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1.  Chromosomal duplications and cointegrates generated by the bacteriophage lamdba Red system in Escherichia coli K-12 
Background
An Escherichia coli strain in which RecBCD has been genetically replaced by the bacteriophage λ Red system engages in efficient recombination between its chromosome and linear double-stranded DNA species sharing sequences with the chromosome. Previous studies of this experimental system have focused on a gene replacement-type event, in which a 3.5 kbp dsDNA consisting of the cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant. The dsDNA was delivered into the cell as part of the chromosome of a non-replicating λ vector, from which it was released by the action of a restriction endonuclease in the infected cell. This study characterizes the genetic requirements and outcomes of a variety of additional Red-promoted homologous recombination events producing Lac+ recombinants.
Results
A number of observations concerning recombination events between the chromosome and linear DNAs were made: (1) Formation of Lac+ and Lac- recombinants depended upon the same recombination functions. (2) High multiplicity and high chromosome copy number favored Lac+ recombinant formation. (3) The Lac+ recombinants were unstable, segregating Lac- progeny. (4) A tetracycline-resistance marker in a site of the phage chromosome distant from cat was not frequently co-inherited with cat. (5) Recombination between phage sequences in the linear DNA and cryptic prophages in the chromosome was responsible for most of the observed Lac+ recombinants. In addition, observations were made concerning recombination events between the chromosome and circular DNAs: (6) Formation of recombinants depended upon both RecA and, to a lesser extent, Red. (7) The linked tetracycline-resistance marker was frequently co-inherited in this case.
Conclusions
The Lac+ recombinants arise from events in which homologous recombination between the incoming linear DNA and both lac and cryptic prophage sequences in the chromosome generates a partial duplication of the bacterial chromosome. When the incoming DNA species is circular rather than linear, cointegrates are the most frequent type of recombinant.
doi:10.1186/1471-2199-5-22
PMCID: PMC545071  PMID: 15596011
2.  Mapping and mutation of the conserved DNA polymerase interaction motif (DPIM) located in the C-terminal domain of fission yeast DNA polymerase δ subunit Cdc27 
Background
DNA polymerases α and δ play essential roles in the replication of chromosomal DNA in eukaryotic cells. DNA polymerase α (Pol α)-primase is required to prime synthesis of the leading strand and each Okazaki fragment on the lagging strand, whereas DNA polymerase δ (Pol δ) is required for the elongation stages of replication, a function it appears capable of performing on both leading and lagging strands, at least in the absence of DNA polymerase ε (Pol ε).
Results
Here it is shown that the catalytic subunit of Pol α, Pol1, interacts with Cdc27, one of three non-catalytic subunits of fission yeast Pol δ, both in vivo and in vitro. Pol1 interacts with the C-terminal domain of Cdc27, at a site distinct from the previously identified binding sites for Cdc1 and PCNA. Comparative protein sequence analysis identifies a protein sequence motif, called the DNA polymerase interaction motif (DPIM), in Cdc27 orthologues from a wide variety of eukaryotic species, including mammals. Mutational analysis shows that the DPIM in fission yeast Cdc27 is not required for effective DNA replication, repair or checkpoint function.
Conclusions
The absence of any detectable phenotypic consequences arising from mutation of the DPIM suggests that despite its evolutionary conservation, the interaction between the two polymerases mediated by this motif is a non-essential one.
doi:10.1186/1471-2199-5-21
PMCID: PMC545490  PMID: 15579205
3.  The Drosophila methyl-DNA binding protein MBD2/3 interacts with the NuRD complex via p55 and MI-2 
Background
Methyl-DNA binding proteins help to translate epigenetic information encoded by DNA methylation into covalent histone modifications. MBD2/3 is the only candidate gene in the Drosophila genome with extended homologies to mammalian MBD2 and MBD3 proteins, which represent a co-repressor and an integral component of the Nucleosome Remodelling and Deacetylase (NuRD) complex, respectively. An association of Drosophila MBD2/3 with the Drosophila NuRD complex has been suggested previously. We have now analyzed the molecular interactions between MBD2/3 and the NuRD complex in greater detail.
Results
The two MBD2/3 isoforms precisely cofractionated with NuRD proteins during gel filtration of extracts derived from early and late embryos. In addition, we demonstrate that MBD2/3 forms multimers, and engages in specific interactions with the p55 and MI-2 subunits of the Drosophila NuRD complex.
