Production of poly-gama-glutamate (PGA) biopolymer by batch and semicontinuous cultures of immobilized Bacilluslicheniformis strain-R

Production of Polyglutamate (PGA) biopolymer by immobilized Bacilluslicheniformis strain-R was intensively investigated. Preliminary experiments were carried out to address the most suitable immobilization methodology. Entrapment of Bacillus cells in alginate-agar led optimal PGA production (36.75 g/l), with 1.32- and 2.18-fold increase in comparison with alginate- or K-carrageenan-immobilized cells, respectively. During semicontinuous cultivation of agar-alginate gel-cell mixture, production of PGA by 10 ml mixture was increased from 2nd to 3rd run whereas, increased till the 4th run using 15ml mixture. Adsorption was the most suitable immobilization technique for production of PGA and the sponge cubes was the preferred matrix recording 43.2 g/l of PGA with the highest cell adsorption. Furthermore, no PGA was detected when B. licheniformis cells were adsorbed on wood and pumice. Although luffa pulp-adsorbed cells recorded the highest PGA production (50.4 g/l), cell adsorption was the lowest. Semicontinuous cultivation of B. licheniformis cells adsorbed on sponge led to increase of PGA production till the 3rd run and reached 55.5 g/l then slightly decreased in the 4th run. The successful use of fixed-bed bioreactor for semicontinuous cultivation of B. licheniformis cells held on sponge cubes (3 runs, 96 hours/run) provides insight for the potential biotechnological production of PGA by immobilized cells.

Efficient procedure of preparation and properties of long uniform G4-DNA nanowires.

Here we describe a novel and efficient procedure for preparation of long uniform G4-DNA wires. The procedure includes (i) enzymatic synthesis of double-stranded DNA molecules consisting of long (up to 10,000 bases), continuous G strands and chains of complementary (dC)20-oligonucleotides, poly(dG)-n(dC)20; (ii) size exclusion HPLC separation of the G strands from the (dC)20 oligonucleotides in 0.1M NaOH; and (iii) folding of the purified G strands into G4-DNA structures by lowering the pH to 7.0. We show by atomic force microscopy (AFM) that the preparation procedure yielded G4-DNA wires with a uniform morphology and a narrow length distribution. The correlation between the total amount of nucleotides in the G strands and the contour length of the G4-DNA molecules estimated by AFM suggests monomolecular folding of the G strands into quadruplex structures. The folding takes place either in the presence or in the absence of stabilizing ions (K+ or Na+). The addition of these cations leads to a dramatic change in the circular dichroism spectrum of the G4-DNA.

Effects of substitutions in a conserved DX(2)GR sequence motif, found in many DNA-dependent nucleotide polymerases, on transcription by T7 RNA polymerase.

The region in bacteriophage T7 RNA polymerase (RNAP) comprising residues 421-425 contains a sequence motif (DX(2)GR) that is conserved among many DNA-dependent nucleotide polymerases. We have found that alterations in this motif result in enzymes that display weaker retention of the RNA product during transcript initiation, a decreased ability to make the transition to a stable elongation complex, and changes in substrate binding and catalytic activity. Many of these defects are coupled with an altered response to the presence or absence of the non-template strand. The observed constellation of defects supports a role for the motif in interacting with and stabilizing the RNA:DNA hybrid during the early stages of transcript initiation. This is consistent with the position of the motif in a T7 RNAP initiation complex. Although a conserved DX(2)GR sequence motif is also observed in multisubunit RNAPs, the structural organization of the motif and the manner in which it interacts with the RNA:DNA hybrid in the latter enzymes is different from that in T7 RNAP. However, another element in the multisubunit RNAPs that contains a highly conserved arginine residue may play the same role as R425 in T7 RNAP. (c) 2002 Elsevier Science Ltd.

Functional importance of the 3'-terminal adenosine of tRNA in ribosomal translation.

