Modification of leaf traits in sugar beet requires a strong leaf specific promoter. With such a promoter, expression in taproots can be avoided which may otherwise take away available energy resources for sugar accumulation.cab11 and Bvcab12) were isolated, linked to reporter genes, and transformed into sugar beet using promoter reporter gene fusions. Transient and transgenic analysis indicate that both promoters direct leaf specific gene expression. A bioinformatic analysis revealed that the Bvcab11 promoter is void of G-box like regulatory elements with a palindromic ACGT core sequence. The data indicate that the presence of a G-box element is not a prerequisite for leaf specific and light induced gene expression in sugar beet.Suppression Subtractive Hybridization (SSH) was utilized to generate an enriched and equalized cDNA library for leaf expressed genes from sugar beet. Fourteen cDNA fragments corresponding to thirteen different genes were isolated. Northern blot analysis indicates the desired tissue specificity of these genes. The promoters for two chlorophyll a/b binding protein genes (BvThis work shows that SSH can be successfully employed for the identification and subsequent isolation of tissue specific sugar beet promoters. These promoters are shown to drive strong leaf specific gene expression in transgenic sugar beet. The application of these promoters for expressing resistance improving genes against foliar diseases is discussed. Beta vulgaris L.) is a biennial plant, a member of the Chenopodiaceae family [Sugar beet  which was recently applied to sugar beet . With thA straight forward approach for the isolation of differentially expressed genes was achieved by the "Suppression Subtractive Hybridization" method (SSH) . SSH is cab genes that are both expressed. To investigate the use of the cab promoters for gene expression, the 5' regulatory sequences were isolated and linked to reporter genes. Transient reporter gene assays indicate that both promoters are expressed in sugar beet leaves. In transgenic sugar beet both promoters are expressed in green tissue. Sequence analysis revealed that the cab11 promoter, in contrast to the cab12 promoter, is void of G-box like regulatory sites with a palindromic ACGT core sequence. A leaf specific promoter in transgenic sugar beets can be employed for biotechnological applications.Here we have employed SSH for the isolation of leaf expressed sugar beet genes. Among the genes isolated was a cDNA fragment for a light-harvesting chlorophyll a/b binding protein (CAB). It is shown that sugar beet genotypes harbor either one or two To isolate cDNA fragments corresponding to leaf expressed genes, poly(A)+RNAs from leaf and taproot were isolated and subjected to cDNA synthesis. Suppression subtractive hybridization was performed as described in Methods. A total of 23 cDNA clones specific for the subtraction for leaf expressed genes were isolated and sequenced (data not shown). Fourteen out of 23 cDNAs were found to be different. Table To further confirm that the enriched cDNA fragments correspond to tissue specific genes, Northern blot hybridizations to total RNA isolated from leaves (L), taproots (R), stems (S), and inflorescences (I) were performed with 7 cDNA fragments. Figure cab gene was isolated and analysed with reporter gene fusions. Compared to the other genes, the expression profile of the gene for SSH fragment L2 showed the strongest tissue specificity during the different time points analysed (data not shown).The expression profile of the gene for SSH fragment L2 encoding a chlorophyll a/b binding protein (CAB) was analysed in more detail. RNA from different tissues and from different developmental stages were used in RNA blot experiments. RNA was isolated from different organs of sugar beets from a field 4, 6, 10, 12, 16 and 19 weeks after sowing. This interval covers the lifetime of a sugar beet in central Europe. Figure cab gene, a complete cDNA clone was isolated (GenBank Acc. Nr. AJ579711, see Methods). A homology search with the encoded 252 amino acid long protein reveals a 87% identity to the cab11 and 85% identity to the cab12 gene from tomato encoding chlorophyll a/b binding protein [For the isolation of a sugar beet promoter corresponding to the EcoRI, HindIII) and four (PstI) hybridizing fragments are detected, while only one (EcoRI) or two (PstI, HindIII) fragments are detected in genotype 4B5421. From this result it is concluded that genotype 4B5421 harbors one and genotype 1K0088 harbors two copies of the cab gene.Prior to the isolation of genomic clones, a gene copy number analysis was performed. DNA from sugar beet genotypes 1K0088 and 4B5421 was digested with different restriction enzymes and hybridized to the L2 fragment (Table Genomic clones for the two different genes were isolated (Methods). Sequence comparison between the cDNA and both genomic clones indicate a very high degree of sequence identity within the coding region. The CAB11 and CAB12 amino acid sequence differ only in one position (data not shown).cab11 promoter and the 3049 bp fragment cab12 promoter. Both fragments harbor 51 base pairs coding region of the CAB protein and 113 (cab11) and 70 (cab12) base pairs upstream untranslated sequence. Upstream of the untranslated region only about 300 bp are homologous between the two promoters while the rest of the sequence is highly divergent (data not shown). Because the cDNA clone isolated before originates from the genotype 4B5421 and corresponds to the cab11 gene, it was investigated if the second gene is also transcribed. Towards these ends 5' RACE amplifications were performed with RNA from genotype 1K0088 and sequenced. This analysis revealed that the cab12 gene is also transcribed (data not shown).From both genes the promoter regions were subcloned into plasmid vectors and sequenced (Methods). Sequence of 1148 and 3049 base pairs, respectively, containing most of the upstream region were deposited to GenBank (Acc. Nr. AX449166 and AX449167). The 1148 bp promoter fragment is designated Photinus pyralis in the vector pGEM-luc were constructed (see Methods). Table cab11 gene and the 342 bp fragment from the cab12 gene harbor all cis-regulatory sequences required for gene expression in sugar beet leaves at least under the conditions of the transient bioassay.To test whether the isolated promoters confer reporter gene expression in sugar beet leaves, a transient assay was performed. Towards these ends translational fusions of promoter fragments with the luciferase gene from cab11 and cab12 promoters from sugar beet drive reporter gene expression in transgenic plants, promoter reporter gene fusions were introduced via Agrobacterium mediated transformation in sugar beet (see Methods). The length of the cab11 promoter fragment is 1097 bp and the length of the cab12 promoter fragment 2998 bp. Leaves of transgenic lines transferred to the greenhouse were analysed for reporter gene activity. The cab11 promoter of 12 independent transformants showed a specific β-glucuronidase (GUS) activity from 9 to 40599 pmol Mu × min-1 × mg-1, respectively . Using the program Patch™ six experimentally verified binding sites for bZIP factors were detected among these twelve sites that harbor the ACGT core in the cab12 promoter  in tomato [cab11 from tomato and Bvcab11 from sugar beet are identical (data not shown).Subtractive hybridization was used to isolate leaf expressed genes from sugar beet. The goal was the identification of a promoter that drives leaf specific gene expression in transgenic sugar beet plants. Among seven analysed genes a cDNA fragment corresponding to a chlorophyll a/b binding protein gene was shown by RNA gel blot hybridization to be highly specific for green tissue Fig.  and 2. Gb12 Fig. . A compln tomato . This ann tomato . Furthercab6a and Lhca4/cab11) expressed in E. coli form a heterodimer in vitro that closely resembles the native LHCI-730 dimer from tomato leaves [LHCI can be subdivided into at least two different chlorophyll-protein complexes, one of which appears to be responsible for the 730 nm fluorescence of PSI (LHCI-730) and the other complex (LHCI-680) fluoresces at lower wavelength . In barlo leaves . Therefocab genes were isolated and linked to reporter genes. Transient gene expression studies in sugar beet indicated that 1097 bp upstream of the ATG from the Bvcab11 gene and 342 bp upstream of the ATG from the Bvcab12 gene are sufficient for leaf specific gene expression in sugar beet (Table The promoters for both sugar beet cab11 and Bvcab12 were stably transformed into sugar beet (Beta vulgaris, var. VRB). In sugar beet both promoters are expressed in leaves  and 900 plants, ) in whiccab11 derived GUS expression is 40599 pmol Mu x min-1 × mg-1 which is comparable with the expression strength of the strong CaMV 35S promoter in tobacco.The highest level of cab promoters from sugar beet. Particularly, the Bvcab11 promoter may be useful to drive strong and specific gene expression in transgenic host plants. The lack of bZIP binding sites harboring the ACGT core sequence could also be advantageous for transient analysis of bZIP transcription factors when using a Bvcab11 reporter gene construct as a transformation control. Furthermore these promoters may be useful to express resistance improving genes against foliar diseases such as Cercospora leaf spot.In summary, this work presents the isolation and expression analysis of two Two different methods for RNA preparation were employed. To isolate RNA for cDNA subtraction, the procedure described below was followed. For some of the Northern blot analyses a method described earlier was employed .g, 10 min.) the nucleic acid containing pellet was dissolved in 20 ml 100 mM NaCl, 10 mM EDTA pH8, 50 mM Tris-HCl pH8, and 0.2% SDS. Afterwards, a phenol:chloroform (1:1) and a chloroform:isoamylalcohol (24:1) extraction was performed. The pH of the aqueous solution was adjusted to about 5 with acidic acid and nucleic acids were precipitated by addition of 0.6 volume isopropanol and 0.05 volume 4 M NaCl and incubation for 2 hrs at -20°C. After centrifugation (20–30 min., 10000 g at 4°C) the nucleic acids containing pellet was resuspended in 10 ml H2ODEPC containing 0.1% SDS. Total RNA was precipitated by addition of 0.25 volume 8 M LiCl and incubation for at least 15 hrs at 4°C with subsequent centrifugation for 20 min at 4°C, 10000 g. Total RNA was resuspended in 400 μl H2ODEPC. After ethanol precipitation (addition of 0.1 volume sodium acetate, 3 M pH4.8, and 2.5 volume ethanol) total RNA was resuspended in a volume of 50–100 μl H2ODEPC. The isolation of poly(A)+ RNA was carried out with the Oligotex Kit according to the manufacturers protocol . Measurements of RNA yield and electrophoretic separation on formaldehyde gels were done following standard protocols [[For RNA isolation plant material was homogenized in liquid nitrogen and resuspended in a solution containing 4 M guanidinthiocyanat, 25 mM Tris-HCl, pH8 und 100 mM β-mercaptoethanol. After centrifugation (4°C, 10 min. at 3300 rcf) nucleic acids in the supernatant were precipitated by addition of 0.03 volume sodium acetate (3 M, pH5) and 0.75 volume ethanol (100%) and incubation over night at -20°C. After centrifugation (4°C, 10000 otocols [, modifieGenomic DNA was isolated from sugar beet genotypes 1K0088 and 4B5421 according to a previous published method . For rec®2.1. For each microgram PCR product approximately 300 recombinant plasmids were obtained. For the cloning of PCR products the Invitrogen T/A Cloning® Kit was employed (Karlsruhe, Germany). Prior to ligation into pCR®2.1 the subtracted PCR cDNA products were subjected to an additional incubation of 1 hour at 72°C with dATP and Taq polymerase  to ensure that the majority of the PCR fragments contain "A-overhangs" for an efficient cloning into the T/A cloning vector.The synthesis of cDNA was performed using the CLONTECH PCR-Select™ cDNA Subtraction Kit (Heidelberg, Germany). Each synthesis was carried out with 8 μg poly(A)+ RNA from sugar beet isolated either from leaves or taproots. Subtractive hybridization was done following the user manual (PT1117-1) of the CLONTECH PCR-Select™ cDNA Subtraction Kit. After the second PCR the amplified fragments from the forward and the reverse subtraction were cloned into the PCR cloning vector pCR® database was employed [The inserts of the plasmids were sequenced with fluorescently labeled M13 reverse and forward (-20) primers using the AutoRead Sequencing Kit (Pharmacia) and the Automated Laser Fluorescent A.L.F.