RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities.

The RegA proteins from the bacteriophage T4 and RB69 are translational repressors that control the expression of multiple phage mRNAs. RegA proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the RB69 RegA protein binds target RNAs with a higher affinity than T4 RegA protein. To study the RNA binding properties of T4 and RB69 RegA proteins more directly, the binding sites of RB69 RegA protein on synthetic RNAs corresponding to the translation initiation region of two RB69 target genes were mapped by RNase protection assays. These assays revealed that RB69 RegA protein protects nucleotides -9 to -3 (relative to the start codon) on RB69 gene 44, which contains the sequence GAAAAUU. On RB69 gene 45, the protected site (nucleotides -8 to -3) contains a similar purine-rich sequence: GAAAUA. Interestingly, T4 RegA protein protected the same nucleotides on these RNAs. To examine the specificity of RNA binding, quantitative RNA gel shift assays were performed with synthetic RNAs corresponding to recognition elements (REs) in three T4 and three RB69 mRNAs. Comparative gel shift assays demonstrated that RB69 RegA protein has an approximately 7-fold higher affinity for T4 gene 44 RE RNA than T4 RegA protein. RB69 RegA protein also binds RB69 gene 44 RE RNA with a 4-fold higher affinity than T4 RegA protein. On the other hand, T4 RegA exhibited a higher affinity than RB69 RegA protein for RB69 gene 45 RE RNA. With respect to their affinities for cognate RNAs, both RegA proteins exhibited the following hierarchy of affinities: gene 44 > gene 45 > regA. Interestingly, T4 RegA exhibited the highest affinity towards RB69 gene 45 RE RNA, whereas RB69 RegA protein had the highest affinity for T4 gene 44 RE RNA. The helix-loop groove RNA binding motif of T4 RegA protein is fully conserved in RB69 RegA protein. However, homology modeling of the structure of RB69 RegA protein reveals that the divergent residues are clustered in two areas of the surface, and that there are two large areas of high conservation near the helix-loop groove, which may also play a role in RNA binding.

Identification of the RNA binding domain of T4 RegA protein by structure-based mutagenesis.

The T4 translational repressor RegA protein folds into two structural domains, as revealed by the crystal structure (Kang, C.-H. , Chan, R., Berger, I., Lockshin, C., Green, L., Gold, L., and Rich, A. (1995) Science 268, 1170-1173). Domain I of the RegA protein contains a four-stranded beta-sheet and two alpha-helices. Domain II contains a four-stranded beta-sheet and an unusual 3/10 helix. Since beta-sheet residues play a role in a number of protein-RNA interactions, one or both of the beta-sheet regions in RegA protein may be involved in RNA binding. To test this possibility, mutagenesis of residues on both beta-sheets was performed, and the effects on the RNA binding affinities of RegA protein were measured. Additional sites for mutagenesis were selected from molecular modeling of RegA protein. The RNA binding affinities of three purified mutant RegA proteins were evaluated by fluorescence quenching equilibrium binding assays. The activities of the remainder of the mutant proteins were evaluated by quantitative RNA gel mobility shift assays using lysed cell supernatants. The results of this mutagenesis study ruled out the participation of beta-sheet residues. Instead, the RNA binding site was found to be a surface pocket formed by residues on two loops and an alpha-helix. Thus, RegA protein appears to use a unique structural motif in binding RNA, which may be related to its unusual RNA recognition properties.

A toxicological assessment of curdlan.

Curdlan was approved for use by the FDA in December 1996 as a formulation aid, processing aid, stabilizer and thickener or texturizer for use in food. It has been evaluated for safety by a series of animal studies and in vitro tests including acute, subchronic and chronic toxicity studies and reproduction and carcinogenicity studies. In addition, nutritional studies in rodents and tolerance and metabolic studies in man have been carried out. The only effects seen in these studies were reductions in weight gain at the higher dietary concentrations due to the replacement of part of the diet by curdlan, which is calorifically inert. No evidence of any toxicity or carcinogenicity nor of any effects on reproduction was seen, although there was an effect on body weights of the pups with the 15% diet, which was shown in additional studies to be due to the reduced food availability in the animals at this dose level. There was no evidence of effects on the nutritional status of the animals nor on the absorption of minerals. This reviews the available toxicological data on curdlan.

