The MITE family Heartbreaker (Hbr): Molecular markers in maize

Transposable elements are ubiquitous in plant genomes, where they frequently comprise the majority of genomic DNA. The maize genome, which is believed to be structurally representative of large plant genomes, contains single genes or small gene islands interspersed with much longer blocks of retrotransposons. Given this organization, it would be desirable to identify molecular markers preferentially located in genic regions. In this report, the features of a newly described family of miniature inverted repeat transposable elements (MITEs) (called Heartbreaker), including high copy number and polymorphism, stability, and preference for genic regions, have been exploited in the development of a class of molecular markers for maize. To this end, a modification of the AFLP procedure called transposon display was used to generate and display hundreds of genomic fragments anchored in Hbr elements. An average of 52 markers were amplified for each primer combination tested. In all, 213 polymorphic fragments were reliably scored and mapped in 100 recombinant inbred lines derived from a cross between the maize inbreds B73 × Mo17. In this mapping population, Hbr markers are distributed evenly across the 10 maize chromosomes. This procedure should be of general use in the development of markers for other MITE families in maize and in other plant and animal species where MITEs have been identified.

A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: Are chromosome knobs megatransposons?

A class of tandemly repeated DNA sequences (TR-1) of 350-bp unit length was isolated from the knob DNA of chromosome 9 of Zea mays L. Comparative fluorescence in situ hybridization revealed that TR-1 elements are also present in cytologically detectable knobs on other maize chromosomes in different proportions relative to the previously described 180-bp repeats. At least one knob on chromosome 4 is composed predominantly of the TR-1 repeat. In addition, several small clusters of the TR-1 and 180-bp repeats have been found in different chromosomes, some not located in obvious knob heterochromatin. Variation in restriction fragment fingerprints and copy number of the TR-1 elements was found among maize lines and among maize chromosomes. TR-1 tandem arrays up to 70 kilobases in length can be interspersed with stretches of 180-bp tandem repeat arrays. DNA sequence analysis and restriction mapping of one particular stretch of tandemly arranged TR-1 units indicate that these elements may be organized in the form of fold-back DNA segments. The TR-1 repeat shares two short segments of homology with the 180-bp repeat. The longest of these segments (31 bp; 64% identity) corresponds to the conserved region among 180-bp repeats. The polymorphism and complex structure of knob DNA suggest that, similar to the fold-back DNA-containing giant transposons in Drosophila, maize knob DNA may have some properties of transposable elements.

Dynamics of mobile element activity in chalcone synthase loci in the common morning glory (Ipomoea purpurea)

Mobile element dynamics in seven alleles of the chalcone synthase D locus (CHS-D) of the common morning glory (Ipomoea purpurea) are analyzed in the context of synonymous nucleotide sequence distances for CHS-D exons. By using a nucleotide sequence of CHS-D from the sister species Ipomoea nil (Japanese morning glory [Johzuka-Hisatomi, Y., Hoshino, A., Mori, T., Habu, Y. & Iida, S. (1999) Genes Genet. Syst. 74, 141–147], it is also possible to determine the relative frequency of insertion and loss of elements within the CHS-D locus between these two species. At least four different types of transposable elements exist upstream of the coding region, or within the single intron of the CHS-D locus in I. purpurea. There are three distinct families of miniature inverted-repeat transposable elements (MITES), and some recent transpositions of Activator/Dissociation (Ac/Ds)-like elements (Tip100), of some short interspersed repetitive elements (SINEs), and of an insertion sequence (InsIpCHSD) found in the neighborhood of this locus. The data provide no compelling evidence of the transposition of the mites since the separation of I. nil and I. purpurea roughly 8 million years ago. Finally, it is shown that the number and frequency of mobile elements are highly heterogeneous among different duplicate CHS loci, suggesting that the dynamics observed at CHS-D are locus-specific.

The evolution of plant nuclear genes

We analyze the evolutionary dynamics of three of the best-studied plant nuclear multigene families. The data analyzed derive from the genes that encode the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS), the gene family that encodes the enzyme chalcone synthase (Chs), and the gene family that encodes alcohol dehydrogenases (Adh). In addition, we consider the limited evolutionary data available on plant transposable elements. New Chs and rbcS genes appear to be recruited at about 10 times the rate estimated for Adh genes, and this is correlated with a much smaller average gene family size for Adh genes. In addition, duplication and divergence in function appears to be relatively common for Chs genes in flowering plant evolution. Analyses of synonymous nucleotide substitution rates for Adh genes in monocots reject a linear relationship with clock time. Replacement substitution rates vary with time in a complex fashion, which suggests that adaptive evolution has played an important role in driving divergence following gene duplication events. Molecular population genetic studies of Adh and Chs genes reveal high levels of molecular diversity within species. These studies also reveal that inter- and intralocus recombination are important forces in the generation allelic novelties. Moreover, illegitimate recombination events appear to be an important factor in transposable element loss in plants. When we consider the recruitment and loss of new gene copies, the generation of allelic diversity within plant species, and ectopic exchange among transposable elements, we conclude that recombination is a pervasive force at all levels of plant evolution.

