Genetic Engineering: Principles and Methods

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English Only. Add to Cart Add to Cart. Add to Wishlist Add to Wishlist. This hypertonically induced apoptosis apparently occurs only in cells that lack regulatory volume increase RVI capability but not in RVIresponsive cells such as COS, HeLa, or L-cells , suggesting an RVI response may act as a survival checkpoint that must be either inhibited or overridden for apoptotic progression. The mechanisms by which cells shrink during apoptosis are largely undefined, although this morphological change is not fully dependent on caspase activities in some death pathways ; Vu and Cidlowski, unpublished data.

The maintenance of cell volume is orchestrated mainly by ion transport, osmolyte accumulation, cytoskeletal organization and enzymatic activities Among these, the efflux of monovalent ions has been demonstrated to be a critical and necessary element for the loss of cell volume during apoptosis , , The primary ionic movement during cell shrinkage is likely dominated by ions, which are the most abundant intracellular ions. A possible relationship between efflux and apoptosis is substantiated by the observation that the ionophore valinomycin can induce cell death In contrast, inhibiting efflux by a high concentration of extracellular ions can completely abrogate Fas-mediated apoptosis in Jurkat cells In cortical neurons, serum depletion- or staurosporine-induced apoptosis is associated with an early enhancement of outward channel activity, resulting in net loss of intracellular ions and cell shrinkage Our laboratory has also shown that many apoptotic features such as nuclease activity and effector caspase activation, mitochondrial collapse and cell shrinkage are restricted to cells with reduced levels , , These findings raise the hypothesis as depicted in Figure 5 that the maintenance of homeostasis is a critical element for cell survival, possibly by preventing inappropriate activation of apoptotic enzymes.

In support of this idea, our laboratory has shown that ions at normal physiological levels can effectively prevent DNA fragmentation and effector caspase activation in rat thymocytes exposed to dexamethasone, thapsigargin and staurosporine , Furthermore, the presence of volume regulatory mechanisms that increase intracellular ions can protect against apoptosis In the latter case, it is possible that perturbation of the intracellular ionic environment may directly or indirectly affect enzymatic activities of proteases and nucleases, along with protein structure and function.

Although it is currently unclear whether the efflux of ions and volume loss function independently or cooperatively to regulate apoptosis, existing evidence implicates that the proper maintenance of intracellular ions and cell volume is likely to have important consequences for cell survival. Bortner, M. Gomez-Angelats and B. Jacobson, M. O'Connor, R. Hengartner, M.

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Uren, A. Fraser, A. G, James, C. Roy, N. Takahashi, R. Yang, Y. Young, S. Genome 10, Orth, K. Krammer, P.


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Wallach, D. Du, C. Verhagen, A. Thome, M. Hu, S. Irmler, M. Goltsev, Y. Shu, H. Inohara, N. Srinivasula, S. Han, D. Rasper, D. Scaffidi, C. Yeh, W. Komiyama, T. Zhou, Q. Garcia-Calvo, M. Smith, K. Ekert, P. G, Silke, J. Bump, N. Xue, D. Bertin, J. Villa, P. Izquierdo, M. Cytokines regulate numerous physiological and pathological events such as lymphocyte development and activation, programmed cell death and inflammatory processes. Research over the past decade has led to the discovery of many new Cytokines and their respective cell surface receptors, and has elucidated the signaling pathways that lead to cytokine receptor-mediated gene induction.

Equal to their complex biological effects, cytokine-induced transcriptional activation is the product of an intricate network of multiple signaling cascades. This article presents an overview of cytokine-mediated transcriptional induction through STAT proteins and two signaling pathways widely utilized among cytokine receptors. In contrast to the more restricted expression of STAT4 in spleen, heart, brain, peripheral blood cells, and testis, most STAT proteins are found rather ubiquitous.

