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Our lab performs studies in functional genomics, genome structure, and homologous meiotic recombination in maize.
Plant transposable elements as tools for functional genomics
Transposons are invaluable tools in genetic analysis. They enable us to clone a gene on the basis of its mutant phenotype, rather than prior knowledge of its function, and to search for genes in organisms with a high content of repetitive DNA, such as maize. Functional analysis of the human-size maize genome appears daunting on the surface. However, genes are concentrated in regions of undermethylated DNA, which also correspond to the genomic component where the transposon Activator(Ac) tends to insert. Thus, genes can be identified as Ac receptor sites and isolated as the DNA adjacent to the transposed Acs (tac sites). This approach generates a sequence that can be compared to existing databases and an insertion library that can be screened intelligently for subtle mutant phenotypes. Our lab is funded by the NSF Plant Genome Program to optimize Ac as a gene searching engine in maize . We developed simple and efficient Ac transposition assays and generated a collection of over 1300 independent Ac transposants. A few of the insertions produce obvious visible phenotypes, but most of them do not, which is not surprising considering that many maize genes exist in duplicate. We isolated the DNA sequences adjacent to several Ac insertions; as expected, most of them correspond to unique DNA. Two of the more interesting mutant phenotypes in our collection are an embryo lethal with no effect on the endosperm, which results from an Ac insertion into a gene encoding the chloroplast ribosomal protein S9, and a defective pollen with corkscrew pollen tubes, which results from an Ac insertion into a homolog of a gene required for root elongation in Arabidopsis. Because Ac has a strong tendency to transpose to nearby sites, about one-half of our tac sites are linked to the donor loci. Clearly, it would be desirable to mobilize Ac from different launching platforms in the genome. Towards that end, we have developed a transformable genetic line and have begun to produce transgenic maize plants carrying a transposon that is modified to facilitate the isolation of tac site DNA. Our construct should integrate at random sites in the genome, providing starting platforms for future transposon mobilization from many locations in the genome. We have mapped the first transgenic platforms and have isolated the sequences adjacent to transposed elements. The transgenic transposon displays a similar preference to insert into genes as the native Ac.
Variability in maize genome structure
It is generally assumed that each gene in one individual will have an allelic counterpart in another individual of the same species. We have found that this assumption does not hold true in maize. We compared the bz genomic region of two different maize inbred lines and found dramatic differences between them. First, the retrotransposon clusters, which comprise most of the repetitive DNA in maize, differ markedly in make-up and pattern of interspersion relative to the genes in the bz region. Second, more importantly, the genes themselves differ between the two lines, demonstrating that genetic microcolinearity can be violated within the same species. Our finding has bearing on the underlying genetic basis of hybrid vigor and recombinational variability in maize. In different maize lines, genes that are members of gene families and are, therefore, expected to have quantitative, rather than qualitative, effects, may be present or absent in certain regions of the genome. Lines lacking different genes would complement one another and show hybrid vigor, whereas lines lacking mostly the same genes would not complement and, in breeding terminology, would fall in the same heterotic group. The number of genes in the bz genomic region appears to vary in different Corn Belt inbred lines. This unexpected finding has propelled us to begin an investigation of the bz genomic region in inbred lines and land races of widely different geographic origin and in some of the wild Mesoamerican relatives of maize. Homologous meiotic recombinationMeiotic recombination is a fundamental mechanism in the creation of novel genotypes in sexually reproducing organisms. We are using the bronze locus of maize, a uniquely