Prohibitin, a putative negative control element present in Pneumocystis carinii.

Little is known about the molecules involved in the regulation of Pneumocystis carinii replication and development in vitro and in vivo. We describe in this report the identification of a P. carinii gene encoding the P. carinii prohibitin protein. In mammals, the prohibitin gene product has been shown to negatively regulate cell proliferation. A cDNA clone encoding the P. carinii prohibitin gene was isolated from a P. carinii cDNA library and identified on the basis of amino acid sequence homology with prohibitin from mammalian sources. Southern blot analysis confirmed that the prohibitin cDNA clone was of P. carinii origin. Western blot analysis of total P. carinii protein indicated that the prohibitin gene is transcribed and translated in vivo. The P. carinii prohibitin gene was expressed in vivo in human fibroblasts and shown to arrest the cell cycle in the G1 phase. The results obtained suggest a potential role for P. carinii prohibitin in the regulation of P. carinii proliferation and development.

Residues at the carboxy terminus of T4 DNA polymerase are important determinants for interaction with the polymerase accessory proteins.

Three T4 DNA polymerase accessory proteins (44P/62P and 45P) stimulate the polymerase (pol) activity and the 3'-5' exonuclease (exo) activity of T4 DNA polymerase (43P) on long, double-stranded DNA substrates. The 44P/62P "clamp loader" facilitates the binding of 45P, the "sliding clamp", to DNA that is primed for replication. Using a series of truncated 43P mutants, we identified a region at the extreme carboxy terminus of the DNA polymerase that is required for its interaction with accessory proteins. Truncation mutants of 43P lacking the carboxy-terminal 3, 6, or 11 residues retained full pol and exo activity on short synthetic primer-templates. However, the ability of the accessory proteins to enhance these activities on long double-stranded DNA templates was drastically reduced, and the extent of the reduction in activity was greater as more residues were deleted. One of the truncation mutants (N881), which had 17 residues removed from the carboxy terminus, showed reduced binding affinity and diminished pol activity but enhanced exo activity upon incubation with a small primer-template. The exo activity of the N881 mutant, on short, single-stranded DNA was unchanged, however, compared to the wild-type enzyme. These results are consistent with inferences drawn from the crystal structure of a DNA polymerase from a related T-even phage, RB69, where the carboxy-terminal 12 residues (equivalent to the 11 residues of 43P from phage T4) protrude from the thumb domain and are free to interact with complementary surfaces of the accessory proteins. The structural integrity of the thumb region in the N881 mutant is probably perturbed and could account for its reduced binding affinity and pol activity when incubated with short, double-stranded DNA substrates.

Bacteriophage T4 regA protein binds RNA as a monomer, overcoming dimer interactions.

The stoichiometry of the complex formed between the T4 translational repressor protein regA and the 16 nt gene 44 recognition element (gene 44RE) RNA has been determined. Under quantitative binding conditions, the association of wild-type regA protein with gene 44RE RNA exhibits saturation at a 1:1 ratio of protein to RNA. It is known that regA protein exists as a dimer in protein crystals. Thus, the stoichiometry may be indicative of a regA dimer bound to two RNAs or a regA monomer bound to one RNA. Gel filtration through Sephadex G-75 revealed that wild-type and R91L regA proteins (14.6 kDa) elute at a mass of 29 kDa, consistent with the mass of a dimer. However, wild-type regA preincubated with gene 44RE (1:1) resulted in a complex that eluted at approximately 20 kDa, consistent with a regA monomer-RNA complex. Covalent crosslinking of surface lysines with glutaraldehyde confirmed that wild-type and R91L proteins exist as dimers and higher oligomers in solution. However, the addition of RNA to wild-type regA protein prior to crosslinking inhibited the formation of crosslinked dimers. Thus, the regA protein-protein interactions observed in solution are disrupted or blocked in the presence of gene 44RE RNA. Together, these studies demonstrate that regA protein binds RNA as a monomer, although free protein exists predominantly as a dimer.