A carboxy-terminal tyrosine residue, which serves as the target for ligand-induced phosphorylation, is crucial for dimerization, nuclear translocation and DNA-binding of STAT molecules. The src-homology 2 SH2 domain of STAT proteins facilitates homoor heterodimerization via reciprocal interaction with the phosphorylated tyrosine residue. In addition, the SH2 domain accounts for the specific interaction of STAT molecules with activated, tyrosine phosphorylated cytokine and growth factor receptors, as well as Jak tyrosine kinases 6.


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Dimerization of STAT proteins allows the centrally-located DNAbinding domains to interact with consensus palindromic sequences that differ only in their core nucleotide sequence 7. The more divergent region carboxy-terminal to the tyrosine phosphorylation site harbors the transactivation domain TAD. It was again the paradigm of the interferon-signaling cascade that led to the discovery of the participation of the Janus tyrosine kinases in STAT-mediated gene transcription The demonstration that mutagenically ablated expression of Tyk2 or Jak1 resulted in unresponsive cells, while similar abrogation of Jak1 and Jak2 expression resulted in the loss of responsiveness, provided the basis for the function of these tyrosine kinases and the identity of this novel signaling pathway 13, Contrary to the ubiquitous expression of these kinases, a fourth family member, Jak3, was identified whose expression is restricted to cells of hematopoietic origin The most striking feature in the structure of Jak family members is the presence of a kinase-like domain of function thus far unclear, in addition to the true kinase domain.

Also noteworthy is the absence of any SH2- or SH3-iike structures that are commonly found in cytoplasmic tyrosine kinases. The relatively large kDa Jak kinases are constitutively associated with many cytokine and growth factor receptors. Nevertheless, this association can be increased after cytokine stimulation.

DAVID The general outline of the sequential events that lead to STAT-mediated gene transcription has been derived mostly from evidence gathered from the interferon signaling paradigm. It is believed that engagement of ligands with their respective receptors results in an increased local concentration of Jak proteins due to receptor aggregation and increased affinity of the receptors for Jak kinases.

Subsequent crossphosphorylation of the Jaks results in the activation of their kinase activity, such that they can phosphorylate tyrosine residues in the receptor chains. These phospho-tyrosine moieties provide the docking sites for the STAT proteins via their SH2-domains, leading to their tyrosine phosphorylation, dimerization and nuclear translocation. In the case of growth factor receptors, not the presence of Jak kinases, but rather the intrinsic tyrosine kinase activity of the receptors is required for the tyrosine phosphorylation of STAT proteins.

As is the case with other signaling pathways, much of our understanding of the diverse functions of Jak and STAT proteins in vivo has been revealed through the generation of knock-out mice As anticipated, STAT1-deficient mice display dramatically increased sensitivity towards viral and microbial pathogens, presumably due to their inability to respond to interferons 17, Disruption of the STAT3 gene results in early embryonic lethality, and only the use of conditional gene targeting revealed an essential role for STAT3 in T cell and macrophage function 19, Jakl-deficient mice fail to nurse, display severely impaired lymphocyte development and die perinatally 24, 25 The function of Jak2 in erythropoietin signaling is evidenced by embryonic lethality due to defective erythropoiesis in its absence Of particular interest are results obtained from Jak3-deficient mice 27 , since mutations in Jak3 have also been identified in humans.

Of equal importance to the activation of a signaling pathway is its spatially and temporally coordinated attenuation. The tyrosine 39 40 K. The SH2-domain containing SOCS proteins have been identified as a family of cytokineinducible inhibitors of Jak kinase activity, thus acting in a classical negative feedback loop Components or products of infectious microorganisms, e.

The binding of these proinflammatory cytokines to their cell surface receptors rapidly induces a genetic program to respond to cellular stress and to generate inflammatory mediators e. Chronic inflammation can result in extensive tissue destruction and disease, such as rheumatoid arthritis. Therefore, it is important that signaling molecules transducing IL-1 and TNF signals not only be triggered quickly but also signal transiently.

