GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions.

Improving the accuracy of prediction of gene starts is one of a few remaining open problems in computer prediction of prokaryotic genes. Its difficulty is caused by the absence of relatively strong sequence patterns identifying true translation initiation sites. In the current paper we show that the accuracy of gene start prediction can be improved by combining models of protein-coding and non-coding regions and models of regulatory sites near gene start within an iterative Hidden Markov model based algorithm. The new gene prediction method, called GeneMarkS, utilizes a non-supervised training procedure and can be used for a newly sequenced prokaryotic genome with no prior knowledge of any protein or rRNA genes. The GeneMarkS implementation uses an improved version of the gene finding program GeneMark.hmm, heuristic Markov models of coding and non-coding regions and the Gibbs sampling multiple alignment program. GeneMarkS predicted precisely 83.2% of the translation starts of GenBank annotated Bacillus subtilis genes and 94.4% of translation starts in an experimentally validated set of Escherichia coli genes. We have also observed that GeneMarkS detects prokaryotic genes, in terms of identifying open reading frames containing real genes, with an accuracy matching the level of the best currently used gene detection methods. Accurate translation start prediction, in addition to the refinement of protein sequence N-terminal data, provides the benefit of precise positioning of the sequence region situated upstream to a gene start. Therefore, sequence motifs related to transcription and translation regulatory sites can be revealed and analyzed with higher precision. These motifs were shown to possess a significant variability, the functional and evolutionary connections of which are discussed.

Leaderless transcripts of the crenarchaeal hyperthermophile Pyrobaculum aerophilum.

We mapped transcription start sites for ten unrelated protein-encoding Pyrobaculum aerophilum genes by primer extension and S(1) nuclease mapping. All of the mapped transcripts start at the computationally predicted translation start codons, two of which were supported by N-terminal protein sequencing. A whole genome computational analysis of the regions from -50 to +50 nt around the predicted translation starts codons revealed a clear upstream pattern matching the consensus sequence of the archaeal TATA box located unusually close to the translation starts. For genes with the TATA boxes that best matched the consensus sequence, the distance between the TATA box and the translation start codon appears to be shorter than 30 nt. Two other promoter elements distinguished were also found unusually close to the translation start codons: a transcription initiator element with significant elevation of C and T frequencies at the -1 position and a BRE element with more frequent A bases at position -29 to -32 (counting from the translation start site). We also show that one of the mapped genes is transcribed as the first gene of an operon. For a set of genes likely to be internal in operons the upstream signal extracted by computer analysis was a Shine-Dalgarno pattern matching the complementary sequence of P. aerophilum 16 S rRNA. Together these results suggest that the translation of proteins encoded by single genes or genes that are first in operons in the hyperthermophilic crenarchaeon P. aerophilum proceeds mostly, if not exclusively, through leaderless transcripts. Internal genes in operons are likely to undergo translation via a mechanism that is facilitated by ribosome binding to the Shine-Dalgarno sequence.

A novel genetic island of meningitic Escherichia coli K1 containing the ibeA invasion gene (GimA): functional annotation and carbon-source-regulated invasion of human brain microvascular endothelial cells.

The IbeA (ibe10) gene is an invasion determinant contributing to E. coli K1 invasion of the blood-brain barrier. This gene has been cloned and characterized from the chromosome of an invasive cerebrospinal fluid isolate of E. coli K1, strain RS218 (018:K1: H7). In the present study, a genetic island of meningitic E. coli containing ibeA (GimA) has been identified. A 20.3-kb genomic DNA island unique to E. coli K1 strains has been cloned and sequenced from an RS218 E. coli K1 genomic DNA library. Fourteen new genes have been identified in addition to the ibeA. The DNA sequence analysis indicated that the ibeA gene cluster was localized to the 98 min region and consisted of four operons, ptnIPKC, cglDTEC, gcxKRCI and ibeRAT. The G+C content (46.2%) of unique regions of the island is substantially different from that (50.8%) of the rest of the E. coli chromosome. By computer-assisted analysis of the sequences with DNA and protein databases (GenBank and PROSITE databases), the functions of the gene products could be anticipated, and were assigned to the functional categories of proteins relating to carbon source metabolism and substrate transportation. Glucose was shown to enhance E. coli penetration of human brain microvascular endothelial cells and exogenous cAMP was able to block the stimulating effect of glucose, suggesting that catabolic regulation may play a role in control of E. coli K1 invasion gene expression. Our data suggest that this genetic island may contribute to E. coli invasion of the blood-brain barrier through a carbon-source-regulated process.

Heuristic approach to deriving models for gene finding.

