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1.  SiteFind: A software tool for introducing a restriction site as a marker for successful site-directed mutagenesis 
Background
Site-directed mutagenesis is a widely-used technique for introducing mutations into a particular DNA sequence, often with the goal of creating a point mutation in the corresponding amino acid sequence but otherwise leaving the overall sequence undisturbed. However, this method provides no means for verifying its success other than sequencing the putative mutant construct: This can quickly become an expensive method for screening for successful mutations. An alternative to sequencing is to simultaneously introduce a restriction site near the point mutation in manner such that the restriction site has no effect on the translated amino acid sequence. Thus, the novel restriction site can be used as a marker for successful mutation which can be quickly and easily assessed. However, finding a restriction site that does not disturb the corresponding amino acid sequence is a time-consuming task even for experienced researchers. A fast and easy to use computer program is needed for this task.
Results
We wrote a computer program, called SiteFind, to help us design a restriction site within the mutation primers without changing the peptide sequence. Because of the redundancy of genetic code, a given peptide can be encoded by many different DNA sequences. Since the list of possible restriction sites for a given DNA sequence is not always obvious, SiteFind automates this task. The number of possible sequences a computer program must search through increases exponentially as the sequence length increases. SiteFind uses a novel "moving window" algorithm to reduce the number of possible sequences to be searched to a manageable level. The user enters a nucleotide sequence, specifies what amino acid residues should be changed in the mutation, and SiteFind generates a list of possible restriction sites and what nucleotides must be changed to introduce that site. As a demonstration of its use, we successfully generated a single point mutation and a double point mutation in the wild-type sequence for Krüppel-like factor 4, an epithelium-specific transcription factor.
Conclusion
SiteFind is an intuitive, web-based program that enables the user to introduce a novel restriction site into the mutated nucleotide sequence for use as a marker of successful mutation. It is freely available from
doi:10.1186/1471-2199-6-22
PMCID: PMC1314889  PMID: 16321147
2.  Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon 
Background
Salmonid fishes are among the most widely studied model fish species but reports on systematic evaluation of reference genes in qRT-PCR studies is lacking.
Results
The stability of six potential reference genes was examined in eight tissues of Atlantic salmon (Salmo salar), to determine the most suitable genes to be used in quantitative real-time RT-PCR analyses. The relative transcription levels of genes encoding 18S rRNA, S20 ribosomal protein, β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), and two paralog genes encoding elongation factor 1A (EF1AA and EF1AB) were quantified in gills, liver, head kidney, spleen, thymus, brain, muscle, and posterior intestine in six untreated adult fish, in addition to a group of individuals that went through smoltification. Based on calculations performed with the geNorm VBA applet, which determines the most stable genes from a set of tested genes in a given cDNA sample, the ranking of the examined genes in adult Atlantic salmon was EF1AB>EF1AA>β-actin>18S rRNA>S20>GAPDH. When the same calculations were done on a total of 24 individuals from four stages in the smoltification process (presmolt, smolt, smoltified seawater and desmoltified freshwater), the gene ranking was EF1AB>EF1AA>S20>β-actin>18S rRNA>GAPDH.
Conclusion
Overall, this work suggests that the EF1AA and EF1AB genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon.
doi:10.1186/1471-2199-6-21
PMCID: PMC1314898  PMID: 16293192
3.  Pseudouridine modification in Caenorhabditis elegans spliceosomal snRNAs: unique modifications are found in regions involved in snRNA-snRNA interactions 
Background
Pseudouridine (Ψ) is an abundant modified nucleoside in RNA and a number of studies have shown that the presence of Ψ affects RNA structure and function. The positions of Ψ in spliceosomal small nuclear RNAs (snRNAs) have been determined for a number of species but not for the snRNAs from Caenorhabditis elegans (C. elegans), a popular experimental model system of development.
Results
As a prelude to determining the function of or requirement for this modification in snRNAs, we have mapped the positions of Ψ in U1, U2, U4, U5, and U6 snRNAs from worms using a specific primer extension method. As with other species, C. elegans U2 snRNA has the greatest number of Ψ residues, with nine, located in the 5' half of the U2 snRNA. U5 snRNA has three Ψs, in or near the loop of the large stem-loop that dominates the structure of this RNA. U6 and U1 snRNAs each have one Ψ, and two Ψ residues were found in U4 snRNA.