The transcription factor fulfills these characteristics. Although a key mediator in the inflammatory response, was first isolated in B cells as a factor necessary for immunoglobulin kappa light chain transcription.

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Later, was found to exist in most cell types Thus, was implicated in innate immunity as well as in the adaptive immune response. The p50 and p52 subunits are first generated as the longer p and p forms, respectively, which are then proteolytically processed into the shorter, active forms. Hence, p50 and p52 homodimers are transcriptionally repressive TNF and IL-1, and LPS itself, activate a pathway which results in the degradation of inhibitors of freeing proteins from the cytosolic tether and exposing the nuclear localization signal NLS.

Similar to transcriptional induction via STAT proteins, the activation of does not require protein synthesis. Once in the nucleus, negatively regulates its own activity by inducing transcription of which enters the nucleus and chaperones molecules out through an active export pathway involving the nuclear export sequence NES The proteins bind to dimers and mask the NLS. The family members and Bc contain ankyrin repeats which bind the an N-terminal regulatory domain and a C-terminal PEST motif for proteolytic degradation Two closely-related proteins with kinase IKK activity were identified as and Additionally, a putative scaffolding subunit was isolated and called NEMO.

Substitution of alanines for these serines abrogated kinase activity; however, mutations of the homologous serines within left TNF and IL-1 induced activity intact, suggesting that is the main IKK kinase subunit through which proinflammatory cytokines signal The and knock-out mice confirmed these earlier biochemical experiments, as embryonic fibroblasts are normal in proinflammatory cytokine-induced activation. In contrast, mice, which die between embryonic day Thus, additional kinases are likely to be involved in activating IKK in particular in vivo situations.

The TNFR1 is a death-domain-containing receptor. The binding of IL-1 to the IL-1R also leads to the development of a signaling scaffold resulting in activation. First, the adapter protein MyD88 myeloid differentiation factor 88 binds to the receptor. It is important to remember that cytokine activation of gene transcription is not the result of a single, linear signaling cascade, but involves a complex network of interacting signal-transduction pathways.

Integration of numerous, often contradicting instructions received by the cell through a variety of cytokine and growth factor receptors determines the qualitative and quantitative transcriptional response. Fu, X. Lamer, A. Schindler, C. Seidel, H. Wen, Z. David, M. Korzus, E. Zhang, J. Velazquez, L. Muller, M. Watling, D. Kawamura, M. Akira, S. Meraz, M. Takeda, K. Kaplan, M. Thierfelder, W. A, Sarawar, S.

Teglund, S. Shimoda, K. A, Chu, C. Neubauer, H. Nosaka, T. Russell, S. Hilton, D. Life Sci. Klingmuller, U. Chen, X. Shuai, K. Ghosh, S. Pahl, H. Karin, M. May, M.

Today 19, Mercurio, F. Lee, F. Israel, A. Arch, R. Barkett, M. Adachi, O. Ninomiya-Tsuji, J. Torrey Pines Rd. As part of glycoproteins, glycolipids and other conjugates, they play key roles in a variety of processes such as signaling and molecular and cellular targeting. Despite the important roles that glycoproteins play in many biological recognition events, the details of these events are generally not well understood.

Research has been stymied by the lack of synthetic and analytical methods available. Recent advances in both classical and enzymatic synthesis are beginning to provide methods for the preparation of homogeneous glycoproteins which were previously inaccessible. Although the number of glycoforms seen in nature is fairly overwhelming, they do fall into a few classes, which are categorized first by the residues to which the sugars are linked. The linkages observed in nature are shown in Figure 1. Genetic Engineering, Volume 23, Edited by J.

N-linked glycans in eukaryotes fall into three categories as shown in Figure 2. Apart from the chitobiose core, the high-mannose type saccharides contain almost entirely mannose. Hybrid-type chains have the characteristics of high mannose glycans on the mannose branch, but look like complex-type chains on the mannose branch. O-linked glycans have more varied core structures. The mucin-type glycoproteins have GalNAc to serine or threonine which can be extended into a variety of basic units, shown in Figure 3.

