Cloning of actin genes from a genomic library of the newt, Notophthalmus viridescens

The regenerating urodele limb is a useful model system in which to study, in vivo, the controls of cell proliferation and differentiation. Techniques are available which enable one to experimentally manipulate mitogenic influences upon the blastema, as well the morphogenesis of the regenerating 11mb. Although classical regeneration studies have generated a wealth of knowledge concerning tissue interactions, little 1s known about the process at the level of gene expression. The aim of this project was to clone potentially developmentally regulated genes from a newt genomic library for use in future studies of gene expression during limb regeneration. We decided to clone the cytoskeletal actin gene for the following reasons: 1. its expression reflects the proliferative and differentiatlve states of cells in other systems 2. the high copy number of cytoplasmic actin pseudogenes in other vertebrates and the high degree of evolutionary sequence conservation among actin genes increased the chance of cloning one of the newt cytoplasmic actin genes. 3. Preliminary experiments indicated that a newt actin could probably be identified using an available chick ~-actln gene for a molecular probe. Two independent recombinant phage clones, containing actin homologous inserts, were isolated from a newt genomic library by hybridization with the chick actin probe. Restriction mapping identified actin homologous sequences within the newt DNA inserts which were subcloned into the plasmid pTZ19R. The recombinant plasmids were transformed into the Escherichia coli strain, DHsa. Detailed restriction maps were produced of the 5.7Kb and 3.1Kb newt DNA inserts in the plasmids, designated pTNAl and pTNA2. The short «1.3 Kb) length of the actin homologous sequence in pTNA2 indicated that it was possibly a reverse transcript pseudogene. Problems associated with molecular cloning of DNA sequences from N. viridescens are discussed with respect to the large genome size and abundant highly repetitive DNA sequences.

The nucleophillic substitution of various chloroanthraquinones of their possible rearrangement via aryne intermediates

The work in this thesis deals mainly with nucleophilic substitution of chloroanthraquinones as a route to various starting materials which might rearrange, via aryne intermediates to afford fused-ring heterocy1ic carboxylic acids. 1-Amino-5-chloroanthraquinone was successfully prepared by reacting 1,5-dichloroanthraquinone with sodium aZide in ref1uxing dimethylsulfoxide (DMSO). It could also be prepared from the same starting material by reaction with ammonia (gas) in DMSO in the presence of potassium fluoride. Treatment of l-amino-5-chloroanthraquinone with potassium amide in liquid ammonia or with potassium t-butoxide in t-butylbenzene returned mainly starting material, although in the latter case some 1-amino-5-hydroxyanthraquinone was also isolated. 1-Hydroxy-5-chloroanthraquinone was ultimately prepared by diazotization of the amino-analog. It was recovered almost quantitatively after treatmenu'with potassium amide in liquid ammonia. The reaction with potassium t-butoxide in t-buty1benzene was anomalous and gave 1-hydroxyanthraquinone as the only iso1able product. Acridines were successfully prepared by the action of 70% sulfuric acid on 1,5-bis(p-toluidino)-anthraquinone and 1-p-toluidino-5- ch10roanthraquinone, and in the latter case, cleavage to give an acridinecarboxylate was attempted. Substituted anthraquinones reacted with sodium azide in sulfuric acid to give azepindiones by -NH insertion. Methods for separating and identifying isomeric mixtures of these compounds were examined. Attempted decarbonylation of selected azepindiones to give acridones gave mainly what were thought to be amino-benzophenone derivatives. Chloroanthraquinones were found to react with hexamethylphosphoramide (HMPA) to give mixtures of the dimethylamino- and methylaminoderivatives. Under the same conditions halogeno-nitrobenzenes and nitrophenols were substituted to give the appropriate dimethyl aminobenzenes, except in two cases. 3-Chloronitrobenzene reacted anomalously to give a small amount of 3,3'-dichloroazobenzene and a trace of 4-dimethylamino-nitrobenzene. Pentachlorophenol reacted to give a pentachlorophenylphosphorodiamidate in good yield.

