scholarly journals Biolistic transformation of Haematococcus pluvialis with constructs based on the flanking sequences of its endogenous alpha tubulin gene

2018 ◽  
Author(s):  
Guanhua Yuan ◽  
Wenlei Zhang ◽  
Xiaoying Xu ◽  
Wei Zhang ◽  
Yulin Cui ◽  
...  
Keyword(s):  
Haematococcus Pluvialis ◽  
Complete Sequence ◽  
Biolistic Transformation ◽  
Selection Marker ◽  
Sequence Information ◽  
Antibiotic Resistant ◽  
Alpha Tubulin ◽  
Flanking Sequences ◽  
Transcriptional Elements ◽  
Flanking Regions

AbstractThe complete sequence information of the alpha tubulin (tub) genes was obtained from both Haematococcus pluvialis NIES144 and SCCAP K0084., Putative transcriptional elements and polyadenylation signals were identified respectively in their 5’ and 3’ flanking regions. Three selection cassettes of tub/aadA, tub/hyr and tub/ble with 3 different antibiotic-resistant genes fused between the 5’ and 3’ flanking sequences of the tub gene were constructed and utilized for biolistic transformation of H.pluvialis. Antibiotic resistant transformants were obtained in the bombardments with the tub/aadA cassette in 2 strains. It was found that, the foreign tub/aadA DNA could be completely transferred and inherited in their genome through non-homologous recombination. Moreover, transcripts of the insert and spectinomycin resistance were identified. Transformation efficiencies up to 3×10-5 per μg DNA could be obtained in H.pluvialis NIES144 or SCCAP K0084 through utilization of a culture with a high percentage of flagellate cells and by optimizing bombarding protocol. The presented selection marker and optimized transforming procedures in this report should strengthen the platform technology for genetical manipulation and modification of H.pluvialis.

scholarly journals Novel Antimicrobial Peptides Derived from Flatfish Genes

2003 ◽  
Vol 47 (8) ◽  
pp. 2464-2470 ◽  
Author(s):  
Aleksander Patrzykat ◽  
Jeffrey W. Gallant ◽  
Jung-Kil Seo ◽  
Jennifer Pytyck ◽  
Susan E. Douglas
Keyword(s):  
Antimicrobial Peptides ◽  
Antimicrobial Activities ◽  
Resistant Organism ◽  
Gram Negative Bacteria ◽  
Genomic Information ◽  
Carboxy Terminus ◽  
Antibiotic Resistant ◽  
Active Peptides ◽  
Flanking Sequences ◽  
Flanking Regions

ABSTRACT We report on the identification of active novel antimicrobials determined by screening both the genomic information and the mRNA transcripts from a number of different flatfish for sequences encoding antimicrobial peptides, predicting the sequences of active peptides from the genetic information, producing the predicted peptides chemically, and testing them for their activities. We amplified 35 sequences from various species of flatfish using primers whose sequences are based on conserved flanking regions of a known antimicrobial peptide from winter flounder, pleurocidin. We analyzed the sequences of the amplified products and predicted which sequences were likely to encode functional antimicrobial peptides on the basis of charge, hydrophobicity, relation to flanking sequences, and similarity to known active peptides. Twenty peptides were then produced synthetically and tested for their activities against gram-positive and gram-negative bacteria and the yeast Candida albicans. The most active peptide (with the carboxy-terminus amidated sequence GWRTLLKKAEVKTVGKLALKHYL, derived from American plaice) showed inhibitory activity over a concentration range of 1 to 8 μg/ml against a test panel of pathogens, including the intrinsically antibiotic-resistant organism Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and C. albicans. The methods described here will be useful for the identification of novel peptides with good antimicrobial activities.

Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mice

1993 ◽  
Vol 13 (9) ◽  
pp. 5266-5275
Author(s):  
R D Palmiter ◽  
E P Sandgren ◽  
D M Koeller ◽  
R L Brinster
Keyword(s):  
Transgenic Mice ◽  
Regulatory Elements ◽  
High Level Expression ◽  
Hypersensitive Sites ◽  
Enhanced Expression ◽  
Rat Growth ◽  
Flanking Sequences ◽  
High Level ◽  
Distal Regulatory Elements ◽  
Flanking Regions

DNA regions of 10 and 7 kb that flank the mouse metallothionein II (MT-II) and MT-I genes, respectively, were combined with a minimally marked MT-I (MT-I*) gene and tested in transgenic mice. This construct resulted in (i) position-independent expression of MT-I* mRNA and copy number-dependent expression, (ii) levels of hepatic MT-I mRNA per cell per transgene that were about half that derived from endogenous MT-I genes, (iii) appropriate regulation by metals and hormones, and (iv) tissue distribution of transgene mRNA that resembled that of endogenous MT-I mRNA. These features were not observed when MT-I* was tested without the flanking regions. These MT-I flanking sequences also improved the expression of rat growth hormone reporter genes, with or without introns, that were under the control of the MT-I promoter. Moreover, they enhanced expression from two of four heterologous promoters/enhancers that were tested. Deletion analysis indicated that regions known to have DNase I-hypersensitive sites were necessary but not sufficient for high-level expression. These data suggest that the DNA regions flanking the mouse MT-I and MT-II genes have functions like the locus control regions described for other genes.

