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PubMed1986

Amino acid sequence of a chicken heat shock protein derived from the complementary DNA nucleotide sequence.

The complete nucleotide sequence for a chicken heat shock protein (hsp108) was determined from cDNA clones isolated from hen oviduct and bursal lymphoma recombinant DNA libraries. This protein has certain biochemical similarities to the progesterone receptor, but it is clearly distinct from it. The initial cDNA clone, isolated from a chicken oviduct cDNA library, was detected by antibody screening and hybrid-selected translation [Zarucki-Schulz, T., Kulomaa, M. S., Headon, D. R., Weigel, N. L., Baez, M., Edwards, D. P., McGuire, W. L., Schrader, W. T., & O'Malley, B. W. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6358-6362]. The earlier clones were used to screen for additional cDNAs, and cDNAs that define the entire mRNA sequence of hsp108 have been obtained. The nucleotide sequence codes for peptides present in hsp108 as determined by protein microsequencing. The 5' end of the mRNA was determined by primer extension studies. The mRNA contains a noncoding region of 101 nucleotides upstream from the predicted initiation codon. The 3' untranslated region contains 244 nucleotides beyond the termination codon, and it contains a predicted polyadenylation signal 26 nucleotides from the end of the complete cDNA. The coding region of 2385 nucleotides corresponds to a polypeptide chain of 795 amino acids, giving a molecular weight of 91,555 for the hsp108 protein. In another paper, evidence is presented that hsp108 shows a high degree of amino acid sequence homology with two heat shock proteins, hsp90 (yeast) and hsp83 (Drosophila), and is indeed inducible by heat shock [Sargan, D. R., Tsai, M.-J., & O'Malley, B. W. (1986) Biochemistry (following paper in this issue)].

Amino Acid Sequence↗

PubMed1998

The upf3 protein is a component of the surveillance complex that monitors both translation and mRNA turnover and affects viral propagation.

The nonsense-mediated mRNA decay pathway functions to degrade aberrant mRNAs that contain premature translation termination codons. In Saccharomyces cerevisiae, the Upf1, Upf2, and Upf3 proteins have been identified as trans-acting factors involved in this pathway. Recent results have demonstrated that the Upf proteins may also be involved in maintaining the fidelity of several aspects of the translation process. Certain mutations in the UPF1 gene have been shown to affect the efficiency of translation termination at nonsense codons and/or the process of programmed -1 ribosomal frameshifting used by viruses to control their gene expression. Alteration of programmed frameshift efficiencies can affect virus assembly leading to reduced viral titers or elimination of the virus. Here we present evidence that the Upf3 protein also functions to regulate programmed -1 frameshift efficiency. A upf3-Delta strain demonstrates increased sensitivity to the antibiotic paromomycin and increased programmed -1 ribosomal frameshift efficiency resulting in loss of the M1 virus. Based on these observations, we hypothesize that the Upf proteins are part of a surveillance complex that functions to monitor translational fidelity and mRNA turnover.

Alleles↗

PubMed1986

Analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin.

Site-directed oligonucleotide mutagenesis has been used to introduce chain termination codons into the cloned DNA sequences encoding the carboxy-terminal transmembrane (27 amino acids) and cytoplasmic (10 amino acids) domains of influenza virus hemagglutinin (HA). Four mutant genes were constructed which express truncated forms of HA that lack the cytoplasmic domain and terminate at amino acids 9, 14, 17, or 27 of the wild-type hydrophobic domain. Analysis of the biosynthesis and intracellular transport of these mutants shows that the cytoplasmic tail is not needed for the efficient transport of HA to the cell surface; the stop-transfer sequences are located in the hydrophobic domain; 17 hydrophobic amino acids are sufficient to anchor HA stably in the membrane; and mutant proteins with truncated hydrophobic domains show drastic alterations in transport, membrane association, and stability.

Amino Acid Sequence↗

PubMed1975

Sequence of the promoter-operator proximal region of the major leftward RNA of bacteriophage lambda.

The sequence of the first 149 nucleotides of the major leftward RNA of bacteriophage lambda has been determined. Preliminary sequence information was also obtained for a portion of the untranscribed area immediately upstream of the point on the template when RNA synthesis normally starts. Several restriction endonuclease sites, deletion endpoints, and single base changes have been localized within the sequence. The first potential translation initiation codon which is not followed by an in-phase termination codon is a GUG located 90 nucleotides from the transcription startpoint.