™ DNA Sequencer from Pharmacia LKB (Freiburg, Germany). The DNA sequence analysis of the genomic and full-length cDNA clones was done by the custom sequencing service of MWG Biotech AG (Ebersberg, Germany). Sequences were subjected to data bank analysis using the BLAST algorithms  and analemployed . DNA seqemployed  and a PCRadioactive probes were generated by the method of random hexamer priming with the Amersham Multiprime DNA Labelling System (Freiburg, Germany). Southern and Northern hybridizations were carried out following standard protocols ,36.32-dATP (6000 Ci/mMol, Amersham Pharmacia Biotech, Freiburg).For genomic Southern blot hybridizations 10 μg of DNA from sugar beet genotypes 1K0088 and 4B5421 was digested with different restriction enzymes. Electrophoretic separation, transfer to Hybond nylon membranes (Amersham Pharmacia Biotech, Freiburg), hybridization to radioactive probes, and exposure of the membrane to X-ray films were done according to standard protocols . Radioaccab cDNA clones were identified after screening of 10000 clones using the SSH fragment L2 as a probe  and cloned into the plasmid vector pCMV Sport 6.0. Screening of the library was done according to standard protocols . Seven pcab loci were isolated.A genomic library from sugar beet genotype 1K0088 was generated in the lambda vector EMBL3 SP6/T7 and screened using standard protocols . Genomiccab11 is present on a ClaI fragment that was subcloned into a plasmid vector and completely sequenced. The fragment is 6294 bp long and contains 51 bp from the coding region of the gene. The resulting plasmid was designated pC1a. Additionally, a 6026 bp large SalI/ClaI fragment was released from the phage clone and subcloned into a Bluescript plasmid and designated pC1b. The promoter for the gene cab12 is present on a PstI fragment that was also subcloned into a plasmid vector and completely sequenced. The fragment is 4002 bp long and the harboring plasmid was designated pC2.The promoter for the gene cab11 promoter and the 3049 bp fragment cab12 promoter.From both genomic clones 1148 and 3049 base pairs containing most of the upstream region were deposited in GenBank (Acc. Nr. AX449166 and AX449167). The 1148 bp promoter fragment is designated Photinus pyralis in the reporter gene vector pGEM-luc (Promega, Mannheim). To introduce a plant polyA addition signal into pGEM-luc the respective fragment was isolated from pBI101.3 (Clontech, Heidelberg) by EcoRI digestion, followed by a Klenow fill in reaction and by redigestion with SacI. This released a 260 bp DNA fragment from the nopaline synthase (nos) gene containing the polyA addition signal. To directionally clone this fragment into pGEM-luc, this plasmid was first linearised with SfiI, treated with T4-polymerase to generate blunt ends and subsequently redigested with SacI. After inserting the nos fragment the resulting plasmid was designated pLuc-nos2. To insert the cab11 promoter fragment, a SalI(fill in)-AviII fragment was cloned into the ApaI linearised and T4-polymerase treated plasmid pLuc-nos2. This plasmid harbors 1145 bp from the cab11 promoter including the coding sequence for the first 16 amino acids of the cab11 gene. This plasmid was designated pC1L-1097. In this plasmid the luciferase gene is translationally fused with the first 16 amino acids from the cab11 gene. A second plasmid was generated which harbors additional upstream sequences. Towards these ends a 6099 bp KpnI fragment was released from the plasmid pC1b (see above) and the ends treated with T4-polymerase. The fragment was redigested with NotI and the desired fragment was directionally cloned as a KpnI(blunt end)-NotI fragment upstream of the cab11 fragment in pC1L-1097. To generate compatible ends pC1L-1097 was digested with HindIII treated with T4-polymerase and redigested with NotI. The resulting plasmid was designated pC1L-7126.For transient gene expression assays, promoter fragments were linked as translational fusions to the luciferase reporter gene from cab12 upstream to the luciferase coding region, the promoter fragment from pC2 was released by NotI/EcoRI digestion and subsequently subjected to a partial digestion with AviII. A 3100 bp long NotI/AviII fragment was purified and subcloned into pLuc-nos2. The plasmid pLuc-nos2 was digested with ApaI, the ends treated with T4-polymerase and redigested with NotI. After ligation the resulting plasmid was designated pC2L-2998. In this plasmid the luciferase gene is translationally fused with the first 16 amino acids from the cab12 gene. To generate 5' promoter deletions pC2L-2998 was (1) digested with KpnI/NotI, T4-polymerase treated, and religated to yield pC2L-1827, (2) digested with SmaI and religated to yield pC2L-989, and (3) digested with NotI and SalI , Klenow polymerase treated, and religated to yield pC2L-342.To clone the promoter for gene cab11 and cab12 promoters were cloned 5' to the β-glucuronidase gene (uidA). Towards these ends a 1.17 kb HindIII/BamHI fragment was released from pC1L-1097 and cloned into the binary vector pBI101.3 (Clontech, Heidelberg). The resulting plasmid pC1G-1097 harbors a translational fusion between the first 16 amino acids of the cab11 gene and the uidA gene. Similarly, the cab12 promoter was released as a PstI fragment from plamid pC2L-2998, treated with T4-polymerase then digested with BamHI and subcloned into pBI101.3 which was linearised with SalI, ends filled in with Klenow and redigested with BamHI. The resulting plasmid was named pC2G-2998.For stable transformation the Renilla reniformis under the control of the doupled CaMV 35S promoter was employed as a second reporter gene [Photinus and Renilla luciferase activity were measured with the dual-luciferase reporter assay system (Promega, Mannheim, Germany) in a Lumat 9501 luminometer (PE Biosystem) according to the protocol of the supplier.The luciferase expression from plasmids pC1L-1097, pC1L-7126, pC2L-2998, pC2L-1827, pC2L-989, and pC2L-342 were measured in sugar beet leaves after biolistic transformation . For bioter gene . For eacAgrobacterium tumefaciens strain GV2260 [Agrobacterium tumefaciens mediated transformation techniques were performed with the binary T-DNA plasmids on sugar beet (Beta vulgaris, var. VRB) according to [-1 × mg-1.For the generation of transgenic plants pC1G-1097 and pC2G-2998 were directly transformed into n GV2260 . Agrobacrding to . Selectirding to  using 4-cis-regulatory elements. RH and DS conceived of the study, and participated in its design and coordination.DS isolated the cDNA and genomic clones, analysed the transcription during different developmental stages and tissues, generated promoter reporter gene constructs, transformed sugar beet, and performed the quantitative reporter gene assays. DUK performed the suppression subtractive hybridization and analysed the homology and tissue specificity of the cDNA fragments. RH identified


Staphylococcus aureus (MRSA) is the leading pathogenic cause of nosocomial infections, especially in bacteraemia and sepsis. The essential therapy for MRSA infection is glycopeptides. Therapeutic failure can be seen with this therapy and the mortality is still high. The aim of this study was to evaluate the additional effect of G-CSF on the traditional antibiotic treatment in an experimental MRSA sepsis.Methicillin resistant 9/ml. Mice were randomised for the study into four group; control group (not receive any therapy), G-CSF group (1000 ng/daily, subcutaneously for 3 d), antibiotic group , and vancomycin+G-CSF group (at the same concentrations and duration). Autopsy was done within one hour after mice died. If mice was still alive at the end of seventh day, they were sacrificed, and autopsy was done. In all groups, the effect of G-CSF therapy on the survival, the number of the MRSA colonies in the lung, liver, heart, spleen, and peritoneal cultures, the histopathology of the lung, liver, heart and spleen was investigated.Experimental sepsis was performed in mice by intraperitoneal injection of MRSA isolate. Inoculum dose was estimated as 6 × 10One hundred and six mice were used. There were no significant differences in survival rates and bacterial eradication in G-CSF group compared with control group, and also in antibiotic +G-CSF group compared with antibiotic alone group. These parameters were all significantly different in antibiotic alone group compared with control group. Histopathologically, inflammation of the lung and liver were significantly reduced in vancomycin (25 mg/kg)+G-CSF and vancomycin (50 mg/kg)+G-CSF subgroups, respectively (p < 0.01). The histopathological inflammation of the other organs was not significantly different in antibiotic+G-CSF group compared with antibiotic group and, also G-CSF group compared with control group.G-CSF treatment had no additional effect on survival and bacterial eradication in MRSA sepsis in nonneutropenic mice; and only a little effect on histopathology. G-CSF treatment is very expensive, likewise glycopeptides. The more interest in infection control measures, and prevent the spread of MRSA infections is more rational. Staphylococcus aureus is an extremely virulent pathogen, and causes serious and deep-seated infections (e.g. endocarditis, osteomyelitis) [S. aureus is the leading pathogenic cause of both community-acquired and nosocomial infections, especially in bacteremia and sepsis [S. aureus sepsis is one of the most important causes of death [S. aureus (MRSA) infections is glycopeptides (vancomycin or teicoplanin). However glycopeptides are intrinsically less effective against staphylococci than are antistaphyloccal β-lactams [yelitis) . In reced sepsis -4. Despiof death . Furtherof death ,7. The e-lactams . This maAdvances in the pathophysiology of sepsis and septic shock suggest new therapeutic agents and approaches. Granulocyte-colony stimulating factor (G-CSF), a potent stimulator of neutrophil counts and functions, is one of these new strategies in sepsis. Despite a number of studies in sepsis, especially in neonatal sepsis, clinical efficacy of G-CSF is still controversial .The aim of this experimental study was to evaluate the role of G-CSF in the treatment of MRSA sepsis.Male BALB-C mice (8–10 weeks old, weighing 20–25 g) were obtained from Erciyes University