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PubMed2003

Powdery mildew-induced Mla mRNAs are alternatively spliced and contain multiple upstream open reading frames.

In barley (Hordeum vulgare), the Mla13 powdery mildew resistance gene confers Rar1-dependent, AvrMla13-specific resistance to Blumeria graminis f. sp. hordei (Bgh). We have identified cDNA and genomic copies of Mla13 and used this coiled-coil nucleotide-binding site leucine-rich repeat protein-encoding gene as a model for the regulation of host resistance to obligate biotrophic fungi in cereals. We demonstrate quantitatively that a rapid increase in the accumulation of Mla transcripts and transcripts of the Mla-signaling genes, Rar1 and Sgt1, is triggered between 16 and 20 h post inoculation, the same time frame that haustoria of avirulent Bgh make contact with the host cell plasma membrane. An abundance of Mla13 cDNAs revealed five classes of transcript leader regions containing two alternatively spliced introns and up to three upstream open reading frames (uORFs). Alternative splicing of introns in the transcript leader region results in a different number of uORFs and variability in the size of uORF2. These results indicate that regulation of Mla transcript accumulation is not constitutive and that induction is coordinately controlled by recognition-specific factors. The sudden increase in specific transcript levels could account for the rapid defense response phenotype conferred by Mla6 and Mla13.

Alternative Splicing↗
PubMed2000

1-Acyldihydroxyacetone-phosphate reductase (Ayr1p) of the yeast Saccharomyces cerevisiae encoded by the open reading frame YIL124w is a major component of lipid particles.

Biosynthesis of phosphatidic acid through the dihydroxyacetone phosphate pathway requires NADPH-dependent reduction of the intermediate 1-acyldihydroxyacetone phosphate before the second step of acylation. Studies with isolated subcellular fractions of the yeast Saccharomyces cerevisiae revealed that lipid particles and the endoplasmic reticulum harbor 1-acyldihydroxyacetone-phosphate reductase (ADR) activity. Deletion of the open reading frame YIL124w (in the following named AYR1) abolished reduction of 1-acyldihydroxyacetone phosphate in lipid particles, whereas ADR activity in microsomes of the deletion strain was decreased approximately 3-fold as compared with the wild-type level. This result indicates that (i) both lipid particles and microsomes harbor Ayr1p, which was confirmed by immunological detection of the protein in these two cellular compartments, and (ii) microsomes contain at least one additional ADR activity. As a consequence of this redundancy, deletion of AYR1 neither results in an obvious growth phenotype nor affects the lipid composition of a haploid deletion strain. When a heterozygous AYR1(+)/ayr1(-) diploid strain was subjected to sporulation; however, spores bearing the ayr1 defect failed to germinate, suggesting that Ayr1p plays an essential role at this stage. Overexpression of Ayr1p at a 5- to 10-fold level of wild type caused growth arrest. Heterologous expression of Ayr1p in Escherichia coli resulted in gain of ADR activity in the prokaryote, confirming that YIL124w is the structural gene of the enzyme and does not encode a regulatory or auxiliary component required for reduction of 1-acyldihydroxyacetone phosphate. Taken together, these results identified Ayr1p of the yeast as the first ADR from any organism at the molecular level.

Amino Acid Sequence↗
PubMed1999

The Mycobacterium tuberculosis recA intein can be used in an ORFTRAP to select for open reading frames.

