Adenovirus Early region 1A(E1A) plays an important role in viral lytic infection and serves as a useful tool in the study of molecular mechanisms of gene regulation and gene expression. In addition to the ability of E1A functions to induce transactivation and transrepression of gene expression, E1A products play a critical role in transformation by adenovirus. A technique employing linker insertion mutagenesis scanning most of the E1A major regions was developed in this laboratory to systematically study the effect of E1A mutations on transcriptional transactivation and trans-repression functions as well as transforming ability (Bautista 1989). Results obtained showed that the unique region was the region primarily for transactivation in agreement with a wealth of other data and that transrepression was sensitive to insertions in CRII (conserved region II) and a region immediately following the unique region at the beginning of Exon II. However, transformation was not affected by repression-sensitive mutants. This thesis describes further studies carried out to create additional insertion mutants within conserved region of E1A to confirm and extend the results obtained by Bautista. A synthetic oligonucleotide of 39 base pair (13 aa) was introduced into two additional restriction sites in this region. As in previous work, the oligonucleotide could be inserted in either of two possible orientations. In one orientation, all three reading frames were open whereas all three reading frames of the other orientation contained stop codons which resulted in truncation of the E1A protein at the site of insertion. In addition, the insertion oligomer was designed with flanking BamHI sites to provide the opportunity of collapsing the full length insertion to a 6 base pair (2 aa) insertion. This allowed comparison to be made between mutations containing 13 aa insertions and those with 2 aa inserts at the same site to see how different oligopeptide segments would influence different functions. Trans-repression assays utilized a novel β-galactosidase assay developed by Bautista for characterizing CRII mutants expressing only the 12S product. The results suggested that CRII was an important region in terms of ability of E1A to transrepress. This negative regulatory function was sensitive to full length 13 aa insertions but not 2 aa inserts for those mutants made to date. Transforming ability of all CRII mutants was more or less impaired. Transactivation activity was not reduced by insertion at CRII except for mutants which were in the reverse orientation and had stop codons in their reading frame thus terminating the translation upstream of the unique region. In an effort to identify functional temperature sensitive mutants, DNA-mediated transformation assays of mutants within E1A functional regions were carried out at different temperatures. Two transformation temperature sensitive mutants were identified which failed to produce transformed colonies at 38.5°C but transformed efficiently at 32°C. No replication temperature sensitive mutants were identified among 61 mutant viruses. / Thesis / Master of Science (MS)

The E1 region of the group C adenoviruses is able to induce oncogenic transformation of rodent cells in culture and is also necessary for the efficient transcription of other viral genes. The proteins of the E1a transcription unit have been shown to play a pivotal role in both of these activities. In order to better understand the regions of the E1a proteins required for transformation and viral growth a program of random insertion mutagenesis was undertaken by D. S. Bautista to help identify important domains. The 39bp linker insertion oligonucleotides were designed to encode a 13 amino acid in frame insertion in one orientation or a closed reading frame insertion in the opposite orientation. As well the insertion mutants could be collapsed by digestion with BamHI to generate a 2 amino acid in frame insertion. Using this method all three types of mutants were generated at 18 different sites within the E1a coding sequences. The purpose of this project was to assay these E1a mutants for the ability to cooperate with EJb in the transformation of primary baby rat kidney cells using DNA-mediated transfection and also to 'rescue' the mutants into infectious virus and study the ability of the mutant virus to replicate on HeLa cells. Results showed that only closed reading frame mutations upstream of the unique region were completely negative for transformation. Conversely, 13aa or 2aa insertions outside of the unique region impaired but did not abolish transformation. However 8 of the 9 insertions in the unique region of the 289R protein of E1a were defective for transformation of BRK cells in E1a plus E1b DNA-mediated transformation assays. To determine whether the unique region played a direct role in the transformation process or if it had an indirect role such as the transactivation of E1b the transformation assay was carried out using selective media that allow~ growth of foci transformed by E1a alone. Results from this assay showed that the unique region mutants combined with E1b were able to transform with about the same efficiency as E1a alone. The transformation assay was also performed using the unique region mutants in an E1a only background cotransfected with the EJ-ras oncogene. Results from these experiments showed that the unique region mutants in an E1a only background could cooperate with ras in transformation as well as wild-type E1a. From these results it was concluded that the unique region does not play a direct role in transformation by E1 but is required for the efficient expression of E1b which results in wild type transforming frequencies. The actual role of E1b in transformation is unknown. The insertion mutants were also 'rescued' back into infectious virus to study their effect on the ability of the viruses to replicate. The results showed that only viruses in which the unique region was either eliminated or altered were defective for growth on HeLa cells. Transactivation assays carried out by D. Bautista showed results which were comparable to results of infectivity assays. Taken together the results suggest that only the unique region is required for transactivation and only the ability of E1a to transactivate is of importance for viral replication in HeLa cells. / Thesis / Master of Science (MS)

