A long stringent sequence signal for programmed chromosome breakage in Tetrahymena thermophila

Programmed chromosome breakage occurs at 50–200 specific sites in the genome of Tetrahymena thermophila during somatic nuclear (macronuclear) differentiation. Previous studies have identified a 15 bp sequence, the Cbs (for chromosome breakage sequence), that is necessary and sufficient to specify these sites. In this study we determined the effects of mutations in the Cbs on its ability to specify the chromosome breakage site and promote new telomere formation in conjugating cells. Twenty-one constructs with single nucleotide substitutions covering all 15 positions of the Cbs were made and tested. Fourteen of them (covering 11 positions) abolished breakage entirely, six (covering six positions, including the remaining four) caused partial loss of breakage function and one showed no detectable effect. This result indicates that the Cbs has an exceptionally long and stringent sequence requirement. It offers no evidence that the Cbs contains a separate domain for promoting new telomere formation. In addition, we found that a partially functional Cbs retained in the macronucleus does not induce chromosome breakage during vegetative growth and that excess copies of this germline-specific sequence in the somatic nucleus have little deleterious effect on cell growth.     

Rapid selection of microbial producers of cholinesterases

A rapid and simple procedure for the screening of microbial producers of cholinesterases has been developed. It is based on the hydrolysis of acetyl- and/or butyryl-thiocholine by the intact cells of cholinesterase positive strains; the released thiocholine reacts with DTNB to form an intensely yellow product.     

Experimental apparatus for selection of adherent microorganisms under stringent growth conditions

A bioreactor apparatus is described for studying bacterial attachment. A cyclic, on-off, flow regime was imposed within the apparatus. Model calculations illustrate the utility of this flow pattern in the selection and maintenance of slow-growing, adherent organisms. The apparatus is believed to have general utility in testing bacterial attachment influenced by many types of experimental or environmental constraints, including variations in fluid dynamics, presence of toxic substances (metals or organics), nature of the substratum surface, concentrations of limiting nutrients, and competition between bacterial strains. As an example application, the apparatus was employed to test 14 bacterial strains for surface attachment in a nutrient-limited growth medium. The medium was developed, using the chemical equilibrium program MINEQL, for planned studies of biofilms in a solution with a chemically defined composition that permits calculation of trace metal speciation. The apparatus was used to select organisms with growth and attachment characteristics that could not be evaluated by conventional batch, or chemostat, culture conditions. When supplied with acetate, pyruvate, or succinate as a carbon and energy source, the gram-negative strains Pseudomonas cepacia 17616 and Zoogloea sp. WGO4 showed superior attachment characteristics to glass surfaces in the chemically defined medium but only moderate fluid-phase growth. The gram-positive Arthrobacter sp. strain 9G4D and gram-negative species P. pickettii and Zoogloea sp. WNJ8, when supplied with pyruvate as a carbon and energy source, were capable of superior growth in the fluid phase but formed only a low to moderate biofilm surface coverage.     

Methods to create a stringent selection system for mammalian cell lines

The efficient establishment of high protein producing recombinant mammalian cell lines is facilitated by the use of a stringent selection system. Here, we describe two methods to create a stringent selection system based on the Zeocin resistance marker. First, we cloned increasingly longer stretches of DNA, encoding a range of 8-131 amino acids immediately upstream of the Zeocin selection marker gene. The DNA stretches were separated from the open reading frame of the selection marker gene by a stopcodon. The idea behind this was that the translation machinery will first translate the small peptide, stop and then restart at the AUG of the Zeocin marker. This process, however, will become less efficient with increasingly longer stretches of DNA upstream of the Zeocin marker that has to be translated first. This would result in lower levels of the Zeocin selection marker protein and thus a higher selection stringency of the system. Secondly, we performed a genetic screen to identify PCR induced mutations in the Zeocin selection protein that functionally impair the selection marker protein. Both the insertion of increasingly longer peptides and several Zeocin selection protein mutants resulted in a decreasing number of stably transfected colonies that concomitantly displayed higher protein expression levels. When the Zeocin mutants were combined with very short small peptides (8-14 amino acids long), this created a flexible, high stringency selection system. The system allows the rapid establishment of few, but high protein producing mammalian cell lines.     