The DNA repair protein RecA of Mycobacterium tuberculosis contains an intein, a self-splicing protein element. We have employed this Mtu recA intein to create a selection system for successful intein splicing by inserting it into a kanamycin-resistance gene so that functional antibiotic resistance can only be restored upon protein splicing. We then proceeded to develop an ORFTRAP, i.e., a selection system for the cloning of open reading frames (ORFs). The ORFTRAP exploits the self-splicing properties of inteins (which depend on full-length in-frame translation of a precursor protein) by allowing protein splicing to occur when DNA fragments encoding ORFs are inserted into the Mtu recA intein, whereas DNA fragments containing non-ORFs are selected against. Regions of the Mtu recA intein that tolerate the insertion of additional amino acids were identified by Bgl II linker scanning mutagenesis, and a respective construct was chosen as the ORFTRAP. To test the maximum insert size that could be cloned into ORFTRAP, DNA fragments of increasing length from the Listeria monocytogenes hly gene as well as a genomic library of Haemophilus influenzae were inserted and it was found that the longest permissive inserts were 425 bp and 251 bp, respectively. The H. influenzae ORFTRAP library also demonstrated the strength (strong selection power) and weakness (insertion of very small fragments) of the system. Further modifications should make the ORFTRAP useful for protein expression, epitope mapping, and antigen screening.

Amino Acid Sequence↗
PubMed1995

Characterization of an open reading frame involved in site-specific integration of filamentous phage Cf1t from Xanthomonas campestris pv. citri.

Cf1t is a single-stranded DNA filamentous phage; a 1.9-kb segment of DNA from Cf1t was found to be responsible for site-specific integration into Xanthomonas campestris pv. citri (XW47), in the absence of any Xanthomonas origin of replication. Deletion analysis and introduction of amber stop codons into this fragment from Cf1t revealed an open reading frame (ORF344) which was involved in the integration function. The predicted amino-acid sequence of ORF344 bears no homology with conserved sequences of the integrase family.

Amino Acid Sequence↗
PubMed1994

Sequencing of a 13.2 kb segment next to the left telomere of yeast chromosome XI revealed five open reading frames and recent recombination events with the right arms of chromosomes III and V.

We report the entire sequence of a 13.2 kb segment next to the left telomere of chromosome XI of Saccharomyces cerevisiae. A 1.2 kb fragment near one end is 91% homologous to the right arm of chromosome III and 0.7 kb of that are 77% homologous to the right arm of chromosome V. Five open reading frames are included in the sequenced segment. Two of them are almost identical to the known YCR104W and YCR103C hypothetical proteins of chromosome III. A third one contains a region homologous to the Zn (2)-Cys (6) binuclear cluster pattern of fungal transcriptional activators. The fourth one, part of which is similar to the mammalian putative transporter of mevalonate, has the structure of membrane transporters. The fifth one is similar to yeast ferric reductase.

Amino Acid Sequence↗
PubMed2006

Analysis of the subgenomic RNAs and the small open reading frames of Beet black scorch virus.

A full-length cDNA of the genome of Beet black scorch virus (BBSV), isolate Ningxia, was constructed and modified by site-directed mutagenesis to permit in vitro transcription of mutant viral RNAs. Two subgenomic (sg) RNAs (sgRNA1 and sgRNA2) appeared during BBSV replication. Mutagenesis revealed that sgRNA1 transcription was initiated at G2209 within the P82 polymerase subunit open reading frame (ORF) and that transcription of sgRNA2 began at G2526 within the nested p7b/p5' ORF. Initiation-codon shifting or premature termination of translation of the three ORFs (P7a, P7b and P5') encoded by sgRNA1 indicated that each of the genes was required for localized movement, accumulation of viral RNAs and formation of local lesions on the leaves of Chenopodium amaranticolor. Microscopic observations of the distribution of green fluorescent protein fused to the N-terminal portion of the capsid protein provided additional evidence that the P7a, P7b and P5' proteins are each required for cell-to-cell movement. In contrast, elimination of sgRNA2 showed that the BBSV coat protein was not required for viral RNA accumulation or the appearance of local lesions on C. amaranticolor. In addition, disruption of the small P5 ORF previously predicted by computer analysis to originate at the C terminus of the P82 ORF had no effect on disease phenotype, suggesting that this ORF may represent a cryptic, non-essential gene. These results show that BBSV has a novel cell-to-cell movement protein organization that differs in size and sequence from that of other viruses.

