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Cold acclimation in temperate, woody plants is a complex phenomenon that involves distinct changes in gene activity and protein synthesis. In previous research, a 60-kDa protein (PCA60), belonging to the dehydrin family of stress-related proteins, was identified in peach bark, and its corresponding gene (ppdhn1) was cloned and characterized. Presently, we report on the results of immunolocalization studies and in vitro cryoprotection assays. Seasonal collections of current-year stems were embedded in LR White or epoxy resin and sections of bark were probed with either a polyclonal antibody directed against a 15 amino acid sequence consensus region of dehydrins or a polyclonal antibody directed against partially-purified PCA60. In vitro cryoprotection assays utilized lactose dehydrogenase (LDH), a cold-labile enzyme. Immunolocalization at the light level indicated that the dehydrin was confined to the cytoplasm and absent in organelles. This localization was preliminarily confirmed at the ultrastructural level. LDH assays indicated cryoprotective activity in total protein extracts collected from winter bark tissues but completely absent in extracts of summer bark tissues. Preliminary LDH assays utilizing purified PCA60 also demonstrated cryoprotective activity. In general, the data further support a role for dehydrins in cold acclimation of woody plants.

Barley and maize seedlings exposed to a non-lethal dehydration treatment were previously shown to synthesise dehydration-induced protein (dehydrins). Antibodies against maize dehydrin react immunologically with polypeptides in dehydrating barley and wheat seedlings. Barley dehydrin cDNA hydridises to RNA present in dehydrating wheat, and other grass seedlings. These results indicate similarity of dehydrin epitopes, mRNA sequences, and gene regulation amongst dehydrins of several grasses, including wheat and barley. Two barley dehydrin genes were assigned to barley chromosome 6 (dhn3 and dhn4) and two to chromosome 7 (dhn1 and dhn2) using barley dehydrin cDNA clones and DNA from wheat-barley addition lines. Genes on the same chromosome have greater similarity in sequence than between chromosomes. Analysis of several different barley cultivars with one of the dehydrin cDNA clones revealed restriction fragment length polymorphism.

A set of broad host range cloning vectors has been constructed from the IncW plasmid pSa. These vectors have been constructed from the transfer defective deletion derivative pSa151, which encodes resistance to kanamycin and spectinomycin–streptomycin. Two of the vectors also contain the chloramphenicol resistance gene of Tn9, and a third contains the chloramphenicol resistance gene of pSa. One of the vectors contains the cos sequence of bacteriophage λ and can be used with in vitro packaging systems in the construction of large recombinant plasmids. Two of these vectors can be mobilized and transferred in the presence of a pBR322 derivative containing the transfer genes of pSa. Together these vectors contain cloning sites for SstII, HindIII, EcoRI, KpnI, PvuII, BamHI, SmaI, and Bg1II, and recombinants at certain of these sites can be detected by insertional inactivation of a drug resistance phenotype. The broad host range properties of the origin of replication of pSa allow the use of these vectors in a variety of gram–negative bacteria.

We isolated a mutant strain of Agrobacterium tumefaciens, designated Ros, that has a pleiotropic phenotype which includes elevated levels of expression of certain genes in the virulence (Vir) region of tumor-inducing plasmid pTiC58. This mutant and others were isolated by fusing the promoter of the Vir bak gene to a promoterless gene (cat) that encodes chloramphenicol acetyltransferase and then selecting for increased expression of cat in A. tumefaciens. The ros mutation is chromosomal in nature and is characterized by a more-than-300-fold increase in the level of expression of bak and a 12-fold increase in the level of expression of an adjacent divergent operon containing the hdv genes, which are involved in some aspect of host specificity. The Ros mutant is fully virulent and is typified by its unusual colony morphology; the colonies have rough surfaces, uneven edges, and a pit in the center at 24 degrees C and vary markedly in appearance from one growth temperature to another. The Ros mutant is not able to form colonies at 12 degrees C, a temperature at which the wild-type strain forms colonies in 14 days. The ros mutation occurs spontaneously with a frequency of 5 X 10(-8). Genetic and biochemical evidence indicates that the product of the ros locus is a negative regulator of Ti plasmid genes and is related to undefined chromosomally encoded functions that are involved in the mutant phenotype.

