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.

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.

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.

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.

The structure of procalcitonin of the salmon as deduced from its cDNA sequence.

Oligonucleotide probes based on the known amino acid sequence of salmon calcitonin were used to screen a cDNA library obtained from ultimobranchial glands of salmon for clones encoding salmon calcitonin. From the cDNA sequence of strongly hybridizing clones the complete primary structure of the calcitonin precursor could be deduced. Its overall structure is identical with the structures of procalcitonins from other vertebrates and has the highest homology with the chicken precursor.

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.

Efficacy of recombinant human granulocyte-macrophage colony-stimulating factor in rhesus monkeys.

The glycosylated and the non-glycosylated recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) expressed in Chinese hamster ovary cells and E. coli, respectively, were administered in rhesus monkeys either by the subcutaneous (three times daily) or intravenous route (6-hr infusions) for seven consecutive days. Within 24 hr peripheral white blood cells (WBC) increased 2-3 fold over normal values. Thereafter, the WBC increased steadily in a dose-dependent manner to reach maximum levels on the last day of or one day after the treatment period. The differential counts showed that neutrophils contributed to 50-80%, eosinophils to 10-20%, monocytes to 2-7%, and lymphocytes to 15-30% of the WBC rise. No effect was found on platelets and erythrocytes. After termination of treatment, WBC counts returned to normal levels within one week. Subcutaneously administered CSF was more effective in inducing leukocytosis than that injected intravenously In addition to the rise in WBC, the administered rh GM-CSF also enhanced the oxidative metabolism and bactericidal activity of the circulating mature granulocytes isolated from the blood of monkeys treated with rh GM-CSF. These results show that glycosylated or non-glycosylated rh GM-CSF is both an effective stimulator of leukocytosis and a potent activator of the functional activity of mature granulocytes in vivo.

[The influence of growth regulators on protein and enzym spectra of explants from roots of cichorium intybus cultured in vitro]. / Der Einfluß von Wachstumsregulatoren auf Protein- und Enzym-Spektren kultivierter Explantate aus Rüben von Cichorium intybus L.

On a simple nutrient medium in explants from roots of Cichorium intybus form shoots visible after about 14 days. Gibberellic acid (GA3) does not influence the spontaneous development of the chicory explants. GA3 in combination with kinetin inhibits shoot formation whereas kinetin alone promotes the process. On the other hand high concentrations of IAA inhibit the regeneration of shoots.The soluble proteins of chicory cultures treated with growth regulators were examined by disc-electrophoresis. It was shown that the proteins detected by staining with Amido black, phosphatases, esterases and glutamate dehydrogenase (GDH) present in the original root tissue remained constant under the different culture conditions during a period of 12 days. The quantitative changes of some of the proteins, phosphatases and esterases observed during the culture period were identical for all the different cultures in spite of the different morphogenetic behaviour. Only the activities of GDH and peroxidase changed after treatment with different growth regulators; however, in these cases, there was also no direct connection with the morphogenetic responses of the cultures.The specific activity of the GDH-band was promoted by IAA and at the same time the formation of peroxidases was inhibited. Kinetin delayed the formation of peroxidases during the first days of the culture period but promoted it later on. There was a repression by IAA of a specific kationic peroxidase. In the tissues treated with GA3 the activity of peroxidases was always higher than in the control tissue. This effect of GA3 can be partly explained by the fact that GA3 inhibits the release of peroxidases of the explants into the nutrient medium.

Suppression of polarity of insertion mutations within the gal operon of E. coli.

Phenotypic revertants of galOP::IS1 and galOP::IS2 mutations have been isolated after mutagenesis with nitrosoguanidine, they are probably caused by mutations in gene suA. The polarity suppressor mutations described in this study and a known mutation in gene suA isolated by D. Morse (Morse and Guertin, 1972) suppress polarity caused by IS1 more effectively than that caused by IS2 or IS4. Furthermore, suppressibility is influenced by the site and orientation of IS integration. The synthesis of the three enzymes in galOP::IS suA double mutants is constitutive and the ratio of the three enzymes is altered in comparison to the wild type. The reasons for constitutive synthesis of the galactose enzymes and for the altered ratio of enzyme synthesis are discussed.