No description available.

No description available.

No description available.

No description available.

No description available.

Understanding water stress responses in maize and the genetic factors controlling these traits has direct application to breeding and genetic study. To understand water stress responses in more detail, this thesis characterizes a novel B73 ethyl methanesulfonate induced wilting maize mutant. This mutant is shown to express a wilting phenotype under both field and greenhouse conditions, exhibiting leaf roll, stunted stem growth and reduced fertility. Germination is unaffected in mutant seeds and no abnormal seedling phenotypes are observed. Onset of the phenotype is between V3 and V6 growth stage, and corresponds with a disruption of stem elongation. To characterize the genetic elements underlying the wilting phenotype, F2 and F3 mapping populations were phenotyped and genotyped. The mutant phenotype was found at variable frequencies between populations; ¼ or less of all mapping population members were observed as wilting, implicating a single genetic factor controlling the mutant phenotype. Background effects of the non-mutant parent and residual EMS mutations are hypothesized to be responsible for the unexpected frequency of wilting observed. Single nucleotide polymorphism and microsatellite markers localized the wilted mutant allele to a 7.4-Megabase region on the long arm of chromosome 7. The phenotype results and rough mapping data provide a base for further study and cloning of the mutant allele.

No description available.

This thesis describes a systematic investigation of the efficiency of misincorporation by Avian Myeloblastosis Virus reverse transcriptase with all possible combinations of dNTP substrate, template nucleotide, and the nucleotide at the 3' terminus of the primer. Each of a series of 16 synthetic oligonucleotide primers was annealed to single-stranded Ml3 DNA templates, and a single dNTP was misincorporated at the primer 3' end using AMV reverse transcriptase. The proportion and pattern of misincorporation and incorporation in all 64 situations were assayed using 5'-labelled primers, and the products were separated on denaturing polyacrylamide gels. Correct incorporations occurred more readily than misincorporations. The efficiency of misincorporation depended on the individual primer but, comparing primers, a clear dependence on the template nucleotide was observed for the preferential misincorporation of different dNTPs. The exact combination of template nucleotide and dNTP was important; although purine:pyrimidine (dNTP substrate: template nucleotide) and pyrimidine:purine misincorporations occurred comparatively readily, some pyrimidine:pyrimidine and purine:purine reactions were equally efficient and yet others were never seen to occur. Some misincorporations were facilitated by subsequent correct incorporations, but despite this the results suggest that the level of misincorporation is limited by the rate of reaction and enzyme inactivation rather than by exonuclease activity. The recovery of point mutants arising from reverse transcriptase-directed misincorporation of single dNTPs onto single oligonucleotide primers is described and discussed. Misincorporation of dNTPs is a form of in vitro mutagenesis which facilitates the generation of a library of point mutations spread throughout a gene. Conditions have been established in this study for the production of a bank of primers with 3' termini distributed over a region of a gene to be mutated. The misincorporation of single dNTPs onto the termini of such a bank of primers should allow the generation of a library of point mutants.