11,153 research outputs found
Utilising Uracil DNA Glycosylase to detect the presence of 5-Methylcytosine
No full textDNA is regularly subjected to endogenous and exogenous reagents that cause mutations that can be detrimental to a cell if they are not repaired. One class of enzymes responsible for DNA repair is the family of DNA glycosylases and their role is to remove damaged bases. Uracil DNA Glycosylase (UDG) is a member of this family and is highly specific, removing only uracil, an RNA base, from DNA. Uracil arises in DNA through misincorporation of deoxyuridine monophosphate (dUMP) creating an A.U base pair, or through deamination of cytosine resulting in a G.U base pair. Though UDG acts on A.U pairs, this is not it’s primarily role as A.U pairings are not mutagenic. However the G.U mispair is highly mutagenic and leads to a G.C to A.T transition on subsequent rounds of replication. UDG only reacts with uracil and has no activity at thymine since the 5-methyl group on the base is excluded from the active site. This thesis examines mutants of UDG that can cleave cytosine but not 5-methylcytosine. Methylation of cytosine at CpG sites leads to gene silencing and is an important epigenetic signal. Knowing the methylation state of cytosines will therefore be important for understanding gene control and may be beneficial for treating many diseases. The most common method for detecting cytosine methylation uses a bisulphite reaction followed by normal DNA sequencing methods. This process has several drawbacks and the aim of this work is to create an enzyme that is capable of distinguishing between5-methylcytosine and cytosine. It has been reported that mutation of a critical asparagine in UDG to an aspartate allows the enzyme to accommodate cytosine into its active site; generating a cytosine DNA glycosylase (CDG). Using the natural ability of UDG to distinguish between uracil and thymine due to the presence of the 5-methyl group, we hypothesised that the mutant enzyme should be able to discriminate between5-methylcytosine and cytosine, which differ by the presence or absence of a methyl group in the same position. E. coli and human CDGs were prepared and their ability to remove cytosine or 5-methylcytosine examined when placed in different sequence contexts. hCDG was generated through complete gene synthesis of hUDG followed by the N204D mutation. The corresponding mutation in E.coli (N123D) generates a highly cytotoxic enzyme that cannot even be cloned in pUC19. As L191 aids base flipping, mutation to alanine (L191A) renders the enzyme inactive; activity can then be rescued using a bulky synthetic nucleoside that occupies the base pair and forces the target base into an extrahelical conformation. The L191A mutation was followed by N123D to generate an expressible and functional eCDG, denoted eCYDG. We demonstrate that these mutants have cytosine glycosylase activity when the cytosine is mispaired or unpaired, but not when paired with guanine, and show no activity against5-methylcytosine in any context. The activity of these CDGs varies with the stability of the base pair, with the fastest cleavage rates being obtained with the least stable base pairs, and also varies with the local sequence context. As CDGs are able to discriminate between cytosine and 5-methylcytosine we began development of a real-time PCR assay for detection of 5-methylcytosine. This employed a hexaethylene glycol (HEG) linker opposite the target cytosine, as this produces one of the fastest cleavage rates and cannot be read by a DNA polymerase
The effect of sequence context on the activity of cytosine DNA glycosylases.
No full textWe have prepared single (N204D) and double (N204D:L272A) mutants of human uracil DNA glycosylase (hUDG), generating two cytosine DNA glycosylases (hCDG and hCYDG). Both these enzymes are able to excise cytosine (but not 5-methylcytosine), when this base is part of a mismatched base pair. hCDG is more active than the equivalent E. coli enzyme (eCYDG) and also has some activity when the cytosine is paired with guanine, unlike eCYDG. hCDG also has some activity against single stranded DNA, while having poor activity towards an unnatural base pair that forces the cytosine into an extrahelical conformation (in contrast to eCYDG for which a bulky base enhances the enzyme's activity). We also examined how sequence context affects the activity of these enzymes, determining the effect of flanking base pairs on cleavage efficiency. An abasic site or a hexaethylene glycol linker placed opposite the target cytosine, also causes an increase in activity compared with an AC mismatch. Flanking an AC mismatch with GC base pairs resulted in a 100-fold decrease in excision activity relative to flanking AT base pairs and the 5'-flanking base pair had a greater effect on the rate of cleavage. However, this effect is not simply due to the stability of the flanking base pairs as adjacent GT mismatches also produce low cleavage efficiency
The 'Burden' of the feminine: Frank Sargeson's encounter with Katherine Mansfield