Functional genomics: Probing plant gene function and expression with transposons

Transposable elements provide a convenient and flexible means to disrupt plant genes, so allowing their function to be assessed. By engineering transposons to carry reporter genes and regulatory signals, the expression of target genes can be monitored and to some extent manipulated. Two strategies for using transposons to assess gene function are outlined here: First, the PCR can be used to identify plants that carry insertions into specific genes from among pools of heavily mutagenized individuals (site-selected transposon mutagenesis). This method requires that high copy transposons be used and that a relatively large number of reactions be performed to identify insertions into genes of interest. Second, a large library of plants, each carrying a unique insertion, can be generated. Each insertion site then can be amplified and sequenced systematically. These two methods have been demonstrated in maize, Arabidopsis, and other plant species, and the relative merits of each are discussed in the context of plant genome research.

The highly recombinogenic bz locus lies in an unusually gene-rich region of the maize genome

The bronze (bz) locus exhibits the highest rate of recombination of any gene in higher plants. To investigate the possible basis of this high rate of recombination, we have analyzed the physical organization of the region around the bz locus. Two adjacent bacterial artificial chromosome clones, comprising a 240-kb contig centered around the Bz-McC allele, were isolated, and 60 kb of contiguous DNA spanning the two bacterial artificial chromosome clones was sequenced. We find that the bz locus lies in an unusually gene-rich region of the maize genome. Ten genes, at least eight of which are shown to be transcribed, are contained in a 32-kb stretch of DNA that is uninterrupted by retrotransposons. We have isolated nearly full length cDNAs corresponding to the five proximal genes in the cluster. The average intertranscript distance between them is just 1 kb, revealing a surprisingly compact packaging of adjacent genes in this part of the genome. At least 11 small insertions, including several previously described miniature inverted repeat transposable elements, were detected in the introns and 3′ untranslated regions of genes and between genes. The gene-rich region is flanked at the proximal and distal ends by retrotransposon blocks. Thus, the maize genome appears to have scattered regions of high gene density similar to those found in other plants. The unusually high rate of intragenic recombination seen in bz may be related to the very high gene density of the region.

Repetitive-DNA elements are similarly distributed on Caenorhabditis elegans autosomes

The positions of ≈4,800 individual miniature inverted-repeat transposable element (MITE)-like repeats from four families were mapped on the Caenorhabditis elegans chromosomes. These families represent 1–2% of the total sequence of the organism. The four MITE families (Cele1, Cele2, Cele14, and Cele42) displayed distinct chromosomal distribution profiles. For example, the Cele14 MITEs were observed clustering near the ends of the autosomes. In contrast, the Cele2 MITEs displayed an even distribution through the central autosome domains, with no evidence for clustering at the ends. Both the number of elements and the distribution patterns of each family were conserved on all five C. elegans autosomes. The distribution profiles indicate chromosomal polarity and suggest that the current genetic and physical maps of chromosomes II, III, and X are inverted with respect to the other chromosomes. The degree of conservation of both the number and distribution of these elements on the five autosomes suggests a role in defining specific chromosomal domains.

DANA elements: a family of composite, tRNA-derived short interspersed DNA elements associated with mutational activities in zebrafish (Danio rerio).

DNA is the first SINE isolated from zebrafish (Danio rerio) exhibiting all the hallmarks of these tRNA-derived elements. DANA is unique in its clearly defined substructure of distinct cassettes. In contrast to generic SINE elements, DANA appears to have been assembled by insertions of short sequences into a progenitor, tRNA-derived element. Once associated with each other, these subunits were amplified as a new transposable element with such a remarkable success that DANA-related sequences comprise approximately 10% of the modern zebrafish genome. At least some of the sequences comprised by the full-length element were capable of movement, forming a new group of mobile, composite transposons, one of which caused an insertional mutation in the zebrafish no tail gene. Being present only in the genus Danio, and estimated to be as old as the genus itself, DANA may have played a role in Danio speciation by massive amplification and genome-wide dispersion. There are extensive DNA polymorphisms between zebrafish populations and strains detected by PCR amplification using primers specific to DANA, suggesting that the DANA element will be useful as a molecular tool for genetic and phylogenetic analyses.