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T C Hall

Publications and source records attributed to T C Hall.

At least 19 recordsLinked to original sources
PubMed1992

Unique nucleotide differences in the conserved 3' termini of brome mosaic virus RNAs are maintained through their optimization of genome replication.

To explore the functionality and conservation of specific base differences in the 3' 200 nucleotides of brome mosaic virus (BMV) RNA-1 (1t) and RNA-2 (2t) with respect to the 3' end of RNA-3 (3t), all possible permutations were used to exchange these regions among the genomic RNAs. When all RNAs bore the 1t promoter, total RNA accumulation was only 15% of wild type; when the 2t or 3t promoter was present on all three RNAs total RNA accumulation was reduced to 30 or 35% of wild type. Two major processes were found to be involved in these dramatic differences. The first reflects the distinct and competitive strengths of the (-)-strand promoters in these sequences, which were shown to have a 3t greater than 1t greater than 2t hierarchy. The second is the importance of the context of upstream sequences in which the 3' promoter is placed. Important contributions of the 3t promoter in preferential amplification of RNA-3 were apparent from changed RNA 1 + 2: 3 ratios and reduced progeny accumulation from transfections using the RNA-3/1t chimera. These interactions contribute to temporal modulation as well as overall optimization of viral RNA functions, leading to selection and maintenance of the specific base differences present in the otherwise highly conserved 3' 200 nucleotides of each genomic RNA component of BMV.

Base Composition↗
PubMed1992

Requirement for ICR-like sequences in the replication of brome mosaic virus genomic RNA.

Previous studies using a brome mosaic virus (BMV) RNA-2 deletion mutant (pRNA-2 M/S) and additional derivatives as reporters established that viral sequences resembling internal control regions (ICRs) 1 and 2 of tRNA gene promoters are vital to (+)-strand replication in protoplasts. Transfer of these mutations to genomic RNA-2 and functional analysis in protoplast, local lesion, and systemic infections revealed a sequence-specific requirement for bases within the ICR2-like motif. Despite the low (generally less than 20% of wild-type) and sometimes undetectable levels of replication of these RNA-2 mutants, sufficient p2a protein was produced to support at least modest levels of RNA-1, -3 and -4 replication in protoplasts. However, only those RNA-2 ICR2 mutants supporting substantial replication of the viral genome in protoplasts were capable of establishing local lesions in Chenopodium hybridum and systemic infections in barley, further establishing the essential role of the ICR-like sequences in viral infectivity. Upon passage through a second set of barley plants, accumulation patterns for progeny from inocula containing certain RNA-2 mutants paralleled those from wild-type inocula, indicating repair of the introduced mutations. RNA stability and translatability were shown to be unaffected by the introduced mutations. BMV RNA-3 contains several ICR-like sequences, each of which was individually deleted. Whereas deletion of the 5'-terminal ICR2-like motif had little effect on RNA-3 accumulation in protoplasts or local lesion formation, it debilitated systemic spread in barley. Deletion of an internal ICR2-like motif at position 1100 decreased (+):(-) strand asymmetry from greater than 100:1 to 14:1, reduced RNA-3 replication in protoplasts to less than 15% of wild-type, and abolished local lesion and systemic infectivity.

Base Sequence↗
PubMed1992

The requirement for a 5' stem-loop structure in brome mosaic virus replication supports a new model for viral positive-strand RNA initiation.

