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Two group I introns with long internal open reading frames in the chloroplast psbA gene of Chlamydomonas moewusii.

We report the nucleotide sequence of the chloroplast psbA gene encoding the 32 kilodalton protein of photosystem II from Chlamydomonas moewusii. Like its land plant homologues, this green algal protein consists of 353 amino acids. The C. moewusii psbA gene is composed of three exons containing 252, 11 and 90 codons and of two group I introns containing 2363 and 1807 nucleotides. Each of the introns features an internal open reading frame (ORF) that potentially encodes a basic protein of more than 300 residues. The primary sequences of the putative intron-encoded proteins are unrelated and none of them shares conserved elements with any of the proteins predicted from the group I intron sequences published so far. The first C. moewusii intron is inserted at the same position as the fourth intron of the psbA gene from Chlamydomonas reinhardtii; the second intron lies at a novel site downstream of this position. On the basis of their RNA secondary structures, the C. moewusii introns 1 and 2 can be assigned to subgroups IA and IB, respectively. However, intron 1 is not typical of subgroup IA introns, its most unusual feature being the location of the ORF in the "loop L5" region. To our knowledge, this is the first time that an ORF is located in this region of the group I intron structure.

Amino Acid Sequence↗

Procedures for enhancing the utility of the metallothionein promoter for the regulated expression of downstream open reading frames.

Procedures which enhance the inducibility of the mouse metallothionein I (mMT-I) transcriptional promoter in mouse C127 cells transformed by bovine papilloma virus have been investigated. These include: (i) induction with Zn2+ at low serum concentration, and (ii) use of a 'superinduction' protocol (presence of 1 microgram/ml of cycloheximide during induction with Zn2+, followed by 2 micrograms/ml of actinomycin D). Use of procedure (i) alone gave a 15- to 20-fold induction of expression of a downstream open reading frame (ORF), which is comparable to the maximum inducibility achieved with mMT-I in other systems. Use of procedures (i) and (ii) in combination allowed a 50-fold induction. Three different reporter ORFs (rabbit ferritin L subunit, human chorionic gonadotropin alpha subunit, and human lutropin beta subunit), in three different chromosomal contexts, responded to these procedures. The maximum rate of expression achieved was estimated at over 10(9) molecules per cell per day, which is 20% of the transformed cell's protein synthetic capacity. At these extremely high levels some of the induced products were cytotoxic.

Animals↗

An intron in the thymidylate synthase gene of Bacillus bacteriophage beta 22: evidence for independent evolution of a gene, its group I intron, and the intron open reading frame.

The thymidylate synthase gene (thy) (EC 2.1.1.45) of Bacillus subtilis bacteriophage beta 22 has a self-splicing, group I intron inserted into a highly conserved region of the coding sequence. The intron is very similar to one that is inserted 21 bp further downstream in the homologous thymidylate synthase gene (td) of Escherichia coli bacteriophage T4. In contrast, the amino acid sequences of the bacteriophage thymidylate synthases are highly divergent. The beta 22 intron has a fragmentary open reading frame (ORF) that encodes a putative helix-turn-helix DNA-binding motif, similar to one at the carboxyl terminus of the homing endonuclease (I-TevI) encoded by the T4 td intron. The td ORF and the thy ORF fragments are inserted into different regions of their respective intron structures. These results suggest that the thymidylate synthase genes, their introns, and their respective intron-ORFs all have separate evolutionary histories and that the acquisition of the intron could not have occurred by a simple homing event.

Amino Acid Sequence↗

E2 of cottontail rabbit papillomavirus is a nuclear phosphoprotein translated from an mRNA encoding multiple open reading frames.

The papillomavirus E2 protein is a transcription trans-activator and as such as a paramount effect on viral functions. We have identified and characterized the cottontail rabbit papillomavirus E2 protein expressed from the late simian virus 40 promoter in COS-7 cells. E2 was shown to be a highly phosphorylated 49-kilodalton protein. Subcellular fractionation and indirect immunofluorescent staining indicated that E2 was located in the nuclei. E2 was expressed from a vector which contained just the open reading frame. ORF E2 and also from vectors which extended farther upstream and also expressed E7 or the short E6 protein. However, the level of E2 was very low in cells transfected with a vector expressing the long E6 protein. Mapping of transcripts with nuclease S1 and exonuclease ExoVII in cells expressing the short E6 and E2 proteins showed that E2 was translated from an RNA which encoded the short E6, E7, and E2 proteins but served as mRNA for only the short E6 and E2 proteins.

