As moscas-das-frutas são consideradas importantes pragas da fruticultura mundial, causando perdas significativas à produção e ao livre trânsito de frutas. Com a preferência por alimentos com baixos níveis de agrotóxicos, pesquisas estão buscando alternativas ecológica, social e econômica viáveis para o controle destas pragas. A Técnica do Inseto estéril (TIE) é uma técnica onde os insetos são criados, esterilizados e liberados em áreas infestadas, reduzindo a fertilidade da população selvagem, além de ser um método não poluente e não agressivo ao ambiente. Com o desenvolvimento de novos estudos da metodologia da TIE, foi introduzida uma nova linhagem mutante a Sensibilidade a Letal Temperatura (tsl) Viena-8, cujas fêmeas são sensíveis à alta temperatura, podendo ser eliminadas já na fase embrionária, no início da criação massal, agregando mais benefícios a este método de controle e/ou supressão de praga. Com diferentes testes de incubação de ovos da linhagem tsl, com temperaturas e tempos combinados e variados, comprovou-se a extrema sensibilidade às mudanças de temperatura. A temperatura ideal para a incubação dos ovos dessa linhagem está entre 24ºC a 26ºC pelo período de 24 a 48 horas. Deve-se tomar o máximo de cuidado com a elevação da temperatura acima de 26ºC durante a manutenção e multiplicação dessa linhagem em condições de laboratório ou massalmente. Acima de 26ºC, há sérios riscos de comprometer a qualidade dos insetos produzidos. O curto período de 6 horas a uma temperatura de 34ºC, já é suficiente para alterar negativamente a proporção de fêmeas de ovos tratados termicamente / The fruit flies are considered important pests of the world horticulture, causing significant loses to the production and the free traffic of fruits. With the preference for foods with low levels of pesticides, researches are looking for alternatives ecological, social and economical viable for the control of these pests. The Sterile Insect Technique (SIT) is a technique where the insects are reared, sterilized and release in infested areas, reducing the fertility of the wild population, and is a method no pollutant and no aggressive to the environment. With the development of new studies of the methodology of SIT, a new mutation, Temperature Sensitive Lethal (tsl) Vienna 8, whose females are sensitive to the high temperature, could already be eliminated in the embryonic phase, in the beginning of the mass-rearing, resulting in more benefits to the control/suppression this pests. Different tests of incubation with eggs of tsl strain, with variation of time and temperature was done and proved a very sensitive to temperature changes. The ideal temperature for the incubation of the eggs is among 24ºC to 26ºC by the period from 24 to 48 hours. The maximum of care should be taken with the elevation of the temperature above 26ºC during the maintenance and multiplication in laboratory conditions or mass-rearing. Above this temperature it can commit the quality of the produced insects. The short period of 6 hours with temperature to 34ºC, is enough to change negatively the proportion of females of eggs in the thermal treatment

Genomic and proteomic investigations have yielded, and continue to produce, a large amount of information about genes and their protein products. In contrast, the evidence bearing on physiological roles of specific proteins is much more scarce. To address the functional part of biological inquiry, one would like to perturb, at will and selectively, the function of any protein of interest in vivo and to analyze the resulting phenotypic effects, thereby probing the protein’s role in a cell. Ideally, a method for doing so should be applicable both to individual gene products and to a large collection of them. Gene knockouts, a powerful tool to study gene function, have limitations in the study of development when the early phenotypes are cell- or organismal- lethal. Conditional mutants are particularly useful for analysis of genes whose functions are essential for the organism’s viability. A conditional mutant retains the function of a gene under one set of conditions, called permissive, and shows an inactive phenotype under a different set of conditions, called nonpermissive; the latter must be still permissive for the wild type (wt) allele of a gene. Conditional mutants make possible the analysis of physiological changes that follow controlled inactivation of a gene or gene product and can be used to address the function of any gene. Temperature sensitive (ts) mutants are an important class of conditional mutants whose phenotype