Never born proteins as a test case for ab initio protein structures prediction

The number of natural proteins although large is significantly smaller than the theoretical number of proteins that can be obtained combining the 20 natural amino acids, the so-called “never born proteins” (NBPs). The study of the structure and properties of these proteins allows to investigate the sources of the natural proteins being of unique characteristics or special properties. However the structural study of NPBs can also been intended as an ideal test for evaluating the efficiency of software packages for the ab initio protein structure prediction. In this research, 10.000 three-dimensional structures of proteins of completely random sequence generated according to ROSETTA and FOD model were compared. The results show the limits of these software packages, but at the same time indicate that in many cases there is a significant agreement between the prediction obtained.     

The maize Knotted1 gene is an effective positive selectable marker gene for Agrobacterium-mediated tobacco transformation

We have assessed the use of a homeobox gene knotted1 (kn1) from maize as a selectable marker gene for plant transformation. The kn1 gene under the control of cauliflower mosaic virus 35S promoter (35S::kn1) was introduced into Nicotiana tabacum cv. Xanthi via Agrobacterium-mediated transformation. Under nonselective conditions (without antibiotic selection) on a hormone-free medium (MS), a large number of transgenic calli and shoots were obtained from explants that were infected with Agrobacterium tumefaciens LBA4404 harboring the 35S::kn1 gene. On the other hand, no calli or shoots were produced from explants that were infected with an Agrobacterium strain harboring pBI121 (nptII selection) or from uninfected controls cultured under identical conditions. Relative to kanamycin selection conferred by nptII, the use of kn1 resulted in a 3-fold increase in transformation efficiency. The transgenic status of shoots obtained was confirmed by both histochemical detection of GUS activity and molecular analysis. The results presented here suggest that kn1 gene could be used as an effective alternative selection marker with a potential to enhance plant transformation efficiency in many plant species. With kn1 gene as a selection marker gene, no antibiotic-resistance or herbicide-resistance genes are needed so that potential risks associated with the use of these traditional selection marker genes can be eliminated.     

Isolation of matrices from maize leaf nuclei: identification of a matrix-binding site adjacent to theAdh1 gene

Nuclear matrices were isolated from maize leaves by the two conventional methods usually employed for the preparation of the corresponding structures of animal origin. It is demonstrated that functionally competent matrices, recognizing and specifically binding the MAR-containing DNA of the mousek-immunoglobulin gene may be prepared by both 2 M NaCl and LIS extractions of maize nuclei.A DNA region with a high affinity for the nuclear matrix was identified at the 5 end of the maizeAdh1-S gene, distal to the promoter region. The presence of sites of reported altered chromatin structure in this particular region is discussed. While the proximity and the cohabitation of MARs with different regulatory elements is a common feature of matrix association regions in animal systems, this is the first plant MAR identified in a region of known significance for gene regulation.     

Genetic control of the opaque-2 gene and background polygenes over some kernel traits in maize (Zea mays L.)

Some kernel traits of agronomical importance in maize are affected by the opaque-2 (o2) gene and background polygenes, which express in different genetic systems such as embryo, endosperm, cytoplasm and maternal plant. A genetic model for seed quantitative traits with the o2 gene effects and polygenic effects as well as their GE interactions was used for protein content, lysine content, oil content and kernel density in maize. The results suggested that the o2 gene was involved in the traits investigated but the effects of the o2 gene were distinctive on various traits. The effects of the o2 gene were large on lysine content and protein content while minor on oil content. There was a substantially wide quantitative variation from polygenes expressing in different genetic systems for the traits evaluated. Significant GE interactions of the o2 gene and background polygenes declared that not only the main effects but also specific expressions depending on environments were responsible for variation of the traits studied. There seemed to have strong maternal heterosis and slight embryo heterosis for kernel density.     

Intron-mediated enhancement of heterologous gene expression in maize

Chimeric genes containing the coding sequence for bacterial chloramphenicol acetyl transferase (CAT) have been introduced by electroporation into maize protoplasts (Black Mexican Sweet) and transient expression monitored by enzyme assays. Levels of CAT expression were enhanced 12-fold and 20-fold respectively by the inclusion of maize alcohol dehydrogenase-1 introns 2 and 6 in the chimeric construct. This enhancement was seen when the intron was placed within the 5 translated region but not when it was located upstream of the promoter or within the 3 untranslated region. Deletion of exon sequences adjacent to intron 2 abolished its ability to mediate enhancement of CAT gene expression. Northern analysis of protoplasts electroporated with intron constructs revealed elevated levels of CAT mRNA. However, this elevation was insufficient to account for the increased enzyme activity. One explanation of these results is that splicing affects both the quantity of mRNA.     

Enhancement of maize transformation efficiency by the use of maize matrix attachment regions

Improving genetic transformation efficiency is a major concern in plant genetic engineering. While various strategies have been investigated, the enhancement of selectable marker gene expression has not been tried extensively. We used maize matrix attachment regions (MARs) to bracket an herbicide resistance transgene, bar. MARs have been reported to enhance transgene expression level and stability. We show here that MARs not only enhance transformation efficiency by 50%, but are also able to increase or decrease relative efficiencies of each step of the regeneration process depending on MAR sequence combinations. Furthermore, we assessed the trans-effect of MARs in co-bombardment experiments with two independent plasmids, one including the MAR sequences and the other one the bar gene. As for simple bombardment, MARs enhanced transformation efficiency by having a positive influence on organogenesis step in the regeneration process.     