Animals↗

Generation of an adenovirus vector lacking E1, e2a, E3, and all of E4 except open reading frame 3.

Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lacking E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressing a beta-galactosidase reporter gene. This vector was generated by transfection of a plasmid carrying the full-length vector sequence into A30.S8 cells that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57BL/6 mice that the Av4orf3nBg vector effected gene transfer with an efficiency comparable to that of the Av3nBg (wild-type E4) vector but that the former exhibited a higher level of beta-galactosidase expression. This observation suggests that E4 ORF3 alone is able to enhance RNA levels from the beta-galactosidase gene when the Rous sarcoma virus promoter is used to drive transgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected with the Av3nBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion background may be beneficial in decreasing immunogenicity and improving safety and toxicity profiles, as well as increasing transgene capacity and expression for liver-directed gene therapy.

Adenovirus E1 Proteins↗

Identification of the human papillomavirus type 6b L1 open reading frame protein in condylomas and corresponding antibodies in human sera.

Genital warts (condylomata acuminata) are among the most frequent sexually transmitted infections. Human papillomavirus type 6 (HPV-6), which is etiologically related to a majority of these lesions, has not been propagated in tissue culture. We generated two forms of HPV-6 viral antigens: a chemically synthesized oligopeptide (referred to as the C-terminal synthetic peptide) corresponding to residues 482 to 495 of the 500-amino-acid-long L1 open reading frame (ORF), and a bacterially expressed 54-kilodalton (kDa) fusion protein containing the N-terminal 13 amino acids encoded by the lambda bacteriophage cII gene followed by one vector-insert junctional residue and 462 amino acids of the L1 ORF sequence (residues 39 to 500). The cII-L1 fusion protein was specifically recognized by an antipeptide serum directed against the N-terminal 13 amino acids derived from the cII gene, an antiserum raised against the C-terminal synthetic peptide, and a genus-specific serum prepared by immunization with disrupted viral capsids. The 54-kDa fusion protein was purified, and the sequence of its first 36 amino acids was determined and found to be as predicted by the DNA sequence. Both the genus-specific anticapsid serum and the antiserum raised against the fusion protein identified authentic L1 ORF proteins in HPV-1-induced (58 kDa) and HPV-6/11-induced (56 kDa) papillomas. The synthetic peptide antiserum recognized the 56- to 58-kDa protein in HPV-6-induced warts, but not in HPV-1- or HPV-11-infected specimens. Using the fusion protein as antigen in immunoassays, we were able to detect the corresponding antibodies in human sera.

Antibodies, Viral↗

Human papillomavirus type 16 transformation of primary human embryonic fibroblasts requires expression of open reading frames E6 and E7.

Human papillomavirus 16 (HPV-16) early genes were tested for their ability to transform primary human embryonic fibroblasts (WI38) or to extend the life span of the fibroblasts. The expression plasmids containing the transcriptional unit for the HPV-16 subgenomic DNA fragments controlled by the simian virus 40 promoter were introduced into WI38 cells with the aid of neomycin selection, and the drug-resistant cells that survived long-term culture were characterized. The fragments containing E6E7 open reading frames (ORFs) intact were found to be capable of extending the life span of WI38 cells. From the results obtained with frameshift mutations in either ORF and from those obtained by cotransfection with the separate plasmids containing E6 and E7 ORFs, it was concluded that HPV-16 transformation (extension of the life span) of human fibroblasts requires expression of genes E6 and E7.

Cell Survival↗

Organization and nucleotide sequence of the broad bean chloroplast genes trnL-UAG, ndhF and two unidentified open reading frames.

We have determined the nucleotide sequence of a 6.9 kbp BamHI-XbaI fragment of broad bean chloroplasts. Part of this fragment (subfragment BglII-ClaI) is known to contain three tRNA genes (trnL-CAA, trnL-UAA and trnF). We have now further identified a gene coding for the third tRNA(Leu) isoacceptor (trnL-UAG) which is located close to trnF. The BamHI-XbaI fragment also contains the gene for subunit 5 of NADH dehydrogenase (ndhF) and two unidentified open reading frames (ORFx and ORF48). ORFx shares a high sequence homology with the long reading frames of tobacco (ORF1708), spinach (ORF2131), and liverwort (ORF2136), while ORF48 shares sequence homology with ORF69 of liverwort and ORF55 of tobacco.

