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Agrobacterium tumefaciens is unique in that it can facilitate the interkingdom transfer of DNA and genetically modify its plant host. While Agrobacterium has been coopted for use in the genetic modification of plants, it is also a major pathogen, causing crown gall disease in the nursery, orchard, and vineyard industries. Pathogenicity in Agrobacterium is the result of two components. First is the Ti plasmid, which carries virulence genes and the transferred T-DNA region. The second component is the chromosome of Agrobacterium, which comprises diverse bacterial lineages and multiple species-level groups. The Ti plasmid can be transferred from strain to strain, diversifying the pathogen and complicating efforts to understand its epidemiology. This system provides an opportunity to study transmission of plasmids and their impact on disease persistence and spread. However, the movement of plasmids, and diversity of chromosomal lineages, means that conventional methods of using whole genome SNPs to track outbreaks are not sufficient, and new techniques must be developed. Additionally, Ti plasmids, like Agrobacterium, are genetically diverse and represent multiple plasmid types. Using a framework of 200 sequenced Agrobacterium genomes isolated from around the world, and a previously developed model of Ti plasmid types, we modelled their epidemiology. Key to this study was that we first separately analyzed plasmids and strain. Combining results revealed links between nurseries, potential horizontal transfer of the plasmid between strains within nurseries, global spread of plasmids, and long-term persistence of plasmids in the agricultural system. Agricultural practices have the potential to promote the diversification of pathogens and the emergence of new pathogen lineages.

Members of the agrobacteria–rhizobia complex (ARC) have multiple and diverse plasmids. The extent to which these plasmids are shared and the consequences of their interactions are not well understood. We extracted over 4000 plasmid sequences from 1251 genome sequences and constructed a network to reveal interactions that have shaped the evolutionary histories of oncogenic virulence plasmids. One newly discovered type of oncogenic plasmid is a mosaic with three incomplete, but complementary and partially redundant virulence loci. Some types of oncogenic plasmids recombined with accessory plasmids or acquired large regions not known to be associated with pathogenicity. We also identified two classes of partial virulence plasmids. One class is potentially capable of transforming plants, but not inciting disease symptoms. Another class is inferred to be incomplete and non-functional but can be found as coresidents of the same strain and together are predicted to confer pathogenicity. The modularity and capacity for some plasmids to be transmitted broadly allow them to diversify, convergently evolve adaptive plasmids and shape the evolution of genomes across much of the ARC. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

Agrobacterium rhizogenes and A. tumefaciens transfer DNA and effector proteins into plant cells, where transferred DNA (T-DNA) is inherited and expressed. Most transgenic plants are created using A. tumefaciens, but transformation by A. rhizogenes yields desirable single-copy transgenes more frequently than A. tumefaciens does. DNA transfer from both species resembles plasmid conjugation, but later events differ between species. Efficient transformation by A. tumefaciens requires single-strand DNA-binding protein VirE2, which A. rhizogenes lacks, so substrates for T-DNA integration differ greatly. In A. rhizogenes, the GALLS proteins substitute for (but do not resemble) VirE2. GALLS proteins occur in two forms: full-length (FL) and a more abundant C-terminal domain (CT). Both have protein-binding domains and type IV secretion signals. GALLS-FL has ATPase/strand transferase and nuclear localization (NLS) domains, allowing it to enter the nucleus and bind VirD2, a pilot protein attached to single-stranded T-DNA (T-strands). GALLS-FL ATPase may pull T-strands into the nucleus. GALLS-CT stimulates an early step in gene transfer to plants; this effector protein alters host gene expression and stimulates T-DNA transfer, apparently by suppressing host defenses. These observations challenge the assumption that A. rhizogenes and A. tumefaciens transform plants and mitigate host defenses by the same mechanisms.

Grapevine rootstock transformed with an Agrobacterium oncogene-silencing transgene was resistant to certain Agrobacterium strains but sensitive to others. Thus, genetic diversity of Agrobacterium oncogenes may limit engineering crown gall resistance. Crown gall disease of grapevine induced by Agrobacterium vitis or Agrobacterium tumefaciens causes serious economic losses in viticulture. To establish crown gall-resistant lines, somatic proembryos of Vitis berlandieri × V. rupestris cv. ‘Richter 110’ rootstock were transformed with an oncogene-silencing transgene based on iaaM and ipt oncogene sequences from octopine-type, tumor-inducing (Ti) plasmid pTiA6. Twenty-one transgenic lines were selected, and their transgenic nature was confirmed by polymerase chain reaction (PCR). These lines were inoculated with two A. tumefaciens and three A. vitis strains. Eight lines showed resistance to octopine-type A. tumefaciens A348. Resistance correlated with the expression of the silencing genes. However, oncogene silencing was mostly sequence specific because these lines did not abolish tumorigenesis by A. vitis strains or nopaline-type A. tumefaciens C58.