Hakan Cetinsaya Experimental and Clinical Research Center. The Animal Care Committee of Erciyes University approved the experimental protocol used in this study. The animals were kept in a cage and allowed to feed and drink water.7/ml bacterial suspension and gradually increased until experimental sepsis developed. Experimental sepsis was defined as the growth of MRSA in two or more organs. The inoculum dose of this study was estimated as 6 × 109/mL.The clinical MRSA isolate, obtained from a patient's blood and pleural effusion admitted in General Surgical Intensive Care Unit of Erciyes University with nosocomial sepsis, was used. The strain was subcultured on blood agar at 37°C over night. On the day of experiment, bacterial suspension was prepared by sodium chloride 0.9% solution and the concentration was adjusted by spectrophotometer. The bacterial account needed for experimental sepsis was determined by a fore study. The fore study was begun with the inoculum dose of 1 × 10First dose of antibiotic was given at the sixth hour of bacterial inoculation. Vancomycin (DBL, 500 mg flacon), diluted in 5% dextrose, was given at 25 mg/kg or 50 mg/kg concentrations intraperitoneally every 12 hours for 7 days. rhG-CSF (Roche, Neupogen flk 30 mu/ 1 mL), diluted in 5% dextrose, was received 1000 ng/daily subcutaneously for 3 days .Animals were randomised to four groups; control group, G-CSF group, vancomycin group, and vancomycin +G-CSF group. The last two groups were divided into two subgroups for different dosage of vancomycin (25 mg/kg or 50 mg/kg). Each group had at least 15 mice and were kept in different cage. In control group, only bacteria suspension was given and no treatment was received.Bacterial suspension was given intraperitoneally to the mice, and when mice died, autopsy was done within one hour in aseptic conditions. If the mice was still alive at the end of seventh day, mice were sacrificed by servical dislocation and autopsy was done. The samples were taken from lung, liver, heart, spleen, and peritoneum for microbiological investigation, and from lung, liver, heart and spleen for histopathological examination. In each group, survival days were noted.Samples were taken from the organs with swab by one rotation on its axis, and were cultured on blood agar over night. The colonies on the agar were counted. The colonies more than 300 cfu in a plate noted as >300 cfu.The same pathologist examined tissue samples stained with hematoxylin and eosin. The degree of inflammation was graded by on a scale of 0 to ++++  . The patSurvival days, semiquantitative bacterial count and histopathologic findings in the tissues of the treatment groups were compared with the control group.Survival was assessed using a log-rank test. Fisher's extract test and chi-square test were used to compare the groups. Mann Whitney U test was used to compare the histopathology. A p value less than 0.05 was considered significant.Esherichia coli sepsis were excluded. 106 mice were evaluated. Six mice in control group, six mice in G-CSF group, 15 mice in vancomycin 25 mg, 15 mice in vancomycin 25 mg+G-CSF group, 18 mice in vancomycin 50 mg group and 14 mice in vancomycin 50 mg+G-CSF group were sacrificed at the end of seventh day. The death day and the survival rate of the mice in the groups are shown by the survival curve in figure This study included 110 mice. Three mice that developed intraabdominal hemorrhage, and one mouse that developed Culture and histopathology results are shown in Table S. aureus strains, isolated from the bloodstream infections [In recent years, MRSA has become widespread around the world, and become highly endemic in some hospitals. In the United States, the proportion of MRSA isolates increased from 2.4% in 1975 to up to 55% in recent years ,12. Simifections . Unfortufections .G-CSF is a cytokine that stimulates myeloid progenitor cell proliferation and increases the bone marrow storage pool and the number of circulating mature neutrophils, which are important component of the host defense . Also, iThe previous experimental therapeutic studies were mostly carried out in gram-negative sepsis, and in this experimental study, we investigated the effect of G-CSF in MRSA sepsis. The survival rates were not significantly different in G-CSF group compared with the control group, and also in antibiotic+G-CSF group compared with antibiotic alone group. Likewise, most of the other experimental studies in gram negative sepsis, showed that prophylactic rhG-CSF administration reduced endotoxemia and serum TNF-α levels and also improved cardiac function and survival, whereas therapeutic rhG-CSF (i.e. administered after the onset of infection) did not improve outcome and at very high dosages appeared harmful ,26. A reIn this study, we did not measure the WBC count, so not have subgroups with neutropenia. In an experimental gram-negative sepsis in rabbits, Smith and colleguaes  showed tBacteria are rapidly cleared from blood and tissues following intravenous antibiotic therapy . In our S. aureus infections are leukocytic infiltration, focal pneumonitis, edema, microabscesses, etc. In an experimental study, significant pathologic changes during and after the elimination of bacteria from the blood and tissues were noted. Also, expression of cytokines (TNF, IL-1, IL-6) was observed in all of the infected tissues, and correlated tissue damage after clearance of bacteria from the blood and tissues[The histopathologic findings of invasive d tissues. HoweverIn conclusion, G-CSF treatment had no additional effect on survival and bacterial eradication in MRSA sepsis in nonneutropenic mice; and only a little effect on histopathology. Furthermore, G-CSF treatment is very expensive, likewise glycopeptides. Because of high mortality and morbidity rates and excess costs, more interest in infection control measures, and prevent the spread of MRSA infections is more rational.This study was supported by Erciyes University Research Fund. There was no non-financial competing interest.EA and SG were the primary researchers. OC and BK are the pathologist and examined tissue samples. MD was the director of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


E. faecalis and E. faecium accounting up to 90% of the clinical isolates. Nevertheless, the incidence of other species of enterococci from clinical sources shows an alarming increase with the properties of intrinsic resistance to several antibiotics including beta-lactams and glycopeptides. Thus proper identification of enterococci to species level is quintessential for management and prevention of these bacteria in any healthcare facility. Hence this work was undertaken to study the prevalence of unusual species of enterococci causing human infections, in a tertiary care hospital in South India.Enterococci tend to be one of the leading causes of nosocomial infections, with The study was conducted in a tertiary care hospital in South India from July 2001 to June 2003. Isolates of enterococci were collected from various clinical specimens and speciated using extensive phenotypic and physiological tests. Antimicrobial susceptibility testing were performed and interpreted as per NCCLS guidelines. Whole cell protein (WCP) fingerprinting of enterococci were done for species validation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed computationally.E. gallinarum (6.2%), 10 E. avium (4.1%), 6 E. raffinosus (2.5%), 6 E. hirae (2.5%), 4 E. mundtii (1.7%), 3 E. casseliflavus-including the two atypical isolates (1.2%) and 2 E. durans (0.8%). The 12 atypical enterococcal species (5%) that showed aberrant sugar reactions in conventional phenotyping were confirmed as E. faecalis, E. faecium and E. casseliflavus respectively by WCP fingerprinting. The antimicrobial susceptibility testing depicted the emergence of high-level aminoglycoside and beta-lactam resistance among different species apart from intrinsic vancomycin resistance by some species, while all the species tested were susceptible for linezolid and teicoplanin.Our study showed the prevalence of unusual  and atypical  species of enterococci as 19% (46 isolates) and 5% (12 isolates) respectively. The 7 unusual species (46 isolates) isolated and confirmed by phenotypic characterization includes: 15 Our study reveals the emergence of multi-drug resistance among unusual species of enterococci posing a serious therapeutic challenge. Precise identification of enterococci to species level enables us to access the species-specific antimicrobial resistance characteristics, apart from knowing the epidemiological pattern and their clinical significance in human infections. Enterococcus faecalis and Enterococcus faecium accounts up to 90% of clinical isolates belonging to this genus [E. faecalis and E. faecium, and only few reports or studies of non-faecalis and non-faecium enterococci are prevalent [Enterococci, generally regarded as normal flora of gastrointestinal and genitourinary tract of humans, have emerged as the etiogen of several nosocomial as well community-acquired infections since last two decades. Globally, many studies have revealed that enterococci tend to be one of the leading causes of several nosocomial infections, with the emergence and spread of multi drug resistance among isolates -3. Sinceis genus . Neverthis genus ,6. But tis genus . Thus prrevalent ,6. HenceThe study was conducted in a 900-bedded tertiary care hospital at Pondicherry, South India from July 2001 to June 2003. Isolates of enterococci were collected over the time period from various clinical specimens such as blood, urine, wound swabs and pus , catheters, ascitic fluid, synovial fluid, by plating them on 5% Sheep Blood agar and Mac-conkey agar, as well on Bile esculin azide agar (Hi-media, Mumbai, India) as per nature of the specimen. Extensive phenotypic and physiological characterization was carried out by the conventional tests devised by Facklam et al . CarbohyAntibiotic susceptibility testing of the clinical isolates along with the quality control strains were performed using BHI agar instead of Muller Hinton agar by disk diffusion method (for the antibiotics: penicillin 10 units], ampicillin [10 μg], gentamicin-high content [120 μg], streptomycin-high content [300 μg], ciprofloxacin [5 μg], nitrofurantoin-for urinary isolates only [300 μg], vancomycin [30 μg], teicoplanin [30 μg] and linezolid [30 μg]), standard agar dilution (for the antibiotics mentioned in Table-0 units, Whole cell protein (WCP) analysis of the enterococcal isolates, including atypical biochemical variants of enterococci and the reference/type strains of enterococci (a kind gift from Dr. Richard.R.Facklam, CDC, Atlanta, GA. USA) were done using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously with minor modifications ,11 for sE. faecalis and E. faecium-data not shown) which included 15 E. gallinarum (6.2%), 10 E. avium (4.1%), 6 E. raffinosus (2.5%), 6 E. hirae (2.5%), 4 E. mundtii (1.7%), 3 E. casseliflavus (1.2%) and 2 E. durans (0.8%). The distribution by site of isolation for the 46 unusual enterococcal species included 30 isolates- 12 E. gallinarum, 6 E. avium, 3 each of E. hirae, E. casseliflavus and E. raffinosus, 2 E. mundtii and 1 E. durans (65.2%) from bloodstream, 6 isolates- 3 E. raffinosus, 2 E. avium and 1 E. mundtii (13 %) from surgical and non-surgical wound swabs, 10 isolates- 3 each of E. hirae and E. gallinarum, 2 E. avium