advantageous system, to attempt to obtain answers to basic questions regarding the process of homologous meiotic recombination in plants. The bronze gene affects seed pigmentation and is at least 100 times more recombinogenic than the average DNA segment in maize, so intragenic recombinants (IGRs) can be generated and identified with relative ease. The high rate of recombination in bz is possibly due to its location within a highly gene-rich region of the genome. Ten genes are contained in a 32-kb stretch of DNA that is uninterrupted by retrotransposons, the most common component of the maize genome. The gene-rich region is flanked proximally and distally by large retrotransposon blocks. In order to monitor recombination in completely homozygous genetic intervals immediately adjacent to bz, we used as markers Ac elements that had transposed from the bz locus to closely linked sites. We found that recombination immediately proximal to bz, a segment consisting mostly of methylated retrotransposon DNA, is at least 100-fold lower than in bz. On the other hand, recombination distal to bz, a gene-rich region consisting exclusively of single-copy DNA, is of the same order of magnitude as within bz . Given our finding that the number of genes in the bz region can vary among lines, we are currently testing if the number of genes between two sites in the region affects the frequency of recombination between them.Dooner Lab Publications (last 10 years) Wang, X. and H.K. Dooner. 2006. Remarkable variation in maize genome structure inferred from haplotype diversity at the bz locus. Proc. Natl. Acad. Sci. USA 103, 17644-17649. Wu, X.R., Z. Chen, A. Shende, H.K. Dooner, and W. R. Folk. 2006. Visualizing bz1 missense suppression in Zea mays: an assay for monocot tRNA expression and utilization. Plant Mol. Biol. 61, 795-798. Xu, Z. and H. K. Dooner. 2006. The maize aberrant pollen transmission 1 (apt1) gene is a SABRE/ KIP homologue required for pollen tube growth. Genetics 172, 1251-1261. Messing, J. and H.K. Dooner. 2006. Organization and variability of the maize genome. Current Opinion in Plant Biology 9, 157-163. Lai, J., Y. Li, J. Messing, and H.K. Dooner. 2005. Gene movement by Helitron transposons contributes to the haplotype variability of maize. Proc. Natl. Acad. Sci. USA 102, 968-973. Xu, Z. and H. K. Dooner. 2005. Mx-rMx , a new family of interacting transposons in the growing hAT superfamily of maize. Plant Cell 17, 375-388. Xu, Z., X. Yan, S. Maurais, H. Fu, D. G. O'Brien, J. Mottinger, and H. K. Dooner. 2004. Jittery, a Mutator distant relative with a paradoxical mobile behavior: excision without reinsertion. Plant Cell 16, 1105-1114. Ma, Z. and H. K. Dooner. 2004. A mutation in the nuclear-encoded plastid ribosomal protein S9 leads to early embryo lethality in maize. Plant Journal 37: 92-103. Fu, H. and H.K. Dooner. 2002. Intraspecific violation of genetic colinearity and its implications in maize. Proc. Natl. Acad. Sci. USA 99: 9573-9578. Fu, H., Z. Zheng, and H.K. Dooner. 2002. Recombination rates between adjacent genic and retrotransposon regions in maize vary by two orders of magnitude. Proc. Natl. Acad. Sci. USA 99: 1082-1087. Fu, H., W. Park, X. Yan, Z. Zheng, B. Shen, and H.K. Dooner. 2001. The highly recombinogenic bz locus lies in an unusually gene-rich region of the maize genome. Proc. Natl. Acad. Sci. USA 98: 8903-8908. Shen, B.,. Z. Zheng, and H.K. Dooner. 2000. A maize sesquiterpene cyclase gene induced by insect herbivory and volicitin: characterization of wild-type and mutant alleles. Proc. Natl. Acad. Sci. USA 97: 14807-14812. Fu, Huihua and H.K. Dooner. 2000. Cloning of large allele-specific NotI fragments from a partial BAC library in maize: isolation of a 230-kb contig of the bronze region. Genome Research 10: 866-873. Yan, X., I.M. Martínez-Férez, S. Kavchok and H.K. Dooner. 1999 Origination of Ds elements from Ac elements in maize: evidence for rare repair synthesis at the site of Ac excision. Genetics 152: 1733-1740. Dooner, H.K. 1998. On the possible occurrence of conversion polarity at the bronze locus. The Plant Cell 10: 646-648. Dooner, H.K. and I.M. Martínez-Férez. 1997.
Germinal excisions of the maize transposable element
Activator do not stimulate meiotic recombination or
homology-dependent repair at the bz locus. Genetics
147: 1923-1932. Lab Support Gregorio Segal, Research Associate |
Research
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