Amino Acid Sequence↗

The first and fourth upstream open reading frames in GCN4 mRNA have similar initiation efficiencies but respond differently in translational control to change in length and sequence.

The third and fourth AUG codons in GCN4 mRNA efficiently repress translation of the GCN4-coding sequences under normal growth conditions. The first AUG codon is approximately 30-fold less inhibitory and is required under amino acid starvation conditions to override the repressing effects of AUG codons 3 and 4. lacZ fusions constructed to functional, elongated versions of the first and fourth upstream open reading frames (URFs) were used to show that AUG codons 1 and 4 function similarly as efficient translational start sites in vivo, raising the possibility that steps following initiation distinguish the regulatory properties of URFs 1 and 4. In accord with this idea, we observed different consequences of changing the length and termination site of URF1 versus changing those of URFs 3 and 4. The latter were lengthened considerably, with little or no effect on regulation. In fact, the function of URFs 3 and 4 was partially reconstituted with a completely heterologous URF. By contrast, certain mutations that lengthen URF1 impaired its positive regulatory function nearly as much as removing its AUG codon did. The same mutations also made URF1 a much more inhibitory element when it was present alone in the mRNA leader. These results strongly suggest that URFs 1 and 4 both function in regulation as translated coding sequences. To account for the phenotypes of the URF1 mutations, we suggest the most ribosomes normally translate URF1 and that the mutations reduce the number of ribosomes that are able to complete URF1 translation and resume scanning downstream. This effect would impair URF1 positive regulatory function if ribosomes must first translate URF1 in order to overcome the strong translational block at the 3'-proximal URFs. Because URF1-lacZ fusions were translated at the same rate under repressing and derepressing conditions, it appears that modulating initiation at URF1 is not the means that is used to restrict the regulatory consequences of URF1 translation to starvation conditions.

Base Sequence↗

The adenovirus early region 4 open reading frame 6/7 protein regulates the DNA binding activity of the cellular transcription factor, E2F, through a direct complex.

Nuclear factor E2F is a cellular protein that binds to the adenovirus E2 promoter and E1A enhancer regions and to the cellular c-myc P2 promoter region. The DNA binding activity of E2F, detected in vitro using nuclear extracts prepared from HeLa cells, is increased by adenovirus infection (termed E2F induction). We demonstrate here that a 19.5-kD protein, encoded by adenovirus early region 4 (E4) open reading frame (ORF) 6/7, is primarily responsible for the induction of E2F DNA binding activity to the E2 promoter region. Viral mutants that contain frame-shift mutations in E4 ORF 6/7 failed to induce E2F binding activity; a virus that carries an E4 ORF 6/7 cDNA in place of the E4-coding sequences induced E2F efficiently. Using gel mobility shift assays, we demonstrate that the E4 ORF 6/7 product induces the binding of E2F to the E2 promoter via a direct complex. The addition of a peptide-specific antiserum, directed against the E4 ORF 6/7 protein, to an in vitro E2F-binding reaction resulted in the formation of a DNA-protein complex with reduced gel mobility compared to the normal, adenovirus-induced E2F-E2 promoter complex. The formation of the E2F-E2 promoter-antibody complex was blocked by the addition of the cognate peptide used to generate the antiserum but not by a nonspecific peptide. Nuclear extracts prepared from adenovirus-infected HeLa cells were cleared of E2F binding activity using the ORF 6/7 peptide-specific serum, but not the preimmune serum, suggesting that E2F and the E4 ORF 6/7 product form a protein-protein complex in solution. The adenovirus E1A proteins are not absolutely required for the induction of E2F binding activity because the infection of HeLa cells with an E1A mutant, dl312, at high multiplicity resulted in E2F induction. Under these conditions of infection, the E4 ORF 6/7 product was synthesized. E2F binding activity was induced, but inefficiently, in cells infected with E4 ORF 6/7 mutants, indicating that an additional pathway may lead to E2F induction.

Adenoviridae↗

A single antigenomic open reading frame of the hepatitis delta virus encodes the epitope(s) of both hepatitis delta antigen polypeptides p24 delta and p27 delta.