Experimental apparatus for selection of adherent microorganisms under stringent growth conditions.

A bioreactor apparatus is described for studying bacterial attachment. A cyclic, on-off, flow regime was imposed within the apparatus. Model calculations illustrate the utility of this flow pattern in the selection and maintenance of slow-growing, adherent organisms. The apparatus is believed to have general utility in testing bacterial attachment influenced by many types of experimental or environmental constraints, including variations in fluid dynamics, presence of toxic substances (metals or organics), nature of the substratum surface, concentrations of limiting nutrients, and competition between bacterial strains. As an example application, the apparatus was employed to test 14 bacterial strains for surface attachment in a nutrient-limited growth medium. The medium was developed, using the chemical equilibrium program MINEQL, for planned studies of biofilms in a solution with a chemically defined composition that permits calculation of trace metal speciation. The apparatus was used to select organisms with growth and attachment characteristics that could not be evaluated by conventional batch, or chemostat, culture conditions. When supplied with acetate, pyruvate, or succinate as a carbon and energy source, the gram-negative strains Pseudomonas cepacia 17616 and Zoogloea sp. WGO4 showed superior attachment characteristics to glass surfaces in the chemically defined medium but only moderate fluid-phase growth. The gram-positive Arthrobacter sp. strain 9G4D and gram-negative species P. pickettii and Zoogloea sp. WNJ8, when supplied with pyruvate as a carbon and energy source, were capable of superior growth in the fluid phase but formed only a low to moderate biofilm surface coverage.     

The use of the protoplast method for the selection of oleandomycin producers

The use of protoplasting with subsequent reversion to the cellular form in improvement of the oleandomycin-producing organism provided a 110% increase in the range of culture variation with respect to the antibiotic production property. A regenerant with a potency of 12 to 20 per cent higher than that of the initial strain which produced 30 per cent lower amounts of dark pigments of melanin nature was isolated. Repeated protoplasting and regeneration of the regenerant provided a very low regeneration frequency i.e. 0.0002%. The potency of all the secondary regenerants was low.     

A composite-likelihood approach for detecting directional selection from DNA sequence data

We present a novel composite-likelihood-ratio test (CLRT) for detecting genes and genomic regions that are subject to recurrent natural selection (either positive or negative). The method uses the likelihood functions of Hartl et al. (1994) for inference in a Wright-Fisher genic selection model and corrects for nonindependence among sites by application of coalescent simulations with recombination. Here, we (1) characterize the distribution of the CLRT statistic (Lambda) as a function of the population recombination rate (R=4Ner); (2) explore the effects of bias in estimation of R on the size (type I error) of the CLRT; (3) explore the robustness of the model to population growth, bottlenecks, and migration; (4) explore the power of the CLRT under varying levels of mutation, selection, and recombination; (5) explore the discriminatory power of the test in distinguishing negative selection from population growth; and (6) evaluate the performance of maximum composite-likelihood estimation (MCLE) of the selection coefficient. We find that the test has excellent power to detect weak negative selection and moderate power to detect positive selection. Moreover, the test is quite robust to bias in the estimate of local recombination rate, but not to certain demographic scenarios such as population growth or a recent bottleneck. Last, we demonstrate that the MCLE of the selection parameter has little bias for weak negative selection and has downward bias for positively selected mutations.     

