Megaprimer Mutagenesis Using Very Long Primers

Lai, Richard; Bekessy, A.; Chen, C. C.; Walsh, Timothy R.; Barnard, Ross

doi:10.2144/03341bm07

Cited by 17 publications

(9 citation statements)

References 4 publications

(10 reference statements)

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“…An IGF-1R 3′UTR mutant for the hsa-mir-99a seed sequence was created using the Megaprimer Mutagenesis assay [41], [42] using primer 1 and primer 3: 5′-TAGAT TA TAAA TA GTCAGTT-3′.…”

Section: Methodsmentioning

confidence: 99%

MiRNA Expression in Psoriatic Skin: Reciprocal Regulation of hsa-miR-99a and IGF-1R

et al. 2011

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BackgroundPsoriasis is a complex disease at the cellular, genomic and genetic levels. The role of microRNAs in skin development was shown in a keratinocyte-specific Dicer knockout mouse model. Considering that two main characteristics of psoriasis are keratinocytes hyperproliferation and abnormal skin differentiation, we hypothesized that aberrant microRNA expression contributes to the psoriatic phenotype. Here, we describe the differential expression of miRNAs in psoriatic involved and uninvolved skin as compared to normal skin, revealing an additional aspect of this complex disorder.Methodology/Principal FindingsExpression arrays were used to compare microRNA expression in normal skin versus psoriatic involved and uninvolved skin. Fourteen differentially expressed microRNAs were identified, including hsa-miR-99a, hsa-miR-150, hsa-miR-423 and hsa-miR-197. The expression of these microRNAs was reevaluated by qPCR. IGF-1R, which is involved in skin development and the pathogenesis of psoriasis, is a predicted target of hsa-miR-99a. In an in situ hybridization assay, we found that IGF-1R and miR-99a are reciprocally expressed in the epidermis. Using a reporter assay, we found that IGF-1R is targeted by hsa-miR-99a. Moreover, over expression of miR-99a in primary keratinocytes down-regulates the expression of the endogenous IGF-1R protein. Over expression of miR-99a also inhibits keratinocyte proliferation and increases Keratin 10 expression. These findings suggest that overexpression of hsa-miR-99a in keratinocytes drives them towards differentiation. In primary keratinocytes grown in high Ca++, miR-99a expression increases over time. Finally, we found that IGF1 increases the expression of miR-99a.Conclusions/SignificanceWe identified several microRNAs that are expressed differentially in normal and psoriatic skin. One of these miRNAs is miR-99a that regulates the expression of IGF-1R. Moreover, miR-99a seems to play a role in the differentiation of keratinocytes. We suggest that miR-99a is one of the regulators of the IGF-1R signaling pathway in keratinocytes. Activation of IGF1 signaling results in elevation of miR-99a which represses the expression of IGF-1R.

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“…An IGF-1R 3′UTR mutant for the hsa-mir-99a seed sequence was created using the Megaprimer Mutagenesis assay [41], [42] using primer 1 and primer 3: 5′-TAGAT TA TAAA TA GTCAGTT-3′.…”

Section: Methodsmentioning

confidence: 99%

MiRNA Expression in Psoriatic Skin: Reciprocal Regulation of hsa-miR-99a and IGF-1R

et al. 2011

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“…The sagCATsag cassette was replaced by the sagBLEsag cassette [48] by Xho I digest swapping. The megaprimer method was used to generate internal deletion or point mutations [49]. The basal IMCs plasmids were designed as follows: ptub-DD-YFP-IMCx/BLE, where x is either 5, 8 or 9 [4].…”