Sequences with strong similarity to internal control regions 1 and 2 (ICR1 and -2; A and B boxes) of tRNA genes are found at the 5' termini of the genomic RNAs of brome mosaic virus (BMV) and other plant viruses. The functionality of these motifs was studied by introducing point mutations into the ICR2-like sequence of pRNA-2 M/S, a BMV RNA-2 deletion mutant that replicates in the presence of RNAs-1 and -2 but does not encode a functional viral protein. The accumulation of positive-strand progeny from pRNAs bearing single and double base substitutions was 70 to 91% lower than that of the wild type, while the addition of single bases at position 8 of this motif reduced replication by 80%. These dramatic decreases in positive-strand synthesis paralleled decreases in transcription seen (C. Traboni, G. Ciliberto, and R. Cortese, Cell 36:179-187, 1984) from a tRNAPro gene containing similar mutations, suggesting comparable functions for the ICR regions in protein factor binding and demonstrating that a wild-type composition of the virus ICR2-like motif is required for proper RNA replication. Substitutions introduced at bases surrounding the ICR2 motif yielded levels of pRNA replication that differed, depending on the maintenance of a putative 5' stem-loop structure in the positive strand of the viral genome. Mutations disrupting this positive-strand stem-loop while maintaining the integrity of the complementary negative-strand structure reduced pRNA replication by 85 to 97%. In contrast, disruption of the negative-strand structure while maintaining the positive-strand stem-loop did not reduce pRNA replication. Similar positive-strand structures can be predicted to form from 5' sequences of cucumber mosaic virus (strain Q) and cowpea chlorotic mottle virus RNAs-1 and -2, supporting the concept that such structures comprise an integral part of virus genomic positive-strand promoters. The requirement of a stem-loop structure present on the positive-strand provided the basis for a new model describing how these sequence and structural elements act in the production of virus positive-strand RNA.

Base Composition↗
PubMed1991

Anthracycline immunoconjugates prepared by a site-specific linkage via an amino-dextran intermediate carrier.

Anthracycline, either daunomycin or doxorubicin, was site specifically attached to the carbohydrate moiety of a monoclonal anticarcinoembryonic antigen antibody by using amino-dextran as the intermediate carrier. The reaction resulted in an immunoconjugate that contains approximately 20 to 25 molecules of drug per molecule of immunoglobulin G. Flow-cytometric studies revealed the retention of the antibody-binding activity. The immunoconjugate was cytotoxic to the target cells, as examined by the 75selenomethionine incorporation studies, and remained efficient for targeting a human colonic tumor (GW-39) in the nude mouse model. The conjugate possessed a greater antitumor activity against the subcutaneous tumor than either the free drug or an irrelevant antibody conjugate, and it was well tolerated by the animals at a much higher dose level than was the unconjugated drug.

Animals↗
PubMed1991

Positive and negative cis-acting DNA domains are required for spatial and temporal regulation of gene expression by a seed storage protein promoter.

Mutations affecting spatial and temporal regulation of a beta-phaseolin gene encoding the major storage protein of bean (Phaseolus vulgaris) were analyzed by stable and transient transformation approaches. The results substantiate the value of transient assays for rapid determination of the functionality of cis-acting sequences and the importance of stable transformation to identify tissue-specific determinants. Spatial information is specified primarily by two upstream activating sequences (UAS). UAS1 (-295 to -109) was sufficient for seed-specific expression from both homologous and heterologous (CaMV 35S) promoters. In situ localization of GUS expression in tobacco embryos demonstrated that UAS1 activity was restricted to the cotyledons and shoot meristem. A second positive domain, UAS2 (-468 to -391), extended gene activity to the hypocotyl. Temporal control of GUS expression was found to involve two negative regulatory sequences, NRS1 (-391 to -295) and NRS2 (-518 to -418), as well as the positive domain UAS1. The deletion of either negative element caused premature onset of GUS expression. These findings indicate combinatorial interactions between multiple sequence motifs specifying spatial information, and provide the first example of the involvement of negative elements in the temporal control of gene expression in higher plants.

Cloning, Molecular↗
PubMed1991

Differential accumulation of four phaseolin glycoforms in transgenic tobacco.

An intron-less phaseolin gene was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical beta-phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (beta wti-) and mutant phaseolin glycoforms (beta dgly1, beta dgly2 and beta dgly1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the beta dgly1 and beta dgly2 mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin beta dgly1,2 gene. Additionally, the profile of 25-29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.

Amino Acid Sequence↗
PubMed1991

Interference in trans with brome mosaic virus replication by RNA-2 bearing aminoacylation-deficient mutants.