Agrobacterium tumefaciens and A. rhizogenes transport single-stranded DNA (ssDNA; T-strands) and virulence proteins into plant cells through a type IV secretion system. DNA transfer initiates when VirD2 nicks border sequences in the tumour-inducing plasmid, attaches to the 5' end, and pilots T-strands into plant cells. Agrobacterium tumefaciens translocates ssDNA-binding protein VirE2 into plant cells where it targets T-strands into the nucleus. Some A. rhizogenes strains lack VirE2 but transfer T-strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant. VirE2 and full-length GALLS (GALLS-FL) contain nuclear localization sequences that target these proteins to the plant cell nucleus. VirE2 binds cooperatively to T-strands allowing it to move ssDNA without ATP hydrolysis. Unlike VirE2, GALLS-FL contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. VirE2 may accumulate in the nucleus and pull T-strands into the nucleus using the force generated by cooperative DNA binding. GALLS-FL accumulates inside the nucleus where its predicted ATP-dependent strand transferase may pull T-strands into the nucleus. These different mechanisms for nuclear import of T-strands may affect the efficiency and quality of transgenic events in plant biotechnology applications.

Healthy, productive farmland separates humans from famine, making wise agricultural practices crucial to everyone. Plant scientists at universities and companies throughout the world endeavour to improve the quality, quantity, safety and sustainability of agricultural production, and their efforts have proven highly successful. Despite the remarkable success of a number of genetically engineered crop plants that have been grown and consumed – without incident – in large quantities since 1996, this technology faces increasing opposition in the United States and Europe. This article will discuss the major crops currently cultivated and provide information necessary to make informed decisions about plant biotechnology. Each new genetically engineered plant variety raises unique issues that must be considered on a case‐by‐case basis. Rejection or acceptance of all genetically engineered plants is unreasonable and will likely harm both the environment and the human condition.

Molecular archaeology brings the tools of molecular biology to bear on fundamental questions in archaeology, anthropology, evolution, and ecology. Ancient DNA research is becoming widespread as evolutionary biologists and archaeologists discover the power of the polymerase chain reaction (PCR) to amplify DNA from ancient plant and animal remains. However, the extraordinary susceptibility of PCR to contamination by extraneous DNA is not widely appreciated. We report the independent observation of DNA from domestic animals in PCR reagents and ancient samples in four separate laboratories. Since PCR conditions used in ancient DNA analyses are extremely sensitive, very low concentrations of contaminating DNA can cause false positives. Previously unidentified animal DNA in reagents can confound ancient DNA research on certain domestic animals, especially cows, pigs, and chickens.

Plant-associated Erwinia include pathogenic and nonpathogenic species . We report the 5.6-Mb genome sequence of Erwinia billingiae OSU19-1, isolated from a canker on a pear tree inoculated with Erwinia amylovora . OSU19-1 and a closely related European isolate, E. billingiae Eb661 T , share many similarities including 40 kb of plasmid sequence.

Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2.

DNA residues may preserve on ancient stone tools used to process animals. We studied 24 stone tools recovered from the Bugas-Holding site in northwestern Wyoming. Nine tools that yielded DNA included five bifaces, two side scrapers, one end scraper, and one utilized flake. The excavators did not handle three of these tools with bare hands, and they were sealed in separate plastic bags to minimize contact with modern DNA. Five sediment samples did not yield mammalian DNA. One tool contained DNA from three species, and these templates competed during polymerase chain reaction (PCR) amplification. DNA identifications suggested a butchery pattern indicative of a human population under resource stress or a broader range of industrial activities at Bugas-Holding. We describe several improvements in DNA residue analysis, including a more effective DNA recovery protocol, methods to measure sensitivity and inhibition of PCR in each sample, and strategies to surmount competition between templates during PCR.