scholarly journals Engineering a Pseudo-26-kDa Schistosoma Glutathione Transferase from bovis/haematobium for Structure, Kinetics, and Ligandin Studies

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1844
Author(s):  
Neo Padi ◽  
Blessing Oluebube Akumadu ◽  
Olga Faerch ◽  
Chinyere Aloke ◽  
Vanessa Meyer ◽  
...  
Keyword(s):  
Glutathione Transferase ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Competitive Inhibitor ◽  
Spectroscopic Studies ◽  
Complete Sequence ◽  
Detoxification Enzymes ◽  
Ligand Docking ◽  
Sequence Information ◽  
Dimer Interface

Glutathione transferases (GSTs) are the main detoxification enzymes in schistosomes. These parasitic enzymes tend to be upregulated during drug treatment, with Schistosoma haematobium being one of the species that mainly affect humans. There is a lack of complete sequence information on the closely related bovis and haematobium 26-kDa GST isoforms in any database. Consequently, we engineered a pseudo-26-kDa S. bovis/haematobium GST (Sbh26GST) to understand structure–function relations and ligandin activity towards selected potential ligands. Sbh26GST was overexpressed in Escherichia coli as an MBP-fusion protein, purified to homogeneity and catalyzed 1-chloro-2,4-dinitrobenzene-glutathione (CDNB-GSH) conjugation activity, with a specific activity of 13 μmol/min/mg. This activity decreased by ~95% in the presence of bromosulfophthalein (BSP), which showed an IC50 of 27 µM. Additionally, enzyme kinetics revealed that BSP acts as a non-competitive inhibitor relative to GSH. Spectroscopic studies affirmed that Sbh26GST adopts the canonical GST structure, which is predominantly α-helical. Further extrinsic 8-anilino-1-naphthalenesulfonate (ANS) spectroscopy illustrated that BSP, praziquantel (PZQ), and artemisinin (ART) might preferentially bind at the dimer interface or in proximity to the hydrophobic substrate-binding site of the enzyme. The Sbh26GST-BSP interaction is both enthalpically and entropically driven, with a stoichiometry of one BSP molecule per Sbh26GST dimer. Enzyme stability appeared enhanced in the presence of BSP and GSH. Induced fit ligand docking affirmed the spectroscopic, thermodynamic, and molecular modelling results. In conclusion, BSP is a potent inhibitor of Sbh26GST and could potentially be rationalized as a treatment for schistosomiasis.

scholarly journals Structure of the SAD mutation and the location of control sites at silent mating type genes in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (7) ◽  
pp. 1278-1285 ◽  
Author(s):  
J Hicks ◽  
J Strathern ◽  
A Klar ◽  
S Ismail ◽  
J Broach
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Control Site ◽  
Left Hand ◽  
Low Level ◽  
Flanking Sequences ◽  
Mating Type Genes ◽  
Weak Expression ◽  
The Right ◽  
Flanking Regions

The SAD mutation, an extra mating type cassette, has been shown to arise from an unequal mitotic crossover between the MAT and HMR loci, resulting in the formation of a hybrid cassette and a duplication of the MAT-HMR interval. The SAD cassette contains the "a" information and left-hand flanking regions from the parental HMRa cassette and the right-hand flanking sequences of the parental MAT cassette. This arrangement of flanking sequences causes a leaky but reproducible mating phenotype correlated with a low-level expression of the cassette as measured by RNA blotting. This weak expression is attributed to the loss of one flanking control site normally present at the silent HM storage loci.

scholarly journals Expression of human alpha-tubulin genes: interspecies conservation of 3' untranslated regions.

1983 ◽  
Vol 3 (10) ◽  
pp. 1738-1745 ◽  
Author(s):  
N J Cowan ◽  
P R Dobner ◽  
E V Fuchs ◽  
D W Cleveland
Keyword(s):  
Fetal Brain ◽  
Complete Sequence ◽  
Untranslated Regions ◽  
Cdna Libraries ◽  
Alpha Tubulin ◽  
Noncoding Regions ◽  
Coding Regions ◽  
Tubulin Genes ◽  
Sequence Relatedness ◽  
Very High

