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At least 811 records · Page 45Linked to original sources
PubMed1993

Human coronavirus OC43 RNA 4 lacks two open reading frames located downstream of the S gene of bovine coronavirus.

Nucleotide sequences between the spike (S) and membrane (M) protein genes of the OC43 strain of human corona-virus were obtained from PCR-amplified viral mRNAs. Sequence analysis of this region revealed the presence of two ORFs encoding proteins of 12.9 and 9.5 kDa. These two proteins were identified as putatively nonstructural (ns) due to their homology to the corresponding BCV ns gene products. Northern blot analysis indicated that each of these two genes was present on a separate mRNA (5 and 5-1, respectively). In vitro translation analyses demonstrated that the HCV-OC43 9.5-kDa protein, like its BCV counterpart, is poorly translated when situated downstream of the 12.9-kDa ORF, although immunofluorescence studies did confirm its presence in infected cells. Sequence analysis showed that a large portion of the 3'-end of the leader sequence is present within the viral genome, upstream of the 12.9-kDa ORF. In addition, two ORFs encoding potential 4.9- and 4.8-kDa ns proteins in BCV are absent in HCV-OC43, although a corresponding mRNA 4 was found at a very low level. These results demonstrate that these two putative ns proteins are not essential for virus replication, at least in HRT-18 cells.

Amino Acid Sequence↗
PubMed1997

The genome-linked protein of potato leafroll virus is located downstream of the putative protease domain of the ORF1 product.

The sequence of the 32 N-terminal amino acids of the protein (VPg) which is covalently linked to the RNA of potato leafroll virus has been determined. The obtained VPg sequence mapped to position 400 to 431 of the PLRV ORF1 product, downstream of the putative protease domain and in front of the RNA-dependent RNA polymerase. Comparison with other viral sequences revealed significant similarities with the ORF1 products of beet western yellows virus, cucurbit aphid-borne yellows virus, and beet mild yellowing virus.

Amino Acid Sequence↗
PubMed1999

Inclusion of the NS2-specific exon in minute virus of mice mRNA is facilitated by an intronic splicing enhancer that affects definition of the downstream small intron.

Alternative splicing of pre-mRNAs plays a critical role in maximizing the coding capacity of the small parvovirus genome. The small-intron region of minute virus of mice (MVM) pre-mRNAs undergoes an unusual pattern of overlapping alternative splicing, using two donors, D1 and D2, and two acceptors, A1 and A2, within a region of 120 nucleotides, that governs the steady-state ratios of the various viral mRNAs. In a previous report we demonstrated that a complex interaction between both donor and acceptor sequences, as well as the constraints of size, defines the small intron and governs its alternative splicing. We also identified a G-rich intronic splicing enhancer sequence (IES) that appeared to function as both an intron- and an exon-defining element. In this report we further examined the components that govern MVM small-intron splicing. In fully processed wild-type mRNAs, A1 is used preferentially over A2. In this report, we show that in the context of the wild-type small intron the position of the downstream acceptor A2 was preferred, and the primary sequence of A1 must be stronger for it to be utilized at wild-type efficiency. Use of A2 in generation of the minor spliced forms D2/A2 required the IES because of a weak A2 polypyrimidine tract and because of the relative strength of A1. The small size of the intron and the relative position of the IES were also shown to play a critical role in donor and acceptor site selection. Finally, we have further characterized how the IES functions as an intronic enhancer of upstream exon definition. When the small intron was expanded, upstream exon inclusion was dependent upon the position of the IES. Within the context of the small intron, alterations of the small intron that overcame the requirement for the IES for splicing to A2 also permitted wild-type levels of upstream exon inclusion in the absence of the IES, suggesting that, in its natural context, the IES facilitates upstream exon inclusion by affecting small-intron definition.

Alternative Splicing↗
PubMed2000

Downstream ribosomal entry for translation of coronavirus TGEV gene 3b.

