Mutations That Improve the ANT Promoter of Salmonella Phage P22

Mutations that increase the activity of the promoter for the antirepressor gene of phage P22 were isolated by pseudoreversion of four severe promoter-down mutations. The sequence changes in these pseudorevertants include single base pair substitutions, single base pair deletions, tandem double base pair deletions and multisite mutations. The single base pair substitutions change nonconsensus base pairs to consensus base pairs at positions -14 and -8. The other mutations provide support for the idea that the length of the spacer region between the conserved -35 and -10 hexamers is an important determinant of promoter strength. Deletions of one or two base pairs in the spacer region apparently activate an alternate -10 hexamer by shifting it from a spacing of 19 base pairs to a spacing of 18 or 17 base pairs, respectively.     

NMR studies of G:A mismatches in oligodeoxyribonucleotide duplexes modelled after ribozymes.

The structures of two oligodeoxyribonucleotide duplexes, the base sequences of which were modelled after both a hammerhead ribozyme and a small metalloribozyme, were studied by NMR. Both duplexes contain adjacent G:A mismatches; one has a PyGAPu:PyGAPu sequence and the other a PyGAPy:PuGAPu sequence. It is concluded on the basis of many characteristic NOEs that in both duplexes G:A base pairs are formed in the unique 'sheared' form, where an amino proton instead of an imino proton of G is involved in the hydrogen bonding, and G and A bases are arranged 'side by side' instead of 'head to head'. A photo-CIDNP experiment, which gives unique and independent information on the solvent accessibility of nucleotide bases, also supports G:A base pairing rather than a bulged-out structure of G and A residues. This is the first demonstration that not only the PyGAPu:PyGAPu sequence but also the PyGAPy:PuGAPu sequence can form the unique sheared G:A base pairs. Taking the previous studies on G:A mismatches into account, the idea is suggested that a PyGA:GAPu sequence is a minimum and essential element for the formation of the sheared G:A base pairs. The sheared G:A base pairs in the PyGAPu:PyGAPu sequence are suggested to be more stable than those in the PyGAPy:PuGAPu sequence. This is explained rationally by the idea proposed above.     

Enzymatic repair of an expanded genetic information system

The excision repair machinery of a thermophilic bacterium has been shown to recognize and repair an expanded genetic base pair. Native Thermus aquaticus DNA polymerase will remove a mispaired natural base and replace it with a non-natural base to form an expanded base pair. In addition, DNA ligase will recognize a nick formed by polymerase between two non-natural base pairs and covalently attach the two strands, thus demonstrating complete repair of a bifurcated base-paired model duplex. These results add evidence to the idea that the cellular replication and repair machinery of an organism containing an expanded genetic alphabet could recognize and properly repair a site containing consecutive unnatural bases.     

An experimental strategy validated to design cost-effective culture media based on response surface methodology

For any fermentation process, the production cost depends on several factors, such as the genetics of the microorganism, the process condition, and the culture medium composition. In this work, a guideline for the design of cost-efficient culture media using a sequential approach based on response surface methodology is described. The procedure was applied to analyze and optimize a culture medium of registered trademark and a base culture medium obtained as a result of the screening analysis from different culture media used to grow the same strain according to the literature. During the experiments, the procedure quantitatively identified an appropriate array of micronutrients to obtain a significant yield and find a minimum number of culture medium ingredients without limiting the process efficiency. The resultant culture medium showed an efficiency that compares favorably with the registered trademark medium at a 95% lower cost as well as reduced the number of ingredients in the base culture medium by 60% without limiting the process efficiency. These results demonstrated that, aside from satisfying the qualitative requirements, an optimum quantity of each constituent is needed to obtain a cost-effective culture medium. Study process variables for optimized culture medium and scaling-up production for the optimal values are desirable.     

Base pairing between the 5' half of epsilon and a cis-acting sequence, phi, makes a contribution to the synthesis of minus-strand DNA for human hepatitis B virus

