Models for prediction and recognition of eukaryotic promoters.
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Animals↗
Biomedical subjects
T Werner
Publications and source records attributed to T Werner.
Radiosynthesis and autoradiographic evaluation of [11C]NAD-299, a radioligand for visualization of the 5-HT1A receptor.
The selective 5-HT1A receptor antagonist NAD-299 ([R]-3-N,N-dicyclobutylamino-8-fluoro-3,4- dihydro-2H-1-benzopyran-5-carboxamide) was labeled with the positron-emitting radionuclide carbon-11. The radioligand was synthesized from NAD-195 ([R]-3-N,N-dicyclobutylamino-8-fluoro-5-trifluoromethylsulfonyl oxy-3,4- dihydro-2H-1-benzopyran) in two radiochemical steps. A palladium-catalyzed reaction of NAD-195 and [11C]cyanide was followed by hydrolysis of the carbon-11-labeled nitrile intermediate with basic hydrogen peroxide. The total radiochemical yield, based on [11C]CO2 and corrected for decay, was 20-40%. The specific radioactivity was 24 GBq/mumol (900 Ci/mmol) at end of synthesis, with a radiochemical purity better than 99% and a total synthesis time of 40-45 min. Autoradiographic examination of [11C]NAD-299 binding in human brain postmortem demonstrated high binding in hippocampus, raphe nuclei, and neocortex. The binding in the hippocampus was higher than in the neocortex. Within the hippocampus, the densest binding was observed in the CA1 region. [11C]NAD-299 binding was inhibited by addition of the 5-HT1A receptor ligands WAY-100635, pindolol, (+/-)-8-OH-DPAT, 5-HT, and buspirone, leaving a low background of nonspecific binding. The results indicate that [11C]NAD-299 binds specifically to 5-HT1A receptors in the human brain in vitro and is a potential radioligand for positron emission tomography (PET) examination of 5-HT1A receptors in vivo.
Autoradiography↗
Increased steady state mRNA levels of the STM and KNAT1 homeobox genes in cytokinin overproducing Arabidopsis thaliana indicate a role for cytokinins in the shoot apical meristem.
This study investigates the consequences of endogenously enhanced biosynthesis of the plant hormone cytokinin in Arabidopsis thaliana (L.) Heynh. Transcriptional control of the bacterial ipt gene by the Drosophila melanogaster hsp70 promoter enabled temperature-dependent increased cytokinin production in transgenic plants. Heat-treated plants accumulated higher levels of unbound and bound zeatin-type cyto-kinins, the latter being preferentially N-conjugated glucosides. Cytokinin overproduction significantly increased the biomass of seedlings. Ipt transgenics had higher steady state mRNA levels of the shoot meristem specifying homeobox genes KNAT1 and STM, similar to the cytokinin-overproducing shoot meristem mutant amp1 (hpt, cop2, pt) This finding, together with previously described phenotypic similarities between transgenic cytokinin-overproducing plants and plants overexpressing the KNAT1 or KN1 genes, suggests that these factors act on the same pathway. We hypothesize that cytokinins act upstream of KNAT1 and STM. The influence of cytokinins on homeobox genes provides a link between the hormone and the developmental genes and indicates a role for cytokinins in the shoot apical meristem.
Alkyl and Aryl Transferases↗
Erectile dysfunction caused by sacral gun-shot injury.
A 22-year-old man suffering from isolated erectile dysfunction associated with damage to the right spinal nerve S2 caused by sacral gun-shot injury. He has no loss of bladder innervation. Treatment has been implantation of a penile prosthesis.
Adult↗
Functional promoter modules can be detected by formal models independent of overall nucleotide sequence similarity.
MOTIVATION: Gene regulation often depends on functional modules which feature a detectable internal organization. Overall sequence similarity of these modules is often insufficient for detection by general search methods like FASTA or even Gapped BLAST. However, it is of interest to evaluate whether modules, often known from experimental analysis of single sequences, are present in other regulatory sequences. RESULTS: We developed a new method (FastM) which combines a search algorithm for individual transcription factor binding sites (MatInspector) with a distance correlation function. FastM allows fast definition of a model of correlated binding sites derived from as little as a single promoter or enhancer. ModelInspector results are suitable for evaluation of the significance of the model. We used FastM to define a model for the experimentally verified NFkappaB/IRF1 regulatory module from the major histocompatibility complex (MHC) class I HLA-B gene promoter. Analysis of a test set of sequences as well as database searches with this model showed excellent correlation of the model with the biological function of the module. These results could not be obtained by searches using FASTA or Gapped BLAST, which are based on sequence similarity. We were also able to demonstrate association of a hypothetical GRE-GRE module with viral sequences based on analysis of several GenBank sections with this module. AVAILABILITY: The WWW version of FastM is accessible at: http://www.gsf.de/cgi-bin/fastm. pl and http://genomatix.gsf.de/cgi-bin/fastm2/fastm.pl
Algorithms↗
Report of the eighth international workshop on the identification of transcribed sequences.