The universally conserved 3'-terminal CCA sequence of tRNA interacts with large ribosomal subunit RNA during translation. The functional importance of the interaction between the 3'-terminal nucleotide of tRNA and the ribosome was studied in vitro using mutant in vitro transcribed tRNA(Val) A76G. Val-tRNA(CCG) does not support polypeptide synthesis on poly(GUA) as a message. However, in a co-translation system, where Val-tRNA(CCG) represented only a small fraction of total Val-tRNA, the mutant tRNA is able to transfer valine into a polypeptide chain, albeit at a reduced level. The A76G mutation does not affect binding of Val- or NAcVal-tRNA(CCG) to the A- or P-sites as shown by efficient peptide bond formation, although the donor activity of the mutant NAcVal-tRNA(CCG) in the peptidyl transfer reaction is slightly reduced compared with wild-type NAcVal-tRNA. Translocation of 3'-CCG-tRNA from the P- to the E-site is not significantly influenced. However, the A76G mutation drastically inhibits translocation of peptidyl-tRNA G(76) from the ribosomal A-site to the P-site, which apparently explains its failure to support cell-free protein synthesis. Our results indicate that the identity of the 3'-terminal nucleotide of tRNA is critical for tRNA movement in the ribosome.

Interrupting the template strand of the T7 promoter facilitates translocation of the DNA during initiation, reducing transcript slippage and the release of abortive products.

We have explored the effects of a variety of structural and sequence changes in the initiation region of the phage T7 promoter on promoter function. At promoters in which the template strand (T strand) is intact, initiation is directed a minimal distance of 5 nt downstream from the binding region. Although the sequence of the DNA surrounding the start site is not critical for correct initiation, it is important for melting of the promoter and stabilization of the initiation complex. At promoters in which the integrity of T strand is interrupted by nicks or gaps between -5 and -2 the enzyme continues to initiate predominately at +1. However, under these conditions there is a decrease in the release of abortive products of 8-10 nt, a decrease in the synthesis of poly(G) products (which arise by slippage of the nascent transcript), and a defect in displacement of the RNA. We propose that unlinking the binding and initiation regions of the T strand changes the manner in which this strand is retained in the abortive complex, reducing or eliminating the need to pack or "scrunch" the strand into the complex during initiation and lowering a thermodynamic barrier to its translocation.

A new G-tailing method for the determination of the poly(A) tail length applied to hepatitis A virus RNA.

To study the role of the poly(A) tail length during the replication of poly(A)-containing plus-strand RNA virus, we have developed a simple reverse transcription polymerase chain reaction (RT-PCR)-based method that substantially improves the previously reported PAT [poly(A) test] assay. In contrast to the PAT assay, the new method is based on the enzymatic 3' elongation of mRNA with guanosine residues, thus immediately preserving the 3' end of the RNA and creating a unique poly(A)-oligo(G) junction. The oligo(G)-protected full-length poly(A) tail is reverse transcribed using the universal anti-sense primer oligo(dC(9)T(6)) and amplified by PCR with a gene-specific sense primer. After sequencing the resulting RT-PCR product the length of the poly(A) tail was unequivocally deduced from the number of adenosine residues between the oligo(G) stretch and the sequence upstream of the poly(A) tail. The efficiency and specificity of the newly developed assay was demonstrated by analysing the poly(A) tail length of the hepatitis A virus (HAV) RNA. We show here that the poly(A) tail of HAV RNA rescued after transfection of in vitro transcripts was elongated in the course of HAV replication.

Reiterative dG addition by Euplotes crassus telomerase during extension of non-telomeric DNA.

Telomerase from the ciliate Euplotes crassus incorporates G4T4telomeric repeats onto both telomeric and non-telomeric single-stranded DNA 3'-ends via reverse transcription of a templating domain in its RNA subunit. Here we describe an unusual mode of template copying that is characteristic of DNA synthesis onto non-telomeric 3'-ends in vitro . When dTTP was eliminated from telomerase reactions, telomeric primers or DNA products generated from the telomerase endonuclease were extended by precise copying of the RNA template. In contrast, telomerase catalyzed the addition of up to 13 dG residues onto primers with non-telomeric 3'-ends under the same reaction conditions. Introducing mismatches in the 3'-terminus of telomeric primers that reduced primer complementarity to the RNA template induced reiterative dG incorporation, indicating that the reaction is influenced by Watson-Crick base pair formation between the primer and the RNA template. Unexpectedly, the reiterative dG addition mode was confined to telomerase derived from developing cells that undergo new telomere formation. This reaction was not observed in vegetatively growing cells. We postulate that indiscriminate dG addition by telomerase occurs by reiterative copying of C residues in the telomerase RNA templating domain and reflects lateral instability of the primer-template interaction during de novo telomere formation.