Conclusion
Our data provide novel insights into the association between Drosophila MBD2/3 and NuRD proteins. Additionally, this work provides a first analysis of the architecture of the Drosophila NuRD complex.
doi:10.1186/1471-2199-5-20
PMCID: PMC529442  PMID: 15516265
4.  Real-time PCR quantitation of glucocorticoid receptor alpha isoform 
Background
The expression of glucocorticoid-receptor (GR) seems to be a key mechanism in the regulation of glucocorticoid (GC) sensitivity and is potentially involved in cases of GC resistance or hypersensitivity. The aim of this study is to describe a method for quantitation of GR alpha isoform (GRα) expression using real-time PCR (qrt-PCR) with analytical capabilities to monitor patients, offering standard-curve reproducibility as well as intra- and inter-assay precision.
Results
Standard-curves were constructed by employing standardized Jurkat cell culture procedures, both for GRα and BCR (breakpoint cluster region), as a normalizing gene. We evaluated standard-curves using five different sets of cell culture passages, RNA extraction, reverse transcription, and qrt-PCR quantification. Intra-assay precision was evaluated using 12 replicates of each gene, for 2 patients, in a single experiment. Inter-assay precision was evaluated on 8 experiments, using duplicate tests of each gene for two patients. Standard-curves were reproducible, with CV (coefficient of variation) of less than 11%, and Pearson correlation coefficients above 0,990 for most comparisons. Intra-assay and inter-assay were 2% and 7%, respectively.
Conclusion
This is the first method for quantitation of GRα expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way.
doi:10.1186/1471-2199-5-19
PMCID: PMC529441  PMID: 15507144
5.  Transcriptional oscillation of canonical clock genes in mouse peripheral tissues 
Background
The circadian rhythm of about 24 hours is a fundamental physiological function observed in almost all organisms from prokaryotes to humans. Identification of clock genes has allowed us to study the molecular bases for circadian behaviors and temporal physiological processes such as hormonal secretion, and has prompted the idea that molecular clocks reside not only in a central pacemaker, the suprachiasmatic nuclei (SCN) of hypothalamus in mammals, but also in peripheral tissues, even in immortalized cells. Furthermore, previous molecular dissection revealed that the mechanism of circadian oscillation at a molecular level is based on transcriptional regulation of clock and clock-controlled genes.
Results
We systematically analyzed the mRNA expression of clock and clock-controlled genes in mouse peripheral tissues. Eight genes (mBmal1, mNpas2, mRev-erbα, mDbp, mRev-erbβ, mPer3, mPer1 and mPer2; given in the temporal order of the rhythm peak) showed robust circadian expressions of mRNAs in all tissues except testis, suggesting that these genes are core molecules of the molecular biological clock. The bioinformatics analysis revealed that these genes have one or a combination of 3 transcriptional elements (RORE, DBPE, and E-box), which are conserved among human, mouse, and rat genome sequences, and indicated that these 3 elements may be responsible for the biological timing of expression of canonical clock genes.
Conclusions
The observation of oscillatory profiles of canonical clock genes is not only useful for physiological and pathological examination of the circadian clock in various organs but also important for systematic understanding of transcriptional regulation on a genome-wide basis. Our finding of the oscillatory expression of canonical clock genes with a temporal order provides us an interesting hypothesis, that cyclic timing of all clock and clock-controlled genes may be dependent on several transcriptional elements including 3 known elements, E-box, RORE, and DBPE.
doi:10.1186/1471-2199-5-18
PMCID: PMC535906  PMID: 15473909
6.  Polyadenylation of ribosomal RNA by Candida albicans also involves the small subunit 
Background
Candida albicans is a polymorphic fungus causing serious infections in immunocompromised patients. It is capable of shifting from yeast to germinating forms such as hypha and pseudohypha in response to a variety of signals, including mammalian serum. We have previously shown that some of the large 25S components of ribosomal RNA in Candida albicans get polyadenylated, and this process is transiently intensified shortly after serum exposure just prior to the appearance of germination changes.
Results
We now present data that this process also involves the small 18S subunit of ribosomal RNA in this organism. Unlike the large 25S subunit, polyadenylation sites near the 3' end are more variable and no polyadenylation was found at the reported maturation site of 18S. Similar to 25S, one or more polyadenylated mature sized 18S molecules get intensified transiently by serum just prior to the appearance of hypha.
