Molecular Biology

Background: Midsegment duplication (dup) of chromatid arms may be symmetric or asymmetric. It can be argued that every dup should yield a discommensured RC with (a) loss of at least one duplicated unit to the template counterpart and; (b) deletion of all sections of the replicating chromatid arm that are distal to both the gap left by the duplicating process and the segment closest to the centromere. Hypothesis: Mechanisms capable of recommensuring the stack of chromatids after topological shifts of duplicated units (dups) are discussed. The mechanics might fail in few cases, which are discussed in terms of statistics and scalability. Conclusion: The dynamics of the highly non-linear processes discussed here may be relevant to duplications of smaller (epsilon) subunits such as telomeric units within malignant genomes.

Background: The availability of genome sequences of numerous organisms allows comparative study of pseudogenes in syntenic regions. Conservation of pseudogenes suggests that they might have a functional role in some instances. Results: We report the first large-scale comparative analysis of ribosomal protein (RP) pseudogenes in four mammalian genomes (human, chimpanzee, mouse and rat). To this end, we have assigned these pseudogenes in the four organisms using an automated pipeline and make the results available online. Each organism has a large number of RP pseudogenes (~1400 to ~2800). The majority of them are processed (generated by retrotransposition). However, we do not see a correlation between the number of pseudogenes associated with a RP gene and its mRNA abundance. Analysis of pseudogenes in syntenic regions between species shows that most are conserved between human and chimpanzee, but very few are conserved between primates and rodents. Interestingly, syntenic pseudogenes have a lower rate of nucleotide substitution than their surrounding intergenic DNA. Moreover, evidence from ESTs indicates that two pseudogenes conserved between human and mouse are transcribed. Detailed analysis shows that one of them, the pseudogene of RPS27, is likely to be a protein-coding gene. This is significant as previous reports indicated there are exactly 80 RP genes encoded by the human genome. Conclusions: Our analysis indicates that processed RP pseudogenes abound in mammalian genomes, but few of these are conserved between primates and rodents. This highlights the large amount of recent retrotranspositional activity in mammals and a relatively larger amount of it in the rodent lineage.

Background: Genomic imprinting is an epigenetic phenomenon that results in monoallelic gene expression. Many hypotheses have been advanced to explain why genomic imprinting evolved in mammals, but few have examined how it arose. The host defence hypothesis suggests that imprinting evolved from existing mechanisms within the cell that act to silence foreign DNA elements that insert into the genome. However, the changes to the mammalian genome that accompanied the evolution of imprinting have been hard to define due to the absence of large scale genomic resources between all extant classes. The recent release of the platypus genome has provided the first opportunity to perform comparisons between prototherian (monotreme; which appear to lack imprinting) and therian (marsupial and eutherian; which have imprinting) mammals. Results: We compared the distribution of repeat elements known to attract epigenetic silencing across the entire genome from monotremes and therian mammals particularly focusing on the orthologous imprinted regions. There was a significant accumulation of certain repeat elements within imprinted regions of therian mammals compared to the platypus. Conclusions: Our analyses show that the platypus has significantly fewer repeats of certain classes in the regions of the genome that have become imprinted in therian mammals. The accumulation of repeats, especially LTRs and DNA elements, in therian imprinted genes and gene clusters is coincident with, and may have been a potential driving force in, the development of mammalian genomic imprinting. These data provide strong support for the host defence hypothesis.

The results of two published genome sequences from marine diatoms provide basic insights into how these remarkable organisms evolved to become one of the most successful groups of eukaryotic algae in the contemporary ocean.

The appointment of two life scientists, including a genome biologist, to the President-elect Obama's President's Council of Advisors on Science and Technology is encouraging.

