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1.
Objectives
To test the applicability of Cpf1 from Francisella novivida in genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae.Results
An easy-to-use vector toolkit, containing a CEN6/ARS4 plasmid expressing Cpf1 from Francisella novivida (FnCpf1) and a 2 μ plasmid for crRNA or crRNA array expressing, was constructed for Cpf1-assisted genomic integration in S. cerevisiae. Our results showed that FnCpf1 allowed for targeted singleplex, doubleplex, and tripleplex genomic integration of in vivo assembled DNA parts with efficiencies of 95, 52, and 43%, respectively.Conclusions
CRISPR-Cpf1 system allows for efficient genomic integration of in vivo assembled DNA parts in S. cerevisiae, and thus provides an alternative CRISPR-Cas method for metabolic pathway engineering in addition to CRISPR-Cas9 system previously reported for yeast.2.
Wenyi Sun Xiaobing Yang Xueying Wang Xinping Lin Yanan Wang Sufang Zhang Yushi Luan Zongbao K. Zhao 《Biotechnology letters》2017,39(7):1001-1007
Objectives
To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method.Results
The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11.Conclusions
Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.3.
Kai Zhang Huijuan Su Muhan Yang Jing Ge Guiyao Li Jun Yi Yang Wang 《Biotechnology letters》2017,39(6):905-909
Objectives
To establish a positive cloning system with a zero background for high-throughput DNA cloning purpose.Results
The cloning vector, pRI857, and the genomic-library construction vector, pRI857-BAC, were constructed based on the mechanism of expression of the thermo-sensitive cI857 repressor gene that can stringently repress the PR promoter and kanamycin resistance gene (PR-kan R ) at 30 °C, but have no effect on PR-kan R gene at 37 °C or at higher temperatures. When the pRI857 vectors were transformed into E. coli with or without a target foreign DNA fragment inserted at the BfrBI site of the cI857 gene, only colonies with the foreign DNA fragment survive. We extended this method to construct a pRI857-BAC vector for genomic library cloning which displays an efficiency of ~107 cfu per µg of genomic DNA, with no empty vectors detected.Conclusions
Cloning by indirect activation of resistance marker gene represents a novel DNA-capturing system, which can be widely applied for high-throughput DNA cloning.4.
Objective
To construct a promoter probe vector, pBE-bgaB, to screen strong promoters from Bacillus amyloliquefaciens.Results
266 colonies containing active promoter elements from the genomic DNA of B. amyloliquefaciens were identified. Among these, promoter P41 exhibited the strongest β-Gal activity in Escherichia coli and B. amyloliquefaciens. Sequence analysis showed that promoter P41 contained P ykuN , a ykuN gene encoding flavodoxin. Optimization of the ribosome-binding site from P41 to P382 improved β-Gal activity by ~ 200%.Conclusion
A new strong promoter for protein expression and genetic engineering of Bacillus species.5.
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Forde BM Neville BA O'Donnell MM Riboulet-Bisson E Claesson MJ Coghlan A Ross RP O'Toole PW 《Microbial cell factories》2011,10(Z1):S13
Background
The genus Lactobacillus is characterized by an extraordinary degree of phenotypic and genotypic diversity, which recent genomic analyses have further highlighted. However, the choice of species for sequencing has been non-random and unequal in distribution, with only a single representative genome from the L. salivarius clade available to date. Furthermore, there is no data to facilitate a functional genomic analysis of motility in the lactobacilli, a trait that is restricted to the L. salivarius clade.Results
The 2.06 Mb genome of the bovine isolate Lactobacillus ruminis ATCC 27782 comprises a single circular chromosome, and has a G+C content of 44.4%. In silico analysis identified 1901 coding sequences, including genes for a pediocin-like bacteriocin, a single large exopolysaccharide-related cluster, two sortase enzymes, two CRISPR loci and numerous IS elements and pseudogenes. A cluster of genes related to a putative pilin was identified, and shown to be transcribed in vitro. A high quality draft assembly of the genome of a second L. ruminis strain, ATCC 25644 isolated from humans, suggested a slightly larger genome of 2.138 Mb, that exhibited a high degree of synteny with the ATCC 27782 genome. In contrast, comparative analysis of L. ruminis and L. salivarius identified a lack of long-range synteny between these closely related species. Comparison of the L. salivarius clade core proteins with those of nine other Lactobacillus species distributed across 4 major phylogenetic groups identified the set of shared proteins, and proteins unique to each group.Conclusions
The genome of L. ruminis provides a comparative tool for directing functional analyses of other members of the L. salivarius clade, and it increases understanding of the divergence of this distinct Lactobacillus lineage from other commensal lactobacilli. The genome sequence provides a definitive resource to facilitate investigation of the genetics, biochemistry and host interactions of these motile intestinal lactobacilli.8.
