Biomedical subjects

S G Rogers

Publications and source records attributed to S G Rogers.

At least 19 recordsLinked to original sources

PubMed1998

Biotechnology and the soybean.

Glyphosate-tolerant soybeans (GTSs), the first biotechnologically improved soybeans to be marketed, became available commercially in 1996. The safety of GTSs was assessed in 2 ways: study of the introduced protein and of the soybean seed and selected processing fractions. Because soybeans are a major source of protein in most farm animals' diets, animal feeding studies in wholesomeness were done to complement the analyses. Analysis of the expressed protein in GTSs [3-phosphoshikimate 1-carboxyvinyltransferase (EC 2.5.1.19)] showed it to be readily digestible and to possess no allergenic concerns. In addition, comparison of the composition of seeds and selected processing fractions from 2 GTS lines with the parental line showed that the lines are equivalent. Feeding studies in various animal species confirmed that the feeding value of GTS lines is comparable with that of the parental line. These studies support the conclusion that GTSs are as safe and nutritious as traditional soybeans marketed currently and can be incorporated safely into feed and food products in the near future.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1995

In vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus.

Replication of the single-stranded DNA genome of geminiviruses occurs via a double-stranded intermediate that is subsequently used as a template for rolling-circle replication of the viral strand. Only one of the proteins encoded by the virus, here referred to as replication initiator protein (Rep protein), is indispensable for replication. We show that the Rep protein of tomato yellow leaf curl virus initiates viral-strand DNA synthesis by introducing a nick in the plus strand within the nonanucleotide 1TAATATT decreases 8AC, identical among all geminiviruses. After cleavage, the Rep protein remains bound to the 5' end of the cleaved strand. In addition, we show that the Rep protein has a joining activity, suggesting that it acts as a terminase, thus resolving the nascent viral single strand into genome-sized units.

Base Sequence↗

PubMed1992

Sequence-specific interaction with the viral AL1 protein identifies a geminivirus DNA replication origin.

The bipartite geminiviruses such as tomato golden mosaic virus (TGMV) and squash leaf curl virus (SqLCV) have two single-stranded circular genomic DNAs, the A and B components, thought to be replicated from double-stranded circular DNA intermediates. Although it has been presumed that the origin sequences for viral replication are located in the highly conserved 200-nucleotide common region (CR) present in both genomic components and that the viral-encoded AL1 protein interacts with these sequences to effect replication, there has been no evidence that this is in fact so. We have investigated these questions, demonstrating selectivity and sequence specificity in this protein-DNA interaction. Simple component switching between the DNAs of TGMV and SqLCV and analysis of replication in leaf discs showed that whereas the A components of both TGMV and SqLCV promote their own replication and that of their cognate B component, neither replicates the noncognate B component. Furthermore, using an in vivo functional replication assay, we found that cloned viral CR sequences function as a replication origin and direct the replication of nonviral sequences in the presence of AL1, with both circular single-stranded and double-stranded DNA being synthesized. Finally, by the creation of chimeric viral CRs and specific subfragments of the viral CR, we demonstrated sequence-specific recognition of the replication origin by the AL1 protein, thereby localizing the origin to an approximately 90-nucleotide segment in the AL1 proximal side of the CR that includes the conserved geminiviral stem-loop structure and approximately 60 nucleotides of 5' upstream sequence. By deletional analysis, we further demonstrated that the conserved stem-loop structure is essential for replication. These studies identify the functional viral origin of replication within the CR, demonstrating that sequence-specific recognition of this origin by the AL1 protein is required for replication.

Base Sequence↗

PubMed1990

Selection for wild type size derivatives of tomato golden mosaic virus during systemic infection.

A chimeric tomato golden mosaic virus (TGMV) A component DNA, which results from replacement of the coding region of the viral coat protein gene (CP) with the larger bacterial beta-glucuronidase coding sequence (GUS), can replicate in agroinoculated leaf discs but is unstable in systemically infected plants (1). We have made similar replacements of the TGMV CP gene with the GUS coding sequence in both the sense and antisense orientations. Both derivatives replicated in leaf discs inoculated via Agrobacterium. However, systemic movement of the GUS substituted vectors was not detected in agroinoculated Nicotiana benthamiana plants. The only TGMV A derivatives detected in systemically infected leaves of inoculated plants were similar in size to the wild type viral component. Sequence analysis of derivatives from six independently inoculated plants revealed that they did not result from internal deletions of the larger replicons detected in leaf discs but, instead, were generated by fusion events occuring within the original T-DNA insert. These results indicate that systemic movement of TGMV in N. benthamiana plants provides a strong selective pressure favoring viral derivatives similar in size to the wild type virus components.