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Animals↗
A pseudoautosomal boundary-like element adjacent to the SSAV1 locus at 18q21.
Pseudoautosomal boundary-like (PABL) elements have been found at transition sites between genomic regions with different GC-contents. A new PAB related sequence was found immediately adjacent to the S71 provirus on human chromosome 18q21.1-2 (officially designated the SSAV1 locus). The S71 PABL element was full-length as defined by comparison with elements identified at the pseudoautosomal boundaries of the sex chromosomes and in the MHC region. The 3'-ends of all PABL elements showed significant homology to functional CpG-islands, indicating that this similarity is a new common feature of PABL elements.
Base Sequence↗
Computer-assisted methods for the identification and characterization of polymerase II promoters.
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Animals↗
In vivo labelling of the mouse brain 5-hydroxytryptamine1A receptor with the novel selective antagonist 3H-NAD-299.
The in vivo labelling of 5-hydroxytryptamine (5-HT)1A receptors in the mouse brain was studied with the novel selective 5-HT1A receptor antagonist, NAD-299 ((R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran- 5-carboxamide hydrogen (2R,3R)-tartrate monohydrate). 3H-NAD-299 was injected in a tail vein and the radioactivity in various brain regions was determined. More than 90% of the radioactivity in hippocampus, 15 min after the injection, was intact NAD-299. At this time the amount of 3H-NAD-299 was highest in hippocampus followed by frontal cortex, mesencephalon, hypothalamus, striatum and cerebellum. The specific accumulation of radioactivity (after subtracting cerebellum values) in frontal cortex and hippocampus was maximal 10 to 30 min after the injection and had almost disappeared after 2 h. Saturation kinetics derived Bmax (pmol/g wet weight tissue) values of 19.6+/-2.0 in frontal cortex and 38.0+/-3.5 in hippocampus. The apparent Kd values expressed in nmol/kg 3H-NAD-299 injected, were 12.3+/-2.2 in frontal cortex and 20.3+/-3.1 in hippocampus. The 5-HT1A receptor antagonist, WAY-100,635 competitively inhibited the specific accumulation of 3H-NAD-299 and was about equipotent with unlabelled NAD-299 with ED50 values of 20-30 nmol/kg s.c. These compounds were about 10 times more potent than the 5-HT1A receptor antagonists, p-MPPI and NDL-249 and 100 times more potent than (S)-UH-301. 5-HT1A receptor agonists, e.g. 8-OH-DPAT and flesinoxan and partial agonists, e.g. pindolol, buspirone and ipsapirone had low potency in this in vivo assay. Spiperone and methiothepin inhibited the 3H-NAD-299 accumulation at 10 micromol/kg s.c. The alpha1-adrenoceptor antagonist, prazosin at 2 micromol/kg s.c. increased significantly the specific accumulation of 3H-NAD-299. Pretreatment of the mice with the non-selective, irreversible receptor antagonist, EEDQ produced a dose related long-lasting decrease in the accumulation of 3H-NAD-299. It is concluded that NAD-299 is a very suitable ligand for studies of 5-HT1A receptors in the brain in vivo.
Alkylating Agents↗
Detection of potential target genes in silico?
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Binding Sites↗
Kappa-casein polymorphisms in Indian dairy cattle and buffalo: a new genetic variant in buffalo.
We screened 57 Sahiwal cattle (Bos indicus) and 53 Murrah, 19 Nili-Ravi and 11 Egyptian buffaloes (Bubalus bubalis) to detect the polymorphisms at kappa-casein (CSN3) gene using the polymerase chain reaction (PCR). CSN3 A and B alleles were identified by PCR-RFLP using the restriction enzymes that detect the underlying nucleotide changes at codon 136 (Taql) and at codon 148 (HindIII or HinfI) in cattle. The frequency of CSN3 B allele in the Sahiwal cattle was estimated as 0.16 with no homozygous BB animal. Using the same set of primers as used in the Sahiwal cattle, a part of exon IV of buffalo CSN3 gene was amplified, but restriction enzyme analysis using HindIII/HinfI and TaqI did not reveal any polymorphism. However, DNA sequencing of the amplified fragment (GenBank Acc. No. U96662) revealed one polymorphism at codon 135 (ThrACC -->I1eATC) in buffalo; the frequencies of 135 Thr/Ile alleles were estimated as 0.88 and 0.12, respectively.