Base Sequence↗
PubMed1999

The open reading frame YAL048c affects the secretion of proteinase A in S. cerevisiae.

Over-expression of the yeast PEP4 gene encoding the vacuolar aspartic protease proteinase A (PrA) leads to saturation of the vacuolar targeting system of the cell and missorting of PrA to the growth medium. In a screen for genes affecting the secretion of over-expressed PrA we found that multiple copies of the open reading frame (ORF) YAL048c enhanced PrA secretion. Since no function has hitherto been ascribed to YAL048c, we undertook further studies of this ORF. Deletion of YAL048c resulted in slightly reduced secretion of over-produced PrA. Furthermore, strains deleted for YAL048c showed a growth inhibition phenotype resulting in wrinkled colony morphology when grown on rich medium containing high concentrations of calcium. YAL048c is predicted to encode a polypeptide of 662 amino acid residues containing two consensus ATP/GTP-binding site motifs and a putative carboxy-terminal transmembrane region. In addition, the amino acid sequence contains two putative calcium-binding domains. The YAL048c protein may be evolutionarily conserved, as homologues exist in humans and Caenorhabditis elegans. We suggest that the YAL048c protein is involved in vesicle transport in the secretory pathway.

Adenosine Triphosphate↗
PubMed1995

Tightly regulated, developmentally specific expression of the first open reading frame from LINE-1 during mouse embryogenesis.

LINE-1 (L1) has achieved its status as a middle repetitive DNA family in mammalian genomes by duplicative transposition. Although transposition may occur in any cell type, expression and transposition of a full-length functional element in the germ line are necessary for evolutionarily significant propagation of L1. An immunohistochemical analysis of adult mouse ovaries and mouse postimplantation embryos revealed expression of L1 open reading frame 1 in the germ line as well as in steroidogenic tissues. These results demonstrate that L1 expression is controlled by a tightly regulated temporal and spatial program of events during development and imply that multiple loci of L1 in the mouse genome are active for expression.

Animals↗
PubMed1993

Alternative mRNA forms and open reading frames of the max gene.

The max gene encodes a heterodimeric partner of Myc. We have recently identified an alternative max mRNA (delta max) that contains an additional internal exon introducing an in-frame translational termination. Here we have studied the expression of human max mRNAs by Northern blotting analysis. In addition to the major 2.3-kb mRNA form, four bands were identified. Our results indicate that these bands represent differentially spliced mRNA forms, which contain altogether three open reading frames. In addition to the previously identified Max and delta Max proteins, sequence analysis of a 3.5-kb mRNA form predicted a protein that resembles delta Max in structure. Like delta Max, this protein enhanced the number of transformed foci in the ras-myc co-transformation assay. Although the 3.5-kb mRNA represents a minor form in actively proliferating cells, a shift from the major 2.3-kb mRNA to the 3.5-kb form was observed in response to high cell density or acidification of the growth medium. Our results indicate the presence of several differentially spliced mRNA forms of the max gene, and suggest a possible mechanism for the production of functionally distinct Max proteins.

Amino Acid Sequence↗
PubMed1994

Identification and characterization of a murine cytomegalovirus gene with homology to the UL25 open reading frame of human cytomegalovirus.

Monoclonal antibody 1B4, previously shown to be protective in vivo and to cross-react with both virally encoded and normal host cell proteins, was used to screen a lambda gt11 cDNA derived from mRNA harvested from mouse embryo fibroblasts 24 hr after infection with murine cytomegalovirus (MCMV). A 700-bp cDNA was identified representing the 5'terminus of a 2460-bp open reading frame (ORF) with significant homology to the human cytomegalovirus UL25 ORF. The UL25 ORF of MCMV potentially encodes an 820 amino acid viral tegument protein with an estimated molecular weight of approximately 90 kDa. Amino acid homology with eukaryotic nucleolins was identified in the acidic N-terminal third of the MCMV UL25 proteins, suggesting that the protein may be involved in transcriptional activation or interactions with chromatin. Northern analysis and S1 nuclease data indicated that the gene is expressed late in infection as an approximately 3-kb transcript and that expression is dependent on viral DNA replication. An epitope recognized by MAb 1B4 was identified using recombinant pGEX plasmids expressing fusion proteins representing the N-terminal region of the MCMV UL25 protein. The identification of the MCMV UL25 ORF as a member of the CMV-specific UL25/UL35 gene family provides an opportunity for the investigation of the role these genes and their products in CMV pathogenesis in an animal model.

