Inheritance of functional foreign genes in plants.

Morphologically normal plants were regenerated from Nicotiana plumbaginifolia cells transformed with an Agrobacterium tumefaciens strain containing a tumor-inducing plasmid with a chimeric gene for kanamycin resistance. The presence of the chimeric gene in regenerated plants was demonstrated by Southern hybridization analysis, and its expression in plant tissues was confirmed by the ability of leaf segments to form callus on media containing kanamycin at concentrations that were normally inhibitory. Progeny derived from several transformed plants inherited the foreign gene in a Mendelian manner.

Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene.

A chimeric gene containing a cloned cDNA of the coat protein (CP) gene of tobacco mosaic virus (TMV) was introduced into tobacco cells on a Ti plasmid of Agrobacterium tumefaciens from which tumor inducing genes had been removed. Plants regenerated from transformed cells expressed TMV mRNA and CP as a nuclear trait. Seedlings from self-fertilized transgenic plants were inoculated with TMV and observed for development of disease symptoms. The seedlings that expressed the CP gene were delayed in symptom development and 10 to 60 percent of the transgenic plants failed to develop symptoms for the duration of the experiments. Increasing the concentration of TMV in the inoculum shortened the delay in appearance of symptoms. The results of these experiments indicate that plants can be genetically transformed for resistance to virus disease development.

Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens.

Transformed Arabidopsis thaliana plants have been produced by a modified leaf disk transformation-regeneration method. Leaf pieces from sterilely grown plants were precultured for 2 days and inoculated with an Agrobacterium tumefaciens strain containing an avirulent Ti (tumor-inducing) plasmid with a chimeric gene encoding hygromycin resistance. After cocultivation for 2 days, the leaf pieces were placed on a medium that selects for hygromycin resistance. Shoots regenerated within 3 months and were excised, rooted, and transferred to soil. Transformation was confirmed by opine production, hygromycin resistance, and DNA blot hybridization of both primary transformants and progeny. This process for producing transgenic Arabidopsis plants should enhance the usefulness of the species for experimental biology.

Engineering herbicide tolerance in transgenic plants.

The herbicide glyphosate is a potent inhibitor of the enzyme 5-enolpyruvylshikimate- 3-phosphate (EPSP) synthase in higher plants. A complementary DNA (cDNA) clone encoding EPSP synthase was isolated from a complementary DNA library of a glyphosate-tolerant Petunia hybrida cell line (MP4-G) that overproduces the enzyme. This cell line was shown to overproduce EPSP synthase messenger RNA as a result of a 20-fold amplification of the gene. A chimeric EPSP synthase gene was constructed with the use of the cauliflower mosaic virus 35S promoter to attain high level expression of EPSP synthase and introduced into petunia cells. Transformed petunia cells as well as regenerated transgenic plants were tolerant to glyphosate.

Plasmid pKC7: a vector containing ten restriction endonuclease sites suitable for cloning DNA segments.

Plasmid pKC7, a derivative of pBR322, specifies resistance to both ampicillin and kanamycin. The DNA of this small plasmid (5.8 kb) contains unique sites for insertion of DNA cleaved with ten different restriction endonucleases. A detailed restriction endonuclease cleavage map is presented. The utility of this plasmid for cloning is discussed.

A thermoinducible lambda phage-ColE1 plasmid chimera for the overproduction of gene products from cloned DNA segments.

Two segments of lambda have been cloned into the multicopy plasmid pBR322. One extends from N through cII (NcII segment, from 71.3 to 81.0% on the physical map) and the other from N through P (NOP segment, from 71.3 to 86.5% on the physical map). Cells carrying these recombinant plasmids express lambda immunity (cIts) and Rex function. In addition, they decrease the efficiency of plating at 32 degrees C of lambdavir and lambdaimm434, but not that of lambdaimm21. Recombinant plasmids with lambdaNOP segments (pKC14, pKC16) differ from recombinant plasmid with labmdaNcII segment (pKC10) in two respects: (i) strains carrying pKC14 or pKC16 are killed at 42 degrees C, and (ii) these strains are thermally inducible for plasmid DNA synthesis, resulting in increase of plasmid copy number from an uninduced level of 50 to more than 130 per chromosome. It was suggested that both these differences are related to functions contained in the lambda DNA segment extending from 81.0 to 86.5%. The usefulness of plasmid pKC16 for overproduction of gene products from cloned DNA segments was demonstrated by cloning the E. coli exonuclease III gene (xth) in pKC16. Thermal induction of this xth plasmid (pSGr) results in a 125-fold increase in exonuclease III activity over that of a control strain lacking the xth gene insert. The extent of exonuclease III overproduction obtained by cloning xth gene in a lambda vector was similar to that obtained with pSGR3.

Cloning of the exonuclease III gene of Escherichia coli.

Overproducers of exonuclease III (exo III) were found within a colony bank containing ColE1-Escherichia coli hybrid plasmids. Through the enzymatic ligation of restriction enzyme fragments, the exo III gene, xth, was transferred to a thermoinducible, integration-proficient lambda phage and to a chimeric ColE1-lambda plasmid that was thermoinducible for lambda-directed DNA replication. Transfer of the xth gene was facilitated by a technique involving prior selection for Tn5 insertions into plasmid, thereby linking the gene to additional restriction sites and to a selectable (drug resistance) marker. After heat induction, cells bearing the thermoinducible ColE1-lambda-xth plasmid produced 120-fold more exo III than did plasmid-free cells. Enzyme production was not further enhanced by any of the following chromosomal mutations: dnaA, recBC, tob, or nusA snu. Several observations suggested that enzyme over-synthesis was the result primarily of lambda-detected replication rather than lambda-directed transcription.