Traces of protein and DNA are preserved on stone tools used to process animals. Previous research documents the identification of protein residues from tools sonicated in 5% ammonium hydroxide, but it remains untested whether the same treatment yields useable DNA. In this study we report both DNA and protein recovery using 5% ammonium hydroxide from residues on stone tools. We extracted 13-year-old residues from experimentally manufactured stone tools used to butcher a single animal. We also show that surface washing procedures typically used to curate stone tools remove only a small fraction of the DNA and protein deposited during animal butchery.

Agrobacterium tumefaciens and Agrobacterium rhizogenes transfer plasmid-encoded genes and virulence (Vir) proteins into plant cells. The transferred DNA (T-DNA) is stably inherited and expressed in plant cells, causing crown gall or hairy root disease. DNA transfer from A. tumefaciens into plant cells resembles plasmid conjugation; single-stranded DNA (ssDNA) is exported from the bacteria via a type IV secretion system comprised of VirB1 through VirB11 and VirD4. Bacteria also secrete certain Vir proteins into plant cells via this pore. One of these, VirE2, is an ssDNA-binding protein crucial for efficient T-DNA transfer and integration. VirE2 binds incoming ssT-DNA and helps target it into the nucleus. Some strains of A. rhizogenes lack VirE2, but they still transfer T-DNA efficiently. We isolated a novel gene from A. rhizogenes that restored pathogenicity to virE2 mutant A. tumefaciens . The GALLS gene was essential for pathogenicity of A. rhizogenes . Unlike VirE2, GALLS contains a nucleoside triphosphate binding motif similar to one in TraA, a strand transferase conjugation protein. Despite their lack of similarity, GALLS substituted for VirE2.

Agrobacterium tumefaciens oncogenes cause transformed plant cells to overproduce auxin and cytokinin. Two oncogenes encode enzymes that convert tryptophan to indole-3-acetic acid (auxin): iaaM (tryptophan mono-oxygenase) and iaaH (indole-3-acetamide hydrolase). A third oncogene (ipt) encodes AMP isopentenyl transferase, which produces cytokinin (isopentenyl-AMP). Inactivation of ipt and iaaM (or iaaH) abolishes tumorigenesis. Because adequate means do not exist to control crown gall, we created resistant plants by introducing transgenes designed to elicit posttranscriptional gene silencing (PTGS) of iaaM and ipt. Transgenes that elicit silencing trigger sequence-specific destruction of the inducing RNA and messenger RNAs with related sequences. Although PTGS has proven effective against a variety of target genes, we found that a much higher percentage of transgenic lines silenced iaaM than ipt, suggesting that transgene sequences influenced the effectiveness of PTGS. Sequences required for oncogene silencing included a translation start site. A transgene encoding a translatable sense-strand RNA from the 5′ end of iaaM silenced the iaaM oncogene, but deletion of the translation start site abolished the ability of the transgene to silence iaaM. Silencing A. tumefaciens T-DNA oncogenes is a new and effective method to produce plants resistant to crown gall disease.

Crown gall disease is an economically significant problem in fruit and nut orchards, vineyards, and nurseries worldwide. Tumors on stems and leaves result from excessive production of the phytohormones auxin and cytokinin in plant cells genetically transformed by Agrobacterium tumefaciens. High phytohormone levels result from expression of three oncogenes transferred stably into the plant genome from A. tumefaciens: iaaM, iaaH, and ipt. The iaaM and iaaH oncogenes direct auxin biosynthesis, and the ipt oncogene causes cytokinin production. In contrast to other tissues, roots do not respond to high cytokinin levels, and auxin overproduction is sufficient to cause tumor growth on roots. Inactivation of iaaM abolished gall formation on apple tree roots. Transgenes designed to express double-stranded RNA from iaaM and ipt sequences prevented crown gall disease on roots of transgenic apple trees.

The effects of selenium on selenoprotein W gene expression were examined in cultured L8 rat skeletal muscle cells. Selenoprotein W contains selenium as selenocysteine in the primary protein structure and levels of this selenoprotein are affected by selenium. Northern blots indicated that there were no significant changes (P

Microcracks produced during the manufacture of stone tools may harbour ancient biological residues. Our studies test this hypothesis using light, scanning electron, fluorescence, and confocal microscopy to characterize microcracks produced in obsidian microblades by pressure flaking. Cell-sized fluorescent latex beads penetrated microcracks to depths exceeding 50 μm below the microblade surface. Fluorescently labelled blood protein and DNA were trapped in microcracks within 5 min of exposure. Washing artifacts with water did not remove blood residues from microcracks. However, 60–80% of trapped blood protein and DNA were released by an 18-hour incubation in 4M guanidine hydrochloride or by sonication in 5% ammonium hydroxide. These experiments suggest that residues trapped in artifact microcracks represent an important and often overlooked source of ancient DNA and protein.

