Evolutionary conserved lineage of Angela-family retrotransposons as a genome-wide microsatellite repeat dispersal agent.

A detailed examination of 45 pea (Pisum sativum L.) simple sequence repeat (SSR) loci revealed that 21 of them included homologous sequences corresponding to the long terminal repeat (LTR) of a novel retrotransposon. Further investigation, including full-length sequencing, led to its classification as an RLC-Angela-family-FJ434420 element. The LTR contained a variable region ranging from a simple TC repeat (TC)(11) to more complex repeats of TC/CA, (TC)(12-30), (CA)(18-22) and was up to 146 bp in length. These elements are the most abundant Ty1/copia retrotransposons identified in the pea genome and also occur in other legume species. It is interesting that analysis of 63 LTR-derived sequences originating from 30 legume species showed high phylogenetic conservation in their sequence, including the position of the variable SSR region. This extraordinary conservancy led us to the proposition of a new lineage, named MARTIANS, within the Angela family. Similar LTR structures and partial sequence similarities were detected in more distant members of this Angela family, the barley BARE-1 and rice RIRE-1 elements. Comparison of the LTR sequences from pea and Medicago truncatula elements indicated that microsatellites arise through the expansion of a pre-existing repeat motif. Thus, the presence of an SSR region within the LTR seems to be a typical feature of this MARTIANS lineage, and the evidence gathered from a wide range of species suggests that these elements may facilitate amplification and genome-wide dispersal of associated SSR sequences. The implications of this finding regarding the evolution of SSRs within the genome, as well as their utilization as molecular markers, are discussed.

Isolation and characterization of the highly repeated fraction of the banana genome.

Although the nuclear genome of banana (Musa spp.) is relatively small (1C approximately 610 Mbp for M. acuminata), the results obtained from other sequenced genomes suggest that more than half of the banana genome may be composed of repetitive and non-coding DNA sequences. Knowledge of repetitive DNA can facilitate mapping of important traits, phylogenetic studies, BAC-based physical mapping, and genome sequencing/annotation. However, only a few repetitive DNA sequences have been characterized in banana. In this work, we used DNA reassociation kinetics to isolate the highly repeated fraction of the banana genome (M. acuminata 'Calcutta 4'). Two libraries, one prepared from Cot

Long-range organization of plant satellite repeats investigated using strand-specific FISH.

The technique of chromosomal orientation and direction fluorescence in situ hybridization (COD-FISH) was adapted for plant chromosomes in order to study long-range organization of two families of satellite repeats, VicTR-B of Vicia sativa and PisTR-B of Pisum sativum. The technique allowed FISH to be performed on mitotic chromosomes in a strand-specific manner, resulting in visualization of the repeat orientation along the chromosomes and with respect to the direction of telomeric repeats. The VicTR-B probe applied to V. sativa chromosomes produced signals on a single chromatid at most regions containing corresponding sequences, thus confirming a presence of long arrays of head-to-tail arranged repeat monomers which is typical for satellite DNA. However, hybridization signals of different or equal intensities on both chromatids were also detected at some loci, suggesting a more complex arrangement of the repeats. Similar observations were made for PisTR-B repeats on P. sativum chromosomes, although the proportion of loci displaying signals on both chromatids was lower. In contrast to VicTR-B, orientation of the PisTR-B clusters with respect to telomeric sequences appeared to be conserved among subtelomeric regions of metacentric chromosomes and of the short arms of acrocentric chromosomes.

Development of a composite map in Vicia faba, breeding applications and future prospects.

A composite map of the Vicia faba genome based on morphological markers, isozymes, RAPDs, seed protein genes and microsatellites was constructed. The map incorporates data from 11 F(2) families for a total of 654 individuals all sharing the common female parent Vf 6. The integrated map is arranged in 14 major linkage groups (five of which were located in specific chromosomes). These linkage groups include 192 loci and cover 1559 cM with an overall average marker interval of 8 cM. By joining data of a new F(2) population segregating for resistance to ascochyta, broomrape and others traits of agronomic interest, have been saturated new areas of the genome. The combination of trisomic segregation, linkage analysis among loci from different families with a recurrent parent, and the analysis of new physically located markers, has allowed the establishment of the present status of the V. faba map with a wide coverage. This map provides an efficient tool in breeding applications such as disease-resistance mapping, QTL analyses and marker-assisted selection.

Multicolor FISH mapping of the dioecious model plant, Silene latifolia.