Alleles↗
DIALIGN: finding local similarities by multiple sequence alignment.
MOTIVATION: DIALIGN is a new method for pairwise as well as multiple alignment of nucleic acid and protein sequences. While standard alignment programs rely on comparing single residues and imposing gap penalties, DIALIGN constructs alignments by comparing whole segments of the sequences. No gap penalty is employed. This point of view is especially adequate if sequences are not globally related, but share only local similarities, as is the case in genomic DNA sequences and in many protein families. RESULTS: Using four different data sets, we show that DIALIGN is able correctly to align conserved motifs in protein sequences. Alignments produced by DIALIGN are compared systematically to the results of five other alignment programs. AVAILABILITY: DIALIGN is available to the scientific community free of charge for non-commercial use. Executables for various UNIX platforms including LINUX can be downloaded at http://www.gsf.de/biodv/dialign.html CONTACT: werner, morgenstern@gsf.de
Algorithms↗
Coagulatory response after femoral instrumentation after severe trauma in sheep.
OBJECTIVE: Pulmonary complications after intramedullary femoral nailing have been attributed to bone marrow fat embolization and a variety of cascade effects. We investigated whether the coagulatory response after intramedullary femoral nailing in merino sheep is altered after severe trauma. METHODS: Adult merino sheep were submitted to hemorrhagic shock (2 hours, 50 mm Hg) and unilateral lung contusion. After recovery (day 3 of the study), reamed femoral intramedullary nailing (RFN), unreamed femoral intramedullary nailing (UFN), or plate osteosynthesis of the femur (P) was performed. Pulmonary artery pressure, central venous levels of factor V, protein C, antithrombin III, and fibrinogen, were determined. At 1 and 3 days before and after femur instrumentation, pulmonary capillary permeability was assessed on the basis of the comparative albumin content in bronchoalveolar lavage fluid. RESULTS: Group RFN, n=8; group UFN, n=7; group P, n=6. A significant (p < 0.05) postoperative increase in the relative albumin content in terms of the BAL/plasma albumin ratio was measured in group RFN (day 3 preoperatively: 0.38+/-0.05 day 3 postoperatively: 0.53+/-0.06, p < 0.05 (RFN vs. P), which contrasted with group UFN (day 3 preoperatively: 0.44+/-0.09, day 3 postoperatively: 0.46+/-0.09, no significant difference). This evidence of increased pulmonary permeability occurred in association with evidence of increased activation of coagulation factors (data presented as percentage of day 3 preoperative baseline values). The data for fibrinogen (15 min postoperatively) is as follows: group RFN, 74+/-9% (p < 0.05 vs. P); group UFN, 83+/-8% (not significant); group P, 98+/-6%. The data for antithrombin III (15 min. postoperatively) is as follows: group RFN, 72+/-6% (p < 0.05 vs. P); group UFN, 79+/-8% (not significant); group P, 92+/-8% (not significant). CONCLUSION: After severe trauma, an increase of pulmonary permeability after reamed femoral nailing was associated with increased consumption of coagulation factors. After unreamed nailing, a similar trend was apparent, but this was not found to be statistically significant. These data provide support for the theory that after severe trauma, unreamed femoral nailing reduces but does not abolish pulmonary sequelae when compared with reamed femoral nailing.
Adolescent↗
Proviral structure, chromosomal location, and expression of HERV-K-T47D, a novel human endogenous retrovirus derived from T47D particles.
We previously described that type B retrovirus-like particles released from the human mammary carcinoma cell line T47D are pseudotypes and package retroviral RNA of different origins (W. Seifarth, H. Skladny, F. Krieg-Schneider, A. Reichert, R. Hehlmann, and C. Leib-Mösch, J. Virol. 69:6408-6416, 1995). One preferentially packaged retroviral sequence, ERV-MLN, has now been used to isolate the corresponding full-length provirus from a human genomic library. The 9,315-bp proviral genome comprises a complete retroviral structure except for a 3' long terminal repeat (LTR) truncation. A lysine tRNA primer-binding site and phylogenetic analyses assign this human endogenous retroviral element, now called HERV-K-T47D, to the HML-4 subgroup of the HERV-K superfamily. The gag, prt, pol, and env genes exhibit 40 to 60% amino acid identity to HERV-K10. HERV-K-T47D is located on human chromosome 10, with five closely related elements on chromosomes 8, 9, 15, 16, and 19 and several hundred HERV-K-T47D-related solitary LTRs dispersed over the human genome. HERV-K-T47D-related sequences are detected in the genomes of higher primates and Old World monkeys but not in those of New World monkeys. High HERV-K-T47D transcription levels were observed in human placenta tissue, whereas transcription in T47D cells was strictly steroid dependent.