Conclusion
The total number of Ψs found in the snRNAs of C. elegans is significantly higher than the minimal amount found in yeasts but it is lower than that seen in sequenced vertebrate snRNAs. When the actual sites of modification on C. elegans snRNAs are compared with other sequenced snRNAs most of the positions correspond to modifications found in other species. However, two of the positions modified on C. elegans snRNAs are unique, one at position 28 on U2 snRNA and one at position 62 on U4 snRNA. Both of these modifications are in regions of these snRNAs that interact with U6 snRNA either in the spliceosome or in the U4/U6 small nuclear ribonucleoprotein particle (snRNP) and the presence of Ψ may be involved in strengthening the intermolecular association of the snRNAs.
doi:10.1186/1471-2199-6-20
PMCID: PMC1276797  PMID: 16236171
4.  The radioresistance kinase TLK1B protects the cells by promoting repair of double strand breaks 
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 missegregation of chromosomes 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 it's resistance to ionizing radiation (IR) or the radiomimetic drug doxorubicin, also likely due to changes in chromatin remodeling. TLK1B is translationally regulated by the availability of the translation factor eIF4E, and its synthesis is activated by IR. The reason for this mechanism of regulation is likely to provide a rapid means of promoting repair of DSBs. TLK1B specifically phosphorylates histone H3 and Asf1, likely resulting in changes in chromatin structure, particularly at double strand breaks (DSB) sites.
Results
In this work, we provide several lines of evidence that TLK1B protects the cells from IR by facilitating the repair of DSBs. First, the pattern of phosphorylation and dephosphorylation of H2AX and H3 indicated that cells overexpressing TLK1B return to pre-IR steady state much more rapidly than controls. Second, the repair of episomes damaged with DSBs was much more rapid in cells overexpressing TLK1B. This was also true for repair of genomic damage. Lastly, we demonstrate with an in vitro repair system that the addition of recombinant TLK1B promotes repair of a linearized plasmid incubated with nuclear extract. In addition, TLK1B in this in vitro system promotes the assembly of chromatin as shown by the formation of more highly supercoiled topomers of the plasmid.
Conclusion
In this work, we provide evidence that TLK1B promotes the repair of DSBs, likely as a consequence of a change in chromatin remodeling capacity that must precede the assembly of repair complexes at the sites of damage.
doi:10.1186/1471-2199-6-19
PMCID: PMC1242231  PMID: 16156902
5.  Analysis of the function of E. coli 23S rRNA helix-loop 69 by mutagenesis 
Background
The ribosome is a two-subunit enzyme known to exhibit structural dynamism during protein synthesis. The intersubunit bridges have been proposed to play important roles in decoding, translocation, and the peptidyl transferase reaction; yet the physical nature of their contributions is ill understood. An intriguing intersubunit bridge, B2a, which contains 23S rRNA helix 69 as a major component, has been implicated by proximity in a number of catalytically important regions. In addition to contacting the small ribosomal subunit, helix 69 contacts both the A and P site tRNAs and several translation factors.
Results
We scanned the loop of helix 69 by mutagenesis and analyzed the mutant ribosomes using a plasmid-borne IPTG-inducible expression system. We assayed the effects of 23S rRNA mutations on cell growth, contribution of mutant ribosomes to cellular polysome pools and the ability of mutant ribosomes to function in cell-free translation. Mutations A1912G, and A1919G have very strong growth phenotypes, are inactive during in vitro protein synthesis, and under-represented in the polysomes. Mutation Ψ1917C has a very strong growth phenotype and leads to a general depletion of the cellular polysome pool. Mutation A1916G, having a modest growth phenotype, is apparently defective in the assembly of the 70S ribosome.
Conclusion
Mutations A1912G, A1919G, and Ψ1917C of 23S rRNA strongly inhibit translation. Mutation A1916G causes a defect in the 50S subunit or 70S formation. Mutations Ψ1911C, A1913G, C1914A, Ψ1915C, and A1918G lack clear phenotypes.
doi:10.1186/1471-2199-6-18
PMCID: PMC1190176  PMID: 16053518
6.  An in vivo analysis of the localisation and interactions of human p66 DNA polymerase δ subunit 
Background
DNA polymerase δ is essential for eukaryotic DNA replication and also plays a role in DNA repair. The processivity of this polymerase complex is dependent upon its interaction with the sliding clamp PCNA and the polymerase-PCNA interaction is largely mediated through the p66 polymerase subunit. We have analysed the interactions of the human p66 DNA polymerase δ subunit with PCNA and with components of the DNA polymerase δ complex in vivo.