On the basis of the complete nucleotide sequence of the single-stranded, covalently closed circular hepatitis delta virus RNA genome (K.-S. Wang, Q.-L. Choo, A. J. Weiner, J.-H. Ou, R. C. Najarian, R. M. Thayer, G. T. Mullenbach, K. J. Denniston, J. L. Gerin, and M. Houghton, Nature [London] 323:508-514, 1986 [Author's correction, 328:456, 1987]), five long open reading frames (ORFs) encoding polypeptides containing a methionine proximal to the amino terminus were expressed in bacteria. Only polypeptides encoded by the antigenomic ORF5 cross-reacted with antisera obtained from patients with hepatitis delta virus infections. Immunological analysis of viral extracts and the recombinant ORF5 polypeptides synthesized in bacteria and yeast cells revealed that ORF5 encodes the immunogenic epitope(s) shared by both hepatitis delta viral polypeptides p27 delta and p24 delta and probably represents the complete structural gene for p27 delta and p24 delta. We also present evidence that ORF5 encodes the hepatitis delta antigen, an antigen originally found in the nuclei of hepatocytes of infected individuals (M. Rizzetto, M. G. Canese, S. Arico, O. Crivelli, F. Bonino, C. G. Trepo, and G. Verme, Gut 18:997-1003, 1977). A comparison of the primary structure of the predicted hepatitis delta antigen polypeptides with that of the core antigen of the hepatitis B virus shows that these polypeptides are very dissimilar.

Amino Acid Sequence↗

The E7 open reading frame of human papillomavirus type 16 encodes a transforming gene.

Prior analysis of bovine papillomavirus has identified two genes, E5 and E6, which induce morphologic transformation of certain established cells. To study the transforming genes of human papillomavirus (HPV) type 16, the HPV type most commonly associated with cervical carcinoma, we examined subgenomic viral DNAs under control of a retroviral long terminal repeat for their capacity to induce cellular transformation of NIH 3T3 cells. Plasmids carrying the entire viral early region, the E6 and E7 (E6/E7) open reading frames (ORFs), or the E2,E4, and E5 (E2-E5) ORFs induced a very low frequency of focal transformation (0.4-1.7 ffu/micrograms DNA). In contrast, the plasmids with the entire early region of E6/E7, when selected by co-transfection with a transformation independent marker (neoR), induced anchorage independent growth with high efficiency. Cells selected after co-transfection with the marker gene and the E2-E5 plasmid grew much less efficiently in agar. Mutational analysis of the E6/E7 plasmid indicated that E7 was the gene primarily responsible for inducing anchorage independent growth. E7 gene therefore represents a third class of PV transforming gene. These results correlate with E7 (along with E6) being selectively retained and expressed in HPV associated cervical carcinomas and cell lines.

Animals↗

Replication of tomato golden mosaic virus DNA B in transgenic plants expressing open reading frames (ORFs) of DNA A: requirement of ORF AL2 for production of single-stranded DNA.

Tomato golden mosaic geminivirus has a genome of two single-stranded (ss) DNA components, A and B. An almost identical 'common' region in DNA A and DNA B is thought to contain sequence elements controlling replication and transcription. Hence investigation of sequences important for DNA replication by in vitro mutagenesis is complicated by possible effects on the transcription of genes for replication proteins. To overcome this problem, transgenic plants expressing open reading frames (ORFs) of DNA A from an enhanced cauliflower mosaic virus 35S RNA promoter were constructed and tested for their ability to support the replication of DNA B and DNA B mutants. The results show that plants transgenic for ORF AL1 are able to support the replication of the double-stranded (ds) forms of DNA B, but that ORF AL2 is required in addition to produce ssDNA B. ORFs AL3, BL1 or BR1 were not required for replication of ds or ssDNA B. To the best of our knowledge this is the first time that essential replication proteins of a geminivirus have been expressed constitutively from a plant genome without giving rise to replicating DNA A molecules, thereby allowing DNA B to replicate alone. Such transgenic plants should enable not only the mutational analysis of sequence elements within the replication origin region, but also the construction of a new generation of vectors for gene amplification in plants, based on a minimal virus replicon.

DNA Probes↗

Spliced RNA from the IR1-U2 region of Epstein-Barr virus: presence of an open reading frame for a repetitive polypeptide.

We have constructed a cDNA library from the cytoplasmic RNAs of Raji cells, a Burkitt's lymphoma cell line latently infected with Epstein-Barr virus. We report here the characterization of a cDNA representing a spliced RNA transcribed from the IR1-U2 region of the viral genome. The cDNA is 1007 bp long. The 5' region contains three tandem repeats of two exons, 66 and 132 bp, which are transcribed from the IR1 repeats. The 3' region is formed from four exons transcribed from U2. An open reading frame extends from the 5' end to position 784, and includes the repeats. This reading frame presumably corresponds to the carboxy-terminal 261 amino acids of a polypeptide containing several repeats of a 66 amino acid sequence. Since it would be encoded by the IR1-U2 region of the viral genome, the putative polypeptide might be involved in the process of growth-transformation of B-lymphocytes.