Effects of gene dosage and sequence modification on the frequency and timing of transposition of the maize element Activator (Ac) in tobacco

The effect of Ac copy number on the frequency and timing of germinal transposition in tobacco was investigated using the streptomycin phosphotransferase gene (SPT) as an excision marker. The activity of one and two copies of the element was compared by selecting heterozygous and homozygous progeny of transformants carrying single SPT::Ac inserts. It was observed that increasing gene copy not only increases the transposition frequency, but also occasionally alters the timing of transposition such that earlier events are obtained. The result is that some homozygous plants generate multiple streptomycin resistant progeny carrying the same transposed Ac (trAc) element. We have also investigated the effect of modification of the sequence in the region around 82 bp downstream of the polyadenylation site and 177 bp from the 3 end of the element on germinal excision frequencies. Alteration of three bases to create a BglII site at this location caused a minor decrease in germinal excision events, but insertion of four bases to create a Cla I site caused a 10-fold decrease in the transposition activity of the Ac element.     

Characterization of the molecular defect in a null allele of the opaque-2 locus of maize

The molecular defect in an opaque-2 (o2) mutant, previously characterized as a null allele, has been identified as containing an insertion of the transposable element of the Bergamo (Bg) family. Restriction mapping and partial sequence analysis of the Bg in the o2 null allele indicates that this element is distinct from the previously described Bg as well as the defective Bg (rbg) of the o2m(r) allele. It is, however, inserted at the same site in O2 as the rbg of 62m(r) and can transpose when Bg is present. This study shows that, depending on genetic background, this allele may not behave as a stable null which could dramatically influence the conclusions drawn from experiments based on this particular mutant.     

Phylogeny of the family hydrocharitaceae inferred fromrbcL andmatK gene sequence data

The family Hydrocharitaceae, with 15 genera and ca. 80 species, shows a remarkable morphological diversity which presumably developed as an adaptation to their aquatic habitat. This is particularly true in the case of the many different kinds of pollination mechanisms. To gather more basic information regarding the adaptive evolution of Hydrocharitaceae, we have carried out a phylogenetic analysis based on the sequences of therbcL andmatK. Our resulting neighbor-joining distance tree provides the following insights: (1) none of the previous classification systems were supported by molecular phylogenetic tree; (2) Najas (Najadaceae), which has never been included in Hydrocharitaceae except in Shaffer-Fehre's (1991) system based on seed coat structures, is an ingroup of Hydrocharitaceae; (3) Limnocharitaceae and Alismataceae are sister groups of Hydrocharitaceae; (4) the three marine genera,Halophila, Enhalus andThalassia, are monophyletic; and (5) a peculiar pollination mechanism specific to Hydrocharitaceae (Hydrocharitaceae-epihydrophily), underwent a parallel evolution.     

Downregulation of genes encoding for subunits of adaptor complex-3 in cervical carcinomas

We explored the expression of four genes encoding for subunits of AP-3 in cervical tumors and cancer cell lines. Using RT-PCR we demonstrated more than twofold decrease in the levels of mRNA of AP3D1, AP3B1, AP3M1, and AP3S1 in 32, 28, 23, and 26% tumors in comparison with normal tissues of uterine cervix, respectively. The level of mRNA of at least one subunit was decreased in 28 out of 47 (60%) of tumors and in four out of five cancer cell lines in comparison to tissues adjacent to tumors. The suppression of expression of any of the subunits was revealed in 15 out of 28 cases (54%). The expression of two and more subunits was decreased simultaneously in different combinations in 13 cases (46%). This fact testifies to the lack of a common mechanism of downregulation of four subunits in tumors. There is a tendency to more frequent suppression of AP-3A expression in tumors associated with lymphatic node metastases as compared with tumors without metastases (P = 0.034). Thus, here we demonstrate for the first time the decrease in expression of genes encoding for AP-3A subunits in tumors.     

RFLP mapping of the sugary enhancer1 gene in maize

RFLP marker data from an F₂₃ population derived from a cross between a sugary1 (su1) and a sugary enhancer1 (su1, sel) inbred were used to construct a genetic linkage map of maize. This map includes 93 segregating marker loci distributed throughout the maize genome, providing a saturated linkage map that is suitable for linkage analysis with quantitative trait loci (QTL). This population, which has been immortalized in the form of sibbed F₂₃ families, was derived from each of the 214 F₂ plants and along with probe data are available to the scientific community. QTL analysis for kernel sucrose (the primary form of sugar) concentration at 20 days after pollination (DAP) uncovered the segregation of seven major QTL influencing sucrose concentration; a locus linked to umc36a described the greatest proportion of the variation (24.7%). Since maltose concentration has previously been reported to be associated with the se1 phenotype, an analysis of probe associations with maltose concentration at 40 DAP was also conducted. The highly significant association of umc36a with maltose and sucrose concentrations provided evidence that this probe is linked to se1. Phenotypic evaluation for the se1 genotype in each F₂₃ family enabled us to map the gene 12.1 cM distal to umc36a. In contrast to previous work where se1 was reported to be located on chromosome four, our data strongly suggest that the sugary enhancer1 locus maps on the the distal portion of the long arm of chromosome 2 in the maize genome.