Engineered Minichromosomes in Plants

Engineered minichromosomes provide the ability to target transgenes to a defined insertion position for predictable expression on an independent chromosome. This technology promises to provide a means to add many genes to a synthetic chromosome in sequential manner. An additional advantage is that the multiple transgenes will not be inserted into the normal chromosomes and thus will not exhibit linkage drag when converging the transgenes to different germplasm nor will they be mutagenic. Telomere truncation coupled with the introduction of site-specific recombination cassettes has proven to be an easy method to produce minichromosomes. Telomere truncation results from the transformation of plasmids carrying a block of telomere repeats at one end. Minichromosomes consisting of little more than a centromere have been produced for B chromosomes of maize. Such small chromosomes have been studied for their meiotic behavior, which differs from normal sized chromosomes in that homologue pairing is rare or nonexistent and sister chromatid cohesion fails at meiosis I. Potential modifications of the minichromosomes that can address these issues are discussed. Minichromosomes can be recovered from transformed plants that are polyploid or that carry an additional chromosome as the preferred target for truncation. Site-specific recombination has been demonstrated to operate on these terminally located sites. By introducing normal B chromosomes into lines with engineered mini-B chromosomes, the latter can be increased in copy number, which provides the potential to augment the expression of the introduced genes. Because the vast majority of plant species have the same telomere sequence, the truncating transgenes should be effective in most plants to generate engineered minichromosomes. Such chromosomes establish the means to add or subtract multiple transgenes, multigene complexes, or whole biochemical pathways to plants to change their properties for agronomic applications or to use plants as factories for the production of foreign proteins or metabolites.     

Centromere Epigenetics in Plants

The centromere is an essential chromosome site at which the kinetochore forms and loads proteins needed for faithful segregation during the cell cycle and meiosis(Houben et al., 1999;Cleveland et al.,2003;Ma et al.,2007;Birchler and Han,2009).Centromere specific sequences such as tandem repeats or transposable elements evolve quickly both within and between the species but have conserved kinetochore proteins(Henikoff and Furuyama,2010).The universal feature     

Genetic polymorphims of estrogen receptor alpha −397 PvuII (T>C) and −351 XbaI (A>G) in a portuguese population: prevalence and relation with breast cancer susceptibility

Estrogen receptor alpha (ERα), that mediates the biologic effects of estrogen in estrogen-sensitive tissues like breast, is genetically polymorphic. To evaluate the association between ?397 PvuII (T>C) and ?351 XbaI (A>G) restriction fragment length polymorphisms (RFLPs) in intron 1 of ERα gene and susceptibility of breast cancer, we undertook a case–control study in BRCA1 185delAG and 5382insC/BRCA2 6174delT negative Portuguese women. The study population consisted of 107 patients with histological diagnosis of breast cancer and 121 women with no history of breast cancer. Genomic DNA was extracted from blood samples and genotyping analyses were performed by PCR–RFLP. XbaI polymorphism was associated with a significant reduced risk of breast cancer for carriers of the x allele in homozygozity (OR 0.178; 95 % CI 0.070–0.456; P < 0.001) or heterozigozity (OR 0.223; 95 % CI 0.089–0.561; P = 0.001). The PvuII polymorphism was associated with a non-significantly reduced risk. The combined analysis of PvuII and XbaI polymorphisms revealed none synergistic effect of the two genotypes, except for simultaneous carriers of pp and xx genotypes, that have a reduced risk of breast cancer (OR 0.226; 95 % CI 0.049–1.035; P = 0.044). The combination of PvuII and XbaI genotypes into haplotypes showed that carriers of two copies of the px (ppxx) haplotype had a reduced risk of breast cancer (OR 0.405; 95 % CI 0.194–0.843; P = 0.014), compared with PX (PPXX + PPXx + PpXX + PpXx) haplotypes. PvuII and XbaI polymorphisms were in linkage disequilibrium both in cases (D = 0.044, r² = 0.049, X² = 5.216, P = 0.022) and controls (D = 0.090, r² = 0.139, X² = 16.819, P < 0.001), but not in the entire sample population analyzed as a whole (D = 0.087, r² = 0.0076, X² = 1.733, P = 0.188). In conclusion, in this case–control study we found that ERα gene XbaI polymorphism may modify individual susceptibility for breast cancer in this population.     