Single feature polymorphisms (SFPs) are microarray-based molecular markers that are detected by hybridization of DNA or cRNA to oligonucleotide probes. With an objective to identify the potential polymorphic markers for drought tolerance in pigeonpea [Cajanus cajan (L.) Millspaugh], an important legume crop for the semi-arid tropics but deficient in genomic resources, Affymetrix Genome Arrays of soybean (Glycine max), a closely related species of pigeonpea were used on cRNA of six parental genotypes of three mapping populations of pigeonpea segregating for agronomic traits like drought tolerance and pod borer (Helicoverpa armigiera) resistance. By using robustified projection pursuit method on 15 pair-wise comparisons for the six parental genotypes, 5,692 SFPs were identified. Number of SFPs varied from 780 (ICPL 8755 × ICPL 227) to 854 (ICPL 151 × ICPL 87) per parental combination of the mapping populations. Randomly selected 179 SFPs were used for validation by Sanger sequencing and good quality sequence data were obtained for 99 genes of which 75 genes showed sequence polymorphisms. While associating the sequence polymorphisms with SFPs detected, true positives were observed for 52.6% SFPs detected. In terms of parental combinations of the mapping populations, occurrence of true positives was 34.48% for ICPL 151 × ICPL 87, 41.86% for ICPL 8755 × ICPL 227, and 81.58% for ICP 28 × ICPW 94. In addition, a set of 139 candidate genes that may be associated with drought tolerance has been identified based on gene ontology analysis of the homologous pigeonpea genes to the soybean genes that detected SFPs between the parents of the mapping populations segregating for drought tolerance. Electronic supplementary material The online version of this article (doi:10.1007/s10142-011-0227-2) contains supplementary material, which is available to authorized users.

The ros chromosomal mutation of Agrobacterium tumefaciens LBA4301 causes increased expression of two promoters in the Vir region and three promoters in the T-region of the plasmid pTiC58. Comparison of the nucleotide sequences of two of these promoters has revealed the presence in both of a region of dyad symmetry containing the sequence ATTG-A. The results suggest that certain promoters in pTiC58 are subject to a form of negative regulation that is inactivated by the ros chromosomal mutation.

A set of four cloning vectors has been constructed from the wide host range IncW plasmid pSa. Although the vectors are transfer defective, three of them can be efficiently transferred into a wide variety of Gram-negative bacteria by a separate mobilizing plasmid. One of the vectors is a cosmid and can be used for cloning large DNA fragments. The vectors, which can be selected on the basis of kanamycin, spectinomycin, or chloramphenicol resistance, contain cloning sites for seven different restriction endonucleases. DNA fragments from the A. tumefaciens plasmid TiC58 have been cloned into these vectors, and used to complement avirulent TiC58::Tn5 mutants.

A DNA fragment encoding the transposon Tn9 chloramphenicol acetyltransferase gene (cat) was inserted into M13 phage and pUC plasmid cloning vehicles. When the cat gene was inserted in the same orientation as the lacZ gene, two new polypeptides were produced. One polypeptide possessed chloramphenicol acetyltransferase activity, while the other expressed beta-galactosidase alpha-donor activity. Both new polypeptides were translated from a hybrid messenger RNA initiating from the lac promoter. These observations may help explain why not all inserts produce white plaques.