Base Sequence↗

PubMed1990

Expression of functional replication protein from tomato golden mosaic virus in transgenic tobacco plants.

The A component of the bipartite genome of the geminivirus tomato golden mosaic virus (TGMV) encodes the viral protein (AL1) that is required for viral DNA replication. We have constructed transgenic Nicotiana benthamiana plants in which the AL1 open reading frame is transcribed under the control of the cauliflower mosaic virus 35S promoter. The transgenic plants, which were phenotypically normal, produced a single transcript from the 35S-AL1 construct and a 40-kDa protein that cross-reacted with a polyclonal antiserum raised against AL1 protein overproduced in Escherichia coli. Six of nine transgenic lines complemented a TGMV A variant with a mutation in AL1 when coinoculated with the B component of the TGMV genome. Single- and double-stranded forms of the B component were synthesized in leaf discs from a complementing, transgenic line in the absence of TGMV A. These results establish that the transgenic plants express functional AL1 protein and show that this viral protein is not only required, but sufficient, for single- and double-stranded replication of TGMV DNA in the presence of host proteins. These results also show that the AL1 protein is not by itself a determinant of disease or pathogenesis.

Journal Article↗

PubMed1989

Maize streak virus genes essential for systemic spread and symptom development.

The entire genome of single component geminiviruses such as maize streak virus (MSV) consists of a single-stranded circular DNA of ~2.7 kb. Although this size is sufficient to encode only three average sized proteins, the virus is capable of causing severe disease of many monocots with symptoms of chlorosis and stunting. We have identified viral gene functions essential for systemic spread and symptom development during MSV infection. Deletions and gene replacement mutants were created by site-directed mutagenesis and insertion between flanking MSV or reporter gene sequences contained in Agrobacterium T-DNA derived vectors. Following Agrobacterium-mediated inoculation of maize seedlings, the mutated MSV DNAs were excised from these binary vectors by homologous recombination within the flanking sequences. Our analyses show that the capsid gene of MSV, while not required for replication, is essential for systemic spread and subsequent disease development. The ;+' strand open reading frame (ORF) located immediately upstream from the capsid ORF and predicted to encode a 10.9 kd protein was also found to be dispensable for replication but essential for systemic spread. By this analysis, MSV sequences that support autonomous replication were localized to a 1.7 kb segment containing the two viral intergenic regions and two overlapping complementary ;-' strand ORFs. Despite the inability of the gene replacement mutants to spread systemically, both inoculated and newly developed leaves displayed chlorotic patterns similar to the phenotype observed in certain developmental mutants of maize. The similarity of the MSV mutant phenotype to these developmental mutants is discussed.

Journal Article↗

PubMed1989

Functional expression of the leftward open reading frames of the A component of tomato golden mosaic virus in transgenic tobacco plants.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. We have constructed Nicotiana benthamiana plants that are transgenic for the three overlapping open reading frames, AL1, AL2, and AL3, from the left side of TGMV A. In the transgenic plants, the AL open reading frames are under the control of the cauliflower mosaic virus (CaMV) 35S promoter. In TGMV infectivity assays, seven of 10 transgenic lines complemented TGMV A variants with mutations in AL1, AL2, or AL3 when co-inoculated with the B component. The 35S-AL construct was transcribed as a single RNA species in the transgenic plants, indicating that AL1, AL2, and AL3 were expressed from a polycistronic mRNA. This differs from the complex transcription pattern in TGMV-infected plants, which contains five AL transcripts. There was no quantitative correlation between the efficiency of complementation in the infectivity assay and the level of expression of transgenic AL RNA in the leaves of a transgenic line. One line that failed to complement defects in AL1, AL2, and AL3 in infectivity assays contained high levels of transgenic AL RNA and functional AL1 protein. These results provide evidence that chromosomal position can affect the cell- and tissue-specific transcription of the 35S promoter in transgenic plants. Comparison of the complementing plants and wild-type infected plants may provide insight into the TGMV infection process and the use of the CaMV 35S promoter for gene expression in transgenic plants.