Background
Deinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored.Results
A segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented. The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF). The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB) folds. The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins. Like the Thermus SSB proteins, the SSBDr functions as a homodimer. The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer.Conclusions
The correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date. The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a homodimer. The Deinococcus radiodurans SSB protein efficiently stimulates Deinococcus radiodurans RecA and also Escherichia coli RecA protein-promoted DNA strand exchange reactions. The identification and purification of Deinococcus radiodurans SSB protein not only allows for greater understanding of the SSB protein family but provides an essential yet previously missing player in the current efforts to understand the extraordinary DNA repair capacity of Deinococcus radiodurans.9.
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Pradeep K Chatterjee Leighcraft A Shakes Naima Stennett Vanessa L Richardson Tennison L Malcolm Ken R Harewood 《BMC research notes》2010,3(1):38
Background
Chromatin adjoining the site of integration of a transgene affects expression and renders comparisons of closely related transgenes, such as those derived from a BAC deletion series retrofitted with enhancer-traps, unreliable. Gene targeting to a pre-determined site on the chromosome is likely to alleviate the problem.Findings
A general procedure to replace the loxP site located at one end of genomic DNA inserts in BACs with lox66 is described. Truncating insert DNA from the loxP end with a Tn10 transposon carrying a lox66 site simultaneously substitutes the loxP with a lox66 sequence. The replacement occurs with high stringency, and the procedure should be applicable to all BACs in the public domain. Cre recombination of loxP with lox66 or lox71 was found to be as efficient as another loxP site during phage P1 transduction of small plasmids containing those sites. However the end-deletion of insert DNA in BACs using a lox66 transposon occurred at no more than 20% the efficiency observed with a loxP transposon. Differences in the ability of Cre protein available at different stages of the P1 life cycle to recombine identical versus non-identical lox-sites is likely responsible for this discrepancy. A possible mechanism to explain these findings is discussed.Conclusions
The loxP/lox66 replacement procedure should allow targeting BACs to a pre-positioned lox71 site in zebrafish chromosomes; a system where homologous recombination-mediated "knock-in" technology is unavailable.12.
Shin-ichi Tsuruta Masumi Ebina Makoto Kobayashi Wataru Takahashi Yoshifumi Terajima 《Molecular breeding : new strategies in plant improvement》2017,37(6):71
Erianthus arundinaceus, a member of the Saccharum complex, is of interest as a potential resource for sugarcane improvement and as a bioenergy crop. Genetic analyses of germplasm collections of E. arundinaceus are being used increasingly. To expand the genomic resources in E. arundinaceus, we aimed at developing simple sequence repeat markers. Using pyrosequencing on the 454 GS FLX system, we sequenced genomic DNA from “JW630” collected in Japan. A total of 1682 candidate loci were used to design the primers, and 1234 primer pairs amplified fragments of the expected size in the primer screening with three wild E. arundinaceus accessions (JW630, “JW4,” and “IJ76-349”). The efficiency of genotyping was validated with a subset of 174 primer pairs and 8 E. arundinaceus accessions. Of these primer pairs, 171 amplified fragments in all accessions tested and 162 detected polymorphic loci. The average values of genetic parameters were estimated as 0.30 (range, 0.09–0.49) for polymorphic information content, 1.65 (0.00–5.87) for marker index, and 2.78 (0.00–8.75) for resolving power. Using these parameters, we selected 61 primer pairs with large discriminatory power for the analyzed loci. Of the 174 primer pairs, 45 (25.9%) were also applicable to Saccharum and 33 (19.0%) to Miscanthus species. These markers would provide a valuable tool for estimating genetic diversity and constructing linkage maps in E. arundinaceus, which would be useful for genetic study and breeding. 相似文献
13.