is similar to that of wt at lower (permissive) temperature, but show low or reduced level of activity above a certain temperature called restrictive temperature, while the wt gene shows a similar phenotype at both the temperatures. Ts mutants provide an extremely powerful tool to study gene expression in vivo and in cell culture. They provide a reversible mechanism to lower the level of a specific gene product simply by changing the temperature of growth of the organism. Ts mutants are typically generated by random mutagenesis; either by ultraviolet light, a chemical mutagen or by error-prone PCR followed by often laborious screening procedures. Therefore, they are cumbersome to make, especially in the case of organisms with long generation times. Keeping in view the importance of ts mutants in biology, Varadarajan et al. 1996, had developed an algorithm to predict ts mutants at predicted, buried sites of a globular protein from its amino acid sequence. Experimental tests of the algorithm were carried out on the CcdB toxin of Escherichia coli to further refine and improve the method (Chakshusmathi et al. 2004). Based on this result simple rules for the design of ts mutants were suggested. This thesis aims at validating and improving on these rules and to find out if ts mutants of a protein can also be generated by perturbing functionally important residues. In addition, it is currently unclear with what frequency ts mutants of a protein isolated in one organism will show a ts phenotype in a completely different organism. This thesis makes preliminary efforts to address this issue. The model system chosen to carry out these studies is a protein called Gal4, which is a yeast transcriptional activator. This protein is biologically relevant as it has been used for ectopic gene expression in diverse organisms including yeast, fruitflies, zebrafish, mice and frogs (Ornitz et al. 1991; Brand and Perrimon 1993; Rahner et al. 1996; Andrulis et al. 1998; Scheer and Camnos-Ortega 1999; Hartley et al. 2002). The introductory chapter (Chapter 1) discusses the importance of ts mutants and our understanding and progress in this field so far, relevant for the work reported in this thesis. Chapter 2 describes generation of ts mutants of Gal4 in yeast. Full length Gal4 (fGal4) is an 881-aa protein. To simplify the construction of ts Gal4, we have designed a functional truncated Gal4 (miniGal4 or mGal4) of 197 residues. Five residues (9, 10, 15, 18 and 23) of the Gal4 DNA binding domain, which are in close contact with the DNA, were randomized in mGal4. Based on average hydrophobicity and hydrophobic moment, 68, 69, 70, 71, and 80 are the only residues in the region 1-150 that are predicted to be buried at the 90% confidence level. Of these five sites, residues 68, 69 and 70 were chosen for mutagenesis. At these three sites, four stereochemically diverse substitutions (Lys, Ser, Ala and Trp) were made. In a separate set of experiments each predicted, buried residues were also individually randomized in both mini and in full length Gal4 (fGal4). In all cases, we have been successful in isolating ts mutants in more than one position. At both permissive and restrictive temperatures, the activity of the Gal4 ts mutants is substantially lower than the wt. However, at the restrictive temperature, the activity of the ts Gal4 is lowered below the threshold required for reporter gene expression. This view of how ts mutants function is quite different from the general notion that the ts and wt behave similarly at permissive temperatures. Chapter 3 deals with transferability of two of the ts constructs mutated at DNA binding residues (R15W and K23P) to Drosophila. Two fGal4 encoding DNA fragments carrying the mutations were cloned into P element vectors under control of Elav and GMR promoters and several transgenic Drosophila lines were generated. These were crossed to various UAS reporter lines and progeny were characterized for reporter gene expression as a function of temperature. We show that both of these yeast ts mutants also show a ts phenotype in Drosophila. We have compared our ts Gal4 system with a popularly used system (TARGET) (McGuire et al. 2003) used for conditional gene expression in Drosophila. Our ts Gal4 mutants appear to provide tighter control at the restrictive temperature and a more uniform and rapid induction of gene expression upon shifting from the restrictive to the permissive temperature than the TARGET system with the promoters and the reporters we have used. Although cold sensitive (cs) mutants are often more useful than