Amino Acid Sequence↗
PubMed1996

Transforming activities of human papillomavirus type 59 E5, E6 and E7 open reading frames in mouse C127 cells.

The DNA sequence from a human papillomavirus type 59 (HPV 59) has been recently determined. The HPV 59 genome consists of 7896 nucleotides (nt). A comparative analysis of this sequence with the sequences of other HPVs revealed the closest homology to HPV 18 (71%). To test the transforming activities of HPV 59 DNA and its gene products, several plasmids expressing HPV 59 open reading frames (ORF) were constructed. The E5, E6, and E7 ORFs of HPV 59 were inserted into pRc/CMV vector containing a promoter of cytomegalovirus to test the transforming activities of these ORFs. When these DNAs were transfected into mouse C127 cells, all three ORFs were independently able to transform C127 cells in the presence of G418, although the full length HPV 59 DNAs failed to induce the focus-formation. The E7 ORF showed the strongest transforming activity and the E5 ORF exhibited the weakest transforming activity. Cell lines transformed by E5, E6, and E7 ORFs were established and they grew anchorage-independently. The presence of HPV 59 ORF DNA was confirmed by polymerase chain reaction (PCR) and Southern blot analysis in HPV 59 ORFs-transformed cell lines.

Animals↗
PubMed1994

The sequence of a 36 kb segment on the left arm of yeast chromosome X identifies 24 open reading frames including NUC1, PRP21 (SPP91), CDC6, CRY2, the gene for S24, a homologue to the aconitase gene ACO1 and two homologues to chromosome III genes.

A 36 kb fragment from the left arm of chromosome X, located at about 50 kb from the telomere, was sequenced and analysed. The segment contains a new putative ARS, a new tRNA for threonine, remnants of a solo delta and 24 open reading frames (ORFs) numbered from J0310 to J0355. Six of them, NUC1, PRP21 (also called SPP91), CDC6, CRY2, the gene encoding the ribosomal protein S24 and the gene coding for a hypothetical protein of 599 amino acids, have been sequenced previously. Three ORFs show high homology to the yeast gene ACO1 encoding mitochondrial aconitase and to the chromosome III genes YCR34W and YCR37C of unknown function. Three other ORFs show lower but significant homology: a first one to UNP, a gene related to the tre-2 oncogene from mouse and to the gene coding for the yeast deubiquitinating enzyme DOA2; a second one to SLY41, a suppressor of the functional loss of YPT1 and a third one to the gene encoding the proline utilization activator PUT3.

Aconitate Hydratase↗
PubMed2006

Nanog retrotransposed genes with functionally conserved open reading frames.

The Nanog gene plays a key role in the pluripotency of early embryonic cells in vitro and in vivo. In this article retrotransposed copies of Nanog, termed NanogPc and NanogPd, are identified on mouse Chromosomes 4 and 7, respectively. In contrast to the two previously characterized mouse Nanog retrogenes that contain multiple frameshifts and point mutations, NanogPc and NanogPd are 98% identical to NANOG within the open reading frame and encode proteins with activity in an embryonic stem cell self-renewal assay. Mutations common to all four retrotransposed genes but distinct from Nanog suggest divergence from a common progenitor that appears likely to be Nanog because transcripts derived from Nanog but not from the retrogenes are detected in germ-line cells. The possibility that expression of Nanog could be erroneously attributed to novel cellular sources is suggested by the high homology among Nanog, NanogPc, and NanogPd. Analysis of distinct Mus species suggests that NanogPc and NanogPd arose between divergence of M. caroli and M. spretus and indicates that Nanog retrotransposition events continue to occur at a high frequency, a property likely to extend to other germ-line transcripts.