Base Sequence↗

PubMed1986

Nucleotide sequence of a portion of the Autographa californica nuclear polyhedrosis virus genome containing the EcoRI site-rich region (hr5) and an open reading frame just 5' of the p10 gene.

The nucleotide sequence of a 1587 bp region lying within the HindIII-Q fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) DNA has been determined. It begins in the EcoRI-S-EcoRI-X region, continues to the HindIII-P/Q boundary and contains an open reading frame that codes for a polypeptide of 240 amino acids (p26). This open reading frame is also included in the 1100 and 1500 base transcripts previously mapped to this region. The sequence reveals that the 5' ends of the 1100 and 1500 base transcripts are located 20 bp downstream from the end of a putative TATA box (TAATTAAAT) and 19 bp upstream from the translation start codon (ATG) of the p26 open reading frame. The translation termination codon (TAA) falls in the immediate 5' flanking region of the major late p10 gene of AcMNPV, 3 bp downstream from the putative TATA box. The probable polyadenylation site for the 1100 base transcript lies 23 bp downstream from the cap site for the 750 and 2500 base transcripts encoding the p10 protein. The 5' flanking region of the p26 open reading frame contains the EcoRI site-rich region, hr5, whose sequence is included here. The EcoRI site-rich region, hr5, consists of six imperfect tandem repeats of a sequence that includes the EcoRI recognition site. These direct repeats also include many inverted repeats.

Amino Acid Sequence↗

PubMed1996

Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824.

A 2.8-kbp DNA region of Clostridium acetobutylicum ATCC 824 containing the putative hydrogenase gene (hydA) was cloned and sequenced. The 1,745-bp hydA encodes a 64,415-Da protein and presents strong identity with the [Fe] hydrogenase genes of Desulfovibrio and Clostridium species. The level of the putative hydA mRNA was high in cells from an acidogenic or an alcohologenic phosphate-limited continuous culture, while it was comparatively very low in cells from a solventogenic phosphate-limited continuous culture. These results were in agreement with the hydrogenase protein level, indicating that expression of hydA is regulated at the transcriptional level. Primer extension analysis identified a major transcriptional start site 90 bp upstream of the hydA start codon. The position of a putative rho-independent transcription terminator immediately downstream of the termination codon is in agreement with the size of the hydA transcript (1.9 kb) determined by Northern (RNA) blot experiments and confirms that the gene is transcribed as a monocistronic operon. Two truncated open reading frames (ORFs) were identified downstream and upstream of hydA and in opposite directions. The amino acid sequence deduced from ORF2 presents strong identity with ortho phosphoribosyl transferases involved in pyrimidine synthesis. The amino acid sequence deduced from ORF3 presents no significant similarity to any sequence in various available databases.

Amino Acid Sequence↗

PubMed1989

Ribosomal protein gene cluster of Halobacterium halobium: nucleotide sequence of the genes coding for S3 and L29 equivalent ribosomal proteins.

A 1643 base pair fragment encoding the S3 and L29 equivalent ribosomal proteins has been sequenced from the archaebacterium Halobacterium halobium. The incomplete open reading frame present upstream from the S3 gene encodes a protein homologous to the eubacterial ribosomal protein L22. The initiation codons of the S3 and L29 genes overlap with the termination codons of the upstream genes. A tight physical organization suggests that these genes are transcribed as a polycistronic operon. Peculiarities of the protein structure and gene organization are discussed.

Amino Acid Sequence↗

PubMed1988

Structure, expression and alternative splicing of the human c-ets-1 proto-oncogene.

