published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

No description available.

The slime mould Dictyostelium discoideum contains a novel cAMP-binding protein, CABPl, that is composed of two subunits. Anti-CABPl monoclonal antibodies were used to isolate a cDNA from a $ lambda$gtll expression library. In hybrid selection experiments, this cDNA was found to be complementary to mRNAs encoding both CABPl subunits and also to share some homology with two polypeptides, p34 and p31, that copurify with CABPl. Further analysis showed that the two CABPl polypeptides are identical except for an additional 37 amino acids present in the larger polypeptide. Both polypeptides are encoded by a single gene and the production of the two CABPl mRNAs is accomplished by an unusual splicing event. The p34 and p31 polypeptides are smaller but highly homologous to the two CABPl polypeptides and are probably produced in a similar fashion. The carboxyl halves of the four polypeptides show a 50% similarity to two polypeptides of a tellurium anion resistance determinant encoded on a bacterial plasmid.

No description available.

Ornithine Aminotransferase (OAT) is expressed at high levels in liver, kidney, and retina. Tissue-specific regulation of OAT has been described for rat kidney and liver. To characterize OAT regulation in retinal lines, we studied OAT synthesis in retinoblastomas RB355 and Y79. / OAT transcription and mRNA levels in the two lines were similar, but 3-fold greater immunoreactive OAT protein and enzyme activity were observed for Y79. Characterization of polysome-associated OAT mRNAs indicated that they were translated less efficiently, due to decreased initiation, in RB355. Initiation factor eIF-4E mRNA and protein were reduced in RB355; eIF-4E overexpression in RB355 increased OAT translation and OAT protein to the level observed in Y79. / Estrogen and thyroid hormone increased OAT expression in both strains. Estrogen increased translational initiation, with no effect on transcription, whereas thyroid hormone was primarily a transcriptional activator. / An alternatively-spliced OAT mRNA was identified in these retinoblastomas; this mRNA was poorly translated and was not affected by eIF-4E overexpression, estrogen, or thyroid hormone. / This study has demonstrated several post-transcriptional regulatory mechanisms for OAT in retinal tissue.

No description available.

The goal of the work presented here was to investigate the hypothesis that cytoplasmic SLBP is required for translation of somatic H1 mRNA, and their translational repression is due to a lack of SLBP in the cytoplasm of oocytes, and early cleavage-stage embryos. To this end, the expression of SLBP in murine oocytes and pre-implantation embryos was characterized by RT-PCR, Western blotting, and immunocytochemical. techniques. / mRNA encoding SLBP was detected throughout oogenesis and pre-implantation development, from small growing oocytes to the late blastocyst stage. SLBP protein was found in the nucleus and cytoplasm of growing and fully-gown prophase I-arrested oocytes. SLBP accumulated to extremely high levels during meiotic maturation in a process requiring translation. The protein remained abundant both in the nucleus and cytoplasm throughout the 1- and 2-cell stages. SLBP was depleted in 4-cell embryos in a process independent of DNA replication, and was not detected again until the late 8-cell stage. From the late 8-cell stage to the early blastocyst stage, SLBP was detected exclusively in the cytoplasm. Interestingly, in late blastocysts, SLBP was translocated to the nucleus. (Abstract shortened by UMI.)

The structure of one member of a family of maize mitochondrial RNA replicons (or RNA plasmids), RNA b, has been characterized. It is shown that RNA b shares a 13 nucleotide sequence with the central conserved region of Cadang-Cadang viroid of coconuts which might be involved in substrate recognition by the RNA replicase during replication. RNA b likely was derived from a larger member in that family, RNA a, through co-linear deletions similar to those involved with the generation of the defective interfering (DI) elements associated with both animal and plant viruses. RNA b may represent a "selfish" RNA optimized for replication as it does not have the capacity to encode its own replicase. Mitochondrial RNA plasmids have been found not to be restricted to the S and RU cytoplasms, as previously reported. Related plasmid RNAs have been detected in maize plants with C, N and T cytoplasms as well as 13 Latin American races. They were not found, however, in teosintes, indicating that the ancestral nucleic acid from which these RNAs evolved was acquired soon after the split between teosintes and maize, before the divergence of the maize races. Distribution of the plasmid RNAs, a maize mRNA (atp6), and a mitochondrial rRNA (26S) in different organs and tissues was investigated by in situ hybridization. These results indicated that mitochondrial gene expression is subjected to an unexpected degree of both spatial and developmental regulation, and that individual mitochondrial genes are subjected to different controls. Similar results were obtained in in situ hybridization studies of Brassica floral buds. Both studies suggested that the levels of mitochondrial genes transcripts are not particularly high in the tapetal cells of both maize and Brassica anthers contrary to common belief. High levels of RNA plasmid were detected in the pollen of S maize anthers prior to pollen abortion, suggesting they might be related to the cytoplasmic male sterility (CMS) trait of S maize

In the context of gene expression, we proposed a nonlinear stochastic delay differential equation as a mathematical model to study the effects of extrinsic noise on a delay induced Hopf bifurcation. We envisaged its direct numerical resolution. Following the example of the noisy oscillator, we first solved a linearized version of the equation, close to the Hopf bifurcation. The numerical scheme used is a combination of a standard algorithm to solve a deterministic delay differential equation and a stochastic Euler scheme. From our calculations we verified that the deterministic behaviour is fully recovered. For the stochastic case, we found that our solution is qualitatively accurate, in the sense that the noise induced shift in the critical value a, follows the trend the known analytic results predict. However, our numerical solution systematically overestimates the value of the shift. This is explained because the accuracy in the numerical estimation of the decay rate of a solution towards the stationary state value is a function of the control parameter a. We believe the mismatch between the numerical solution and the analytic results is due to a lack of convergence of our scheme, rather than to lack of accuracy. As our numerical scheme is an hybrid, the convergence problem can be improved, both at the deterministic and at the stochastic parts of the scheme. In this work we left our numerical results on the nonlinear case out, because before proceeding to the investigation of the nonlinear equation, the convergence must be assured in the linear case.

Transposons are mobile genetic elements that have the potential to cause mutations as a result of different aspects of their life cycle. Because most of these mutations are detrimental and due to a selfish life cycle seemingly aiming at increasing their numbers, transposons have been regarded as "junk" DNA that contributes no evolutionary function to the host organism. In this thesis, I argue that although most of the transposon-induced mutations are detrimental, some may lead to benefits for the host and become adaptive. I discuss recent evidence from the literature that points to several mechanisms by which transposons and transposon activity may lead to the diversification of host genes and gene functions. I also provide evidence that transposons have inserted in the 5' flanking region of the gene coding for maize and teosinte auxin-binding protein 1 (Abp1). These transposons do not change the structure of the Abp1 transcripts but may modulate Abp1 gene expression quantitatively. In addition, I document the first case outside of oncogenic retroviruses, of a retrotransposon (called Bs1) that transduced portions from three different maize genes. I show that the Bs1-associated transduction events resulted in the formation of one uninterrupted open reading frame that is both transcribed and translated in reproductive tissues. I suggest that, such as in the case of Bs1, gene transduction by retroelements may be a general mechanism for the evolution of new hybrid genes by the shuffling of host sequences. I conclude that transposons are better regarded as means for the generation of gene and genomic diversity. Natural selection will favor and fix adaptive variants whereas detrimental mutations are likely to be purged out of the population.