3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures.

DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3'-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (T(m)) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5'-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same T(m)(65 degrees C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe T(m)and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.

The effects of recombinant human thrombomodulin on endotoxin-induced multiple-system organ failure in rats.

Activation of the coagulation system is postulated to play an important role in the pathogenesis of endotoxin-induced tissue injury. Thrombomodulin (TM) is an endothelial cell membrane glycoprotein receptor for thrombin. Once bound to TM, thrombin loses its procoagulant activity, which results in decreased clotting. In addition, the binding of thrombin to TM activates the endogenous anticoagulant pathway through protein C. We studied the effect of recombinant human TM (rh-TM) on endotoxin-induced multiple-system organ failure (MSOF) in Sprague-Dawley rats weighing 400 to 450 g: 2 mg/kg of rh-TM was injected (T1/2 = 4.5 h) 30 min prior to intravenous injection of 20 mg/kg of Escherichia coli endotoxin. The study presented here consisted of three separate experiments. Experiment 1: 24-h survival study. Experiment 2: multiple-system organ microthrombi study in which 125I-human fibrinogen was injected 30 min prior to an endotoxin or saline injection and tissue microthrombi formation was assessed by measuring the percentage of organ radioactivity (lung, heart, liver, and kidney) against total injected radioactivity (microthrombi index, MI) 2.25 h after an endotoxin or saline injection. Experiment 3: endotoxin-induced MSOF study in which 125I-rat albumin was injected 5 h after an endotoxin or saline injection, and endotoxin-induced organ injury was evaluated by measuring tissue wet-to-dry ratios (W/D) and tissue-to-plasma 125I-rat albumin concentration ratios (T/P) 8 h after the endotoxin or saline injection. Blood contamination in samples from Experiments 2 and 3 was corrected by using 131I-rat albumin measurements. Pretreatment with rh-TM improved the survival from 12 h through 23 h as compared with that of the endotoxin control group (p < 0.05). However, at 24 h, after essentially all injected rh-TM had been eliminated, there was no difference in survival. Significant reductions in MI, W/D, and T/P in the organs sampled were observed in the rh-TM pretreated groups (p < 0.05). In conclusion, rh-TM improved short-term but not overall survival and decreased MSOF in endotoxemic rats.

Prevention of autoimmune demyelination in non-human primates by a cAMP-specific phosphodiesterase inhibitor.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system that serves as a model for the human disease multiple sclerosis. We evaluated rolipram, a type IV phosphodiesterase inhibitor, for its efficacy in preventing EAE in the common marmoset Callithrix jacchus. In a blinded experimental design, clinical signs of EAE developed within 17 days of immunization with human white matter in two placebo-treated animals but in none of three monkeys that received rolipram (10 mg/kg s.c. every other day) beginning 1 week after immunization. In controls, signs of EAE were associated with development of cerebrospinal fluid pleocytosis and cerebral MRI abnormalities. In the treatment group, there was sustained protection from clinical EAE, transient cerebrospinal fluid pleocytosis in only one of three animals, no MRI abnormality, and marked reduction in histopathologic findings. Rolipram-treated and control animals equally developed circulating antibodies to myelin basic protein. Thus, inhibition of type IV phosphodiesterase, initiated after sensitization to central nervous system antigens, protected against autoimmune demyelinating disease.

Reiterative copying by E.coli RNA polymerase during transcription initiation of mutant pBR322 tet promoters.

The major in vitro transcripts from the tet promoter of pBR322 derivatives pTA22 and pTA33 have heterogeneous 5' ends consisting of variable lengths of oligo(A). Their structure is 5'pppAnU..., where n ranges from 1 to greater than 12, but the template strand can encode at most four A residues at the site of transcription initiation. The abundance of additional A residues at the 5' end of the pTA22 and pTA33 tet transcripts could be reduced by elevating the concentration of UTP, but even at high concentrations (greater than 1 mM) non-cognate A residues were still observed. Aberrant initiation was not artifactual since the major and minor transcripts of the pBR322 tet promoter region, and other transcripts arising from minor promoters on pTA22 or pTA33 DNA all had unique 5' termini. Mixing experiments showed that RNA polymerase did not utilize pppA2-4-OH produced by abortive initiation as primers. The data suggest that the initial nascent RNA chain 'slips' in the 5' direction during elongation opposite T4 on the template strand causing RNA polymerase to reiteratively add A residues to the 5' end of the transcript. The generality and possible significance of this mechanism is discussed.