Enrichment technique for the selection of catabolite repression-resistant mutants of Trichoderma as producers of cellulase

Abstract The enrichment technique for the preparation of catabolite repression-resistant producers of cellulase from Trichoderma reesei is based on the submerged cultivation of mutagenized conidia on 2% (w/v) cellobiose or carboxymethyl-cellulose and in the presence of 0.5% (w/v) 2-deoxyglucose as the catabolite repressor. Conidia that are resistant towards the catabolite repressor can produce enzymes necessary for hydrolysis of used substrates and grow under the given conditions. They can be separated from the ungerminated conidia by filtration and used for the production of new conidia which are already enriched with catabolite repression-resistant mutants.     

A practical and robust sequence search strategy for structural genomics target selection

MOTIVATION: Target selection strategies for structural genomic projects must be able to prioritize gene regions on the basis of significant sequence similarity with proteins that have already been structurally determined. With the rapid development of protein comparison software a robust prioritization scheme should be independent of the choice of algorithm and be able to incorporate different sequence similarity thresholds. RESULTS: A robust target selection strategy has been developed that can assign a priority level to all genes in any genome. Structural assignments to genome sequences are calculated at two thresholds and six levels (1-6) describe the prioritization of all whole genes and partial gene regions. This simple two-threshold approach can be implemented with any fold recognition or homology detection algorithms. The results for 10 genomes are presented using the SSEARCH and PSI-BLAST programs. AVAILABILITY: Programs are available on request from the authors.     

Automated constraint-based nucleotide sequence selection for DNA computation

We present techniques for automating the design of computational systems built using DNA, given a set of high-level constraints on the desired behavior and performance of the system. We have developed a program called SCAN that exploits a previously implemented computational melting temperature primitive to search a 'nucleotide space' for sequences satisfying a pre-specified set of constraints, including hybridization discrimination, primer 5' end and 3' end stability, secondary structure reduction, and prevention of oligonucleotide dimer formation. The first version of SCAN utilized 24 h of computer time to search a space of over 7.5 billion unary counter designs and found only nine designs satisfying all of the pre-specified constraints. One of SCAN's designs has been implemented in the laboratory and has shown a marked improvement in performance over the products of previous attempts at manual design. We conclude with some novel ideas for improving the overall speed of the program that offer the promise of an efficient method for selecting optimal nucleotide sequences in an automated fashion.     

琥珀酸产生菌的快速选育

以牛瘤胃内容物为菌源,在高浓度CO2下向培养基额外添加莫能菌素和富马酸钠实现选择性富集培养.溴甲酚绿中性平板变色圈作为筛选标记,以变色区域大小初步筛选得到500株产酸菌株.结合TLC法快速地确定了其中含有28株产琥珀酸菌株.在HPLC和HPCE对发酵液检测的对比过程中优选HPCE法,检测发酵液得到6株优良菌株,其中发现15号菌株琥珀酸产量达11.5g/L,有较少的甲酸和乙酸产生.验证试验表明:该方法对牛瘤胃中的琥珀酸产生菌可以实现快速、有效的分离筛选,具有很好的重现性.     

In vitro selection of an RNA sequence that interacts with high affinity with thymidylate synthase

Previous studies have shown that the repressive effect of thymidylate synthase (TS) mRNA translation is mediated by direct binding of TS itself to two cis-acting elements on its cognate mRNA. To identify the optimal RNA nucleotides that interact with TS, we in vitro synthesized a completely degenerate, linear RNA pool of 25 nt and employed in vitro selection to isolate high affinity RNA ligands that bind human TS protein. After 10 rounds of selection and amplification, a single RNA molecule was selected that bound TS protein with nearly 20-fold greater affinity than native, wild-type TS RNA sequences. Secondary structure analysis of this RNA sequence predicted it to possess a stem–loop structure. Deletion and/or modification of the UGU loop element within the RNA sequence decreased binding to TS by up to 1000-fold. In vivo transfection experiments revealed that the presence of the selected RNA sequence resulted in a significant increase in the expression of a heterologous luciferase reporter construct in human colon cancer H630 and TS-overexpressing HCT-C:His-TS+ cells, but not in HCT-C18 cells expressing a functionally inactive TS. In addition, the presence of this element in H630 cells leads to induced expression of TS protein. An immunoprecipitation method using RT–PCR confirmed a direct interaction between human TS protein and the selected RNA sequence in transfected human cancer H630 cells. This study identified a novel RNA sequence from a degenerate RNA library that specifically interacts with TS.     