Conclusions
The transient increase in polyadenylation of both the large and the small subunits of ribosomal RNA just prior to the appearance of hypha, raises the possibility of a role in this process.
doi:10.1186/1471-2199-5-17
PMCID: PMC522811  PMID: 15461824
7.  Glycogen Synthase Kinase-3 regulates IGFBP-1 gene transcription through the Thymine-rich Insulin Response Element 
Background
Hepatic expression of several gene products involved in glucose metabolism, including phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) and insulin-like growth factor binding protein-1 (IGFBP-1), is rapidly and completely inhibited by insulin. This inhibition is mediated through the regulation of a DNA element present in each of these gene promoters, that we call the Thymine-rich Insulin Response Element (TIRE). The insulin signalling pathway that results in the inhibition of these gene promoters requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase). However, the molecules that connect PI 3-kinase to these gene promoters are not yet fully defined. Glycogen Synthase Kinase 3 (GSK-3) is inhibited following activation of PI 3-kinase. We have shown previously that inhibitors of GSK-3 reduce the activity of two TIRE-containing gene promoters (PEPCK and G6Pase), whose products are required for gluconeogenesis.
Results
In this report we demonstrate that in H4IIE-C3 cells, four distinct classes of GSK-3 inhibitor mimic the effect of insulin on a third TIRE-containing gene, IGFBP-1. We identify the TIRE as the minimum requirement for inhibition by these agents, and demonstrate that the target of GSK-3 is unlikely to be the postulated TIRE-binding protein FOXO-1. Importantly, overexpression of GSK-3 in cells reduces the insulin regulation of TIRE activity as well as endogenous IGFBP-1 expression.
Conclusions
These results implicate GSK-3 as an intermediate in the pathway from the insulin receptor to the TIRE. Indeed, this is the first demonstration of an absolute requirement for GSK-3 inhibition in insulin regulation of gene transcription. These data support the potential use of GSK-3 inhibitors in the treatment of insulin resistant states such as Type 2 diabetes mellitus, but suggest that it will be important to identify all TIRE-containing genes to assess potential side effects of these agents.
doi:10.1186/1471-2199-5-15
PMCID: PMC517930  PMID: 15350195
GSK-3; Insulin; IGFBP-1 gene transcription; TIRE; CHIR99021
8.  Coactivators p300 and PCAF physically and functionally interact with the foamy viral trans-activator 
Background
Foamy virus Bel1/Tas trans-activators act as key regulators of gene expression and directly bind to Bel1 response elements (BRE) in both the internal and the 5'LTR promoters leading to strong transcriptional trans-activation. Cellular coactivators interacting with Bel1/Tas are unknown to date.
Results
Transient expression assays, co-immunoprecipitation experiments, pull-down assays, and Western blot analysis were used to demonstrate that the coactivator p300 and histone acetyltransferase PCAF specifically interact with the retroviral trans-activator Bel1/Tas in vivo. Here we show that the Bel1/Tas-mediated trans-activation was enhanced by the coactivator p300, histone acetyltransferases PCAF and SRC-1 based on the crucial internal promoter BRE. The Bel1/Tas-interacting region was mapped to the C/H1 domain of p300 by co-immunoprecipitation and pull-down assays. In contrast, coactivator SRC-1 previously reported to bind to the C-terminal domain of p300 did not directly interact with the Bel1 protein but nevertheless enhanced Bel1/Tas-mediated trans-activation. Cotransfection of Bel1/Tas and p300C with an expression plasmid containing the C/H1domain partially inhibited the p300C-driven trans-activation.
Conclusions
Our data identify p300 and PCAF as functional partner molecules that directly interact with Bel1/Tas. Since the acetylation activities of the three coactivators reside in or bind to the C-terminal regions of p300, a C/H1 expression plasmid was used as inhibitor. This is the first report of a C/H1 domain-interacting retroviral trans-activator capable of partially blocking the strong Bel1/Tas-mediated activation of the C-terminal region of coactivator p300. The potential mechanisms and functional roles of the three histone and factor acetyltransferases p300, PCAF, and SRC-1 in Bel1/Tas-mediated trans-activation are discussed.
doi:10.1186/1471-2199-5-16
PMCID: PMC517496  PMID: 15350211
9.  Sequence periodicity of Escherichia coli is concentrated in intergenic regions 
Background
Sequence periodicity with a period close to the DNA helical repeat is a very basic genomic property. This genomic feature was demonstrated for many prokaryotic genomes. The Escherichia coli sequences display the period close to 11 base pairs.