A system has been developed to transcriptionally map plasmid DNA molecules in vivo. This system is based upon the activity of the Tn 9 Cm r gene whose product, chloramphenicol acetyltransferase (CAT), can be easily assayed enzymatically and identified phenotypically in Escherichia coli . The Cm r gene was excised as a single DNA fragment from the plasmid cloning vector, pBR328, and its ends were converted to hexamer-specific restriction enzyme recognition sites. These modified Cm r gene fragments were designated “CAT cartridges”. The CAT cartridges were expressed at high levels only when inserted in the proper orientation, downstream of a plasmid promoter. Using DNA restriction endonuclease cleavage sites as insertion points, the CAT cartridges were inserted into plasmid pBR327, and the levels of CAT activity produced in cells containing the various plasmids were determined. The CAT activities at each position and orientation were used to construct a transcriptional map of plasmid pBR327. The positions and strengths of promoters demonstrated in vivo using the CAT cartridge coincided with those predicted by in vitro studies. Using the CAT cartridges it should be possible to study gene expression on any extrachromosomal element in the appropriate host.

Agrobacterium tumefaciens plasmid pTiC58 contains a cluster of genes spanning approximately 30 kb that are essential for virulence (Vir). The Vir region of pTiC58 is composed of at least six complementation groups much like the Vir region of octopine Ti plasmids (Klee et al., 1983; Hille et al., 1984; Hooykaas et al., 1984, Lundquist et al, 1984). These groups are designated virA, virB, virG, virC, virD, and virE, and occur in the same order in octopine and nopaline Ti plasmids (Fig. 1). Each Open image in new window complementation group seems so far to be organized as an operon, and genes that have been compared at the level of DNA sequence or antibody cross reaction show considerable homologies (Hagiya, et al., 1985; Hirooka & Kado, 1986; Close et al., in press; Yanofsky et al., in press; Winans et al., in press; Hooykaas et al., unpublished). One function of the Vir genes is to process a section of the pTi plasmid that is transferred and integrated into plant genomes (T-DNA) (Alt-Moerbe et al., 1986).

SUMMARY: Genetic and reversion analyses of a thermosensitive pleiotropic mutant strain of Klebsiella pneumoniae with defects in nitrogen fixation and nitrogen metabolism have shown that the pleiotropic behaviour of the mutant is due to a single mutation in a gene designated nim. This gene is cotransducible with trp at a frequency of about 30% (using bacteriophage P1) and with cys at a frequency of about 14%. The gene order is cys, trp, nim. The defect in the nim mutant is complemented by the E. coli F’ element, F’148.

To obtain bacterial-mediated oncogenic transformation of plants, the transferred DNA (T-DNA) of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens is transferred to its plant host cells during infection. The initial phases of transformation involve the processing of the T-DNA in the bacterial cell after induction of the vir genes located on the Ti plasmid. The kinetics and conditions of this processing were examined and upon induction with acetosyringone up to 40% of the left and right borders of the T-DNA were cleaved. This cleavage was dependent upon virA, virG, and VirD and was rec-independent. Processed T-DNA was observed within 30 min after induction and was delayed by an increased concentration of phosphate in the induction medium. When DNA was isolated in the absence of protease treatment, the DNA fragment corresponding to the left side of the cut at both the left and right border region exhibited gel retardation, suggesting one or more "pilot" proteins may be involved in T-DNA transfer. Although the relative abundance of a processed product does not necessarily imply relative importance, the preponderance of double-stranded cleavage products suggests that double-stranded T-DNA should be considered as a possible intermediate in T-DNA transfer.

Several cDNAs related to an ABA-induced cDNA from barley aleurone were isolated from barley and corn seedlings that were undergoing dehydration. Four different barley polypeptides with sizes of 22.6, 16.2, 14.4 and 14.2 kDa and a single corn polypeptide with a size of 17.0 kDa were predicted from the nucleotide sequences of the cDNAs. These dehydration-induced proteins (dehydrins) are very similar to each other and to a previously identified rice protein induced by ABA and salt, and have at least some similarity to a previously identified cotton embryo protein. Each dehydrin is extremely hydrophilic, glycine-rich, cysteine- and tryptophan-free and contains repeated units in a conserved linear order. A lysine-rich repeating unit occurs twice in each protein, once at the carboxy terminus and once partway through the polypeptide, adjacent to a succession of serines. This repeating unit and the adjacent flanking run of serines are conserved with minimal variation among all dehydrins. Another repeating unit is flanked by the two copies of the lysine-rich unit, and varies in number from one to five copies. This latter repeating unit is less conserved than the former, varying even within a singly dehydrin. The messenger RNAs corresponding to each cDNA are abundant in dehydrating, but not in well-watered seedlings. The amino acid sequence of tryptic peptides from purified dehydration-induced proteins of corn established that the corn cDNAs correspond to a protein that is produced in abundance during the response of corn seedlings to dehydration.

