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PubMed1994

A unique structural feature of rabbit DNA repair methyltransferase as revealed by cDNA cloning.

cDNA encoding rabbit O6-methylguanine-DNA methyltransferase that repairs DNA damaged by alkylating agents was isolated, using as a probe a fragment of mouse cDNA coding for a region containing the active site of the enzyme. The nucleotide sequence of the cDNA revealed that the rabbit methyltransferase is a 181-amino acid polypeptide with a mol. wt of 19,385. Expression of the cDNA in a methyltransferase-deficient Escherichia coli mutant resulted in appearance of a 23 kDa polypeptide with methyltransferase activity, and this rendered the E.coli cells resistant to N-methyl-N'-nitro-N-nitrosoguanidine, in terms of both cell killing and induction of mutation. The rabbit methyltransferase is highly homologous to this enzyme in human, mouse, rat and hamster, but is 26-30 amino acid residues shorter as compared with methyltransferases in other mammalian species. Based on a comparison of the nucleotide sequences for the C-terminal regions of these proteins, we propose that a single base substitution, which would generate a TGA termination codon, was introduced into the sequence for the rabbit enzyme during the process of evolution. Existence of the naturally occurring truncated form of methyltransferase suggests that the longer C-terminal tails of other mammalian methyltransferases may have no significant role in exerting functions of the enzyme in vivo.

Amino Acid Sequence↗

PubMed1977

Structure of human hemoglobin messenger RNA and its relation to hemoglobinopathies.

1. One-fifth to 1/4 of globin mRNA is untranslated sequence other than polyadenylic acid. 2. The untranslated sequences of mRNA vary markedly in their sequence and in their length. 3. Globin mRNAs demonstrate a marked bias in codon selection. 4. Viral mRNA shows a quite different pattern of codon selection; therefore, the selection of codons is not uniform for all mRNAs functioning in animal cells. 5. Elongated hemoglobin chains can be accounted for by frame-shift mutations, or point mutations within the normal termination codon. The additional amino acids are then coded for by sequences that are normally untranslated. 6. Certain hemoglobin deletion mutants occur at sites where there are partially reiterated sequences within the heomoglobin messenger RNA.

Base Sequence↗

PubMed1983

Comparison of the lipoprotein gene among the enterobacteriaceae. DNA sequence of Morganella morganii lipoprotein gene and its expression in Escherichia coli.

A DNA sequence of 532 base pairs encompassing the entire Morganella morganii lipoprotein gene (lpp) was determined. Sequence comparisons of the M. morganii lpp gene with the lpp genes from Escherichia coli, Serratia marcescens, and Erwinia amylovora reveal that the M. morganii lpp gene is more distantly related to the E. coli lpp gene than any of the other lpp genes examined. Between the E. coli and M. morganii lpp genes, the following homologies were found: 44% in the promoter region (bases, -45 to -1), 88% in the 5'-end untranslated region of the mRNA, 58% in the signal sequence coding region, 75% in the coding region for the first 51 and 43% for the last 7 amino acid residues. Upstream of the promoter region and downstream of the termination codon, there are extensive insertions, deletions, and base substitutions. In spite of the differences in the DNA sequences, the lipoprotein structure was found to be highly conserved except for the carboxyl-terminal sequence of 7 amino residues. The coding region of the M. morganii lpp gene including the signal sequence was inserted into an expression cloning vector so that the production of the M. morganii lipoprotein could be induced in E. coli by a lac inducer, isopropyl-beta-D-thioglactoside. It was found that when induced, the M. morganii prolipoprotein was apparently secreted normally across the E. coli cytoplasmic membrane, modified with glycerol and palmitic acid, processed to the mature lipoprotein, and assembled in the E. coli outer membrane. The bound form covalently linked to the peptidoglycan was also found.

Base Sequence↗

PubMed2000

Evolutionarily conserved features of the arginine attenuator peptide provide the necessary requirements for its function in translational regulation.

Neurospora crassa arg-2 mRNA contains an evolutionarily conserved upstream open reading frame (uORF) encoding the Arg attenuator peptide (AAP) that confers negative translational regulation in response to Arg. We examined the regulatory role of the AAP and the RNA encoding it using an N. crassa cell-free translation system. AAPs encoded by uORFs in four fungal mRNAs each conferred negative regulation in response to Arg by causing ribosome stalling at the uORF termination codon. Deleting the AAP non-conserved N terminus did not impair regulation, but deletions extending into the conserved region eliminated it. Introducing many silent mutations into a functional AAP coding region did not eliminate regulation, but a single additional nucleotide change altering the conserved AAP sequence abolished regulation. Therefore, the conserved peptide sequence, but not the mRNA sequence, appeared responsible for regulation. AAP extension at its C terminus resulted in Arg-mediated ribosomal stalling during translational elongation within the extended region and during termination. Comparison of Arg-mediated stalling at a rare or common codon revealed more stalling at the rare codon. These data indicate that the highly evolutionarily conserved peptide core functions within the ribosome to cause stalling; translational events at a potential stall site can influence the extent of stalling there.