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG RP4 ) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans -acting Dtr functions and TraG RP4 . A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG RP4 was expressed in the donors. Mutations in traG RP4 with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG RP4 nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG RP4 mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG RP4 -mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG RP4 and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.

VirD2 is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer. In addition to its endonuclease domain, VirD2 contains a bipartite C-terminal nuclear localization sequence (NLS) and a conserved region called omega that is important for virulence. Previous results from our laboratory indicated that the C-terminal, bipartite NLS and the omega region are not essential for nuclear uptake of T-DNA, and further suggested that the omega domain may be required for efficient integration of T-DNA into the plant genome. In this study, we took two approaches to investigate the importance of the omega domain in T-DNA integration. Using the first approach, we constructed a T-DNA binary vector containing a promoterless gusA-intron gene just inside the right T-DNA border. The expression of beta-glucuronidase (GUS) activity in plant cells transformed by this T-DNA would indicate that the T-DNA integrated downstream of a plant promoter. Approximately 0.4% of the tobacco cell clusters infected by a wild-type A. tumefaciens strain harboring this vector stained blue with 5-bromo-4-chloro-3-indolyl beta-D-glucuronic acid (X-gluc). However, using an omega-mutant A. tumefaciens strain harboring the same binary vector, we did not detect any blue staining. Using the second approach, we directly demonstrated that more T-DNA is integrated into high-molecular-weight plant DNA after infection of Arabidopsis thaliana cells with a wild-type A. tumefaciens strain than with a strain containing a VirD2 omega deletion/substitution. Taken together, these data indicate that the VirD2 omega domain is important for efficient T-DNA integration. To determine whether the use of the T-DNA right border is altered in those few tumors generated by A. tumefaciens strains harboring the omega mutation, we analyzed DNA extracted from these tumors. Our data indicate that the right border was used to integrate the T-DNA in a similar manner regardless of whether the VirD2 protein encoded by the inciting A. tumefaciens was wild-type or contained an omega mutation. In addition, a mutant VirD2 protein lacking the omega domain was as least as active in cleaving a T-DNA border in vitro as was the wild-type protein. Finally, we investigated the role of various amino acids of the omega and bipartite NLS domains in the targeting of a GUS-VirD2 fusion protein to the nucleus of electroporated tobacco protoplasts. Deletion of the omega domain, or mutation of the 10-amino-acid region between the two components of the bipartite NLS, had little effect upon the nuclear targeting of the GUS-VirD2 fusion protein. Mutation of both components of the NLS reduced, but did not eliminate, targeting of the fusion protein to the nucleus.

In the first unit, students prepare plasmid DNA, construct a restriction map of the plasmid, and transform it into E. coli . The plasmid contains a luciferase ( lux ) reporter gene from Vibrio fischeri , which introduces the concept of reporter genes through first-hand experience. Students learn to digest DNA with a restriction endonuclease and analyze the restriction fragments by agarose gel electrophoresis.

In the third unit, students isolate bacteria from environments that they choose. Each student selects one unknown bacterium to culture, examine by light microscopy, and identify by DNA sequence analysis. Students isolate genomic DNA from the bacterium and use polymerase chain reaction (PCR) to amplify 16S ribosomal RNA (rRNA) genes. They purify the PCR products and analyze these amplicons by agarose gel electrophoresis. Students submit the purified PCR products for sequence analysis, and they use the Basic Local Alignment Search Tool (BLAST) program ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) to identify bacterial species with similar or identical 16S rRNA genes.

In the fourth unit, students extract genomic DNA from uncultured bacterial populations from environments that they choose, and they use polymerase chain reaction (PCR) to amplify bacterial 16S ribosomal RNA (rRNA) genes. The PCR product synthesized will likely contain a mixture of amplicons derived from 16S rRNA genes from each of the bacterial species in the sample. The terminal restriction fragment length polymorphism (T-RFLP) method uses one fluorescently labeled primer and one unlabeled primer so that one end of the amplicon has a fluorescent tag. The students digest the amplicons with a restriction endonuclease and use capillary electrophoresis to detect restriction fragments containing the labeled primer. T-RFLP analysis indicates the complexity of a bacterial population, and differences (or similarities) between two bacterial populations may become obvious by comparing their T-RFLP patterns.