Results
Using the two-hybrid system, we have mapped the interaction domains for binding to the p50 polymerase δ subunit and with PCNA to the N-terminus and the C-terminus of p66, respectively. Co-immunoprecipitation experiments confirm that these interaction domains are functional in vivo. Expression of EGFP-p66 shows that it is a nuclear protein which co-localises with PCNA throughout the cell cycle. p66 is localised to sites of DNA replication during S phase and to repair foci following DNA damage. We have identified a functional nuclear localisation sequence and shown that localisation to replication foci is not dependent upon active nuclear import. Sub-domains of p66 act as dominant negative suppressors of colony formation, suggesting that p66 forms an essential structural link between the p50 subunit and PCNA. Analysis of the C-terminal PCNA binding motif shows that deletion of the QVSITGFF core motif results in a reduced affinity for PCNA, while deletion of a further 20 amino acids completely abolishes the interaction. A reduced affinity for PCNA correlates with reduced targeting to replication foci. We have confirmed the p66-PCNA interaction in vivo using fluorescence resonance energy transfer (FRET) techniques.
Conclusion
We have defined the regions of p66 required for its interaction with PCNA and the p50 polymerase subunit. We demonstrate a functional link between PCNA interaction and localisation to replication foci and show that there is a direct interaction between p66 and PCNA in living cells during DNA replication. The dominant negative effect upon growth resulting from expression of p66 sub-domains confirms that the p66-PCNA interaction is essential in vivo.
doi:10.1186/1471-2199-6-17
PMCID: PMC1187890  PMID: 16000169
7.  Cloning and characterization of cDNAs encoding putative CTCFs in the mosquitoes, Aedes aegypti and Anopheles gambiae 
Background
One of the many ascribed functions of CCCTC-binding factor (CTCF) in vertebrates is insulation of genes via enhancer-blocking. Insulation allows genes to be shielded from "cross-talk" with neighboring regulatory elements. As such, endogenous insulator sequences would be valuable elements to enable stable transgene expression. Recently, CTCF joined Su(Hw), Zw5, BEAF32 and GAGA factor as a protein associated with insulator activity in the fruitfly, Drosophila melanogaster. To date, no known insulators have been described in mosquitoes.
Results
We have identified and characterized putative CTCF homologs in the medically-important mosquitoes, Aedes aegypti and Anopheles gambiae. These genes encode polypeptides with eleven C2H2 zinc fingers that show significant similarity to those of vertebrate CTCFs, despite at least 500 million years of divergence. The mosquito CTCFs are constitutively expressed and are upregulated in early embryos and in the ovaries of blood-fed females. We have uncovered significant bioinformatics evidence that CTCF is widespread, at least among Drosophila species. Finally, we show that the An. gambiae CTCF binds two known insulator sequences.
Conclusion
Mosquito CTCFs are likely orthologous to the widely-characterized vertebrate CTCFs and potentially also serve an insulating function. As such, CTCF may provide a powerful tool for improving transgene expression in these mosquitoes through the identification of endogenous binding sites.
doi:10.1186/1471-2199-6-16
PMCID: PMC1174870  PMID: 15985163
8.  Zinc-finger domains of the transcriptional repressor KLF15 bind multiple sites in rhodopsin and IRBP promoters including the CRS-1 and G-rich repressor elements 
Background
In the retina, many of the genes that encode components of the visual transduction cascade and retinoid recycling are exclusively expressed in photoreceptor cells and show highly stereotyped temporal and spatial expression patterns. Multiple transcriptional activators of photoreceptor-specific genes have been identified, but little is known about negative regulation of gene expression in the retina. We recently identified KLF15, a member of the Sp/Krüppel-like Factor family of zinc-finger containing transcription factors, as an in vitro repressor of the promoters of the photoreceptor-specific genes rhodopsin and IRBP/Rbp3. To gain further insight into the mechanism of KLF15-mediated regulation of gene expression, we have characterized the binding characteristics and specificity of KLF15's DNA binding domains and defined the KLF15 binding sites in the rhodopsin and IRBP promoters.