THE COMBINED USE OF WHOLE CUPHEA SEEDS CONTAINING MEDIUM CHAIN FATTY ACIDS AND AN EXOGENOUS LIPASE IN PIGLET NUTRITION

In search for an alternative for nutritional antimicrobials in piglet feeding, the effects of adding whole Cuphea seeds, as a natural source of medium chain fatty acids (MCFA), with known antimicrobial effects, and an exogenous lipase to a weaner diet were studied. The foregut flora, the gut morphology, some digestive parameters and the zootechnical performance of weaned piglets were investigated. Thirty newly weaned piglets, initial weight 7.0 ± 0.4 kg, were divided according to litter, sex and weight in two groups (control diet; Cuphea+lipase diet). The Cuphea seeds (lanceolata and ignea) (50 g kg^?1) were substituted for soybean oil (15 g kg^?1), Alphacell (25 g kg^?1) and soy protein isolate (10 g kg^?1) in the control diet. Also 500 mg kg^?1 microbial lipase was added to the Cuphea diet. The piglets were weighted individually on days 0, 3, 7, 14 and 16. Feed intake was recorded per pen during days 0 to 3, 3 to 7, 7 to 14 and 14 to 16. On day 7 five piglets of each experimental group were euthanized for counting the gastric and small intestinal gut flora and for gut morphology at two sites of the small intestine (proximal, distal). The results indicate a trend towards improved performances parameters by feeding Cuphea + lipase. The enzymic released MCFA (1.7 g kg^?1 fresh gastric contents) tended to decrease the number of Coliforms in the proximal small intestine, but increased the number in the stomach and distal small intestine. With Cuphea, the number of Streptococci was significantly lower in small intestine, but not in the stomach, while the number of Lactobacilli was significantly lower in the distal small intestine and tended to be lower in the stomach and proximal small intestine. No differences between the diets were noted for the total anaerobic microbial load in the stomach or in the gut. Feeding Cuphea+lipase resulted in a significantly greater villus height (distal small intestine) and a lesser crypt depth (proximal and distal small intestine) and greater villus/crypt ratio depth (proximal and distal small intestine). The intra-epithelial lymphocyte (IEL) counts per 100 enterocytes were significantly decreased in the proximal small intestine and tended to decrease in the distal small intestine by feeding the Cuphea+lipase diet. Both phenomena are indicative for a more healthy and better functional state of the mucosa. Present results are in line with foregoing research, showing that manipulation of the gut ecosystem by the enzymic in situ released MCFA in the stomach and foregut can result in improved performances of the piglets, which makes the concept a potential alternative for in-feed nutritional antibiotics.     

Phenotypic and gene expression analyses of a ploidy series of maize inbred Oh43

Polyploidization has repeatedly occurred during plant evolution. Although autopolyploidy is the best model to characterize the polyploidization effects in a highly controlled manner, there are limited studies on autopolyploids compared to allopolyploids. To improve our understanding of autopolyploidy effects in maize, we developed an inbred Oh43 ploidy series consisting of the diploid (2X), tetraploid (4X) and hexaploid (6X) lines and compared their phenotypes and gene expression in the mature adult leaf tissue. Our phenotypic study showed that plants of higher ploidy exhibit increased cell size but slower growth rate, later flowering, fewer tassel branches, reduced stature and fertility. Two-dimensional difference gel electrophoresis (2D DIGE) and gel electrophoresis followed by liquid chromatography and mass spectrometry (GeLC-MS) assays of the leaf proteomes revealed ~40 and 26% quantitative differentially expressed (DE) proteins, respectively, at the per genome level. A small number of qualitative DE proteins were also identified in the GeLC-MS assay. The majority of the quantitative DE proteins found in the 2D DIGE assay were present in either the 4X versus 6X or the 2X versus 6X comparison but not the 2X versus 4X comparison. Aneuploidy in some 6X plants might contribute to the more extensive changes of gene expression per genome in the 6X. Most changes of the protein expression per genome are less than twofold. Less than 5% of the DE genes exhibit a positive or negative continuous correlation through the ploidy series between their protein expression per genome, and the genome copy number. Hence, in the Oh43 ploidy series, expression for most proteins in a cell increases linearly with ploidy.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation.

