Coevolution of apomixis and genome size within the genus<Emphasis Type="Italic"> Hypericum</Emphasis>

Trends concerning coevolution of mode of reproduction and genome size were elucidated by screening both components in 71 species/subspecies of the genus Hypericum. Two independent agamic complexes were identified (sections Ascyreia with ten, and Hypericum with five apomictic species). In the phylogenetically younger section Hypericum, the relative DNA content of apomicts is increased solely by polyploidy. The apomicts of the evolutionarily older section Ascyreia have significantly larger genomes than all other species due to polyploidization and higher DNA content per chromosome. An accumulation of retroelements might be one reason for the larger genomes. The male fertility of the apomicts was reduced compared to sexuals, although all apomicts were facultative pseudogamous, forming reduced male gametes. Another form of apomixis (obligate pseudogamous with unreduced male gametes), probably indicating an escape from interspecific sterility, was found in H. scabrum, the only case of asexual seed formation outside of sections Ascyreia and Hypericum. The described scenario for evolution of apomixis in relation to genome size deserves consideration in harnessing of apomixis.     

ClpE from Lactococcus lactis promotes repression of CtsR-dependent gene expression

The heat shock response in bacterial cells is characterized by rapid induction of heat shock protein expression, followed by an adaptation period during which heat shock protein synthesis decreases to a new steady-state level. In this study we found that after a shift to a high temperature the Clp ATPase (ClpE) in Lactococcus lactis is required for such a decrease in expression of a gene negatively regulated by the heat shock regulator (CtsR). Northern blot analysis showed that while a shift to a high temperature in wild-type cells resulted in a temporal increase followed by a decrease in expression of clpP encoding the proteolytic component of the Clp protease complex, this decrease was delayed in the absence of ClpE. Site-directed mutagenesis of the zinc-binding motif conserved in ClpE ATPases interfered with the ability to repress CtsR-dependent expression. Quantification of ClpE by Western blot analysis revealed that at a high temperature ClpE is subjected to ClpP-dependent processing and that disruption of the zinc finger domain renders ClpE more susceptible. Interestingly, this domain resembles the N-terminal region of McsA, which was recently reported to interact with the CtsR homologue in Bacillus subtilis. Thus, our data point to a regulatory role of ClpE in turning off clpP gene expression following temporal heat shock induction, and we propose that this effect is mediated through CtsR.     

Dlx5/6-enhancer directed expression of Cre recombinase in the pharyngeal arches and brain

Dlx5 and Dlx6, two members of the Distalless gene family, are required for development of numerous tissues during embryogenesis, including facial and limb development. This gene pair is expressed in tandem, transcribed toward each other and separated by a short intergenic region containing multiple putative enhancers. Targeted inactivation of Dlx5 and Dlx6 in mice results in multiple developmental defects in craniofacial and limb structures, suggesting that these genes are crucial for aspects of both neural crest and nonneural crest development. To further investigate potential developmental roles of Dlx5 and Dlx6, we used one of the Dlx5/6 intergenic enhancers to drive Cre recombinase expression in transgenic mice. Crossing Dlx5/6-Cre transgenic mice with mice from the R26R strain results in beta-galactosidase staining in the apical ectodermal ridge, brain, and neural crest-derived mesenchyme of the pharyngeal arches, with staining in term embryos observed in the facial skeleton and specific brain structures. However, in contrast to endogenous expression patterns of Dlx5 and Dlx6, Cre expression within the pharyngeal arches occurs during a very narrow window in early development. Our studies suggest that Dlx5/6-Cre mice may prove useful both in further understanding the function and regulation of Distalless genes during development and in studies of gene function in conditional knockout mice.     

Proteome analysis of the purine stimulon from Lactococcus lactis

A comparative expression proteome analysis was carried out by analyzing differential expression patterns of pulse-labelled proteins on two-dimensional gels under standard conditions and during purine nucleotide starvation, followed by mass spectrometric identification of regulated proteins. Based upon the expression patterns, three stimulons could be identified in Lactococcus lactis subsp. cremoris. The Psu proteins (purine starvation up-regulated) had increased synthesis during purine depletion in a purine auxotroph. Among these proteins were enzymes of the purine biosynthesis pathways (PurE, PurS, PurM, PurL), and enzymes involved in the generation of C1 units (GlyA, Fhs). C1 units are primarily required for purine biosynthesis. Upon analysis of the nucleotide sequence preceding the structural genes for these proteins in the L. lactis IL1403 genome sequence showed that all contained PurBox-Pribnov box structures resembling the PurR activated promoters for the purDEK and purCSQLF operons. Most, and possibly all members of the Psu stimulon are thus members of the PurR regulon. Five Psu proteins could not be identified. The second stimulon, the Psd stimulon (purine starvation decreased), whose members are down-regulated during purine depletion, contained proteins related to protein synthesis (PpsB, EF-TS, trigger factor), or to GTPases (FtsZ, EF-TS); or are involved in energy metabolism (GapB, CcpA). No common regulatory elements could be found for members of this stimulon. Two Psd proteins escaped identification. The last, Dcu (decoynine up-regulated), stimulon contained proteins whose synthesis escaped the severe general depression during inhibition of the GMP synthetase by decoynine. This regulon was comprised of mostly glycolytic enzymes (fructose bisphosphate aldolase, enolase, pyruvate kinase) and translation elongation factors (GTPases: EF-TU, EF-G). Two Dcu proteins could not be identified. Out of 28 proteins subjected to mass spectrometry, 19 could be readily identified despite the fact that only the genome sequence of a strain of L. lactis subsp. lactis was available. The two subspecies share about 85% sequence identity, comparable to the genetic distance between Escherichia coli and Salmonella typhimurium. A success rate of 68% indicates that it may be feasible to perform proteomics based upon genomic sequences of relatives outside the genus.     

The GP problem: quantifying gene-to-phenotype relationships

In this paper we refer to the gene-to-phenotype modeling challenge as the GP problem. Integrating information across levels of organization within a genotype-environment system is a major challenge in computational biology. However, resolving the GP problem is a fundamental requirement if we are to understand and predict phenotypes given knowledge of the genome and model dynamic properties of biological systems. Organisms are consequences of this integration, and it is a major property of biological systems that underlies the responses we observe. We discuss the E(NK) model as a framework for investigation of the GP problem and the prediction of system properties at different levels of organization. We apply this quantitative framework to an investigation of the processes involved in genetic improvement of plants for agriculture. In our analysis, N genes determine the genetic variation for a set of traits that are responsible for plant adaptation to E environment-types within a target population of environments. The N genes can interact in epistatic NK gene-networks through the way that they influence plant growth and development processes within a dynamic crop growth model. We use a sorghum crop growth model, available within the APSIM agricultural production systems simulation model, to integrate the gene-environment interactions that occur during growth and development and to predict genotype-to-phenotype relationships for a given E(NK) model. Directional selection is then applied to the population of genotypes, based on their predicted phenotypes, to simulate the dynamic aspects of genetic improvement by a plant-breeding program. The outcomes of the simulated breeding are evaluated across cycles of selection in terms of the changes in allele frequencies for the N genes and the genotypic and phenotypic values of the populations of genotypes.