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Ginkgo biloba breeding commonly concentrates on the selection of superior trees that have high flavonoid contents. The flavonoids present in Ginkgo leaves have strong medicinal implications, including anti-dengue, anti-HIV, anticancer, antioxidant and anti-inflammatory properties. Flavonoids play important roles in plant immune responses; however, the molecular mechanisms underlying these interesting responses remain unclear. To obtain a comprehensive understanding, we performed the transcriptome sequencing of Ginkgo with different flavonoid contents. Using an Illumina sequencing platform, we obtained approximately 533,952,528 clean reads. After the sequences were filtered and assembled, the transcriptome data generated 37,625 unigenes, of which 21,472 (57.07%) were successfully annotated in five public databases. Among those genes, many candidates were involved in flavonoid biosynthesis, transport and regulation. Expression profiles were generated, and 457 genes were found to be significantly differentially expressed between the Sample_GB_FH1/2/3 (FH) and Sample_GB_FL1/2/3 (FL) libraries; 246 (53.83%) genes were up-regulated, and 211 (46.17%) were down-regulated. These genes included 14 genes that were enriched in flavonoid transport, 1 MYB gene that encoded a putative transcription factor (TF), and 1 dihydroflavonol reductase (DFR) gene that was involved in the flavonoid pathway. Our results provide comprehensive gene expression information about the Ginkgo transcriptome and can facilitate our understanding of the molecular mechanisms of flavonoid development in Ginkgo. Furthermore, our results markedly expand both the available Ginkgo genetic library and analyses of the species and provide valuable information to the Ginkgo-related pharmaceutical industry.

Protein-metal hybrids are functional materials with various industrial applications. For example, a redox enzyme immobilized on a platinum electrode is a key component of some biofuel cells and biosensors. To create these hybrid materials, protein molecules are bound to metal surfaces. Here, we report the selection of a novel platinum-binding sequence in a loop of a four-helix bundle protein, the Lac repressor four-helix protein (LARFH), an artificial protein in which four identical α-helices are connected via three identical loops. We created a genetic library in which the Ser-Gly-Gln-Gly-Gly-Ser sequence within the first inter-helical loop of LARFH was semi-randomly mutated. The library was then subjected to selection for platinum-binding affinity by using the T7 phage display method. The majority of the selected variants contained the Tyr-Lys-Arg-Gly-Tyr-Lys (YKRGYK) sequence in their randomized segment. We characterized the platinum-binding properties of mutant LARFH by using quartz crystal microbalance analysis. Mutant LARFH seemed to interact with platinum through its loop containing the YKRGYK sequence, as judged by the estimated exclusive area occupied by a single molecule. Furthermore, a 10-residue peptide containing the YKRGYK sequence bound to platinum with reasonably high affinity and basic side chains in the peptide were crucial in mediating this interaction. In conclusion, we have identified an amino acid sequence, YKRGYK, in the loop of a helix-loop-helix motif that shows high platinum-binding affinity. This sequence could be grafted into loops of other polypeptides as an approach to immobilize proteins on platinum electrodes for use as biosensors among other applications.

Long-read sequencing, coupled to cDNA capture, provides an unrivaled view of the transcriptome of chromosome 21, revealing surprises about the splicing of long noncoding RNAs.

Combatting antibiotic resistance is critical to our ability to treat infectious diseases. Here, we identified and characterized diverse antimicrobial resistance genes, including potentially mobile elements, from synthetic wastewater treatment microcosms exposed to the antibacterial agent triclosan. After seven weeks of exposure, the microcosms were subjected to functional metagenomic selection across 13 antimicrobials. This was achieved by cloning the combined genetic material from the microcosms, introducing this genetic library into E. coli, and selecting for clones that grew on media supplemented with one of the 13 antimicrobials. We recovered resistant clones capable of growth on media supplemented with a single antimicrobial, yielding 13 clones conferring resistance to at least one antimicrobial agent. Antibiotic susceptibility analysis revealed resistance ranging from 4 to >50 fold more resistant, while one clone showed resistance to multiple antibiotics. Using both Sanger and SMRT sequencing, we identified the predicted active gene(s) on each clone. One clone that conferred resistance to tetracycline contained a gene encoding a novel tetA-type efflux pump that was named TetA(62). Three clones contained predicted active genes on class 1 integrons. One integron had a previously unreported genetic arrangement and was named In1875. This study demonstrated the diversity and potential for spread of resistance genes present in human-impacted environments.