ts mutants, for reasons currently unclear, cs mutants are much more difficult to isolate than ts mutants. In Chapter 4, we have attempted to convert the ts phenotypes observed with Gal4 mutants in Drosophila and CcdB mutants in E. coli (Chakshusmathi et al. 2004) to cs phenotypes by increasing the expression level of these mutant proteins selectively at higher temperature. Several ts mutants of CcdB have been previously reported (Chakshusmathi et al. 2004). For converting the ts phenotype observed by E. coli toxin CcdB mutants (Chakshusmathi et al. 2004) to a cs phenotype, the arabinose inducible plasmid pBAD24CcdB and its mutant derivatives were used. By inducing expression of the mutant protein at higher temperature with arabinose, while keeping the basal level of expression without arabinose at lower temperature, we have been able to show cold sensitive behavior by these CcdB ts mutants in E. coli. For producing a cs phenotype with Gal4 mutants in Drosophila, we have used a P element vector where the GMR element is placed in-between hsp70 binding sites. This driver results in enhanced expression of downstream genes at 30 relative to 18°C because of the presence of the hsp elements (Kramer and Staveley 2003). Ts mutants at DNA binding and buried residues of fGal4 were cloned into this vector and several transgenic lines for each construct were obtained. The Gal4 mutants at exposed DNA binding residues but not at buried residues show a cs phonotype when they were crossed to various UAS-reporters lines. The buried residue mutants are likely to be destabilized and their degradation pathway might differ in yeast and in Drosophila. Because of this, these mutants might not be showing the desired cs phenotype in Drosophila. Although mGal4 and fGal4 have very similar activities in yeast, it was necessary to examine if they also had identical activities in Drosophila. Determining their relative activities in Drosophila is the aim of Chapter 5. To this end, mGal4 was cloned into P element vectors under control of hsp70 or GMRhs promoters and transgenic flies were generated. The transgenic lines were crossed to various UAS-reporters and reporter gene activities in the progeny were characterized. Although mGal4 and fGal4 showed similar activity in yeast, in Drosophila for reasons that are currently unclear, mGal4 was considerably less active than fGal4. As some of the fGal4 mutants showed a cs phenotype under GMRhs driver as shown in the earlier chapter (Chapter 4), several ts mutants of mGal4 in yeast in buried and as well as at the DNA binding residues were transferred to Drosophila under hs and GMRhs promoter. The transgenic lines obtained were tested for cold sensitivity by crossing with various UAS-reporter lines. However, in all cases mutant mGal4 showed an inactive phenotype in Drosophila. We suggest that this is because the intrinsic activity of these mGal4 mutants is substantially weaker than wt mGal4 even at permissive temperature in yeast. The further lowering of activity in Drosophila pushes the activity below the threshold required for reporter gene expression resulting in an inactive phenotype. The concluding chapter (Chapter 6) summarizes the conclusions drawn from this entire study and provides insights into possible mechanisms responsible for ts and cs phenotypes. The mutant phenotypes of Gal4 in yeast and in Drosophila suggest that ts phenotypes appear to result from a threshold effect. Such mutations lower the activity and/or level of the protein relative to the wt at all temperatures. Since maximal stability temperatures are rarely in excess of room temperature, with an increase in temperature, the activity of an already marginally active mutant can fall below the threshold required for function resulting in a temperature sensitive phenotype. The strategies we used for producing ts mutants have several advantages over standard approaches of generating ts alleles by random mutagenesis. We anticipate that conclusions of this study would be useful for generation of ts mutants of other globular proteins in diverse organisms. We also show that exposed, functional residues involved in protein: ligand or protein: protein interactions appear to be attractive candidate sites for generating ts mutants that are transferable between organisms. In addition, the active site mutants of fGal4 in Drosophila, which show ts and cs phenotypes depending on the Drosophila promoter chosen for expression, can be used for conditional and reversible expression of a number of other genes using the Gal4-UAS system (Brand and Perrimon 1993).