Gene 3b (ORF 3b) in porcine transmissible gastroenteritis coronavirus (TGEV) encodes a putative nonstructural polypeptide of 27.7 kDa with unknown function that during translation in vitro is capable of becoming a glycosylated integral membrane protein of 31 kDa. In the virulent Miller strain of TGEV, ORF 3b is 5'-terminal on mRNA 3-1 and is presumably translated following 5' cap-dependent ribosomal entry. For three other strains of TGEV, the virulent British FS772/70 and Taiwanese TFI and avirulent Purdue-116, mRNA species 3-1 is not made and ORF 3b is present as a non-overlapping second ORF on mRNA 3. ORF 3b begins at base 432 on mRNA 3 in Purdue strain. In vitro expression of ORF 3b from Purdue mRNA 3-like transcripts did not fully conform to a predicted leaky scanning pattern, suggesting ribosomes might also be entering internally. With mRNA 3-like transcripts modified to carry large ORFs upstream of ORF 3a, it was demonstrated that ribosomes can reach ORF 3b by entering at a distant downstream site in a manner resembling ribosomal shunting. Deletion analysis failed to identify a postulated internal ribosomal entry structure (IRES) within ORF 3a. The results indicate that an internal entry mechanism, possibly in conjunction with leaky scanning, is used for the expression of ORF 3b from TGEV mRNA 3. One possible consequence of this feature is that ORF 3b might also be expressed from mRNAs 1 and 2.

Animals↗
PubMed2002

Murine coronavirus-induced apoptosis in 17Cl-1 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and bid cleavage.

Mouse hepatitis virus (MHV) infection in murine 17Cl-1 cells results in apoptotic cell death. Inhibition of MHV-induced apoptosis by the pancaspase inhibitor Z-VAD-FMK promoted virus production late in infection, indicating that apoptosis could be a host response to limit the production of viral progeny. Activation of the mitochondria-mediated apoptotic pathway was indicated by the activation of caspase-9 and delay of apoptosis by Bcl-2 overexpression. Analyses of the subcellular distribution of cytochrome c, procaspase-9, and Apaf-1 suggested an aberrant apoptosome formation in the vicinity of the mitochondria, which could be a cell type-specific event. An increase in the amount of Fas (APO-1/CD95), caspase-8 activation, caspase-8-mediated Bid cleavage, and subsequent translocation of truncated Bid to mitochondria, all of which relate to the Fas-mediated pathway, also occurred in MHV-infected 17Cl-1 cells, whereas the formation of the death-inducing signaling complex, a direct indication of the activation of Fas-mediated pathway, was undetectable. Caspase-8 and Bid activation appeared to be downstream of mitochondria, because Bcl-2 overexpression suppressed both events, suggesting that infected 17Cl-1 cells might have activated a receptor-mediated "type II" signaling pathway, in which primary and low levels of receptor-mediated pathway activation lead to the activation of the mitochondria-mediated pathway. All our data indicate that a mitochondria-mediated pathway played a major regulatory role in apoptosis in MHV-infected 17Cl-1 cells.

Animals↗
PubMed1999

Transgenic plants for therapeutic proteins: linking upstream and downstream strategies.

We have described two very different and innovative plant-based production systems--postharvest production and recovery of recombinant product from tobacco leaves using an inducible promoter and oleosin-mediated recovery of recombinant product from oilseeds using a seed-specific promoter. Both base technologies are broadly applicable to numerous classes of pharmaceutical and industrial proteins. As with any emerging technology, the key to success may lie in identifying those products and applications that would most benefit from the unique advantages offered by each system. The postharvest tobacco leaf system appears effective for proteins requiring complex posttranslational processing and endomembrane targeting. Because of the remarkable fecundity and biomass production capacity of tobacco, biomass scale-up is very rapid and production costs are low. Clearly the development of equally cost-effective extraction and purification technologies will be critical for full realization of the commercial opportunities afforded by transgenic plant-based bioproduction. The recovery of protein from tobacco leaves or oleosin-partitioned proteins by oil-body separations represent significant break-throughs for cost-effective commercialization strategies. Additional low-cost, high-affinity separation technologies need to be developed for effective scale-up purification of plant-synthesized recombinant proteins. Clearly successful commercialization of plant-synthesized biopharmaceuticals must effectively link upstream strategies involving gene and protein design with downstream strategies for reproducible GMP-level recovery of bioactive recombinant protein. Both the tobacco and oilseed systems are uniquely designed to address issues of biomass storage, product recovery, quality assurance, and regulatory scrutiny in addition to issues of transgene expression and protein processing.