and 1 each of E. durans and E. mundtii (21.8%) from miscellaneous sites, including muscle tissues sent for anaerobic culture, catheter tips, peritoneal fluid, ear swab and urine. Of the 46 persons from whom unusual enterococci were obtained, 56.5% were males and 43.5% were females including newborn/neonates. The infections were polymicrobial in 6 (13%) of the 46 cases from which unusual enterococci were isolated, including 2 (6.7%) of 30 bloodstream infections. The 12 atypical enterococcal strains (5%) showing aberrant sugar reactions included 6 mannitol negative variant E. faecalis like species, 1 arginine negative variant E. faecalis like species, 3 mannitol negative variant E. faecium like species and, 2 arginine negative variant E. casseliflavus like species. The WCP analysis by SDS-PAGE confirmed the species identities of seven different species. Atypical strains showed a similar banding pattern like the reference strains from CDC  except for minor quantitative differences, with no qualitative difference. The computational analysis of the WCP gel images of atypical strains were performed by Dice coefficient, and the dendrogram constructed using unweighted pair group method using arithmetic averages (UPGMA) as shown in Figure-E. faecalis, E. faecium and E. casseliflavus respectively. The 2  isolates of arginine negative variant E. casseliflavus like species after taxonomic validation were included as an unusual species of enterococci accounting to 3 E. casseliflavus isolated overall.We isolated a total of 242 enterococci during our 2-year study period from different clinical samples. The biochemical phenotyping results revealed 46 isolates 19%) belonging to 7 different unusual species of enterococci , 2 E. durans (n = 2), 11 E. gallinarum (n = 15), 2 E. hirae (n = 6), 2 E. mundtii (n = 4) and, 3 E. raffinosus (n = 6). Only E. casseliflavus (n = 3) exhibited 100% susceptibility to ciprofloxacin. None of the 46 isolates was positive for β-lactamase, but resistance for β-lactam agents were prevalent variably among different species. The results of MICs for penicillin, ampicillin, high-level gentamicin and high-level streptomycin resistance were in accordance with the disk diffusion testing results except for vancomycin. Disk diffusion testing showed vancomycin resistance for 6 isolates , but the agar screening method exhibited vancomycin resistance for 11 isolates .The antimicrobial susceptibilities of the isolates given in Table-E. faecalis and E. faecium as the only prevalent species [E. faecalis and E. faecium) prevalence of unusual species of enterococci from catheterized patients with urinary tract infections. E. mundtii and E. durans were not reported in their study, whose prevalence was 1.7% and 0.8% respectively in our study. E. gallinarum (6.2%) and E. avium (4.1%) were the most commonly identified species, which markedly differs in isolation rate (0.3–1.2%) from other studies [E. gallinarum isolated during our study period were from pediatrics unit, while 7 of the 10 isolates exhibiting a similar antibiotype were isolated from the same ward within a span of 2 months. The remaining 3 of the 10 E. gallinarum were isolated from the same ward in the preceding 3 months, one of which showed an antibiotype similar to the cluster of 7 isolates. The same was the case of 3 E. casseliflavus isolated from the same pediatrics unit within a span of 2 months in the preceding year. Most of these  isolates were from cases of septicemia. Although molecular epidemiological studies have not been done to compare the genetic similarities of these isolates, the data depicts the nosocomial spread of these species. WCP analysis by SDS-PAGE had been proven to assist in validating the species identities as well, to identify strains that do not exhibit phenotypic characteristics identical to the type strains of each species [Our study reveals that the prevalence of unusual species of enterococci as 19% in our clinical setup in South India. Many studies and reviews show the prevalence of non-faecalis and non-faecium enterococci as 2–10% ,6,12. Pr species -16, whic species ,17. The  species  showing  studies . The incE. avium .1% were  species . We wereE. casselifalvus) which proves that it may be successful only in treating enterococcal urinary tract infections [E. gallinarum and, 1 E. casseliflavus isolates, which may go undetected by disk diffusion testing [E. durans and two E. mundtii (MIC ≤ 6 μg/ml). The genotypic basis of vancomycin resistance for these 3 isolates yet to be studied, will give us a definitive picture regarding its clinical significance, since studies have reported the prevalence of vancomycin resistance in these two species, and its transferable nature from E. durans to E. faecium [Ciprofloxacin resistance was 63% among isolates  low-level vancomycin resistance by disk diffusion testing  emphasizThe author(s) declare that they have no competing interests.PVP designed the study and carried out the experimental works and analysis, and drafted the manuscript. RSR supervised and participated in the design of the study and coordination, and helped to draft the manuscript. SCP participated in the coordination of the study and helped to draft the manuscript.The pre-publication history for this paper can be accessed here:


The discovery of cis-regulatory modules in metazoan genomes is crucial for understanding the connection between genes and organism diversity. It is important to quantify how comparative genomics can improve computational detection of such modules.We run the Stubb software on the entire D. melanogaster genome, to obtain predictions of modules involved in segmentation of the embryo. Stubb uses a probabilistic model to score sequences for clustering of transcription factor binding sites, and can exploit multiple species data within the same probabilistic framework. The predictions are evaluated using publicly available gene expression data for thousands of genes, after careful manual annotation. We demonstrate that the use of a second genome (D. pseudoobscura) for cross-species comparison significantly improves the prediction accuracy of Stubb, and is a more sensitive approach than intersecting the results of separate runs over the two genomes. The entire list of predictions is made available online.in silico. The future availability of additional fruitfly genomes therefore carries the prospect of highly specific genome-wide predictions using Stubb.Evolutionary conservation of modules serves as a filter to improve their detection  Berman et al. , Markste [et al.  and Half [et al.  predicte [et al. , called  [et al. ). Most pet al. [et al. [The Stubb algorithm  extende [et al. ). The seD. melanogaster and D. pseudoobscura) using Stubb can lead to a significant improvement in the accuracy of genome-wide CRM prediction. To our knowledge, this is the first direct evaluation of the effect of cross-species comparison on CRM prediction on a genome-wide scale. Another important contribution of this paper is to present a benchmark for evaluating genome-wide CRM prediction tools, collected from the BDGP database and the literature, and curated by manual inspection of several hundred expression patterns. Using the same benchmark, we evaluate the effect of varying how background sequence information is incorporated in the algorithm, since this is the only tunable parameter in the Stubb program, other than the module length. We are thus able to suggest the optimal parameter settings for genome-wide CRM prediction using Stubb. Finally, we report all genome-wide predictions for cis-regulatory modules involved in anterior-posterior patterning in the early fly embryo, using both single-species and two-species Stubb, many of which make a strong case for experimental validation.In this paper, we present evidence that the exploitation of cross-species comparison  axis during early -Volhard ; Rivera--Volhard ; Furriol-Volhard ). The maet al. [et al. [et al. [et al. [et al. [The segmentation paradigm has been used as a test system for the computational detection of CRMs by us and others . Here,  [et al. ), we useD. melanogaster and D. pseudobscura have been sequenced, and Stubb was used to predict CRM's in the D. melanogaster genome. This was done in two modes – (i) STUBBSS, where Stubb is run on D. melanogaster genomic sequence alone, and (ii) STUBBMS, where Stubb uses orthologous sequence data from D. pseudobscura to help predict CRM's in D. melanogaster. For each mode of execution, we obtain a separate list of predicted CRM's, sorted in order of confidence in the prediction. The ideal test for our purpose would be to compare the accuracy of these two sorted lists. However, the set of experimentally verified CRM's involved in this system is sparse compared to the size of the system – roughly 50 CRM's are known  The Berkeley Drosophila Genome Project (BDGP) has catalogued the expression patterns of a large number of genes in D. melanogaster, at various stages of development. We considered such a catalogue of 2167 genes, obtained from BDGP and from the literature.  Visualst Genes .) Hence,We note that some accuracy is lost in the translation of a list of predicted CRM's to the predicted genes it is mapped to, as per the mapping defined above. For instance, it is known that CRM's may control a gene located at large distances, i.e., further than the distance threshold of 20 Kb used in the mapping procedure. Also, it is possible that a CRM is located close to two genes, and directs the expression of both genes, or only of the farther gene, being somehow insulated from the nearer one. To address these concerns, we repeat our evaluation with a slightly different mapping from the one described above. A caveat that remains is that there may be genomic sequences that are functional, in the sense that they are capable of directing a specific blastoderm pattern in reporter gene constructs, but whose activity is 'silenced' in native genomic context and does not translate to patterning of any gene. Also, the CRM may direct expression of the gene only at post-blastodermal stages, so that the gene is not included in the "positive" test set of blastoderm patterned genes. Conversely, it may also happen that a predicted CRM lies close to a patterned gene, thereby being counted as a true positive, but the predicted CRM is not the sequence responsible for the gene's regulation. We assume that such effects are not biased against either algorithm.st order background for STUBBSS and global, 2nd order background for STUBBMS.  