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A novel automated image collection and analysis system was used to compare two new soybean (Glycine max (L.) Merr.) promoters with the cauliflower mosaic virus 35S (CaMV35S) promoter, which was used as an expression standard. For expression comparisons, various permutations of a soybean polyubiquitin (Gmubi) promoter, a soybean heat shock protein 90-like (GmHSP90L) promoter and the CaMV35S promoter were placed upstream of a green fluorescent protein (gfp) gene. DNA constructs were introduced via particle bombardment into excised cotyledons of germinating lima bean (Phaseolus lunatus L.) seeds, which were arranged in Petri dishes for automated image capture and image analysis. The automated system allowed monitoring and quantification of gfp gene expression in the same piece of tissue over time. The Gmubi promoter, with its intronic region intact, showed the highest expression that was over five times stronger than the CaMV35S promoter. When an intronic region was removed from the Gmubi promoter, GFP expression was reduced, but was still over two times greater than with the CaMV35S promoter. The full-length soybean GmHSP90L promoter was four times stronger than the CaMV35S promoter. Truncation of the GmHSP90L promoter resulted in stepwise decreases in promoter strength, which appear to correspond to removal of regulatory elements. Automated image capture and analysis allowed the rapid and efficient evaluation of these new promoters.

Moderately repetitive DNA sequences in Lilium (cv Enchantment) which undergo a meiotic-specific repair synthesis during pachytene (P-DNA) were previously shown to exist as families of very low internal sequence divergence. The present study concerns P-DNA sequence preservation among higher plants. The relative abundance of these sequences in a variety of plant species and their divergence relative to Enchantment P-DNA was determined through C0t analysis and thermal denaturation of hybrid duplexes. Nearly all of the P-DNA sequence families of Enchantment were found to be present in the genomes of a number of monocot species and the dicot Vicia faba. Sequence content is highly conserved, with less than 6% divergence between Lilium and distantly related species such as Zea mays and Secale cereale. However, the number of repeats per P-DNA family varies considerably in different species, being particularly low among the Poales. P-DNA differs from most high thermal stability (HTS) sequence families of Enchantment which, although exhibiting a high degree of internal homology, are not present as repetitive DNA in the genomes of the other species examined. For most HTS families, the lack of internal divergence probably reflects their fairly recent introduction into the moderately repetitive DNA class, while P-DNA sequences represent evolutionarily ancient families which are the products of strong selective pressure for an indispensable meiotic function.

The genome of the homosporous fern Thelypteris normalis contains a large middle-repetitive component, essentially a single second-order kinetic class, which exhibits heterogeneity in the precision of pairing of the reformed duplexes upon remelting. There are two possible models to explain this observed sequence heterogeneity. Either different families of the middle-repetitive class exhibit different degrees of sequence divergence and Tm reduction (inter familial heterogeneity), or else all are equally diverged, and the broad melt is the sum of thousands of equally broad melts for all the families (intra familial heterogeneity). To differentiate between these two hypotheses, iodinated Thelypteris DNA, reannealed through middle-repetitive C0t, was thermally fractionated on hydroxyapatite into low (65–75° C), medium (75–85° C), and high (85–95° C) thermal stability classes. When reannealed with excess cold DNA, each class remelted over its characteristic temperature range. C0t curves of these thermal fractions reannealed with cold driver demonstrated that all were from the middle-repetitive class. It was shown that these results were not due to G + C differences nor to artifacts of the labeling technique. Therefore it was concluded that, although all families consisted of approximately the same number of repeats, the families ranged from those with virtually no sequence divergence to those barely able to reanneal at the criterion used, in accordance with the model of inter familial heterogeneity. Though this model may have wide applicability to middle-repetitive DNAs, a different pattern appears to prevail in Selaginella, a heterosporous pteridophyte. Some evolutionary implications are discussed.

Moderately repetitive sequences of Lilium DNA synthesized during pachytene consist of families that have a considerably lower divergence than those of total genomic middle repeat DNA, the latter having an average divergence of 10%. 80% of the sequences synthesized during the early phase of pachytene and 100% of those synthesized during the latter phase of pachytene reassociate with perfect or near-perfect fidelity. Except for the small amount of DNA synthesized during early pachytene, pachytene middle repeat sequences are non-divergent and thus constitute a distinctive subset of total moderately repetitive DNA. The modal length of pachytene and total middle repeat sequences are similar. In contrast to earlier measurements based on isotope incorporation, the modal length is of the order of 1500–2000 base pairs, ten times the previously estimated size. Calculations based on the new length lead to the conclusion that pachytene middle repeat sequences account for 1% of the genome.

RecA-like proteins have been purified from somatic and meiotic cells of mouse and lily. The rec proteins have been designated "s-rec" and "m-rec" to indicate their respective tissues of origin. The two proteins differ in molecular weight and in their response to temperature, the latter being consistent with the optimal temperature for physiological function of their tissues of origin. There is a major increase in m-rec protein with the entry of cells into meiosis, the peak of activity being early pachytene. Extracts of the cells and also those of yeast (Saccharomyces cerevisiae) have been prepared that have the capacity to catalyze homologous recombination. These extracts behave similarly to the m-rec proteins upon entry of cells into meiosis. Yeast transferred to sporulation medium displays a 100-fold increase in the recombination activity of the extract at about the time of entry into meiosis. The occurrence of peak levels of m-rec and recombination activity in extracts from cells in early pachytene points strongly to that stage as the time at which the enzymatic phase of recombination occurs.

Publisher Summary This chapter discusses the role of moderately repetitive DNA in evolution. Moderately repetitive sequences are found as dispersed segments among unrelated, often single-copy DNA sequences throughout the genome, while simple-sequence DNAs occur in long tandemly repeated clusters, frequently at the centromeric or telomeric regions of chromosomes. The chapter examines data on the organization of the moderately repetitive component in representatives of a spectrum of eukaryote lineages, paying attention to the differences in groups of relatively established phyletic affinity. Information is derived from studies involving direct experimental comparisons within taxa and genomes because this bears on the evolution of moderately repetitive DNA components in different groups. Thus, group-specific or convergent peculiarities may be readily identified. The chapter describes the approaches that can be made using the reassociation and sizing techniques as well as the types of information they have revealed in some prototype studies. The aspects of population genetics and speciation processes which may influence the evolution of moderately repetitive components are also considered.