Amino Acid Sequence↗

PubMed1997

The human glutaredoxin gene: determination of its organization, transcription start point, and promoter analysis.

A genomic clone for the human glutaredoxin gene was isolated and sequenced. An intron was located within the coding region and began 211 nt downstream of the initiator codon. Except for this intron, the genomic sequence shares 100% identity to the published glutaredoxin cDNA sequence. A second intron was located in the 3' UTR 6 bp downstream of the terminator codon. The tsp of the glutaredoxin gene was determined by primer extension and confirmed by S1 mapping analysis. Analysis of the 5'-flanking region of the gene revealed that the promoter sequences TATA and CCAAT were 30 and 160 bp upstream, respectively, from the tsp. Other potential transcription factor binding sites included NF-E1, HNF-5, P2II and AP-1. Glutaredoxin promoter constructs inserted into a reporter plasmid for firefly luciferase were transfected into fibroblasts, and luciferase activity was 8-10-fold higher compared with controls lacking glutaredoxin promoter. These data indicate that the promoter region of the isolated glutaredoxin gene is functional.

Amino Acid Sequence↗

PubMed2000

Structural and functional studies on the leukemia inhibitory factor receptor (LIF-R): gene and soluble form of LIF-R, and cytoplasmic domain of LIF-R required for differentiation and growth arrest of myeloid leukemic cells.

The leukemia inhibitory factor receptor (LIF-R) subunit is a component of cell-surface receptor complexes for the multifunctional cytokines, LIF, cardiotrophin-1, ciliary neurotrophic factor, and human oncostatin M. The structure of the human LIF-R gene is similar to that of the mouse gene. The transmembrane receptor is encoded by 19 exons. Two distinct 5' non-coding exons are present, indicating the existence of alternative promoters. An extra-exon specific to the mouse soluble receptor contains a stop codon and polyadenylation signals in a B2 repetitive element. On the other hand, LIF-R mRNAs containing unspliced introns are abundantly present in human tissues. These intronic sequences introduce a termination codon before the transmembrane domain. Human choriocarcinoma cells expressing these mRNAs release soluble LIF-R. The cytoplasmic domain of LIF-R can generate the signals for growth arrest and differentiation of mouse myeloid leukemic cells when they are induced to form a homodimer of the cytoplasmic domain independently of gp130. Two membrane-distal tyrosines on the YXXQ motif of LIF-R are critical not only for STAT3 activation but also for growth arrest and macrophage differentiation of WEHI-3B D+ cells.

Amino Acid Motifs↗

PubMed1987

Activities of herpes simplex virus type 1 (HSV-1) ICP4 genes specifying nonsense peptides.

Synthetic oligonucleotide linkers containing translational termination codons in all possible reading frames were inserted at various positions in the cloned gene encoding the herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein, ICP4. It was determined that the amino-terminal 60 percent of the ICP4 gene was sufficient for trans-induction of a thymidine kinase promoter-CAT chimera (pTKCAT) and negative regulation of an ICP4 promoter-CAT chimera (pIE3CAT); however, it was relatively inefficient in complementing an ICP4 deletion mutant. The amino-terminal ninety amino acids do not appear to be required for infectivity as reflected by the replication competence of a mutant virus containing a linker insertion at amino acid 12. The size of the ICP4 molecule expressed from the mutant virus was consistent with translational restart at the next methionine codon corresponding to amino acid 90 of the deduced ICP4 amino acid sequence.

Amino Acid Sequence↗

PubMed1991

Effects of the nucleotide 3' to an amber codon on ribosomal selection rates of suppressor tRNA and release factor-1.