Transcription initiation at the tet promoter and effect of mutations.

We have identified the startpoint for transcription in vitro of the tetracycline resistance gene (tet) of pBR322 and several deletion and insertion mutations which alter tet promoter structure. Tetracycline resistance in host bacteria correlates qualitatively with the efficiency of DNA fragments from these plasmids to promote tet transcription in vitro. Only in active promoters could we find by computer analysis promoter structures in which the -10 and -35 sequences and the relative spacing of the two regions agree with consensus sequence determinants. These data support the current model of the E. coli promoter sequence. Two promoter mutants gave heterogeneous 5' termini with additional A residues not encoded by the DNA sequence.

Determinants for protein localization: beta-lactamase signal sequence directs globin across microsomal membranes.

A hybrid gene containing 182 codons of Escherichia coli beta-lactamase at the amino terminus of the corresponding protein and 141 codons of alpha-globin at the carboxyl terminus was generated by inserting chimpanzee alpha-globin cDNA into the Pst I site of plasmid pBR322. RNA transcribed in vitro from this plasmid gave a corresponding hybrid protein in a wheat germ cell-free translation system. The hybrid protein was protected from tryptic digestion and the pre-beta-lactamase signal peptide was removed when dog pancreas membrane vesicles were present during translation. A deletion mutant containing 23 codons of pre-beta-lactamase signal sequence and 5 codons of mature beta-lactamase fused to the alpha-globin cDNA gave a shorter hybrid protein that behaved similarly. However, a mutation that removed essentially all of the pre-beta-lactamase sequence gave a protein that was neither protected nor processed. Hence, at most, only the signal peptide and the first 5 amino acids of beta-lactamase were necessary to convert alpha-globin (a cytoplasmic protein) into a secretory protein.

A stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems.

We have combined molecular genetic and cell-free reconstitution approaches to study the mechanism of membrane assembly. The coding region for the carboxy-terminal transmembrane sequence of membrane IgM heavy chain has been inserted between the coding regions for lactamase and globin domains of a fusion protein previously shown to be completely translocated across microsomal membranes in a cell-free transcription-linked translation system. The resulting fusion protein behaves as an integral transmembrane protein of predicted asymmetry: all of the membrane integrated copies display lactamase within the lumen and globin on the cytoplasmic face of the vesicles. In another construction, this transmembrane coding region replaces that of the signal sequence. The resulting fusion protein is not translocated across membranes. These data provide strong evidence that there are stop transfer sequences whose ability to arrest chain translocation and achieve asymmetric transmembrane orientation is independent of the size of the subsequent carboxy-terminal domain to be localized in the cytosol; and that signal and stop transfer sequences are functionally distinct.

Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli.

We cloned the DNA containing the Escherichia coli hemolysin determinant on a small, high-copy plasmid. We generated plasmids containing fragments of this DNA and used them either alone or in two-plasmid complementation systems to define the limits of the structural genes. This system also allowed us to partially characterize the function of each of the gene products in the production and transport of hemolysin. Taken with previously published data, the present experiments indicate the following. (i) At least three cistrons, hlyC, hlyA, and hlyB (these were previously designated cisC, etc. [Noegel et al., Mol. Gen. Genet. 175:343-350, 1979]), contain the specific genetic information for the hemolytic phenotype, (ii) hlyA encodes a 107,000-kilodalton protein, which seems to be an inactive precursor of hemolysin. (iii) Normal amounts of hemolysin activity inactive precursor of hemolysin. (iii) Normal amounts of hemolysin activity require only the products of hlyA and hlyC. This activity was found in the periplasm; very little hemolysin activity was found in the cytoplasm, suggesting that the hlyC product is required for transport or activation of the hlyA product or both. (iv) Active hemolysin remains in the periplasm in the absence of hlyB function, hence the hlyB product seems to be necessary for the transport of hemolysin to the exterior of the cell. We further show that overproduction of the hlyA product is lethal, probably causing lysis of the cell.

Analysis of lambda receptor and beta-lactamase synthesis and export using cloned genes in a minicell system.

