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Biomedical subjects

A G Hunt

Publications and source records attributed to A G Hunt.

At least 37 records · Page 2Linked to original sources
PubMed1992

Purification and Characterization of a 70-Kilodalton Polyadenylate-Binding Protein from Pea (Pisum sativum).

A polyadenylate-binding protein (PABP) was purified from cell-free extracts prepared from pea seedlings (Pisum sativum) by ammonium sulfate precipitation and Affi-Gel Blue and polyadenylate-Sepharose 4B affinity chromatography. The final preparation from polyadenylate-Sepharose 4B columns contained a single 70-kilodalton polypeptide with high polyadenylate-binding activity. The purified protein was active over a broad range of ionic strengths and showed temperature and pH optima of 37 degrees C and pH 6.5, respectively. Specificity studies indicated that the pea PABP was most active with polyadenylic acids, showed some activity with polyguanylic acid, and did not bind to polycytidylic acid. Moreover, longer polyadenylate molecules were bound more effectively than shorter ones. Because these properties are similar to PABPs isolated from other sources, we conclude that we have identified, purified, and characterized a plant PABP analogous to those described in yeast and animal systems.

Journal Article↗
PubMed1992

Several distinct types of sequence elements are required for efficient mRNA 3' end formation in a pea rbcS gene.

We have conducted an extensive linker substitution analysis of the polyadenylation signal from a pea rbcS gene. From these studies, we can identify at least two, and perhaps three, distinct classes of cis element involved in mRNA 3' end formation in this gene. One of these, termed the far-upstream element, is located between 60 and 120 nt upstream from its associated polyadenylation sites and appears to be largely composed of a series of UG motifs. A second, termed the near-upstream element, is more proximate to poly(A) sites and may be functionally analogous to the mammalian polyadenylation signal AAUAAA, even though the actual sequences involved may not be AAUAAA. The third possible class is the putative cleavage and polyadenylation site itself. We find that the rbcS-E9 far-upstream element can replace the analogous element in another plant polyadenylation signal, that from cauliflower mosaic virus, and that one near-upstream element can function with either of two poly(A) sites. Thus, these different cis elements are largely interchangeable. Our studies indicate that a cellular plant gene possesses upstream elements distinct from AAUAAA that are involved in mRNA 3' end formation and that plant genes probably have modular, multicomponent polyadenylation signals.

Base Sequence↗
PubMed1991

Characterization of the polyadenylation signal from the T-DNA-encoded octopine synthase gene.

We have characterized the polyadenylation signal from the octopine synthase (ocs) gene. This signal directs mRNA 3' end formation at a number of distinct sites. A combination of deletion and linker-substitution analyses revealed that each of these sites is controlled by multiple upstream sequence elements. Upstream sequences relatively far (greater than 80 nt) from the ocs poly[A] sites were found to be needed for functioning of these sites. Upstream sequences nearer to poly [A] sites were also found to be involved in mRNA 3' end formation in the ocs gene. In addition, a set of novel elements that mediates 3' end choice was uncovered by deletion analysis of sequences downstream from the ocs polyadenylation sites. Our experiments indicate mRNA 3' end formation in the ocs is controlled by a complex series of cis-acting signals, and suggest that the process of mRNA 3' end formation might be linked to transcription termination.

Agrobacterium tumefaciens↗
PubMed1991

The SV40 small t intron is accurately and efficiently spliced in tobacco cells.

We have introduced the SV40 small t intron into tobacco cells as part of a cauliflower mosaic virus 35S promoter-chloramphenicol acetyltransferase-SV40 transcription unit. We find that the small t intron is efficiently and accurately spliced in transgenic tobacco cells that carry this transcription unit. Our results indicate that there is substantial conservation of RNA processing signals between plants and animals, more than has been previously assumed. They also suggest that pre-mRNA processing in plants requires multiple branch sites for efficient processing.

