It has been indicated through a review of the literature that wood by-products have a potential as a dietary source of energy for the ruminant animal. However, lignin constitutes a barrier to the proper utilization of cellulose. Generally, any treatment to remove or alter lignin makes the cellulose within lignocellulose materials more susceptible to the activity of the cellulolytic enzymes. Therefore, an efficient biological treatment would require a system to solubilize or to remove lignin from the lignin-carbohydrate complex. Cellulose within ruminant feeds forms an effective substrate for eventual conversion to body protein. There are several methods available for delignification. This study was carried out using the thermophilic aerobic oxidation method for which swine manure was used both as the source inocula and initial culture media. In the process of degradation, a part of the energy produced was utilized by the bacteria for cell function and multiplication. The remainder of the available energy was released as heat energy. In this method the heat necessary to maintain the temperature in the thermophilic range was derived from both mechanical and from microbial activity. Thermophilic activity is considered to reduce the time required for organic waste digestion over that experienced by mesophilic digestion. The rate of the destruction of pathogenic bacteria, virus and other organisms is increased as a result of the high temperatures fermentation. Batch studies conducted to delignify alder sawdust by the use of the aerobic thermophilic oxidation method demonstrated that the lignin content of sawdust can be reduced by as much as 74%, and crude bacterial protein was generated by approximately 17%. Constant supply of small amounts of swine manure ensures high temperature maintenance. Periodical addition of 2 kg sawdust within thermophilic temperature range results in better delignification. / Land and Food Systems, Faculty of / Graduate

Call number: LD2668 .T4 1978 M63 / Master of Science

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The specificity of guinea pig liver transglutamlnase was Investigated by the determination of modification sites within polypeptides and proteins of known sequence and (In some cases) folded structure. It was shown that some globular proteins have substrate properties for transglutamlnase In conformations which resemble their native states. Novel substrate sites for transglutamlnase were determined within the following proteins: 1) bovine B-Iactoglobulln. 2) the Hls3~ Gln388 mutant form of yeast phosphoglycerate kinase. 3) bovine B-caseln. 4) porcine pepsin. Despite the high exposure of many glutamlnyl residues within these proteins only a small fraction of these residues were observed to be reactive towards transglutamlnase. This Is taken to Indicate that features such as the chemical nature of the amino acid side chains In the local vicinity of unreactive glutamlnyl residues strongly determine the specificity of transglutamlnase. When structural models were available for substrates of transglutamlnase, the local secondary structure associated with substrate sites could be assessed. When no such models were available computer based methods were used to predict the local secondary structures associated with these sites. This approach allows substrate sites to be classified according to their local conformational preference Into conformationally flexl ble (type A substrates) and more conformationally restricted (type B substrates). Since diverse amino acid sequences are observed to surround the reactive glutamlnyl residues of many of the non-physiological substrates of transglutamlnase, It was assumed that the glutamlnyl residues within these sequences were probably reactive due to having; 1) a favourable stereochemistry during modification by transglutaminase 2) a lack of Inhibitory features. In order to determine why some exposed glutamlnyl residues were reactive and others not, It was necessary to find features which were present In unreactive sequences but absent In reactive ones. Through the use of this approach an "anti-consensus sequence" motif was Identified. This was based on the observation that exposed glutamlnyl residues which were unreactive towards transglutamlnase often have charged residues within their surrounding sequences. The distribution of allowed/disallowed residues within substrate sequences, together with what Is known concerning the conformational preference of transglutamlnase for its substrates was built Into a preliminary set of "rules'·. These rules may provide a basis for understanding the observed specificity of transglutamlnase. The application of these rules to a number of model systems has resulted In the correct prediction of both reactive and unreactive glutamlnyl side chains within a number of proteins. The demonstration of the substrate properties of the Hls3~ln388 mutant of phosphoglycerate kinase Illustrates the feasibility of Introducing a novel substrate site for transglutamlnase Into a protein using recombinant DNA technology.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1981. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE. / Bibliography: leaves 174-182. / by Daniel Jeffrey Capon. / Ph.D.

Thesis. 1975. Ph.D.--Massachusetts Institute of Technology. Dept. of Biology. / Vita. / Bibliography: leaves 187-195. / Ph.D.