Στην παρούσα διατριβή μελετήθηκε μια φυλοσύνδετη θερμοευαίσθητη μετάλλαξη, η wiser, η οποία προέκυψε από μια δυσγενική διασταύρωση με το 23.5 hobo MRF στέλεχος και οφείλεται σε ένθεση Ρ στοιχείου στη θέση 7Ε του Χ χρωμοσώματος. Μελέτες βιωσιμότητας έδειξαν ότι τα κρίσιμα στάδια στα οποία η μετάλλαξη επιφέρει το θάνατο των ατόμων είναι της προνύμφης και της νύμφης. Στο στάδιο της νύμφης στους 29οC τα άτομα πεθαίνουν λίγο πριν ή κατά τη διάρκεια της εκκόλαψης των ακμαίων ατόμων. Η Ρ ένθεση εντοπίζεται στην 5’ ρυθμιστική περιοχή του γονιδίου CG32711. Το γονίδιο CG32711 δίνει δύο μετάγραφα που προκύπτουν από εναλλακτική συρραφή και όχι ένα όπως προβλέπεται στη Flybase. Η προβλεπόμενη μεταφραζόμενη περιοχή είναι κοινή και για τα δύο μετάγραφα. Η ανάλυση κατά Western αποκάλυψε ότι το γονίδιο μεταφράζεται και δίνει δύο πρωτεϊνικές ισομορφές με μοριακό βάρος περίπου 8 και 9 kD. Με βάση τη δομή της πρωτεΐνης, η ισομορφή των 9 kD πρέπει να οφείλεται σε μετα-μεταφραστική τροποποίηση. Στα κανονικά άτομα στα στάδια της προνύμφης εκφράζεται η ισομορφή των 8 kD, της νύμφης και οι δύο και στα ακμαία η ισομορφή των 9 kD. Ο μεταλλαγμένος φαινότυπος στο στέλεχος wiser πρέπει να οφείλεται στην μη σωστή έκφραση της ισομορφής των 9 kD κατά τη διάρκεια της ανάπτυξης. Από τα αποτελέσματα της RNA in situ υβριδοποίησης, της ανοσοϊστοχημείας και της εκτοπικής έκφρασης προκύπτει ότι το γονίδιο εμπλέκεται στη γλοιογένεση, την ανάπτυξη του τραχειακού συστήματος, των αιμοκυττάρων, τη μορφογένεση των φτερών, των ποδιών και των ματιών και είναι απαραίτητο για τη βιωσιμότητα των ατόμων. Η πρωτεΐνη CG32711 μπορεί να δρα ως ενδιάμεσο μόριο σε ένα ευρύτερο μονοπάτι ενεργοποίησης άλλων μορίων που επηρεάζουν τις παραπάνω λειτουργίες. Εκτοπική έκφραση της προβλεπόμενης μεταφραζόμενης περιοχής του γονιδίου οδήγησε σε λειτουργική πρωτεΐνη. Εκφραζόμενη υπό τον έλεγχο της ρυθμιστικής περιοχής του γονιδίου apmd544 οδήγησε στην εκκόλαψη ακμαίων ατόμων wiser στους 29οC. Υπερέκφραση της προβλεπόμενης μεταφραζόμενης περιοχής του γονιδίου CG32711 υπό τον έλεγχο των ρυθμιστικών περιοχών των αλληλομόρφων των γονιδίων apmd544 και elavC155 οδήγησε σε υπερέχουσες φαινοτυπικές ανωμαλίες στα φτερά, τα πόδια, τα μάτια και το θώρακα. Γεγονός που δείχνει ότι η έκφραση του γονιδίου είναι δοσοευαίσθητη. Τέλος, στην 5΄ρυθμιστική περιοχή του γονιδίου CG32711 υπάρχουν περιοχές με διαφορετικές ιδιότητες. Δύο άλλες Ρ ενθέσεις (οι PL26 και PL28) σε διαφορετικές θέσεις της οδηγούν σε θανατογόνες μεταλλάξεις με διαφορετικές ιδιότητες ως προς το πρότυπο έκφρασης. / In the present study we describe the genetic, molecular and developmental properties of an X-linked temperature sensitive mutation in Drosophila melanogaster. The mutant flies emerged at 19oC have incised wings, smaller and rough eyes. For this reason we have named the mutation wiser (wings scalloped-eyes rough). At 29oC the mutation is lethal. The lethality occurs at the stage of late pupae, just before or during the eclosion of imago. The mutation occurred from a dysgenic cross with the 23.5 hobo MRF strain and is due to a P element insertion at the locus 7E. Viability studies showed that the mutation causes the death of the mutants also at the stage of 3rd instar larvae at 29oC and acts as semi-lethal. The P insertion is located 490bp upstream the predicted coding region of the gene CG32711. The gene is transcribed in two mRNA, not in one as predicted in Flybase. The two transcripts have shown quantitative differences among the developmental stages that where examined in both the mutant strain wiser and the wild-type Canton-s both in 19oC and 29oC. The longer transcript derives from alternative splicing in 260nt of the first intron. The predicted translated region is identical to both transcripts. Western analysis revealed that the gene is translated in two protein isoforms of 8 kD and 9 kD probably due to post-translational modification. The pattern expression of the two isoforms in the wild-type strain is the following: at the stages of 3rd instar larvae and 1st instar pupae the isoform of 8 kD, at the stages of 2nd and 3rd instar pupae both isoforms and in adults just the protein isoform of 9 kD is expressed. The mutant phenotype and properties are due to the abnormal expression of the protein isoform of 9 kD during the development of the mutants. RNA in situ hybridization, immunohistochemistry and ectoping expression of the predicted translated region of the gene CG32711 revealed that the gene plays a functional role in gliogenesis, tracheal and hemocyte development, wing, eye and leg formation and is essential for the