Results
Here we demonstrate that practically only ApA/TpT dinucleotides contribute to overall dinucleotide periodicity in Escherichia coli. The noncoding sequences reveal this periodicity much more prominently compared to protein-coding sequences. The sequence periodicity of ApC/GpT, ApT and GpC dinucleotides along the Escherichia coli K-12 is found to be located as well mainly within the intergenic regions.
Conclusions
The observed concentration of the dinucleotide sequence periodicity in the intergenic regions of E. coli suggests that the periodicity is a typical property of prokaryotic intergenic regions. We suppose that this preferential distribution of dinucleotide periodicity serves many biological functions; first of all, the regulation of transcription.
doi:10.1186/1471-2199-5-14
PMCID: PMC516772  PMID: 15333140
10.  Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent 
Background
Serum Response Factor (SRF) is a transcription factor that is required for the expression of many genes including immediate early genes, cytoskeletal genes, and muscle-specific genes. SRF is activated in response to extra-cellular signals by its association with a diverse set of co-activators in different cell types. In the case of the ubiquitously expressed immediate early genes, the two sets of SRF binding proteins that regulate its activity are the TCF family of proteins that include Elk1, SAP1 and SAP2 and the myocardin-related MKL family of proteins that include MKL1 and MKL2 (also known as MAL, MRTF-A and -B and BSAC). In response to serum or growth factors these two classes of co-activators are activated by different upstream signal transduction pathways. However, it is not clear how they differentially activate SRF target genes.
Results
In order to identify the serum-inducible SRF target genes that are specifically dependent on the MKL pathway, we have performed microarray experiments using a cell line that expresses dominant negative MKL1. This approach was used to identify SRF target genes whose activation is MKL-dependent. Twenty-eight of 150 serum-inducible genes were found to be MKL-dependent. The promoters of the serum-inducible genes were analyzed for SRF binding sites and other common regulatory elements. Putative SRF binding sites were found at a higher rate than in a mouse promoter database but were only identified in 12% of the serum-inducible promoters analyzed. Additional partial matches to the consensus SRF binding site were found at a higher than expected rate in the MKL-dependent gene promoters. The analysis for other common regulatory elements is discussed.
Conclusions
These results suggest that a subset of immediate early and SRF target genes are activated by the Rho-MKL pathway. MKL may also contribute to the induction of other SRF target genes however its role is not essential, possibly due to other activation mechanisms such as MAPK phosphorylation of TCFs.
doi:10.1186/1471-2199-5-13
PMCID: PMC516031  PMID: 15329155
11.  Periodicity of DNA in exons 
Background
The periodic pattern of DNA in exons is a known phenomenon. It was suggested that one of the initial causes of periodicity could be the universal (RNY)npattern (R = A or G, Y = C or U, N = any base) of ancient RNA. Two major questions were addressed in this paper. Firstly, the cause of DNA periodicity, which was investigated by comparisons between real and simulated coding sequences. Secondly, quantification of DNA periodicity was made using an evolutionary algorithm, which was not previously used for such purposes.
Results
We have shown that simulated coding sequences, which were composed using codon usage frequencies only, demonstrate DNA periodicity very similar to the observed in real exons. It was also found that DNA periodicity disappears in the simulated sequences, when the frequencies of codons become equal.
Frequencies of the nucleotides (and the dinucleotide AG) at each location along phase 0 exons were calculated for C. elegans, D. melanogaster and H. sapiens. Two models were used to fit these data, with the key objective of describing periodicity. Both of the models showed that the best-fit curves closely matched the actual data points. The first dynamic period determination model consistently generated a value, which was very close to the period equal to 3 nucleotides. The second fixed period model, as expected, kept the period exactly equal to 3 and did not detract from its goodness of fit.