Computer methods of accurate gene finding in DNA sequences require models of protein coding and non-coding regions derived either from experimentally validated training sets or from large amounts of anonymous DNA sequence. Here we propose a new, heuristic method producing fairly accurate inhomogeneous Markov models of protein coding regions. The new method needs such a small amount of DNA sequence data that the model can be built 'on the fly' by a web server for any DNA sequence >400 nt. Tests on 10 complete bacterial genomes performed with the GeneMark.hmm program demonstrated the ability of the new models to detect 93.1% of annotated genes on average, while models built by traditional training predict an average of 93.9% of genes. Models built by the heuristic approach could be used to find genes in small fragments of anonymous prokaryotic genomes and in genomes of organelles, viruses, phages and plasmids, as well as in highly inhomogeneous genomes where adjustment of models to local DNA composition is needed. The heuristic method also gives an insight into the mechanism of codon usage pattern evolution.

IL-8 derivatives with a reduced potential to form homodimers are fully active in vitro and in vivo.

Interleukin 8 (IL-8) is a member of the CXC subfamily of chemokines which attracts and activates preferentially neutrophilic granulocytes. At nanomolar concentrations monomeric and dimeric forms of the molecule are in equilibrium, with the monomer being the prevalent form. Five amino acids from position 23 to 29 of the 72-amino acid IL-8 sequence form the dimer interface, with Leu25 and Val27 being highly conserved among the CXC chemokines. To investigate the contribution of these amino acids to the dimerization of IL-8, we produced in escherichia coli IL-8 derivatives with phenylalanine substitutions at position 25 or 27, or both 25 and 27. All three recombinant proteins were characterized by a significantly impaired potential to form dimers in solution as seen in chemical crosslinking experiments. IL-8 Val27 also could not be crosslinked as a dimer on its receptors. Receptor affinities and in vitro chemotactic activities, however, were not significantly different between wild-type and IL-8 with single mutations. The dimerization deficient IL-8 analogue had also full inflammatory activity in vivo. Thus, the monomer is the biologically active form of IL-8.

Structure and activity of a chimeric interleukin-8-melanoma-growth-stimulatory-activity protein.

A 72-amino-acid chimeric protein, Chi1, was constructed from the N-terminal part of interleukin 8, IL-8-(1-53), and the C-terminal part of melanoma growth stimulatory activity, MGSA-(54-72). Chi1 protein showed receptor-binding specificity and biological activity similar, but not identical to IL-8 and decidedly different from MGSA. The structure of Chi1 was determined in solution by two-dimensional NMR and molecular-dynamics calculations. The structure resembled the structures of MGSA and IL-8 closely, containing a triple-stranded beta-sheet in the IL-8 part and an amphipathic alpha-helix in the MGSA part. Chi1 formed dimers at millimolar concentrations via the first strand from the N-terminus, analogous to IL-8 and MGSA. In contrast to the latter molecules, however, the alpha-helix of Chi1 did not pack against the beta-sheet part, but was an independent structural element. This structural difference could be explained mainly by the modulation of hydrophobic interactions between the helix and the rest of the protein in Chi1 as compared to IL-8 and MGSA. It is concluded that tight helix packing is not required for receptor binding and biological activity of Chi1.

Monomer-dimer equilibria of interleukin-8 and neutrophil-activating peptide 2. Evidence for IL-8 binding as a dimer and oligomer to IL-8 receptor B.

By chemical cross-linking experiments we show that at physiologically relevant concentrations IL-8 and NAP-2 monomers are in an equilibrium with dimers and even oligomers (KD approximately 300-800 nM). Oligomerization seems to be more prevalent for IL-8 than for NAP-2. The form in which IL-8 and NAP-2 bind to their specific receptors was analyzed in binding experiments with COS-1 cells expressing IL-8 receptor A or B in recombinant forms. Both receptors were cloned from the human myeloid leukemic cell line AML-193. Type A receptor had high affinity for IL-8 (KD approximately 4 nM) and low affinity for NAP-2 (KD > or = 700 nM), whereas the type B receptor was of equally high affinity (KD approximately 2 nM) for both IL-8 and NAP-2. However, IL-8 receptor B could bind specifically three to four times more IL-8 than NAP-2, and NAP-2 was a weak competitor for IL-8 binding to the same receptor. In addition, IL-8, but not NAP-2, could be cross-linked to dimers when bound to IL-8 receptor B. We suggest from these findings that IL-8, but not NAP-2, binds as a dimer and oligomer to IL-8 receptor.

Cross-linking of human neutrophil surface proteins to iodinated interleukin 8 or neutrophil activating peptide-2 results in at least four separable proteins.