Guillermina Isla Constanza G. Taverna Wanda Szusz Walter Vivot Guillermo García-Effron Graciela Davel 《Current fungal infection reports》2017,11(4):203-208
Purpose of review
This review summarizes the information available of Candida haemulonii sensu lato, and updates the in vitro susceptibility profile of the isolates of these species from Argentina. C. haemulonii sensu lato causes fungemia in low frequency in adults and children; however, the knowledge about this emerging yeast is relevant since it is considered as a multi-resistant pathogen.Recent findings
The discovery of new antifungal molecules, the determination of epidemiological cutoff value and clinical breakpoints for some yeasts of clinical impact, and the update of techniques to determine the in vitro susceptibility profile to yeast have generated some available information, although, for C. haemulonii sensu lato, this information is not always useful for clinical application.Summary
We determined the susceptibility profile of C. haemulonii sensu lato strains isolated in Argentina, as a first step to establish the epidemiological cutoff values in our region. We also review the current situation in other countries.14.
Background
The genus Burkholderia consists of species that occupy remarkably diverse ecological niches. Its best known members are important pathogens, B. mallei and B. pseudomallei, which cause glanders and melioidosis, respectively. Burkholderia genomes are unusual due to their multichromosomal organization, generally comprised of 2-3 chromosomes.Results
We performed integrated genomic analysis of 127 Burkholderia strains. The pan-genome is open with the saturation to be reached between 86,000 and 88,000 genes. The reconstructed rearrangements indicate a strong avoidance of intra-replichore inversions that is likely caused by selection against the transfer of large groups of genes between the leading and the lagging strands. Translocated genes also tend to retain their position in the leading or the lagging strand, and this selection is stronger for large syntenies. Integrated reconstruction of chromosome rearrangements in the context of strains phylogeny reveals parallel rearrangements that may indicate inversion-based phase variation and integration of new genomic islands. In particular, we detected parallel inversions in the second chromosomes of B. pseudomallei with breakpoints formed by genes encoding membrane components of multidrug resistance complex, that may be linked to a phase variation mechanism. Two genomic islands, spreading horizontally between chromosomes, were detected in the B. cepacia group.Conclusions
This study demonstrates the power of integrated analysis of pan-genomes, chromosome rearrangements, and selection regimes. Non-random inversion patterns indicate selective pressure, inversions are particularly frequent in a recent pathogen B. mallei, and, together with periods of positive selection at other branches, may indicate adaptation to new niches. One such adaptation could be a possible phase variation mechanism in B. pseudomallei.15.
16.
FLOWERING LOCUS T (FT), a major effect gene, regulates flowering time in Arabidopsis. We analyzed evolutionary changes distinguishing two FT homeologous loci in B. rapa, described genetic variation in homologs isolated and reported expression pattern of FT in B. juncea. Synteny analysis confirmed presence of two FT genomic copies in B. rapa ssp. pekinensis and resolved pre-existing anomalies regarding copy number in “AA” genome. Synteny analysis of B. rapa homeologous regions CR1 (129 kb) and CR2 (232 kb) revealed differential gene fractionation and wide-spread re-arrangements. Seven genomic DNA (gDNA) variants (2.1–2.2 kb) and 10 complementary DNA (cDNA) variants (528 bp) were isolated from 6 Brassica species. The gDNA variants shared 72–99 % similarity within Brassica and 58–60 % between Arabidopsis and Brassica. FT cDNA variants shared 92–100 % similarity within Brassica and 87 % between Arabidopsis and Brassica. Phylogenetic analysis of FT gDNA, cDNA and protein sequences revealed two major clades, differentiating homologs derived from species containing shared “BB” and “CC” genomes. Phylogram based on Brassica FT gDNA differentiated homeologs derived from AA-LF (Least fractioned) and AA-MF1 (Moderately fractioned) sub-genomes. Analysis of FT expression pattern in B. juncea revealed increasing levels correlating with attainment of physiological maturity; highest levels were detected in older leaves implying conservation in spatio-temporal expression pattern vis-à-vis Arabidopsis. In conclusion, our study reveals that polyploidy in Brassicas resulted in expansion of FT gene copies with homologs charting independent evolutionary course through accumulation of mutations. However, expression domains of FT remained conserved across Brassicaceae to preserve the critical function of FT in controlling flowering time. 相似文献
17.
