Transparent Testa Glabra 1 (TTG1) and TTG1-like genes in Matthiola incana R. Br. and related Brassicaceae and mutation in the WD-40 motif.

TTG1 (Transparent Testa Glabra 1), a WD-40 repeat protein, is involved in regulation of flavonoid/anthocyanin biosynthesis, seed coat (mucilage) development/pigmentation and trichome formation in leaves. Here, we characterized the TTG1 gene of Matthiola incana wild type (e locus), showing 85.3% similarity to TTG1 of A. thaliana on the nucleotide level and 96.2% on the protein level. A white-flowered and glabrous mutant, line 17, of M. incana exhibits one nucleotide change, leading to an amino acid substitution directly in the WD motif (W158R). Correspondingly, the DFR (dihydroflavonol 4-reductase) gene, in which the expression is known to be dependent on TTG1, is not expressed in Matthiola mutant lines 17 (and 19). Comparison of the GC content of the Matthiola TTG1 (54.1%) and Arabidopsis TTG1 (46.1%) genes revealed a strong difference, mostly obtained by neutral substitutions (C to T transitions). To examine whether this is an ecologically influenced trend, a fragment of TTG1 was characterized from another Matthiola species (M. tricuspidata) and from Malcolmia flexuosa subsp. naxensis from the eastern Mediterranean, near a beach with sandy and salty soils. Both Matthiola species have a higher GC content in the TTG1 gene than Arabidopsis and the closer-related Malcolmia, indicating that the GC content is rather an evolutionary than an ecological signal. A similar WD-40 repeat protein gene (containing no intron in the 3' untranslated region) with high similarity to the Arabidopsis TTG1-like (AtAN11) gene was found in Matthiola.

Dynamic changes in the distribution of a satellite homologous to intergenic 26-18S rDNA spacer in the evolution of Nicotiana.

An approximately 135-bp sequence called the A1/A2 repeat was isolated from the transcribed region of the 26-18S rDNA intergenic spacer (IGS) of Nicotiana tomentosiformis. Fluorescence in situ hybridization (FISH) and Southern blot analysis revealed its occurrence as an independent satellite (termed an A1/A2 satellite) outside of rDNA loci in species of Nicotiana section Tomentosae. The chromosomal location, patterns of genomic dispersion, and copy numbers of its tandemly arranged units varied between the species. In more distantly related Nicotiana species the A1/A2 repeats were found only at the nucleolar organizer regions (NOR). There was a trend toward the elimination of the A1/A2 satellite in N. tabacum (tobacco), an allotetraploid with parents closely related to the diploids N. sylvestris and N. tomentosiformis. This process may have already commenced in an S(3) generation of synthetic tobacco. Cytosine residues in the IGS were significantly hypomethylated compared with the A1/A2 satellite. There was no clear separation between the IGS and satellite fractions in sequence analysis of individual clones and we found no evidence for CG suppression. Taken together the data indicate a dynamic nature of the A1/A2 repeats in Nicotiana genomes, with evidence for recurrent integration, copy number expansions, and contractions.

Molecular genetic characterization of different Trypanosoma cruzi strains and comparison of their development in Mus musculus and Calomys callosus.

Trypanosoma cruzi populations are characterized by diverse morphology, heterogeneous biological behavior, high genetic variability, and distinctly different clinical courses. The first objective of this work was to characterize different strains of T. cruzi with various molecular markers [simple-sequence-repeat PCR, randomly amplified polymorphic DNA (RAPD)-PCR, mini-exon genes]. All examined strains could be divided into two major lineages. Only one strain showed a different banding pattern in RAPD-PCR, which could be a further indication of the existence of a third lineage. The second aim was to examine the biological behavior of the different strains. Two animal models, Calomys callosus and Mus musculus, were infected. The results provide strong evidence that the biological behavior of the strains is not only lineage-specific. It appears that all factors, such as the infecting strain belonging to a certain lineage, the predominant morphological form of the isolate, and the immune response of the respective infected host, play an important role in the course of this infection.

Molecular diversity and physical mapping of 5S rDNA in wild and cultivated oat grasses (Poaceae: Aveneae).