survival of the individuals. Ectoping expression of the predicted translated region of the gene CG32711 revealed that the translated protein is functional and when expressed under control of the regulatory region of the gene apterous rescues the mutant flies at 29oC. The function of the gene CG32711 is also dosage-sensitive due to the fact that many individuals carrying phenotype abnormalities in the wings, thorax, eyes and legs were developed by the ectoping expression. Two other lethal mutations PL26 and PL28 are located at the 5´ regulatory region of the gene CG32711. The differences concerning the pattern expression and the properties of these two mutations in relation to those of the wiser mutation, revealed that the three mutations affect the same gene but in a different way.

As moscas-das-frutas são consideradas importantes pragas da fruticultura mundial, causando perdas significativas à produção e ao livre trânsito de frutas. Com a preferência por alimentos com baixos níveis de agrotóxicos, pesquisas estão buscando alternativas ecológica, social e econômica viáveis para o controle destas pragas. A Técnica do Inseto estéril (TIE) é uma técnica onde os insetos são criados, esterilizados e liberados em áreas infestadas, reduzindo a fertilidade da população selvagem, além de ser um método não poluente e não agressivo ao ambiente. Com o desenvolvimento de novos estudos da metodologia da TIE, foi introduzida uma nova linhagem mutante a Sensibilidade a Letal Temperatura (tsl) Viena-8, cujas fêmeas são sensíveis à alta temperatura, podendo ser eliminadas já na fase embrionária, no início da criação massal, agregando mais benefícios a este método de controle e/ou supressão de praga. Com diferentes testes de incubação de ovos da linhagem tsl, com temperaturas e tempos combinados e variados, comprovou-se a extrema sensibilidade às mudanças de temperatura. A temperatura ideal para a incubação dos ovos dessa linhagem está entre 24ºC a 26ºC pelo período de 24 a 48 horas. Deve-se tomar o máximo de cuidado com a elevação da temperatura acima de 26ºC durante a manutenção e multiplicação dessa linhagem em condições de laboratório ou massalmente. Acima de 26ºC, há sérios riscos de comprometer a qualidade dos insetos produzidos. O curto período de 6 horas a uma temperatura de 34ºC, já é suficiente para alterar negativamente a proporção de fêmeas de ovos tratados termicamente / The fruit flies are considered important pests of the world horticulture, causing significant loses to the production and the free traffic of fruits. With the preference for foods with low levels of pesticides, researches are looking for alternatives ecological, social and economical viable for the control of these pests. The Sterile Insect Technique (SIT) is a technique where the insects are reared, sterilized and release in infested areas, reducing the fertility of the wild population, and is a method no pollutant and no aggressive to the environment. With the development of new studies of the methodology of SIT, a new mutation, Temperature Sensitive Lethal (tsl) Vienna 8, whose females are sensitive to the high temperature, could already be eliminated in the embryonic phase, in the beginning of the mass-rearing, resulting in more benefits to the control/suppression this pests. Different tests of incubation with eggs of tsl strain, with variation of time and temperature was done and proved a very sensitive to temperature changes. The ideal temperature for the incubation of the eggs is among 24ºC to 26ºC by the period from 24 to 48 hours. The maximum of care should be taken with the elevation of the temperature above 26ºC during the maintenance and multiplication in laboratory conditions or mass-rearing. Above this temperature it can commit the quality of the produced insects. The short period of 6 hours with temperature to 34ºC, is enough to change negatively the proportion of females of eggs in the thermal treatment

A comparison was made of bacteriophage MS2 RNA translation in infected Escherichia coli cells and in a defined cell-free system. A number of temperature-sensitive mutants were used as hosts for viral RNA translation at permissive and restrictive temperatures. The amount of viral coat protein synthesis was determined after gel electrophoresis of proteins from the cell lysates. These results were compared to those obtained with cell-free translation assays conducted with ribosomes isolated from the same mutants. Compared with control cells, a reduced activity in vivo and in vitro was found for each mutant examined at elevated temperatures. A good correlation between the two types of translational assays was observed. These findings are discussed in terms of the translational defects known to be a characteristic of some of these mutant strains.