The virulence (vir) region of pTiC58 was screened for promoter activities by using gene fusions to a promoterless lux operon in the broad-host-range vector pUCD615. Active vir fragments contained the strongly acetosyringone-inducible promoters of virB, virC, virD, and virE and the weakly inducible promoters of virA and virG. Identical induction patterns were obtained with freshly sliced carrot disks, suggesting that an inducer is released after plant tissue is wounded. Optimal conditions for vir gene induction were pH 5.7 for 50 microM acetosyringone or sinapic acid. The induction of virB and virE by acetosyringone was strictly dependent on intact virA and virG loci. An increase in the copy number of virG resulted in a proportional, acetosyringone-independent increase in vir gene expression, and a further increase occurred only if an inducing compound and virA were present.

The origin of replication of the IncW plasmid pSa has been cloned and the function of this origin in Escherichia coli examined. A 1.9-kb region of DNA is required for efficient autonomous replication, and a 0.47-kb fragment within this region can initiate replication only in the presence of an autonomously replicating derivative of pSa. An Mr 35,000 protein (repA) is encoded adjacent to the origin and is required for efficient initiation of replication. The derivatives examined provide information suggesting a direct role of partition factors in plasmid replication and incompatibility.

Directed genetic modification of plants requires the transmission of specific foreign DNA to the host plant, where the DNA is incorporated and stably maintained in the plant nuclear genome. Although there are several DNA transmission techniques, the transfer of DNA mediated through the natural transmission of T-DNA of Ti plasmids of Agrobacterium tumefaciens has proven relatively efficient and practical. The initial processing and transmission of the T-DNA requires genes situated on the Ti plasmid and on the bacterial chromosome. The T-DNA processing genes on the Ti plasmid are located in the virulence (Vir) region. The virD2 gene of the virD operon, which appears to be involved in the formation of T-DNA intermediates by encoding a specific endonuclease, is constitutively synthesized in a permissive mutant ros of A. tumefaciens. Gene fusions to reporter genes such as the luciferase cassette of Vibrio fischeri made it possible to measure the expression of Vir genes during the on-going interaction between A. tumefaciens and carrot disks. Using phenolic inducers such as acetosyringone, virB, virC, virD and virE were identified as inducible operons of the Vir region in the presence of virA, virG and inducer. virC and virD are also controlled by the ros gene of the chromosome as mutations in the ros gene results in the constitutive expression of these operons. This has enabled us to study the formation of double stranded T-DNA intermediates in the absence of acetosyringone or other phenolic inducers. Integration of the T-DNA was directly observed in Haplopappus gracilis chromosome A by in situ hybridization analysis.