In the second unit, students express, purify, and analyze affinity-tagged Staphylococcus aureus fibronectin-binding protein (FnbA), which allows S. aureus to bind host cells. Students use an E. coli strain that expresses the fnbA gene from an inducible promoter. The gene has six histidine codons fused to its 5’ end. The six histidines at the amino-terminus of FnbA comprise a nickel-binding domain, which allows purification of the histidine-tagged protein by chromatography over a nickel resin. Unit 2 concludes with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to assess yield and purity of the 6-His-tagged FnbA protein.

The T-DNA portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events generate linear single-stranded VirD2-bound DNA molecules that include the entire T-DNA (T-strands). VirD2 protein contains a nuclear localization signal (NLS) near the C terminus and may direct bound T-strands to plant nuclei. We constructed mutations in virD2 and showed that the NLS was important for tumorigenesis, although T-strand production occurred normally in its absence. A tobacco etch virus NLS, substituted for the VirD2 NLS, restored tumor-inducing activity. Amino acids (the omega sequence) at the C terminus of VirD2, outside the NLS and the endonuclease domain, contributed significantly to tumorigenesis, suggesting that VirD2 may serve a third important function in T-DNA transfer.

Humann, J., Andrews, S., and Ream, W. 2006. VirE1-mediated resistance to crown gall in transgenic Arabidopsis thaliana. Phytopathology 96:105110. Crown gall disease, caused by Agrobacterium tumefaciens, remains a serious agricultural problem despite current biocontrol methods. Agrobacterium tumefaciens transfers single-stranded DNA (T-strands) into plant cells along with several virulence proteins, including a singlestranded DNA-binding protein (VirE2). In plant cells, T-strands are protected from nucleases and targeted to the nucleus by VirE2, which is essential for efficient transmission (transfer and integration) of T-strands. VirE1 is the secretory chaperone for VirE2; it prevents VirE2 from forming aggregates and from binding the T-strands in bacterial cells. Therefore, we hypothesized that sufficient quantities of VirE1 expressed in plant cells might block T-DNA transmission by preventing VirE2 from binding T-strands. Here we show that root explants from Arabidopsis thaliana plants that expressed virE1 formed 3.5-fold fewer tumors than roots from plants without virE1. Also, this resistance was specific for VirE2-mediated Agrobacterium transformation. Plants that have been genetically altered to resist crown gall may prove more effective than biological control.

Agrobacterium tumefaciens and Agrobacterium rhizogenes are related pathogens that cause crown gall and hairy root diseases, which result from integration and expression of bacterial genes in the plant genome. Single-stranded DNA (T strands) and virulence proteins are translocated into plant cells by a type IV secretion system. VirD2 nicks a specific DNA sequence, attaches to the 5′ end, and pilots the DNA into plant cells. A. tumefaciens translocates single-stranded DNA-binding protein VirE2 into plant cells where it likely binds T strands and may aid in targeting them into the nucleus. Although some A. rhizogenes strains lack VirE2, they transfer T strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant for tumor formation. Unlike VirE2, full-length GALLS (GALLS-FL) contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. GALLS-FL and VirE2 contain nuclear localization signals (NLS) and secretion signals. Mutations in any of these domains abolish the ability of the GALLS gene to substitute for v irE2 . Here, we show that the GALLS gene encodes two proteins from one open reading frame: GALLS-FL and a protein comprised of the C-terminal domain, which initiates at an internal in-frame start codon. On some hosts, both GALLS proteins were required to substitute for VirE2. GALLS-FL tagged with yellow fluorescent protein localized to the nucleus of tobacco cells in an NLS-dependent manner. In plant cells, the GALLS proteins interacted with themselves, VirD2, and each other. VirD2 interacted with GALLS-FL and localized inside the nucleus, where its predicted helicase activity may pull T strands into the nucleus.

