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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.

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).

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 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.

Subrepeats located in the rDNA intergenic spacer are also present as independently occurring, tandemly arranged satellite DNA clusters in the genome of Vigna radiata (mung bean). These 174-bp satellite repeats are identified as non-rDNA repeats by the presence of an AluI site. In the closely related Vigna angularis (adzuki bean), 174-bp repeats characterized by an AluI site occur in the rDNA with high sequence homology to the V. radiata rDNA subrepeats. A part of the 174-bp element that shows high similarity to a Xenopus terminator box (T2/T3) is slightly modified in V. angularis. However, a characteristic stem-loop structure can be formed, as in the case of V. radiata. Two highly conserved 12-bp regions occur within the 174-bp rDNA repeats of the two plants investigated. One of these 12-bp stretches exhibits some sequence identity to an element repeated twice in the 325-bp repeats in the intergenic spacer region of Vicia faba (broad bean).

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.