Base Sequence↗

PubMed1988

Transient expression of heterologous RNAs using tomato golden mosaic virus.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. The A component contains the only virally-encoded function required for autonomous replication in infected plant cells. We used agroinoculation of petunia leaf discs with the A component to develop a transient expression system which permits direct examination of viral transcripts by S1 nuclease protection. The AR1 gene, which encodes the TGMV coat protein, was transcribed transiently in leaf discs after agroinoculation of TGMV a DNA. Synthesis of AR1 RNA was dependent on T-DNA transfer and TGMV DNA replication, demonstrating that it is a plant transcription product. The AL open reading frames of TGMV A were also expressed transiently in leaf discs. The ratio between AR1 RNA and the major leftward RNA was constant and was used to normalize AR1 transcription for viral DNA copy number. The bacterial genes encoding chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS) were transiently expressed in leaf discs from the AR1 promoter in TGMV A. The levels of AR1 and GUS RNAs were similar in leaf discs after adjusting for viral DNA copy number, while CAT RNA was less abundant. The geminivirus transient expression system allows rapid analysis of RNAs transcribed from foreign genes and can serve as a preliminary screen in the construction of transgenic plants.

DNA Replication↗

PubMed1988

Genetic analysis of the tomato golden mosaic virus. II. The product of the AL1 coding sequence is required for replication.

Tomato golden mosaic virus (TGMV) belongs to the geminivirus subgroup that is characterized by a split genome consisting of two single-stranded circular DNAs. The TGMV A genome component encodes the virus coat protein as well as all of the functions necessary for viral DNA replication. Analysis of the nucleotide sequence indicates that the TGMV A component has, in addition to the coat protein encoding ORF, four overlapping open reading frames (ORFs) with the potential to encode proteins of greater than 10 kD. We have investigated the functions of these putative proteins in both symptom formation and DNA replication by creating mutations in each of the ORFs. Our results show that the AL4 ORF, which is encoded within the N-terminal region of ORF AL1, is not essential for normal virus infection. In contrast, we find that disruption of the AL3 ORF results in delay and attenuation of symptom formation. We also report that the products of the AL1 and AL2 ORFs are absolutely required for symptom formation. Studies of DNA replication show that only the AL1 open reading frame is essential for viral DNA synthesis. The significance of these results for the development of vectors from the geminiviruses is discussed.

DNA Mutational Analysis↗

PubMed1988

In vitro transcription and translational efficiency of chimeric SP6 messenger RNAs devoid of 5' vector nucleotides.

A plasmid containing the bacteriophage SP6 promoter, designated pHSTO, permits in vitro transcription of RNAs devoid of vector-derived nucleotides. This vector has been characterized for relative transcriptional activity using constructs which alter the conserved nucleotides extending beyond the SP6 transcriptional initiation site. SP6 polymerase efficiently transcribes cDNA inserts which contain a guanosine (G) nucleotide at position +1 relative to the SP6 promoter; however, inserts with an adenosine (A) or pyrimidine at position +1 are not transcribed. Several cellular and viral cDNAs have been transcribed into translatable messenger RNA using this vector; however, SP6 polymerase will not transcribe the A-T rich untranslated leader from alfalfa mosaic virus RNA 4 efficiently unless the viral mRNA cap site is separated from the transcriptional initiation site by twelve base pairs of vector DNA. Chimeric messenger RNAs were created by linking the untranslated leader sequence of several viral mRNAs to the coding region of barley alpha-amylase, and the resultant mRNAs were translated in a wheat germ extract to determine relative translational efficiencies. The untranslated leader sequences of turnip yellow mosaic virus coat protein mRNA and black beetle virus RNA 2 did not increase translational efficiency, while the tobacco mosaic virus leader stimulated translation significantly. The results indicate that substitution of a cognate untranslated leader sequence with a leader derived from a highly efficient mRNA does not necessarily predict enhanced translational efficiency of the chimeric mRNA.