Conclusions
Conclusion can be drawn that DNA periodicity in exons is determined by codon usage frequencies. It is essential to differentiate between DNA periodicity itself, and the length of the period equal to 3. Periodicity itself is a result of certain combinations of codons with different frequencies typical for a species. The length of period equal to 3, instead, is caused by the triplet nature of genetic code. The models and evolutionary algorithm used for characterising DNA periodicity are proven to be an effective tool for describing the periodicity pattern in a species, when a number of exons in the same phase are analysed.
doi:10.1186/1471-2199-5-12
PMCID: PMC516030  PMID: 15315715
Codon usage; DNA periodicity; simulated sequences; evolutionary algorithm
12.  Co-transcriptional folding is encoded within RNA genes 
Background
Most of the existing RNA structure prediction programs fold a completely synthesized RNA molecule. However, within the cell, RNA molecules emerge sequentially during the directed process of transcription. Dedicated experiments with individual RNA molecules have shown that RNA folds while it is being transcribed and that its correct folding can also depend on the proper speed of transcription.
Methods
The main aim of this work is to study if and how co-transcriptional folding is encoded within the primary and secondary structure of RNA genes. In order to achieve this, we study the known primary and secondary structures of a comprehensive data set of 361 RNA genes as well as a set of 48 RNA sequences that are known to differ from the originally transcribed sequence units. We detect co-transcriptional folding by defining two measures of directedness which quantify the extend of asymmetry between alternative helices that lie 5' and those that lie 3' of the known helices with which they compete.
Results
We show with statistical significance that co-transcriptional folding strongly influences RNA sequences in two ways: (1) alternative helices that would compete with the formation of the functional structure during co-transcriptional folding are suppressed and (2) the formation of transient structures which may serve as guidelines for the co-transcriptional folding pathway is encouraged.
Conclusions
These findings have a number of implications for RNA secondary structure prediction methods and the detection of RNA genes.
doi:10.1186/1471-2199-5-10
PMCID: PMC514895  PMID: 15298702
13.  The p68 and p72 DEAD box RNA helicases interact with HDAC1 and repress transcription in a promoter-specific manner 
Background
p68 (Ddx5) and p72 (Ddx17) are highly related members of the DEAD box family and are established RNA helicases. They have been implicated in growth regulation and have been shown to be involved in both pre-mRNA and pre-rRNA processing. More recently, however, these proteins have been reported to act as transcriptional co-activators for estrogen-receptor alpha (ERα). Furthermore these proteins were shown to interact with co-activators p300/CBP and the RNA polymerase II holoenzyme. Taken together these reports suggest a role for p68 and p72 in transcriptional activation.
Results
In this report we show that p68 and p72 can, in some contexts, act as transcriptional repressors. Targeting of p68 or p72 to constitutive promoters leads to repression of transcription; this repression is promoter-specific. Moreover both p68 and p72 associate with histone deacetylase 1 (HDAC1), a well-established transcriptional repression protein.
Conclusions
It is therefore clear that p68 and p72 are important transcriptional regulators, functioning as co-activators and/or co-repressors depending on the context of the promoter and the transcriptional complex in which they exist.
doi:10.1186/1471-2199-5-11
PMCID: PMC514542  PMID: 15298701
14.  Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors 
Background
RNA interference (RNAi) can potently reduce target gene expression in mammalian cells and is in wide use for loss-of-function studies. Several recent reports have demonstrated that short double-stranded RNAs (dsRNAs), used to mediate RNAi, can also induce an interferon-based response resulting in changes in the expression of many interferon-responsive genes. Off-target gene silencing has also been described, bringing into question the validity of certain RNAi-based approaches for studying gene function. We have targeted the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA using lentiviral vectors for delivery of U6 promoter-driven PAI-2-targeted short hairpin RNA (shRNA) expression. PAI-2 is reported to have anti-apoptotic activity, thus reduction of endogenous expression may be expected to make cells more sensitive to programmed cell death.
Results
As expected, we encountered a cytotoxic phenotype when targeting the PAI-2 mRNA with vector-derived shRNA. However, this predicted phenotype was a potent non-specific effect of shRNA expression, as functional overexpression of the target protein failed to rescue the phenotype. By decreasing the shRNA length or modifying its sequence we maintained PAI-2 silencing and reduced, but did not eliminate, cytotoxicity. ShRNA of 21 complementary nucleotides (21 mers) or more increased expression of the oligoadenylate synthase-1 (OAS1) interferon-responsive gene. 19 mer shRNA had no effect on OAS1 expression but long-term selective pressure on cell growth was observed. By lowering lentiviral vector titre we were able to reduce both expression of shRNA and induction of OAS1, without a major impact on the efficacy of gene silencing.