No full textThis essay is situated in relation to the critical commonplace that the contrasting literary modes and prose styles of Frank Sargeson and Katherine Mansfield -- of hard-edged realist writing and the miniaturist ‘subjectivist’ writing of impressionism -- laid the foundation for the two traditions in New Zealand prose. I suggest that significant similarities can be found in the writers' artistic orientation, traceable to their critique of colonial culture and society: namely, an aesthetics of fragmentation, resistance to normative gender constructions of colonial society and their use of symbolic modes of representation. Furthermore, I argue that Mansfield can be traced as an intertextual presence in Sargeson’s work, alongside an implied gendered critique of her female voice, values and attitudes, and that he developed his repertoire by adapting her techniques of impressionism and impersonation to his ambivalently gendered viewpoint in order to nuance masculine vulnerability and unrequited love. This specific influence of Mansfield upon Sargeson will be illustrated with reference to his story, ‘A Man and his Wife’ (1939), in which I suggest he surreptitiously draws on Mansfield’s last story, ‘The Canary’ (1923), ‘writing back’ in a rural colonial context and voice to her metropolitan discourse
Letter From William Bell Scott to Mr Chambers
No full textabstract: Concerning Scott's thanks, his writings about his own works, and a manuscript of "The Nightingale Unheard."Seller's Description: Reads "A.L.S. from Author to Mr. Chambers explaining how busy he is... The sonnet is printed in the book. Fredeman: 56.7 £87.50"Handwritten Note: Unknown handwriting at top right reads "June 1st 1877."Publication Details: "The Nightingale Unheard" published in "Poems" by William Bell Scott.Creation Date Details: Undated range is the author's lifespan.Provenance: Removed from:
Poems / by William Bell Scott. Ballads, studies from nature, sonnets, etc. / illustrated by seventeen etchings by the author and L. Alma Tadema. Publisher London : Longmans, Green, 1875. CALL #
HAYDEN SPECIAL COLL SPEC PRB-13
Scott Belsky in Conversation with DesignByThem - Part One.mp4
No full textWatch here as Scott Belsky (CEO/Founder Behance + Author Making Ideas Happen) joins Sydney design duo Sarah Gibson and Nicholas Karlovasitis from DesignByThem to discuss some of the challenges facing creative companies as they try to grow their business. In part one Nick and Sarah get some great advice from Scott about what makes an effective partnership and how to learn from the challenges that can arise. Scott also explains the Behance technique of ?Concepting Teams? with representatives from all areas of the business to brainstorm issues or new ideas
Cardozo AELJ Author Interview Series: Scott Semaya, Class of 2023
No full textThe Cardozo AELJ Author Interview Series seeks to give our readers further insight into the Articles and Notes published in the Cardozo Arts & Entertainment Law Journal. In this interview, Scott Semaya discusses his Note, Name, Image and Likeness: Giving College Athletes the Clearest Guidance to Best Profit off Their NIL, which was published in Volume 41, Issue 2.
This post was originally published on the Cardozo Arts & Entertainment Law Journal website on June 6, 2023. The original post can be accessed via the Archived Link button above
Cardozo AELJ Author Interview Series: Scott Semaya, Class of 2023
No full textThe Cardozo AELJ Author Interview Series seeks to give our readers further insight into the Articles and Notes published in the Cardozo Arts & Entertainment Law Journal. In this interview, Scott Semaya discusses his Note, Name, Image and Likeness: Giving College Athletes the Clearest Guidance to Best Profit off Their NIL, which was published in Volume 41, Issue 2.
This post was originally published on the Cardozo Arts & Entertainment Law Journal website on June 6, 2023. The original post can be accessed via the Archived Link button above
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Cardozo AELJ Author Interview Series: Scott Semaya, Class of 2023
No full textThe Cardozo AELJ Author Interview Series seeks to give our readers further insight into the Articles and Notes published in the Cardozo Arts & Entertainment Law Journal. In this interview, Scott Semaya discusses his Note, Name, Image and Likeness: Giving College Athletes the Clearest Guidance to Best Profit off Their NIL, which was published in Volume 41, Issue 2.
This post was originally published on the Cardozo Arts & Entertainment Law Journal website on June 6, 2023. The original post can be accessed via the Archived Link button above
Scott Belsky in Conversation with DesignByThem - Part Two.mp4
No full textSarah Gibson and Nicholas Karlovasitis from DesignByThem continue their conversation with Scott Belsky (CEO/Founder Behance + Author Making Ideas Happen). They discuss the relationship between taking financial risks in creative enterprise whilst valuing professional standards in their work
Article entitled "Emmett J. Scott, Author and Business Man, Dies"
No full textNewspaper article entitled "Emmett J. Scott, Author and Business Man, Dies." Mr. Scott died on Dec. 12, 1957
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