Antigens, Polyomavirus Transforming↗
PubMed1991

A tyrosine residue in the small nuclear inclusion protein of tobacco vein mottling virus links the VPg to the viral RNA.

The identity of the amino acid residue that links the VPg of the potyvirus tobacco vein mottling virus (TVMV) to the viral RNA was determined. 32P-labeled TVMV RNA was digested with RNase A and micrococcal nuclease. The resulting 32P-labeled VPg was isolated and partially hydrolyzed with 6 N HCl at 110 degrees C for 2 h. Analysis by thin-layer electrophoresis revealed the presence of [32P]phosphotyrosine but not [32P]phosphoserine or [32P]phosphothreonine. Another preparation of TVMV RNA was treated with endoproteinase Lys-C, and the resulting peptide-RNA was purified by sodium dodecyl sulfate-sucrose gradient centrifugation. The sequence of the N-terminal 15 amino acid residues of the peptide, when compared with the RNA-derived amino acid sequence of the TVMV polyprotein, demonstrated that the peptide occurs in the small nuclear inclusion protein. These data suggest that Tyr-1860 of the polyprotein is the amino acid residue that links the TVMV VPg to the viral RNA.

Amino Acid Sequence↗
PubMed1990

The VPg of tobacco etch virus RNA is the 49-kDa proteinase or the N-terminal 24-kDa part of the proteinase.

Preparations of tobacco etch virus (TEV) RNA which were purified by sucrose gradient centrifugation, digested with RNase, and analyzed by SDS-polyacrylamide gel electrophoresis contained proteins of 49, 32, and 24 kDa. The 49- and 24-kDa proteins reacted with polyclonal antiserum to the TEV 49-kDa proteinase while the 32-kDa protein reacted with anti-TEV serum. Further purification of the RNA by centrifugation through CsCl removed the coat protein (32 kDa), but not the 49- and 24-kDa proteins. The 49- and 24-kDa proteins did not migrate into a polyacrylamdie gel when the RNA was not digested with RNase. These results indicate that the VPg of TEV is either the 49-kDa proteinase or the 24 kDa that represents the amino-terminal half thereof.

Centrifugation, Density Gradient↗
PubMed1990

Upstream sequences other than AAUAAA are required for efficient messenger RNA 3'-end formation in plants.

We have characterized the upstream nucleotide sequences involved in mRNA 3'-end formation in the 3' regions of the cauliflower mosaic virus (CaMV) 19S/35S transcription unit and a pea gene encoding ribulose-1,5-bisphosphate carboxylase small subunit (rbcS). Sequences between 57 bases and 181 bases upstream from the CaMV polyadenylation site were required for efficient polyadenylation at this site. In addition, an AAUAAA sequence located 13 bases to 18 bases upstream from this site was also important for efficient mRNA 3'-end formation. An element located between 60 bases and 137 bases upstream from the poly(A) addition sites in a pea rbcS gene was needed for functioning of these sites. The CaMV -181/-57 and rbcS -137/-60 elements were different in location and sequence composition from upstream sequences needed for polyadenylation in mammalian genes, but resembled the signals that direct mRNA 3'-end formation in yeast. However, the role of the AAUAAA motif in 3'-end formation in the CaMV 3' region was reminiscent of mRNA polyadenylation in animals. We suggest that multiple elements are involved in mRNA 3'-end formation in plants, and that interactions of different components of the plant polyadenylation apparatus with their respective sequence elements and with each other are needed for efficient mRNA 3'-end formation.

Base Sequence↗
PubMed1989

Expression of a potyvirus non-structural protein in transgenic tobacco.