Dietary selenium deficiency causes white muscle disease, which results in calcification of skeletal and cardiac muscle in sheep, and is associated with a cardiomyopathy called Keshan disease in humans. This prompted us to search for selenoproteins abundant in skeletal and cardiac muscle, which led to the discovery of selenoprotein W (SelW). This small (87 amino acid) protein contains one selenocysteine residue and exists in four forms. One type has glutathione bound to a specific cysteine residue, which suggests that SelW has an antioxidant function, as several other selenoproteins do. Another SelW form contains an unknown 41 MW moiety, SelW is also present with both adducts or with none. SelW contains a putative calcium-binding domain near its carboxy terminus, although there is no evidence for a direct association between SelW and calcium. SelW is highly conserved (83% identical) among the six mammals studied to date (human, monkey, rat, mouse, sheep, and pig) suggesting that SelW plays an important role in the health of skeletal and cardiac muscle. SelW is abundant in skeletal and cardiac muscle and brain in primates and sheep, but it is not abundant in rodent heart muscle. In contrast to primates and sheep, rodent cardiac muscle does not deteriorate upon selenium depletion. Dietary selenium affects SelW mRNA and protein accumulation, but selenium levels do not affect the transcription rate of the SelW gene. Instead, adequate selenium is required to stabilize SelW mRNA, and selenium depletion reduces the half life of SelW mRNA. Although the metabolic function of SelW remains unknown, selenium deficiency eliminates SelW from skeletal and cardiac muscle, suggesting a link between SelW and healthy muscle tissues.

Agrobacterium tumefacienstransfers single-stranded DNA (ssDNA) into plants. Efficient tumorigenesis requires VirE1-dependent export of ssDNA-binding (SSB) protein VirE2. VirE1 binds VirE2 domains involved in SSB and self-association, and VirE1 may facilitate VirE2 export by preventing VirE2 aggregation and the premature binding of VirE2 to ssDNA.

Selenoprotein W was purified from monkey skeletal muscle to investigate its binding of glutathione. The purification was accomplished by concentration of the cytosol with an Amicon cell, gel filtration using Sephadex G-50, cation-exchange chromatography with CM-Sephadex, and reverse-phase high-pressure liquid chromatography using a C-18 Vydac column. Selenoprotein W was monitored during purification by slot blots. These steps resulted in an electrophoretically pure selenoprotein W preparation that was estimated by gel filtration to have molecular weight of about 10 kDa. N-terminal amino acid sequencing was used to confirm that the pure proteins were selenoprotein W. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) revealed that the proteins existed in three masses of 9635 +/- 7, 9371 +/- 11, and 9330 +/- 5 Da. The theoretical mass of the protein predicted from the cDNA sequence is 9330 Da. The 9635-Da form of the protein was shown to contain bound glutathione (306 Da), which could be released by reduction with dithiothreitol at 50 degreesC. The form with a mass of 9371 Da is assumed to result from binding of an unidentified 41-Da moiety to the 9330-Da form of the protein. MALDI peptide mapping with endoproteinase Glu-C suggested that glutathione is bound to the 36th amino acid (cysteine) of selenoprotein W.

Publisher Summary Chain termination with dideoxynucleoside triphosphates (ddNTPs) is used in automated sequencers and for sequencing at the bench. Dideoxy sequencing occurs in a set of four reactions, each of which contains the DNA template to be sequenced; an oligonucleotide primer complementary to DNA 3´ of the area to be sequenced; DNA polymerase; the four deoxynucleoside triphosphates (dNTPs), one of which is radioactively labeled to allow detection; and a small amount of one of the four ddNTPs. Either a single-stranded template is used or double-stranded DNA is denatured to allow the primer to anneal. During primer extension, a ddNTP may sometimes incorporate instead of the corresponding dNTP. Because the ddNTPs lack a 3´-OH, which is required to add the next nucleotide, the incorporation of a ddNTP terminates a growing chain at that position. The result is a mixture of DNA chains of different lengths, complementary to the template, each of which was terminated at the position of a specific nucleotide in the sequence. When the four reactions are loaded in four wells of a denaturing polyacrylamide gel and separated by electrophoresis, the dideoxy-terminated segments form a ladder of bands. The gel is exposed to X-ray film, and the sequence (complementary to the template) is read off the film. Automated sequencing reactions are labeled with fluorescent dyes, which are detected as they travel past a laser beam during electrophoresis. Four reactions must be run, with four different dye labels, one for each dNTP. The fluors are attached either to the primer or to the ddNTPs. This chapter also illustrates how to sequence the putative mutant virD2 gene created in site-directed mutagenesis experiments using α-35S-labelled dATP.