Animals↗
PubMed1993

The Rubisco activase (rca) gene is located downstream from rbcS in Anabaena sp. strain CA and is detected in other Anabaena/Nostoc strains.

A gene encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (rca) was found downstream from the rbcLrbcS operon in the heterocystous cyanobacterium Anabaena sp. strain CA. Two unknown open reading frames were shown to be located between rbcS and rca in strain CA and all the genes, rbcLrbcS, ORF1, ORF2, and rca were in the same transcriptional orientation. The deduced amino acid sequence of the Anabaena Rubisco activase showed both similarities and differences to the plant enzyme with considerable differences at the carboxy and amino termini. Proposed ATP-binding sites were conserved in the cyanobacterial protein. Recombinant cyanobacterial Rubisco activase, however, reacted with antisera to spinach Rubisco activase. Hybridization studies, using the Anabaena sp. strain CA rca gene as a heterologous probe, detected homologous sequences in heterocystous Anabaena/Nostoc strains but not in unicellular or nonheterocystous filamentous cyanobacteria, suggestive of a close evolutionary relationship of chloroplasts and heterocystous cyanobacteria.

Amino Acid Sequence↗
PubMed1991

Transcriptional activation by upstream activator sequences requires distinct interactions with downstream elements in the yeast TRP1 promoter.

The interactions between different upstream activator sequences (UAS) and the downstream transcriptional elements of the TRP1 promoter were studied. We have inserted the UAS from the PGK gene into a series of TRP1 promoter deletions such that the PGK UAS is positioned at various distances upstream from or replaces the TRP1 UAS (UAST1). We show that activation of the TRP1 transcription unit I by the PGK UAS shows a marked position dependence, which is solely a function of the position of the PGKUAS relative to sequences involved in the determination of the RNA initiation sites in the TRP1 promoter. No cooperative activation is seen when both UASs are present in the promoter; the PGK UAS is dominant and is not repressed by the TRP1 negative element. In addition, we show that the PGK and TRP1 UASs interact differently with TATA sequence at the TRP1 RNA initiation site. Our results suggest that these UASs are functionally distinct because they use different mechanisms for activating heterologous promoters.

Aldose-Ketose Isomerases↗
PubMed1993

The ORF encoding a putative ferredoxin-like protein downstream of the vnfH gene in Azotobacter vinelandii is involved in the vanadium-dependent alternative pathway of nitrogen fixation.

An open reading frame (ORF) in the same operon as, but downstream of, vnfH in Azotobacter vinelandii can code for a ferredoxin-like protein. The role this ORF may play in the vnf (vanadium-dependent alternative) pathway of nitrogen fixation was investigated. Site-directed mutagenesis was used to alter one base in each of the codons specifying amino acids 18 and 19 generating a unique Bg/II site. A kanamycin resistance cartridge was cloned into the Bg/II site. This construct was mobilized into A. vinelandii CA12 (delta nifHDK) strain by conjugation and the mutation was introduced into the genome by marker exchange. The resulting mutant was unable to fix nitrogen under conditions in which the vnf pathway of nitrogen fixation operates. This suggests that this ORF is functional and is essential for the vanadium-dependent alternative pathway of nitrogen fixation in A. vinelandii.

Amino Acid Sequence↗
PubMed1992

Downstream activating sequence within the coding region of a yeast gene: specific binding in vitro of RAP1 protein.