There may be a variety of other modifications in addition: sulfation, methylation, phosphorylation, Oacetylation, and addition of GlcNAc-, mannose-, or GalNAcphosphate. A well-researched review of the biological consequences of glycosylation was written by Varki 2. At the molecular level, there are examples where glycosylation affects nearly every property of a protein. There are a host of cases in which it has been demonstrated to increase the stability of proteins toward denaturation or protease degradation.

Glycosylation can also alter the physical characteristics of the glycoprotein solution, as in the case of mucins, which are responsible for the sliminess of mucous secretions. The activity of proteins can be modulated by their glycosylation states. O-GlcNAcylation is a recently-discovered transient modification of cytoplasmic and nuclear proteins that is thought to have a regulatory role 3.

Recognition of glycoproteins is often based on the saccharides they carry. Many immunoglobulins recognize foreign saccharide epitopes. It is the recognition of that is responsible, at least in part, for many cases of xenograft rejection, and N-glycans fucosylated at the core may contribute to bee sting anaphylaxis 4. Some proteins cannot fold properly when unglycosylated, frequently aggregating instead, so in these cases glycosylation may help both to improve solubility and aid folding possibly by nucleating "kinks" in the protein.

Glycosylation also allows the binding of folding chaperones such as calnexin 5. There are many examples in which glycosylation affects protein targeting. Proteins labeled with mannose6-phosphate are shuttled to lysosomes, for example, and the presence of on some pituitiary hormones is thought to play a part in their retrieval from the blood by hepatic endothelial cells 6. This occurs because the saccharide that a protein receives reflects the cumulative effort of many glycosidases and transferases, and the action of some of these will preclude the action of others.

The glycan produced will be determined by many factors 7, 8 including the local protein structure around the glycosylation site, the relative amounts of glycoprocessing enzymes produced in the cell and their location, the rate of transit of the protein through the endoplasmic reticulum and Golgi apparatus, and so on. Many of these factors also vary with the cell line, so a glycoprotein produced in one cell line will have different glycosylation than the same protein produced in another cell line.

A variety of techniques are becoming available for the preparation of homogeneous glycoproteins. Protecting group chemistry with milder conditions such as hydrogenation, oxidation, or treatment with weak base compatible with the glycosidic linkage, has been worked out, however, and so good yields can be obtained for short peptides with small to moderate amounts of sugar 9, As the peptide and sugar moieties become longer, of course, yields still drop off, and so for larger glycoproteins and peptides it becomes desirable to do much of the synthesis biologically.

Fermentation, as mentioned above, necessarily produces a population of many different glycoforms of a given protein. This mixture, however, can be used as a starting point in a variety of schemes in which the glycoprotein is digested down to a simple, homogeneous core and then re-elaborated enzymatically Figure 4. One option is to remove the glycosylated sections with proteases, and then reattach short, chemically-synthesized glycopeptides in their place.

This ligation can be accomplished via "native peptide ligation" strategies, via the use of proteases, or with inteins in which case the peptide segment to be replaced is substituted at the genetic level with the sequence encoding the intein. Alternatively, N-glycosylated proteins can have the glycans digested down to the innermost N-acetylglucosamine with endonucleases, thus converting a heterogenous 50 P. These simple glycoproteins can then be elaborated enzymatically to increase the size and complexity of the glycan with the use of glycosyltranferases or endoglycosidase-catalyzed transglycosylation.

Protein Hydrolysis and Glycopeptide Ligation with Proteases One starting point for this method is to produce either a heterogeneous glycoprotein population from eukaryotic cell culture or a non-glycosylated protein from prokaryotic cell fermentation. Proteases may then be used in an aqueous medium to remove the glyco peptide to be substituted. A replacement glycopeptide is synthesized chemically and then ligated onto the protein fragment with proteases again, this time under conditions that favor peptide synthesis e. Clearly, the fewer segments one must reassemble, the more useful this approach will be.