5S rDNA repeats studied in five genera of Aveneae have lengths between 285 and 329 bp (Avena sativa, Avena macrostachya, 26 species of Helictotrichon, Pseudarrhenatherum longifolium, Lagurus ovatus, and Trisetum spicatum). In only a single species (Helictotrichon aetolicum) an additional repeat of 456 bp occurs infrequently. Variation is largely due to insertions or deletions in the nontranscribed spacer as determined from sequences of 163 independent clones. The 5S gene of the Aveneae studied is conserved in length and sequence except for Helictotrichon bromoides and Helictotrichon marginatum in which duplications occur at two different sites. This new type of duplication and all duplications reported to date in 5S genes of angiosperms are shown to center on defined palindromic sequences. The "uncommon" 5S gene sequences detected in some Aveneae are not necessarily nonfunctional as pseudogenes because the essential features of the internal control region are maintained even after such duplication events. In each instance such gene sequences have spacers with unmodified structure, indicating that change in gene sequence is not necessarily coupled with change in adjacent spacers. The value of 5S spacer sequences for genomic identifications in Aveneae is exemplified in A. macrostachya (perennial), A. sativa (annual), and several diploid taxa of the genus Helictotrichon.

Tobacco ribosomal DNA spacer element stimulates amplification and expression of heterologous genes.

Here we show that the cis-acting genetic element aps (amplification-promoting sequence), isolated from the nontranscribed spacer region of tobacco ribosomal DNA (rDNA), increases the level of expression of recombinant proteins. Transgenic tobacco plants, transformed with expression cassettes containing the herbicide-resistant acetolactate synthase (hr-ALS) gene or the green fluorescent protein (GFP) gene fused to the aps sequence, had greater levels of corresponding messenger RNAs (mRNAs) and proteins compared to transformants lacking aps. Analysis of transgenic plants showed that aps increased the copy number and transcription of the adjacent heterologous genes and, in the case of hr-ALS, enhanced the herbicide resistance phenotype. Both the increased transgene copy number and enhanced expression were stably inherited. These data provide the first evidence that the aps sequence can be used for gene amplification in transgenic plants and possibly other multicellular organisms.

Elimination and rearrangement of parental rDNA in the allotetraploid Nicotiana tabacum.

Origin and rearrangement of ribosomal DNA repeats in natural allotetraploid Nicotiana tabacum are described. Comparative sequence analysis of the intergenic spacer (IGS) regions of Nicotiana tomentosiformis (the paternal diploid progenitor) and Nicotiana sylvestris (the maternal diploid progenitor) showed species-specific molecular features. These markers allowed us to trace the molecular evolution of parental rDNA in the allopolyploid genome of N. tabacum; at least the majority of tobacco rDNA repeats originated from N. tomentosiformis, which endured reconstruction of subrepeated regions in the IGS. We infer that after hybridization of the parental diploid species, rDNA with a longer IGS, donated by N. tomentosiformis, dominated over the rDNA with a shorter IGS from N. sylvestris; the latter was then eliminated from the allopolyploid genome. Thus, repeated sequences in allopolyploid genomes are targets for molecular rearrangement, demonstrating the dynamic nature of allopolyploid genomes.

Molecular evolution of the internal transcribed spacers (ITS1 and ITS2) and phylogenetic relationships among species of the family Cucurbitaceae.

Phylogenetic relationships of different members of the family Cucurbitaceae were estimated from sequences of the internal transcribed spacer (ITS1 and ITS2) regions of the nuclear ribosomal RNA genes. Twenty-six species of different genera belonging to different tribes and several subtribes were analyzed. The whole ITS regions were amplified by PCR technique and cloned, and three to five different clones of each species were sequenced; for some species PCR products were sequenced directly. ITS1 and ITS2 regions are slightly variable in length, with each length appearing genus-specific. A substitution rate of 3.62 x 10(-9) substitutions per site per year was calculated assuming 40 MYA separation time. Phylogenetic relationships inferred from ITS sequences of some species is in agreement with morphological data, but deviations to the taxonomic classification were also observed. A polyphyletic origin of the New World species must be considered. In the genus Cucurbita different "types" of ITS sequences within one species exist, possibly due to the high frequency of introgression during domestication or due to polyploidization events; in contrast, low intraspecific variability was detectable in the genus Cucumis, indicating different stages of speciation.

Structural analysis of rDNA in the genus Nicotiana.

The nucleotide sequence of the intergenic spacer (IGS) region between the 25S and the 18S rRNA coding regions has been determined for tobacco (Nicotiana tabacum). The IGS (5140 bp in length) can be subdivided into several regions (I-VII) two of which, upstream and downstream of the putative transcription initiation site (TIS), contain prominent subrepeats (A and C). The unique sequence in the central part of the IGS (region IV) preceding the TIS is extremely AT-rich. The distance from the putative TIS to the 5' end of the 18S rRNA gene is 3005 bp. The IGS sequences are compared with potato (Solanum tuberosum) and tomato (Lycopersicon esculentum) IGS. Restriction mapping of 13 Nicotiana species shows that considerable rDNA repeat length heterogeneity in this genus is probably due to different numbers of A and C subrepeats.