Mutagenesis of the COOH-terminal region of bacteriophage T4 regA protein.

The bacteriophage T4 regA protein is a translational repressor that regulates the synthesis of > 12 T4 proteins. Earlier studies demonstrated that photocross-linking of the 122-residue regA protein to (dT)16 occurs at two sites, with the major site occurring at Phe-106. Amino acid substitutions were introduced at Phe-106 to evaluate its role in nucleic acid binding. Binding affinities of mutants F106C, F106V, and F106Y for nonspecific and specific RNA ligands indicated little difference between the Kapp of the mutants and wild type regA protein, for either poly(U) or for a specific gene 44 oligoribonucleotide. Thus, Phe-106 does not contribute measurably to the overall free energy of binding. Partial proteolysis of regA protein was carried out to further probe its domain structure. Chymotryptic cleavage produced a fragment of 11,095 Da that has reduced affinity for poly(U) and that contains the first 93 residues of regA protein. Interestingly, proteolysis of regA protein is reduced in the presence of the specific target, gene 44 RNA. Two deletion mutants, 1-->94 and 1-->109, have also been cloned and purified. The binding affinities of these deletion mutants indicated a 100-1000-fold reduction in their affinities for poly(U). These studies indicate the last 13 amino acids in regA protein make a significant contribution to RNA binding.

A survey of nucleic acid services in core laboratories.

Core facility services related to DNA synthesis and sequencing were surveyed by the Association of Biomolecular Resource Facilities. Responses from 85 facilities offering DNA synthesis and 37 facilities offering DNA sequencing were obtained. Data on instrumentation, volume, number of users, cost, methodology and a number of other criteria were obtained. The volume of work performed by these centralized core facilities was quite substantial (combined synthesis output of 4 million bases per year and a combined sequencing output of 35 million bases per year). The large number of users supported by these facilities and the high sample throughput make these core resource facilities good indicators of technological trends.

Gene for an extracellular matrix receptor protein from Pneumocystis carinii.

An initial and crucial step in the establishment of many microbial infections is the attachment of the pathogen to the host cells. Thus, adherence of Pneumocystis carinii (Pc) to type I pneumocytes is believed to be important in the induction of Pc pneumonia. Little is known about the nature of the attachment of Pc to type I cells, although extracellular matrix (ECM) proteins, such as fibronectin and laminin, have been implicated in the process. We report here the isolation of a Pc gene encoding a receptor protein that binds both fibronectin and laminin in vitro. A cDNA clone encoding the Pc ECM receptor was isolated from a Pc cDNA library and identified on the basis of sequence homology to the human colon carcinoma laminin receptor. Southern blot analysis of Pc genomic DNA confirmed that the cDNA was of Pc origin. Northern blot analysis of Pc total RNA showed a predominant mRNA of approximately 1400 nucleotides that hybridized to the ECM receptor gene. The ECM receptor predicted from the cDNA sequence is 295 amino acid residues long, with a molecular mass of 32.8 kDa. The C-terminal third of the polypeptide is highly negatively charged, whereas the N-terminal two-thirds contains hydrophobic segments that may play a role in membrane association. Sequence analysis and alignment of the N terminus with the laminin receptor cDNA sequence of human colon carcinoma support the conclusion that the Pc ECM receptor cDNA clone is a full-length clone. A Western blot of the overexpressed ECM receptor protein bound both laminin and fibronectin in vitro. Antibodies raised to the overexpressed receptor protein interacted with a 33-kDa protein in total Pc cell lysates. These findings raise the possibility that the Pc ECM receptor protein may mediate the organism's attachment to type I pneumocytes and, thus, may play a crucial role in Pc pathogenesis.

Oncogenicity studies of the cognition-enhancing agent nefiracetam in mice and rats.

