Weichang Yu

Construction and behavior of engineered minichromosomes in maize

Engineered minichromosomes were constructed in maize by modifying natural A and supernumerary B chromosomes. By using telomere-mediated chromosomal truncation, it was demonstrated that such an approach is feasible for the generation of minichromosomes of normal A chromosomes by selection of spontaneous polyploid events that compensate for the deficiencies produced. B chromosomes are readily fractionated by biolistic transformation of truncating plasmids. Foreign genes were faithfully expressed from integrations into normal B chromosomes and from truncated miniB chromosomes. Site-specific recombination between the terminal transgene on a miniA chromosome and a terminal site on a normal chromosome was demonstrated. It was also found that the miniA chromosome did not pair with its progenitor chromosomes during meiosis, indicating a useful property for such constructs. The miniB chromosomes are faithfully transmitted from one generation to the next but can be changed in dosage in the presence of normal B chromosomes. This approach for construction of engineered chromosomes can be easily extended to other plant species because it does not rely on cloned centromere sequences, which are species-specific. These platforms will provide avenues for studies on plant chromosome structure and function and for future developments in biotechnology and agriculture.

Telomere-mediated chromosomal truncation in maize

Direct repeats of Arabidopsis telomeric sequence were constructed to test telomere-mediated chromosomal truncation in maize. Two constructs with 2.6 kb of telomeric sequence were used to transform maize immature embryos by Agrobacterium-mediated transformation. One hundred seventy-six transgenic lines were recovered in which 231 transgene loci were revealed by a FISH analysis. To analyze chromosomal truncations that result in transgenes located near chromosomal termini, Southern hybridization analyses were performed. A pattern of smear in truncated lines was seen as compared with discrete bands for internal integrations, because telomeres in different cells are elongated differently by telomerase. When multiple restriction enzymes were used to map the transgene positions, the size of the smears shifted in accordance with the locations of restriction sites on the construct. This result demonstrated that the transgene was present at the end of the chromosome immediately before the integrated telomere sequence. Direct evidence for chromosomal truncation came from the results of FISH karyotyping, which revealed broken chromosomes with transgene signals at the ends. These results demonstrate that telomere-mediated chromosomal truncation operates in plant species. This technology will be useful for chromosomal engineering in maize as well as other plant species.

Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors

High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant l-cysteine alone or l-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.

Minichromosome Analysis of Chromosome Pairing, Disjunction, and Sister Chromatid Cohesion in Maize

With the advent of engineered minichromosome technology in plants, an understanding of the properties of small chromosomes is desirable. Twenty-two minichromosomes of related origin but varying in size are described that provide a unique resource to study such behavior. Fourteen minichromosomes from this set could pair with each other in meiotic prophase at frequencies between 25 and 100%, but for the smaller chromosomes, the sister chromatids precociously separated in anaphase I. The other eight minichromosomes did not pair with themselves, and the sister chromatids divided equationally at meiosis I. In plants containing one minichromosome, the sister chromatids also separated at meiosis I. In anaphase II, the minichromosomes progressed to one pole or the other. The maize (Zea mays) Shugoshin protein, which has been hypothesized to protect centromere cohesion in meiosis I, is still present at anaphase I on minichromosomes that divide equationally. Also, there were no differences in the level of phosphorylation of Ser-10 of histone H3, a correlate of cohesion, in the minichromosomes in which sister chromatids separated during anaphase I compared with the normal chromosomes. These analyses suggest that meiotic centromeric cohesion is compromised in minichromosomes depending on their size and cannot be maintained by the mechanisms used by normal-sized chromosomes.

Centromere Function and Nondisjunction Are Independent Components of the Maize B Chromosome Accumulation Mechanism

Supernumerary or B chromosomes are selfish entities that maintain themselves in populations by accumulation mechanisms. The accumulation mechanism of the B chromosome of maize (Zea mays) involves nondisjunction at the second pollen mitosis, placing two copies of the B chromosome into one of the two sperm. The B chromosome long arm must be present in the same nucleus for the centromere to undergo nondisjunction. A centromere, containing all of the normal DNA elements, translocated from the B chromosome to the short arm of chromosome 9 was recently found to be epigenetically silenced for centromeric function. When intact B chromosomes were added to this genotype, thus supplying the long arm, the inactive centromere regained the property of nondisjunction causing the translocation chromosome 9 to be differentially distributed to the two sperm or resulted in chromosome breaks in 9S, occasionally producing new translocations. Translocation of the inactive B centromere to chromosome 7 transferred the nondisjunction property to this chromosome. The results provide insight into the molecular and evolutionary basis of this B chromosome accumulation mechanism by demonstrating that nondisjunction is caused by a process that does not depend on normal centromere function but that the region of the chromosome required for nondisjunction resides in the centromeric region.

Plant engineered minichromosomes and artificial chromosome platforms

The introduction of telomere sequences during transformation of maize will cause chromosomal truncation. This technique has been used to create minichromosomes. With the simultaneous introduction of site specific recombination cassettes, the ability to add additional genes to the newly formed engineered minichromosome becomes possible. Targeting the supernumerary B chromosome produces truncations at a very high rate and produces minichromosomes with the additional property that they can be dosage manipulated by the reintroduction of full length B chromosomes. The uses of engineered minichromosomes are discussed.

Cytological Visualization of DNA Transposons and Their Transposition Pattern in Somatic Cells of Maize

Global genomic analysis of transposable element distributions of both natural (En/Spm, Ac–Ds, and MuDR/Mu) and modified (RescueMu) types was performed by fluorescence in situ hybridization (FISH) on somatic chromosomes coupled with karyotyping of each chromosome. In lines without an active transposable element, the locations of silent En/Spm, Ac–Ds, and MuDR/Mu elements were visualized, revealing variation in copy number and position among lines but no apparent locational bias. The ability to detect single elements was validated by using previously mapped active Ac elements. Somatic transpositions were documented in plants containing an engineered Mutator element, RescueMu, via use of the karyotyping system. By analyzing the RescueMu lines, we found that transposition of RescueMu in root-tip cells follows the cut-and-paste type of transposition. This work demonstrates the utility of FISH and karyotyping in the study of transposon activity and its consequences.