Distribution and complex organization of satellite DNA sequences in Aveneae species.

Distribution, organization, and molecular analysis of four unrelated satellite DNA components in Aveneae species are described. Highly repeated DNA elements were cloned from Helictotrichon convolutum (CON1 and CON2) and Helictotrichon compressum (COM1 and COM2). The lengths of the repeat monomers are 365 bp (CON1), 562 bp (CON2), 346 bp (COM1), and 476 bp (COM2). Similar repeats were detected by dot blots, Southern blots, and by DNA sequencing in other species of the genus Helictotrichon, in Aveneae species, and in species of the tribes Andropogoneae and Oryzeae. All four satellite DNAs are differently distributed in the taxonomic groups mentioned above. Remarkably, the longer elements are built up in a complex pattern of either shorter subrepeats arranged in tandem (COM2) or by duplications inserted into an original 369-bp element (CON2). Shorter representatives, 190 bp, similar to CON1 elements occur in Holcus species. In Koeleria species, COM1-related repeats are only 180 bp in length. No similarity was found among the sequences CON2, COM1, and COM2 or with sequences of other repetitive DNA elements of the grasses, but CON1 shows sequence similarity to an A genome specific repetitive DNA of Oryza (rice).

Somatic hybrids between the cultivated potato Solanum tuberosum L. and the 1EBN wild species Solanum pinnatisectum Dun.: morphological and molecular characterization.

Interspecific somatic hybrids between the 1EBN-wild species Solanum pinnatisectum (S. pnt) and four different diploid breeding lines of Solanum tuberosum (S. tbr) were produced by electrofusion. S. pnt exhibits resistance to Phytophthora infestans and Erwinia blackleg. Somatic hybrids were identified by RFLP analysis using the oligonucleotide (GATA)4 as a probe. In three of four combinations all regenerates obtained were somatic hybrids. All 86 somatic hybrids between the breeding line H256/1 and S. pnt were analyzed in detail with respect to morphological and molecular characters; 50% of the somatic hybrids showed normal intermediate leaf morphology. Tubers of somatic hybrid plants grown in the greenhouse as well as in the field were evenly shaped and remarkably similar to those of the S. tbr breeding line. Analysis of relative DNA content by flow cytometry revealed that 75% of the somatic hybrids were tetraploid, some were hypotetraploid and others polyploid or mixoploid. Slotblot and RFLP analyses were carried out using repetitive and some single-copy DNA probes. The genome portion of the S. tbr breeding line was determined by slot-blot analysis using the species-specific repetitive probe pSA287. Obviously, most somatic hybrids contain the complete genomes of both fusion partners. In some of the somatic hybrids, a significantly lower intensity of the S. pnt-specific hybridization signal indicated a certain degree of asymmetry.

Differential homogenization and amplification of two satellite DNAs in the genus Cucurbita (Cucurbitaceae).

Two different satellite DNAs exist in the genus Cucurbita which are different with respect to repeat length (350 bp and 170 bp), array size, and sequence homogenization. Whereas the 350-bp satellite DNA is prominent and very homogeneous in all species investigated except for C. maxima and C. lundelliana, the 170-bp satellite is rather evenly distributed in all species. In C. maxima and C. lundelliana the 350-bp satellite is present only in small amounts, but detectable by the sensitive PCR method. These repeats are also very homogeneous, reflecting a silent stage of satellite DNA. In contrast, the 170-bp satellite DNA is intra- and interspecifically heterogeneous. It is striking that the species with no detectable amount of 350-bp satellite contain 170-bp satellite DNA clusters with the highest degree of homogeneity. The evolution of satellite DNA repeats within cultivated and wild species in the genus Cucurbita is elucidated using the sequence data of both satellite DNAs from all species investigated. The value of satellite DNA for phylogenetic analysis between closely related species is discussed.

Distribution of novel and known repeated elements of Solanum and application for the identification of somatic hybrids among Solanum species.