Using a gel retardation assay, a protein factor that specifically interacts with a 33 bp intragenic sequence of the highly expressed and glucose-inducible SRP1 gene of Saccharomyces cerevisiae has been detected. This binding site is located in a transcribed region and within the open reading frame (positions +710 to +743 relative to the first base of the initiation codon). A mutant strain carrying a deletion of this binding site showed a dramatic decrease in steady-state levels of SRP1 transcripts. This decline is not the result of a decrease in mRNA stability, since expression of hybrid genes in which the SRP1 promoter was replaced by the heterologous CYC1 promoter was not affected by the binding site deletion. These findings suggest that the 33 bp sequence contains a cis-acting downstream activating element which is involved in the transcriptional activation of the SRP1 promoter. Sequence comparisons showed similarities between a site located within the 33 bp sequence and the high-affinity consensus binding site of the RAP1/GRF1 (also named TUF) factor and methylation interference experiments confirmed that this site was involved in the protein-DNA interaction. Both the results of competition experiments with upstream activating sequences of ribosomal protein genes (UASrpg), which are targets for RAP1 binding, and determination of the apparent molecular weight of the affinity-purified DNA-binding protein indicated that RAP1 factor recognized the SRP1 33 bp element. The 33 bp sequence was found to be unable to provide UAS activity when placed upstream of the TATA box and transcription start site.

Amino Acid Sequence↗
PubMed1989

Differential inhibition of downstream gene expression by the cauliflower mosaic virus 35S RNA leader.

The effect of the 600 nucleotide-long CaMV 35S RNA 5' leader sequence on the expression of downstream genes was analyzed both in plant protoplasts and in vitro. For transient expression studies in protoplasts derived from host and nonhost plants, the bacterial chloramphenicol acetyl transferase (CAT) gene was fused to the initiation codon of ORF VII. The leader sequence reduced CAT expression two- to four-fold in protoplasts derived from three host species, but 10- to 50-fold in protoplasts derived from three different nonhost species. For in-vitro studies the 35S promoter was replaced by the SP6 promoter. The leader reduced in-vitro translation of SP6 transcripts approximately six-fold, indicating that at least part of the inhibition observed in protoplasts is directly due to the interference of the leader sequence with translation. Other steps in gene expression that may also be affected are discussed.

Chloramphenicol O-Acetyltransferase↗
PubMed1989

Transcription of a region downstream from lambda ori is required for replication of plasmids derived from coliphage lambda.

DNA replication of lambda phage depends on transcriptional activation at or around the lambda ori region by RNA polymerase. To elucidate the function of the transcriptional activation, we constructed several plasmids carrying lambda ori and lacP, whose relative locations and directions were different from each other, and studied replication activity of these recombinant plasmids. Transcription in a region immediately downstream from lambda ori, but not in the lambda ori region, was found to be essential for plasmid replication. Transcription proceeding over a certain minimal length was required and only rightward-directed transcription was effective for the activation.

Bacteriophage lambda↗
PubMed1983

Tn5 carries a streptomycin resistance determinant downstream from the kanamycin resistance gene.

In Rhizobium meliloti, Tn5 conferred resistance not only to kanamycin but to streptomycin, as well, in Escherichia coli, however only to kanamycin. Using in vitro recombinant DNA techniques, it was shown that the streptomycin resistance determinant was located downstream from the kanamycin resistance gene in the unique central region of Tn5. Expression of various cloned fragments of Tn5 suggested that both kanamycin and streptomycin resistance genes were transcribed from the same promoter. E. coli mutants allowing the expression of streptomycin resistance from Tn5 were isolated. The differential expression of the streptomycin resistance gene provides a simple selection/counterselection criterion, using only streptomycin in transfer experiments of Tn5 between E. coli and R. meliloti.

Chromosome Mapping↗
PubMed1986

The Escherichia coli heat shock regulatory gene is immediately downstream of a cell division operon: the fam mutation is allelic with rpoH.