Transcription of insertion elements IS1 and IS2 in vitro.

Insertion elements IS1 and IS2 integrated within the gal operator-promoter region, an IS1 element in gene galT and insertions IS1 and IS2 integrated in the xycIIOP region of phage lambda were transcribed in vitro with E. coli RNA-polymerase. The insertion elements are transcribed exclusively by polymerase molecules started at the gal promoter and the lambdaPR promoter respectively. No promoter exists on IS1 or IS2 which can be recognized by RNA-polymerase in the pure in vitro transcription system used. Both insertions apparently are transcribed with a lower elongation rate than gal operon DNA or lambdaDNA. RNAs transcribed from the termini of IS1 and IS2 respectively were analysed by hybridization experiments. They are different in sequence.

The structure of unstable constitutive revertants of mutant galOP-308::IS2-I.

The isolation and characterization of three unstable and constitutive revertants of mutant galOP-308 of E. coli is described. In this mutant an IS2 element is integrated between the promoter and the first structural gene of the galactose operon, and exerts a strong polar effect on the expression of the three galactose genes. In the three revertants under investigation it was observed that relief of polarity and constitutive expression of the gal-operon were accompanied by the deletion of 90% of the IS2 sequence and of various lengths of the adjacent sequences including the gal-promoter. We conclude from this result that the transcription termination signals causing strong polarity were located on the deleted part of IS2, and that in our revertants the galactose genes are now under the control of a new promoter which is apparently unstable.

Deletions and DNA rearrangements within the transposable DNA element IS2. A model for the creation of palindromic DNA by DNA repair synthesis.

Three derivatives of mutant ga10P-308::IS2-I of Escherichia coli were characterized by DNA sequence analysis. Deletions and DNA sequence rearrangements were observed which apparently were initiated at short A-T rich inverted repeats within IS2. Two of the mutants carried newly synthesized DNA sequences which were inverted copies of already existing IS2 sequences. Thus long stretches with twofold symmetry were formed. It is discussed whether these inverted repeats were formed by DNA repair synthesis which was initiated at the A-T rich palindromes of IS2.

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.

The isolation of IS1 and IS2 DNA.

DNA of the IS-elements IS1 and IS2 was prepared by digestion of appropriate heteroduplex molecules with endonuclease S1, followed by sucrose gradient centrifugation or gel electrophoresis. The material obtained is homogeneous with regard to size. The length of IS1 DNA is 820 +/- 65 nucleotides, the length of IS2 DNA is 1,350 +/- 70 nucleotides. IS1 DNA is not cleaved by the restriction endonucleases Eco R1, Hind II or Hind III. IS2 DNA is cleaved once by each of the two latter enzymes. The buoyand density determined by equilibrium centrifugation of Hg-complexes in Cs2so4 corresponds to a GC content of approximately 50%. Labelling with polynucleotide kinase indicates that both IS DNA's have a guanosyl residue at both of their 5'-termini.

Isolation and characterization of phi80dgal transducing phages that carry gal operator-promoter insertion mutations.

phi80dgal transducing bacteriophages have been isolated by the F-fusion technique of Press et al. (1971) and gal-operator-promoter insertion mutations have been introduced by homogenate formation. Five different phi80dgal isolates have been studied in more detail. One of the phi80 phages transduces the gal operon and gene aroG as well as at least part of the trp-operon; the gal operon of another phi80dgal transducing phage is inverted with respect to the phi80dgal sequences. Heteroduplex DNA mapping indicates that one of the phi80dgal isolates in addition to the gal operon and a portion of the adjacent chromosomal region carries an IS2-element which is derived from the F'gal episome. The isolated phi80dgal phages may be utilized for preparing pure gal mRNA and insertion-RNA as well as pure gal operon DNA.

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.

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.

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.