The tRNA-like domain present at the 3' end of each of the three genomic RNAs of brome mosaic virus (BMV) encompasses the (-)-strand promoter essential for replication. The replicative competence of two BMV RNA-2 transcripts bearing mutations delta 5' and 5'AGA in the tRNA-like domain (previously shown by in vitro assays to be deficient in tyrosylation) was evaluated in barley protoplasts. Transfection of protoplasts with low (2 micrograms) amounts of delta 5'RNA-2, together with transcripts of wild-type RNA-1 and -3, not only incapacitated the replication of RNA-2 but also significantly interfered in trans with the synthesis and accumulation of the other viral RNAs. In contrast, RNA-2 mutants bearing either 5'AGA or M4 (a mutation yielding enhanced minus-strand replication activity in vitro) were inhibitory to viral replication only when present at a relatively high level (12 micrograms). Coinoculation of protoplasts with high levels (12 micrograms) of each of the three RNA-2 mutants and transcripts corresponding to wild-type RNA-1, -2 and -3 (2 micrograms each) revealed that the mutants were capable of competing in trans, resulting in greatly reduced accumulation of the viral RNA and suggesting that their expression from constitutive promoters in transgenic plants may provide protection against viral infection.

Base Sequence↗
PubMed1991

Regulation of (+):(-)-strand asymmetry in replication of brome mosaic virus RNA.

Transfection of barley protoplasts with brome mosaic virus (BMV) RNAs 1 + 2 in the absence of RNA-3 yielded a molar ratio for (+):(-)-strand progeny at 24 hr postinoculation near unity, whereas over 100-fold more (+)- than (-)-strand progeny accumulated in its presence. The presence of RNA-3 enhanced total (+)-strand RNA production 205-fold and that of RNAs 1 + 2 by 29-fold. In contrast, total (-)-strand RNA accumulation decreased by 68% and that for (-)RNAs 1 + 2 by 79% in the presence of RNA-3. Transfections containing an RNA-3 mutant (Gsgi----U RNA-3) that is incapable of yielding RNA-4 as a result of a single nucleotide substitution at the subgenomic RNA initiation site yielded only 66% of the (+):(-) asymmetry seen in the presence of wild-type RNA-3. Only 1.8-fold excess of (+)-over (-)-strand production was obtained for transfections that included delta SGP RNA-3, a deletion that includes the subgenomic promoter core and extends 43 nt into the RNA-4 sequence. Transfections containing RNA-3 mutants bearing frameshifts or deletions in the coat protein cistron yielded levels of asymmetry similar to those seen for Gsgi----U RNA-3. These findings implicate the subgenomic promoter and other sequences in the intercistronic region of RNA-3 as the primary determinants of asymmetric replication, although the coat protein may be an additional factor enhancing the accumulation of (+)-strand RNA.

Capsid↗
PubMed1991

Non-replicating deletion mutants of brome mosaic virus RNA-2 interfere with viral replication.

Naturally occurring defective interfering RNAs (DI-RNAs) and satellite RNAs greatly reduce the accumulation of their helper virus in vivo, but often modulate symptom expression in an unpredictable manner. Deletion mutants Nc/S, Na/M and Sa/Nc + M/S, derived from brome mosaic virus (BMV) RNA-2, failed to replicate when co-inoculated with BMV RNAs-1 and -2 to barley protoplasts. However, the inoculum RNA corresponding to these deletion mutants was extremely stable and could have been mistaken for plus-strand progeny had minus-strand progeny analysis been omitted. These results accentuate the need for such tests in evaluating the ability of mutant viral sequences to replicate. One of the mutants, Nc/S, effectively interfered with the accumulation of BMV RNAs-1 and -2 in barley protoplasts. This non-replicating interfering RNA was termed NRI RNA-2 Nc/S. When present with RNAs-1 and -2 at low inoculum amounts (1 microgram), NRI RNA-2 Nc/S reduced replication of RNA-2, the parental RNA, by 63% and preferentially interfered with minus-strand RNA accumulation. At higher levels (4 micrograms), it completely displaced replication of both RNAs-1 and -2. Mutations eliminating translation of a truncated p2a protein from NRI RNA-2 Nc/S did not alleviate the interference effect, demonstrating that a defective replicase protein was not responsible for the decreased accumulation of genomic RNA. At an NRI RNA: genomic RNA inoculum molar ratio of 1:1, NRI RNA-2 Nc/S reduced the accumulation of all helper virus RNAs by 55%. Since this reduction was seen for both wild-type RNA-3 and delta SGP RNA-3, a deletion mutant of RNA-3 that lacks the subgenomic promoter necessary for coat protein expression, it was evident that the effective interference mediated by NRI RNA-2 Nc/S was not mitigated by encapsidation. The ability of the NRI RNAs to mimic satellite DI RNAs in depressing helper virus replication suggests that their expression in transgenic plants may provide a new and widely applicable approach for inducing resistance to viral infection.