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.

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.

Bacteriophage T5-induced endonucleases that introduce site-specific single-chain interruptions in duplex DNA.

Four site-specific endodeoxyribonucleases have been partially purified from extracts of bacteriophage T5-infected Escherichia coli by gel filtration and affinity chromatography on single- and double-stranded DNA. The enzymes were detected and characterized by agarose gel electrophoresis of alkali-denatured digestion products. None of the four is found in uninfected cells. In the presence of a divalent cation, all four endonucleases make ligase-repairable, single-chain interruptions at specific sites in the duplex DNA of several bacteriophages (lambda, T7, and T5) and a mammalian virus (adenovirus 2). These activities are not stimulated by ATP. None of the four is active on single-stranded DNA. The fragments produced by each enzyme from ligase-repaired T5 DNA do not correspond to those derived from mature T5 DNA. Each of the enzymes is able to cleave the intact strand of T5 DNA.

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.

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.

Interruption-deficient mutants of bacteriophage T5. II. Properties of a mutant lacking a specific interruption.

An examination was made of the properties of T5HA4, a mutant of bacteriophage T5 that lacks the single-chain interruption that occurs at 7.9% from the left end of the genome. The DNAs of T5HA4 and the wild type were compared by electrophoresis in agarose gels of both single-stranded fragments produced by denaturation and duplex fragments generated by sequential treatment with exonuclease III and SI nuclease. These studies demonstrated that T5HA4 also lacks an interruption that occurs at 99.6% in wild-type DNA. The interruptions at 7.9 and 99.6% therefore occur within the 8.3% of T5 DNA that is terminally repetitious. Evidence on the location of other interruptions within the terminal repetition was also obtained. Analysis of T5HA4 with a restriction endonuclease indicated that the interruption deficiency is not due to a deletion or addition mutation. The injection of T5HA4 DNA into a host bacterium was found to occur, as with the wild type, in a two-step manner. The interruption at 7.9% is therefore not required for stopping DNA transfer after the initial 8% segment has been injected.

A DNase for apurinic/apyrimidinic sites associated with exonuclease III of Hemophilus influenzae.

An endonuclease purified from Hemophilus influenzae made single strand breaks in DNA containing apurinic or apyrimidinic sites but had no detectable endonuclease activity on untreated native DNA. The new 5'-termini created at the cleavage sites were base-free deoxyribose 5-phosphate residues. The enzyme preparation also catalyzed the exonucleolytic release of 5'-mononucleotides from bihelical DNA and the hydrolysis of DNA 3'-terminal phosphomonoesters. The phosphatase-exonuclease activity was indistinguishable from that reported by Gunther and Goodgal (J. Biol. Chem. (1970) 245, 5341-5349) and resembled that of exonuclease III of Escherichia coli. The endonucleolytic and exonucleolytic activities could not be separated by electrophoresis, sedimentation, or gel filtration, and they were also affected simultaneously by mutation. The enzymatic activities appear to be functions of a single monomeric protein (M(r) = 30,000).

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.

Nucleotide sequence of the tmr locus of Agrobacterium tumefaciens pTi T37 T-DNA.

The nucleotide sequence of the tmr locus from the nopaline-type pTi T37 plasmid of Agrobacterium tumefaciens was determined. Examination of this sequence allowed us to identify an open reading frame of 720 nucleotides capable of encoding a protein with a derived molecular weight of 27025 d. Comparison of the pTi T37 tmr sequence with the published sequence of the pTi Ach5 tmr locus shows over 88% homology in the 240 bases 5' to the translational initiation codon and over 91% homology in the coding sequences. The 3' nontranslated regions show less than 50% homology as expected for the 3' regions of divergent related genes. The possible significance of areas of conserved sequences, particularly in the 5' regulatory regions, is discussed.

Independent encapsidation of tomato golden mosaic virus A component DNA in transgenic plants.

Tomato golden mosaic virus (TGMV), a member of the geminivirus group, has a genome consisting of two DNA molecules designated the A and B components. Both are required for infectivity in healthy plants, although the former has been shown to replicate independently in transgenic plants containing tandem direct repeats of the A genome component. In the studies presented here, petunia plants transgenic for either both components (A×B hybrids) or the A component alone were examined for the presence of virus particles and encapsidated, single stranded viral DNA. The results of DNase protection experiments and direct observation of extracts from transgenic plants by electron microscopy indicate that single stranded TGMV DNA is in both cases packaged into paired particles identical to those obtained from virus-infected plants. DNase-treated virions isolated from A×B hybrid petunia are infectious when inoculated onto healthy Nicotiana benthamiana. Likewise, virions obtained from transgenic A petunia are infectious for plants transgenic for the B component.Our observations of TGMV replication in transgenic plants indicate that TGMV A DNA encodes all viral functions necessary for the replication and encapsidation of viral DNA. The possible role of the B component in TGMV replication is discussed.

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.