Synthesis of minus-strand DNA of human hepatitis B virus (HBV) can be divided into three phases: initiation of DNA synthesis, the template switch, and elongation of minus-strand DNA. Although much is known about minus-strand DNA synthesis, the mechanism(s) by which this occurs has not been completely elucidated. Through a deletion analysis, we have identified a cis-acting element involved in minus-strand DNA synthesis that lies within a 27-nucleotide region between DR2 and the 3' copy of DR1. A subset of this region (termed Phi) has been hypothesized to base pair with the 5' half of epsilon (H. Tang and A. McLachlan, Virology, 303:199-210, 2002). To test the proposed model, we used a genetic approach in which multiple sets of variants that disrupted and then restored putative base pairing between the 5' half of epsilon and phi were analyzed. Primer extension analysis, using two primers simultaneously, was performed to measure encapsidated pregenomic RNA (pgRNA) and minus-strand DNA synthesized in cell culture. The efficiency of minus-strand DNA synthesis was defined as the amount of minus-strand DNA synthesized per encapsidation event. Our results indicate that base pairing between phi and the 5' half of epsilon contributes to efficient minus-strand DNA synthesis. Additional results are consistent with the idea that the primary sequence of phi and/or epsilon also contributes to function. How base pairing between phi and epsilon contributes to minus-strand DNA synthesis is not known, but a simple speculation is that phi base pairs with the 5' half of epsilon to juxtapose the donor and acceptor sites to facilitate the first-strand template switch.     

Ideas are not replicators but minds are

An idea is not a replicator because it does not consist of coded self-assembly instructions. It may retain structure as it passes from one individual to another, but does not replicate it. The cultural replicator is not an idea but an associatively-structured network of them that together form an internal model of the world, or worldview. A worldview is a primitive, uncoded replicator, like the autocatalytic sets of polymers widely believed to be the earliest form of life. Primitive replicators generate self-similar structure, but because the process happens in a piecemeal manner, through bottom-up interactions rather than a top-down code, they replicate with low fidelity, and acquired characteristics are inherited. Just as polymers catalyze reactions that generate other polymers, the retrieval of an item from memory can in turn trigger other items, thus cross-linking memories, ideas, and concepts into an integrated conceptual structure. Worldviews evolve idea by idea, largely through social exchange. An idea participates in the evolution of culture by revealing certain aspects of the worldview that generated it, thereby affecting the worldviews of those exposed to it. If an idea influences seemingly unrelated fields this does not mean that separate cultural lineages are contaminating one another, because it is worldviews, not ideas, that are the basic unit of cultural evolution.     

Complexes of DNA hairpins and a single-stranded oligonucleotide detected by affinity chromatography and mung bean nuclease cleavage

The emergence of a simple translation device consisting of an assembler strand (primordial mRNA) and RNA hairpins (primordial tRNA) is presumed to be an important step leading to the origin of life. The assumption of a non-enzymatic interaction of primordial tRNA and mRNA is experimentally approached. DNA hairpins containing five or more adenosine residues in the loop are able to bind to complementary oligonucleotides covalently bound to cellulose. The exact number of base pairs formed between the hairpins and the assembler strand is determined by two methods applied to DNA hairpin/assembler complexes. The melting temperature of a complex is measured and the cleavage pattern by nuclease from mung bean is determined. The loop of the smallest hairpin able to bind consists of five adenosine residues and only three base pairs are formed. This supports the idea of a primordial recognition similar to the contemporary codon-anticodon interaction.     

Biological degradation of 2,4-dichlorophenoxyacetic acid: chloride mass balance in stirred tank reactors.

A mass balance was developed for the degradation of 2,4-dichlorophenoxyacetic acid by a mixed culture. Batch culture experiments showed the degradation to be an acid-producing step. Inorganic chloride concentration consistently correlated with the expected value and with base consumption to maintain a constant pH.     

Biological degradation of 2,4-dichlorophenoxyacetic acid: chloride mass balance in stirred tank reactors

A mass balance was developed for the degradation of 2,4-dichlorophenoxyacetic acid by a mixed culture. Batch culture experiments showed the degradation to be an acid-producing step. Inorganic chloride concentration consistently correlated with the expected value and with base consumption to maintain a constant pH.     

Recognition of DNA structure by 434 repressor.

In complexes of bacteriophage 434 binding sites with 434 repressor the central 4 bp of the 14 bp site are not contacted by the protein, although changes in these bases alter binding site affinity for the repressor. Our previous data suggested that the ability of the non-contacted central bases to be overtwisted in repressor-DNA complexes governs affinity of the binding site for 434 repressor. This idea was tested by examining the affinity of two central sequence variant 434 binding sites for 434 repressor as a function of binding site average twist. The 434 repressor preferred the relatively overwound binding site to the two more underwound forms. The greatest affinity enhancement resulting from increasing twist was observed with a binding site that is relatively underwound and more resistant to twisting deformation. Consistent with the idea that 434 repressor overtwists its binding site upon DNA binding, we show that 434 repressor is capable of binding to sites bearing a single base insertion in their center (a 15mer), but binds poorly to binding sites bearing central base deletions (12mer and 13mer). The N-terminal dimer interface plays a large role in determining 434 repressor central base preferences. Mutations in this interface eliminate central base discrimination and/or site size preferences. These mutations also lead to changes in the size of the repressor footprint on the various sized DNA sites that are consistent with their binding characteristics.     

