[17]Gene splicing by overlap extension

doi:10.1016/0076-6879(93)17067-F

Methods in Enzymology

Volume 217, 1993, Pages 270-279

https://doi.org/10.1016/0076-6879(93)17067-F Get rights and content

Publisher Summary

Conventional methods of engineering recombinant DNA make use of restriction enzymes to cut molecules apart at specific nucleotide sequences and ligases to rejoin the parts. A significant limitation of this technology is that restriction enzymes are sequence dependent and these recognition sequences appear more or less randomly in DNA. That is, restriction enzymes cut where recognition sites are located and not necessarily at optimal positions along the gene for purposes of genetic engineering. The polymerase chain reaction (PCR) has made possible a sequence-independent engineering method that is referred to as “gene splicing by overlap extension” or “SOE.” This technology is especially useful in complicated constructions that require precise recombination points—such as joining two coding sequences in frame—and it also provides a straightforward way of performing site-directed mutagenesis.

References (17)

R.M. Horton et al.
Gene
(1989)
S.N. Ho et al.
Gene
(1989)
B. Berkhout et al.
Cell
(1990)
C.T. Wittwer et al.
Anal. Biochem.
(1990)
R.M. Horton et al.
BioTechniques
(1990)
K. Mullis and F. Faloona, this series, Vol. 155, p....
D.H. Jones et al.
BioTechniques
(1990)
A.R. Schuldiner et al.
Anal. Biochem.
(1990)

There are more references available in the full text version of this article.

Cited by (438)