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<p>This thesis describes results from fundamental studies of liposomes intended for drug delivery and pH or temperature triggered release. In addition, the effect of lipid composition on bilayer disc formation and a potential application of the bilayer discs were investigated.</p><p>The lower pH encountered by endocytosed liposomes can be utilized to trigger drug release. The mechanisms behind cytosolic drug delivery were investigated using two different kinds of pH-sensitive liposomes. The results indicate that incorporation of non-lamellar forming lipids into the endosome membrane may allow for drug escape into the cytosol.</p><p>Temperature-sensitive liposomes containing lysolipid (LTSL) release their content almost instantly when heated to temperatures close to the gel to liquid crystalline phase transition temperature (T<i>C</i>). Morphological changes of the liposomes in response to temperature cycling were studied. Temperature cycling induced liposome openings and disintegration of the liposomes into bilayer discs. Incubation of LTSL in the presence of multilamellar liposomes (MLVs) resulted in relocalisation of lysolipid into the MLVs, which affected the rapid release from LTSL. We propose that the presence of micelle-forming components, such as lysolipids and PEG-lipids, facilitates the formation of defects and membrane openings during the initial phase of membrane melting, resulting in the observed rapid release. Similar to added lysolipids, also hydrolysis generated lysolipids induce disc-formation upon heating through T<i>C</i> of the lipid mixture.</p><p>Two fundamentally different micelles may form in PEG-lipid/lipid mixtures. We found that discoidal structures are preferred over cylindrical micelles when the mixture contains components that reduce the spontaneous curvature, increase the monolayer bending modulus, or reduce PEG-lipid/lipid miscibility. The large discoidal micelles found at low PEG-lipid content are better described as bilayer discs. We evaluated such discs as model membranes in drug partitioning studies, and suggest that they, in some cases, produce more accurate data than liposomes.</p>

The unique native structure is a basic requirement for normal functioning of most proteins. Many diseases stem from mutations in proteins that destabilize the protein structure thereby resulting in impairment or loss of function (Sunyaev et al. 2000). Therefore, it is important from both fundamental and applied points of view, to elucidate the sequence determinants of protein structure and function. With the advent of recombinant DNA techniques for modifying protein sequences, studies on the effect of amino acid replacements on protein structure and function have acquired momentum. It is well established from previous mutagenesis studies that buried residues in a protein are important determinants of protein structure or stability while surface residues are involved in protein function (Rennell et al. 1991; Terwilliger et al. 1994; Axe et al. 1998). Inspite of this, there is no universally accepted definition and probe to distinguish and identify buried residues from exposed residues. A part of this thesis aims to examine the feasibility of using scanning mutagenesis to distinguish between buried and exposed positions in the absence of three-dimensional structure and also to arrive at an experimental definition of the appropriate accessibility cut-off to distinguish between buried and exposed residues. Proline, being an unusual amino acid is usually exploited to determine sites in a protein important for protein stability (Sauer et al. 1992). This thesis also explores the use of proline scanning mutagenesis to make inferences about protein structure and stability. Temperature sensitive mutant proteins, which result from single amino acid substitutions, are particularly useful in elucidating the determinants of protein folding and stability (Grutter et al. 1987; Sturtevant et al. 1989). Temperature sensitive (ts) mutants are an important class of conditional mutants which are widely used to study gene function in vivo and in cell culture (Novick and Schekman 1979; Novick and Botstein 1985). They display a marked drop in the level or activity of the gene product when the gene is expressed above a certain temperature (restrictive temperature). Below this