Template-dependent biosynthesis of poly(G) x poly (C) and its antiviral activity in vitro and in vivo.

Experimental conditions for poly(G) synthesis from GTP on a poly(C) template with the aid of Escherichia coli DNA-dependent RNA polymerase were investigated. The reaction product was purified without the use of RNase. On the basis of spectral data, gel permeation chromatography, affinity adsorption and electron microscopic visualization, the poly(G) x poly(C) product was assumed to possess a high degree of structural regularity. Its in vitro and in vivo antiviral activities were compared with those of traditional poly(G) x poly(C) and poly(I) x poly(C). Template-dependent poly(G) x poly(C) was similar in its in vitro activity to poly(I) x poly(C) or even surpassed it, whereas the 'traditional' poly(G) x poly(C) was only slightly active in vitro. However, 'traditional' poly(G) x poly(C) and poly(I) x poly(C) had similar activity in vivo, whereas template-dependent poly(G) x poly(C) was much less active in vivo. The role of intramolecular structural regularity in the in vitro and in vivo antiviral activity of polyribonucleotide duplexes is discussed.

Transdominant inhibition of Moloney murine leukemia virus proliferation by defective mutants of reverse transcriptase.

To investigate the dominant-negative inhibition of Moloney murine leukemia virus (Mo-MuLV) proliferation by polymerization-defective mutants of reverse transcriptase (RT), we constructed several plasmids harboring the Mo-MuLV RT gene in which the YVDD sequence, one of the conserved sequences in RNA-dependent polymerases, was altered, and then transformed mouse NIH3T3 cells and Escherichia coli with the mutant plasmids. Mouse NIH3T3 cells expressing these mutant RT genes were highly resistant to Mo-MuLV proliferation. The mutant RT expressed in E. coli exhibited no polymerization activity, but it retained its binding activity to the template RNA and inhibited in vitro poly(dG) synthesis occurring with the wild-type RT. These results suggest that the competition for binding of the two types of enzymes to the template is responsible for the resistance to Mo-MuLV proliferation and that the YVDD sequence of Mo-MuLV may be a good target for dominant-negative inhibition of retroviral proliferation.

A study on the function of the glycine residue in the YGDD motif of the RNA-dependent RNA polymerase beta-subunit from RNA coliphage Q beta 1.

Q beta replicases in which the Gly residue of the beta-subunit in the motif sequence, YGDD, was replaced with Ala, Ser, Pro, Met, or Val lost their replicase activity in vivo. In an in vitro Mg(2+)-dependent RNA-synthesizing system using poly(rC) or MDV-poly(+) RNA (a derivative of the naturally occurring small RNA that accumulates in the cells during Q beta phage infection) as templates, the lysates from the cells expressing such defective replicases exhibited only 2-6% of the enzyme activity of the lysate from those expressing wild-type replicase. However, the defective replicases, especially A357, with Ala substituted for the Gly, recovered enzyme activity when Mn2+ was added to the reaction mixture. Furthermore, the characteristics of the MDV-poly(+) RNA-dependent RNA synthesis by A357 replicase were similar to those by wild-type replicase in the presence of Mn2+. Gel retardation assay showed that all of the defective replicases could bind MDV-poly(+) RNA. These results suggest that the Gly residue in this motif of Q beta replicase is involved in Mg(2+)-catalyzed polymerization. In the Mn(2+)-catalyzed polymerization, A357 and S357 replicases can act as well as the wild-type replicase.

Inhibition of HIV-1 reverse transcriptase by defined template/primer DNA oligonucleotides: effect of template length and binding characteristics.

The interaction of partially double stranded DNA oligonucleotides with HIV-1 RT was studied by investigating their ability to inhibit the homopolymeric poly(rC) directed (dG) synthesis reaction. A 20/18mer oligonucleotide, with a sequence based on the Lys3-tRNA primer region, showed stronger inhibition of the homopolymeric RT reaction than a G/C rich oligonucleotide series lacking or possessing a hairpin moiety. Interaction of the enzyme with the G/C rich oligonucleotides, as determined by IC50 measurements, was insensitive to the extent of the unpaired template region at the 3' or 5' position. Addition of a hairpin moiety, composed of four thymidine bases, onto G/C rich oligonucleotides increase their inhibitory potency (at least six times) and shifted the mode of inhibition of RT to competitive with respect to poly (rC).(dG), which was otherwise mixed (competitive/noncompetitive) for the linear G/C rich and 20/18mer oligonucleotides. The results indicate that interaction of the enzyme with the primer/template stem, but not with the unpaired template region, is an important step in complex formation.