Amino Acid Sequence↗
PubMed1996

Borrelia burgdorferi supercoiled plasmids encode multicopy tandem open reading frames and a lipoprotein gene family.

DNA sequencing and Southern blot analyses of a Borrelia burgdorferi DNA fragment encoding a signal sequence led to the discovery of a genetic locus, designated 2.9, which appears to be present in at least seven copies in virulent B. burgdorferi 297. DNA sequence analysis of these regions revealed that each 2.9 locus contained an operon of four genes (ABCD) and open reading frames designated rep+ (positive strand) and rep- (negative strand) which encoded multiple repeat motifs. Downstream of the rep+ gene(s) in six of the completely cloned and sequenced 2.9 loci also were lipoprotein (LP) genes possessing highly similar signal sequences but encoding variable mature polypeptides. The lipoproteins could he separated into two classes on the basis of hydrophilicity profiles, sequence similarities, and reactivity with specific antibodies. The 2.9 loci were localized to two (20- and 30-kb) supercoiled plasmids in B. burgdorferi 297. Northern (RNA) blot analysis established that the 2.9 ABCD operon was only minimally expressed, whereas the rep- gene(s) and at least three of the seven LP genes were expressed by B. burgdorferi in vitro. A single putative promoter element was identified by RNA primer extension analysis upstream of the ABCD operon, whereas a number of potential promoter regions existed upstream of the LP genes. The combined data indicate that the ABCD operon, rep+ and rep- genes, and LP genes are separately transcribed during in vitro growth. The 2.9 loci possess a repetitiveness, diversity, and complexity not previously described for B. burgdorferi; differential expression of these genes may facilitate the spirochete's ability to survive in diverse host environments.

Amino Acid Sequence↗
PubMed2000

Fission yeast retrotransposon Tf1 integration is targeted to 5' ends of open reading frames.

Target site selection of transposable elements is usually not random but involves some specificity for a DNA sequence or a DNA binding host factor. We have investigated the target site selection of the long terminal repeat-containing retrotransposon Tf1 from the fission yeast Schizosaccharomyces pombe. By monitoring induced transposition events we found that Tf1 integration sites were distributed throughout the genome. Mapping these insertions revealed that Tf1 did not integrate into open reading frames, but occurred preferentially in longer intergenic regions with integration biased towards a region 100-420 bp upstream of the translation start site. Northern blot analysis showed that transcription of genes adjacent to Tf1 insertions was not significantly changed.

Binding Sites↗
PubMed1997

X-I and X-II open reading frames of HTLV-I are not required for virus replication or for immortalization of primary T-cells in vitro.

In contrast to other retroviruses of the oncovirinae subgroup, the primate and bovine leukemia viruses (HTLV, STLV, and BLV) encode genes in the X-region of the genome, between the env gene and the 3' long terminal repeat. In HTLV-I, two overlapping open reading frames (ORFs) in the distal half of the X-region encode tax and rex genes, while two ORFs (X-I and X-II) in the proximal half of this region potentially encode proteins designated p12(XI) (or rof) and p30(XII) (or tof). The biological functions and mechanisms of tax and rex have been studied extensively whereas the roles of the other ORFs have not yet been established. To identify possible functions for ORFs X-I and X-II, an infectious molecular clone of HTLV-I and a mutant provirus lacking these ORFs were compared with respect to virus replication, gene expression, and ability to immortalize primary T-cells. When transiently transfected into 293 cells, both intact and deleted proviruses directed the synthesis of virus mRNAs and proteins that were quantitatively and qualitatively identical. These viruses were also indistinguishable in their abilities to infect and replicate in DBS-FRhL cells, which are permissive for HTLV-I propagation. Immortalized T-cell lines were established after cell-free or coculture methods for infection of activated, human peripheral blood or cord blood lymphocytes with each of the cloned viruses. The growth kinetics, cytokine dependence, and cell surface markers of the infected T-cell cultures were similar for each provirus clone. Thus, ORFs X-I and X-II are not essential for virus infectivity, replication, gene expression, or T-cell immortalization in vitro.