Blotting, Northern↗
PubMed1991

Artificial defective interfering RNAs derived from brome mosaic virus.

Naturally occurring defective interfering RNAs (DI-RNAs) greatly reduce the accumulation of their helper virus in vivo, but are rarely associated with plant positive-strand RNA viruses. Deletion mutants pRNA-2 M/S and pRNA-2 E/S, derived from brome mosaic virus (BMV) genomic RNA-2, replicated in a manner dependent on BMV RNA-1 and -2, and effectively interfered with their accumulation in barley protoplasts. Based on their mode of replication, these mutant RNAs have been termed parasitic RNAs (pRNAs). When present with RNA-1 and -2 at low inoculum amounts, pRNA-2 M/S and pRNA-2 E/S reduced the level of replication of RNA-2, the parental RNA, by 37% and 64%, respectively. Greater amounts of pRNA in the inoculum completely eliminated the replication of both RNA-1 and -2. Mutations that prevented translation of truncated proteins from the pRNAs did not affect interference, but those that reduced pRNA replication decreased their ability to interfere with genomic RNA replication. At a molar pRNA: genomic RNA inoculum ratio of 1.5:1, pRNA-2 E/S reduced the accumulation of all helper virus RNAs by greater than 60%. This occurred in the presence of wild-type RNA-3 or delta SGP RNA-3, a deletion mutant of RNA-3 that lacks the subgenomic promoter necessary for coat protein expression, demonstrating that the interference mediated by the pRNAs was not effected by encapsidation. These data indicate that the expression of pRNAs that function as artificial DI-RNAs in transgenic plants may be an approach for inducing resistance to virus infection which is applicable to a wide range of plant viruses.

Blotting, Northern↗
PubMed1991

Targeting, dosimetry, and radioimmunotherapy of B-cell lymphomas with iodine-131-labeled LL2 monoclonal antibody.

Sixteen patients with non-Hodgkin's lymphoma were infused with 6.2 to 58.2 mCi (0.2 to 3.9 mg) doses of radioactive iodine (131I)-labeled LL2 immunoglobulin G (IgG) or F(ab')2, in order to study antibody distribution, pharmacokinetics, dosimetry, toxicity, tumor targeting, and therapy. LL2 is a murine IgG2a monoclonal antibody (MAb) reactive with B cells and non-Hodgkin's B-cell lymphoma. In a series of five assessable therapy patients, doses as small as 30 mCi 131I-LL2 IgG or F(ab')2 resulted in tumor responses (two partial remissions, two mixed and minor responses, and one no response), while one patient receiving diagnostic doses as low as 6.2 mCi showed a partial remission for 1 year and a complete remission after a second low radiation dose. No acute toxicities were noted, and only myelotoxicity accompanied therapeutic doses, with grade IV marrow toxicity seen in three of seven patients receiving total doses of about 50 mCi. Dosimetry calculations showed spleen and tumor dose rules of about 4.6 cGy/mCi, which was three to four times the dose to other organs. Despite the administration of relatively low doses of LL2 (0.2 to 3.9 mg), 82% of 60 known extrasplenic lymphoma sites were imaged. Serum clearance showed an average distribution half-life (T1/2) of 2.1 hours and an elimination T1/2 of 32.0 hours. The average total-body clearance T1/2 was 43 to 45 hours. LL2's antigenic target does not appear to be shed in high amounts into the circulation. Three of eight patients having at least two injections showed a human antimouse antibody response. These patients may have been presensitized to animal protein. An interesting observation in this study was the marked drop in circulating B lymphocytes after the administration of radioiodinated LL2 or anticarcinoembryonic antigen MAbs, suggesting that this is a nonspecific radiation effect and not necessarily related to the binding of MAb to normal B cells.