The virC (formerly bak) complementation group of the nopaline-type Ti plasmid pTiC58 encodes two proteins, VirC1 and VirC2. According to the primary structure of the polypeptides predicted by the nucleotide sequence, VirC1 is composed of 231 amino acids with a total molecular mass of 25.5 kilodaltons, and VirC2 is composed of 202 amino acids with a molecular mass of 22.1 kilodaltons. The pTiC58 VirC1 and VirC2 polypeptides are equal in length to VirC1 and VirC2 of the octopine-type plasmid pTiA6NC. VirC1 proteins of pTiC58 and pTiA6NC are identical at 202 (87.4%) of the amino acid residues, and this homology is distributed fairly evenly throughout the protein. VirC2 identities occur at 142 residues (70.3%), but fall predominantly into two blocks of higher homology (84.6 and 78.5%) separated by a 41-residue segment of much lower homology (29.3%). Mutations in virC resulted in attenuated virulence on all hosts tested, the severity of attenuation varying markedly depending on the type of plant inoculated. For example, the attenuation was more pronounced on Kalanchoe than on sunflower or jimson weed. Virulence was restored to normal on all hosts by in-trans complementation with corresponding nonmutant DNA fragments of pTiC58 or of the octopine-type plasmid pTi15955. Two oligopeptides from within the predicted pTiC58 VirC1 polypeptide were synthesized and used to raise antibodies. These antibodies were used to detect the VirC1 product of both pTiC58 and pTi15955. In both cases, virC was expressed constitutively in the Agrobacterium tumefaciens ros mutant. The homology between virC genes of octopine- and nopaline-type Ti plasmids thus includes a conservation of genetic regulatory control mechanisms as well as considerable conservation of the primary structure of the protein products.

Mutants with Tn5 insertions in the vir region of the Agrobacterium tumefaciens TiC58 plasmid are unable to form crown-gall tumors. Complementation tests of these vir region mutants were carried out by constructing merodiploids in a recombination-deficient strain. Each merodiploid possessed a mutant TiC58 plasmid and a recombinant plasmid containing either the homologous wild-type DNA region or the homologous region containing a second Tn5 insertion. The analysis identified six complementation groups. Mutations in one of these complementation groups were not complemented in trans and represent a cis-dominant locus. The mutation in one complementation group showed variation in host range.

Motivation: The deconvolution of the relationships between BAC clones and genes is a crucial step in the selective sequencing of the regions of interest in a genome. It usually requires combinatorial pooling of unique probes obtained from the genes (unigenes), and the screening of the BAC library using the pools in a hybridization experiment. Since several probes can hybridize to the same BAC, in order for the deconvolution to be achievable the pooling design has to be able to handle a large number of positives. As a consequence, smaller pools need to be designed which in turn increases the number of hybridization experiments possibly making the entire protocol unfeasible. Results: We propose a new algorithm that is capable of producing high accuracy deconvolution even in the presence of a weak pooling design, i.e., when pools are rather large. The algorithm compensates for the decrease of information in the hybridization data by taking advantage of a physical map of the BAC clones. We show that the right combination of combinatorial pooling and our algorithm not only dramatically reduces the number of pools required, but also successfully deconvolutes the BAC-gene relationships with almost perfect accuracy. Availability: Software available on request from the rst author.

Background Bread wheat is an allopolyploid species with a large, highly repetitive genome. To investigate the impact of selection on variants distributed among homoeologous wheat genomes and to build a foundation for understanding genotype-phenotype relationships, we performed population-scale re-sequencing of a diverse panel of wheat lines. Results A sample of 62 diverse lines was re-sequenced using the whole exome capture and genotyping-by-sequencing approaches. We describe the allele frequency, functional significance, and chromosomal distribution of 1.57 million single nucleotide polymorphisms and 161,719 small indels. Our results suggest that duplicated homoeologous genes are under purifying selection. We find contrasting patterns of variation and inter-variant associations among wheat genomes; this, in addition to demographic factors, could be explained by differences in the effect of directional selection on duplicated homoeologs. Only a small fraction of the homoeologous regions harboring selected variants overlapped among the wheat genomes in any given wheat line. These selected regions are enriched for loci associated with agronomic traits detected in genome-wide association studies. Conclusions Evidence suggests that directional selection in allopolyploids rarely acted on multiple parallel advantageous mutations across homoeologous regions, likely indicating that a fitness benefit could be obtained by a mutation at any one of the homoeologs. Additional advantageous variants in other homoelogs probably either contributed little benefit, or were unavailable in populations subjected to directional selection. We hypothesize that allopolyploidy may have increased the likelihood of beneficial allele recovery by broadening the set of possible selection targets. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0606-4) contains supplementary material, which is available to authorized users.