Animals↗
PubMed1992

Open reading frames encoding a protein kinase, homolog of glycoprotein gX of pseudorabies virus, and a novel glycoprotein map within the unique short segment of equine herpesvirus type 1.

DNA sequence analysis of the unique short (Us) segment of the genome of equine herpesvirus type 1 Kentucky A strain (EHV-1) by our laboratory and strains Kentucky D and AB1 by other workers identifies a total of nine open reading frames (ORF). In this report, we present the DNA sequence of three of these newly identified ORFs, designated EUS 2, EUS 3, and EUS 4. The EUS 2 ORF is 1146 nucleotides (nt) in length and encodes a potential protein of 382 amino acids. Cis-regulatory sequences upstream of the putative ATG start codon include a G/C box 112 nt upstream and two potential TATA-like elements located between 15 and 90 nt before the ATG. The EUS 2 translation product exhibits significant homology to Ser/Thr protein kinases encoded within the Us segments of other herpesviruses, such as herpes simplex virus (26% homology) and pseudorabies virus (PRV), (45% homology), and possesses sequence domains conserved in protein kinases of cellular and viral origin. The EUS 3 ORF begins 127 nt downstream from the EUS 2 stop codon and ends at a stop codon 1119 nt further downstream. A single TATA-like element maps 61 nt upstream of the ORF. This ORF encodes a potential protein of 373 amino acids and is a homolog of glycoprotein gX of PRV, as judged by overall homology of amino acid residues, cysteine displacement, and presence of potential glycosylation sites and signal sequence. Interestingly, the EUS 4 ORF encodes a potential membrane glycoprotein that does not exhibit homology to any reported protein sequence. The EUS 4 ORF encodes a 383 amino acid polypeptide with a sequence indicative of a signal sequence at its amino terminal end, glycosylation sites for N-linked oligosaccharides, and a transmembrane domain near its carboxyl terminus. Several cis-acting regulatory sequences lie upstream of this ORF. These findings support the observation that the short region of alphaherpesviruses show considerable variation in their genetic content and gene organization.

Amino Acid Sequence↗
PubMed1989

Sequences that surround the stop codons of upstream open reading frames in GCN4 mRNA determine their distinct functions in translational control.

Translational control of the GCN4 gene of Saccharomyces cerevisiae requires at least two of the four short upstream open reading frames (URFs) in the leader of GCN4 mRNA. URF4 is a strong negative element that is sufficient for repression of GCN4 expression in normal growth conditions. URF1 is approximately 30-fold less effective as a translational barrier when it is the single URF present in the mRNA leader and is required upstream from URF4 for efficient derepression of GCN4 expression under amino acid starvation conditions. We show that the last codon plus 10 bp immediately after the stop codon of URF4 are sufficient to convert URF1 into a strong translational barrier when it is present as a solitary URF. This result suggests that the characteristics of translation termination at URF4 are responsible largely for its strong inhibitory effect on translation initiation at the GCN4 AUG codon. Introduction of the same URF4 sequences at URF1 also reduces GCN4 expression under derepressing conditions when URF1 is upstream from URF4. This fact suggests that URF1 translation normally is compatible with efficient scanning and initiation downstream and that this property is required for its ability to overcome the translational barrier at URF4. These findings are consistent with the idea that ribosomes must first translate URF1 and then resume scanning in order to traverse URF4 sequences under starvation conditions. Our results indicate that nucleotides located 3' to the stop codon can be as important as those located 5' to the start site in determining the inhibitory effect of an URF on translation initiation downstream.

Base Sequence↗