temperature (permissive temperature), the level or activity of the mutant is very similar to that of the wild type. Inspite of their widespread use, little is known about the molecular mechanisms responsible for generating a Ts phenotype. A part of this thesis discusses a set of sequence/structure-based strategies for the successful design and isolation of ts mutants of a globular protein, inferred from saturation mutagenesis of CcdB. The experimental system, CcdB (Controller of Cell Division or Death B protein), is a 101 residue, homodimeric protein encoded by F plasmid. The protein is an inhibitor of DNA gyrase and is a potent cytotoxin in E.coli (Bernard et al. 1993). Crystallographic structures of CcdB in the free and gyrase bound forms (Loris et al. 1999; Dao-Thi et al. 2005) are also available. Expression of the CcdB functional protein results in cell death, thus providing a rapid and easy assay for the protein (Chakshusmathi et al. 2004). This dissertation focuses on understanding the determinants of globular protein stability and temperature sensitivity using saturation mutagenesis of E.coli CcdB. Towards this objective, we attempted to replace each of the 101 residues of CcdB with 19 other amino acids using high throughput mutagenesis tools. A total of 1430 (~75%) of all possible single site mutants of the CcdB saturation mutagenesis library could be isolated. These mutants were characterized in terms of their activity at different expression levels. The correlation between the observed mutant phenotypes with residue burial, nature of substitution and expression level was examined. The introductory chapter (Chapter 1) describes the use of mutagenesis as a tool to understand the relationship between protein sequence, structure and function. It represents an overview of previous large scale mutagenesis studies from the literature. It also addresses the motivation behind this work and problems which we have attempted to address in these studies. Chapter 2 discusses mutagenesis based definitions and probes for residue burial in proteins as derived from alanine and charged scanning mutagenesis of CcdB. Every residue of the 101 amino acid E. coli toxin CcdB was substituted with Ala, Asp, Glu, Lys and Arg using site directed mutagenesis. The activity of each mutant in vivo was characterized as a function of CcdB transcriptional level. The mutation data suggest that an accessibility value of 5% is an appropriate cutoff for definition of buried residues. At all buried positions, introduction of Asp results in an inactive phenotype at all CcdB transcriptional levels. The average amount of destabilization upon substitution at buried positions decreases in the order Asp>Glu>Lys>Arg>Ala. Asp substitutions at buried sites in two other proteins, MBP and Thioredoxin were also shown to be severely destabilizing. Ala and Asp scanning mutagenesis, in combination with dose dependent expression phenotypes, was shown to yield important information on protein structure and activity. These results also suggest that such scanning mutagenesis data can be used to rank order sequence alignments and their corresponding homology models, as well as to distinguish between correct and incorrect structural alignments. When incorporated into a polypeptide chain, Proline (Pro) differs from all other naturally occurring amino acids in two important respects. The  dihedral angle of Pro is constrained to values close to –65o and Pro lacks an amide hydrogen. Chapter 3 describes a procedure to accurately predict the effects of proline introduction on protein stability. 77 of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to proline and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or non-perturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties, including main chain dihederal angle and main chain amide hydrogen bonds, were determined from 3D models of the mutant proteins built using MODELLER. The performance of the decision tree was assessed on 74 nsSNPs and 37 other proline substitutions from the literature. The overall accuracy of this algorithm was found to be 89% in case of CcdB, 71% in case of nsSNPs and 83% in case of other proline substitution data. Contrary to previous assertions, Proline scanning mutagenesis cannot be reliably used to make secondary structural assignments in proteins. The studies will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design. Mutants of CcdB were also characterized in terms of their activity at two different temperatures (30oC and 37oC) to screen for temperature sensitive (ts) mutants. The isolation and structural analysis of Ts mutants of CcdB is dealt with in Chapter 4. Of the total 1430 single site mutants, 12% showed a ts phenotype and were mapped onto the crystal structure of the protein. Almost all the ts mutants could be interpreted in terms of the wild type, native structure. ts mutants were found at all buried sites and all active sites (except one). ts mutants were also obtained at sites in close proximity to active site residues where polar side-chains were involved in H-bonding interaction with active site residues. Several proline substitutions also displayed a ts phenotype. The effect of expression level on ts phenotype was also studied. 