Silene latifolia is a key plant model in the study of sex determination and sex chromosome evolution. Current studies have been based on genetic mapping of the sequences linked to sex chromosomes with analysis of their characters and relative positions on the X and Y chromosomes. Until recently, very few DNA sequences have been physically mapped to the sex chromosomes of S. latifolia. We have carried out multicolor fluorescent in situ hybridization (FISH) analysis of S. latifolia chromosomes based on the presence and intensity of FISH signals on individual chromosomes. We have generated new markers by constructing and screening a sample bacterial artificial chromosome (BAC) library for appropriate FISH probes. Five newly isolated BAC clones yielded discrete signals on the chromosomes: two were specific for one autosome pair and three hybridized preferentially to the sex chromosomes. We present the FISH hybridization patterns of these five BAC inserts together with previously described repetitive sequences (X-43.1, 25S rDNA and 5S rDNA) and use them to analyze the S. latifolia karyotype. The autosomes of S. latifolia are difficult to distinguish based on their relative arm lengths. Using one BAC insert and the three repetitive sequences, we have constructed a standard FISH karyotype that can be used to distinguish all autosome pairs. We also analyze the hybridization patterns of these sequences on the sex chromosomes and discuss the utility of the karyotype mapping strategy presented to study sex chromosome evolution and Y chromosome degeneration.

Zaba: a novel miniature transposable element present in genomes of legume plants.

A novel family of miniature transposable elements, named Zaba, was identified in pea (Pisum sativum) and subsequently also in other legume species using computer analysis of their DNA sequences. Zaba elements are 141-190 bp long, generate 10-bp target site duplications, and their terminal inverted repeats make up most of the sequence. Zaba elements thus resemble class 3 foldback transposons. The elements are only moderately repetitive in pea (tens to hundreds copies per haploid genome), but they are present in up to thousands of copies in the genomes of several Medicago and Vicia species. More detailed analysis of the elements from pea, including isolation of new sequences from a genomic library, revealed that a fraction of these elements are truncated, and that their last transposition probably did not occur recently. A search for Zaba sequences in EST databases showed that at least some elements are transcribed, most probably due to their association with genic regions.

Molecular and cytogenetic analysis of repetitive DNA in pea (pisum sativum L.).

A set of pea DNA sequences representing the most abundant genomic repeats was obtained by combining several approaches. Dispersed repeats were isolated by screening a short-insert genomic library using genomic DNA as a probe. Thirty-two clones ranging from 149 to 2961 bp in size and from 1,000 to 39,000/lC in their copy number were sequenced and further characterized. Fourteen clones were identified as retrotransposon-like sequences, based on their homologies to known elements. Fluorescence in situ hybridization using clones of reverse transcriptase and integrase coding sequences as probes revealed that corresponding retroelements were scattered along all pea chromosomes. Two novel families of tandem repeats, named PisTR-A and PisTR-B, were isolated by screening a genomic DNA library with Cot-1 DNA and by employing genomic self-priming PCR, respectively. PisTR-A repeats are 211-212 bp long, their abundance is 2 x 10(4) copies/lC, and they are partially clustered in a secondary constriction of one chromosome pair with the rest of their copies dispersed on all chromosomes. PisTR-B sequences are of similar abundance (10(4) copies/lC) but differ from the "A" family in their monomer length (50 bp), high A/T content, and chromosomal localization in a limited number of discrete bands. These bands are located mainly in (sub)telomeric and pericentromeric regions. and their patterns, together with chromosome morphology, allow discrimination of all chromosome types within the pea karyotype. Whereas both tandem repeat families are mostly specific to the genus Pisum, many of the dispersed repeats were detected in other legume species, mainly those in the genus Vicia.

Microarray-based survey of repetitive genomic sequences in Vicia spp.

A modified DNA microarray-based technique was devised for preliminary screening of short fragment genomic DNA libraries from three Vicia species (V. melanops, V. narbonensis, and V. sativa) to isolate representative highly abundant DNA sequences that show different distribution patterns among related legume species. The microarrays were sequentially hybridized with labeled genomic DNAs of thirteen Vicia and seven other Fabaceae species and scored for hybridization signals of individual clones. The clones were then assigned to one of the following groups characterized by hybridization to: (1) all tested species, (2) most of the Vicia and Pisum species, (3) only a few Vicia species, and (4) preferentially a single Vicia species. Several clones from each group, 65 in total, were sequenced. All Group I clones were identified as rDNA genes or fragments of chloroplast genome, whereas the majority of Group II clones showed significant homologies to retroelement sequences. Clones in Groups III and IV contained novel dispersed repeats with copy numbers 10(2)-10(6)/1C and two genus-specific tandem repeats. One of these belongs to the VicTR-B repeat family, and the other clone (S12) contains an amplified portion of the rDNA intergenic spacer. In situ hybridization using V. sativa metaphase chromosomes revealed the presence of the S12 sequences not only within rDNA genes, but also at several additional loci. The newly identified repeats, as well as the retroelement-like sequences, were characterized with respect to their abundance within individual genomes. Correlations between the repeat distributions and the current taxonomic classification of these species are discussed.