Results
In EMSA and DNAseI footprinting assays, a KLF15-GST fusion protein containing the C-terminal zinc-finger domains (123 amino acids) showed zinc-dependent and sequence-specific binding to a 9 bp consensus sequence containing a core CG/TCCCC. Both the bovine rhodopsin and IRBP promoters contained multiple KLF15 binding sites that included the previously identified CRS-1 and G-rich repressor elements. KLF15 binding sites were highly conserved between the bovine, human, chimp and dog rhodopsin promoters, but less conserved in rodents. KLF15 reduced luciferase expression by bRho130-luc (containing 4 KLF15 sites) and repressed promoter activation by CRX (cone rod homeobox) and/or NRL (neural retina leucine zipper), although the magnitude of the reduction was smaller than previously reported for a longer bRho225-luc (containing 6 KFL15 sites).
Conclusion
KLF15 binds to multiple 9 bp consensus sites in the Rhodospin and IRBP promoters including the CRS-1 and G-rich repressor elements. Based on the known expression pattern of KLF15 in non-photoreceptor cells, we hypothesize an in vivo role for KLF15 in repressing photoreceptor-specific gene expression in the inner retina.
doi:10.1186/1471-2199-6-15
PMCID: PMC1182371  PMID: 15963234
9.  Comparative 3-D Modeling of tmRNA 
Background
Trans-translation releases stalled ribosomes from truncated mRNAs and tags defective proteins for proteolytic degradation using transfer-messenger RNA (tmRNA). This small stable RNA represents a hybrid of tRNA- and mRNA-like domains connected by a variable number of pseudoknots. Comparative sequence analysis of tmRNAs found in bacteria, plastids, and mitochondria provides considerable insights into their secondary structures. Progress toward understanding the molecular mechanism of template switching, which constitutes an essential step in trans-translation, is hampered by our limited knowledge about the three-dimensional folding of tmRNA.
Results
To facilitate experimental testing of the molecular intricacies of trans-translation, which often require appropriately modified tmRNA derivatives, we developed a procedure for building three-dimensional models of tmRNA. Using comparative sequence analysis, phylogenetically-supported 2-D structures were obtained to serve as input for the program ERNA-3D. Motifs containing loops and turns were extracted from the known structures of other RNAs and used to improve the tmRNA models. Biologically feasible 3-D models for the entire tmRNA molecule could be obtained. The models were characterized by a functionally significant close proximity between the tRNA-like domain and the resume codon. Potential conformational changes which might lead to a more open structure of tmRNA upon binding to the ribosome are discussed. The method, described in detail for the tmRNAs of Escherichia coli, Bacillus anthracis, and Caulobacter crescentus, is applicable to every tmRNA.
Conclusion
Improved molecular models of biological significance were obtained. These models will guide in the design of experiments and provide a better understanding of trans-translation. The comparative procedure described here for tmRNA is easily adopted for the modeling the members of other RNA families.
doi:10.1186/1471-2199-6-14
PMCID: PMC1168896  PMID: 15958166
10.  Nuclear distribution and chromatin association of DNA polymerase α-primase is affected by TEV protease cleavage of Cdc23 (Mcm10) in fission yeast 
Background
Cdc23/Mcm10 is required for the initiation and elongation steps of DNA replication but its biochemical function is unclear. Here, we probe its function using a novel approach in fission yeast, involving Cdc23 cleavage by the TEV protease.
Results
Insertion of a TEV protease cleavage site into Cdc23 allows in vivo removal of the C-terminal 170 aa of the protein by TEV protease induction, resulting in an S phase arrest. This C-terminal fragment of Cdc23 is not retained in the nucleus after cleavage, showing that it lacks a nuclear localization signal and ability to bind to chromatin. Using an in situ chromatin binding procedure we have determined how the S phase chromatin association of DNA polymerase α-primase and the GINS (Sld5-Psf1-Psf2-Psf3) complex is affected by Cdc23 inactivation. The chromatin binding and sub-nuclear distribution of DNA primase catalytic subunit (Spp1) is affected by Cdc23 cleavage and also by inactivation of Cdc23 using a degron allele, implying that DNA polymerase α-primase function is dependent on Cdc23. In contrast to the effect on Spp1, the chromatin association of the Psf2 subunit of the GINS complex is not affected by Cdc23 inactivation.