The avian acute leukemia virus E26 induces a mixed erythroid-myeloid leukemia in chicken and carries two distinct oncogenes, v-myb and v-ets. It was shown that there exist two ets loci in humans, c-ets-1 and c-ets-2, which were mapped to human chromosome 11 and 21, respectively. The human c-ets-1 proto-oncogene locus was shown to translocate from chromosome 11 to 9 or 4 in certain patients with acute monocytic leukemia. In order to understand the functional role of c-ets-1 proto-oncogene, we have made a cDNA library from MOLT-4 cells and isolated seven c-ets-1 positive cDNA clones. Analysis of two cDNA clones revealed a complete open reading frame of the c-ets-1 proto-oncogene. Except for 81 nucleotides at the 5'-end (downstream of initiation codon) and 39 nucleotides at the 3'-end (upstream of termination codon), the c-ets-1 cDNA is approximately 82% homologous to v-ets oncogene sequences and encodes a 441-residue protein (c-ets-1a) which contains contiguous 5' and 3' regions of the v-ets oncogene as in the case of chicken. Sequence analysis also revealed alternative splicing, giving rise to another related c-ets-1 polypeptide (c-ets-1b) lacking 87 amino acids. Our results suggest that differential RNA splicing allows the synthesis of at least two related c-ets-1 polypeptides from a single c-ets-1 proto-oncogene.

Amino Acid Sequence↗

PubMed1995

Cerebrin-50, a human cerebrospinal fluid protein whose mRNA is present in multiple tissues but predominantly expressed in the lymphoblastoid cells and the brain.

A full-length cDNA of 2295 bp coding for a human cerebrospinal fluid protein, designated cerebrin-50, has been isolated from a human brain cDNA expression library. Nucleotide sequence analysis of this cDNA revealed that it codes for a 435 amino acid polypeptide which starts with an ATG initiation codon from the 5'-end and ends with a TGA termination codon with a calculated molecular weight of 51,484 daltons. A 10-amino acid peptide of NH2-SGDLETRYWG based on the deduced amino acid sequence of the cerebrin-50 cDNA was synthesized, conjugated to bovine serum albumin and was used to raise a monospecific polyclonal antibody in a rabbit. Immunoblot analysis using this antibody indicated the presence of a 52 kD protein in the human cerebrospinal fluid, brain cytosol, T lymphoblastoid cell-conditioned medium, and human serum; it is also noted that the concentration of this 52 kD protein is several order of magnitude higher in the cerebrospinal fluid than in other biological fluids. The presence of its mRNA in the brain, T lymphoblastoid cells, spleen, liver, and testis was confirmed by sequential use of reverse-transcription and polymerase chain reaction (RT-PCR). Preliminary analysis by quantitative RT-PCR has noted that the expression of the cerebrin-50 mRNA is higher in the lymphoblastoid cells and brain than the spleen, liver, and testis.

Amino Acid Sequence↗

PubMed1986

Nucleotide sequence corresponding to five chemotaxis genes in Escherichia coli.

The nucleotide sequence of DNA which contains five chemotaxis-related genes of Escherichia coli, cheW, cheR, cheB, cheY, and cheZ, and part of the cheA gene was determined. Molecular weights of the polypeptides encoded by these genes were calculated from translated amino acid sequences, and they were 18,100 for cheW, 32,700 for cheR, 37,500 for cheB, 14,100 for cheY, and 24,000 for cheZ. Nucleotide sequences which could act as ribosome-binding sites were found in the upstream region of each gene. After the termination codon of the cheW gene, a typical rho-independent transcription termination signal was observed. There are no other open reading frames long enough to encode polypeptides in this region except those which code for the two previously reported genes tar and tap.

Base Sequence↗

PubMed1994

Expression of modified human cytochrome P450 1A1 in Escherichia coli: effects of 5' substitution, stabilization, purification, spectral characterization, and catalytic properties.

Human cytochrome P450 (P450) 1A1 is primarily an extrahepatic enzyme and is important because of its roles in the activation of polycyclic hydrocarbons and other xenobiotic chemicals. Purification of active enzyme from human tissues has not been successful. We report the expression and purification of the recombinant enzyme from Escherichia coli. A full-length cDNA of human cytochrome P450 1A1 and several modified constructs were engineered into a pCW vector and used to transform E. coli cells. Little expression was observed with the native sequence and several modified constructs, but successful expression (20-25 nmol membrane-bound P450 1A1 per liter of culture) was achieved with a construct in which the Ala codon GCT was placed in the second position and the 5'-terminal codons were maximized for AT content and minimized for the potential of secondary structure formation of the mRNA transcript. alpha-Naphthoflavone was found to protect against denaturation by detergents during solubilization and was added to buffers used for purification. The recombinant P450 1A1 was purified to electrophoretic homogeneity after two ion-exchange chromatography steps in approximately 50% yield. N-Terminal amino acid sequence analysis verified the expected first 21 residues, with the exception of the terminal Met. The isolated human ferric P450 1A1 was predominantly in the high spin state, in contrast to the orthologous rat and rabbit enzymes. Recombinant P450 1A1 catalyzed 7-ethoxyresorufin O-deethylation and benzo[a]pyrene 3-hydroxylation with Km values of 0.58 and 15 microM and Vmax values of 8.3 and 2.5 nmol min-1 (nmol P450 1A1)-1, respectively. The successful expression and purification of human P450 1A1 should increase the availability of this enzyme and the generation of antibodies for further biochemical and other biological studies.