Protein synthesis, or translation, is a complex, multi-step process that requires regulatory and quality control mechanisms to ensure the accurate production of proteins. Two major challenges during bacterial protein synthesis are maintaining the accuracy of translation during the elongation stage and resolving stalled ribosomal complexes. Interestingly, bacteria have evolved two mechanisms, a post-peptidyl transfer quality control (post PT QC) and a ribosome rescue mechanism, to counter these challenges. Both of these mechanisms make use of a protein factor that normally functions during translation termination, Release Factor 2 (RF2), along with an additional protein factor, Release Factor 3 (RF3) for post PT QC and Alternative ribosome-rescue factor A (ArfA) for ribosome rescue, to achieve these non-canonical functions. The mechanistic role of RF3 and ArfA in these two pathways remains unclear; however, they may play a role in regulating RF2 in context of these non-canonical pathways. As a step toward understanding the role of RF3 and ArfA in post PT QC and ribosome rescue and, in particular, their role in the regulation of RF2, I sought to determine the effect of RF3 and ArfA on the binding kinetics of RF2 in post PT QC and ribosome rescue pathways. Using a single-molecule fluorescence resonance energy transfer (smFRET) signal between the P-site peptidyl-tRNA and RF2, the binding and dissociation of RF2 can be directly monitored in the absence or in the presence of RF3 or ArfA. In Chapter 2, I describe the development of smFRET signals using different chromophores, cyanine 3 to cyanine 5 (Cy3-Cy5) or to a fluorescence quencher (Cy3-QSY9). The Cy3-Cy5 and Cy3-QSY9 smFRET signals complement each other for monitoring RF2 binding; whereas Cy3-Cy5 is suitable for observing stable binding using low substrate concentration, Cy3-QSY9 is suitable for observing transient binding using high substrate concentration. The RF2 binding and dissociation to ribosomal complexes was first examined in the absence of other factors thus providing the foundation for studying the regulation of RF2 binding by RF3 or ArfA. In the bacterial post PT QC mechanism, RF3 enhances the rate of RF2-mediated peptide release to catalyze premature termination of miscoded protein, thus ultimately increasing the fidelity of protein synthesis1. Without addition of RF3, the rate of RF2-mediated peptide release is too slow to compete with the rate of protein synthesis. In Chapter 3, the role of RF3 on RF2 binding kinetics in post PT QC was investigated using both an fMet-Lys-tRNALys(Cy3) to RF2(Cy5) smFRET signal and an fMet-Lys-tRNALys(Cy3) to RF2(QSY9) smFRET signal. The ArfA-RF2 ribosome rescue pathway is a backup mechanism for trans-translation, which relieves stalled ribosomal complexes by providing an open reading frame coding for both a degradation tag and a stop codon2. Because the expression of ArfA is under strict control by trans-translation, the ArfA-RF2 pathway only functions in the absence of active trans-translation. More importantly, deletion of both the trans-translation and ArfA-RF2 pathways leads to synthetic lethality in E. coli, highlighting the critical role of ribosome rescue in vivo3. In Chapter 4, I used an fMet-Phe-tRNAPhe(Cy3) to RF2(Cy5) smFRET signal to evaluate the role of ArfA on RF2 binding and dissociation in the ribosome rescue pathway. Collectively, these studies survey the regulation of RF2 binding kinetics by RF3 or ArfA in performing non-canonical functions such as post PT QC and ribosome rescue in bacteria.

Thesis. 1976. Ph.D.--Massachusetts Institute of Technology. Dept. of Biology. / Microfiche copy available in Archives and Science. / Vita. / Bibliography: leaves 231-249. / Ph.D.

Protein translation has emerged as an important mediator of cellular activity. Here, we discuss efforts to develop and apply new technologies designed to gain insights into translational control. We begin with the application of ribosome profiling to a RiboTag Glioma mouse model which enables translational profiling of transformed cellular populations. This approach demonstrates a number of abnormalities of translation in transformed cells. We go on to report the development of an inexpensive and rapid library preparation methodology which enables high-throughput sequencing of ribosome-protected footprints from small amounts of input material. We apply this technique to a CAMKII RiboTag mouse model to make new insights into cell-type specific translation. Finally, we describe efforts to investigate translation regulatory networks through the development of a technique which couples large-scale perturbation with a genome-wide readout of translation. Molecular dissection of tissues through the ectopic expression of modified ribosomal proteins commonly relies on tissue-specific genes which act as drivers. In the case of glioma, a gene specific to transformed tissue, but not expressed in normal brain tissue, has not been identified. Chapter 2 focuses on efforts to bypass this through the development of a RiboTag Glioma mouse model which allows for concurrent transformation and the expression of an epitope-tagged ribosomal protein in virally infected cells. This model made possible the isolation of translating mRNA from transformed cellular populations and was used to demonstrate the existence of a number of translational abnormalities in transformed cells. Conventional ribosome profiling is a powerful tool which allows for the identification of ribosome-protected mRNA footprints. However, it is time-consuming, expensive, and difficult to implement. Based on our experiences with conventional ribosome profiling, we sought to develop a method which could decrease the overall number of enzymatic reactions and purification steps, thereby reducing the time and cost associated with the procedure; these efforts are discussed in Chapter 3. Utilizing a ligation-free library preparation process, which incorporates poly(A)-polymerase, template switching and bead-based purification, we reduced the time, costs and input requirements required to generate a ribosome profiling library while maintaining high library complexity. We applied our ligation-free ribosome profiling technique to a CAMKII RiboTag mouse model which enabled us to identify patterns of cell-type specific translation and the effects of mTOR inhibition in CAMKII-expressing excitatory neurons. Regulation of protein expression is an essential and highly complex cellular activity. Aberrations of translational control are central to a host of pathologies and have direct clinical relevance. However, our knowledge of the networks which control translation is limited. Chapter 4 details our efforts to develop a highly-scalable technology which enables the identification of gene-specific translational alteration in response to perturbation. Coupled with a large-scale perturbation screen, this technique could lead to the generation of a network for translational control, similar to efforts previously undertaken to understand transcriptional control. By combining the recently developed PLATE-Seq method, which utilizes unique barcode identifiers and pooled library construction, with a technique for the identification and isolation of ribosome associated mRNA, we are able to rapidly and inexpensively determine genome-wide translational states in a highly scalable