Oncogenicity studies of nefiracetam (N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide, DM-9384, CAS 77191-36-7), a new cognition-enhancing agent, were carried out in male and female mice and rats. The compound was administered in diet for 104 weeks at dosage levels of 30, 90 and 270 mg/kg/d for mice and of 200, 600 and 1800 ppm for rats. The administration of nefiracetam produced no effects on survival, appearance or behavior. Body weights of the high dose male mice and rats were occasionally significantly decreased when compared to the controls. When calculated on a g/animal/d basis, food consumption was sometimes decreased for these male groups. At necropsy, there was no evidence of treatment related changes, nor were these seen on histopathological examination. All microscopic changes seen in mice and rats were of the usual type commonly occurring in untreated aged B6C3F1 mice and F344 rats. In conclusion, the administration of nefiracetam for 24 months to B6C3F1 mice and F344/DuCrj rats produced only slight effects on body weight in the high dose males with a no-effect level of 90 mg/kg/d for mice or 600 ppm for rats. There was no evidence of an oncogenic effect of nefiracetam.

Identification of amino acid residues at the interface of a bacteriophage T4 regA protein-nucleic acid complex.

The bacteriophage T4 regA protein (M(r) = 14,6000) is a translational repressor of a group of T4 early mRNAs. To identify a domain of regA protein that is involved in nucleic acid binding, ultraviolet light was used to photochemically cross-link regA protein to [32P]p(dT)16. The cross-linked complex was subsequently digested with trypsin, and peptides were purified using anion exchange high performance liquid chromatography. Two tryptic peptides cross-linked to [32P]p(dT)16 were isolated. Gas-phase sequencing of the major cross-linked peptide yielded the following sequence: VISXKQKHEWK, which corresponds to residues 103-113 of regA protein. Phenylalanine 106 was identified as the site of cross-linking, thus placing this residue at the interface of the regA protein-p(dT)16 complex. The minor cross-linked peptide corresponded to residues 31-41, and the site of cross-linking in the peptide was tentatively assigned to Cys-36. The nucleic acid binding domain of regA protein was further examined by chemical cleavage of regA protein into six peptides using CNBr. Peptide CN6, which extends from residue 95 to 122, retains both the ability to be cross-linked to [32P]p(dT)16 and 70% of the nonspecific binding energy of the intact protein. However, peptide CN6 does not exhibit the binding specificity of the intact protein. Three of the other individual CNBr peptides have no measurable affinity for nucleic acid, as assayed by photo-cross-linking or gel mobility shifts.

The evaluation of 6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl)amino] benzoate dimethanesulfonate (FUT-187) by oral administration in cynomolgus monkeys in a 13 week subacute toxicity study.

To assess the subacute toxicity of 6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl)amino] benzoate dimethanesulfonate (FUT-187) a 13 week subacute toxicity study by gavage was done in Cynomolgus monkeys at dosage levels of 0, 15, 45 and 135 mg/kg/day. Deaths were seen in the 135 mg/kg/day group; with associated debility. The animals that died had high plasma levels of FUT-187. Little weight gain was seen in the 135 mg/kg/day group. There were no clear treatment related effects on ophthalmoscopy or electrocardiography or on hematological, biochemical or urinalysis. There were no effects noted at necropsy or on histopathology and the causes of death for each of the 3 animals that died were suppurative glomerulonephritis, bone marrow depression and possible gavage injury respectively. The no-effect level for toxicology was considered to be 45 mg/kg/day.

A rapid method for purification of synthetic oligoribonucleotides.

A procedure for large-scale purification of synthetic oligoribonucleotides has been developed that has significant advantages over gel purification techniques currently in use. Synthesis was performed using commercially available 2'-O-silylated ribonucleoside 3'-O-phosphoramidites, and coupling efficiencies were consistently greater than 97% for oligoribonucleotides up to 31 residues in length. Using C4 reverse-phase chromatography to remove material not deprotected by treatment with tetrabutylammonium fluoride, we have eliminated reactants in which the 2'-O-silyl group is only partly removed, thus ensuring a homogeneous population of oligoribonucleotide.