This chapter explains how the plasmid pGEX2–VirD2 (the polymerase chain reaction [PCR] product virD 2 inserted into pGEX 2 ) expresses the fusion protein VirD 2 . Transcription of this hybrid gene comes from the tac promoter located upstream of glutathione- S -transferase ( gst ). The tac promoter is a hybrid of the E. coli trp and lac promoters; it is a very strong promoter regulated in the same manner as the lac promoter. The lacI gene encodes the Lac repressor, which binds to lac promoter/operator regions and inhibits transcription. In lacI+ cells, a single copy lac (or tac ) promoter will be repressed by lacI unless an inducer, such as lactose or IPTG (isopropylthiogalactoside), is present. In multicopy plasmids (for example, pUC or pGEX), multiple copies of the tac promoter titrate all of the Lac repressor made in lacI+ strains. To regulate tac promoters on these multicopy plasmids, cells must overproduce the LacI protein. Strains carrying the mutant allele lacI q overexpress LacI at levels sufficient to regulate the tac promoter on multicopy plasmids. Full repression of the gene is not necessary because the Gst–VirD 2 fusion protein is not deleterious to E. coli. Various problems that could be faced during this experiment have also been highlighted in the chapter. Finally, crude extracts are prepared and fusion protein is purified, which are then analyzed by SDS-polyacrylamide gel electrophoresis.

This chapter uses Northern blot analysis to estimate the amount and molecular weight of a specific messenger RNA (mRNA) within a preparation of total RNA. The Northern blot procedure measures mRNA accumulation, which reflects both the rate of transcription and the stability of the RNA in question. A hybridization probe can detect a particular mRNA among a population of RNAs separated by electrophoresis and transferred to a membrane. To demonstrate the Northern blot technique, this chapter utilizes expression of the Rbs S gene in tobacco leaves as an example. This light-regulated nuclear gene encodes the small subunit of the photosynthetic enzyme, RuBisCO. The large subunit of the enzyme is encoded by the chloroplast DNA. The RuBisCO holoenzyme catalyzes the addition of CO 2 to ribulose-1,5-bisphosphate—the first step in the Calvin cycle. RbsS mRNA is the most abundant message in tobacco leaves, making it easily detectable by Northern blot analysis.

Oligonucleotide-directed mutagenesis allows altering DNA in a specific location by hybridizing a complementary oligonucleotide primer containing the desired mutation to a circular single-stranded vector containing the target sequence. DNA polymerase extends the primer in vitro ; DNA ligase covalently joins the ends of the newly extended strand. This heteroduplex DNA now contains the mutation in its newly synthesized strand and the original sequence in the template strand. On transformation into E. coli, DNA repair and replication resolve this mismatch to produce colonies with either mutant or wild-type target DNA. Several methods can increase the chances of recovering mutant-containing clones. This chapter also illustrates how 58 bp can be deleted from virD2 and replaced with a 7-bp sequence that includes an NruI site.

Southern blots are used to identify a specific restriction fragment (containing a DNA sequence of interest) among a large heterogeneous population of DNA fragments. First, agarose gel electrophoresis is used to separate restriction fragments according to size. Next, the DNA is denatured in situ (in the gel) and transferred to a membrane. The DNA is bound to the membrane by UV cross-linking. Finally, the DNA sequence of interest is detected using a hybridization probe that must be complementary (or nearly complementary) to its target. After a prehybridization treatment to block sites on the membrane where the hybridization probe may bind nonspecifically, the membrane is incubated with the probe. Posthybridization washes remove all probes except those annealed to the target DNA bound to the filter. The probe–DNA fragment complex is then detected by autoradiography or a colorimetric method. To demonstrate the Southern (DNA) blot technique, this chapter also describes how to isolate genomic DNA from several different strains of Agrobacterium tumefaciens and examine restriction fragment length polymorphisms (RFLPs) in their virD2 genes.

Publisher Summary The yeast two-hybrid system is a way to analyze protein–protein interactions in vivo. The system is based on the observation that many eukaryotic transcription factors consist of two separable domains: one domain binds a specific DNA sequence and the other activates transcription. DNA-binding and activation domains from different transcription factors often retain their functions when combined. The two-hybrid system uses the transcription of a reporter gene in yeast to assay the interaction between two proteins, one of which is fused to an activator domain and the other is fused to a DNA-binding domain. If the two proteins in question interact, they will bring together the DNA-binding and the transcription-activation domains, and transcription of the reporter gene will occur. If the two proteins do not interact, transcription of the reporter gene will not occur because although the DNA-binding domain can find its DNA target, it requires the activation domain to stimulate transcription. Two uses of yeast two-hybrid screening are common: it is used to screen for unknown genes encoding proteins that interact with a protein encoded by a known gene, and it is used to determine whether two known proteins interact and to study the molecular aspects of the interaction. This chapter also uses the yeast two-hybrid system to determine whether two Agrobacterium virulence proteins, VirE1 and VirE2, interact.