Thermodynamics of A:G mismatch poly(dG) synthesis by human immunodeficiency virus 1 reverse transcriptase.

Human immunodeficiency virus 1 (HIV-1) reverse transcriptase has been found to conduct error-prone synthesis on DNA and RNA templates. We find here that tolerance of an A:G mispair with poly(rA) as template is particularly strong, such that extensive poly(dG) synthesis is conducted. This type of extensive misincorporation is not observed with several reference DNA polymerases. Surprisingly, HIV reverse transcriptase processivity and kcat for dGMP misincorporation and normal dTMP incorporation are about the same. However, the Km value for dGTP in poly(dG) synthesis is approximately 1000-fold higher than the Km for dTTP in poly(dT) synthesis. Comparison of thermodynamic parameters for dGMP misincorporation and normal dNMP incorporation indicates a lower energy of activation for dGMP misincorporation than for normal dNMP incorporation. Entropy of activation (delta S*) for normal dTMP incorporation is positive (approximately 10 cal/kmol), whereas delta S* for dGMP misincorporation is negative (-36 cal/kmol). Since differences in delta S* are usually considered to reflect differences in solvation for the transition state complex, these results are consistent with the interpretation that the active site of HIV reverse transcriptase is flexible enough to misincorporate dGMP without the usual dispersion of water molecules.

Extensive synthesis of poly[r(G-C)] using Escherichia coli RNA polymerase.

Conditions have been found that allow for extensive synthesis of poly[r(G-C)] using Escherichia coli RNA polymerase with a poly[d(I-C) X poly[d(I-C)] template. The extensive synthesis, in which many copies of the template were produced by the enzyme, continued for 24 h. Repeated addition of the template was necessary for synthesis to continue, as the poly[r(G-C)] appeared to be binding to the template, inhibiting transcription by the polymerase. Four hundred ODU, or 20 mg of the poly[r(G-C)] has been prepared using 0.3 mg of protein.

Purification of a primase activity associated with DNA polymerase alpha from HeLa cells.

Highly purified preparations of eukaryotic DNA polymerase alpha have been shown to contain primase activity (Kaguni, L.S., Rossignol, J-M., Conaway, R.C. Banks, G.R., and Lehman, I.R. (1983) J. Biol. Chem. 258, 9037-9039; Yagura, T., Kozu, T., and Seno, T. (1982) J. Biol. Chem. 257, 11121-11127; Shioda, M., Nelson, E.M., Bayne, M.L., and Benbow, R.M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7209-7213). We have investigated the de novo synthesis of DNA by a primase-DNA polymerase alpha preparation isolated from human HeLa cells using the synthetic homopolymers poly(dT) and poly(dC) as templates. In the presence of poly(dT), synthesis of poly(dA) required ATP in addition to dATP while synthesis of poly(dG) in the presence of poly(dC) required GTP in addition to dGTP. The primase activity required a much lower GTP concentration (Km = 0.1 mM) than ATP (Km = 0.8 mM) for the synthesis of DNA. Guanosine 5'-O-(3-thiotriphosphate), 5'-guanylyl-beta, gamma-imidodiphosphate, and 5'-guanylyl methylenediphosphonate substituted for GTP but the corresponding ATP analogues did not substitute for ATP. Furthermore, ATP and ATP analogues inhibited the GTP-dependent reaction while GTP and GTP analogues inhibited the ATP-dependent reaction. DNase treatment of products labeled with [alpha-32P] GTP revealed that an RNA oligomer was covalently linked to newly synthesized DNA. Alkaline hydrolysis of these products yielded GMP and pppGp, indicating that the primer was initiated with GTP. Alkaline hydrolysis of [alpha-32P]dGTP-labeled products yielded 2'- and 3'-GMP showing that DNA chains are covalently linked to the 3' ends of RNA chains. The primase activity could not be separated from DNA polymerase alpha through a 200-fold enrichment involving phosphocellulose, DNA-cellulose, hydroxylapatite, DEAE-cellulose and glycerol gradient purification steps. However, primase activity was found to be less stable than DNA polymerase alpha activity under a variety of conditions.