We study the problem of merging genetic maps, when the individual genetic maps are given as directed acyclic graphs. The problem is to build a consensus map, which includes and is consistent with all (or, the vast majority of) the markers in the individual maps. When markers in the input maps have ordering conflicts, the resulting consensus map will contain cycles. We formulate the problem of resolving cycles in a combinatorial optimization framework, which in turn is expressed as an integer linear program. A faster approximation algorithm is proposed, and an additional speed-up heuristic is developed. According to an extensive set of experimental results, our tool is consistently better than JOINMAP, both in terms of accuracy and running time.

The use of DNA sequence-based comparative genomics for evolutionary studies and for transferring information from model species to crop species has revolutionized molecular genetics and crop improvement strategies. This study compared 4485 expressed sequence tags (ESTs) that were physically mapped in wheat chromosome bins, to the public rice genome sequence data from 2251 ordered BAC/PAC clones using BLAST. A rice genome view of homologous wheat genome locations based on comparative sequence analysis revealed numerous chromosomal rearrangements that will significantly complicate the use of rice as a model for cross-species transfer of information in nonconserved regions.

The virulence genes of nopaline (pTiC58) and octopine (pTiA6NC) Ti plasmids are similarly affected by the Agrobacterium tumefaciens ros mutation. Of six vir region complementation groups (virA, virB, virG, virC, virD, and virE) examined by using fusions to reporter genes, the promoters of only two (virC and virD) responded to the ros mutation. For each promoter that was affected by ros, the level of expression of its associated genes was substantially elevated in the mutant. This increase was not influenced by Ti plasmid-encoded factors, and the mutation did not interfere with the induction of pTiC58 vir genes by phenolic compounds via the VirA/VirG regulatory control mechanism. The effects of the ros mutation and acetosyringone were cumulative for all vir promoters examined. The pleiotropic characteristics of the ros mutant include the complete absence of the major acidic capsular polysaccharide.

Background A typical genetical genomics experiment results in four separate data sets; genotype, gene expression, higher-order phenotypic data and metadata that describe the protocols, processing and the array platform. Used in concert, these data sets provide the opportunity to perform genetic analysis at a systems level. Their predictive power is largely determined by the gene expression dataset where tens of millions of data points can be generated using currently available mRNA profiling technologies. Such large, multidimensional data sets often have value beyond that extracted during their initial analysis and interpretation, particularly if conducted on widely distributed reference genetic materials. Besides quality and scale, access to the data is of primary importance as accessibility potentially allows the extraction of considerable added value from the same primary dataset by the wider research community. Although the number of genetical genomics experiments in different plant species is rapidly increasing, none to date has been presented in a form that allows quick and efficient on-line testing for possible associations between genes, loci and traits of interest by an entire research community. Description Using a reference population of 150 recombinant doubled haploid barley lines we generated novel phenotypic, mRNA abundance and SNP-based genotyping data sets, added them to a considerable volume of legacy trait data and entered them into the GeneNetwork http://www.genenetwork.org. GeneNetwork is a unified on-line analytical environment that enables the user to test genetic hypotheses about how component traits, such as mRNA abundance, may interact to condition more complex biological phenotypes (higher-order traits). Here we describe these barley data sets and demonstrate some of the functionalities GeneNetwork provides as an easily accessible and integrated analytical environment for exploring them. Conclusion By integrating barley genotypic, phenotypic and mRNA abundance data sets directly within GeneNetwork's analytical environment we provide simple web access to the data for the research community. In this environment, a combination of correlation analysis and linkage mapping provides the potential to identify and substantiate gene targets for saturation mapping and positional cloning. By integrating datasets from an unsequenced crop plant (barley) in a database that has been designed for an animal model species (mouse) with a well established genome sequence, we prove the importance of the concept and practice of modular development and interoperability of software engineering for biological data sets.