Background: Polydnaviruses, double-stranded DNA viruses with segmented genomes, have evolved as obligate endosymbionts of parasitoid wasps. Virus particles are replication deficient and produced by female wasps from proviral sequences integrated into the wasp genome. These particles are co-injected with eggs into caterpillar hosts, where viral gene expression facilitates parasitoid survival and, thereby, survival of proviral DNA. Here we characterize and compare the encapsidated viral genome sequences of bracoviruses in the family Polydnaviridae associated with Glyptapanteles gypsy moth parasitoids, along with near complete proviral sequences from which both viral genomes are derived. Results: The encapsidated Glyptapanteles indiensis and Glyptapanteles flavicoxis bracoviral genomes, each comprised of 29 different size segments, total ~517 and 594 kbp, respectively. They are generated from a minimum of 7 distinct loci in the wasp genome. Annotation of these sequences revealed numerous novel features for polydnaviruses, including insect-like sugar transporter genes and transposable elements. Evolutionary analyses suggest that positive selection is widespread among bracoviral genes. Conclusions: The structure and organization of Glyptapanteles indiensis and Glyptapanteles flavicoxis bracovirus proviral segments as multiple loci containing one to many viral segments, flanked and separated by wasp gene-encoding DNA, is confirmed. Rapid evolution of bracovirus genes supports the hypothesis of bracovirus genes in an "arms race" between bracovirus and caterpillar. Phylogenetic analyses of the bracoviral genes encoding sugar transporters provides the first robust evidence of a wasp origin for some polydnavirus genes. We hypothesize transposable elements, such as those described here, could facilitate transfer of genes between proviral segments and host DNA.

When DNA replication is slowed down, normally dormant replication origins are activated. Recent work demonstrates that cells adapt by changing the organization of chromatin loops and maintaining the new pattern of origin use in subsequent cell cycles.

The transcription factor Prox1 is the master regulator of lymphatic endothelial cell differentiation and its expression initiates the morphogenesis of the lymphatic vasculature in the early embryo. Two new studies now answer some fundamental questions concerning Prox1 biology.

The development of second-generation biofuels - those that do not rely on grain crops as inputs - will require a diverse set of feedstocks that can be grown sustainably and processed cost-effectively. Here we review the outlook and challenges for meeting hoped-for production targets for such biofuels in the United States.

Background: With the exception of vertebrates, most organisms have plasma membrane associated ammonium transporters which primarily serve to import a source of nitrogen for nutritional purposes. Dictyostelium discoideum has three ammonium transporters, Amts A, B and C. Our present work used fluorescent fusion proteins to determine the cellular localization of the Amts and tested the hypothesis that the transporters mediate removal of ammonia generated endogenously from the elevated protein catabolism common to many protists. Results: Using RFP and YFP fusion constructs driven by the actin 15 promoter, we found that the three ammonium transporters were localized on the plasma membrane and on the membranes of subcellular organelles. AmtA and AmtB were localized on the membranes of endolysosomes and phagosomes, with AmtB further localized on the membranes of contractile vacuoles. AmtC also was localized on subcellular organelles when it was stabilized by coexpression with either the AmtA or AmtB fusion transporter. The three ammonium transporters exported ammonia linearly with regard to time during the first 18 hours of the developmental program as revealed by reduced export in the null strains. The fluorescently tagged transporters rescued export when expressed in the null strains, and thus they were functional transporters. Conclusions: Unlike ammonium transporters in most organisms, which import NH3/NH4+ as a nitrogen source, those of Dictyostelium export ammonia/ammonium as a waste product from extensive catabolism of exogenously derived and endogenous proteins. Localization on proteolytic organelles and on the neutral contractile vacuole suggests that Dictyostelium ammonium transporters may have unique subcellular functions and play a role in the maintenance of intracellular ammonium distribution. A lack of correlation between the null strain phenotypes and ammonia excretion properties of the ammonium transporters suggests that it is not the excretion function that is important for coupling ammonia levels to the slug versus culmination choice, but rather a sensor and/or signaling function of these proteins that is important.

Background: The renal (kNBC1) and intestinal (pNBC1) electrogenic Na+/HCO3- cotransporter variants differ in their primary structure, transport direction, and response to secretagogues. Previous studies have suggested that regulatory differences between the two subtypes can be partially explained by unique consensus phosphorylation sites included in the pNBC1, but not the kNBC1 sequence. After having shown activation of NBC by carbachol and forskolin in murine colon, we now investigated these pathways in HEK293 cells transiently expressing a GFP-tagged pNBC1 construct. Results: Na- and HCO3-dependent pHi recovery from an acid load (measured with BCECF) was enhanced by 5-fold in GFP-positive cells compared to the control cells in the presence of CO2/HCO3-. Forskolin (10-5 M) had no effect in untransfected cells, but inhibited the pHi recovery in cells expressing pNBC1 by 62%. After preincubation with carbachol (10-4 M), the pHi recovery was enhanced to the same degree both in transfected and untransfected cells, indicating activation of endogenous alkalizing ion transporters. Acid-activated Na/HCO3 cotransport via pNBC1 expressed in renal cells is thus inhibited by cAMP and not affected by cholinergic stimulation, as opposed to the findings in native intestinal tissue. Conclusion: Regulation of pNBC1 by secretagogues appears to be not solely dependent on its primary structure, but also on properties of the cell type in which it is expressed.