The x-axis is the number of unique genes that are predicted by the algorithm (by progressively decreasing its score threshold) and are in the set of 2167 genes with expression information. On the y-axis we plot how many of those predicted genes are in the "positive" set (i.e., have an ap blastoderm pattern.) Thus, the y-axis is the specificity of the algorithm. We observe that STUBBMS performs significantly better than STUBBSS. For instance, to predict 100 genes correctly, STUBBSS has to make 343 predictions, while STUBBMS only has to make 267 predictions. Figure Figure -12 and <10-37 respectively, Binomial Proportions test). Further examination of the top 102 gene predictions made by each algorithm revealed that 24 true positives are common to both lists. STUBBMS reports 37 true positives not discovered by STUBBSS, while the latter reports 15 true positives not found by the former. Similar results are seen for the top 311 predictions , STUBBSS reports 39 positives, while STUBBMS scores 61 hits, an improvement of over 56%. In comparison, the random expectation is ~13.5 hits. Thus the predictions of both STUBBSS and STUBBMS are significantly enriched in patterned genes (P < 10We next examined separately the following three sets of genes: (i) INTERSECTION  (ii) MS-ONLY (predicted only by STUBBMS) and (iii) SS-ONLY (predicted only by STUBBSS). Table D. melanogaster, but intersecting these predictions with a similar number of top predictions in D. pseudoobscura gives only 68 unique genes, 33 of which are patterned. Of the top 68 predictions made in D. melanogaster alone, 29 are patterned. Thus the "intersection" strategy yields only a modest improvement over the single-species search, and does so at the price of significantly reducing the total number of predictions. Similar results were obtained when intersecting modules instead of gene predictions.One possible strategy that uses two-species sequence is to make predictions using STUBBSS on each of the two genomes separately and then intersect the respective lists. We found this strategy to be very restrictive – for instance, with a particular score threshold, STUBBSS predicts 205 unique genes in et al. [et al. [We have noted above that the evaluation method is influenced by the way we map the predicted CRM's to predicted genes. To offset potential biases induced by this mapping, we repeated our analysis with a slightly different evaluation procedure, borrowing from the approach of Grad et al. . We now  [et al. , is thatet al. [et al. [The default mapping from CRM's to genes used in our evaluations predicts a gene to be patterned only if its proximal end is less than 20 Kb from the CRM. Schroeder et al.  studied  [et al.  observe Genes in the segmentation hierarchy often have multiple aspects to their expression pattern, with more than one CRM regulating them. We therefore measured how the Stubb predictions fare if we required that each predicted gene be evidenced by at least two predicted CRM's. This heuristic improves the performance of STUBBSS more prominently than that of STUBBMS, though much fewer predictions are made by either algorithm.  ubiquitously expressed at all developmental stages, (ii) not expressed at any stage, or (iii) only maternally expressed.  Incluset al. [I to I+19 were examined, with I being incremented in steps.) In each step, we computed the fraction of the 20 genes that belonged to the following three non-exclusive categories: (i) genes with dv (in addition to ap) modulation, (ii) genes with strong expression pattern, and (iii) genes in the "core" set of 48 genes. These values are reported in Figure Our annotations of the blastoderm patterned genes also include whether the gene expression is strong, weak or of intermediate strength; if it has a dorsal-ventral (dv) modulation in addition to the primary anterior-posterior pattern; and if the gene belongs to a "core" set of 48 genes that have been shown experimentally to be required for the segmentation of the embryo . We wer1. Genes with dorsal-ventral aspects to their blastoderm pattern are more frequent at lower ranks of prediction; i.e., the top predictions are enriched in genes with anterior-posterior patterns only.2. Core genes are predominantly found in the top predictions.3. Genes found at higher ranks are somewhat more likely to be strongly expressed.The first two observations imply that the genes more directly involved in the ap axis formation are recovered at better ranks, and that the lower rank genome-wide predictions are richer in derivative patterns characteristic of genes with more complex regulatory inputs (pair-rule factors, dv factors etc.). The same trends were found for the correct predictions made by STUBBSS. (Data not shown.)k for this parameter means that local correlations are assumed to be present at the level of (k+1)-mers, i.e., the random probability of seeing a particular base at a position depends on the bases seen at the previous k positions. th order background in the terminology of this paper.) We vary this parameter to take the values k = 1 and k = 2, in different runs. The other parameter is the actual sequence used by Stubb to measure background nucleotide frequencies. Here the two options are (i) to use the current sequence window as background, or (ii) to use a pre-specified sequence (or collection of sequences) as background. We call these two the "local" and "global" background models respectively. For the "global" model, we input into Stubb 150 Kb of sequence from non-coding regions of the D. melanogaster genome, collected from the five chromosome arms 2L, 2R, 3L, 3R, and X.We next evaluate the effect of varying how background sequence information is incorporated in the Stubb algorithm. This is the only configurable aspect of the program, other than the module length.  One important parameter is the "Markov order" of background. A value of y et al.  and Schrst order background, though the other parameter values produce only slightly different results. On the other hand, the effect of background parameters on two-species Stubb is more pronounced, with the best choice being a global, 2nd order background. Using a global 1st background order gives almost identical results (data not shown), hence we infer that a global background is the optimal choice for STUBBMS.Figure et al. [st and 2nd order Markov backgrounds), and comparing its performance to that of Stubb.  Figure As mentioned earlier, the STUBBSS program implements the same class of algorithm as the Ahab algorithm of Rajewsky et al. , with soAll the above runs were on genomic sequence with tandem repeats masked by the Tandem Repeats Finder program of Benson . We haveThe Stubb program is an extension of Ahab, with the important feature that it can handle two-species data within its probabilistic framework. The two programs differ in their underlying optimization method, with Stubb using an Expectation-Maximization approach in contrast to Ahab's conjugate gradient method. Performance evaluation of the two programs shows little difference between them, implying that the algorithm is robust to the actual optimization method used. Another technical difference between Ahab and Stubb is in the manner that orientation of binding sites is treated. While Stubb assumes a uniform prior on the orientation of a binding site, Ahab picks the best orientation for each site, with the caveat that probabilities are not strictly normalized.et al. [et al. [et al. [An important component of Stubb is the alignment step where the two species are aligned (using LAGAN) and blocks of high sequence similarity are extracted. (See Methods.) The parameters used in LAGAN runs were obtained from Emberly et al. , who der [et al. versus SM [et al. ) for CRMa priori how tandem repeats should affect module detection – repeats similar to binding sites of the system may improve sensitivity when they occur in CRM's; but if repeats resembling binding sites occur by chance in non-functional regions, prediction specificity may suffer. The occurrence of tandem repeats marks statistical deviation from Stubb's probabilistic model of sequence generation. In our tests, we found that repeats distract the algorithm more than they help, as manifested in better performance on repeat-masked sequence. , first  [et al. report anRegarding the recovery of patterned genes by Stubb, several observations can be made. Of the 286 genes with ap patterns, we recover roughly half at a score cut-off of 10, using STUBBMS. Why is the other half not found? While it is obvious that lowering the cut-off will detect more patterned genes, there are other reasons why a patterned gene may be missed by Stubb. Some genes are likely to be lost due to the distance filter we have imposed (CRM to nearest gene <20 kb), since the regulatory regions of some genes (e.g., homeotic genes) are likely to be larger than that. More importantly, most of the patterned genes that are not part of the core transcriptional machinery have derivative patterns that reflect a more complex input (binding sites for pair rule factors, d-v factors etc.) and thus will only be recovered to the extent their input has a solid maternal/gap component. Conversely, there are at least two reasons for reporting false positives (roughly two thirds at a score cutoff of 10). The presence of an insulator could prevent the interaction between a CRM and its nearest basal promoter. More likely is a scenario where the predicted CRM's do drive expression but at post-blastoderm stages. All gap factors are active in multiple tissues in later development and therefore CRM's with dominant or exclusive gap input may well be active in these later contexts. These caveats affect all current CRM detection algorithms, and accounting for such additional axes of information as genomic context and module composition rules will be a difficult but important challenge for the future.A very interesting observation comes from the analysis in Table changes in terms of their binding site compositions. This leads to a powerful bioinformatic tool to predict regulatory changes between the two fly species. We can thus obtain hypotheses about changes in expression patterns, which can be verified experimentally. We have examined a representative collection of CRM's, and experimentally verified several of the changes predicted by Stubb, thereby building a catalogue of the different modes of cis-regulatory evolution. The results of this study will be reported in the near future.The Stubb program not only predicts cis-regulatory modules genome-wide, it additionally outputs the binding site profile of each predicted CRM, i.e., the locations and probabilities of binding sites in the CRM. Schroeder et al  use the We have seen that the use of a second fly genome significantly improves genome-wide module prediction. Since STUBBMS uses a natural "two-species" extension of the algorithm of STUBBSS, this finding is largely a statement about the inherent potential of cross-species comparison as a paradigm for improving functional genomics. The STUBBMS program also has a natural extension to incorporate more than two genomes, and it will be very interesting to see how much of a difference a third genome makes. The genome of D. yakuba is expected to be sequenced soon, and since this species is closer to D. melanogaster, it may help better discriminate conserved regulatory modules.D. melanogaster sequences were obtained from Flybase Release 3. The analysis was limited to the five chromosome arms 2L, 2R, 3L, 3R, and X. D. pseudobscura contigs were obtained from  (February 2003 Release). Based on Blast results, we created a mapping, called "CONTIGMAP", between regions of the D. melanogaster genome and D. pseudobscura contigs, each region typically being tens of Kb long. This mapping is many to many, i.e., different regions of D. melanogaster may map to the same contig, and the same (or overlapping) region in D. melanogaster may map to two or more D. pseudobscura contigs. For each entry (M, P) in CONTIGMAP, where M is the D. melanogaster region and P is the D. pseudobscura contig, the LAGAN alignment program  was runD. melanogaster genome with a sliding window of length 500 bp, in shifts of 50 bp. The input weight matrices for the maternal and gap transcription factors Bcd, Hb, Cad, Kni, Kr, Tll, Dstat