Section: Methodsmentioning

confidence: 99%

A Toxoplasma MORN1 Null Mutant Undergoes Repeated Divisions but Is Defective in Basal Assembly, Apicoplast Division and Cytokinesis

et al. 2010

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The membrane occupation and recognition nexus protein 1 (MORN1) is highly conserved among apicomplexan parasites and is associated with several structures that have a role in cell division. Here we dissected the role of MORN1 using the relatively simple budding process of Toxoplasma gondii as a model. Ablation of MORN1 in a conditional null mutant resulted in pronounced defects suggesting a central role for MORN1 in apicoplast segregation and in daughter cell budding. Lack of MORN1 resulted in double-headed parasites. These Janus-headed parasites form two complete apical complexes but fail to assemble a basal complex. Moreover, these parasites were capable of undergoing several more budding rounds resulting in the formation of up to 16-headed parasites conjoined at the basal end. Despite this segregation defect, the mother's cytoskeleton was completely disassembled in every budding round. Overall this argues that successful completion of the budding is not required for cell cycle progression. None of the known basal complex components, including a set of recently identified inner membrane complex (IMC) proteins, localized correctly in these multi-headed parasites. These data suggest that MORN1 is essential for assembly of the basal complex, and that lack of the basal complex abolishes the contractile capacity assigned to the basal complex late in daughter formation. Consistent with this hypothesis we observe that MORN1 mutants fail to efficiently constrict and divide the apicoplast. We used the null background provided by the mutant to dissect the function of subdomains of the MORN1 protein. This demonstrated that deletion of a single MORN domain already prevented the function of MORN1 whereas a critical role for the short linker between MORN domains 6 and 7 was identified. In conclusion, MORN1 is required for basal complex assembly and loss of MORN1 results in defects in apicoplast division and daughter segregation.

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“…Escherichia coli DH5α was the host, and the new plasmids were re-sequenced before use. The G220A, I221A, Y222A, G235A and K245A variants were created using megaprimer PCR (28). The megaprimer was synthesized in 50 μl-reactions containing 15 ng pI-CreII, 100 pmol of mutagenic and reverse primers, and the Pfx polymerase (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Biochemical and mutagenic analysis of I-CreII reveals distinct but important roles for both the H-N-H and GIY-YIG motifs

Corina¹,

Qiu²,

Desai³

et al. 2009

Nucleic Acids Research

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Homing endonucleases typically contain one of four conserved catalytic motifs, and other elements that confer tight DNA binding. I-CreII, which catalyzes homing of the Cr.psbA4 intron, is unusual in containing two potential catalytic motifs, H-N-H and GIY-YIG. Previously, we showed that cleavage by I-CreII leaves ends (2-nt 3′ overhangs) that are characteristic of GIY-YIG endonucleases, yet it has a relaxed metal requirement like H-N-H enzymes. Here we show that I-CreII can bind DNA without an added metal ion, and that it binds as a monomer, akin to GIY-YIG enzymes. Moreover, cleavage of supercoiled DNA, and estimates of strand-specific cleavage rates, suggest that I-CreII uses a sequential cleavage mechanism. Alanine substitution of a number of residues in the GIY-YIG motif, however, did not block cleavage activity, although DNA binding was substantially reduced in several variants. Substitution of conserved histidines in the H-N-H motif resulted in variants that did not promote DNA cleavage, but retained high-affinity DNA binding—thus identifying it as the catalytic motif. Unlike the non-specific H-N-H colicins, however; substitution of the conserved asparagine substantially reduced DNA binding (though not the ability to promote cleavage). These results indicate that, in I-CreII, two catalytic motifs have evolved to play important roles in specific DNA binding. The data also indicate that only the H-N-H motif has retained catalytic ability.

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Megaprimer Mutagenesis Using Very Long Primers

Cited by 17 publications

References 4 publications

MiRNA Expression in Psoriatic Skin: Reciprocal Regulation of hsa-miR-99a and IGF-1R

MiRNA Expression in Psoriatic Skin: Reciprocal Regulation of hsa-miR-99a and IGF-1R

A Toxoplasma MORN1 Null Mutant Undergoes Repeated Divisions but Is Defective in Basal Assembly, Apicoplast Division and Cytokinesis

Biochemical and mutagenic analysis of I-CreII reveals distinct but important roles for both the H-N-H and GIY-YIG motifs

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