Amino Acid Sequence↗
Muscle actin genes: a first step towards computational classification of tissue specific promoters.
Tissue-specific gene expression is governed by enhancer and promoter sequences determining the specificity most probably by their internal organization of transcription factor binding sites. In case of muscle-specific gene expression excellent compilations of sequence regions responsible for the tissue-specificity are available. We took advantage of such a compilation in order to elucidate organizational features that are directly correlated with promoter specificity. We chose a systematic approach solely based on a sequence collection known to consist of specific regulatory regions which can in principle be applied to every precompiled set of such sequences. We were able to show that these sequences contained a detectable subgroup (actin promoters) for which it was possible to construct a highly specific promoter model recognizing the majority of all known actin sequences. The model was robust with respect to different training sets, almost 100% specific and sensitive enough to be suitable for database searches. We believe this pilot study demonstrates the general applicability of our approach as well as the concept of modular promoter organization.
Actins↗
A novel method to develop highly specific models for regulatory units detects a new LTR in GenBank which contains a functional promoter.
Functional promoters are composed of individual modules (e.g. transcription factor binding sites, secondary structure elements, repeats) arranged in distinct patterns. Recognition of such patterns is essential for identification of promoters in non-coding sequences. However, this is difficult due to the absence of overall sequence similarity in promoters even if they are regulated in a similar way. We implemented simple formal representations of general features of regulatory regions into an algorithm capable of developing complex models reflecting both the element composition and the functional organization of individual elements (ModelGenerator). Though ModelGenerator requires a very simple initial model (e.g. two modules and their relative order) it will generate a much more sophisticated model by analysis of the training set of at least ten sequences. We show ModelGenerator to successfully model different retroviral long terminal repeat (LTR) classes (Lentivirus as well as avian and mammalian C-type) which contain functional promoters. Database searches with the program ModelInspector demonstrated the high specificity of these models and no apparent false negatives were detected. We also verified one match from GenBank to the mammalian C-type LTR model experimentally and showed this sequence to contain an active promoter. Thus, the concept of modular organization of functional regulatory DNA regions (e.g. promoters) could be successfully implemented into a set of computer tools which might be flexible and specific enough to be suitable for prospective analysis of new genomic DNA sequences.
Algorithms↗
Identification of endogenous retroviral sequences based on modular organization: proviral structure at the SSAV1 locus.
The current genome sequencing projects reveal megabases of unknown genomic sequences. About 1% of these sequences can be expected to be of retroviral origin. These are often severely deleted or mutated. Therefore, identification of the retroviral origin of these sequences can be very difficult due to the absence of convincing overall sequence similarity. There are also many copies of solo-LTRs (long terminal repeats) distributed throughout genomic sequences. LTR and envelope sequences in general are among the most divergent parts of the retroviral genome and thus especially hard to detect in mutated endogenous sequences. We took advantage of the fact that these retroviral sections contain short highly conserved sequence regions providing retroviral hallmarks even after loss of overall similarity. We defined several sequence elements and peptide motifs within LTR and Env sequences and used these elements to construct models for LTRs and Env proteins of mammalian C-type retroviruses. We then used this strategy to identify successfully the hitherto missing LTRs and an env-like region in the S71 human retroviral sequence. Our approach provides a new strategy for identifying remotely related retroviral sequences in genomic DNA (especially human DNA), of potential significance for the interpretation of genomic sequences obtained from the current large-scale sequencing projects.
Amino Acid Sequence↗
Molecular cloning of ozone-inducible protein from Pinus sylvestris L. with high sequence similarity to vertebrate 3-hydroxy-3-methylglutaryl-CoA-synthase.
We have isolated a Pinus sylvestris cDNA encoding a globular protein of 474 amino acids with a predicted molecular weight of 52,995 Da. The deduced amino acid sequence showed 41.9% identity and 13.6% similarity to mammalian cytosolic 3-hydroxy-3-methylglutaryl-CoA-synthase (HMGS). Treatment of Scots pine seedlings with ozone resulted in a transient increase of a 1.95 kb transcript, whereas a 1.2 kb mRNA decreased transiently, indicating a possible influence of ozone on isoprenoid biosynthesis.
Amino Acid Sequence↗