78% of the mutants that showed an inactive phenotype at the lowest expression level and an active phenotype at highest expression level, resulted in a ts phenotype at an intermediate expression level. The molecular determinant responsible for the ts phenotype of buried site ts mutant is suggested to be the thermodynamic destabilization of the protein which results in a reduced steady state in vivo level of soluble, functional protein relative to wild type. The active site ts mutants probably lower the specific activity of the protein and hence the total activity relative to wild type. However these effects might be less severe at lower temperature. Specific structure/function based mutagenesis strategies are suggested to design ts mutant of a protein. These studies will simplify the design of ts mutants for any globular protein and will have applications in diverse biological systems to study gene function in vivo. Chapter 5 represents the structural and sequence correlations of a CcdB saturation mutagenesis library which was obtained by replacing each of 101 amino acid residues with 19 other amino acids. Polar substitutions i.e. Asn, Gln, Ser, Thr and His were poorly tolerated at buried sites at lower expression levels. Aromatic substitutions and Gly were also not well tolerated at buried positions at lower expression levels. Trp was poorly tolerated at residues with accessibility <15%. However, most of the surface exposed residues with accessibility >40% (except functional ones) could tolerate all kinds of substitutions. Chapter 6 deals with the thermodynamic characterization of monomeric and dimeric forms of CcdB. The stability and aggregation state of CcdB have been characterized as a function of pH and temperature. Size exclusion chromatography revealed that the protein is a dimer at pH 7.0, but a monomer at pH 4.0. CD analysis and fluorescence spectroscopy showed that the monomer is well folded, and has similar tertiary structure to the dimer. Hence intersubunit interactions are not required for folding of individual subunits. The oligomeric status of CcdB at pH 7.0 at physiologically relevant low concentrations of protein, was characterized by labeling the protein with two different pairs of donor and acceptor fluorescent dyes (Acrylodan-Pyrene and IAF-IAEDANS) separately and carrying out fluorescence resonance energy transfer (FRET) measurements by mixing them together. CcdB exists in a dimeric state even at nanomolar concentrations, thus indicating that the dimeric form is likely to be the physiologically active form of CcdB. The stability of the dimeric form at pH 7.0 and the monomeric form at pH 4.0 was characterized by isothermal denaturant unfolding and calorimetry. The free energies of unfolding were found to be 9.2 kcal/mol (1 cal=4.184 J) and 21 kcal/mol at 298 K for the monomer and dimer respectively. The denaturant concentration at which one-half of the protein molecules are unfolded (Cm) for the dimer is dependent on protein concentration, whereas the Cm of the monomer is independent of protein concentration, as expected. Although thermal unfolding of the protein in aqueous solution is irreversible at neutral pH, it was found that thermal unfolding is reversible in the presence of GdnCl (guanidinium chloride). Differential scanning calorimetry in the presence of low concentrations of GdnCl in combination with isothermal denaturation melts as a function of temperature were used to derive the stability curve for the protein. The value of Cp (representing the change in excess heat capacity upon protein denaturation) is 2.8 ± 0.2 kcalmol-1K-1 for unfolding of dimeric CcdB, and only has a weak dependence on denaturant concentration. These studies advanced the understanding of protein folding of oligomeric proteins. The concluding section summarizes all the chapters in a nutshell and addresses the future directions provided by these investigations.