In order for this method to work, one must be able to find a set of conditions under which the peptides can be reassembled by the same enzymes that normally degrade them. Peptide synthesis with the use of proteases has been a topic of interest to many There are two different approaches by which normally proteolytic enzymes can be coaxed into running the synthetic reaction, as illustrated in Figure 5.

In some cases, the product precipitates in water and so the reaction is pulled to the peptide product even in aqueous solution. In most cases, however, the reaction is driven toward the peptide by conducting the reaction in nearly dry organic solvents. Unfortunately, under very low water conditions, many enzymes are minimally active. In particular, the solvents in which enzyme suspensions are most active and stable hydrophobic solvents are the same solvents in which the peptide substrates are minimally soluble.

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The more polar solvents such as DMF and DMSO, which are better able to dissolve the peptide substrates, are poor solvents with regard to enzymatic activity and stability Also, the substrate specificity 16 and enantioselectivity 17 can be hampered by the addition of solvents. Finally, the activity and stability of the enzyme suspension is exquisitely sensitive to many variables 18 , including the type, concentration, and pH of the buffer from which it was lyophilized 19 , the amount of water in the solvent 20, 21 and particle shearing.

This approach suffers from problems of its own. The enzymes are often less stable in high concentrations of solvent than in water, and this instability is exacerbated at elevated temperature Another approach is to freeze the reaction mixture. Frozen solutions have solutes concentrated in water "channels" between ice crystals, and this freeze concentration effect is at least in part responsible for the increased yield observed in frozen reactions 23, The addition of cosolvent enhances synthesis by removing much of the competing nucleophile water and suppressing the ionization of the amine, but appears to have additional effects on the active site of the serine protease subtilisin.

X-ray crystallography and NMR studies have provided evidence for a flip in the orientation of the active-site histidine Figure 6 How this would contribute to an enhanced rate of aminolysis is not clear, although the flipped orientation is reminiscent of that of the activesite modified enzymes methylsubtilisin and methylchymotrypsin, in which the Nmethylated active-site histidine is also thought to be flipped during catalysis 26, These enzymes also display an improved aminolysis:hydrolysis ratio, which has been attributed to the displacement of the nucleophilic water molecule due to the new active-site geometry An increase in the concentration of cosolvent can have negative effects on enzyme activity and stability, and for this reason many groups have been improving the stability of several key proteases toward both thermal and organic-cosolvent-mediated denaturation and evolving them for better activity under elevated concentrations of cosolvent.

The subtilisins, a set of serine proteases from Bacillus species, have been the subject of intensive studies in this regard. These enzymes have been favored because of their broad 52 P. In order to suppress the hydrolysis further, the enzyme active site can be modified. The replacement of the active site serine of serine proteases with a cysteine 32 or selenocysteine 33 can dramatically improve the aminolysis:hydrolysis ratio.

This modification, which can be made chemically Figure 7A 34 or at the genetic level 35 produces a thiolenzyme that can have an improvement in the aminolytic:hydrolytic ratio of several orders of magnitude Figure 7B. Reactions with the thiolenzyme can, in fact, be run in water without cosolvent at all to obtain good yields of the peptide product.

The enzyme is somewhat crippled, however, and works best with activated ester substrates such as cyanomethyl esters. Under these conditions, thiolsubtilisin BPN' would quickly deactivate, while the serine protease would primarily hydrolyze the substrate. Wells and coworkers took a different tactic to improve thiolsubtilisin activity. Rather than raising the reaction temperature, they discovered that an additional modification near the active site ProAla would pull the active site thiol away from the histidine slightly, providing more room for the larger sulfur atom and restoring much of the activity of the enzyme toward ester hydrolysis at room temperature, though the enzyme still retains the highly diminished protease activity characteristic of thiolsubtilisin Many proteases are commercially available for use in the degradation and reassembly of glycoproteins.