To examine the sequence complexity and differential expression of human alpha-tubulin genes, we constructed cDNA libraries from two unrelated tissue types (epidermis and fetal brain). The complete sequence of a positively hybridizing alpha-tubulin clone from each library is described. Each is shown to represent an abundantly expressed gene from fetal brain and keratinocytes, respectively. Although the coding regions are extensively homologous (97%), the 3' untranslated regions are totally dissimilar. This property has been used to dissect the human alpha-tubulin multigene family into members bearing sequence relatedness in this region. Surprisingly, each of these noncoding regions shares very high (65 to 80%) interspecies homology with the 3' untranslated region of one of the two rat alpha-tubulin genes of known sequence. These unexpected homologies imply the existence of selective pressure on the 3' untranslated regions of some cytoskeletal genes which maintains sequence fidelity during the course of evolution, perhaps as a consequence of an as yet unidentified functional requirement.

scholarly journals Remodeling of Yeast CUP1 Chromatin Involves Activator-Dependent Repositioning of Nucleosomes over the Entire Gene and Flanking Sequences

2001 ◽  
Vol 21 (2) ◽  
pp. 534-547 ◽  
Author(s):  
Chang-Hui Shen ◽  
Benoit P. Leblanc ◽  
Jennifer A. Alfieri ◽  
David J. Clark
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Structure ◽  
Yeast Cells ◽  
Chromatin Domain ◽  
Sliding Mechanism ◽  
Flanking Sequences ◽  
Flanking Regions ◽  
Entire Gene

ABSTRACT The yeast CUP1 gene is activated by the copper-dependent binding of the transcriptional activator, Ace1p. An episome containing transcriptionally active or inactiveCUP1 was purified in its native chromatin structure from yeast cells. The amount of RNA polymerase II on CUP1 in the purified episomes correlated with its transcriptional activity in vivo. Chromatin structures were examined by using the monomer extension technique to map translational positions of nucleosomes. The chromatin structure of an episome containing inactive CUP1 isolated from ace1Δ cells is organized into clusters of overlapping nucleosome positions separated by linkers. Novel nucleosome positions that include the linkers are occupied in the presence of Ace1p. Repositioning was observed over the entire CUP1 gene and its flanking regions, possibly over the entire episome. Mutation of the TATA boxes to prevent transcription did not prevent repositioning, implicating a chromatin remodeling activity recruited by Ace1p. These observations provide direct evidence in vivo for the nucleosome sliding mechanism proposed for remodeling complexes in vitro and indicate that remodeling is not restricted to the promoter but occurs over a chromatin domain including CUP1 and its flanking sequences.

scholarly journals Assexon: Assembling Exon Using Gene Capture Data

2019 ◽  
Vol 15 ◽  
pp. 117693431987479 ◽  
Author(s):  
Hao Yuan ◽  
Calder Atta ◽  
Luke Tornabene ◽  
Chenhong Li
Keyword(s):  
De Novo ◽  
Phylogenetic Signal ◽  
Read Depth ◽  
Model Organisms ◽  
Coding Regions ◽  
Exon Capture ◽  
Flanking Sequences ◽  
Phylogenetic Divergence ◽  
Lepisosteus Oculatus ◽  
Flanking Regions

Exon capture across species has been one of the most broadly applied approaches to acquire multi-locus data in phylogenomic studies of non-model organisms. Methods for assembling loci from short-read sequences (eg, Illumina platforms) that rely on mapping reads to a reference genome may not be suitable for studies comprising species across a wide phylogenetic spectrum; thus, de novo assembling methods are more generally applied. Current approaches for assembling targeted exons from short reads are not particularly optimized as they cannot (1) assemble loci with low read depth, (2) handle large files efficiently, and (3) reliably address issues with paralogs. Thus, we present Assexon: a streamlined pipeline that de novo assembles targeted exons and their flanking sequences from raw reads. We tested our method using reads from Lepisosteus osseus (4.37 Gb) and Boleophthalmus pectinirostris (2.43 Gb), which are captured using baits that were designed based on genome sequence of Lepisosteus oculatus and Oreochromis niloticus, respectively. We compared performance of Assexon to PHYLUCE and HybPiper, which are commonly used pipelines to assemble ultra-conserved element (UCE) and Hyb-seq data. A custom exon capture analysis pipeline (CP) developed by Yuan et al was compared as well. Assexon accurately assembled more than 3400 to 3800 (20%-28%) loci than PHYLUCE and more than 1900 to 2300 (8%-14%) loci than HybPiper across different levels of phylogenetic divergence. Assexon ran at least twice as fast as PHYLUCE and HybPiper. Number of loci assembled using CP was comparable with Assexon in both tests, while Assexon ran at least 7 times faster than CP. In addition, some steps of CP require the user’s interaction and are not fully automated, and this user time was not counted in our calculation. Both Assexon and CP retrieved no paralogs in the testing runs, but PHYLUCE and Hybpiper did. In conclusion, Assexon is a tool for accurate and efficient assembling of large read sets from exon capture experiments. Furthermore, Assexon includes scripts to filter poorly aligned coding regions and flanking regions, calculate summary statistics of loci, and select loci with reliable phylogenetic signal. Assexon is available at https://github.com/yhadevol/Assexon .

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