Conclusions
Our data demonstrate a rapid cytotoxic effect of shRNAs expressed in human tumor cell lines. There appears to be a cut-off of 21 complementary nucleotides below which there is no interferon response while target gene silencing is maintained. Cytotoxicity or OAS1 induction could be reduced by changing shRNA sequence or vector titre, but stable gene silencing could not be maintained in extended cell culture despite persistent marker gene expression from the RNAi-inducing transgene cassette. These results underscore the necessity of careful controls for immediate and long-term RNAi use in mammalian cell systems.
doi:10.1186/1471-2199-5-9
PMCID: PMC514603  PMID: 15291968
15.  High-level gene expression in Aedes albopictus cells using a baculovirus Hr3 enhancer and IE1 transactivator 
Background
Aedes aegypti is the key vector of both the Yellow Fever and Dengue Fever viruses throughout many parts of the world. Low and variable transgene expression levels due to position effect and position effect variegation are problematic to efforts to create transgenic laboratory strains refractory to these viruses. Transformation efficiencies are also less than optimal, likely due to failure to detect expression from all integrated transgenes and potentially due to limited expression of the transposase required for transgene integration.
Results
Expression plasmids utilizing three heterologous promoters and three heterologous enhancers, in all possible combinations, were tested. The Hr3/IE1 enhancer-transactivator in combination with each of the constitutive heterologous promoters tested increased reporter gene expression significantly in transiently transfected Aedes albopictus C7-10 cells.
Conclusions
The addition of the Hr3 enhancer to expression cassettes and concomitant expression of the IE1 transactivator gene product is a potential method for increasing the level of transgene expression in insect systems. This mechanism could also potentially be used to increase the level of transiently-expressed transposase in order to increase the number of integration events in transposon-mediated transformation experiments.
doi:10.1186/1471-2199-5-8
PMCID: PMC487899  PMID: 15251037
16.  Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro 
Background
Incorporation of exon 11 of the insulin receptor gene is both developmentally and hormonally-regulated. Previously, we have shown the presence of enhancer and silencer elements that modulate the incorporation of the small 36-nucleotide exon. In this study, we investigated the role of inherent splice site strength in the alternative splicing decision and whether recognition of the splice sites is the major determinant of exon incorporation.
Results
We found that mutation of the flanking sub-optimal splice sites to consensus sequences caused the exon to be constitutively spliced in-vivo. These findings are consistent with the exon-definition model for splicing. In-vitro splicing of RNA templates containing exon 11 and portions of the upstream intron recapitulated the regulation seen in-vivo. Unexpectedly, we found that the splice sites are occupied and spliceosomal complex A was assembled on all templates in-vitro irrespective of splicing efficiency.
Conclusion
These findings demonstrate that the exon-definition model explains alternative splicing of exon 11 in the IR gene in-vivo but not in-vitro. The in-vitro results suggest that the regulation occurs at a later step in spliceosome assembly on this exon.
doi:10.1186/1471-2199-5-7
PMCID: PMC481066  PMID: 15233842
17.  Post-translational generation of constitutively active cores from larger phosphatases in the malaria parasite, Plasmodium falciparum: implications for proteomics 
Background
Although the complete genome sequences of a large number of organisms have been determined, the exact proteomes need to be characterized. More specifically, the extent to which post-translational processes such as proteolysis affect the synthesized proteins has remained unappreciated. We examined this issue in selected protein phosphatases of the protease-rich malaria parasite, Plasmodium falciparum.
Results
P. falciparum encodes a number of Ser/Thr protein phosphatases (PP) whose catalytic subunits are composed of a catalytic core and accessory domains essential for regulation of the catalytic activity. Two examples of such regulatory domains are found in the Ca+2-regulated phosphatases, PP7 and PP2B (calcineurin). The EF-hand domains of PP7 and the calmodulin-binding domain of PP2B are essential for stimulation of the phosphatase activity by Ca+2. We present biochemical evidence that P. falciparum generates these full-length phosphatases as well as their catalytic cores, most likely as intermediates of a proteolytic degradation pathway. While the full-length phosphatases are activated by Ca+2, the processed cores are constitutively active and either less responsive or unresponsive to Ca+2. The processing is extremely rapid, specific, and occurs in vivo.
