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At least 415 records · Page 23Linked to original sources
PubMed2001

Bovine adenovirus type 3 E1B(small) protein is essential for growth in bovine fibroblast cells.

In order to study the function of bovine adenovirus type 3 (BAV-3) E1A and E1B(small) proteins, we constructed two mutants: (a) BAV102A carries an in-frame deletion in the coding region for the E1A protein (nt 831-1080); (b) BAV102B carries an insertion of triple stop codons in the E1B region (nt 1654, 178 bp downstream of the E1B(small) start codon), which stops the translation of the E1B(small) gene. BAV102A virus could grow to the wild-type BAV-3 titer in transformed cell line VIDO R2 (HAV-5 E1 transformed) cells, but no progeny virus could be found in fetal bovine retina cells (FBRC). RT-PCR and Western blot analysis showed that neither mRNA transcripts nor protein expression of early genes [E1B(small) and DNA binding protein (DBP)] could be detected in BAV102A infected FBRC. The BAV102B grew 1.5 log less than wild-type BAV-3 in FBRC; however, no BAV102B progeny virus could be observed in bovine fibroblast (BFB) cells. No appreciable difference was observed in DBP transcript synthesis between wild-type BAV-3- or BAV102B-infected FBRC. However, compared to wild-type BAV-3, BAV102B viral DNA synthesis and fiber gene expression were found to be slightly reduced in FBRC. In contrast, compared to wild-type BAV-3, DBP transcripts and viral DNA synthesis were drastically reduced in BAV102B-infected BFB cells. In addition, no fiber gene expression could be detected in BAV102B-infected BFB cells. These results suggest that BAV-3 E1A is essential for virus replication and is required for activating the transcription of other BAV-3 early genes. However, the requirement for E1B(small) protein for BAV-3 replication appears to be cell type-dependent.

Adenovirus E1A Proteins↗
PubMed1990

Molecular analysis of structural protein genes of the Yamagata-1 strain of defective subacute sclerosing panencephalitis virus. II. Nucleotide sequence of a cDNA corresponding to the P plus M dicistronic mRNA.

The nucleotide sequence of a cloned cDNA corresponding to the P + M dicistronic mRNA of a subacute sclerosing panencephalitis (SSPE) virus was determined and compared with data of measles virus (MV). The dicistronic mRNA of the SSPE virus consisted of the 3' proximal 626 nucleotides of P mRNA, intercistronic trinucleotides, a full length of M mRNA, and 75 poly A nucleotides. The part encoding the P protein had a high homology to MV, except at the noncoding region. The terminating consensus sequence of the P gene and the intercistronic trinucleotides of the SSPE virus were CTAC(A)6 and CCT; in MV they are TTAT(A)6 and CTT, respectively. In the M gene, the starting consensus sequence was exactly the same as MV, but at the 5' proximal end, one third of this gene was different: The first ATG codon of the MV M gene signaling opening of the reading frame was changed to ACG in the SSPE virus and one long open reading frame started from the third ATG codon. The stop codon (TAG) of the MV M gene was also changed to CAG in the SSPE virus. Thus, the deduced SSPE-virus M protein lacked 50 amino acids at the amino terminal and had 15 extra amino acids at the carboxyl end when compared with the MV M protein.

Amino Acid Sequence↗
PubMed1989

Codon reassignment (codon capture) in evolution.

The genetic code, once thought to be "frozen," shows variations from the universal code. Variations are found in mitochondria, Mycoplasma, and ciliated protozoa. The variations result from reassignment of codons, especially stop codons. The reassignments take place by disappearance of a codon from coding sequences, followed by its reappearance in a new role. Simultaneously, a changed anticodon must appear. We discuss the role of directional mutation pressure in the events, and we also describe the possibility that such events have taken place during early evolution of the genetic code and can occur during its present evolution.

Animals↗
PubMed2003

Analysis of dystrophin mRNA from skeletal muscle but not from lymphocytes led to identification of a novel nonsense mutation in a carrier of Duchenne muscular dystrophy.