A cDNA fragment encoding the cytoplasmic inclusion protein of tobacco vein mottling virus was inserted into the plant expression cassette of a Ti plasmid-based binary vector. The vector was transferred to Agrobacterium tumifaciens, and following a modified leaf disc procedure, transformed tobacco plants were obtained. Analysis of poly(A)+ RNA from transgenic plants revealed a novel RNA of approximately 2100 nucleotides possessing tobacco vein mottling virus sequences. Also, immunoprecipitation of protein extracts of [35S]methionine-labeled transformed callus using anti-cytoplasmic inclusion protein antiserum revealed a polypeptide of approximately 70 kDa. This size is consistent with that predicted from the inserted tobacco vein mottling virus coding sequences. Together these data demonstrate the expression of the cytoplasmic inclusion protein in the absence of viral infections.

Blotting, Northern↗
PubMed1989

Deletion analysis of the polyadenylation signal of a pea ribulose-1,5-bisphosphate carboxylase small-subunit gene.

The polyadenylation signal of a pea gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS) has been analyzed by deletion mutagenesis and Ti plasmid-mediated gene transfer. Sequences between 6 and 137 bases upstream from the normal polyadenylation sites in this gene (bases -6 to -137) are required for functioning of these sites. In addition, bases -111 to -235 can affect 3' end formation by altering the pattern of 3' termini seen in various transcription units. Sequences between 37 and 95 bases upstream from a cryptic polyadenylation site in this gene [A. G. Hunt, DNA 7: 329-336 (1988)] are necessary for mRNA 3' end formation at this site. At least two different parts of the 3' region of this rbcS gene can serve as a downstream element for polyadenylation at the normal poly(A) addition sites in this gene. Our studies indicate that: 1. the upstream sequences required for polyadenylation in plants are different from those defined in mammalian RNA polymerase II transcription units; 2. sequences 100 or more bases upstream and downstream from poly(A) addition sites in this gene can affect poly(A) addition site choice; and 3. there are apparently redundant downstream elements for polyadenylation in this gene.

Base Sequence↗
PubMed1989

Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus.

Full-length cDNA copies of tobacco vein mottling virus (TVMV) RNA were constructed downstream from bacteriophage T7 or T3 RNA polymerase promoters. The plasmids were designed to produce in vitro transcripts containing, respectively, one or two guanosine residues at the 5' terminus not derived from the TVMV sequence and a single cytidine residue at the 3' terminus following the poly(A) tail. Introduction of transcripts from either plasmid into tobacco mesophyll protoplasts resulted in the accumulation of TVMV coat protein and RNA. Neither coat protein nor viral RNA accumulated in protoplasts inoculated with linearized cDNA or with in vitro transcripts synthesized in the absence of 7-methylguanosine(5')triphospho(5')guanosine (m7GpppG). Tobacco seedlings inoculated with native TVMV RNA developed symptoms a few days before those inoculated with in vitro transcripts; however, 3 weeks after inoculation, the symptoms produced by the two inocula were indistinguishable.

Blotting, Western↗
PubMed1989

Expression in transgenic plants of a viral gene product that mediates insect transmission of potyviruses.

Helper component (HC) is a virus-encoded nonstructural protein that is required for transmission of potyviruses by their aphid vectors. As a prelude to studies on the molecular basis of HC activity, a cDNA clone (pPB-3) was constructed that contained the first three cistrons (34 kDa-HC-42 kDa) of the RNA genome of the potyvirus tobacco vein mottling virus, the first six nucleotides of the adjacent cylindrical inclusion body protein cistron, and a synthetic translation termination codon. This construction was introduced into tobacco cells via a Ti plasmid-based vector. Northern blot analysis of transgenic plants demonstrated the presence of an RNA of the size expected from the construction of pPB-3, and Western blot analysis revealed the presence of a protein that comigrated with authentic HC, indicating that the proteolytic activity necessary to produce mature-sized HC was encoded by pPB-3. The HC produced in the transgenic plants was demonstrated to be active in a virus transmission bioassay with aphids.

Animals↗
PubMed1989

Inheritance and expression of the mouse metallothionein gene in tobacco: impact on cd tolerance and tissue cd distribution in seedlings.