Conclusions
Post-translational cleavage efficiently degrades complex full-length phosphatases in P. falciparum. In the course of such degradation, enzymatically active catalytic cores are produced as relatively stable intermediates. The universality of such proteolysis in other phosphatases or other multi-domain proteins and its potential impact on the overall proteome of a cell merits further investigation.
doi:10.1186/1471-2199-5-6
PMCID: PMC459218  PMID: 15230980
18.  A Xenopus Dbf4 homolog is required for Cdc7 chromatin binding and DNA replication 
Background
Early in the cell cycle a pre-replicative complex (pre-RC) is assembled at each replication origin. This process involves the sequential assembly of the Origin Recognition Complex (ORC), Cdc6, Cdt1 and the MiniChromosome Maintenance (Mcm2-7) proteins onto chromatin to license the origin for use in the subsequent S phase. Licensed origins must then be activated by S phase-inducing cyclin-dependent kinases (S-CDKs) and the Dbf4/Cdc7 kinase.
Results
We have cloned a Xenopus homologue of Dbf4 (XDbf4), the sequence of which confirms the results of Furukhori et al. We have analysed the role of XDbf4 in DNA replication using cell-free extracts of Xenopus eggs. Our results indicate that XDbf4 is the regulatory subunit of XCdc7 required for DNA replication. We show that XDbf4 binds to chromatin during interphase, but unlike XCdc7, its chromatin association is independent of pre-RC formation, occurring in the absence of licensing, XCdc6 and XORC. Moreover, we show that the binding of XCdc7 to chromatin is dependent on the presence of XDbf4, whilst under certain circumstances XDbf4 can bind to chromatin in the absence of XCdc7. We provide evidence that the chromatin binding of XDbf4 that occurs in the absence of licensing depends on checkpoint activation.
Conclusions
We have identified XDbf4 as a functional activator of XCdc7, and show that it is required to recruit XCdc7 to chromatin. Our results also suggest that XCdc7 and XDbf4 are differentially regulated, potentially responding to different cell cycle signals.
doi:10.1186/1471-2199-5-5
PMCID: PMC446192  PMID: 15222894
19.  Essential role for poly (ADP-ribosyl)ation in mouse preimplantation development 
Background
Poly (ADP-ribosyl)ation is a covalent modification of many nuclear proteins. It has a strong chromatin modifying potential involved in DNA repair, transcription and replication. Its role during preimplantation development is unknown.
Results
We have observed strong but transient synthesis of poly ADP-ribose polymers on decondensing chromosomes of fertilized and parthenogenetically activated mouse oocytes. Inhibition of this transient upregulation with a specific enzyme inhibitor, 3-aminobenzamide, has long-term effects on the postimplantation development of the embryos. In addition, inhibition of poly (ADP-ribosyl)ation at the 4–8 cell stage selectively blocks morula compaction.
Conclusion
These observations suggest that poly (ADP-ribosyl)ation is involved in the epigenetic chromatin remodeling in the zygote.
doi:10.1186/1471-2199-5-4
PMCID: PMC441376  PMID: 15200685
20.  The expression of HSP83 genes in Leishmania infantum is affected by temperature and by stage-differentiation and is regulated at the levels of mRNA stability and translation 
Background
Exposure of Leishmania promastigotes to the temperature of their mammalian hosts results in the induction of a typical heat shock response. It has been suggested that heat shock proteins play an important role in parasite survival and differentiation.
Results
Here we report the studies on the expression of the heat shock protein 83 (HSP83) genes of Leishmania infantum. Confirming previous observations for other Leishmania species, we found that the L. infantum HSP83 transcripts also show a temperature-dependent accumulation that is controlled by a post-transcriptional mechanism involving sequences located in the 3'-untranslated region (3'-UTR). However, contrary to that described for L. amazonensis, the accumulation of the HSP83 transcripts in L. infantum is dependent on active protein synthesis. The translation of HSP83 transcripts is enhanced during heat shock and, as first described in L. amazonensis, we show that the 3'-UTR of the L. infantum HSP83 gene is essential for this translational control. Measurement of the steady-state levels of HSP83 transcripts along the promastigote-to-amastigote differentiation evidenced a specific profile of HSP83 RNAs: after an initial accumulation of HSP83 transcripts observed short after (2 h) incubation in the differentiation conditions, the amount of HSP83 RNA decreased to a steady-state level lower than in undifferentiated promastigotes. We show that this transient accumulation is linked to the presence of the 3'-UTR and flanking regions. Again, an 8-fold increase in translation of the HSP83 transcripts is observed short after the initiation of the axenic differentiation, but it is not sustained after 9 h.