Conclusion
An important function of Cdc23 in the elongation step of DNA replication may be to assist in the docking of DNA polymerase α-primase to chromatin.
doi:10.1186/1471-2199-6-13
PMCID: PMC1182370  PMID: 15941470
11.  Characterization of TRZ1, a yeast homolog of the human candidate prostate cancer susceptibility gene ELAC2 encoding tRNase Z 
Background
In humans, mutation of ELAC2 is associated with an increased risk of prostate cancer. ELAC2 has been shown to have tRNase Z activity and is associated with the γ-tubulin complex.
Results
In this work, we show that the yeast homolog of ELAC2, encoded by TRZ1 (tRNase Z 1), is involved genetically in RNA processing. The temperature sensitivity of a trz1 mutant can be rescued by multiple copies of REX2, which encodes a protein with RNA 3' processing activity, suggesting a role of Trz1p in RNA processing in vivo. Trz1p has two putative nucleotide triphosphate-binding motifs (P-loop) and a conserved histidine motif. The histidine motif and the putative nucleotide binding motif at the C-domain are important for Trz1p function because mutant proteins bearing changes to the critical residues in these motifs are unable to rescue deletion of TRZ1. The growth defect exhibited by trz1 yeast is not complemented by the heterologous ELAC2, suggesting that Trz1p may have additional functions in yeast.
Conclusion
Our results provide genetic evidence that prostate cancer susceptibility gene ELAC2 may be involved in RNA processing, especially rRNA processing and mitochondrial function.
doi:10.1186/1471-2199-6-12
PMCID: PMC1156898  PMID: 15892892
12.  Investigation of factors responsible for cell line cytoplasmic expression differences 
Background
Previous work has described a novel cytoplasmic expression system that results in a 20-fold increase in the levels of gene expression over a standard CMV-based nuclear expression system, as compared with a 2–3 fold increase seen with previous similar systems. While this increase was seen with BHK and Neuro-2a cells, further studies revealed that some cell lines, such as COS-7, demonstrated relatively poor levels of cytoplasmic expression. The objective of this study was to determine what factors were responsible for the different expression levels between BHK (a high expressing cell line) and COS-7 (a low expressing cell line).
Results
The main findings of this work are that the individual elements of the cytoplasmic expression system (such as the T7 RNAP gene and Internal Ribosome Entry Sequence) are functioning similarly in both cell types. Both cell types were found to have the same amount of cytosolic nuclease activity, and that the cells appeared to have differences in the intra-cellular processing of DNA -cationic lipid complexes.
Conclusion
After exploring many factors, it was found that differences in the intra-cellular processing of the DNA-cationic lipid complex was the most probable factor responsible for the difference in cytoplasmic gene expression.
doi:10.1186/1471-2199-6-11
PMCID: PMC1134659  PMID: 15888202
13.  Relative transcript quantification by Quantitative PCR: Roughly right or precisely wrong? 
Background
When estimating relative transcript abundances by quantitative real-time PCR (Q-PCR) we found that the results can vary dramatically depending on the method chosen for data analysis.
Results
Analyses of Q-PCR results from a salmon louse starvation experiment show that, even with apparently good raw data, different analytical approaches [1,2] may lead to opposing biological conclusions.
Conclusion
The results emphasise the importance of being cautious when analysing Q-PCR data and indicate that uncritical routine application of an analytical method will eventually result in incorrect conclusions. We do not know the extent of, or have a universal solution to this problem. However, we strongly recommend caution when analysing Q-PCR results e.g. by using two or more analytical approaches to validate conclusions. In our view a common effort should be made to standardise methods for analysis and validation of Q-PCR results.
doi:10.1186/1471-2199-6-10
PMCID: PMC1090581  PMID: 15854230
14.  Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR 
Background
Preparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target.
Results
We investigated the efficiency of two DNase I based protocols for eliminating DNA contaminations from RNA samples obtained from yeast cells. Both procedures are very efficient in eliminating DNA contamination from RNA samples and entail three main steps, which involve treating of RNA samples with DNase I, inhibition of the enzyme by EDTA and its subsequent inactivation at 65°C. The DNase I treated samples were further purified with phenol: chloroform followed by precipitation with ice-cold ethanol (protocol I) or, alternatively, they were directly used in RT-PCR reactions (protocol II). Transcripts from ACT1, PDA1, CNA1, CNA2, TPS1 and TPS2 analyzed after each treatment showed that all mRNAs tested can be amplified if total RNA was extracted and purified after DNase I treatment, however, only TPS1, TPS2 and ACT1 mRNAs were amplified without extraction/purification step.