The human neutrophil activating peptides-1 and -2 (NAP-1/IL-8, NAP-2) are two structurally and functionally related members of the chemokine cytokine family. They are chemoattractants and activators of neutrophils and exert their effects by binding to specific receptors which are expressed on responsive cells. Two closely related IL-8 receptors of neutrophils have been characterized recently by molecular cloning. We show here that NAP-1/IL-8 and NAP-2 can be cross-linked to at least four protein bands from human neutrophil surfaces with apparent molecular masses of 55, 65, 71 and 81 kDa. The two cross-linked proteins with lower masses were associated with high, the two with the higher masses with low affinity binding of NAP-2, NAP-1/IL-8 was bound to all bands with high affinity. NAP-1/IL-8 and NAP-2 could also be cross-linked to form dimers when bound to cells and in solution. Our results show that more than two NAP-1/IL-8 receptors, or more than two forms of the known receptors exist. Alternatively, the four protein bands can be explained by cross-linking of ligand monomers and dimers, respectively, to the known receptors of neutrophils.

Endogenous serum levels and surface receptor expression of GM-CSF and IL-3 in patients with myelodysplastic syndromes.

The majority of patients suffering from myelodysplastic syndromes (MDS) die of complications due to cytopenia. Clinical trials have demonstrated that in an essential number of MDS patients cytopenia can be ameliorated by exogenously supplied growth factors. We investigated endogenous serum levels of GM-CSF and IL-3 in 15 healthy individuals and 34 patients suffering from MDS. No circulating growth factors were detected in the serum of healthy controls, nor was IL-3 measurable in MDS patients. GM-CSF serum levels, however, were elevated in a significant number of patients (26.5%). Levels did not correlate with FAB classification, leukocyte count or chromosomal abnormalities. No significant differences in GM-CSF or IL-3 receptor expression were detected between healthy individuals and MDS patients. One patient with increased endogenous GM-CSF serum level and normal surface receptor expression responded to exogenously applied GM-CSF. In the light of these results, a functional alteration of growth factor receptors or disturbances of signal transduction pathways must be discussed for MDS.

Neutrophil activating peptide-2 binds with two affinities to receptor(s) on human neutrophils.

Neutrophil receptor(s) for neutrophil activating peptides 1 and 2 were studied by competition binding experiments with radiolabeled NAP-1 and NAP-2 preparations. NAP-1 bound with one affinity, NAP-2 with two quite different affinities, to common receptor(s) on neutrophils. Concentrations of NAP-2 needed to induce exocytosis of beta-glucosaminidase corresponded to the higher dissociation constant of the two binding equilibria. Thus, the binding of NAP-2 to PMN with high affinity does not activate the cells.

Interleukin-4 counteracts pyrogen-induced downregulation of thrombomodulin in cultured human vascular endothelial cells.

Inflammatory mediators such as tumor necrosis factor (TNF) or interleukin-1 (IL-1) and bacterial lipopolysaccharides (LPS) were shown to shift the hemostatic balance of the endothelial cell (EC) surface in favor of procoagulant activities by inducing tissue factor (TF) expression and downregulation of thrombomodulin (TM). In the present study, the effects of IL-4 on these regulatory mechanisms were investigated using cultured human umbilical vein EC. TM downregulation induced by the pyrogens IL-1 (100 U/mL), TNF (500 U/mL), and LPS (20 micrograms/mL) to less than 50% of TM activity of untreated cells during a 12-hour incubation period was completely neutralized when these mediators were coincubated with IL-4 (100 U/mL). In accordance with TM surface activity, TM messenger RNA was decreased by IL-1, TNF, and LPS to less than 40% of untreated cells; this effect was in part antagonized by IL-4. No influence of IL-4 on EC tissue factor induction by IL-1, TNF, and LPS was found. Binding studies using 125I-radiolabeled IL-4 suggest that EC express a single class of high-affinity binding sites (kd = 3.2 pmol/L; 2,000 to 2,500 receptors per cell). These results show that IL-4, in part, protects the EC surface against pyrogen-induced procoagulant changes. Transcriptional regulatory mechanisms seem to be involved in EC surface TM regulation.

Interleukin 4 promotes expression of mast cell ICAM-1 antigen.

Cell recognition molecules play a crucial role in the regulation of immune cells. We recently found that mast cells (MCs) express leukocyte recognition molecules, including ICAM-1 antigen, a natural ligand of LFA-1. We here report that interleukin 4 (IL-4), a pleiotropic cytokine and mast cell differentiation factor, selectively promotes expression of surface ICAM-1 antigen and ICAM-1 mRNA in human MCs. IL-4 also up-regulates ICAM-1 antigen in cells of monocyte/macrophage lineage but has no effect on ICAM-1 antigen expressed on basophils, fibroblasts, or lymphocytes. The increase in expression of mast cell/macrophage ICAM-1 antigen induced by IL-4 may contribute to the accumulation of leukocytes and facilitate cell-contact-dependent regulation of immune cells in inflamed tissues.