Amino Acid Sequence↗

PubMed1993

A programmed translational frameshift is required for the synthesis of a bacteriophage lambda tail assembly protein.

Two proteins, one of 31 kDa and one of 16 kDa, are encoded by a segment of the phage lambda tail gene region that contains two overlapping reading frames, neither of which is long enough to encode the larger protein. We show that the abundant 16-kDa protein (gpG) is encoded by the upstream open reading frame, gene G. The 31-kDa protein, gpG-T, is encoded jointly by gene G and the overlapping downstream T open reading frame. gpG-T is synthesized as the result of a translational frameshift that occurs when a ribosome translating the G gene slips back by one nucleotide at a position six codons from the C terminus of the gene and thereby bypasses the G termination codon to continue on in the T open reading frame. The resulting protein shares 135 residues of N-terminal amino acid sequence with gpG, followed by 144 amino acid residues of unique sequence. The frameshift event occurs with a frequency of approximately 4% at the sequence G GGA AAG, which encodes the dipeptide -Gly-Lys- in both the zero and -1 reading frames. The frameshift frequencies of point mutants in this "slippery sequence" argue that codon-anticodon interactions with both the glycyl and the lysyl-tRNA are important for frameshifting to occur. We find no clear evidence for a pausing mechanism to enhance frameshifting, as is seen in other well-characterized frameshifts. No simple secondary structure has been predicted for the region downstream from the slippery sequence, but this downstream sequence does contribute to the frameshifting rate. Our results together with those of Katsura and Kühl show that the frameshift product, gpG-T, has an essential role in lambda tail assembly, acting prior to tail shaft assembly. The role of gpG in tail assembly is not known. We find that both gpG and the gpG-T are absent from mature virions.

Amino Acid Sequence↗

PubMed1991

ApoB gene nonsense and splicing mutations in a compound heterozygote for familial hypobetalipoproteinemia.

Two novel apoB gene mutations were identified in a patient (CM) with phenotypic homozygous hypobetalipoproteinemia. Haplotype analysis of the apoB alleles from this patient and his family members revealed him to be a genetic compound for the disease. In contrast to previous studies of other hypobetalipoproteinemic patients, no clues existed as to where in the apoB gene the molecular defects resided. Therefore, it was necessary to characterize the apoB genes of the patient by sequence analysis. The apoB gene contains 29 exons and is 43 kb in length. The gene encodes a 14.1 kb mRNA and a 4563 amino acid protein. Both apoB alleles from the patient were cloned via 26 sets of polymerase chain reactions (PCR). These clones contained a total of approximately 24 kb of apoB gene sequence, including regions 5' and 3' to the coding region, 29 exons, and the intron/exon junctions. Complete DNA sequence analysis of these clones showed that each apoB allele had a mutation. In the paternal apoB allele, there was a splicing mutation. The first base of the dinucleotide consensus sequence (GT) in the 5' splice donor site in intron 5 was replaced by a T. It is likely that this base substitution interferes with proper splicing and results in the observed absence of plasma apoB. In the maternal apoB allele, there was a nonsense mutation. The first base of the Arg codon (CGA) at residue 412 in exon 10 was replaced by a T, resulting in a termination codon (TGA). The nonsense mutation is likely to terminate translation after residue 411 resulting in a severely truncated protein only 9% of the length of B-100.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMedDate not supplied

Initiation of translation at internal AUG codons in mammalian cells.