Adult↗
PubMed1990

Clinical studies of cancer radioimmunodetection with carcinoembryonic antigen monoclonal antibody fragments labeled with 123I or 99mTc.

Seventy-three patients with diverse cancers containing carcinoembryonic antigen received 123I-labeled anti-carcinoembryonic antigen monoclonal antibody F(ab')2 fragment [38 patients], 99mTc-labeled anti-carcinoembryonic antigen monoclonal antibody Fab' fragment [23 patients], or both reagents at different times [6 patients] for evaluation of antibody targeting and imaging [radioimmunodetection (RAID)], using planar and single-photon emission computed tomography. The results indicated that antibody fragments are preferred for early tumor imaging (within 24 h). Rapid targeting and clearance from blood and normal organs of the antibody fragments (blood median t1/2 elimination of 26.5 and 13.2 h for the F(ab')2 and Fab' fragments respectively) permitted the use of short-lived radionuclides, such as 123I (13.3 h) and 99mTc (6 h), and confirmed that selective antibody accretion in tumors occurred very soon after administration, such as between 2 and 5 h. Scan interpretations at 24 h for the 123I-labeled F(ab')2 and at 2-5 h for the 99mTc-labeled Fab' revealed overall sensitivities, on a tumor site basis, of 95.9 and 94.9%, respectively. On a site basis, the overall accuracies were 94.2 and 93.8% for the 123I and 99mTc immunoconjugates, respectively. In the 6 patients studied with both radioimmunoconjugates, a high concordance in detection was found. Both imaging agents also revealed a high number of putatively new tumor sites not disclosed by other radiological methods at the time of the RAID studies, of which 40.0 and 20.5% were subsequently confirmed as tumor for the 123I and 99mTc agents, respectively, within an 11-month follow-up period. This represented 24 proven occult tumor sites in 19 patients given the 123I-immunoconjugate and 16 proven occult tumor sites in 9 patients receiving the 99mTc agent. The new lesions were found up to 17 and 7 months earlier for 123I-RAID and 99mTc-RAID, respectively, than with other detection methods. The smallest tumors identified were below 0.5 cm, especially with the 99mTc immunoconjugate and single-photon emission computed tomography imaging. The findings of this study confirm previous evidence that RAID is a safe and a potentially useful new method of cancer detection. Despite the excellent results with the 123I-F(ab')2 antibody fragment, its poor availability and high cost limit its clinical use. Therefore, the 99mTc agent, which is made by an instant, 1-step, 1-vial, direct labeling method, appears to be the method of choice for rapid and accurate detection of cancer by RAID.

Adolescent↗
PubMed1990

Opportunities for bioactive compounds in transgenic plants.

A variety of bioactive compounds have now been introduced into plants through recombinant DNA techniques. Early examples included genes encoding proteins conferring herbicide tolerance and insect or virus resistance. More recently, pharmacologically useful compounds such as enkephalin and immunoglobulin have been produced in transgenic plants. Modification of existing compounds to provide better nutritional value or improved functional properties is exemplified in the case of seed storage proteins. The value of RNAs as bioactive compounds for suppression of undesirable products and viral infection has now been demonstrated in plants. The developmentally regulated expression of novel bioactive compounds in defined tissues, and their targeting to specific subcellular locations, is becoming of ever increasing economic and sociological importance as knowledge of the molecular mechanisms involved accumulates.

Endopeptidases↗
PubMed1990

Histochemical analysis of CaMV 35S promoter-beta-glucuronidase gene expression in transgenic rice plants.