Reverse transcriptase associated with avian sarcoma-leukosis viruses. II. Comparison of subunit structure and catalytic properties.

The three enzyme forms (alpha, alpha beta-, and beta-form) of the RNA-dependent DNA polymerase (the reverse transcriptase) from three strains of avian sarcoma virus (ASV B77, ASV tsLA334, and ASV QV2) and one exogenous (avian myeloblastosis virus (AMV)) and one endogenous avian leukosis virus (Rous-associated virus type-0 (RAV-0) were compared with each other in subunit structure and catalytic properties. Despite the gross similarity in the subunit molecular weight (Mr(alpha) = 65,000 and Mr(beta) = 92,000), minor differences were found in the molecular size of the subunit as determined by SDS-gel electrophoresis, the order being ASV tsLA334 less than or equal to ASV QV2 less than ASV B77 less than or equal to RAV-0 = AMV. The structural differences were supported by analysis of peptide fragments after treatment with S. aureus V8 protease. Although the general catalytic properties of the purified enzymes from the five virus strains were similar, the selectivety of template-primer differed in the RAV-0 enzymes. The template-primer selectivity of the reverse transcriptases from all five virus strains tested was also found to be greatly influenced by the reaction temperature for DNA synthesis, resulting in a temperature-dependent increase of poly(dG) synthesis over [(A)m] . [(dT)12-18]-dependent poly(dT) synthesis. The RAV-0 enzymes required a lower temperature for DNA synthesis, particularly for [(C)n] . [(dG)12-18]-dependent poly(dG) synthesis.

The stimulation of Escherichia coli stringent factor-dependent synthesis of guanosine 3',5'-polyphosphate [(p)ppGpp] by rat liver ribosomal proteins.

The effect of groups of proteins from rat liver ribosomes on the Escherichia coli stringent factor-catalyzed synthesis of (p)ppGpp was tested. Most groups were capable of supporting (p)ppGpp synthesis; the exceptions were A40, B140, B240 and B160 which contain proteins which are relatively less basic than those in the active groups. The capacity of 30 individual rat liver ribosomal proteins to activate stringent factor was assessed; most sustained the synthesis of (p)ppGpp. Proteins S12, S21, L12, P1, and P2 (which are acidic or relatively acid) had no activity; proteins S6, S8, and L3 were the most active: the others had moderate activity.

Polyribonucleotides containing thiopurines. Synthesis and properties of poly(1-methyl-6-thioguanylic acid).

The synthesis of 1-methyl-6-thioguanosine 5'-diphosphate and its conversion to poly(1-methyl-6-thioguanylic acid) by means of polynucleotide phosphorylase are described. The polymer exhibited cooperative behavior (Tm = 294 K in the absence of added NaCl) characteristic of a highly stacked single-stranded helical array. In a high salt environment (0.5 M NaCl) the melting was much less cooperative and gave a higher Tm (313 K); this is suggestive of interstrand aggregation involving hydrogen bonding. The polynucleotide exhibited a remarkably high pKa (6.2) compared to that of the mononucleotide (2.6), and formed a very stable acid structure (Tm = 356 K in 50% ethylene glycol). Comparisons with poly(1-methyl-6-thioinosinic acid) and poly(6-thioguanylic acid) establish that both the 2-amino group and the 1-methyl group are required for the formation of the stable acid structure.

An efficient synthesis of polyguanylic acid by a thermophilic polynucleotide phosphorylase.

Polyguanylic acid (poly(G)) was synthesized from GDP in a yield of 60-75% by Thermus thermophilus polynucleotide phosphorylase (polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8) at 70 degrees C, pH 8.5 in the presence of Mg2+. The yield was dependent on the ratio of GDP to Mg2+, but was independent of the concentrations of enzyme or substrate. The maximal rate of GDP polymerization was obtained when the ratio of GDP to Mg2+ was 3:1. However, by prolonged incubation, the higher initial ratio of over 4:1 was preferred because of the rapid consumption of GDP in the reaction mixture. Poly(G) prepared by 1 h incubation was heterogeneous in size from 5 S to over 23 S, but by prolonged incubation of 19 h the size of product converged to 9 S as judged by sucrose density gradient centrifugation. Its chain length was determined by terminal nucleoside analysis to be 200 nucleotides long.