Fluorescence imaging of neuron activity would be greatly facilitated by the availability of brightly fluorescent genetically encoded voltage indicators with large and fast responses to membrane potential changes. The Cohen group has recently described a new class of genetically encoded voltage indicators based on the microbial rhodopsin protein archaerhodopsin-3 (Arch) with excellent properties. However, this class of voltage indicators suffers from poor quantum efficiency that limits the range of potential applications. To address this shortcoming, we developed a strategy for directed evolution for brighter Arch-based voltage indicators. Briefly, a random genetic library of Arch is fused to the N-terminus of the fluorescent protein mOrange2 and is expressed in E. coli. Hundreds of colonies on a Petri dish are ratiometrically imaged (i.e., the ratio of Arch to mOrange2 fluorescence) using a custom imaging system. The colonies with the highest ratio are picked and cultured in liquid media. The fluorescence of the overnight cultures is then recorded with a microplate reader. The brightest Arch variants are expressed in Hela cells and their voltage sensitivities are evaluated in a fluorescent microscope by electric field stimulation. The brightest functional variants are then used as the library template for the next round of directed evolution. Several iterative rounds of this screening procedure combined with further screening of site-directed mutagenesis libraries resulted in identification of our current best voltage-sensitive Arch (vArch) variant, vArch1.0. vArch1.0 is voltage sensitive, fast and several-fold brighter than wild-type Arch in mammalian cells. Unlike Arch, vArch1.0 doesn't generate photo-induced current that perturbs membrane potential of cells during imaging. vArch1.0 is capable of resolving electrically triggered action potentials in neurons in single trials with optical signal-to-noise ratio >30 and is a promising tool for optical interrogation of complex neural circuit.

We have determined the sequence requirements for the N-terminal protein hinge of the active-site lid of triosephosphate isomerase. The codons for the hinge (PVW) were replaced with a genetic library of all possible 8000 amino acid combinations. The most active of these 8000 mutants were selected using in vivo complementation of a triosephosphate isomerase-deficient strain of Escherichia coli, DF502. Approximately 0.3 % of the mutants complement DF502 with an activity that is between 10 and 70 % of wild-type activity. They all contain Pro at the first position. Furthermore, the sequences of these hinge mutants reveal that hydrophobic packing is very important for efficient formation of the enediol intermediate. However, the reduced catalytic activities observed are not due to increased rates of loop opening. To explore the relationship between the N-terminal and C-terminal hinges, three semi-active mutants from the N-terminal hinge selection experiment (PLH, PHS and PTF), and six active C-terminal hinge mutants from previous work (NSS, LWA, YSL, KTK, NPN, KVA) were combined to form 18 “double-hinge” mutants. The activities of these mutants suggest that the N-terminal and C-terminal hinge structures affect one another. It appears that specific side-chain interactions are important for forming a catalytically active enzyme, but not for preventing release of the unstable enediol intermediate from the active site of the enzyme. The independence of intermediate release on amino acid sequence is consistent with the absence of a “universal” hinge sequence in structurally related enzymes

Sphingomonas paucimobilis var. EPA505 utilizes fluoranthene (FLA), naphthalene (NAP), and phenanthrene (PHE) as sole carbon sources for energy and growth. A genetic library of EPA505 was constructed using mini-Tn5 promoter reporter genes encoding for tetracycline resistance (tc(p-)) or luminescence (luxAB(p-)). Out of 2250 Tn5 mutants, ten were deficient in utilization of FLA, NAP, and/or PHE as sole carbon sources. Three classes of Tn5 mutants were defined: classI (nap(-)phe(-)fla(-)), classII (nap(-)phe(-)), and classIII (fla(-)). Four of five mutants in classI did not express dioxygenase function, whereas one classI mutant and all classII and classIII mutants retained dioxygenase activity. In Tn5 tc(p-) classI mutants 200 and 394 (dioxygenase negative) and classII mutant 132 (dioxygenase positive), promoter reporter was expressed when induced with FLA, NAP, PHE, other polycyclic aromatic hydrocarbons (PAHs), and several proposed PAH-derived catabolites. The Tn5 tc(p-) derived classIII mutant 104 was induced only with PAHs and not with PAH-derived catabolites. DNA sequence analysis of cloned regions of classI mutant 200 revealed that Tn5 inserted into a gene that shared (96%) DNA sequence homology with 2,3-dihydroxybiphenyl 1,2-dioxygenase that is designated pbhA. Nucleotide sequences downstream of pbhA shared (84%) homology to a Rieske-type ferredoxin subunit gene of a multicomponent dioxygenase designated pbhB. The Tn5 tc(p-) in classII mutant 132 occurred within sequences that shared (74%) homology with a trans-o-hydroxybenzylidene-pyruvate hydratase-aldolase gene (pbhC). Sequence analysis of the region proximal to this gene revealed a putative promoter that contained a binding site for a LysR transcriptional activator. In classIII mutant 104, the Tn5 tc(p-) resided within a region that shared 94% nucleotide homology to that of a pyruvate phosphate dikinase gene known to be involved in cellular uptake of glucose. The FLA-specific catabolic gene disrupted in mutant 104 was designated phbD. Functional and sequence analyses of promoter probe mutants allowed identification of four genes necessary for the utilization of PAHs that are controlled by at least two promoters that are affected by a wide range of aromatic compounds.