Conclusion
Although more laborious and requiring a higher initial amount of material, the inclusion of an extraction and purification step allows to prepare RNA samples that are free from DNA and from low molecular contaminants and can be applied to amplify any Saccharomyces cerevisiae mRNA by RT-PCR.
doi:10.1186/1471-2199-6-9
PMCID: PMC1097730  PMID: 15833107
15.  Use of adenoviral E1A protein to analyze K18 promoter deregulation in colon carcinoma cells discloses a role for CtBP1 and BRCA1 
Background
The promoter of the keratin 18 (K18) gene is 5- to 10-fold more active in tumorigenic (T-type) cell clones derived from the SW613-S human colon carcinoma cell line than in non-tumorigenic (NT-type) clones. We have reported previously that the mechanism responsible for this differential activity is acting on the minimal K18 promoter (TATA box and initiation site). This mechanism does not require the binding of a factor to a specific site on the DNA but involves the acetylation of a non-histone substrate. To get further insight into this mechanism, we investigated the effect of the adenovirus E1A protein on the activity of the K18 promoter, both in T and NT cells.
Results
Wild type adenovirus E1A protein and C-terminal deletion mutants inhibit the K18 promoter, specifically in T-type cells. The domain responsible for this inhibitory effect is located in the 12–25 region of the viral protein. E1A mutants that have lost this region but retain the PLDLS motif (the C-terminal binding site for CtBP1) stimulate the K18 promoter, specifically in NT cells. The inhibitory or stimulatory effects of the different E1A mutants are not dependent on a particular sequence of the promoter. An E1A N-terminal deletion mutant carrying point mutations in the PLDLS motif cannot stimulate the K18 promoter. CtBP1 interacts with CtIP, which is a known partner of BRCA1, itself a component of the RNA polymerase II holoenzyme. The stimulatory effect of two BRCA1 mutants, specifically in NT cells, implicates a tripartite BRCA1-CtIP-CtBP1 complex in the regulation of the K18 promoter.
Conclusion
Since we have shown previously that the K18 promoter is stimulated by deacetylase inhibitors, specifically in NT cells, we conclude that the activity of the promoter is repressed in NT cells by a mechanism involving the recruitment, by a BRCA1/CtIP complex, of CtBP1 and associated deacetylases to the preinitiation complex. We propose a model depicting the mechanism responsible for the differential activity of the K18 promoter between T and NT cells of the SW613-S cell line.
doi:10.1186/1471-2199-6-8
PMCID: PMC1087485  PMID: 15831101
16.  Cloning and transcriptional analysis of the mouse receptor activity modifying protein-1 gene promoter 
Background
Receptor activity modifying protein-1 (RAMP-1) is a single transmembrane-domain protein required for the functional expression of calcitonin gene-related peptide (CGRP) receptors. To date, little is known about the molecular mechanism(s) that activate/inhibit RAMP-1 gene expression. Such mechanism(s) are likely to play a major role in modulating the responsiveness of tissues to CGRP.
Results
To initiate studies on the transcriptional regulation of the mouse RAMP-1 gene, RAMP-1 transcriptional initiation sites were mapped in a variety of tissues. Analysis of RAMP-1 expression in C2C12 myoblasts demonstrated that RAMP-1 mRNA is expressed at greatest levels in confluent myoblasts verses non-confluent and fused myoblasts. Transfection of confluent C2C12 myoblasts and NIH 3T3 cells with RAMP-1 promoter/luciferase deletion constructs revealed that 4.7 kb of RAMP-1 5' flanking region demonstrated optimal promoter activity while 343 bp of 5' flanking region was defined as a minimal RAMP-1 promoter. In non-RAMP-1 expressing HEK293 cells, constructs containing 4.7 kb to 782 bp of RAMP-1 5' flanking region were transcriptionally inactive. However, deletion of sequences -782 to -343 activated RAMP-1 promoter activity.