Dystrophin mRNA expressed in peripheral lymphocytes of individuals with X-linked Duchenne muscular dystrophy (DMD) has been used as a source material for mutation analysis. Here we present the first report of failure of isolation of nonsense dystrophin mRNA in lymphocytes but success in skeletal muscle in a female carrier of DMD. The mutation responsible for dystrophin-negative muscle fibers of the carrier was analysed by direct sequencing of the reverse transcription PCR product of dystrophin mRNA. In her peripheral lymphocytes, no nucleotide change was detected in the 14 kb long mRNA. Remarkably, a novel nucleotide change of C1682T in exon 12, changing glutamine codon to stop codon (Q492X) was found to be present in her skeletal muscle. This change was heterozygous. Analysis of her genomic DNA disclosed heterozygous C and T nucleotides at nt 1682, confirming the genomic origin of the nonsense mutation. Although dystrophin cDNA prepared from lymphocytes was sequenced again after subcloning, mutation-retaining clone could not be isolated. This lymphocyte-specific disappearance of nonsense mRNA strongly suggested tissue-specific skewing of X-inactivation. However, both paternal and maternal dystrophin alleles were shown to be equally expressed in lymphocytes as well as in muscle, indicating no skewing of X-inactivation in lymphocytes. We concluded that the dystrophin mRNA of the DMD carrier was destabilized in lymphocytes. Our results indicated that analysis of mRNA in lymphocytes is not enough for exact carrier diagnosis of Duchenne muscular dystrophy.

Adult↗
PubMed1996

Tissue-specific alternative splicing of mouse brain type ryanodine receptor/calcium release channel mRNA.

We detected alternative splicing of the mouse brain type ryanodine receptor (RyR3) mRNA. The splicing variant was located in the transmembrane segment. The non-splicing type (RyR3-II) included a stretch of 341 bp, and that of the 13th codon was stop codon TAA. Reverse transcription-polymerase chain reaction (RT-PCR) analysis shows that RyR3-II mRNA was expressed in various peripheral tissues and brain at all developmental stages. However, interestingly, the splicing type (RyR3-I) mRNA was detected only in the cerebrum. These findings suggest that the splicing variants RyR3-I and RyR3-II may generate functional differences of RyR3 in a tissue-specific manner.

Aging↗
PubMed1988

Expression of rubella virus cDNA coding for the structural proteins.

A cDNA clone encoding the precursor polypeptide (Mr 115,000) to the nucleocapsid C (Mr 30,000) and two envelope glycoproteins E1 (Mr 58,000) and E2 (Mr 42,000-47,000) of rubella virus was inserted into a simian virus 40-derived eukaryotic expression vector. When the plasmid was introduced into COS cells, three proteins were synthesized. The expressed proteins were antigenically similar and identical in size to the authentic structural proteins of rubella virus. Expression in the presence of tunicamycin confirmed that E1 and E2 are glycoproteins. Unglycosylated E1 and E2 had Mrs of about 53,000 and 30,000, respectively. The mobility of the nucleocapsid protein was unaffected by tunicamycin. The locations of the translation start and stop codons for synthesis of the precursor to the structural proteins of rubella virus were determined by in vitro and in vivo expression studies. It was found that the first AUG codon at the 5' end of the rubella virus 24S cDNA acts as a start codon for translation. The stop codon was found to be 3183 bp from the start codon.

Animals↗
PubMed1991

Chicken poly(ADP-ribose) synthetase: complete deduced amino acid sequence and comparison with mammalian enzyme sequences.

The complete nucleotide (nt) sequence of the cDNA encoding the chicken poly(ADP-ribose) synthetase has been determined. Positive clones overlapping the 5' region or the 3' region of the cDNA have been isolated from a lambda gt 10 hen oviduct cDNA library using two human cDNA probes. The missing middle portion has been obtained by the polymerase chain reaction procedure. A single 3033-nt open reading frame from start codon to stop codon encodes a sequence of 1011 amino acid residues. The alignment of this sequence with those from human and mouse reveals overall identities of 79% and 77%, respectively. However, an identity of about 82% is obtained in the DNA-binding domain within the two zinc fingers, and an even higher similarity (85-87%) is observed in the NAD-binding domain. The isolated clones consistently hybridize on chicken Northern blots to an mRNA species of about 4 kb, whereas they do not cross-hybridize with RNA blots of Drosophila melanogaster. Thus, it appears that, even if the functional properties of the enzyme are maintained, the cDNA identity will be much decreased in nonvertebrate organisms.

Amino Acid Sequence↗
PubMed2002

Two new molecular bases for the Dombrock null phenotype.