and the torRE binding factor were obtained from Rajewsky et al. [et al. [D. melanogaster sequence M, in shifts of 50 bp. Thus, STUBBMS was not run on regions of D. melanogaster that are not aligned with some D. pseudoobscura contig. The weight matrices used were the same as in STUBBSS runs. The locations of the blocks computed in the alignment step (above) were input to STUBBMS, and the input value of the neutral mutation rate was 0.5, the value being chosen due to its better performance over alternatives tested.Tandem repeats in the input sequences were masked with the Tandem Repeat Finder program of Benson , with pay et al.  and SchrEach genome-wide run of Stubb produces, for each starting position of the sliding window, a score that measures the likelihood of the sequence having a cluster of binding sites. The next step is to extract the coordinates of each window that scores better than all other windows overlapping it. Such windows correspond to local "peaks" in the score profile along the genome. All such "peak" windows with scores above a certain threshold are sorted in decreasing order of their score, to produce a sorted list of predicted CRM's. Each window in this list is annotated with useful information including the identity and relative location of its neighboring genes. The list is then filtered to retain only those predicted CRM's where Stubb predicts occurrences of at least two weight matrices. This is a heuristic that incorporates the combinatorial nature of CRM's, i.e., their tendency to have sites for multiple transcription factors (activators as well as repressors.) Finally, any predicted CRM that overlaps with an exon is removed from the list before evaluation. The predictions made by STUBBMS and STUBBSS are listed in the files "Predicted CRM's – two species"  and "Pre lists as showing expression during blastoderm (embryonic stages 4–6) were visually inspected. From this list, we removed genes with ubiquitous expression , extremely faint or irreproducible expression (31), or expression in pole cells or yolk nuclei only (64), as well as genes whose expression is modulated along the dv axis only (13). The remaining 258 genes show patterned expression in the somatic portion along the ap axis of the blastoderm embryo; 28 known segmentation genes not captured in the BDGP expression database were added to the list, for a total of 286 genes showing ap patterned blastoderm expression. These genes were further categorized by expression level (strong, intermediate, weak) and type of pattern (ap, ap+dv, dv+ap). ap includes gap, pair rule and segment polarity-like patterns (e.g., Kr, fkh, eve); ap+dv denotes ap pattern with some dv modulation (e.g., kni, so, en); dv+ap denotes dv pattern with some ap modulation (e.g., neur).The 792 genes which the BDGP expression database SS and EDS worked out the details of genome-wide Stubb runs. SS performed the Stubb runs, collected all statistics from the runs, and drafted the manuscript. EDS suggested several analyses reported. MDS, UU, and UG annotated the genes for expression pattern, suggested many of the analyses in the Results and Discussion sections, and wrote parts of the manuscript. All authors read and approved the final manuscript.et al: Genome Biol 2004, 5:R61, published 20 August 2004.) also evaluates the effect of cross-species comparison on CRM prediction in Drosophila.A recently published paper (Berman Test Genes List of 2167 genes with expression information. (Source BDGP + literature.)Click here for filePatterned Genes List of 286 genes with blastoderm pattern, as determined by manual inspection of in-situ expression pattern. (Source BDGP + literature.)Click here for fileAdditional Genes List of 2065 genes that are ubiquitous, maternal only, or not expressed. (Source BDGP.)Click here for filend order background, sorted in decreasing order of Stubb score.Predicted CRM's – two species List of predicted CRM's using two-species Stubb with a global, 2Click here for filest order background, sorted in decreasing order of Stubb score. This list of predictions, as well as that in Additional File 4 (above), is meant to be as inclusive as possible; therefore, the specificity of the lowest ranked predictions may be poor.Predicted CRM's – single species List of predicted CRM's using single-species Stubb with a local, 1Click here for file


DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants.We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1). After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes.This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction. Molecular biological studies of plants require high-quality DNA. Several DNA extraction procedures for isolating genomic DNA from various plant sources have been described, including the salt extraction method and the cetyltrimethyl ammonium bromide (CTAB) method  and its e.g., the Wizard genomic DNA purification system; Promega, USA). However, these methods are not time efficient for consistently obtaining PCR-quality DNA from calluses and plants, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc.Most methods require the use of liquid nitrogen  or freez2) of leaf tissue.In our laboratory, we investigate the stability of transgenes expressed in calluses or plants transformed by nuclear or chloroplast transformation in tobacco, lettuce, potato, etc. In addition, we need high-quality genomic DNA for Southern blot analysis to confirm homologous recombination in chloroplast transformation . For our2O, and PCR was performed using 1 μl of the diluted supernatant as a template . An aliquot of the supernatant (~5 μl) was diluted 5 fold with sterile dH. An aliqDNA samples prepared using the three different extraction procedures  [To examine the presence of bar ,12 or thbar  at a dirme Table , lane 1,2450 bp)   were between 1.7 and 2.0. However, the DNA concentrations from rice, maize, and poplar were relatively low (< 3 μg). This may be because homogenization using a hand-operated homogenizer with a plastic tip is incomplete, since the leaves of these plants are stronger than the leaves of tobacco, potato, cabbage, lettuce, and Siberian ginseng. Genomic DNA from various plant sources was electrophoresed on 1% agarose gels, and high-molecular-weight DNA was obtained  is put in a 1.5-ml microfuge tube and homogenized directly; consequently, a very small sample is required for DNA extraction. There is no sample waste with our method, whereas much larger samples are required when plant samples are ground in a mortar and pestle with liquid nitrogen and transferred to a tube. Previously reported techniques require several steps [2 was used in the extraction, co-extracts (e.g., chlorophyll) were extracted alongside the DNA and inhibited the PCR. On the contrary, our protocol does not require appropriate sample size to extract DNA. Warner et al. [There are many advantages in using our genomic DNA extraction method to obtain template for PCR amplification. Many different plants could be amplified using the same DNA extraction method and the same PCR protocol. Using this protocol, we successfully amplified DNA repeatedly from all eight plant sources examined. Our procedure is not only very simple, but is also time and cost effective. After homogenization in DNA extraction buffer using a hand-operated homogenizer, the template DNA for PCR could be extracted by phenol/chloroform/isopropyl alcohol treatment. Since this method does not require liquid nitrogen, expensive commercial DNA extraction kits, or ethanol precipitation to produce DNA template for PCR, we can save considerable time and expense. The time required for our DNA extraction method is less than 30 min, which is extraordinary compared with other genomic DNA extraction methods. With our procedure, leaf tissue , potato (Solanum tuberosum), cabbage (Brassica oleracea), rice (Oryza sativa), lettuce (Lactuca sativa), maize (Zea mays), poplar (Populus nigra), and Siberian ginseng (Eleutherococcus senticosus). The plants used for genomic DNA extraction were grown in a culture room or greenhouse. Tobacco, potato, cabbage, lettuce, and Siberian ginseng were grown in a culture room. Seeds were surface-sterilized with 70% ethanol for 3 min, and then with 10% sodium hypochlorite for 15 min. The seeds were washed five times in sterile water and placed in Petri dishes containing 4.6 g l-1 MS salts [-1 sucrose, and 7.5 g l-1 bactoagar at pH 5.7. The seeds were grown in a controlled environment at 25°C on a 16-h continuous light and 8-h dark daily cycle. Rice, maize, and poplar plants were grown in a greenhouse for genomic DNA extraction. Transgenic tobacco plants and Siberian ginseng calluses were also used to extract genomic DNA and to confirm foreign gene insertion by PCR amplification.We examined plant material from plant collections commonly used for foreign gene expression: tobacco , using a hand-operated homogenizer (Sigma, Z35997-1) with a plastic pestle, for 15~20 s. After an initial homogenization, another 150 μl of DNA extraction buffer were added and homogenized with the same homogenizer for 15~20 s. Then, 20 μl of 20% SDS were added and vortexed for 30 s. The samples were incubated at 65°C for 10 min for cell lysis. At this point, three different DNA extraction protocols were used for PCR amplification. Protocol 1: An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to the samples, mixed by vortexing for 30 s, and then centrifuged at 10,000 g for 3 min at 4°C. The supernatant was diluted 5 fold, and 1 μl of the supernatant was used as the DNA template for PCR analysis. Protocol 2: The supernatant from protocol 1 was transferred to a fresh tube and extracted one more time with phenol/chloroform/isoamyl alcohol (25:24:1) and then with chloroform. The supernatant was diluted 5 fold, and 1 μl of the supernatant was used as the DNA template for PCR analysis. Protocol 3: The supernatant from protocol 2 was transferred to a fresh tube, and a double volume of ethanol was added to each sample, mixed well, and the samples were incubated at -20°C for 30 min. The samples were then centrifuged at 10,000 g for 10 min at 4°C. The pellet was washed with 70% ethanol, dried, and resuspended in sterile dH2O containing 20 μg/ml DNase-free RNase A. The concentration and purity were determined from the A260/A280 ratio using a spectrophotometer. Five micrograms of each genomic DNA sample were incubated at 37°C for 3 h for complete digestion with 20 U of EcoRI and HindIII (Life Technologies, USA) in a total volume of 100 μl and analyzed on 1.0% agarose gels using 15 μl aliquots of the reaction mixture.We tested three different variations of the genomic DNA extraction procedure. About 0.5 cmEcoRI and HindIII in a total volume of 100 μl and analyzed on 1.0% agarose gels using 15 μl aliquots of the reaction mixture. By using the genomic DNA isolated from the leaves or calluses of wild-type and transgenic plants, PCR amplifications were performed on a Perkin Elmer GeneAmp PCR System 2400 (Biorad, USA) in a total volume of 25 μl containing 1 × PCR buffer, 0.2 mM dNTP, 10 pmol of each primer (Table Five micrograms of each genomic DNA sample measured by spectrophotometer were incubated at 37°C for 3 h for complete digestion with 20 U of TJK developed the method and performed majority of the experiments. MSY provided technical assistance, funding and supervision for the work. All authors have read and approved the final manuscript.