Several mutations which affect critical cell functions in Escherichia coli map at 76 min on the chromosome. The genes which map in this region are the cell division genes fts Y, E, X and S, the heat shock regulatory gene rpoH/htpR/hin, the lipoprotein biogenesis gene fam and another essential gene dnaM. We determined the relative positions of most of these genes and show that the rpoH gene lies immediately downstream of the last gene (ftsX) of a cell division operon and is transcribed in the same direction. We also show that the fam-715 mutation is allelic with rpoH and so the conditional lipoprotein deficiency of the fam mutation must be due to the pleiotropic nature of the heat shock response.

Alleles↗
PubMed1988

A downstream activator sequence regulates the expression of the yeast transposon.

The regulation of transcription of the class II yeast transposon Ty 1-17 has been examined using a Ty::LEU2 fusion gene. Deletion analysis has defined a 251 bp region within the coding sequence of the TY A gene which is essential for Ty transcription and is also required for diploid control of Ty expression. This downstream activator sequence (DAS) contains two sequence blocks which show homology to the SV40 enhancer core and three with homology to the diploid-regulation-site from the yeast MAT gene.

Amino Acid Sequence↗
PubMed1992

The non-dosage compensated LSP1-alpha gene of Drosophila melanogaster lies immediately downstream of the dosage compensated L12 gene.

The X-linked gene LSP1-alpha of Drosophila melanogaster, expressed in the third larval instar, does not exhibit dosage compensation at its normal locus but does compensate when it is relocated to ectopic sites on the X chromosome. A transcription unit designated L12, which is active in the second larval instar and capable of encoding a putative protein of 28.5 kDa, lies immediately downstream from LSP1-alpha. We have determined that L12 is dosage compensated by measuring the steady-state level of its transcript in male and female larvae. The difference in response of these two adjacent genes should be taken into consideration when models of the mechanism of dosage compensation are formulated.

Animals↗
PubMed1994

Sequence of the region downstream of the Vitreoscilla hemoglobin gene: vgb is not part of a multigene operon.

The 1668 base pairs (bp) downstream of the Vitreoscilla hemoglobin gene were sequenced in the hope of finding related genes that might be part of an operon. Instead, a sequence was found that constituted an open reading frame (ORF) of 569 amino acids (apparently the carboxy-terminal part of a larger ORF), in the direction opposite to the hemoglobin gene. This sequence was found to have 64% similarity with the 1685 bp at the 3' end of the Escherichia coli uvrA gene. The inferred amino acid sequence of the Vitreoscilla DNA has 69% similarity with the corresponding sequence of the E. coli uvrA protein, with similarities of 90, 100, and 85% in the helix-turn-helix, C-terminal ATP binding, and C-terminal zinc finger domains, respectively. The distance between the 3' ends of the Vitreoscilla hemoglobin and uvrA genes is 63 bp.

Adenosine Triphosphatases↗
PubMed1995

Does luminal release of EDRF contribute to downstream microvascular tone?

The contribution of luminally released endothelium-derived relaxing factor (EDRF) transported with the flowing blood to the control of smooth muscle tone of downstream arterioles was studied. The dilatory response of arterioles in the spinotrapezius muscle of the rat to superfusion of acetylcholine (ACH) was investigated by intravital microscopy before and after blockade of blood flow by micropipette occlusion upstream of the site of observation. Vessels were studied without treatment, after topical application of indomethacin (INDO, 3 10(-5) M) to inhibit prostanoid production and after local treatment with NG-nitro-L-arginine (LNNA, 2.9 10(-3) M) to suppress local production of EDRF. In untreated vessels and after INDO, responses to ACH were not significantly different in the presence or absence of blood flow. After LNNA, the responses to ACH were reduced to about 50%, but were again similar with or without flow. These results indicate that luminally transported EDRF does not play a significant role in controlling arteriolar tone in small skeletal muscle arterioles.

Acetylcholine↗