Red blood cells (RBCs) with the Do(null) phenotype lack all antigens in the Dombrock blood group system, i.e. Do(a), Do(b), Gy(a), Hy and Jo(a). Sequence analysis of DNA from one proband with the Do(null) phenotype revealed a single nucleotide mutation of t to c in the donor splice site of DO (IVS1 + 2t > c), with outsplicing of exon 2. Analysis of a second proband revealed a homozygous nonsense mutation 442 C > T in exon 2 predicting a premature stop codon (Gln148 Stop). The molecular bases described in these two probands provide an explanation for the lack of Do glycoprotein on their RBCs.

Base Sequence↗
PubMed2000

A decamer duplication in the 3' region of the BRI gene originates an amyloid peptide that is associated with dementia in a Danish kindred.

Familial Danish dementia (FDD), also known as heredopathia ophthalmo-oto-encephalica, is an autosomal dominant disorder characterized by cataracts, deafness, progressive ataxia, and dementia. Neuropathological findings include severe widespread cerebral amyloid angiopathy, hippocampal plaques, and neurofibrillary tangles, similar to Alzheimer's disease. N-terminal sequence analysis of isolated leptomeningeal amyloid fibrils revealed homology to ABri, the peptide originated by a point mutation at the stop codon of gene BRI in familial British dementia. Molecular genetic analysis of the BRI gene in the Danish kindred showed a different defect, namely the presence of a 10-nt duplication (795-796insTTTAATTTGT) between codons 265 and 266, one codon before the normal stop codon 267. The decamer duplication mutation produces a frame-shift in the BRI sequence generating a larger-than-normal precursor protein, of which the amyloid subunit (designated ADan) comprises the last 34 C-terminal amino acids. This de novo-created amyloidogenic peptide, associated with a genetic defect in the Danish kindred, stresses the importance of amyloid formation as a causative factor in neurodegeneration and dementia.

3' Untranslated Regions↗
PubMed1997

Initiation codon scanthrough versus termination codon readthrough demonstrates strong potential for major histocompatibility complex class I-restricted cryptic epitope expression.

Accumulating evidence shows that the repertoire of major histocompatibility complex class I-restricted epitopes extends beyond conventional translation reading frames. Previously, we reported that scanthrough translation, where the initiating AUG of a primary open reading frame is bypassed, is most likely to account for the presentation of cryptic epitopes from alternative reading frames within the influenza A PR/8/34 nucleoprotein gene. Here, we confirm and extend these findings using an epitope cassette construct that features two well-defined CD8(+) T cell (TCD8+) epitopes in alternative reading frames, each preceded by a single start codon. Expression of one epitope depends on scanning of the ribosome over the first AUG with translation initiation occurring at the second AUG. We find that scanthrough translation has great potency in our system, with its impact being modulated, as predicted, by the base composition surrounding the first initiation codon, the number of start codons preceding the point of alternate reading frame initiation, and the efficiency with which the epitope itself is generated. Additionally, we investigated the efficiency of eukaryotic translation termination codons, to assess codon readthrough as a mechanism for cryptic epitope expression from 3' untranslated regions. In contrast with initiation codons, eukaryotic stop codons appear to be highly efficient at preventing expression of epitopes encoded in 3' untranslated regions, suggesting that 3' untranslated regions are not a common source of cryptic epitope substrate. We conclude that scanthrough is a powerful mechanism for the expression of epitopes encoded in upstream alternative open reading frames that may contribute significantly to TCD8+ responses and to tolerance induction.

Animals↗
PubMed2006

A periodic pattern of mRNA secondary structure created by the genetic code.

Single-stranded mRNA molecules form secondary structures through complementary self-interactions. Several hypotheses have been proposed on the relationship between the nucleotide sequence, encoded amino acid sequence and mRNA secondary structure. We performed the first transcriptome-wide in silico analysis of the human and mouse mRNA foldings and found a pronounced periodic pattern of nucleotide involvement in mRNA secondary structure. We show that this pattern is created by the structure of the genetic code, and the dinucleotide relative abundances are important for the maintenance of mRNA secondary structure. Although synonymous codon usage contributes to this pattern, it is intrinsic to the structure of the genetic code and manifests itself even in the absence of synonymous codon usage bias at the 4-fold degenerate sites. While all codon sites are important for the maintenance of mRNA secondary structure, degeneracy of the code allows regulation of stability and periodicity of mRNA secondary structure. We demonstrate that the third degenerate codon sites contribute most strongly to mRNA stability. These results convincingly support the hypothesis that redundancies in the genetic code allow transcripts to satisfy requirements for both protein structure and RNA structure. Our data show that selection may be operating on synonymous codons to maintain a more stable and ordered mRNA secondary structure, which is likely to be important for transcript stability and translation. We also demonstrate that functional domains of the mRNA [5'-untranslated region (5'-UTR), CDS and 3'-UTR] preferentially fold onto themselves, while the start codon and stop codon regions are characterized by relaxed secondary structures, which may facilitate initiation and termination of translation.

