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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.

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.

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)

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)

The large intergenic spacer (IGS) of potato rDNA was sequenced and compared to the IGS sequence of tomato. Both spacers exhibit similar length and architecture. Absence of repeated elements down-stream of the putative transcription initiation site (TIS) in potato is compensated by the larger number of subrepeats upstream of the TIS. Especially high level of similarity (86% and 86.5%, respectively) is found in the AT-rich domain containing the TIS and the region approx. 800 bp upstream of the 18S rRNA gene.

The length homogeneous part of the intergenic spacer (IGS) of the 18S-25S ribosomal RNA genes of cucumber (Cucumis sativus) was characterized by sequencing 2389 bp preceding the 18S rRNA coding region of a 12.5 kbp repeat type. This part of the IGS is composed of repeated elements and shows a very complex structural organization. Most obvious is a 119 bp element which is repeated seven times. A single transcription initiation site (TIS) was detected by a 'T4 polymerase stop' experiment upstream of these repetitions giving rise to a 2013 bp 5' external transcribed spacer (ETS) for cucumber. Nuclease mapping showed several transcription termination sites (TTS): the first one is located 350 bp downstream of the 25S rRNA coding region, the others are found within the duplications of this region accounting for the length heterogeneity of cucumber rDNA. Therefore, the TTS is repeated two or three times in the IGS depending on the length of the respective repeat classes and the rRNA precursor is heterogeneous in length varying from approx. 8000 to 11000 nts.

A cDNA clone (pcM12) of the chalcone synthase (CHS) of Matthiola incana R. Br. (Brassicaceae) was isolated from a cDNA library, sequenced and analysed. It comprises the complete coding sequence for the CHS and 5' and 3' untranslated regions. The deduced amino acid sequence shows that the Matthiola incana CHS consists of 394 amino acid residues. Comparison with CHS amino acid sequences of other plants indicates more than 82% homology.

The internal transcribed spacer (ITS) 1 and 2, the 5.8S rRNA gene, and adjacent 18S rRNA and 25S rRNA coding regions of two Cucurbitaceae (Cucurbita pepo, zucchini, ITS 1: 187 bp, and ITS 2: 252 bp in length, and Cucumis sativus, cucumber, ITS 1: 229 bp, and ITS 2: 245 bp in length) have been sequenced. The evolutionary pattern shown by the ITSs of these plants is different from that found in vertebrates. Deletions, insertions, and base substitutions have occurred in both spacers; however, it is obvious that some selection pressure is responsible for the preservation of stem-loop structures. The dissimilarity of the 5' region of ITS 2 found in higher plants has consequences for proposed models on U3 snRNA-ITS 2 interaction in higher eukaryotes. The two investigated Cucurbitaceae species show a G + C content of ITS 1 that nearly equals that of ITS 2. An analysis of the ITS sequences reveals that in 19 out of 20 organisms published, the G + C content of ITS 1 nearly equals that of ITS 2, although it ranges from 20% to 90% in different organisms (GC balance). Moreover, the balanced G + C content of the ITSs in a given species seems to be similar to that of so-called expansion segments (ESs) in the 25/28S rRNA coding region. Thus, ITSs show a phenomenon called molecular coevolution with respect to each other and to the ESs. In the ITSs of Cucurbitaceae the balanced G + C composition is at least partly achieved by C to T transitions, via deamination of 5-methylcytosine. Other mutational events must be taken into account. The appearance of this phenomenon is discussed in terms of functional constraints linked to the structures of these spacers.