The Smiles rearrangement in hydrazidic systems and related syntheses /|nG. A. Pawelchak. -- 260 St. Catharines, Ont. : [s. n.],

This research was directed towards the investigation of the Smiles rearrangement in hydrazidic systems and the synthesis of related heterocyclic compounds. The work can be conveniently divided into two main sections. Section 1 of the thesis relates to the synthesis and examination of the O+N migration of phenoxy- derivatives of hydrazidic halides. In general, hydrazidic halides were found to react with 2-nitrophenol and 4-nitrophenol to give corresponding a-nitrophenoxy- compounds. These a-nitrophenoxy- compounds were found to rearrange in warm base to give the corresponding N-benzoyl compounds via a proposed five-membered transition state. Experiments conducted in styrene revealed no radical contribution to the rearrangement. Cross-over product analysis indicated the rearrangement as intramolecular and consistent with the Smiles rearrangement. The preparation of N-a-chlorobenzylidene-N'-2-nitrophenyl- -N'-(2,4-dibromophenyl)hydrazine from N-benzoyl-N'-2-nitrophenyl- N'-(2,4-dibromophenyl)hydrazine was accomplished using phosphorus oxychloride. Examination of this hydrazidic chloride indicated a marked decrease .in reactivity as compared to the N-a-chlorobenzylidene-N'-phenylhydrazine case. Section 2 concerns itself with the preparation of heterocyclic compounds using an analogy of the five-membered transition state present in the Smiles rearrangement of a substituted benzylidene derivatives A new preparation of 2,4-phenyl1,3,4- oxadiazol-S-one using N-benzoyl-N'-phenylhydrazine and ethyl thiochloroformate is reported. Two new preparations of N-a-thiobenzoyl-N'-(2,4-dibromophenylhydrazine are reported using sodium hydrosulfide in conjunction with N-a-bromobenzylidene-N'-(2,4-dibromophenyl)hydrazine in the first, and phosphorus pentasulfide with N-benzoylN'-( 2,4-dibromophenyl)hydrazine in the second. The latter is preferred due to the formation of a sulfide co-product in the former. Two preparations of 2-phenyl-4-(2,4-dibromophenyl)-1,3,4- thiadiazol-S-one are reported using N-thiobenzoyl-N'-(2,4-dibromophenyl) hydrazine and ethyl chloroformate and ethyl thiochloroformate Two rapid and easy preparations of 2-phenyl-4-(2,4-dibromophenyl)- 1,3,4-triazol-S-one are reported using ethyl chloroformate and ethyl thiochloroformate. Sodium cyanate in conjunction with a-aminobenzylidene-N'-(2,4-dibromophenyl)hydrazine also provided 2-phenyl-4-(2,4-dibromophenyl)-1,3,4-triazol-S-one Section 2 concludes with an examination of two possible mechanistic routes to the prepared heterocycles.

Isolation and characterization of genomic DNA sequences that enhance the stability of plasmid DNA in mammalian cells /

The ease of production and manipulation has made plasmid DNA a prime target for its use in gene transfer technologies such as gene therapy and DNA vaccines. The major drawback of plasmid however is its stability within mammalian cells. Plasmid DNA is usually lost by cellular mechanisms or as a result of mitosis by simple dilution. This study set out to search for mammalian genomic DNA sequences that would enhance the stability of plasmid DNA in mammalian cells.Creating a plasmid based genomic DNA library, we were able to screen the human genome by transfecting the library into Human Embryonic Kidney (HEK 293) Cells. Cells that contained plasmid DNA were selected, using G418 for 14 days. The resulting population was then screened for the presence of biologically active plasmid DNA using the process of transformation as a detector.A commercially available plasmid DNA isolation kit was modified to extract plasmid DNA from mammalian cells. The standardized protocol had a detection limit of -0.6 plasmids per cell in one million cells. This allowed for the detection of 45 plasmids that were maintained for 32 days in the HEK 293 cells. Sequencing of selected inserts revealed a significantly higher thymine content in comparison to the human genome. Sequences with high A/T content have been associated with Scaffold/Matrix Attachment Region (S/MAR) sequences in mammalian cells. Therefore, association with the nuclear matrix might be required for the stability of plasmids in mammalian cells.