3' Untranslated Regions↗
PubMed2004

Combinatorial codon-based amino acid substitutions.

Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time. A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 beta-lactamase. Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols. This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues. Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides. Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the beta-lactamase omega loop region.

Amino Acid Substitution↗
PubMed1996

A second nonsecretor allele of the blood group alpha(1,2)fucosyl-transferase gene (FUT2).

While screening Le(a+b+)Polynesian DNA samples for a candidate Se(w) allele, a point mutation (C571-->T) resulting in a new stop codon (Arg191-->stop) in the alpha(1,2)fucosyltransferase gene (FUT2) was identified. This point mutation resulted in the gaining of a new restriction enzyme cleavage site (DdeI), which allowed restriction enzyme cleavage screening of 40 selected Polynesians and 42 random Caucasians. The nonsecretor phenotype in two of the three nonsecretor Polynesians analyzed was due to homozygosity for the 'new' mutation, whereas the third Polynesian nonsecretor (with Caucasian ancestors) was due to homozygosity of the 'old' (Trp143-->stop) mutation. The nonsecretor phenotype in all Caucasians analyzed was a consequence of homozygosity for the 'old' mutation. Both the new and the old nonsecretor mutations were identified in the heterozygous state in some secretor-positive Polynesians, while only the old mutation was found in the heterozygous state in Caucasians of the same phenotype.

Alleles↗
PubMed2003

Human papillomavirus type 31 E5 protein supports cell cycle progression and activates late viral functions upon epithelial differentiation.

The function of the E5 protein of human papillomaviruses (HPV) is not well characterized, and controversies exist about its role in the viral life cycle. To determine the function of E5 within the life cycle of HPV type 31 (HPV31) we first constructed HPV31 mutant genomes that contained an altered AUG initiation codon or stop codons in E5. Cell lines were established which harbored transfected wild-type or E5 mutant HPV31 genomes. These cell lines all maintained episomal copies of HPV31 and revealed similar phenotypes with respect to growth rate, early gene expression, and viral copy number in undifferentiated monolayer cultures. Following epithelial differentiation, genome amplification and differentiation-dependent late gene expression were observed in mutant cell lines, but at a rate significantly reduced from that observed in cells containing the wild-type genomes. Organotypic raft cultures indicated that E5 does not effect the expression of differentiation markers but does reduce expression of late viral proteins. Western analysis and immunofluorescence staining for cyclins during epithelial differentiation revealed a decreased expression of cyclin A and B in E5 mutant cells compared to HPV wild-type cells. Using a replating assay, a significant reduction in colony-forming ability was detected in the absence of E5 expression when cells containing wild-type or E5 mutant HPV genomes were allowed to proliferate following 24 h in suspension-induced differentiation. This suggests that HPV E5 modifies the differentiation-induced cell cycle exit and supports the ability of HPV31-positive keratinocytes to retain proliferative competence. In these studies, E5 was found to have little effect on the levels of the epidermal growth factor receptor (EGFR) or on its phosphorylation status. This indicates that EGFR is not a target of E5 action. Our results propose a role for high risk HPV E5 in modulation of late viral functions through activation of proliferative capacity in differentiated cells. We suspect that the primary target of E5 is a membrane protein or receptor that then acts to alter the levels or activities of cell cycle regulators.

Cell Cycle↗
PubMed2004

Molecular cloning of goat 20alpha-hydroxysteroid dehydrogenase cDNA.

20Alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which catalyzes the conversion of progesterone to its inactive form 20alpha-dihydroprogesterone, is expressed in murine placenta and has been suggested to play roles in maintaining pregnancy. To understand the role of 20alpha-HSD during pregnancy in the goat, as a first step, cloning and sequencing of 20alpha-HSD cDNA were performed. The full nucleotide sequence of 20alpha-HSD cDNA was determined on samples obtained from the corpus luteum at the luteal phase of the estrous cycle and the placenta in late pregnancy by RT-PCR and 3' and 5' RACE systems. Cloned 20alpha-HSD cDNA consisted of 1124 bp and belonged to the aldo-keto reductase superfamily. From the start codon to stop codon there were 323 amino acids, the same as in other species. To verify whether the protein derived from goat 20alpha-HSD cDNA had 20alpha-HSD activity, the cDNA was expressed by bacteria. Bacterially expressed goat 20alpha-HSD protein showed 20alpha-HSD enzyme activity. A tissue distribution study demonstrated that 20alpha-HSD was expressed in the placenta, but not in the adrenal gland, liver and spleen during pregnancy. The present study suggests that goat 20alpha-HSD is another member of the aldo-keto reductase superfamily and that it plays a role in the placenta during pregnancy.