The percentage of DNA from the crucifer Matthiola incana coding for different types of RNA was measured by filter saturation hybridisation experiments using RNA labelled in vivo. In addition, the melting curves of the various DNA - RNA hybrids formed and the buoyant densities of the DNA sequences complementary to different types of RNA were measured. 1. The RNA preparations used were 25, 18, and 5 S rRNA and 4 S RNA, purified by gel electrophoresis, and poly(A)-containing RNA purified by oligo-(dT)-cellulose chromatography. The molecular weights of the 25 S and 18 S rRNAs, calculated from the mobility in formamide-acrylamide gels relative to Escherichia coli RNA, are 1.25 - 10(6) and 0.64 - 10(6). The rRNA precursor has a molecular weight of approx. 2.1 - 10(6) and the average molecular weight of the poly(A)-containing RNA from both cotyledons and roots is 4 - 10(5). 2. The percentage of the genome, calculated on the basis of double-stranded DNA, coding for these RNAs and the estimated number of genes per haploid DNA amount are approximately 0.46% and 1100 for 25 S plus 18 S rRNA, 0.032% and 3600 for 5 S rRNA and 0.072% and 13 000 for 4 S RNA. In filter hybridisation experiments very little hybridisation of poly(A)-containing RNA was found. A rapidly-hybridising component is attributed to small amounts of contaminating rRNA. 3. M. incana DNA has a main band at 1.697 g - ml-1 in CsCl and a satellite constituting approximately 3% of the DNA, at 1.708 g - ml-1 - 25 and 18 S rRNA hybridise to DNA with a buoyant density of 1.701--2 g - ml-1. The buoyant density of 5 S DNA is slightly less at 1.700--1 g - ml-1. 4. S RNA hybridises to at least two separate regions, one within the main-band DNA and a second lighter component. None of the RNAs tested hybridised to the satellite DNA. The Tm of the DNA - RNA hybrids in 1 X SSC is 89 degrees C for 25 S rRNA, 85 degrees C for 5 S rRNA and 82 degrees C for 4 S RNA. 4. 5 and 4 S RNA preparations contain fragments which hybridise to sequences complementary to high-molecular-weight rRNA. This spurious hybridisation can be eliminated by competition with unlabelled high-molecular-weight RNA.

The tandemly arranged and clustered highly repeated 5S rRNA genes are investigated for two plants belonging to different higher plant families: Matthiola incana (Brassicaceae, Dilleniidae, Rosidae; 3600 5S rRNA genes/n) shows a homogeneous repeat size of 510 bp, whereas Vigna radiata (mung bean, former Phaseolus aureus, Fabaceae, Rosidae; approx. 4300 5S rRNA genes) has a repeat size of 215 bp. The mung-bean 5S rRNA coding region starts 5' with AGG and ends with CCT; Matthiola starts with GGG and ends with CCC. Striking is the strict occurrence of a 'TATA' box starting at nucleotide-28 similar to Neurospora crassa 5S rRNA genes. The 3' end is followed by CTTTT or GTTT stretches present in different numbers in the non-transcribed spacer suggesting a function in termination.

The water plant Lemna perpusilla has been incubated with the E. coli plasmids pMB9 and pBR325, respectively. Uptake of plasmids has been shown by subsequent transformation of E. coli cells to tetracycline resistance after treatment with Lemna DNA from plasmid-incubated plants. In 7 out of 15 assays we found stable transformants. From the transformation rate an amount of 10(-4) to 10(-6) micrograms plasmid DNA per 10 micrograms of plant DNA can be calculated.

Isolated T4 phage DNA (sigma=1.694 g/ml) is applied to seedlings of the crucifer Matthiola incana (DNA density sigma=1.698 g/ml). The phage DNA can partly be reextracted from the plants in a specific DNA fraction, which is predominantly characterized by its unusual high density (high density complex=HDC; sigma=1.724 g/ml). DNA:DNA hybridization studies show that phage specific DNA sequences are preserved in the HDC. Results of BrdUrd labeling of the plant DNA before and during incubation with T4DNA suggest that the HDC is composed of T4DNA and a plant DNA component of high density. The analysis of ultrasonicated HDC confirms this suggestion. The ability of plant cells to recognize and handle T4 DNA specifically is discussed.

Restriction enzyme analysis and cloning of the 18S, 5.8S, and 25S ribosomal RNA genes (rDNA) of the mung bean (Vigna radiata = Phaseolus aureus) reveal length heterogeneity in the repeating units (10-11 kbp) localized within two different regions in the ribosomal spacer. The 1.5-2.0 kbp region flanking the 3' end of the 25S rRNA contains various numbers (8-10) of tandemly arranged 180 bp subrepeats. After DNA sequencing a complex organized length heterogeneous 5' external spacer built up of different numbers of 340 bp subrepeats, each flanked by 52 bp direct repeats, is detected and described for the first time for plant ribosomal DNA repeating units. Sequences occurring in front of and within this repeated structure (elements II-IV) can be combined with the motifs P1, P2, and P3. These exhibit a strong similarity to transcription initiation sites specific for RNA polymerase I described for other plant and animal rDNA investigated to date. Transcription products complementary to the complex repeated structures are detected by hybridization to total nuclear RNA. The 9 bp element V located in fron of the first 340 bp region appears in duplicated form as a direct repeat with sequence similarity to SV40 (or RNA polymerase II) enhancer sequences.