Breast pain and tenderness affects 70% of women at some time. These symptoms have been attributed to stretching of the nerves with increase in breast size, but tissue mechanisms are poorly understood.Eighteen patients (n = 12 breast reduction and n = 6 breast reconstruction) were recruited and assessed for breast pain by clinical questionnaire. Breast skin biopsies from each patient were examined using immunohistological methods with specific antibodies to the capsaicin receptor TRPV1, related vanilloid thermoreceptors TRPV3 and TRPV4, and nerve growth factor (NGF).TRPV1-positive intra-epidermal nerve fibres were significantly increased in patients with breast pain and tenderness . Nerve Growth Factor, which up-regulates TRPV1 and induces nerve sprouting, was present basal keratinocytes: some breast pain specimens also showed NGF staining in supra-basal keratinocytes. TRPV4-immunoreactive fibres were present in sub-epidermis but not significantly changed in painful breast tissue. Both TRPV3 and TRPV4 were significantly increased in keratinocytes in breast pain tissues; TRPV3, median [range] – no pain group, n = 6, 0.75 [0–2]; pain group, n = 11, 2 [8; p = 0.Increased TRPV1 intra-epidermal nerve fibres could represent collateral sprouts, or re-innervation following nerve stretch and damage by polymodal nociceptors. Selective TRPV1-blockers may provide new therapy in breast pain. The role of TRPV3 and TRPV4 changes in keratinocytes deserve further study. Breast pain is a common problem, which can affect up to 70% of women . Breast The mechanisms of breast pain in the majority of women are not well understood at the cellular or molecular level. We hypothesized a relationship between clinical breast pain, nerve growth factor (NGF) and its regulated ion channels or receptors expressed by nociceptor fibres. Estrogens upregulate NGF receptor mRNA in sensory neurons , and enhEighteen patients were recruited  at Chelsea and Westminster, Charing Cross, Ravenscourt Park Hospitals in London and Broomfield Hospital in Essex were recruited. Breast reduction patients had no previous surgery. The breast reconstruction patients had Latissimus dorsi flap reconstructions after previous mastectomies, and had implants. Patients below 18 years or above 70 years, with any local skin inflammation, infection or cancerous skin changes were excluded. The Research Ethics Committee of Hammersmith Hospitals Trust and Mid Essex Hospitals Trust gave ethical permission for the study. Informed consent was obtained prior to the clinical examination and questionnaire administration.Age, parity, height, weight and menstrual data were collected. Details of current surgery, any previous breast surgery and breast disease were also recorded. A questionnaire which included questions on breast and period pain was administered, a diagram to indicate painful and tender areas and the 78 pain descriptors from the McGill Pain Questionnaire  were proFull thickness skin biopsies were collected from each patient along the incision line of about 2 mm depth. Samples were coded, frozen on site and stored at -70°C. The skin samples were mounted in embedding medium (Tissue-Tek OCT compound, Sakura Finetek, USA). Frozen tissue sections (10 μm) were collected onto poly-L-lysine-coated (Sigma Poole Dorset UK) glass slides and post-fixed in freshly prepared, 4% w/v paraformaldehyde in phosphate buffered saline . After washing in PBS, endogenous peroxidase was blocked by incubation with 0.3% w/v hydrogen peroxide in methanol. After a further wash in PBS the tissue sections were incubated overnight with affinity purified antibodies to TRPV1 , TRPV3 , TRPV4 , recombinant human NGF  or marker of large and some small calibre nerve fibres . Methodological controls included omission of primary antibodies, or their replacement with pre-immune serum. Specificity of antibodies has been described in previous publications ,20. SiteA non-parametric, two tailed test (Mann Whitney U) was used. Commercially available statistical software was used to perform the test (Prism 3™).Pain Rating Indices (PRI) and Visual Analogue Scores (VAS) were used to group patients with (n = 10) and without (n = 8) breast pain: pain group; PRI -Median (range) 12 (4–30); Mean (SEM) 13.44(3.07) and VAS – Median (range) 5(3.7–6.7); Mean (SEM) 5.14(0.31)]; in patients without breast pain, PRI and VAS were zero. Only 2 patients reported thermal pain descriptors (burning, hot), while most reported ache and tenderness. The numbers of breast reduction and reconstruction patients with pain were 7 out of 12 and 3 out of 6 respectively, and all reported pain of duration greater than 6 months. The presence (n = 8) or absence (n = 10) of dysmenorrhoea was also recorded. There was no association between breast pain and the presence of dysmenorrhoea.TRPV1-immunoreactive fibres were present mainly in the sub-epidermis in normal (pain free) skin Fig. . In breaTRPV3 immunoreactivity was detected in basal keratinocytes and occasional suprabasal cells throughout the epidermis. Quantification of immunostaining showed a significant (*P = 0.011) increase with breast pain  and the expression of the capsaicin receptor 1 (TRPV1) in nociceptor fibres, as these are key molecules in pain and hypersensitivity. Our finding, that TRPV1-positive intra-epidermal fibres were significantly increased in patients with breast pain and tenderness, is both novel and important. The increased and abnormal "clusters" of intra-epidermal fibres were shown in patients who had no previous breast surgery, and no known episodes of mastitis. Hence this may be a surrogate marker for "idiopathic" or macromastia related breast pain in some patients. Studies are in progress to correlate changes in TRPV1 nerve fibres with quantitative sensory perception thresholds.The cause of the increased intra-epidermal fibres is not known. Given the trend to thinning of the epidermis, normally associated with denervation , in the Our recent studies have demonstrated increased TRPV1-immunoreactive nerve fibres in inflammatory bowel disease , and in Little is known of the roles of TRPV3 and TRPV4 in human pain pathophysiology and keratinocyte function. While we have previously demonstrated TRPV3 in human sensory neurons , no TRPVBreast pain and tenderness appears to be associated with abnormal intra-epidermal innervation. This may reflect re-innervation of skin following nerve stretch damage, and/or collateral sprouting. While further studies are necessary to establish functional links between the TRPV1, TRPV3 and TRPV4 immunohistological changes and breast pain, our findings indicate a path for increasing understanding and treatment of breast pain.TRPV = transient receptor potential vanilloid; NGF = Nerve Growth Factor; VAS = Visual Analogue Score; PRI = Pain Rating IndexThe author(s) declare that they have no competing interests.PG and EW recruited patients, collected biopsies and participated in immunohistology studies. PF participated in immunohistology and coordination of the study. AH participated in design of the study and recruitment of patients. JD, GS and CB provided antibodies and helped draft the manuscript. PA conceived the original study, its design and coordination, and helped with the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholders It is indeed regrettable that a distinguished and thoughtful scientist such as Elizabeth Blackburn should have been dismissed from the President's Council on Bioethics. Scientific perspectives such as hers are surely needed on this committee.Her dismissal was apparently the consequence of her disagreement with some of the text of the Council's report, “Beyond Therapy: Biotechnology and the Pursuit of Happiness” (2003). The thrust of this report is that some of the directions of current biological research will, if carried to fulfillment, result in major changes in the nature of human life—changes that the report regards with foreboding.In their essay, Drs. I would suggest that as scientists we should face these issues forthrightly. We should not seek refuge in presentday uncertainties. The authors of the report are not naïve nor ignorant. Yes, if these lines of research are successful, their outcome will change the nature of human life.As an example, consider current research into the causes of aging. Clearly, we do not at present know how to achieve major increases in the human life span . But it is plausible that we will learn how to do so. And surely a, say, doubling of the human life span would change the nature of human life.Likewise, if we learn to modify the human gene pool so as to produce exceptional individuals or to alter human capabilities, or if powerful drugs are developed that may commandeer the human psyche, the nature of human life will be altered.But so be it. The nature of human life has changed repeatedly and profoundly in the past—with the invention of agriculture, with the invention of writing, with the development of machines and mechanical power, with the advent of modern science and medicine. The nature of human life is different in 2004 a.d. from what it was in 1000 a.d. or 46 b.c. or 5000 b.c. or 10,000 b.c., and it will change again in the future.The concerns expressed in the report are earnest, and they should be confronted in earnest.