A photobioreactor with pH control: demonstration by growth of the marine diatom Skeletonema costatum

A photobioreactor with pH control for cultivation of algae isdescribed. The magnetically stirred culture flask is connectedto separate reservoirs for medium and for acid and base (dilutedHCl and NaOH, respectively). A pH electrode is inserted intothe culture flask and coupled to a pH controller, which activatesacid and base titration at set points of pH 8.1 and 7.8, respectively.Illumination is provided by light tubes with a diel light :dark cycle. The use of the photobioreactor in batch mode isillustrated by showing pH curves in different growth phasesof the marine diatom Skeletonema costatum. The photobioreactorcan also be run as a semicontinuous or continuous reactor withslight modifications.     

Mismatch base pair detection by fluorescence spectral change upon addition of metal cation--toward efficient analysis of single nucleotide polymorphism

Addition of mercury (II) cation to fluorescent-labeled duplex involving a T:T mismatch base pair and silver (I) cation to fluorescent-labeled duplex involving a C:C mismatch base pair significantly changed the fluorescence intensity, but no significant change in the fluorescence intensity was observed for duplexes involving the other base pairs. The fluorescence spectral change upon addition of the metal cation can discriminate T:T and C:C mismatch base pairs from the other base pairs. Our results certainly support the idea that the fluorescence spectral change upon addition of the metal cation could be a convenient strategy for the mismatch base pair detection by the heteroduplex analysis, and may eventually lead to progress in single nucleotide polymorphism genotyping.     

A model for messenger RNA sequences maximizing secondary structure due to code degeneracy.

The general occurrence of stable self-complementary mRNAs in the cells of higher organisms is assumed. As an example the amino acid sequence of human α-globin was “retranslated” into a hypothetical polynucleotide sequence, corrected as much as possible by mutant globins and completed by the chain termination variant “Constant Spring”. Under the idea of optimizing secondary structures due to code degeneracy models for α-globin mRNA with base paired regions were constructed on the basis of thermodynamic data. They were chosen by hand and by a Fortran program. Preservation of mRNA conformations is discussed as a possible function of code degeneracy during evolution.     

Codon choice and potential complementarity between mRNA downstream of the initiation codon and bases 1471-1480 in 16S ribosomal RNA affects expression of glnS.

A cis-acting expression mutation, GAG to GAA, in the third codon of the glnS gene is analyzed. Both codons code for glutamic acid but the mutation is known to increase gene expression by four fold. We show that the mutation has an effect only if it is located in the beginning of a gene but not if located internally. Data are presented that suggest that the reason for the increased expression by the mutation is the potential formation of one more base pair between the mRNA and 16S ribosomal RNA. Gene expression varies about 16 fold as the number of potential base pairs within the sequence 1471-1480 in 16S RNA increase from two to ten. We also give evidence that supports the idea that the presence of rare codons near the beginning of the mRNA can affect expression.     

罗汉果组织培养中愈伤组织和腋生枝的形成

用罗汉果茎段为外植体,培养在MS+BA1.0+IBA0.1毫克/升培养基上,诱导愈伤组织和芽形成。观察了罗汉果茎段愈伤组织的生长以及腋生枝的形成。罗汉果茎段培养5天后,潜伏腋芽开始萌动和生长。培养10天后罗汉果茎段的基部一端开始膨大。培养20天后产生大量白色疏松的愈伤组织,这时腋生枝已经长成3—6厘米长。培养30天后基部的愈伤组织中有少量瘤状小突起,但再分化形成芽的频率极低。结果表明,罗汉果茎段组培形成的苗均是从腋芽产生的。     

The effect of adriamycin on Z-DNA formation and DNA synthesis.

The ability of adriamycin to inhibit Z-DNA formation induced by a high-salt environment was investigated. ADM inhibited this conversion, such that in poly (dG-dC) total inhibition was observed at 1 ADM: 9 base pairs and in eukaryotic DNA (calf thymus) at 1 ADM: 11,5 base pairs. Even at low ADM concentration, 1 ADM: 160 base pairs, some inhibition was observed. At similar ADM:DNA concentrations, an inhibition in DNA synthesis in cells in culture was observed, which showed some parallel with the inhibition of Z-DNA formation. A model is proposed where Z-DNA formation precedes DNA synthesis and where inhibition of the former could explain the antineoplastic nature of adriamycin.     