20-alpha-Hydroxysteroid Dehydrogenase↗
PubMed2006

Extensive RNA editing in transcripts from the PsbB operon and RpoA gene of plastids from the enigmatic moss Takakia lepidozioides.

RNA editing is a post-transcriptional process that changes individual nucleotides in transcripts, and usually occurs in the plastids of land plants. The number of RNA editing sites in a plastid is significantly divergent in bryophytes, ranging from zero in liverworts to almost 1,000 sites in hornworts. In this study, we identified 132 RNA editing sites in the transcripts of six genes from the psbB operon and the rpoA of the moss Takakia lepidozioides. This is the highest number of RNA editing sites known in this region among land plant species. All were cytidine-to-uridine conversions. More than 91% of RNA editing occurred at the first or second codon positions, and it altered amino acid identity. Six editing sites created new translation initiation codons or stop codons. Thirty-two sites were commonly observed in the hornwort Anthoceros angustus. This finding suggests that the enigmatic bryophyte Takakia is closely related to hornworts with respect to RNA editing events.

Base Sequence↗
PubMed2004

[Molecular cloning and analysis of a monocot mannose-binding agglutinin from Zephyranthes grandiflora (family Amaryllidaceae)].

The monocot mannose-binding lectin can inhibit HIV from infecting the target cells. The total RNA of Zephyranthes grandiflora was extracted and reversely transcribed into cDNA. Degenerate primers were designed based on the conserved regions of other monocot mannose-binding agglutinins by homology alignment. The 694bp full-length cDNA of Zephyranthes grandiflora agglutinin (ZGA) was cloned by RT-PCR, 3' and 5' RACE (rapid amplification of cDNA ends). The start codon and stop codon of ZGA were at 37-39bp and 529-531bp respectively. The NCBI Blast analysis result showed that ZGA gene encoded a protein precursor with signal peptide, mature protein and C-terminal cleavage sequence. The mature ZGA protein contained 106 amino acids residues and its molecular weight was 11.6KD. The percentages of identity of the deduced mature ZGA protein with those of Galanthus nivalis agglutinin, Narcissus hybrid cultivar agglutinin, Lycoris radiate agglutinin and Clivia miniata agglutinin were 71.8%, 81%, 81.8% and 84.5%, respectively. Blocks analysis revealed that ZGA had three functional domains and three mannose-binding boxes (QDNY).

Agglutinins↗
PubMed1995

Four new mutations in the NADH-cytochrome b5 reductase gene from patients with recessive congenital methemoglobinemia type II.

Recessive congenital methemoglobinemia (RCM) due to NADH-cytochrome b5 reductase (cytb5r) deficiency leads to two different types of diseases. In the type I form, cyanosis is the only symptom, and the soluble enzyme is defective in red blood cells. In the type II form, cyanosis is associated with severe mental retardation and neurologic impairment; the enzymatic defect is systemic, involving both soluble and membrane-bound isoforms. We characterized mutations responsible for cytb5r deficiency in three unrelated patients with severe RCM type II. The first patient presented a homozygous exon 5 skipping. The only mutation detected was a homozygous G to C transversion at position +8, downstream from the 5' splice site of exon 5. We suggest that this unusual mutation might be responsible for the abnormal splicing of the primary transcripts, resulting in frameshift with premature STOP codon. The second mutation found corresponds to a homozygous C to T transition changing the Arg-218 codon to a premature STOP codon in exon 8. The third case was a compound heterozygote, carrying two different mutant alleles in the cyb5r gene. One allele presented a missense mutation with replacement of Cys-203 (TGC) by Arg (CGC) in exon 7. The second allele carried a 3-bp deletion (TGA) of nucleotides 815 to 817, modifying two contiguous codons in exon 9 of the cDNA with loss of Met-272. These results confirm the genetic polymorphism of cytb5r gene mutations identified in RCM type II, as observed for the mutations described in the RCM type I, and shed light on the molecular bases of the two different diseases associated with cytb5r deficiency.

Amino Acid Sequence↗