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

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.     

Engineered minichromosomes in plants

Genetic engineering for complex or combined traits requires the simultaneous expression of multiple genes, and has been considered as the bottleneck for the next generation of genetic engineering in plants. Minichromosome technology provides one solution to the stable expression and maintenance of multiple transgenes in one genome. For example, minichromosomes can be used as a platform for efficient stacking of multiple genes for insect, bacterial and fungal resistances together with herbicide tolerance and crop quality traits. All the transgenes would reside on an independent minichromosome, not linked to any endogenous genes; thus linkage drag can be avoided. Engineered minichromosomes can be easily constructed by a telomere-mediated chromosomal truncation strategy. This approach does not rely on the cloning of centromere sequences, which are species-specific, and bypasses the any complications of epigenetic components for centromere specification. Thus, this technique can be easily extended to all plant species. The engineered minichromosome technology can also be used in combination with site-specific recombination systems to facilitate the stacking of multiple transgenes.     

A hemicentric inversion in the maize line knobless Tama flint created two sites of centromeric elements and moved the kinetochore-forming region

A maize line, knobless Tama flint (KTF), was found to contain a version of chromosome 8 with two spatially distinct regions of centromeric elements, one at the original genetic position and the other at a novel location on the long arm. The new site of centromeric elements functions as the kinetochore-forming region resulting in a change of arm length ratio. Examination of fluorescence in situ hybridization markers on chromosome 8 revealed an inversion between the two centromere sites relative to standard maize lines, indicating that this chromosome 8 resulted from a hemicentric inversion with one breakpoint approximately 20 centi-McClintocks (cMc) on the long arm (20% of the total arm length from the centromere) and the other in the original cluster of centromere repeats. This inversion moved the kinetochore-forming region but left the remainder of the centromere repeats. In a hybrid between a standard line (Mo17) and KTF, both chromosome 8 homologues were completely synapsed at pachytene despite the inversion. Although the homologous centromeres were not paired, they were always correctly oriented at anaphase and migrated to opposite poles. Additionally, recombination on 8L was severely repressed in the hybrid.     

Progressive heterosis in genetically defined tetraploid maize

Progressive heterosis, i.e., the additional hybrid vigor in double-cross tetraploid hybrids not found in their single-cross tetraploid parents, has been documented in a number of species including alfalfa,potato, and maize. In this study, four artificially induced maize tetraploids, directly derived from standard inbred lines, were crossed in pairs to create two single-cross hybrids. These hybrids were then crossed to create double-cross hybrids containing genetic material from all four original lines. Replicated fieldbased phenotyping of the materials over four years indicated a strong progressive heterosis phenotype in tetraploids but not in their diploid counterparts. In particular, the above ground dry weight phenotype of double-cross tetraploid hybrids was on average 34% and 56% heavier than that of the single-cross tetraploid hybrids and the double-cross diploid counterparts, respectively. Additionally,whole-genome resequencing of the original inbred lines and further analysis of these data did not show the expected spectrum of alleles to explain tetraploid progressive heterosis under the complementation of complete recessive model. These results underscore the reality of the progressive heterosis phenotype,its potential utility for increasing crop biomass production, and the need for exploring alternative hypothesis to explain it at a molecular level.