Failure to detect IL-3-binding sites on human mast cells.

IL-3, a pleiotropic lymphokine, has been termed mast cell growth factor because it promotes growth and differentiation of murine mast cells. Murine mast cells, in turn, express cell surface receptors for IL-3. Human rIL-3 has been shown to induce proliferation and differentiation of human basophils and to activate basophils via high affinity binding sites. To investigate whether human mast cells express IL-3R, binding studies with 125I-radiolabeled human rIL-3 were performed on HMC-1, a novel human mast cell line, and on pure populations (i.e., 93 to 99% purity) of human tissue mast cells obtained with mAb and C from dispersed lung (n = 2). Unexpectedly, neither enriched human lung mast cells nor HMC-1 cells bound radiolabeled human rIL-3 specifically. Moreover, human rIL-3 failed to promote uptake of [3H]thymidine, synthesis of histamine, histamine releasability, or changes in expression of mast cell differentiation Ag (YB5B8, CD54/ICAM-1, CD9/p24, CD33/gp67) on either human lung mast cells or HMC-1 cells. It is hypothesized that the fundamental difference in the biologic response to IL-3 between human and murine mast cells is due to a loss during evolution of mast cell high affinity IL-3 binding sites.

Human basophils express interleukin-4 receptors.

Interleukin-4 (IL-4), a multipotential lymphokine reputed to play an important role in the regulation of immune responses, interacts with a variety of hemopoietic target cells through specific cell surface membrane receptors. The present study was designed to investigate whether human basophils express IL-4 binding sites. For this purpose, basophils were enriched to homogeneity (93% and 98% purity, respectively) from the peripheral blood of two chronic granulocytic leukemia (CGL) donors using a cocktail of monoclonal antibodies (MoAbs) and complement. Purified basophils bound 125I-radiolabeled recombinant human (rh) IL-4 in a specific manner. Quantitative binding studies and Scatchard plot analysis revealed the presence of a single class of high affinity IL-4 binding sites (280 +/- 40 sites per cell in donor 1 and 640 +/- 45 sites per cell in donor 2) with an apparent dissociation constant, kd, of 7.12 x 10(-11) +/- 2.29 x 10(-11) and 9.55 +/- 3.5 x 10(-11) mol/L, respectively. KU812-F, a human basophil precursor cell line, was found to express a single class of 810 to 1,500 high affinity IL-4 binding sites with a kd of 2.63 to 5.54 x 10(-10) mol/L. No change in the numbers or binding constants of IL-4 receptors was found after exposure of KU812-F cells to rhIL-3 (a potent activator of basophils) for 60 minutes. No effect of rhIL-4 on 3H-thymidine uptake, release or synthesis of histamine, or expression of basophil differentiation antigens (Bsp-1, CD11b, CD25, CD40, CD54) on primary human CGL basophils or KU812-F cells was observed.

In vitro and in vivo activity of human recombinant granulocyte-macrophage colony-stimulating factor in dogs.

Although it is well documented that human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production and functional activity of human and nonhuman primate granulocytes and macrophages, relatively little is known about its effects on cells obtained from other species. The molecular cloning of the complementary DNA for human GM-CSF has made it possible to determine the cross-reactivity of the purified recombinant human material (rhGM-CSF) on cells of other species. The results presented herein show that specific receptors for human GM-CSF exist on dog bone marrow cells and mature circulating dog granulocytes. The number of the receptors and the apparent binding affinity of the rhGM-CSF to its receptors on granulocytes were similar to those observed either on human or monkey cells. In cultures of dog bone marrow cells, rhGM-CSF was capable of promoting colony formation in a dose-dependent manner. Human GM-CSF also primed dog granulocytes for increased production of reactive oxygen metabolites in response to either phorbolmyristic acetate-or zymosan-activated dog serum. In vivo, s.c. administration to healthy dogs of rhGM-CSF in daily doses of 15, 50, or 150 micrograms/kg body weight over a period of 7-20 days induced a dose-dependent rise of up to a maximum of a fourfold increase in peripheral WBC counts. The rise in WBC counts was mainly due to elevated neutrophil levels, but an increase in the numbers of monocytes and eosinophils was also observed. However, the rhGM-CSF-induced leukocytosis in dogs was not as dramatic as that observed in nonhuman primates. In all rhGM-CSF-treated dogs, circulating platelet counts dropped to nadir levels of about 20%-30% of normal numbers. Dogs that were treated with 150 micrograms/kg rhGM-CSF developed specific antibodies after about 10-12 days of treatment. These antibodies were able to neutralize the effect of rhGM-CSF in in vitro assays. In vivo WBC counts began to decline when specific antibodies developed, but they never dropped below normal levels. Taken together, the results suggest that human GM-CSF does not appear to exhibit absolute species specificity.