Background: Some years ago we established an ENU screen for modifiers of transgene variegation in the mouse and a preliminary description of the first six mutant lines, named MommeD1-D6, has been published. We have reported the underlying genes in three cases; MommeD1 is a mutation in SMC hinge domain containing 1 (Smchd1), a novel modifier of epigenetic gene silencing, MommeD2 is a mutation in DNA methyltransferase 1 (Dnmt1) and MommeD4 is a mutation in Smarca 5 (Snf2h), a known chromatin remodeller. The identification of Dnmt1 and Smarca5 attest to the effectiveness of the screen design. Results: We have now extended the screen and have identified four new modifiers, MommeD7-D10. Here we show that all ten MommeDs link to unique sites in the genome, that homozygosity for the mutations is associated with severe developmental abnormalities and that heterozygosity results in phenotypic abnormalities and reduced reproductive fitness in some cases. In addition, we have now identified the underlying gene for MommeD5 and MommeD10. MommeD5 is a mutation in Hdac1, which encodes histone deacetylase 1, and MommeD10 is a mutation in Baz1b (also known as Williams syndrome transcription factor), which encodes a transcription factor containing a PHD-type zinc finger and a bromodomain. We show that reduction in the level of Baz1b in the mouse results in craniofacial features reminiscent of Williams syndrome. Conclusions: These results demonstrate the importance of dosage-dependent epigenetic reprogramming in the development of the embryo and the power of the screen to provide mouse models to study this process.

Background: During flowering central anther cells switch from mitosis to meiosis, ultimately forming pollen containing haploid sperm. Four rings of surrounding somatic cells differentiate to support first meiosis and later pollen dispersal. Synchronous development of many anthers per tassel and within each anther facilitates dissection of carefully staged maize anthers for transcriptome profiling. Results: Global gene expression profiles of seven stages representing 29 days of anther development are analyzed using a 44K oligonucleotide array querying ~80% of maize protein-coding genes. Mature haploid pollen containing just two cell types expresses 10,000 transcripts. Anthers contain five major cell types and express >24,000 transcript types: each anther stage expresses ~10,000 constitutive and ~10,000 or more transcripts restricted to one or a few stages. The lowest complexity is present during meiosis. Large suites of stage-specific and co-expressed genes are identified through GO and clustering analyses as functional classes for pre-meiotic, meiotic, and post-meiotic anther development. MADS box and zinc finger transcription factors with constitutive and stage-limited expression are identified. Conclusions: We propose that the extensive gene expression of anther cells and pollen represents the key test of maize genome fitness, permitting strong selection against deleterious alleles in diploid anthers and haploid pollen. Because flowering plants show a substantial bias for male-sterile compared to female-sterile mutations, we propose that this fitness test is general. Because both somatic and germinal cells are transcriptionally quiescent during meiosis, we hypothesize that successful completion of meiosis is required to trigger maturation of anther somatic cells.

Background: The ErbB3 binding protein-1 (Ebp1) belongs to a family of DNA/RNA binding proteins implicated in cell growth, apoptosis and differentiation. However, the physiological role of Ebp1 in the whole organism is not known. Therefore, we generated Ebp1-deficient mice carrying a gene trap insertion in intron 2 of the Ebp1 (pa2g4) gene. Results: Ebp1 -/- mice were on average 30% smaller than wild type and heterozygous sex matched littermates. Growth retardation was apparent from Day 10 until Day 30. IGF-1 production and IGBP-3 and 4 protein levels were reduced in both embryo fibroblasts and adult knock-out mice. The proliferation of fibroblasts derived from Day 12.5 knock out embryos was also decreased as compared to that of wild type cells. Microarray expression analysis revealed changes in genes important in cell growth including members of the MAPK signal transduction pathway. In addition, the expression or activation of proliferation related genes such as AKT and the androgen receptor, previously demonstrated to be affected by Ebp1 expression in vitro, was altered in adult tissues. Conclusions: These results indicate that Ebp1 can affect growth in an animal model, but that the expression of proliferation related genes is cell and context specific. The Ebp1-/- mouse line represents a new in vivo model to investigate Ebp1 function in the whole organism.