Flow analysis and sorting of plant chromosomes.

The use of flow cytometry for evaluation of plant chromosomes requires some specialized attention to preparation and instrumentation. This unit deals exclusively with plant cytogenetics and presents an outline of this area as well as methods for accumulation of cells in metaphase, preparation of chromosome suspensions, flow analysis and sorting of chromosomes, and processing of the sorted chromosomes. Each method is described in tremendous detail because in many aspects dealing with plant cells is quite different from dealing with mammalian cells. Supporting histograms are presented as well as a range of special hints on dealing with plant material and a discussion of the utility of sorted chromosomes for plant genome mapping.

Analysis of nuclear DNA content and ploidy in higher plants.

This is the first of a series of units discussing the application of cytometry to plant material. Techniques commonly used for mammalian nuclei evaluation need considerable modification to be successful with plant material. David Galbraith and his colleagues bring together many years of knowledge in plant cytometry. Their unit provides detailed protocols on measuring DNA content, ploidy, and cell cycle status of plant tissue using both conventional laser based instruments as well as arc lamp cytometers. This unit provides an excellent starting point for those interested in doing cytometry with plants.

Nuclear dynamics in Arabidopsis thaliana.

The nucleus is a definitive feature of eukaryotic cells, comprising twin bilamellar membranes, the inner and outer nuclear membranes, which separate the nucleoplasmic and cytoplasmic compartments. Nuclear pores, complex macromolecular assemblies that connect the two membranes, mediate communication between these compartments. To explore the morphology, topology, and dynamics of nuclei within living plant cells, we have developed a novel method of confocal laser scanning fluorescence microscopy under time-lapse conditions. This is used for the examination of the transgenic expression in Arabidopsis thaliana of a chimeric protein, comprising the GFP (Green-Fluorescent Protein of Aequorea victoria) translationally fused to an effective nuclear localization signal (NLS) and to beta-glucuronidase (GUS) from E. coli. This large protein is targeted to the nucleus and accumulates exclusively within the nucleoplasm. This article provides online access to movies that illustrate the remarkable and unusual properties displayed by the nuclei, including polymorphic shape changes and rapid, long-distance, intracellular movement. Movement is mediated by actin but not by tubulin; it therefore appears distinct from mechanisms of nuclear positioning and migration that have been reported for eukaryotes. The GFP-based assay is simple and of general applicability. It will be interesting to establish whether the novel type of dynamic behavior reported here, for higher plants, is observed in other eukaryotic organisms.

Two new families of tandem repeats isolated from genus Vicia using genomic self-priming PCR.

A modified genomic self-priming technique was used for rapid isolation of tandem repeats from several Vicia species. Based on homologies of their nucleotide sequences the newly isolated clones were assigned to two repeat families named VicTR-A and VicTR-B. Both families are rich in AT (74%) and are organized as long blocks of tandemly repeated units. The VicTR-A repeats are characterized by a monomer size of 69 bp, whereas the VicTR-B repeat monomer is about 38 bp long, and the two families do not share significant sequence homology. VicTR sequences show different degrees of amplification (up to 10(6)-10(7) copies/haploid genome) in individual Vicia species and are not amplified in other legumes. The abundances of these repeats do not correlate with genome sizes but are similar in species that belong to the same taxonomic section within the genus Vicia. Primed in situ (PRINS) labeling of metaphase chromosomes of V. pannonica revealed that VicTR-A sequences are located predominantly in the telomeric regions of the short arms of all chromosomes. In contrast, labeling of VicTR-B repeats in V. sativa resulted in mainly intercalary bands of various intensities and only weak telomeric signals.

The agrobacterium tumefaciens C58-6b gene confers resistance to N(6)-benzyladenine without modifying cytokinin metabolism in tobacco seedlings

The 6b gene of Agrobacterium tumefaciens has been demonstrated to modify the activity of the plant growth regulators, auxin and cytokinin. To study the possible mode of action of the gene, tobacco (Nicotiana tabacum L. cv. Samsun) plants were transformed with the A. tumefaciens C58-6b gene. Seeds obtained from morphologically normal transgenic as well as wild-type plants were germinated on media supplemented with growth-inhibitory levels of cytokinin, N(6)-benzyladenine (BA). The transgenic seedlings showed increased resistance to cytokinins, as reflected by continuous shoot development, whereas further growth of the wild-type plants beyond the cotyledonary stage was inhibited. Concurrently, the levels of 6b gene transcripts in transgenic seedlings increased greatly upon BA treatment. Since glucosylation of BA represents the main inactivation mechanism of the hormone, we analyzed BA glucoside formation during the early stages of seedling growth. Intracellular levels of the major BA metabolite, N(6)-benzyladenine-7-glucoside (80-92%), as well as other BA-derived components were found to be comparable in transgenic and wild-type seedlings. Therefore, increased resistance of the C58-6b transgenic seedlings to cytokinins could not be directly attributed to enhanced BA glucosylation and subsequent hormone inactivation.