Genetically engineered seedlings obtained from self-fertilized transgenic tobacco (Nicotiana tabacum) contained and expressed the mouse metallothionein and kanamycin resistance marker genes and were more tolerant to cadmium stress than untransformed controls. Cadmium accumulation in leaves of transgenic seedlings exposed to a low, field-like Cd concentration (0.02 micromolar) was about 20% lower than that in untransformed controls. Genetic analysis of R1 and R2 progeny showed inheritance of the marker gene to be as a dominant Mendelian trait. These results suggest the possibility of developing transgenic plants with modified tolerance to heavy metal stress and food crops having lower Cd content.

Journal Article↗
PubMed1988

Seed-transmissible expression of mammalian metallothionein in transgenic tobacco.

A binary plasmid was constructed to contain the mouse metallothionein c-DNA, the constitutive 35S promoter from cauliflower mosaic virus, the polyadenylation signal from the pea rbcS-E9 gene and several selectable markers. The plasmid was transferred to Agrobacterium tumefaciens and the leaf disc method was used to transform tobacco. Callus and shoots were regenerated in the presence of kanamycin and transformed plants were obtained. Southern, Northern and Western blot analysis demonstrated integration and expression of the metallothionein gene in transformed callus and transgenic plants. The gene is transmitted to and expressed in seed derived progeny as a dominant Mendelian trait.

Animals↗
PubMed1988

Identification and characterization of cryptic polyadenylation sites in the 3' region of a pea ribulose-1,5-bisphosphate carboxylase small subunit gene.

The polyadenylation signal of a pea ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS) gene has been studied using in vitro mutagenesis and Ti plasmid-mediated transformation of tobacco. Analysis of a mutant that is lacking sequences upstream from -6 (relative to the "normal" site of polyadenylation of RNAs from the rbcS-E9 gene) reveals a number of alternate polyadenylation sites located downstream from the normal poly(A) site. Transcripts carrying these sequences end at any one of at least seven sites between 20 and 300 bases downstream from the normal site. These sites are seen in populations of transgenic plant cells, and also in independent transgenic plants.

Base Sequence↗
PubMed1987

Design and construction of a versatile system for the expression of foreign genes in plants.

We have built a series of vectors to allow the constitutive or light-regulated expression of foreign genes in plants. These vectors carry expression cassettes consisting of either the cauliflower mosaic virus 35S promoter or the pea rbcS-E9 promoter, a multiple cloning site derived from M13um20, and the rbcS-E9 polyadenylation site. These cassettes have been incorporated into pBR322-based or RK2-based replicons to facilitate direct DNA uptake or Agrobacterium tumefaciens-mediated gene transfer. Their application for the expression of a bacterial gene is described.

Base Sequence↗
PubMed1983

Phosphorus-31 nuclear magnetic resonance investigation of membrane vesicles from Escherichia coli.

Phosphorus-31 nuclear magnetic resonance studies of isolated membrane vesicles prepared from Escherichia coli PSM116 as described by Hunt and Hong [Hunt, A. G., & Hong, J.-S. (1981) J. Biol. Chem. 256, 11988-11991; Hunt, A. G., & Hong, J.-S. (1983) Biochemistry 22, 844-850] are detailed here. This strain harbored a recombinant plasmid containing the phosphoglycerate transport system from Salmonella typhimurium (pJH7). Evidence indicating a surprising metabolic diversity, such as the presence of the enzymes enolase and phosphoglycerate mutase, is presented. The nature of the energization of these membrane vesicles for transport as described by Hugenholtz et al. [Hugenholtz, J., Hong, J.-S., & Kaback, H. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3446-3449] is also discussed. Membrane vesicles prepared from the PSM116 strain do not form a transmembrane pH gradient when phosphoenolpyruvate is added. The present results show that phosphorus-31 nuclear magnetic resonance spectroscopy is an excellent tool to investigate the metabolism of membrane vesicles.

Cell Membrane↗