They receive signals from various sources (e.g. cell surface receptors) and control the level of transcription initiation, which largely determines gene expression. In vertebrates, transcription start sites and surrounding regulatory elements are often poorly defined. To support promoter analysis, we present CORG The automated annotation of promoter regions integrates information of two kinds. First, statistically significant cross-species conservation within upstream regions of orthologous genes is detected. Pairwise as well as multiple sequence comparisons are computed. Second, binding site descriptions (position-weight matrices) are employed to predict conserved regulatory elements with a novel approach. Assembled EST sequences and verified transcription start sites are incorporated to distinguish exonic from other sequences.. The benefits of our framework are exemplarily shown in the context of phylogenetic profiling of transcription factor binding sites and detection of microRNAs close to transcription start sites of our gene set.As of now, we have included 5 species in our analysis pipeline (man, mouse, rat, fugu and zebrafish). We characterized promoter regions of 16,127 groups of orthologous genes. All data are presented in an intuitive way via our web site. Users are free to export data for single genes or access larger data sets via our DAS server The CORG platform is a versatile tool to support analyses of gene regulation in vertebrate promoter regions. Applications for CORG cover a broad range from studying evolution of DNA binding sites and promoter constitution to the discovery of new regulatory sequence elements (e.g. microRNAs and binding sites). Applications range from grouping of sequences (e.g. protein sequences into families) to Speaking of gene regulation, it has been known for a long time that there is considerable sequence conservation between species in non-coding regions of the genome. A comprehensive explanation of this observation is still elusive. However, sequence conservation within promoter regions of genes often stems from transcription factor binding sites that are under selective pressure (see  for a remir-10 and mir-196 which are located in the (short) intergenic regions in the Hox gene clusters of vertebrates [Conserved sequence elements of other types have recently caught much attention. Not all non-coding conserved DNA in the vicinity of a gene's transcription start site necessarily functions at the level of transcriptional regulation. For example, most known methylation-guide snoRNAs are intronencoded and processed from transcripts of housekeeping genes . A few mtebrates -7.A second class of conserved sequence elements exert their function as regulatory motifs in the untranslated region (UTR) of the primary transcript or the mature mRNA. The UTRsite database , for exaThe CORG framework aims at detecting and describing regulatory elements that are proximal to the transcription start site. In this context, the comparison of upstream regions of orthologous genes is particularly valuable. This concept is called "phylogenetic footprinting" and an overview of this approach can be found in .Phylogenetic footprinting in a strict sense is carried out on orthologous promoter regions. Local sequence similarities can then be directly interpreted as related regions harboring conserved functional elements. We denote these similarities as Conserved Non-coding Blocks (CNBs).Homo sapiens, Mus musculus, Rattus norvegicus, Danio rerio and Fugu rubripes. Multiple alignments are built from pairwise CNBs as described in the subsequent section.Comparative approaches gain power from the inclusion of sequences from more than two species . Multi-sIn this work, we take a gene-centered view of phylogeny. Homology among proteins and thus genes is often concluded on the basis of sequence similarity. The EnsEMBL database  allows tVarious recent experimental efforts supply information about the position of transcriptional start sites in the human and mouse genome. Table Some repositories offer genomic coordinates for their start site entries. Existing genomic mapping information was incorporated unless the underlying genome assembly build differed. The remaining data were projected onto the genome with SSAHA (Sequence Search and Alignment by Hashing Algorithm), a rapid near-exact alignment algorithm .The notion of "promoter region" deserves some further explanation in the context of our approach. Typically, though not exclusively, we expect conserved regulatory regions to appear in the vicinity of the transcription start site of a gene. Since we do not know the precise location of the start of transcription for each and every gene, we chose to compare the sequence regions upstream of the start of translation from orthologous genes. If verified transcription start sites are known, we define a sequence window that is large enough to hold both, translation and transcription start sites, plus 5 kB upstream sequence. In case we lack this information, our observations on known transcription start sites indicate that most promoter regions should be captured in a sequence window of 10 kb size  in two sequences are computed with an implementation of the Waterman-Eggert algorithm. We have already given an account of the algorithm and statistics in ,20. The Q is the transition rate matrix. We left out the elements on the diagonal, which are constrained by the requirement that the sum of all elements in a row equals zero.where πi are the stationary nucleotide frequencies, their sum is constrained to be one. Although the two genomes under consideration are in general not in their stationary state with respect to the substitutional process we take the mean  of the two observed nucleotide frequencies, , to be the best estimate of the stationary base composition.The πi we have to allow for deviation from those ratios. We do this by choosing for example α ∝ (R(A → T)/πT + R(T → A)/πA)/2, where R(i → j) is either 1, 3, or 5 depending on the process under consideration. At the end we scale the matrix Q, such that the PAM distance [From other studies we have further knowledge about the relative rates between transversions, the transition A:T→G:C, and the transition G:C→A:T, which occur in roughly in the ratio 1:3:5 along vertebrate lineages . These rdistance  of the sSince we were mainly interested in highly conserved regulatory elements, we demanded an average similarity level at least as high as the average exon conservation between the species under comparison.i and j is then s(i, j) = log(P(i, j)/(πiπj)) where P(i, j) is the probability of finding the pairing of i and j under the above substitution model [The score for aligning two nucleotides on model .O = (V, E) with vertices V and edges E is constructed, which represents the species-internal overlap of CNBs on the genomic coordinate level. Each vertex a ∈ V represents a footprint, which is a pairwise local alignment between two species. An undirected edge is placed between two vertices if the corresponding CNBs have only one species in common and show an overlap of at least 10 bp on the sequence level.All CNBs from pairwise sequence alignments are split up into groups as defined by gene homology. For each group a graph O, cliques of minimal size three are detected with an implementation of the Bron-Kerbosh algorithm [In our graph lgorithm . Only thlgorithm  who applN and edges E, which are one-way edges and form no cycles.Partial order graphs belong to the class of directed acyclic graphs (DAGs). A DAG is a graph consisting of a set of nodes The multiple alignment problem is then reduced to to subsequent alignment steps of individual sequences to a growing multiple alignment graph. If the sequences to be aligned share substantial sequence similarity, the number of bifurcation points within the POG stays low and allows rapid computation of the multiple alignment.Alignment results are subsequently trimmed to encompass the leftmost and rightmost ungapped block of at least 6 nucleotides.GENENEST [-4 is applied.Promoter regions in CORG always extend upstream from the most downstream coding start (ATG). As a consequence, promoter regions may contain exons that are not translated. Our way of detecting such exons is a similarity search of man-mouse footprints versus GENENEST , a databGENENEST . An E-vaThe TRANSFAC database  is a repS: one is given by the signal profile F and the other one by the background model B. Under both models the distribution of weight matrix scores can conveniently be calculated by convolution, since the score is a sum of independent random variables. Probability mass distributions of PF (Score(S)) as well as PB(Score(S)) can be computed by dynamic programming if column scores are reasonably discretized. This allows a fine tuning of the proportions of false positives and negatives for each TRANSFAC weight matrix. Both error levels were set to be equal. All details are given in [Potential binding sites are detected with TRANSFAC weight matrices by the method of . Here, tgiven in .We now present an overview of the web interface of the database and several example applications. and offers a redesigned web interface. From the search page one can quickly jump to gene loci via EnsEMBL or other standard identifers (e.g. HUGO symbol, LocusLink identifier, ...). The search query is processed according to the chosen reference source and a list of all matching database entries is returned to the user. This list serves as a springboard to a summary page where the genomic context of the selected gene and its similarities to other upstream regions is visualized as in Figure The CORG database is accessible via its home page Pairwise as well as multiple comparisons are displayed on demand at this stage with a JAVA applet that complies with the JDK 1.1 standard. Alternatively, upstream region sequence and corresponding annotation can be exported in EMBL format . The JAVA applet should run on all JAVA-compatible web browsers. Detailed information about the conserved non-coding block structure are simultaneously shown for multiple upstream regions of different species. If available, annotation information on putative binding sites of transcription factors and EST matches are displayed for the query sequence. The applet facilitates zooming into sequence and annotation. In addition, web links are assigned to sequence features that relate external data sources to the corresponding annotation.DAS, [ constitutes such an external data source. Position information on all conserved non-coding blocks and mapped promoters is accessible from this DAS server. Each DAS sequence feature provides a link to the corresponding CORG database entry. New DAS sources can be easily added to the ENSEMBL display. A small tutorial on installing external DAS data sources is available on our web page .CORG data may be also embedded into other viewers or programs via the distributed annotation system (DAS, ). DAS faAdditionally, tools for on-site batch retrieval of CORG data will be added to the web portal in the near future.One potential application of CORG is phylogenetic profiling of promoter regions. We define phylogenetic profiling in the context of gene regulation as comparative analysis of presence/absence patterns of binding sites in promoter regions. Here, we consider conserved predicted binding sites and contrast them with validated ones.6GG) in target gene promoters and by recruiting different co-factors. SRF regulates transcription downstream of MAPK signaling in association with ternary complex factors (TCFs) . TCFs bFigure We get a better idea on the cause of sequence conservation by browsing the multiple alignment. Textual information can be obtained by clicking on the alignment boxes. Then, the alignment appears in a pop-up window and may be copied to another destination. In Figure L ≥ 50 against the Rfam database [Non-coding RNA can be classified as transcribed regulatory elements. Non-coding RNAs are also accessible to the user via the CORG database. Since we were primarily interested in non-coding RNAs rather than small mRNA motifs we restricted our search here to long CNBs. A blast search of our multiple alignments with length database  and the database  identifiThe snoRNA U93 is an unusual mammalian pseudouridinylation guide RNA which accumulates in Cajal (coiled) bodies and it is predicted to function in pseudouridylation of the U2 spliceosomal snRNA . It appemir10 and the mir196 precursors are located at specific positions in the Hox gene clusters [mir-196 family regulates Hox8 and Hox7 genes, the function of mir10 is unknown.The known microRNAs belong to four different groups. The clusters -7. The mmir10 is a good example of this typical substitution pattern, which gives rise to a hairpin structure. The pairwise correlation structure of nucleotides is depicted on top of the multiple alignment in Figure SLCA1, a member of the sodium transporter family. This time the substitution pattern does not meet the minimal length of the microRNA definition above. Nevertheless, it is conserved across all vertebrate species as shown in Figure For a microRNA we expect a subsequence of about 20 nt that is almost absolutely conserved among vertebrates (the mature miRNA) and a well-conserved complementary sequence forming the other side of the stem from which the mature microRNA is excised. In contrast, the substitution rate should be much larger in the loop region of the hairpin . mir10 iWe have improved and extended our framework of comparative analysis and annotation of vertebrate promoter regions over previous releases (see ). The fo• Mapping of validated promoter regions and proper adjustment of the extent of upstream regions.• Multiple alignments from significant local pair wise alignments.• Novel approach to predict transcription factor binding sites.• Web site offers now a genomic context view .We presented selected application examples from the realm of vertebrate gene regulation. Conserved regulatory elements of different kinds (binding sites, microRNAs and UTR elements) are readily accessible to CORG users. New genomes and annotation will be continuously added to CORG.. Programs, scripts and MySQL database dumps are available from the authors upon request.The database is freely accessible through the website Christoph Dieterich built the entire pipeline and some parts of the web interface. Steffen Grossmann annotated transcription factor binding sites and provided parts of the web interface. Andrea Tanzer analyzed known and novel RNA elements in the multiple alignments of the CORG database. Stefan Röpcke set up our database of binding site descriptions. Peter F. Arndt worked on an appropriate alignment scoring scheme. Peter F. Stadler and Martin Vingron initiated this work and provided all necessary infrastructure.Distribution of distance between start of transcription and translation. Histogram of observed genomic distances between start sites of transcription and translation in man for 1,700 entries from the EPD. The red and blue line indicates the 90% and 95% quantiles, respectively. Distances greater than 106 bp were exluded from the analysis as they mostly occur due to mismappings in the ENSEMBL database.Click here for file