Conclusion
These findings suggest that tissue specificity of RAMP-1 gene expression is mediated by a negative acting transcription factor that represses RAMP-1 gene expression in non-RAMP-1 expressing tissues. This transcription factor is therefore likely to play an important role in modulating the responsiveness of tissues to CGRP.
doi:10.1186/1471-2199-6-7
PMCID: PMC1079840  PMID: 15790393
17.  Recognition and binding of mismatch repair proteins at an oncogenic hot spot 
Background
The current investigation was undertaken to determine key steps differentiating G:T and G:A repair at the H-ras oncogenic hot spot within the nuclear environment because of the large difference in repair efficiency of these two mismatches.
Results
Electrophoretic mobility shift (gel shift) experiments demonstrate that DNA containing mismatched bases are recognized and bound equally efficiently by hMutSα in both MMR proficient and MMR deficient (hMLH1-/-) nuclear extracts. Competition experiments demonstrate that while hMutSα predictably binds the G:T mismatch to a much greater extent than G:A, hMutSα demonstrates a surprisingly equal ratio of competitive inhibition for both G:T and G:A mismatch binding reactions at the H-ras hot spot of mutation. Further, mismatch repair assays reveal almost 2-fold higher efficiency of overall G:A repair (5'-nick directed correct MMR to G:C and incorrect repair to T:A), as compared to G:T overall repair. Conversely, correct MMR of G:T → G:C is significantly higher (96%) than that of G:A → G:C (60%).
Conclusion
Combined, these results suggest that initiation of correct MMR requires the contribution of two separate steps; initial recognition by hMutSα followed by subsequent binding. The 'avidity' of the binding step determines the extent of MMR pathway activation, or the activation of a different cellular pathway. Thus, initial recognition by hMutSα in combination with subsequent decreased binding to the G:A mismatch (as compared to G:T) may contribute to the observed increased frequency of incorrect repair of G:A, resulting in the predominant GGC → GTC (Gly → Val) ras-activating mutation found in a high percentage of human tumors.
doi:10.1186/1471-2199-6-6
PMCID: PMC555755  PMID: 15766387
18.  Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin 
Background
Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs), a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor.
Results
In this study, we show stable transfection and expression of the T. vaginalis ap65 gene in T. foetus from an episomal pBS-ap65-neo plasmid. Expression of the gene and protein was confirmed by RT-PCR and immunoblots, respectively. AP65 in transformed T. foetus bound to host cells. Specific mAbs revealed episomally-expressed AP65 targeted to the parasite surface and hydrogenosome organelles. Importantly, surface-expression of AP65 in T. foetus paralleled increased levels of adherence of transfected bovine trichomonads to human VECs.
Conclusion
The T. vaginalis AP65 adhesin was stably expressed in T. foetus, and the data obtained using this heterologous system strongly supports the role of AP65 as a prominent adhesin for T. vaginalis. In addition, the heterologous expression in T. foetus of a T. vaginalis gene offers an important, new approach for confirming and characterizing virulence factors.
doi:10.1186/1471-2199-6-5
PMCID: PMC1079839  PMID: 15748280
19.  Selection of reference genes for gene expression studies in human neutrophils by real-time PCR 
Background
Reference genes, which are often referred to housekeeping genes, are frequently used to normalize mRNA levels between different samples. However the expression level of these genes may vary among tissues or cells, and may change under certain circumstances. Thus the selection of reference gene(s) is critical for gene expression studies. For this purpose, 10 commonly used housekeeping genes were investigated in isolated human neutrophils.
Results
Initial screening of the expression pattern demonstrated that 3 of the 10 genes were expressed at very low levels in neutrophils and were excluded from further analysis. The range of expression stability of the other 7 genes was (from most stable to least stable): GNB2L1 (Guanine nucleotide binding protein, beta polypeptide 2-like 1), HPRT1 (Hypoxanthine phosphoribosyl transferase 1), RPL32 (ribosomal protein L32), ACTB (beta-actin), B2M (beta-2-microglobulin), GAPD (glyceraldehyde-3-phosphate dehydrogenase) and TBP (TATA-binding protein). Relative expression levels of the genes (from high to low) were: B2M, ACTB, GAPD, RPL32, GNB2L1, TBP, and HPRT1.