Rates of ribosomal selection of both release factor 1 (RF1) and a suppressor tRNA (Su7C33) were studied at an amber codon at which the 3' neighbor was permuted. Rates of RF1 selection vary 2.6-fold among contexts. The 3' neighbor-dependent variation of RF1 action correlates very strongly with the non-random frequencies of 3' neighbors at UAG terminators (r = 0.97), which argues that the rate of RF1 selection is an important determinant 3' neighbor choice at termination codons. The data are consistent with a model for RF1 selection in which RF1 makes a specific contact(s) to the 3' neighbor and that this interaction is most favorable to uridylic acid. Measured rates of Su7C33 selection vary fivefold among 3' contexts. We also develop a method to calculate rates of selection for other suppressors, based on the assumption that rates of RF1 selection at each 3' context can be generalized to other sites that have the same 3' neighbor. Rates for various suppressors appear to vary from two- to fivefold depending on the 3' neighbor. Generally, the rate of selection of suppressors at different contexts correlates with the stacking strength of the 3' neighbor as measured in vitro. The two- to fivefold range of 3' neighbor effects on rate of aminoacyl-tRNA selection is greater than that previously observed within sets of codons read by the same tRNA. It is suggested that the choice of codons to achieve favorable contexts may be more important than the choice of a common codon at some message sites.

Base Sequence↗

PubMed1992

cDNA sequence of the small subunit of the hamster ribonucleotide reductase.

Ribonucleotide reductase activity is markedly elevated in cell lines selected for resistance to hydroxyurea, a cytotoxic drug known specifically to inhibit ribonucleotide reductase. From a cDNA library constructed from a highly hydroxyurea-resistant hamster lung cell line, 600H in which the activity is elevated more than 80-fold, we have isolated a full length cDNA for the small subunit of the reductase. The cDNA is 3.48 kb long with an open reading frame of 1158 nucleotides and a long 3' flanking region of 2169 nucleotides from the termination codon. The derived polypeptide sequence is closely similar to the small subunit of the mouse, differing from it in 20 amino acid positions. Most of these replacements occur in the N-terminal segment of the protein. The hamster subunit does not contain 4 amino acid residues found in the mouse small subunit near the C-terminal end. RNA blots probed with the cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.

Amino Acid Sequence↗

PubMed2004

SMG7 acts as a molecular link between mRNA surveillance and mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that eliminates mRNAs containing premature termination codons (PTCs). The proteins UPF1, SMG5, SMG6, and SMG7 are essential NMD factors in metazoa. SMG5 and SMG7 form a complex with UPF1 and interact with each other via their N-terminal domains. Here we show that SMG5 and SMG7 colocalize in cytoplasmic mRNA decay bodies, while SMG6 forms separate cytoplasmic foci. When SMG7 is tethered to a reporter transcript, it elicits its degradation, bypassing the requirement for a PTC, UPF1, SMG5, or SMG6. This activity is mediated by the C-terminal domain of SMG7. In contrast, SMG5 requires SMG7 to trigger mRNA decay and to localize to decay bodies. Our findings indicate that SMG7 provides a link between the NMD and the mRNA degradation machinery by interacting with SMG5 and UPF1 via its N-terminal domain and targeting bound transcripts for decay via its C-terminal domain.

Blotting, Western↗

PubMed1992

Structural studies of the acidic transactivation domain of the Vmw65 protein of herpes simplex virus using 1H NMR.

We have overproduced and purified the carboxy-terminal transactivation domain of Vmw65 (VP16) of herpes simplex virus, and studied potential folding of the domain by 1H NMR. Two species of the acidic domain were obtained from the bacterial expression system, and we demonstrate that one of these represents read-through of the natural amber termination codon of the Vmw65 reading frame producing a larger polypeptide. Additional residues in the read-through product were identified by total amino acid analysis and by NMR. Study of the correctly terminated product by 1D NMR gave resonances which were clustered into groups around their random-coil chemical shift positions, and 2D NMR demonstrated that, even in mixed solvents containing up to 80% MeOH, there was very little evidence of secondary structure. Together these results indicate that the isolated acid domain has little if any alpha-helical content of any stable nature. We discuss these results with reference to the demonstrated activity of the acidic domain in a wide variety of polypeptide contexts.

Amino Acid Sequence↗

PubMed1981

Sequence and organization of the human mitochondrial genome.

The complete sequence of the 16,569-base pair human mitochondrial genome is presented. The genes for the 12S and 16S rRNAs, 22 tRNAs, cytochrome c oxidase subunits I, II and III, ATPase subunit 6, cytochrome b and eight other predicted protein coding genes have been located. The sequence shows extreme economy in that the genes have none or only a few noncoding bases between them, and in many cases the termination codons are not coded in the DNA but are created post-transcriptionally by polyadenylation of the mRNAs.