Wenyi Sun Xiaobing Yang Xueying Wang Xiang Jiao Sufang Zhang Yushi Luan Zongbao K. Zhao 《Biotechnology letters》2018,40(6):933-940
Objectives
To establish a recombinase flippase (FLP) and flippase recognition target (FRT) system-mediated protocol for post-integration excision of exogenous DNA fragments in the oleaginous yeast Rhodosporidium toruloides.Results
Binary vectors were constructed to harbor FLP expressing cassette together with the hygromycin-resistance marker. Results showed that R. toruloides transformants produced FLP, but failed to mediate removal of the bleomycin-resistance marker within two FRT sites. When FLP was fused with a native nuclear localization signal (NLS) peptide, the system was found functional. Moreover, R. toruloides recombinant strains expressing the NLS-fused FLP under the control of PADH2, an promoter of alcohol dehydrogenase 2 gene (RHTO_03062), were obtained to realize homologous recombination upon growing in glucose-deficient medium.Conclusions
We have devised a homologous recombination method for R. toruloides based on the FLP/FRT system, which may facilitate further metabolic engineering and designing advanced cell factories for value-added chemicals.18.
Vasiliki?Chondrou Eleana?F.?Stavrou Georgios?Markopoulos Alexandra?Kouraklis-Symeonidis Vasilios?Fotopoulos Argiris?Symeonidis Efthymia?Vlachaki Panagiota?Chalkia George?P.?Patrinos Adamantia?Papachatzopoulou Argyro?Sgourou
Background
We aimed to clarify the emerging epigenetic landscape in a group of genes classified as “modifier genes” of the β-type globin genes (HBB cluster), known to operate in trans to accomplish the two natural developmental switches in globin expression, from embryonic to fetal during the first trimester of conception and from fetal to adult around the time of birth. The epigenetic alterations were determined in adult sickle cell anemia (SCA) homozygotes and SCA/β-thalassemia compound heterozygotes of Greek origin, who are under hydroxyurea (HU) treatment. Patients were distinguished in HU responders and HU non-responders (those not benefited from the HU) and both, and in vivo and in vitro approaches were implemented.Results
We examined the CpG islands’ DNA methylation profile of BCL11A, KLF1, MYB, MAP3K5, SIN3A, ZBTB7A, and GATA2, along with γ-globin and LRF/ZBTB7A expression levels. In vitro treatment of hematopoietic stem cells (HSCs) with HU induced a significant DNA hypomethylation pattern in ZBTB7A (p*, 0.04) and GATA2 (p*, 0.03) CpGs exclusively in the HU non-responders. Also, this group of patients exhibited significantly elevated baseline methylation patterns in ZBTB7A, before the HU treatment, compared to HU responders (p*, 0.019) and to control group of healthy individuals (p*, 0.021), which resembles a potential epigenetic barrier for the γ-globin expression. γ-Globin expression in vitro matched with detected HbF levels during patients’ monitoring tests (in vivo) under HU treatment, implying a good reproducibility of the in vitro HU epigenetic effect. LRF/ZBTB7A expression was elevated only in the HU non-responders under the influence of HU.Conclusions
This is one of the very first pharmacoepigenomic studies indicating that the hypomethylation of ZBTB7A during HU treatment enhances the LRF expression, which by its turn suppresses the HbF resumption in the HU non-responders. Its role as an epigenetic regulator of hemoglobin switching is also supported by the wide distribution of ZBTB7A-binding sites within the 5′ CpG sequences of all studied human HBB cluster “modifier genes.” Also, the baseline methylation level of selective CpGs in ZBTB7A and GATA2 could be an indicator of the negative HU response among the β-type hemoglobinopathy patients.19.