An NMR characterization of the regA protein-binding site of bacteriophage T4 gene 44 mRNA.

The conformations of two RNA dodecamers that differ markedly in affinity for the regA protein from bacteriophage T4 have been examined by NMR to see if the ability of that protein to discriminate between mRNAs is based on pre-existing differences in their three-dimensional structures. One of the RNAs examined has the same sequence as the site where regA protein binds when it inhibits the expression of gene 44's mRNA. The second RNA differs from the first in having a U instead of a G at position -9; it binds regA protein 100 times less tightly. The NMR data indicate that both RNAs have similar single-stranded conformations and that they each resemble an isolated strand of a double helix. They also show that most, if not all of the ribose rings in both molecules have appreciable 2'-endo puckering. It is unlikely that regA protein distinguishes between these two molecules on the basis of differences in their global conformations in solution.

DNA polymerization in the absence of exonucleolytic proofreading: in vivo and in vitro studies.

Classical genetic selection was combined with site-directed mutagenesis to study bacteriophage T4 DNA polymerase 3'----5' exonuclease activity. A mutant DNA polymerase with very little (less than or equal to 1%) 3'----5' exonuclease activity was generated. In vivo, the 3'----5' exonuclease-deficient DNA polymerase produced the highest level of spontaneous mutation observed in T4, 500- to 1800-fold above that of wild type. The large reduction in 3'----5' exonuclease activity appears to be due to two amino acid substitutions: Glu-191 to Ala and Asp-324 to Gly. Protein sequence similarities have been observed between sequences in the Escherichia coli DNA polymerase I 3'----5' exonuclease domain and conserved sequences in eukaryotic, viral, and phage DNA polymerases. It has been proposed that the conserved sequences contain metal ion binding ligands that are required for 3'----5' exonuclease activity; however, we find that some proposed T4 DNA polymerase metal binding residues are not essential for 3'----5' exonuclease activity. Thus, our T4 DNA polymerase studies do not support the hypothesis by Bernad et al. [Bernad, A., Blanco, L., Lazaro, J.M., Martin, G. & Salas, M. (1989) Cell 59, 219-228] that many DNA polymerases, including T4 DNA polymerase, share an extensively conserved 3'----5' exonuclease motif. Therefore, extrapolation from E. coli DNA polymerase I sequence and structure to other DNA polymerases for which there is no structural information may not be valid.

Characterization of bacteriophage T4 regA protein-nucleic acid interactions.

The bacteriophage T4 regA protein is a translational repressor of a group of T4 early mRNAs. We have characterized the binding of regA protein to polynucleotides and to specific RNAs. Binding to nucleic acids was monitored by the quenching of the intrinsic tryptophan fluorescence of regA protein. regA protein exhibited differential affinities for the polynucleotides examined, with the order of affinity being poly(rU) greater than poly(dT) greater than poly(dU) = poly(rG) greater than poly(rC) = poly(rA). The binding site size calculated for regA protein binding to poly(rU) was n = 9 +/- 1 nucleotides. Cooperativity was observed in binding to multiple-site oligonucleotides, with a cooperativity parameter (omega) value of 10-22. To study the specific interaction between regA protein and T4 gene 44 mRNA, the affinity of regA protein for synthetic gene 44 RNA fragments was measured. The association constant (Ka) for regA protein binding to gene 44 RNA fragments was 100-fold higher than for binding to nontarget RNA. Study of variant gene 44 RNA fragments indicated that the nucleotides required for specific binding are contained within a 12-nucleotide sequence spanning -12 to -1, relative to the AUG codon. The bases of five nucleotides (indicated in upper case type) are critical for specific regA protein interaction with the gene 44 recognition element, 5'-aaUGAGgAaauu-3'. These studies further showed that formation of a regA protein-RNA complex involves a maximum of 2-3 ionic interactions and is primarily an enthalpy-driven process.