We have cloned lamB, the gene for lambda receptor (an outer membrane protein), on a small plasmid which also carries the gene for beta-lactamase (a periplasmic protein). We have identified a promoter in the region of malK, the gene immediately preceding lamB, which is active in minicells but relatively inactive in vitro. Using a minicell system, we have found that both lambda receptor and beta-lactamase are made as full length precursors which are subsequently processed. We also show that the lambda receptor precursor can be exported to the outer membrane before it is processed. Mature beta-lactamase is found only in the periplasm, suggesting that processing may be a requirement for export to the periplasm.

Sequence analysis of mutations that prevent export of lambda receptor, an Escherichia coli outer membrane protein.

The amino-terminal signal sequence is required for initiation of transmembrane protein transfer of the Escherichia coli lambda receptor protein. Mutations leading to insertion of charged amino acids into or deletion of amino acids from the hydrophobic segment of this sequence prevent export of this outer membrane protein.

DNA sequence encoding the NH2-terminal peptide involved in transport of lambda receptor, an Escherichia coli secretory protein.

lamB encodes the lambda receptor of Escherichia coli, an outer membrane protein. We have identified the beginning of the lamB gene by correlating DNA nucleotide sequence with a partial sequence of the primary translation product of lamB. We show that lambda receptor is synthesized as a precursor containing an extra 25 amino acids at its NH2 terminus. These amino acids are predominately hydrophobic and probably comprise a structure required for initiation of transport of lambda receptor from the cytoplasm to the outer membrane.

Synthesis and maturation of lambda receptor in Escherichia coli K-12: in vivo and in vitro expression of gene lamB under lac promoter control.

The lambda receptor is an outer membrane protein from Escherichia coli K-12 lamB, its structural gene, is part of the maltose regulon. We have cloned this gene in a phage so that it is under the control of the lac promoter. The phage was devised in such a way that it can infect lamB mutants and that chromosomal lamB mutations can be transferred to it. In vivo, the lambda receptor is expressed under lac promoter control and is exported normally to the outer membrane, independently of the expression of the other genes of the maltose regulon. In vitro, DNA of the phage allows efficient synthesis of the lamB product. The protein--or pre-lambda-receptor--made in vitro contains an NH2-terminal sequence of about 25 amino acids not found in the lambda receptor. We have detected no inactivation of phage lambda by the pre-lambda-receptor. Conversion of the pre-lambda-receptor to a form that has the apparent molecular weight of the mature lambda receptor was achieved. A lamB mutation that blocks export in vivo also blocks conversion in vitro.

[DNA sequence encoding the signal peptide of the lambda receptor in E. coli K 12]. / Séquence de l'ADN correspondant à la région du peptide signal pour le récepteur de lambda E. coli K12.

A 181 base pairs DNA fragment from E. coli K 12 has been sequenced. This allows determination of the sequence of the signal peptide of the precursor for the lambda receptor, an outer membrane protein.

Temperature-sensitive mutation in the initiation codon of the rIIB gene of bacteriophage T4.

We have determined the sequence of a ribosome-protected region of T4 rIIB mRNA labeled in vivo. The rIIB mutant HD263, which is temperature sensitive for translation, has an altered initiation codon (AUA instead of AUG). Because wild-type and HD263 proteins have the same fMet peptide, this AUA is used to initiate translation in the mutant rIIB mRNA. We have also identified a six-base sequence which is complementary to the 3" end of the 16S rRNA.

Lambda phage promoter used to enhance expression of a plasmid-cloned gene.

A 50-fold (or greater) increase in the production of phage 21 repressor was obtained by construction of a plasmid in which the 21cI (repressor) gene could be transcribed from lambdaPL. The enhancement due to increased 21cI gene copy number and transcription from lambdaPL were at least five-fold and ten-fold, respectively. The plasmid was constructed in vitro by recombination of EcoRI-generated DNA fragments. The use of the DNA fragment containing lambdaPL in obtaining expression of cloned genes is discussed.

Oligo(A) not coded by DNA generating 3'-terminal heterogeneity in a lambda phage RNA.

An RNA species from Escherichia coli infected with phage lambda was purified by hybridization to lambda 1-strand DNA and shown to have variable length. This variability was due to a variable number of adenylate residues attached to the 3' end of the molecule. Pancreatic RNase treatment of the RNA-DNA hybrid removed the 3'-terminal adenylate residues, generating a homogeneous RNA molecule terminating with -Up. The results indicate the presence of adenylate residues not coded by the DNA template at the terminus of this transcript from intact cells.