Synthetic dual hormone-responsive promoters enable engineering of plants with broad-spectrum resistance
2023, Plant Communications
In plant immunity, the mutually antagonistic hormones salicylic acid (SA) and jasmonic acid (JA) are implicated in resistance to biotrophic and necrotrophic pathogens, respectively. Promoters that can respond to both SA and JA signals are urgently needed to engineer plants with enhanced resistance to a broad spectrum of pathogens. However, few natural pathogen-inducible promoters are available for this purpose. To address this problem, we have developed a strategy to synthesize dual SA- and JA-responsive promoters by combining SA- and JA-responsive cis elements based on the interaction between their cognate trans-acting factors. The resulting promoters respond rapidly and strongly to both SA and Methyl Jasmonate (MeJA), as well as different types of phytopathogens. When such a synthetic promoter was used to control expression of an antimicrobial peptide, transgenic plants displayed enhanced resistance to a diverse range of biotrophic, necrotrophic, and hemi-biotrophic pathogens. A dual-inducible promoter responsive to the antagonistic signals auxin and cytokinin was generated in a similar manner, confirming that our strategy can be used for the design of other biotically or abiotically inducible systems.
Orthopedia expression during Drosophila melanogaster nervous system development and its regulation by microRNA-252
2022, Developmental Biology
During brain development of Drosophila melanogaster many transcription factors are involved in regulating neural fate and morphogenesis. In our study we show that the transcription factor Orthopedia (Otp), a member of the 57B homeobox gene cluster, plays an important role in this process. Otp is expressed in a stable pattern in defined lineages from mid-embryonic stages into the adult brain and therefore a very stable marker for these lineages. We determined the abundance of the two different otp transcripts in the brain and hindgut during development using qPCR. CRISPR/Cas9 generated otp mutants of the longer protein form significantly affect the expression of Otp in specific areas. We generated an otp enhancer trap strain by gene targeting and reintegration of Gal4, which mimics the complete expression of otp during development except the embryonic hindgut expression. Since in the embryo, the expression of Otp is posttranscriptionally regulated, we looked for putative miRNAs interacting with the otp 3′UTR, and identified microRNA-252 as a candidate. Further analyses with mutated and deleted forms of the microRNA-252 interacting sequence in the otp 3′UTR demonstrate an in vivo interaction of microRNA-252 with the otp 3′UTR. An effect of this interaction is seen in the adult brain, where Otp expression is partially abolished in a knockout strain of microRNA-252. Our results show that Otp is another important factor for brain development in Drosophila melanogaster.
A newly identified flavoprotein disulfide reductase Har protects Streptococcus pneumoniae against hypothiocyanous acid
2022, Journal of Biological Chemistry
Hypothiocyanous acid (HOSCN) is an antimicrobial oxidant produced from hydrogen peroxide and thiocyanate anions by heme peroxidases in secretory fluids such as in the human respiratory tract. Some respiratory tract pathogens display tolerance to this oxidant, which suggests that there might be therapeutic value in targeting HOSCN defense mechanisms. However, surprisingly little is known about how bacteria protect themselves from HOSCN. We hypothesized that tolerant pathogens have a flavoprotein disulfide reductase that uses NAD(P)H to directly reduce HOSCN, similar to thioredoxin reductase in mammalian cells. Here, we report the discovery of a previously uncharacterized flavoprotein disulfide reductase with HOSCN reductase activity, which we term Har (hypothiocyanous acid reductase), in Streptococcus pneumoniae, a bacterium previously found to be tolerant of HOSCN. S. pneumoniae generates large amounts of hydrogen peroxide that can be converted to HOSCN in the respiratory tract. Using deletion mutants, we demonstrate that the HOSCN reductase is dispensable for growth of S. pneumoniae in the presence of lactoperoxidase and thiocyanate. However, bacterial growth in the HOSCN-generating system was completely crippled when deletion of HOSCN reductase activity was combined with disruption of GSH import or recycling. Our findings identify a new bacterial HOSCN reductase and demonstrate a role for this protein in combination with GSH utilization to protect S. pneumoniae from HOSCN.
Tandem repeats in precursor protein stabilize transcript levels and production levels of the fungal ribosomally synthesized and post-translationally modified peptide ustiloxin B
2022, Fungal Genetics and Biology
Citation Excerpt :
The DNA fragment containing a 30-bp sequence after the stop codon of ustA, pyrG, and ∼ 1-kb ustA downstream was amplified from the plasmid constructed for ustAyaig1 preparation. The three DNA fragments were concatenated into one DNA construct by overlap extension PCR (Horton et al., 1993) for transformation. As a control, a transformant with the pyrG marker downstream of native ustA was prepared in ustROE pyrG- by using a DNA construct composed of ∼ 1-kb ustA upstream, ustA, pyrG, and ∼ 1-kb ustA downstream (Fig. S3).