The cauliflower mosaic virus promoter is commonly used to drive transcription of chimeric genes in transgenic plants, including the cereals. To determine the tissue and cell types of cereal plants that the promoter functions in, transgenic rice plants containing a CaMV 35S promoter/GUS chimeric gene were analyzed for GUS activity. Insertion of a 35S/GUS chimeric gene at low copy number into chromosomal DNA of plants regenerated from electroporated protoplasts was confirmed by gel blot hybridization analysis of uncut and endonuclease-digested DNA. Quantitative measurement showed that GUS activity was some tenfold higher in rice leaves than in tobacco leaves whereas activities obtained for rice roots were similar to those reported for tobacco roots. Histochemical localization of GUS activity confirmed that the CaMV 35S promoter functions in cells of the leaf epidermis, mesophyll and vascular bundle. It is also active in the cortex and vascular cylinder of the root, but only marginally active in the root epidermis. The generally similar distribution and levels of GUS activity obtained in differentiated tissue of stably transformed rice plants indicates the value of the CaMV 35S promoter as a positive control for studies in gene activity in transgenic monocots and dicots.

Cloning, Molecular↗
PubMed1990

Point mutations in the ICR2 motif of brome mosaic virus RNAs debilitate (+)-strand replication.

Sequences at the 5' termini of the genomic RNAs of brome mosaic virus (BMV) and other (+)-stranded RNA viruses have been shown (L.E. Marsh and T.C. Hall, 1987, Cold Spring Harbor Symp. Quant. Biol. 52, 331-341) to resemble the ICRs 1 and 2 (A and B boxes) of tRNA genes, with the complementary sequences at the 3' termini of the (-) strands resembling the ICR2 motif of methionine initiator tRNA genes (L.E. Marsh, G.P. Pogue, and T.C. Hall, 1989, Virology 172, 415-427). In order to examine the role of these sequences in viral replication, point mutations have been introduced into the ICR2-like sequence of a BMV RNA-2 deletion mutant, pRNA delta M/S (parasitic RNA), that does not encode a functional viral protein but replicates in the presence of genomic RNA-1 and -2. Single-base substitutions introduced at positions A7 or T8 of the (+)-sense ICR2-like motif reduced pRNA delta M/S replication by 70-82%, the primary effect being shown by kinetic analyses to be debilitation of (+)-strand synthesis. Whether these motifs act in their (+)-sense orientation in a manner analogous to tRNA genes or through the tRNA(Meti)-like sequence on the 3' (-) strand remains to be determined, but the data clearly demonstrate that the base composition within the ICR-like region of BMV RNAs contributes greatly to (+)-strand promoter function.

Base Sequence↗
PubMed1990

Analysis of RNA stability and (-) strand content in viral infections using biotinylated RNA probes.

Non-radioactive biotinylated RNA probes specific for plus (+) and minus (-) sense RNAs of brome mosaic virus (BMV) were synthesized in vitro from a plasmid bearing a 200 base pair fragment complementary to the 3' terminus of each of the three genomic RNAs of the virus. Using virion RNAs isolated from BMV infected barley plants, the sensitivity of biotinylated RNAs as hybridization probes was compared with that of 32P-labeled probes in Northern hybridization assays. Although the sensitivity of biotinylated and 32P-labeled probes is similar (approximately 5 pg), the time required to detect the RNA bands was much less than for autoradiography; (-) sense RNAs could be detected in 30 min whereas 48 h or more were required by autoradiography. The value of biotinylated probes for following RNA stability was exemplified by the detection of supplied inocula in protoplasts 24 h post-inoculation. Quantitation of relative accumulation of progeny (+) and (-) sense RNAs by densitometry of the Northern blots probed with biotinylated RNAs paralleled that of radiolabeled probes. The application of these probes was extended to the detection of RNAs in barley protoplasts and BMV infected plant sap by dot hybridization. In these tests, viral RNAs were detected in as few as 250 protoplasts and sap dilutions up to 1:2000. The merits of these non-radioactive probes in monitoring the replication events by the detection and quantitation of mutant progeny RNAs of BMV are discussed.

Biotin↗