Species-specific repetitive DNA probes are a useful tool for the molecular identification of somatic hybrids. Therefore, the distribution of three repetitive DNA elements of Solanum was investigated in Solanum wild species, Solanum breeding lines, and in more distantly related species of the genera Lycopersicon, Nicotiana, and Datura. The clone pSCH15, obtained from S. circaeifolium, represents a new 168-bp repetitive element; it shows 73-79% sequence similarity to repetitive elements of S. brevidens and Lycopersicon species. The 163-bp element in pSBH6, cloned from S. bulbocastanum, turned out to be very similar (95% sequence homology) to the Lycopersicon element pLEG15/TGRI previously regarded to be present only in species of the genus Lycopersicon and in S. lycopersicoides. Lower sequence similarity of approximately 80% was observed to repetitive elements of S. brevidens which are organized differently. The repeats exhibited different degrees of specificity: by Southern hybridization the element represented by the clone pSBH6 could be detected in almost all Solanum species investigated here but only after long exposure to X-ray film. The previously described "Solanum-specific" element represented by the clone pSA287 was also found, although in a very low copy number, in Lycopersicon esculentum. Therefore, detection of the repetitive elements pSA287 and pSBH6 in those species in which the respective repeat is less represented depends on exposure time. In contrast, the element pSCH15 is prominently present only in a small number of Solanum wild species and - to some extent - in the diploid breeding lines as revealed after long exposure. Use of these repeated elements for the identification of specific genomes in protoplast-fusion hybrids between Solanum wild species and Solanum breeding lines, or between two breeding lines, was evaluated.

Characterization of a highly repeated DNA component of perennial oats (Helictotrichon, Poaceae) with sequence similarity to a A-genome-specific satellite DNA of rice (Oryza).

The taxonomic relationships among perennial oats (Helictotrichon Besser ex Schultes & Schultes, Aveninae, Aveneae, Poaceae) have been studied using highly repeated satellite DNA as a molecular marker. Highly repetitive sequences were isolated from restriction endonuclease digests of nuclear DNA of Helictotrichon convolutum, and satellite repeats (approximately 365 bp in length) were cloned, sequenced and compared among each other. They exhibited an intraspecific sequence variability of 6-9%. This satellite DNA, CON1, is differentially distributed within the genus Helictotrichon. In species of the subgenus Helictotrichon a high copy number is detectable, whereas in representatives of the subgenera Pratavenastrum and Pubavenastrum the number of copies per genome is rather low. Surprisingly, the satellite DNA repeat CON1 shows 74% sequence similarity to an A-genome specific repetitive DNA of Oryza (rice).

A specific oligonucleotide of the 5S rDNA spacer and species-specific elements identify symmetric somatic hybrids between Solanum tuberosum and S. pinnatisectum.

The nucleotide sequences of the 5S rRNA genes (5S rDNA) of two Solanum tuberosum breeding lines (R1 and B15) and of the Mexican wild species S. pinnatisectum were determined and compared with each other and to the 5S rDNA of other Solanaceae species (Lycopersicon esculentum, Nicotiana rustica and Petunia hybrida). The 5S rDNA repeats of the Solanum species are 324-329 bp in length, and they exhibit 91-95% sequence identity. Sequence variability is mainly located in a short region of the spacer separating the 5S rRNA coding regions. A synthetic 28-mer oligonucleotide constructed according to this region can be used as a specific hybridization probe to distinguish symmetric somatic hybrids between S. tubersosum breeding line B15 and S. pinnatisectum produced by protoplast fusion. Interestingly, the two Solanum breeding lines R1 and B15 differ also in this spacer region.

Pattern and degree of methylation in ribosomal RNA genes of Cucurbita pepo L.

Methylation with respect to its degree and distribution throughout the 18S, 5.8S and 25S rRNA gene clusters (rDNA) and within single rDNA repeats in seedlings of the higher plant Cucurbita pepo L. (zucchini) was investigated. In this plant, which is characterized by several thousand repeats, at least 70% are completely or nearly completely methylated in CpGs and to a lower degree in CpNpGs. Detailed methylation analysis revealed that a fraction of about 3-4% of all repeats is hypomethylated near the transcription initiation site (TIS) which may indicate the fraction of active repeats in C. pepo. However, a different fraction (3-4% of all repeats) which is not methylated in all sites tested (including those at the TIS) is present in C. pepo and may thus represent active but differentially methylated rDNA. The results are discussed in context of recent models on methylation and gene activity.

Comparison of nuclear ribosomal RNA genes among Solanum species and other Solanaceae.