Ustiloxin B is a ribosomally synthesized and post-translationally modified peptide (RiPP) first reported in Ascomycetes. Its biosynthetic pathway was recently identified in the filamentous fungus Aspergillus flavus. The precursor protein of ustiloxin B, UstA, has a signal peptide to the endoplasmic reticulum at its N-terminal and a subsequent tandemly highly repeated segment cleaved at Lys-Arg dipeptides by Kex2 protease; such proteins are called Kex2-processed repeat proteins (KEPs). RiPP biosynthetic pathways using KEPs as precursor proteins are widely distributed in the Fungi kingdom, with high diversity of precursor protein sequences. UstA in A. flavus has a 16-fold tandemly repeated segment containing the core peptide Tyr-Ala-Ile-Gly, which forms the ustiloxin B backbone structure, but it is unknown why such a costly-to-maintain highly repeated sequence is retained. Here, we replaced ustA, the gene encoding the ustiloxin B precursor protein, with synthetic genes encoding 1-, 3-, 5-, 7-, and 11-fold tandem-repeat segments in A. flavus, to investigate the relationship between the repeat number and ustiloxin B production. Ustiloxin B production increased quadratically with increasing repeat number in ustA variants, although it dropped in a previously constructed ustA variant that had a substituted synthetic gene encoding a 16-fold repeat segment probably because of the presence of the many rare codons in the sequence. We also examined the transcript levels of substituted synthetic genes in ustA variants, and surprisingly we found that the transcript levels of the synthetic genes increased linearly with increasing repeat number. This result implies that an unknown mechanism stabilizes ustA transcripts via the highly repeated structure in a feedback manner. We also constructed a transformant without the intron in native ustA, but no effect of intron removal was observed on either ustiloxin B production or the precursor gene transcript level. The costly-to-maintain highly repeated sequence in KEPs probably serves the purpose of maintaining stable transcripts and thus increasing the amount of substrate.
Removal of single-site N-linked glycans on factor VIII alters binding of domain-specific monoclonal antibodies
2022, Journal of Thrombosis and Haemostasis
A portion of individuals with hemophilia A develop neutralizing antibodies called inhibitors to glycoprotein factor VIII (FVIII). There are multiple risk factors that contribute to the risk of inhibitor formation. However, knowledge of the role of FVIII asparagine (N)‐linked glycosylation in FVIII immunity is limited.
To evaluate the effect of site‐specific N‐linked glycan removal on FVIII biochemical properties, endocytosis by murine bone marrow‐derived dendritic cells (BMDCs), and antibody responses.
Four recombinant B domain‐deleted (BDD) FVIII variants with single‐site amino acid substitutions to remove N‐linked glycans were produced for experimental assays.
BDD FVIII‐N41G, FVIII‐N239A, FVIII‐N1810A, and FVIII‐N2118A with confirmed removal of N‐linked glycans and similar glycosylation profiles to BDD FVIII were produced. There were no differences in thrombin activation or von Willebrand factor binding of FVIII variants compared with BDD FVIII; however, reduced FVIII expression, activity, and specific activity was observed with all variants. BDD FVIII‐N41G and FVIII‐N1810A had reduced uptake by BMDCs, but there were no differences in antibody development in immunized hemophilia A mice compared with BDD FVIII. Half of a repertoire of 12 domain‐specific FVIII MAbs had significantly reduced binding to ≥1 FVIII variant with a 50% decrease in A1 domain MAb 2‐116 binding to FVIII‐N239A.
Modifications of FVIII N‐linked glycans reduced FVIII endocytosis by BMDCs and binding of domain‐specific FVIII MAbs, but did not alter de novo antibody production in hemophilia A mice, suggesting that N‐glycans do not significantly contribute to inhibitor formation.
The mycobacterial proteasomal ATPase Mpa forms a gapped ring to engage the 20S proteasome
2021, Journal of Biological Chemistry
Although many bacterial species do not possess proteasome systems, the actinobacteria, including the human pathogen Mycobacterium tuberculosis, use proteasome systems for targeted protein removal. Previous structural analyses of the mycobacterial proteasome ATPase Mpa revealed a general structural conservation with the archaeal proteasome-activating nucleotidase and eukaryotic proteasomal Rpt1–6 ATPases, such as the N-terminal coiled-coil domain, oligosaccharide-/oligonucleotide-binding domain, and ATPase domain. However, Mpa has a unique β-grasp domain that in the ADP-bound crystal structure appears to interfere with the docking to the 20S proteasome core particle (CP). Thus, it is unclear how Mpa binds to proteasome CPs. In this report, we show by cryo-EM that the Mpa hexamer in the presence of a degradation substrate and ATP forms a gapped ring, with two of its six ATPase domains being highly flexible. We found that the linkers between the oligonucleotide-binding and ATPase domains undergo conformational changes that are important for function, revealing a previously unappreciated role of the linker region in ATP hydrolysis–driven protein unfolding. We propose that this gapped ring configuration is an intermediate state that helps rearrange its β-grasp domains and activating C termini to facilitate engagement with proteasome CPs. This work provides new insights into the crucial process of how an ATPase interacts with a bacterial proteasome protease.

View all citing articles on Scopus

View full text

[17]Gene splicing by overlap extension

Publisher Summary

Gene

Gene

Cell

Anal. Biochem.

BioTechniques

BioTechniques

Anal. Biochem.