bleeding history, the sister of a single, severely affected haemophilia B patient requested carrier detection and prenatal diagnosis. In Southern blots, using Taq I digested DNA and a factor-IX cDNA probe, a normal invariant band at 1.6 kb was missing in the haemophiliac suggesting the loss of the Taq I site at the 5' end of exon h. A 162 bp sequence which includes the suspected mutant region was amplified by the polymerase chain reaction in each DNA. Two oligonucleotide probes were synthesized and differed by only one base pair which substituted a T for C in the normal Taq I recognition sequence. The amplified DNA was dot-blotted and hybridized with the labelled probes. The altered sequence hybridized to DNA from the affected individual, his sister and her fetus and not to DNA from the normals. The mutation, involving the haemophiliac, his mother, his sister and her fetus, transforms a CGA codon that encodes for arginine in the catalytic domain of the protein into a UGA stop codon.

Adult↗
PubMed1987

Nucleotide sequence of the gene encoding the 11-kDa subunit of the ubiquinol-cytochrome-c oxidoreductase in Saccharomyces cerevisiae.

The nucleotide sequence of the gene encoding the 11-kDa subunit VIII of the ubiquinol-cytochrome-c oxidoreductase in Saccharomyces cerevisiae has been determined. The coding sequence has a length of 330 bp and is preceded at a distance of 361 bp by another reading frame, coding for a protein of as yet unknown function. The 11-kDa gene is transcribed independently of the URFx gene and transcription of both is sensitive to catabolite repression. Multiple 5' and 3' termini of transcripts of the gene for the 11-kDa subunit were identified by S1 nuclease protection analysis of DNA X RNA hybrids. The 5' termini map 52 +/- 2 and 60 +/- 2 nucleotides upstream of the initiation codon whereas the 3' termini map 336 +/- 2 and 350 +/- 2 nucleotides downstream of the stop codon. The subunit VIII reading frame encodes a protein with a molecular mass of 12.4 kDa and a polarity of 37.6%. It is predicted to contain a high content of beta-sheet segments, which may be capable of forming a barrel-like structure in a lipid bilayer. A comparison of the sequence with those of the small subunits of the beef heart complex reveals similarity with the 9.5-kDa subunit VII (core-linked protein) characterized by Borchart et al. (1986) FEBS Lett. 200, 81-86. The significance of this is discussed.

Animals↗