A novel, high stringency selection system allows screening of few clones for high protein expression

To obtain highly productive mammalian cell lines, often large numbers of clones need to be screened. This is largely due to low selection stringencies, creating many, but low protein producing clones. To remedy this problem, a novel, very stringent selection system was designed, to create few, but high protein producing clones. In essence, a selection marker with a startcodon that confers attenuated translation initiation frequency was placed upstream of the gene of interest with a startcodon that confers optimal translation initiation. From the transcribed bicistronic mRNA, the selection marker is translated at a low frequency, and the protein of interest at a high frequency. This selection system is so stringent that clones form only rarely. However, application of anti-repressor elements, which increase promoter activity, did induce the formation of clones that expressed proteins at high levels. When combined with anti-repressor elements, this novel selection system can be a valuable tool to rapidly create few, but highly productive mammalian cell lines.     

Typing and selection of wild strains of Trichoderma spp. producers of extracellular laccase

Using the ITS region and the gene tef1, 23 strains of the genus Trichoderma were identified as belonging to the species T. harzianum (n = 14), T. olivascens (n = 1), T. trixiae (n = 1), T. viridialbum (n = 1), T. tomentosum (n = 2), T. koningii (n = 1), T. atroviride (n = 1), T. viride (n = 1), and T. gamsii (n = 1). Strains expressing extracellular laccase activity were selected by decolorization/oxidation assays in solid media, using azo, anthraquinone, indigoid, and triphenylmethane dyes, and the phenolic substances tannic acid and guaiacol. No strain decolorized Direct Blue 71 or Chicago Blue 6B, but all of them weakly oxidized guaiacol, decolorized Methyl Orange, and efficiently oxidized tannic acid. Based in decolorization/oxidation assays, strains CMU‐1 (T. harzianum), CMU‐8 (T. atroviride), CMU‐218 (T. viride), and CMU‐221 (T. tomentosum) were selected for evaluating their extracellular laccase activity in liquid media. Strain CMU‐8 showed no basal laccase activity, while strains CMU‐1, CMU‐218, and CMU‐221 had a basal laccase activity of 1,313.88 mU/mL, 763.88 mU/mL, and 799.53 mU/mL, respectively. Addition of sorghum straw inhibited laccase activity in strain CMU‐1 by 34%, relative to the basal culture, while strains CMU‐8, CMU‐21, and CMU‐221 increased their laccase activity by 1,321.5%, 64%, and 47%, respectively. These results show that assayed phenolic substrates are good tools for selecting laccase producer strains in Trichoderma. These same assays indicate the potential use of studied strains for bioremediation processes. Straw laccase induction suggests that analyzed strains have potential for straw delignification in biopulping and other biotechnological applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:787–798, 2016     

Computational analysis of sequence selection mechanisms

Mechanisms leading to gene variations are responsible for the diversity of species and are important components of the theory of evolution. One constraint on gene evolution is that of protein foldability; the three-dimensional shapes of proteins must be thermodynamically stable. We explore the impact of this constraint and calculate properties of foldable sequences using 3660 structures from the Protein Data Bank. We seek a selection function that receives sequences as input, and outputs survival probability based on sequence fitness to structure. We compute the number of sequences that match a particular protein structure with energy lower than the native sequence, the density of the number of sequences, the entropy, and the "selection" temperature. The mechanism of structure selection for sequences longer than 200 amino acids is approximately universal. For shorter sequences, it is not. We speculate on concrete evolutionary mechanisms that show this behavior.