The ability to detect activation of signaling pathways based solely on gene expression data represents an important goal in biological research. We tested the sensitivity of singular value decomposition (SVD)-based regression by focusing on functional interactions between the Ras and TGFbeta signaling pathways. Our findings demonstrate that this approach is sufficiently sensitive to detect the secondary activation of endogenous signaling pathways as it occurs through cross-talk following ectopic activation of a primary pathway.

KEGG spider is a web-based tool for interpretation of experimentally derived gene lists in order to gain understanding of metabolism variations at a genomic level. KEGG spider implements a "pathway-free" framework which overcomes a major bottleneck of enrichment analyses: it provides global models uniting genes from different metabolic pathways. Analysing a number of experimentally derived gene lists, we demonstrate that KEGG spider provides deeper insights into metabolism variations in comparison to existing methods.

Little is known about the structure and distinguishing features of core promoters in Plasmodium falciparum. In this work, we describe the first method to computationally identify core promoters in this AT rich genome. This prediction algorithm uses solely DNA physicochemical properties as descriptors. Our results add to a growing body of evidence that a physicochemical code for eukaryotic genomes plays a crucial role in core promoter recognition.

Bioinformatics has become too central to biology to be left to specialist bioinformaticians. Biologists are all bioinformaticians now.

Background: Malaria is a one of the most important infectious diseases and is caused by parasitic protozoa of the genus Plasmodium. Previously, the quantitative characterization of the P. falciparum transcriptome demonstrated that the strictly controlled progression of these parasites through their intra-erythrocytic developmental cycle is accompanied by a continuous cascade of gene expression. Although such analyses have proven immensely useful, the correlations between abundance of transcripts and their cognate proteins remain poorly characterized. Results: Here, we present a quantitative time-course analysis of relative protein abundance for schizont-stage parasites (34-46 hours post-invasion) based on two-dimensional differential gel electrophoresis (2D-DIGE) of protein samples labeled with fluorescent dyes. For this purpose we analyzed parasite samples taken at four-hour intervals from a tightly synchronized culture and established more than 500 individual protein abundance profiles with high temporal resolution and quantitative reproducibility. Approximately half of all profiles exhibit a significant change in abundance and 12% display an expression peak during the observed 12-hour time interval. Intriguingly, identification of 54 protein spots by mass spectrometry revealed that 58% of the corresponding proteins including actin-I, enolase, eIF4A, eIF5A, and several heat shock proteins are represented by more than one isoform, presumably due to post-translational modifications, with the various isoforms of a given protein frequently showing different expression patterns. Furthermore, comparisons with transcriptome data generated from the same parasite samples reveal evidence of significant post-transcriptional gene expression regulation. Conclusions: Together, our data indicate that both post-transcriptional and post-translational events are widespread and of presumably great biological significance during the intra-erythrocytic development of P. falciparum.

Background: As an obligatory intracellular parasite, Trypanosoma cruzi, the etiological agent of Chagas' disease, must invade and multiply within mammalian cells. Cytokeratin 18 (CK18) is among the host molecules that have been suggested as a mediator of important events during T. cruzi-host cell interaction. Based on that possibility, we addressed whether RNA interference (RNAi)-mediated down regulation of the CK18 gene could interfere with the parasite life cycle in vitro. HeLa cells transiently transfected with CK18-RNAi had negligible levels of CK18 transcripts, and significantly reduced levels of CK18 protein expression as determined by immunoblotting or immunofluorescence. Results: CK18 negative or positive HeLa cells were invaded equally as well by trypomastigotes of different T. cruzi strains. Also, in CK18 negative or positive cells, parasites recruited host cells lysosomes and escaped from the parasitophorous vacuole equally as well. After that, the growth of amastigotes of the Y or CL-Brener strains, was drastically arrested in CK18 RNAi-treated cells. After 48 hours, the number of amastigotes was several times lower in CK18 RNAi-treated cells when compared to control cells. Simultaneous staining of parasites and CK18 showed that in HeLa cells infected with the Y strain both co-localize. Although the amastigote surface protein-2 contains the domain VTVXNVFLYNR previously described to bind to CK18, in several attempts, we failed to detect binding of a recombinant protein to CK-18. Conclusions: The study demonstrates that silencing CK18 by transient RNAi, inhibits intracellular multiplication of the Y and CL strain of T. cruzi in HeLa cells, but not trypanosome binding and invasion.