The goal of this experiment is to fuse virD2 (a virulence gene from Agrobacterium tumefaciens ) to glutathione- S -transferase gene ( gst ) to express and purify the fusion protein. To accomplish this, it is necessary to create restriction sites on either side of virD2. Polymerase chain reaction (PCR) is used to make copies of virD2 flanked by the required restriction sites. These restriction sites allow one the insertion of virD2 into the expression vector pGEX2. To start this and the subsequent experiments, several different plasmids are required, which are to be purified. The experiment includes steps like preparing plasmid by cesium chloride density gradient centrifugation, using PCR to synthesize virD2 flanked with restriction sites, later performing restriction digests of plasmid pGEX2 and PCR products, and then purifying DNA fragments from agarose. After purification, the PCR product is ligated to pGEX2 and E. coli is transformed with ligated plasmid. The final steps involve making small-scale preparation of plasmid DNA from broth cultures and performing restriction digests of DNAs.

SECIS elements form stem-loop structures in the 3′ untranslated regions (UTR) of eukaryotic mRNAs that encode selenoproteins. These elements direct incorporation of selenocysteine at UGA codons, provided the SECIS element lies a sufficient distance from the UGA. The cDNAs encoding skeletal muscle selenoprotein W from human, rhesus monkey, sheep, rat, and mouse contained highly similar SECIS elements that retained important features common to all known SECIS elements. Comparative analysis of these SECIS elements showed that in some regions both predicted secondary structure and nucleotide sequences were conserved, in other areas secondary structure was maintained using different primary sequence, and in still other portions, base pairing was not conserved. The rodent and sheep selenoprotein W mRNAs used UGA as a stop codon and as a selenocysteine codon. Thus, UGA specified both selenocysteine incorporation and termination in a single mRNA. The selenoprotein W SECIS elements contained an additional highly conserved base-paired stem that may prevent inappropriate selenocysteine incorporation at the UGA stop codons.

Agrobacterium tumefaciens transfers single-stranded DNAs (T strands) into plant cells. VirE1 and VirE2, which is a single-stranded DNA binding protein, are important for tumorigenesis. We show that T strands and VirE2 can enter plant cells independently and that export of VirE2, but not of T strands, depends on VirE1.

This review considers four fundamental questions that comprise the heart of the agrobacterium story. How does interkingdom genetic exchange occur? How do the host and pathogen communicate, and what do they say to each other? How do enzymes encoded by the T-DNAs of A. tumefaciens and A. rhizogenes transform host cells into tumor cells? How does the plant-agrobacterium interaction influence the ecology and evolution of each participant? We have partial answers to each of these questions

Agrobacterium tumefaciens incites crown gall tumors on many dicotyledonous plants when viable bacteria infect wounded tissue (2, 22). Virulent strains contain a 190 kilobase (kb) tumor-inducing (Ti) plasmid that carries genes essential for tumorigenesis (8, 38). During tumorigenesis a specific segment of the Ti plasmid, the T-DNA, integrates into plant nuclear DNA (5, 19). Plant tumor cells express T-DNA genes responsible for tumorous growth, but T-DNA transmission does not require tumorigenesis or T-DNA encoded proteins (20, 29). Similar 23 bp direct repeats lie at both ends of four different T regions (1, 33, 40), and these repeats signal the T-DNA borders since T-DNA ends occur in or near these repeats in several different tumors (11, 32, 34, 41). Deletions that remove the T region right border severely attenuate virulence on most plants even though the tumor maintenance genes remain intact (11, 15, 24, 26-27, 31, 39). Such deletion mutants provide an assay for right border function: we can reintroduce T-DNA borders to the right of the T region and measure their ability to restore virulence. T-DNA transmission requires only a right border repeat in cis (26–27, 39), but Ti plasmid sequences that lie to the right of the right border repeat greatly stimulate its function (13, 25–27).