IL-3 promotes basophilic differentiation of KU812 cells through high affinity binding sites.

The myeloid precursor cell line KU812 exhibits a constitutive potential to differentiate into basophilic cells. In the present study, the influence of recombinant human (rh)IL-2, rhIL-3, and recombinant human granulocyte-macrophage-CSF on basophilic differentiation of KU812-F cells was studied. Of all cytokines tested, rhIL-3 induced a significant increase in formation of metachromatically granulated cells (from 10% in control cultures up to 30% in cultures supplemented with 100 U/ml of rhIL-3) as well as dose-dependent (1.5- to 3 fold) increase in cellular histamine in KU812-F cell cultures. In addition, KU812-F cells exposed to rhIL-3 bound more IgE antibody than cells cultured in control medium with up to 3.3-fold increases in the mean fluorescence intensity on days 2 and/or 5 compared with control (p less than 0.001). RhIL-3 failed to induce significant changes in expression of the Tac-reactive subunit of the IL-2R (CD25), surface aminopeptidase N (CD13), ICAM-1 Ag (CD54), or CD40 Ag on KU812-F cells. To investigate the mechanism of IL-3 action on KU812-F cells, receptor analyses were performed by using 125I-radiolabeled rhIL-3. Quantitative binding studies and Scatchard plot analyses revealed the presence of a single class of 1910 to 2460 high affinity IL-3-binding sites per KU812-F cell with an apparent dissociation constant of 1.22 to 2.35 x 10(-9) M. Together, these results show that rhIL-3 promotes basophilic differentiation of KU812-F cells through a specific receptor.

Specific binding, internalization, and degradation of human neutrophil activating factor by human polymorphonuclear leukocytes.

The interaction of 125I-labeled recombinant human neutrophil activating factor (NAF) with polymorphonuclear leukocytes (PMN) was studied by means of a radioreceptor assay. The binding was characterized by a rapid transition (t1/2 less than or equal to 1 min) from a pH 3-sensitive state at 4 degrees C to pH 3 resistance at 37 degrees C. This was not caused by internalization of NAF since pH 3-resistant bound iodinated NAF could still be exchanged by an excess of nonlabeled NAF, i.e. was dissociable. Internalized iodinated NAF was processed into trichloroacetic acid-soluble forms. Scatchard transformation of binding isotherms at 4 and 37 degrees C led to nonlinear curves, a finding which is consistent with the expression of two receptor populations, one with high (KD = 11-35 pM) and the other with lower affinity (KD = 640-830 pM) at 4 degrees C. Numbers of the low affinity binding sites were approximately 34,000, and those with high affinity were 5,200/PMN when estimated at 4 degrees C. Binding of iodinated NAF to PMN was specific since it could be competed by an excess of nonlabeled NAF but not by two other activators of PMN function, formylmethionyl-leucyl-phenylalanine or human recombinant granulocyte-macrophage colony-stimulating factor. In addition to human PMN, NAF also bound specifically to two human monocytic cell lines; however, only the low affinity binding site could be detected on these cells.

Interleukin 3 activates human blood basophils via high-affinity binding sites.

Pure populations of human basophilic granulocytes were obtained from chronic myeloid leukemia (CML) blood by negative selection using a mixture of monoclonal antibodies and complement. 125I-radiolabeled recombinant human interleukin 3 (rhIL-3) bound to purified basophils in a specific manner. Quantitative binding studies and Scatchard plot analyses performed on samples from two donors revealed the presence of a single class of high-affinity IL-3 binding sites (500 and 2100 sites per cell; dissociation constant at equilibrium, 230 and 160 pmol/liter, respectively). Purified CML basophils maintained in suspension in the presence of rhIL-3 (100 units/ml) incorporated up to 12 times more [3H]thymidine than basophils in control cultures. Furthermore, after preincubation in vitro with rhIL-3 (100 units/ml) for 30 min, normal blood basophils released 2- to 3-fold more histamine than basophils pretreated with control medium when exposed to various concentrations of an anti-IgE antibody. Together, these results show that rhIL-3 binds to a specific receptor on blood basophils and is a regulator of basophil function.