Conclusion
Our data suggest that GNB2L1, HPRT1, RPL32, ACTB, and B2M may be suitable reference genes in gene expression studies of neutrophils.
doi:10.1186/1471-2199-6-4
PMCID: PMC551605  PMID: 15720708
20.  Role of Leishmania (Leishmania) chagasi amastigote cysteine protease in intracellular parasite survival: studies by gene disruption and antisense mRNA inhibition 
Background
The parasitic protozoa belonging to Leishmania (L.) donovani complex possess abundant, developmentally regulated cathepsin L-like cysteine proteases. Previously, we have reported the isolation of cysteine protease gene, Ldccys2 from Leishmania (L.) chagasi. Here, we have further characterized this cysteine protease gene and demonstrated its role during infection and survival of Leishmania (L.) chagasi within the U937 macrophage cells.
Results
The amastigote specific Ldccys2 genes of L. (L.) chagasi and L. (L.) donovani have identical gene organization, as determined by southern blots. In vivo expression analyses by Northern blots showed that Ldccys2 is amastigote specific. Western blot using anti-Ldccys2 antibody confirmed the amastigote specific protein expression. Recombinant expression of Ldccys2, a 30 kDA protein, was functionally active in a gelatin assay. Results from Ldccys2 heterozygous knockout mutants showed its role during macrophage infection and in intra-macrophage survival of the parasites. Since attempts to generate null mutants failed, we used antisense RNA inhibition to regulate Ldcccys2 gene expression. Not surprisingly, the results from antisense studies further confirmed the results from heterozygous knockout mutants, reiterating the importance of amastigote specific cysteine proteases in Leishmania infection and pathogenesis.
Conclusions
The study shows that Ldccys2 is a developmentally regulated gene and that Ldccys2 is expressed only in infectious amastigote stages of the parasite. The collective results from both the heterozygous knockout mutants and antisense mRNA inhibition studies shows that Ldccys2 helps in infection and survival of L. (L.) chagasi amastigotes within the macrophage cells. Finally, antisense RNA technique can be used as an alternate approach to gene knockout, for silencing gene expression in L. (L.) chagasi, especially in cases such as this, where a null mutant cannot be achieved by homologous recombination.
doi:10.1186/1471-2199-6-3
PMCID: PMC549197  PMID: 15691375
21.  cAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene 
Background
The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE).
Results
The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP) family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA) in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2), known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene.
Conclusions
Using a constitutively active CREB2/CREB fusion protein and a mutant of the PKA catalytic subunit that is targeted to the nucleus, we have shown that the glucose-6-phosphatase gene has two distinct genetic elements that function as bona fide CRE. This study further shows that the expression vectors encoding C2/CREB and catalytic subunit of PKA are valuable tools for the study of CREB-mediated gene transcription and the biological functions of CREB.
doi:10.1186/1471-2199-6-2
PMCID: PMC548273  PMID: 15659240
22.  A minor alternative transcript of the fumarylacetoacetate hydrolase gene produces a protein despite being likely subjected to nonsense-mediated mRNA decay 
Background
Coupling of alternative splicing with nonsense-mediated mRNA decay (NMD) may regulate gene expression. We report here the identification of a nonsense alternative transcript of the fumarylacetoacetate hydrolase (fah) gene, which produces a protein despite the fact that it is subject to NMD.
Results
During the characterization of the effects of the W262X nonsense mutation on FAH mRNA metabolism, two alternative transcripts (del100 and del231) of the fah gene were identified. Del100 lacks exon 8 and as a consequence, the reading frame is shifted and a premature termination codon appears at the 3'end of exon 10. Exons 8 and 9 are skipped in del231, without any disruption of the reading frame. Specific amplification of these transcripts demonstrate that they are produced through minor alternative splicing pathways, and that they are not caused by the W262X mutation per se. As shown with an antiserum raised against the C-terminal part of the putative DEL100 protein, the del100 transcript produces a protein, expressed at different levels in various human tissues. Interestingly, the del100 transcript seems to be subjected to nonsense-mediated mRNA decay, as its level was stabilized following a cycloheximide treatment.
Conclusions
The del100 and del231 transcripts arise due to minor alternative splicing pathways and del100 is likely subjected to nonsense-mediated mRNA decay. However the remaining amount of transcript seems sufficient to produce a protein in different human tissues. This suggests that NMD has a broader role than simply eliminating aberrant transcripts and when coupled to alternative splicing, may act to modulate gene expression, by allowing the production of low amounts of protein.
doi:10.1186/1471-2199-6-1
PMCID: PMC546004  PMID: 15638932

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