Base Sequence↗

PubMed2001

Specific interaction between the ribosome recycling factor and the elongation factor G from Mycobacterium tuberculosis mediates peptidyl-tRNA release and ribosome recycling in Escherichia coli.

Once the translating ribosomes reach a termination codon, the nascent polypeptide chain is released in a factor-dependent manner. However, the P-site-bound deacylated tRNA and the ribosomes themselves remain bound to the mRNA (post-termination complex). The ribosome recycling factor (RRF) plays a vital role in dissociating this complex. Here we show that the Mycobacterium tuberculosis RRF (MtuRRF) fails to rescue Escherichia coli LJ14, a strain temperature-sensitive for RRF (frr(ts)). More interestingly, co-expression of M.tuberculosis elongation factor G (MtuEFG) with MtuRRF rescues the frr(ts) strain of E.coli. The simultaneous expression of MtuEFG is also needed to cause an enhanced release of peptidyl-tRNAs in E.coli by MtuRRF. These observations provide the first genetic evidence for a functional interaction between RRF and EFG. Both the in vivo and in vitro analyses suggest that RRF does not distinguish between the translating and terminating ribosomes for their dissociation from mRNA. In addition, complementation of E.coli PEM100 (fusA(ts)) with MtuEFG suggests that the mechanism of RRF function is independent of the translocation activity of EFG.

Amino Acid Sequence↗

PubMed1998

Mutations in hamster single-strand break repair gene XRCC1 causing defective DNA repair.

The molecular basis for the DNA repair dysfunction observed in mutant Chinese hamster ovary cell lines of X-ray repair cross complementing group 1 (XRCC1) is unknown and the exact role of the XRCC1 protein remains unclear. To help clarify the role of the XRCC1 gene we analyzed four mutant cell lines of this complementation group and a revertant cell line for XRCC1 protein content and for sequence alterations in the XRCC1 coding region. Immunoblot analysis of cellular extracts indicated that each of four mutant lines was lacking XRCC1 protein, whereas the repair-proficient revertant line derived from one of these mutants contained a normal level of XRCC1. Although each of these cell lines expressed XRCC1 mRNA, we found in all cases a distinct point mutation resulting in crucial alterations in the encoded XRCC1 protein sequence of 633 amino acids. Two of the mutations cause non-conservative amino acid changes, Glu102-->Lys and Cys390-->Tyr, at positions that are invariant among hamster, mouse and human XRCC1 sequences and are located in putative functional domains. A third debilitating mutation disrupts RNA splicing, generating multiple transcripts of different length that contain deletions spanning a region of >100 amino acids in the midsection of the XRCC1 coding sequence. A fourth mutation results in a termination codon that shortens the open reading frame to 220 amino acids, however, in the revertant cell line a further mutation in the same codon, Stop221-->Leu, permits translation of a full-length functional variant protein. These mutational data indicate the importance of the putative functional regions in XRCC1, such as the BRCA1 C-terminal (BRCT) domain found in common with BRCA1 and other DNA repair and cell cycle checkpoint proteins, and also regions necessary for interaction with DNA polymerase beta and DNA ligase III.

Alternative Splicing↗

PubMed1989

Identification and nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4.

The nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4 (EHV-4) was determined. The gene was located within a BamHI genomic library by a combination of Southern and dot-blot hybridization with probes derived from the herpes simplex virus type 1 (HSV-1) gB DNA sequence. The predominant portion of the coding sequences was mapped to a 2.95-kilobase BamHI-EcoRI subfragment at the left-hand end of BamHI-C. Potential TATA box, CAT box, and mRNA start site sequences and the translational initiation codon were located in the BamHI M fragment of the virus, which is located immediately to the left of BamHI-C. A polyadenylation signal, AATAAA, occurs nine nucleotides past the chain termination codon. Translation of these sequences would give a 110-kilodalton protein possessing a 5' hydrophobic signal sequence, a hydrophilic surface domain containing 11 potential N-linked glycosylation sites, a hydrophobic transmembrane domain, and a 3' highly charged cytoplasmic domain. A potential internal proteolytic cleavage site, Arg-Arg/Ser, was identified at residues 459 to 461. Analysis of this protein revealed amino acid sequence homologies of 47% with HSV-1 gB, 54% with pseudorabies virus gpII, 51% with varicella-zoster virus gpII, 29% with human cytomegalovirus gB, and 30% with Epstein-Barr virus gB. Alignment of EHV-4 gB with HSV-1 (KOS) gB further revealed that four potential N-linked glycosylation sites and all 10 cysteine residues on the external surface of the molecules are perfectly conserved, suggesting that the proteins possess similar secondary and tertiary structures. Thus, we showed that EHV-4 gB is highly conserved with the gB and gpII glycoproteins of other herpesviruses, suggesting that this glycoprotein has a similar overall function in each virus.