Amino Acid Sequence↗

Structural features of the mdg1 lineage of the Ty3/gypsy group of LTR retrotransposons inferred from the phylogenetic analyses of its open reading frames.

The increasing amount of data generated in recent years has opened the way to exhaustive studies of the relationships among different members of the Ty3/gypsy group of LTR retrotransposons, a widespread group of eukaryotic transposable elements. Former research led to the identification of several independent lineages within this group. One of the worse represented of them is that of mdg1, integrated so far only by the Drosophila retrotransposons mdg1 and 412. Our exhaustive database searches indicate the existence of three other Drosophila members of this lineage. Two of them correspond to elements already known, namely, Stalker and blood, but the third one is a new element, which we have called Pilgrim. This element is well represented within the D. melanogaster genome, as revealed by our Southern blot analysis of different strains. The case of Stalker is particularly remarkable, since its phylogenetic relationships clearly point to the mosaic origin of its genome. Finally, our analysis of the evolution of a small ORF preserved within the 5' leader region of these elements indicates different evolutionary rates, presumably as a result of distinct selective constraints.

Amino Acid Sequence↗

Equine infectious anemia virus gene expression: characterization of the RNA splicing pattern and the protein products encoded by open reading frames S1 and S2.

The utilization of predicted splice donor and acceptor sites in generating equine infectious anemia virus (EIAV) transcripts in fetal donkey dermal cells (FDD) was examined. A single splice donor site identified immediately upstream of the gag coding region joins the viral leader sequence to all downstream exons of spliced EIAV transcripts. The predominant 3.5-kb transcript synthesized in EIAV-infected FDD cells appears to be generated by a single splicing event which links the leader sequence to the first of two functional splice acceptor sites near the 5' end of the S1 open reading frame (ORF). The translation products encoded by the 3.5-kb transcript were examined by producing in vitro transcripts from a cDNA corresponding to this RNA followed by in vitro translation in wheat germ extracts. These transcripts directed the synthesis of three proteins: the virus trans-activator protein (EIAV Tat) encoded by ORF S1, a protein of unknown function encoded by ORF S2, and the virus envelope glycoprotein. When transfected into FDD cells, this cDNA also directed expression of EIAV Tat. Amino-terminal sequence analysis of the in vitro-synthesized S1 protein supports the suggestion that translation of EIAV Tat is initiated at a CUG codon within the virus leader region. Both in vitro-synthesized S2 protein and synthetic peptides corresponding to S2 are shown to react positively with sera obtained from EIAV-infected horses, providing the first direct evidence of expression of this protein in infected animals.

Amino Acid Sequence↗

Nucleotide sequence of the human c-myc locus: provocative open reading frame within the first exon.

The nucleotide sequence of a HindIII-EcoRI DNA fragment, 8 kbp long, of a lambda recombinant containing the whole human c-myc gene has been deduced by the method of Maxam and Gilbert. This fragment encodes the complex c-myc locus and the sequence provides information relative to the 2.7 kb long c-myc transcript. It appears that although exons 2 and 3 would code for a 48-K protein homologous to the myc domain of the viral p110 gag-myc protein, the first exon, which has a large open reading frame ending with a stop codon just upstream from the donor splice site, could code on its own for a 20-K protein. Speculations about the role of that putative protein on the regulation of the expression of exons 2 and 3 are made.

Base Sequence↗

Shufflon: multi-inversion of four contiguous DNA segments of plasmid R64 creates seven different open reading frames.

The IncI alpha plasmid R64 was found to bear a highly mobile DNA segment which was designated as a clustered inversion region (J. Bacteriol. 165, 94-100, 1986). The clustered inversion region consists of four DNA segments designated respectively as A, B, C and D which differ in molecular size and restriction sites. The four DNA segments invert independently or in groups resulting in a complex DNA rearrangement. We now show the nucleotide sequence of the clustered inversion region of R64. The present results suggest that the clustered inversion region is a biological switch to select one of seven open reading frames whose primary structures at the region proximal to N-termini are constant while those at the C-terminal region are variable. A name, "Shufflon" was proposed to call this kind of the clustered inversion region.

Base Sequence↗