A genetic library of tributyltin chloride (TBTC1)-resistant marine bacterium, Alteromonas sp. M-1, was constructed using plasmid vector pUC 19. Three positive clones were obtained from E. coli JM 109 transformed with the plasmids by the method of replica plating to LB medium containing 1 mM TBTC1. These clones could grow in LB liquid medium containing 100 microM TBTC1. Plasmids harbouring genes of Alteromonas sp. M-1 were designated pTBT1, pTBT2 and pTBT3 which contain 1.8 kb Hind III-fragment, 4.8 kb Pst I-fragment and 7.8 kb Pst I-fragment, respectively. Nucleotide sequence of the shortest fragment, 1.8 kb Hind III-fragment was determined, revealing an open reading frame (ORF) was contained in the fragment. The ORF was 324 bp (108 amino acids). The 48.5% of the amino acids encoded was hydrophobic, suggesting that the product relating to TBTC1 resistance might be membrane related protein. Homology search in amino acids alignment indicated that the product has homology with transport proteins.

Three cloned Klebsiella pneumoniae nitrogenase gene probes were used to perform genomic Southern hybridization analysis and to screen an Anabaena variabilis-Charon 30 genetic library for Anabaena nitrogenase genes. One of ten phage clones that were isolated using a Klebsiella nifHD gene probe was studied in detail. This clone (ABN 10) contains a 9 kb Eco R1 fragment which was observed in genomic Southerns. ABN 10 does not hybridize with a Klebsiella nifK gene probe. A 2.8 kb Hind III fragment that lies within the 9 kb Eco R1 fragment contains the linked Anabaena nifH and nifD genes; the nifK gene lies at least 4 kb away. Partial sequencing of the nifH and nifD genes confirmed their identity and showed strong conservation of amino acid sequence in comparison to Anabaena 7120, Klebsiella pneumoniae, Azotobacter vinlandii, and Clostridium pasteurianum. When the cloned Anabaena nif genes were put into Klebsiella cells on plasmids, they were not expressed, suggesting that the Anabaena promoters are not recognized by Klebsiella transcription factors.

The genetic library of Tribolium castaneum contains some 125 mutations described so far. Close to 50 of these are sex-linked (recessive or dominant) or autosomal (recessives, semi-dominants or dominants). Fourteen of the 50 mutations affecting a wide variety of body parts were examined in the hybrids resulting from crosses between T. castaneum (Herbst) and T. freemani Hinton, a recently rediscovered species of the castaneum species group. The behavior of the mutations in the hybrids was identical in phenotypic expression and action as those previously observed and reported in T. castaneum , attesting to the similar genetics of the two species. Where hybridization of beetles is possible, this technique provides a means of identifying wild types alleles in common, i.e. homologous, in the two species.

The gtfA gene of Streptococcus mutons GS-5, which encodes a 55-kDa glucosyltransferase has been isolated from a genetic library in an Escherichia coli-Bacillus subtilis shuttle vector, pMK3. The construction containing the gene enables E. coli JM83 and a sucrase-deficient mutant of B. subtilis to grow on sucrose as the sole carbohydrate source. The gene is expressed under its own control in both organisms. The level of biochemical activity detectable in B. subtilis carrying the clone is approx. 50% ofthat found in E. coli harboring the same construction. In Bacillus, the gene is expressed through exponential and stationary phases of growth with a decrease in activity as the culture enters stationary phase, corresponding to increases in intracellular protease levels. The enzyme produced in E. coli or B. subtilis harboring the cloned gene is identical to the enzyme produced by S. mutans GS-5 as determined by migration in native polyacrylamide gels.