Digestion of plant chromatin from Brassica pekinensis and Matthiola incana with staphylococcus nuclease leads to a DNA repeat of 175 plus or minus 8 and a core size of 140 base pairs. DNase I digestion results in multiples of 10 bases. Ribosomal RNN genes were studied as a model system for active plant chromatin because of their great redundancy and their high transcriptional activity in growing and differentiating tissues. The actively transcribed genes were identified by nascent RNA of ribosomal origin still attached to its matrix DNA. Hybridization techniques were used to demonstrate that even transcriptionally active gene sequences are present in nuclease generated chromatin subunits. Comparison of the DNase I kinetics of chromatin digestion with the amount of ribosomal RNA genes which is available for hybridization at the given times indicated that ribosomal RNA genes are digested, but not preferentially degraded by DNase I.

Two higher plant systems are presented which allow to study coordinated gene expression of the light-induced metabolic pathway of flavonoid biosynthesis: tissue culture cells of Petroselinum hortense (Apiaceae) and different developmental stages of various genotypes of Matthiola incana (Brassicaceae). The gene structure of the chalcone synthase is mainly studied. A cDNA clone (pLF56) of parsley has been constructed and characterized conferring the chalcone synthase gene sequence. Strong cross hybridization between the parsley cDNA and Matthiola DNA allowed to identify a HindIII fragment (6000 bp) identical in size for parsley and different Matthiola wild type lines and a mutant line.

The use of intergenic spacer (IGS) fragments of plant ribosomal DNA (rDNA) for the differentiation between genera and species is tested by cross-hybridization experiments with different IGS probes of two Cucurbitaceae, Cucurbita pepo (zucchini) and Cucumis sativus (cucumber). Hybridization with cloned fragments of different parts of the IGS of ribosomal DNA exhibit a different degree of conservation within and between the Cucurbitaceae genera. In general, Cucurbita species seem to be closer related to each other than the Cucumis species. A repetitive element of the external transcribed spacer (ETS) shows a more genus-specific pattern, reacting only with the respective genera; the region preceding the ETS is conserved between the Cucurbita species but also cross-hybridizes weakly with the Cucumis species. A GC-rich element of the Cucumis sativus IGS ("Cfo-cluster") is present in small amounts in Cucumis melo (melon) and even less represented in other genera of the Cucurbitaceae.

The prominent satellites of the Cucurbitaceae Cucumis melo (melon) and Cucumis sativus (cucumber) have been characterized, in actinomycin/CsCl gradients where the satellite sequences can be separated from ribosomal, organelle, and main band DNA the location of the satellites is different indicating a different GC content. The purified satellite of C. melo is cut by HindIII into a repeat unit of 380 bp; AluI digestion gives rise to two bands (about 80 and 220 bp in size). The HindIII repeat unit if cloned into pBR325 exhibits new recognition sites for HpaII leaving two bands with 150 and 80 bp suggesting methylation of the C/CGG cutting site in the uncloned material. The restriction pattern indicates an internal sequence repeat within the 380 bp HindIII fragment. The C. sativus satellite is cut by AluI to a repeat unit of 180 bp showing no other recognition site for the restriction enzymes tested so far. About 10% sequence homology has been determined between the C. melo and C. sativus satellites by cross hybridization studies. A high methylation degree of cytosines has been measured for both satellites and the ribosomal DNA of C. sativus (about 30%). No transcription products of the C. melo satellite were found during seedling development.

Seedlings of Matthiola incana (crucifer) are able to take up exogenous homologous DNA by the roots. DNA homogenously labelled with [3H]adenine and 5-bromodeoxyuridine is incorporated into the plants in a macromolecular form. Intact donor DNA and a fraction with a buoyant density intermediate between that of the donor and the recipient DNA can be recovered. Analysis of this intermediate fraction by ultrasonication and alkali treatment allows the suggestion that homologous DNA is integrated as a double-stranded DNA which becomes covalently linked to the recipient DNA. Control experiments in which seedlings were incubated in a mixture simulating donor DNA degradation products in the presence and absence of unlabelled competitors suggest that these results are not due to the breakdown of donor DNA and reincorporation of the products during DNA synthesis in the recipient plants. When ultrasonicated or thermally denatured DNA is applied to the plants it may be degraded and reused for recipient DNA synthesis but it is not recovered in a macromolecular form. The possibility that the intermediate DNA fraction arises by bacterial contamination of the plants can be excluded by several arguments. Autoradiographic studies show that at least part of the radioactivity of the donor DNA taken up by the plants is associated with the cell nucleus.