Bacteriophages↗

PubMed1988

Genetic analysis of tomato golden mosaic virus: the coat protein is not required for systemic spread or symptom development.

The geminiviruses are a unique group of higher plant viruses that are composed of twin isometric particles which contain circular, single-stranded DNA. Tomato golden mosaic virus (TGMV), a whitefly-transmitted agent, belongs to the subgroup of geminiviruses whose members possess a bipartite genome. The TGMV A genome component has the capacity to encode at least four proteins. One of these is the viral coat protein, as inferred by homology with coat-protein, genes of other geminiviruses and by the observation of typical geminate particles in transgenic plants that contain inserts of TGMV A DNA. We have investigated the role of the coat protein in TGMV replication and report here that its coding sequence may be interrupted or substantially deleted without loss of infectivity. However, certain coat-protein mutants showed reproducible delays in time of symptom appearance as well as reduced symptom development, when inoculated onto transgenic Nicotiana benthamiana plants containing the TGMV B component. The most attenuated symptoms were seen with a mutant in which the coat-protein coding sequence was almost entirely deleted. The significance of these findings for the development of plant vectors from TGMV DNA is discussed.

Journal Article↗

PubMed1988

Evaluation of selectable markers for obtaining stable transformants in the gramineae.

Cell suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) resistance genes. Resistant colonies were obtained at low frequencies (1 x 10(-5) to 2 x 10(-6)) when selected under conditions which were growth inhibitory to protoplasts electroporated without DNA. Southern blot hybridization confirmed stable integration of plasmid DNA into T. monococcum using hygromycin vectors and P. maximum using the methotrexate vector with 1 to 10 copies integrated per haploid genome.

Journal Article↗

PubMed1988

Development of a plant transformation selection system based on expression of genes encoding gentamicin acetyltransferases.

The development of selectable markers for transformation has been a major factor in the successful genetic manipulation of plants. A new selectable marker system has been developed based on bacterial gentamicin-3-N-acetyltransferases [AAC(3)]. These enzymes inactivate aminoglycoside antibiotics by acetylation. Two examples of AAC(3) enzymes have been manipulated to be expressed in plants. Chimeric AAC(3)-III and AAC(3)-IV genes were assembled using the constitutively expressed cauliflower mosaic virus 35S promoter and the nopaline synthase 3' nontranslated region. These chimeric genes were engineered into vectors for Agrobacterium-mediated plant transformation. Petunia hybrida and Arabidopsis thaliana tissue transformed with these vectors grew in the presence of normally lethal levels of gentamicin. The transformed nature of regenerated Arabidopsis plants was confirmed by DNA hybridization analysis and inheritance of the selectable phenotype in progeny. The chimeric AAC(3)-IV gene has also been used to select transformants in several additional plant species. These results show that the bacterial AAC(3) genes will serve as useful selectable markers in plant tissue culture.

Journal Article↗

PubMed1987

Bacterial expression and isolation of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase.

5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase, EPSPS), an in vivo enzyme target of the herbicide glyphosate (N-phosphonomethyl glycine), was purified from a Petunia hybrida suspension culture line, MP4-G, by a small-scale high-performance chromatographic purification procedure. The cDNA encoding the mature petunia EPSPS (lacking the chloroplast transit sequence) was cloned into a plasmid, pMON342, for expression in Escherichia coli. This clone complemented the EPSPS deficiency of an E. coli aroA- mutant, and the plant enzyme constituted approximately 1% of the total extractable protein. Large-scale purification of the enzyme from E. coli cells resulted in a highly active protein which was homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino terminal sequencing. Antibodies raised against the purified enzyme also reacted with the E. coli EPSPS in Western analyses. The availability of large quantities of the plant enzyme will significantly facilitate mechanistic investigations as well as a comparative study with EPSPS from bacteria and fungi.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1987

5' proximal sequences of a soybean ribulose-1,5-bisphosphate carboxylase small subunit gene direct light and phytochrome controlled transcription.