Nuclear expressed sequence Tag (NEST) analysis: a novel means to study transcription through amplification of nuclear RNA.

We describe a novel concept and corresponding methods for the analysis of transcription in higher plant cells. The concept is that an examination of the presence of different polyadenylated transcripts within isolated nuclei reflects the state of gene expression at a given moment more precisely than do conventional techniques using total cellular mRNA. The methods involve isolation of polyadenylated nuclear transcripts from flow-sorted nuclei, reverse transcription, amplification using the polymerase chain reaction, and analysis of the products through gel electrophoresis and sequencing. By using specific primers, we have demonstrated detection of selected gene products in nuclei from transgenic plants. We also employed a technique for analysis of individual transcripts based on the length polymorphisms of restriction fragments derived from their 3' ends. Because the technique does not require a priori knowledge of the analyzed sequences, it is suitable for displaying the complete spectra of RNA transcripts present in nuclei at the moment of their isolation. These fragments can be easily isolated and sequenced and the sequence information used for assignment of putative function of corresponding genes. These techniques have been used to identify leaf-, root-, and cell cycle-specific transcripts. In principle, they should be applicable to the tissues of any eukaryotic species that contain transcriptionally active nuclei.

Adapting the Biomek 2000 Laboratory Automation Workstation for printing DNA microarrays.

The Biomek 2000 Laboratory Automation Workstation is used for liquid handling and other repetitive operations in many laboratories. Since it has very good spatial positioning capabilities, we have modified this workstation to deliver samples at high densities onto microscope slides to produce DNA microarrays. The workstation tool, originally designed for bacterial colony replication, was adapted to carry special printing pins and was further modified to improve its positional accuracy. Software written in the Tool Command Language was concurrently developed to control the movements of the workstation arm during the process of printing. With these modifications, the workstation can reliably deliver individual samples at a spacing of 0.5 mm, corresponding to a total of more than 3000 samples on a single slide. Arrays prepared in this way were successfully tested in hybridization experiments.

Analysis of T-DNA-mediated translational beta-glucuronidase gene fusions.

Three random translational beta-glucuronidase (gus) gene fusions were previously obtained in Arabidopsis thaliana, using Agrobacterium-mediated transfer of a gus coding sequence without promoter and ATG initiation site. These were analysed by IPCR amplification of the sequence upstream of gus and nucleotide sequence analysis. In one instance, the gus sequence was fused, in inverse orientation, to the nos promoter sequence of a truncated tandem T-DNA copy and translated from a spurious ATG in this sequence. In the second transgenic line, the gus gene was fused to A. thaliana DNA, 27 bp downstream an ATG. In this line, a large deletion occurred at the target site of the T-DNA. In the third line, gus is fused in frame to a plant DNA sequence after the eighth codon of an open reading frame encoding a protein of 619 amino acids. This protein has significant homology with animal and plant (receptor) serine/threonine protein kinases. The twelve subdomains essential for kinase activity are conserved. The presence of a potential signal peptide and a membrane-spanning domain suggests that it may be a receptor kinase. These data confirm that plant genes can be tagged as functional translational gene fusions.

Construction of chromosome-specific DNA libraries covering the whole genome of field bean (Vicia faba L.).

Recombinant DNA libraries were constructed for seven chromosome types isolated from two translocation lines of field bean (Vicia faba L.) with reconstructed karyotypes. The chromosomes were selected so that the set of libraries covers the whole V. faba genome more than once. Individual chromosome types were highly purified by flow sorting, and their DNA was amplified by degenerate oligonucleotide-primed (DOP) polymerase chain reaction (PCR) and cloned into a plasmid vector. The choice of restriction site present in PCR primer and refinement of cloning protocol resulted in high cloning efficiency and allowed generation of libraries consisting of about 10(5) clones from 250 or 1000 sorted chromosomes. The insert size ranged between 50 and 2200 bp and the mean length estimated in individual libraries varied between 310 and 487 bp. Hybridization of cloned fragments with labelled genomic DNA showed that about 60% of inserts represented unique or low-copy sequences. The suitability of the libraries for genome mapping was demonstrated by isolation of clones containing microsatellite motifs.