Conclusions
This transient expression of HSP83 genes could be relevant for the differentiation of Leishmania, and the underlying regulatory mechanism may be part of the developmental program of this parasite.
doi:10.1186/1471-2199-5-3
PMCID: PMC436058  PMID: 15176985
21.  Glucocorticoids synergize with IL-1β to induce TLR2 expression via MAP Kinase Phosphatase-1-dependent dual Inhibition of MAPK JNK and p38 in epithelial cells 
Background
Despite the importance of glucocorticoids in suppressing immune and inflammatory responses, their role in enhancing host immune and defense response against invading bacteria is poorly understood. Toll-like receptor 2 (TLR2) has recently gained importance as one of the major host defense receptors. The increased expression of TLR2 in response to bacteria-induced cytokines has been thought to be crucial for the accelerated immune response and resensitization of epithelial cells to invading pathogens.
Results
We show that IL-1β, a key proinflammatory cytokine, greatly up-regulates TLR2 expression in human epithelial cells via a positive IKKβ-IκBα-dependent NF-κB pathway and negative MEKK1-MKK4/7-JNK1/2 and MKK3/6-p38 α/β pathways. Glucocorticoids synergistically enhance IL-1β-induced TLR2 expression via specific up-regulation of the MAP kinase phosphatase-1 that, in turn, leads to dephosphorylation and inactivation of both MAPK JNK and p38, the negative regulators for TLR2 induction.
Conclusion
These results indicate that glucocorticoids not only suppress immune and inflammatory response, but also enhance the expression of the host defense receptor, TLR2. Thus, our studies may bring new insights into the novel role of glucocorticoids in orchestrating and optimizing host immune and defense responses during bacterial infections and enhance our understanding of the signaling mechanisms underlying the glucocorticoid-mediated attenuation of MAPK.
doi:10.1186/1471-2199-5-2
PMCID: PMC419700  PMID: 15125785
22.  Translation of the radioresistance kinase TLK1B is induced by γ-irradiation through activation of mTOR and phosphorylation of 4E-BP1 
Background
The mammalian protein kinase TLK1 is a homologue of Tousled, a gene involved in flower development in Arabidopsis thaliana. The function of TLK1 is not well known, although knockout of the gene in Drosophila, or expression of a dominant negative mutant in mouse cells causes loss of nuclear divisions and chromosome missegregation probably due to alterations in chromatin remodeling capacity. Overexpression of TLK1B, a spliced variant of the TLK1 mRNA, in a model mouse cell line increases their resistance to ionizing radiation, also likely through changes in chromatin remodeling. The TLK1B mRNA is translationally repressed by its 5'UTR and is regulated by the availability of eIF4E. We now report that radiation or doxorubicin result in an increase in the translation of TLK1B, and we have uncovered the likely mechanism for this effect.
Results
Radiation causes a shift in the polysomal distribution of TLK1B mRNA, from the untranslated region and small polysomes to the large polysomes, concomitant with an increase in the expression of TLK1B protein. This change is preceded by an increase in phosphorylation of the eIF4E inhibitory protein 4E-BP1, which releases eIF4E when it is phosphorylated. The phosphorylation of 4E-BP1 depends on mTOR, since rapamycin blocked the increase in phosphorylation induced by radiation, and prevented the increase in TLK1B protein expression. The activation of mTOR was likely due to the rapid activation of Akt following radiation. The activation of Akt could be inhibited with wortmannin, an inhibitor of PI3 kinase, hence placing PI3 kinase upstream of Akt as a very early event following radiation. Wortmannin also inhibited translation of TLK1B mRNA following activation by IR. This was shown both by western blot and by measuring the initiation capacity of the mRNA, as indicated by its distribution on polysomes.
Conclusions
The translational upregulation of TLK1B elicited by DNA double strand breaks represents an interesting mechanism of translational regulation of a protein involved in radioprotection and highlights a novel mechanism of the stress response following radiation.
doi:10.1186/1471-2199-5-1
PMCID: PMC406498  PMID: 15070431

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