Bacteriophage T4 regA protein binds to the Shine-Dalgarno region of gene 44 mRNA.

We have overproduced and purified wild type regA protein, a translational repressor encoded by bacteriophage T4. The repressor activity of the cloned regA protein has been tested on four known regA target genes (T4 genes: 44, 45, rpbA and regA) using in vitro coupled transcription-translation reactions. We have demonstrated the sensitivity of two additional T4 genes coding for alpha- and beta-glucosyltransferases to regA protein in vitro. The regA target site on the gene 44 messenger RNA has been identified through deletion analysis and RNase protection assays, using plasmids containing gene 44-lacZ fusions. The effect of regA protein on expression of 44P-beta-galactosidase fusion proteins was assayed in vitro, in coupled transcription-translation reactions. Analysis of deletion mutants of gene 44-lacZ localized the regA recognition region to between nucleotides -11 and +9 of the mRNA. RNase protection assays of g44-lacZ transcripts further defined the site of regA protein interaction to between nucleotides -10 and +2 of the mRNA. This region overlaps the gene 44 Shine-Dalgarno region and the A and U of the initiation codon.

The 44P subunit of the T4 DNA polymerase accessory protein complex catalyzes ATP hydrolysis.

The genes encoding all three T4 DNA polymerase accessory proteins have been cloned into overexpression plasmids. Induction of cells harboring these plasmids results in the synthesis of each accessory protein at levels that approach 10% of the total cellular protein. The solubility of the accessory proteins after induction at 42 degrees C ranges from about 60% to greater than 95%. A plasmid that allows overexpression of the 44P/62P complex has been manipulated further to overexpress selectively the 44P subunit without 62P, permitting us to assess how each subunit contributes to the properties of the 44P/62P complex. A comparison of 44P and 44P/62P by conventional hydrodynamic techniques shows that 44P forms a subcomplex nearly as large as the 44P/62P complex. In addition, 44P catalyzes DNA-dependent ATP hydrolysis with a specific activity similar to that of the 44P/62P ATPase. However, unlike the 44P/62P complex, the ATPase activity of 44P alone is only slightly stimulated by 45P. This suggests that one role of the 62P subunit is to facilitate a productive interaction of 44P and 45P.

Primary structure of T4 DNA polymerase. Evolutionary relatedness to eucaryotic and other procaryotic DNA polymerases.

Bacteriophage T4 gene 43 codes for the viral DNA polymerase. We report here the sequence of gene 43 and about 70 nucleotides of 5'- and 3'-flanking sequences, determined by both DNA and RNA sequencing. We have also purified T4 DNA polymerase from T4 infected Escherichia coli and from E. coli containing a gene 43 overexpression vector. A major portion of the deduced amino acid sequence has been verified by peptide mapping and sequencing of the purified DNA polymerase. All these results are consistent with T4 DNA polymerase having 898 amino acids with a calculated Mr = 103,572. Comparison of the primary structure of T4 DNA polymerase with the sequence of other procaryotic and eucaryotic DNA polymerases indicates that T4 DNA polymerase has regions of striking similarity with animal virus DNA polymerases and human DNA polymerase alpha. Surprisingly, T4 DNA polymerase shares only limited similarity with E. coli polymerase I and no detectable similarity with T7 DNA polymerase. Based on the location of specific mutations in T4 DNA polymerase and the conservation of particular sequences in T4 and eucaryotic DNA polymerases, we propose that the NH2-terminal half of T4 DNA polymerase forms a domain that carries out the 3'----5' exonuclease activity whereas the COOH-terminal half of the polypeptide contains the dNTP-binding site and is necessary for DNA synthesis.

Cloning and expression of T4 DNA polymerase.

The structural gene coding for bacteriophage T4 DNA polymerase (gene 43) has been cloned into inducible plasmid vectors, which provide a source for obtaining large amounts of this enzyme after induction. The T4 DNA polymerase produced in this fashion was purified by an innovative three-step procedure and was fully active.