Amino Acid Sequence↗

PubMed2006

Analysis of the COL17A1 in non-Herlitz junctional epidermolysis bullosa and amelogenesis imperfecta.

Non-Herlitz junctional epidermolysis bullosa (nH-JEB) disease manifests with skin blistering, atrophy and tooth enamel hypoplasia. The majority of patients with nH-JEB harbor mutations in COL17A1, the gene encoding type XVII collagen. Heterozygotes with a single COL17A1 mutation, nH-JEB defect carriers, may exhibit only enamel hypoplasia. In this study, to further elucidate COL17A1 mutation phenotype/ genotype correlations, we examined two unrelated families with nH-JEB. Furthermore, we hypothesized that COL17A1 mutations might underlie or worsen the enamel hypoplasia seen in amelogenesis imperfecta (AI) patients that are characterized by defects in tooth enamel formation without other systemic manifestations. We therefore conducted COL17A1 mutational analysis in three patients from two AI families. One nH-JEB patient showed no COL17A1 expression and was a compound heterozygote for the novel premature termination codon (PTC) mutations 1285delA and Q1387X. In addition, reduced COL17A1 expression was found in a second nH-JEB patient who was homozygous for the novel PTC mutation 4335delC, the most carboxyl terminal PTC mutation thus far identified. Due to nonsense mediated mRNA decay, the position of these PTC mutations is thought not to influence the effect of COL17A1 transcript loss and hence the severity of the nH-JEB phenotype. This study is the first to suggest that type XVII collagen carboxyl PTC mutations lead to restoration of truncated polypeptide expression and to a milder clinical disease severity in nH-JEB. Conversely, we failed to detect any pathogenic COL17A1 defects in AI patients, in either exon or within the intron-exon borders of AI patients. This study furthers the understanding of mutations in COL17A1 causing nH-JEB, and clearly demonstrates that the mechanism of enamel hypoplasia differs between nH-JEB and AI diseases.

Adult↗

PubMed1987

Cloning and characterization of an abundant Plasmodium knowlesi antigen which cross reacts with Gambian sera.

A 110 kDa Plasmodium knowlesi antigen, termed PK110, has been identified on the basis of messenger RNA abundance in late schizonts. Most Plasmodium genes previously cloned have been identified by immune sera, which have selected immunodominant antigens composed of repeating epitopes. Although PK110 was not selected by immune sera, it also contains amino acid repeats, indicating that this structure may be a common feature of malarial proteins. Determination of 296 codons in the PK110 gene revealed the presence of thirteen tandem repeats of twelve amino acids whose consensus sequence is E E T Q K T V E P E Q T. A termination codon interrupts the fourteenth repeat, indicating that these repeats are at the C-terminus of the protein. Indirect immunofluorescence experiments with sera raised against the lambda gt11 fusion protein indicate that PK110 is present in intra-erythrocytic late schizonts. Cloned PK110 is recognized by Gambian sera, and shares epitopes with Plasmodium ovale. PK110 does not cross react immunologically or by DNA hybridization with Plasmodium falciparum.

Amino Acid Sequence↗

PubMed1977

Nucleotide sequence of 3' untranslated portion of human alpha globin mRNA.

We have determined the nucleotide sequence of 75 nucleotides of the 3'-untranslated portion of normal human alpha globin mRNA which corresponds to the elongated amino acid sequence of the chain termination mutant Hb Constant Spring. This was accomplished by sequence analysis of cDNA fragments obtained by restriction endonuclease or T4 endonuclease IV cleavage of human globin cDNA synthesized from globin mRNA by use of viral reverse transcriptase. Analysis of cRNA synthesized from cDNA by use of RNA polymerase provided additional confirmatory sequence information. Possible polymorphism has been identified at one site of the sequence. Our sequence overlaps with, and extends the sequence of 43 nucleotides determined by Proudfood and coworkers for the very 3'-terminal portion of human alpha globin mRNA. The complete 3'-untranslated sequence of human alpha globin mRNA (112 nucleotides including termination codon) shows little homology to that of the human or rabbit beta globin mRNAs except for the presence of the hexanucleotide sequence AAUAAA which is found in most eukaryotic mRNAs near the 3'-terminal poly (A).

Anemia, Sickle Cell↗