Two closely related ribulose-1,5-bisphosphate carboxylase small subunit (SSU) genes, SRS1 and SRS4, are transcribed at high levels in soybean plants in response to light. Transgenic petunia plants containing 5' sequences from SRS1 or SRS4 fused to the polypeptide encoding region of a neomycin phosphotransferase (NPTII) gene exhibit selectable kanamycin resistance. Deletion of three ATG codons from the region preceding the normal NPTII translation start site has little effect on the levels of kanamycin resistance in transformed plants. Run-on transcription assays in isolated nuclei demonstrate that transcription of the SRS1/NPTII chimera and the native petunia SSU11A gene subfamily is light regulated and under phytochrome control in leaves of transgenic plants. In young expanding leaves of fully light grown plants, transcription of these genes is markedly reduced within minutes of far-red treatment, while ribosomal DNA and actin gene transcription remains unchanged. This is analogous to the transcriptional response we observed for SRS1 and SRS4 in soybean seedlings. These data suggest (1) that transcription of SSU genes in both soybean and petunia require the continued presence or synthesis of phytochrome in the Pfr form and (2) that 5' sequences are sufficient to direct the phytochrome controlled transcriptional response of the SRS1 gene. In fully expanded mature leaves we found the transcription rates of the native SSU11A gene subfamily, the chimeric SRS1/NPTII gene, the rDNA genes, and several other control genes to be reduced markedly after far-red treatment or after extended periods of darkness. The contrast between results in young and mature leaves is discussed.

Chlorophyll↗

PubMed1987

Comparison of cauliflower mosaic virus 35S and nopaline synthase promoters in transgenic plants.

We have compared the level of expression of the Cauliflower Mosaic Virus 35S promoter and the nopaline synthase promoter when fused to a common reporter gene. A cassette containing the neomycin phosphotransferase (type II) coding sequence followed by the nopaline synthase 3' nontranslated region was used for transcriptional and translational evaluation of the two different promoters. These chimeric genes were introduced into petunia plants and the copy number of the gene, the steady state level of NPTII transcript and the levels of NPTII enzyme activity were determined. In this paper, we report that the NPT II transcript levels are on the average 30 fold higher in plants containing CaMV 35S promoter and leader sequences than in plants containing the same reporter gene but nopaline synthase promoter and leader sequences. Similarly, plants containing the CaMV 35S promoter had an average of 110 fold higher levels of NPTII enzyme activity than those containing the nopaline synthase promoter. The significance of these results for expression of foreign genes in plants is discussed. In addition, we describe the construction of a convenient plant expression cassette vector (pMON316) which utilizes the CaMV 35S promoter.

Amino Acid Oxidoreductases↗

PubMed1987

Expression of mouse dihydrofolate reductase gene confers methotrexate resistance in transgenic petunia plants.

Transgenic petunia plants containing an altered (Leu22----Arg22) mouse dihydrofolate reductase gene fused to the cauliflower mosiac virus 35S (CaMV 35S) promoter and nopaline synthase (nos) polyadenylation site were obtained by transforming petunia leaf disks with an Agrobacterium tumefaciens strain carrying the chimeric gene. Transformants were directly selected for and rooted on medium containing 1 microM methotrexate (MTX). The chimeric gene was present in the regenerated plants at one to three copies and produced the expected 950-nucleotide-long transcript based on Southern and Northern hybridization analyses, respectively. Leaf pieces from the regenerated transgenic plants were able to form callus when cultured on medium containing 1 microM MTX and were able to incorporate 32P into high-molecular-weight DNA in the presence of greater than 100 microM MTX, thus demonstrating that the chimeric mouse dhfr gene was fully functional and useful as a selectable marker in plant transformation experiments. To date, this is the first report of successful expression of a vertebrate gene in transformed plant cells.

Animals↗

PubMed1986

Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants.

Phenotypically normal petunia plants carrying chromosomal inserts of either the tomato golden mosaic virus (TGMV) A or the B component DNA, as single or tandem inserts, were obtained using an Agrobacterium tumefaciens Ti plasmid-based transformation system. Southern hybridization analysis revealed that the tandem, direct-repeat A plants contained free single and double stranded A component DNAs. No free B component DNA was detected in plants carrying tandem repeats of the B component. Progeny of self-fertilized plants appeared normal. In contrast, one-quarter of the progeny from tandem A by tandem B plant crosses showed chlorotic lesions on their leaves similar to virus symptoms. The significance of these results and the use of this method for the study of virus functions involved in TGMV replication and symptom production are discussed.

DNA Replication↗