The organization of the nuclear-encoded 18S, 5.8S, and 25S ribosomal RNA genes (ribosomal DNA; rDNA) of 21 New World species from different sections of the genus Solanum, of two Old World Solanum species, and of representatives of other Solanaceae (Nicotiana, Atropa, Datura, Physalis, and Capsicum) was analyzed by restriction enzyme mapping using different rDNA specific hybridization probes. All Solanum species investigated exhibited rDNA repeats between 8.7 and 9.3 kb in length; the only exception was S. neorossii with a repeat length of 10.3 kb. Sequence heterogeneity was observed mostly in the intergenic spacer (IGS) region. Restriction sites for EcoRI and DraI in the spacer sequences were found to be characteristic for the New World species of the genus Solanum and for Lycopersicon esculentum. An additional XbaI site was detected in the spacer region of two nontuber-bearing species, S. brevidens and S. etuberosum (subsection Estolonifera Hawkes; series Etuberosa), as well as in the primitive tuber-bearing species of the series Pinnatisecta and Polyadenia (subsection Potatoe G. Don), thus demonstrating that these Mexican species are separated from the other tuber-bearing species but are closely linked to the nontuber-bearing Estolonifera group. Two EcoRI sites mapped at the 3' end of the 25S rRNA coding region seem to be characteristic for members of the Solanaceae; the first EcoRI site is apparently methylated in approximately 50% of the rDNA repeats. Southern hybridization with an IGS fragment of Solanum tuberosum as hybridization probe and nucleotide sequence analysis of the phylogenetically informative 3' end of the 25S rDNA support the assumption that the New World species of the genus Solanum are closely related to Lycopersicon (tomato) in contrast with other Solanaceae investigated, Nicotiana, Atropa, Datura, Physalis, and Capsicum.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for nucleosomal phasing and a novel protein specifically binding to cucumber satellite DNA.

The nucleosomal organization and the protein-binding capability of highly repeated and methylated satellite DNA of cucumber (Cucumis sativus L.), comprising approx. 30% of the genome, were analyzed. Nucleosomal core DNA from satellite type I was prepared after micrococcal nuclease digestion of chromatin and sequenced. Most of the core sequences obtained could be grouped in two main (A and B) and two minor groups (C and D) indicating a specific and complex phasing of nucleosomes on this satellite DNA. In vitro, gel retardation assays with cloned satellite DNA repeats (types I-IV) demonstrated a specific binding of nuclear proteins. These specific binding effects are also obtained with genomic, in vivo methylated and sequence heterogeneous (1 to 10% diversity) satellite type I DNA. For the first time in plants, a satellite DNA-binding protein with an apparent molecular weight of 14 kDa (SAT 14) was identified.

Nuclear proteins interact with RNA polymerase I promoter and repeated elements of the 5' external transcribed spacer of the rDNA of cucumber in a single-stranded stage.

Directly repeated elements have been characterized downstream of the transcription initiation site (TIS) in the 5' external transcribed spacer (5' ETS) of the rRNA genes of cucumber (Cucumis sativus). In order to show that these repeated elements are also involved in transcriptional regulation processes of RNA polymerase I while being single-stranded during transcription, DNA-protein binding assays were performed with synthetic oligonucleotides prepared from the promoter region as well as from the repeated elements. The single-stranded DNA of the upstream binding element (from -164 to -105), the core promoter (from -41 to +16) and a loop-forming sequence (LRE) of the repeated elements interact with the same nuclear proteins whereas another region of the repeated elements (XRE) cooperates with a different nuclear protein. Remarkably, both complementary strands show identical protein binding.

Molecular evolution of the intergenic spacer in the nuclear ribosomal RNA genes of cucurbitaceae.

The intergenic spacer (IGS) of a 10-kbp repeat (clone pRZ7D) of the nuclear 18S, 5.8S, and 25S ribosomal RNA genes of Cucurbita pepo (zucchini) was sequenced and compared to the IGS sequences of two other Cucurbitaceae, Curcurbita maxima (squash), and Cucumis sativus (cucumber). The nucleotide sequence and the structural organization of the IGS of C. pepo and C. maxima are rather similar (between 75 and 100% sequence similarity depending on the region compared). The IGS are mainly composed of three different repeated elements interspersed into unique sequences: GC-rich clusters, a 422-bp AT-rich element including the transcription initiation site (TIS) for RNA polymerase I, and 260-bp repeats in the 5' external transcribed spacer (D repeats). The TIS is duplicated in the 10-kbp repeat class of C. pepo, as it is also described for the 11.5-kbp rDNA repeat of C. maxima. The IGS of Cucumis sativus is also composed of different repeated elements; however, obvious sequence identity to the Cucurbita species only occurs around the TIS and the preceding AT-rich region. GC-rich clusters with different primary sequences are present in the IGS of all three plants. Remarkably, the repeated elements in the 5'ETS accumulate TpG and TpNpG motifs, whereas CpG and CpNpG motifs less frequently occur. This accumulation might be caused by the transition of methylated cytosines (in mCpG or mCpNpG motifs) into thymidine via deamination in a previously GC-rich ancestor. The following singular region exhibits 50% G + C in C. pepo, 53% G+C in C. maxima, and 63% G + C in C. sativus.(ABSTRACT TRUNCATED AT 250 WORDS)