Those used for peptide ligation are typically serine or thiolproteases, for which a covalent acyl enzyme intermediate is formed that may then undergo aminolysis to form the amide product. Proteases range from the exquisitely specific and thus of limited utility for this purpose , such as enterokinase, to the very nonspecific. An exhaustive discussion of the substrate specificities of these enzymes is not feasible here, but an excellent handbook was recently published that tabulates substrate preferences and other information on the known proteases Notably, subtilisin and papain are frequently used because of their ready availability, good stability and broad specificity.

Subtilisin, for example, prefers hydrophobic sequences, but will accept a very wide range of amino acid residues around the scissile bond Figure 9. Other proteases that have been used in synthesis include trypsin, chymotrypsin, elastase and thermolysin. An early synthesis of the octapeptide dynorphin was accomplished with a several proteases in succession 11 , while human insulin is prepared from the porcine hormone by coupling the desoctapeptide B -insulin with a synthetic peptide corresponding to positions of the human hormone The artificial sweetener aspartame is made by coupling cbz-L-glutamic acid with L-phenylalanine methyl ester, catalyzed by thermolysin in aqueous solution Most proteases have preferred cleavage sequences, but it is worth noting that the tertiary structure of the protein substrate has so much influence on the ability of the protease to cleave it that even the broadly-specific enzymes may only cleave the substrate at one or two sites.

Subtilisin, despite its broad substrate acceptance, only cleaves a single site of ribonuclease after four hours at room temperature, and that site is not flanked by the "preferred" residues for subtilisin cleavage. This site is, however, on the surface of the protein, and thus highly accessibly to proteases. Figure 10 56 P. This is a fairly typical observation.

For this reason, the choice of a protease for cleavage and religation will require a certain amount of trial and error. When sugars are added to the equation, matters change even further, if the sugars are near the scissile bond. Glycosylation has been recognized for many years to promote resistance to proteolysis. The ability of subtilisin to accept glycosylated peptide substrates has been investigated in this laboratory via the systematic placement of single sugars at the and positions nomenclature according to Schecter and Berger 41 , where and refer to the peptide residues and enzyme subsites, respectively, to the N-terminal side of the scissile bond and and refer to those on the C-terminal end.

Subtilisin would not accept sugars immediately adjacent to the scissile bond, and accepted the sugars better as they were moved further away Figure 11 They frequently are, due to enzyme specificity issues, but an interesting strategy for ligating peptides in which the resulting amide is not a substrate for hydrolysis has been presented, and is called the "inverse substrate" 43 or "substrate mimetic" 44 principle.

With this strategy, the acyl donor contains a small residue and a leaving group that satisfies the substrate requirements of the subsite. The leaving group wraps around into the site, eliminating the need of the residue to fill that site. This has been illustrated in Figure 12 for trypsin, which prefers basic residues at the position. With the use of p-guanidinophenyl ester, the necessity of having a basic residue can be avoided. This substrate can serve as an acyl donor, but once the leaving group is gone, the resulting amide product is no longer a substrate for the enzyme, avoiding the problem of further hydrolysis of the peptide product.

Intein-Mediated Glycopeptide Ligation A recent alternative to protease-catalyzed fragment condensation has been made available since the discovery of self-splicing inteins, segments of proteins that excise themselves post-translationally. The general mechanism of a self-splicing intein is shown in Figure 13 The intein is flanked by the two exteins to be ligated. Publisher: Springer , This specific ISBN edition is currently not available.

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View all copies of this ISBN edition:. Synopsis About this title Presents the latest research in genetic engineering. Topics include agrobacterium -mediated horizontal gene transfer, detection of single nucleotide variations, the ribosome as a vehicle for antisense RNA, cloning and expression of large mammalian cDNAs, the use of genetically engineered cells in dr "synopsis" may belong to another edition of this title. About the Author : Jane K.

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