Synthetic hydrogel niches that promote hMSC viability.

Photopolymerized poly(ethylene glycol) (PEG) hydrogels were used as a base platform for the encapsulation and culture of human mesenchymal stem cells (hMSCs). The base PEG formulation presents an environment completely devoid of cell-matrix interactions. As such, viability of hMSCs in unmodified PEG hydrogels is very low. This formulation was modified to contain pendant phosphate groups to facilitate the sequestering of osteopontin within the gel, as well as pendant cell-adhesive RGD peptide sequences, which are found in osteopontin and other cell adhesion proteins. The survivability of hMSCs was examined with culture time and as a function of the gel chemistry to examine the role of cell-matrix interactions in promoting long-term viability. In the absence of any adhesive ligands, hMSC viability drops to 15% after 1 week in culture. However, by incorporating the RGD sequence or pendant phosphate groups this low viability was rescued to 75% and 97%, respectively. It is believed that the phosphate groups promote mineralization of the hydrogel network, and this mineral phase sequesters cell-secreted osteopontin, resulting in enhanced cell-matrix interactions and improved cell viability.     

Mixed culture biotechnology for bioenergy production

Mixed culture biotechnology (MCB) could become an attractive addition or alternative to traditional pure culture based biotechnology for the production of chemicals and/or bioenergy. On the basis of ecological selection principles, MCB-based processes can be established that generate a narrow product spectrum from a mixed substrate. Three example processes are briefly discussed in this paper: anaerobic digestion aimed at the production of methane-containing biogas, mixed culture fermentation for the production of solvents or biohydrogen, and a two-step process for the production of polyhydroxyalkanoates. These examples give an idea of the potential contribution of mixed culture biotechnology to sustainable production of bioenergy from waste.     

Somatic hypermutagenesis in immunoglobulin genes. II. Influence of neighbouring base sequences on mutagenesis.

A new approach for the analysis of hotspots of mutations is described. It is based on the classification of hotspot site sequences. Using this approach, the consensuses RGYW and TAA of hotspot sites were revealed in the V gene. Correlation between somatic mutations and these consensuses is investigated by the statistical weight method in 323 somatic substitutions in 14 V genes. Assuming the absence of any correlation, the probability of observing such data in the sample would be very low (0.0003). These results support the idea that emergence of somatic mutation is significantly influenced by neighbouring base sequences. This idea was also supported by the analysis of 296 somatic mutations in flanking sequences of V genes. It is supposed that this influence is an important feature of somatic hypermutagenesis.     

Time-resolved photointermediate changes in rhodopsin glutamic acid 181 mutants

The role of glutamic acid 181 in the bovine rhodopsin retinylidene chromophore pocket was studied by expressing E181 mutants in COS cells and measuring, as a function of time, the absorbance changes produced after excitation of lauryl maltoside pigment suspensions with 7 ns laser pulses. All mutants studied except E181D showed accelerated decay of bathorhodopsin compared to wild type. Even for E181D, an anomalously large blue shift was observed in the absorption spectrum of the bathorhodopsin decay product, BSI. These observations support the idea that E181 plays a significant role in the earliest stages of receptor activation. E181 mutations have a pronounced effect on the decay of the lumirhodopsin photointermediate, primarily affecting the size of the red shift that occurs in the lumirhodopsin I to lumirhodopsin II transition that takes place on the 10 micros time scale after wild-type photoexcitation. While the spectral change that occurs in the lumirhodopsin I to lumirhodopsin II transition in wild-type rhodopsin is very small ( approximately 2 nm), making it difficult to detect, it is larger in E181D ( approximately 6 nm), making it evident even in the lower signal-to-noise ratio measurements possible with rhodopsin mutants. The change seen is even larger for the E181F mutant where significant amounts of a deprotonated Schiff base intermediate are produced with the 10 micros time constant of lumirhodopsin II formation. The E181Q mutant shows lumirhodopsin decay more similar to wild-type behavior, and no lumirhodopsin I to lumirhodopsin II transition can be resolved. The addition of chloride ion to E181Q increases the lumirhodopsin I-lumirhodopsin II spectral shift and slows the deprotonation of the Schiff base. The latter result is consistent with the idea that a negative charge at position 181 contributes to protonated Schiff base stability in the later intermediates.