Initiation of translation of eukaryotic mRNAs typically occurs at the first AUG triplet from the 5' end of the message, although several notable exceptions have been described. Using vectors which efficiently express the gene encoding the surface antigen of hepatitis B virus in monkey cells, we have studied the consequences of inserting ATG triplets in all three reading frames upstream of the usual translational initiation codon of this gene. In agreement with the scanning model for eukaryotic translation initiation, these additional codons can severely depress the initiation of translation at the 'authentic' start codon, although the extent of inhibition depends on sequences flanking the upstream AUG. Such inhibition can, however, be at least partially suppressed by the presence of a translation termination codon in-frame with the upstream AUG. These results raise the possibility that mammalian ribosomes can reinitiate translation at an AUG codon after previously initiating, and terminating, at an upstream site.

Animals↗

PubMed1980

The nucleotide sequence of the cloned tufA gene of Escherichia coli.

The 4 kb (8.5 % lambda units) EcoRI fragment harboring the tufA gene of Escherichia coli was cloned using plasmid pTUA1 (Shibuya et al., 1979) and its structure was analyzed. The nucleotide sequence of about 1500 base pairs, covering the C-terminal portion of elongation factor EF-G (fus gene), the intercistronic region between fus and tufA, the entire structural gene for tufA with the GUG initiation and UAA termination codons, and the 3' flanking region of tufA, was determined. Comparison of the tufA nucleotide sequence with the tufB sequence (An and Friesen, 1980) and the known amino acid sequence of EF-Tu (Arai et al., 1980) revealed that the products of genes tufA and tufB are identical except for one amino acid at the C-terminal, i.e., glycine for tufA and serine for tufB. Nucleotide differences between tufA and tufB were found at 13 positions. Among them, one in the initiation codon and the other one in the C-terminal amino acid codon had replacements at the first letter of the codons. The other eleven changes were in the third codon positions, which did not affect the amino acid coding. The pattern of codon usage in tufA and tufB is highly nonrandom, and remarkably similar to that in ribosomal protein genes, with the codons for the most abundant species of isoaccepting tRNAs being preferentially utilized (Post et al., 1979; Post and Nomura, 1980).

Bacterial Proteins↗

PubMed1996

Factor XIII(A) subunit deficiency due to a homozygous 13-base pair deletion in exon 3 of the A subunit gene.

We investigated the molecular basis of factor XIII(A) subunit deficiency in a Greek family. Each of the 15 exons of the A subunit gene were individually amplified by polymerase chain reaction, using previously reported oligoprimers. The proband with severe deficiency was found to have a homozygous 13-base pair deletion in the 3' half of exon 3. The deleted sequence, extending from codons 82-86, results in a frameshift and generates a downstream termination codon in exon 4. Single strand conformation polymorphism (SSCP) analysis detected no additional mutations in the coding or consensus splice sequences of the A subunit gene. Both parents of the proband were heterozygous for the defect. Only one previous microdeletion (AG dinucleotide) has been reported in the A subunit gene, and was located at the intron B-exon 3 boundary. Further studies are necessary to determine whether this region of the gene is a "hot spot" for microdeletion mutations.

Amino Acid Sequence↗

PubMed1993

Similarity of the VP4 protein of human rotavirus strain 116E to that of the bovine B223 strain.

Rotavirus strain 116E was isolated from the fecal specimen of a newborn infant from New Delhi who had no symptoms of diarrhea. The strain could not be P typed using the PCR method recently developed in our laboratory so we have sequenced the 4th gene segment that encodes the VP4 protein using viral transcript RNA prepared in vitro. This gene is 2353 base pairs in length and codes for a protein 772 amino acids long which begins with a methionine initiation codon at nucleotides 11 to 13 and ends with a single termination codon at nucleotides 2327 to 2329. Northern blot analysis demonstrates that the VP4 protein of this strain is encoded by genome segment 4. This gene is closely related to the VP4 gene of the bovine B223 strain (P type 11) at both the nucleotide (90.8% identity) and amino acid (92.2% identity) levels, but is unique from those of strains from P types 1 to 10. The close relatedness of the VP4 proteins of strains 116E and B223 is demonstrated by the amino acid composition of the potential trypsin cleavage sites and their flanking sequences, the size of the cleavage fragments, and conservation of most cysteine and proline residues. Comparative amino acid analyses of the variable regions thought to be important in VP4 antigenicity are consistent with the hypothesis that strain 116E may represent the first reported human P type 11 strain.

Amino Acid Sequence↗

PubMed1993

Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker.

The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes.

Alleles↗