I describe the application of new methods for reverse genetic analyses in Drosophila melanogaster to genes expressed in the nervous system. The methods can be devided into two classes: tools for molecular analysis and tools for genetic analysis.
\r\n\r\nThe first set of methods is designed to facilitate rapid, large-scale molecular analysis of cDNA clones. This set of tools begins with a family of bacteriophage \u03bb cDNA cloning vectors and E. coli host cell strains that allow automatic plasmid subcloning by in vivo site-specific recombination. A high density filter hybridization and diagnostic PCR assay technique applied to size-selected libraries then substantially simplifies the isolation of full-length cDNA clones in these vectors. Next, a transposon \u03b3\u03b4-facilitated DNA sequencing procedure minimizes the labor required to isolate a nested set of initiation sites for chain termination sequencing of each strand of a cloned DNA segment. I also describe the characterization of 250 cDNA clones isolated from adult heads on the basis of gross expression patterns in the embryonic ventral nerve cord and larval fat body.
\r\n\r\nThe second set of procedures facilitates the isolation of mutations in the chromosomal genes that correspond to isolated cDNA clones. I describe three such experiments. A plasmid rescue and hybridization strategy allowed the isolation of PlacW elements inserted adjacent to 4 cloned genes in an array of nearly 700. Modifications of this procedure to take advantage of P-element local transposition allowed the isolation and characterization of an apparent null mutation in the receptor-linked protein tyrosine phosphatase gene DPTP99A. In a pilot study, I demonstrate the feasibility of isolating chemically induced mutations that remove targeted restriction enzyme cleavage sites with a PCR-based assay. In addition, I describe the serendipitous isolation of a PlacW-induced mutation, encumbered, that affects the morphogenesis of imaginal wing discs as well as adult longevity, activity, and fertility.
\r\n", "doi": "10.7907/aqes-7j89", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:7334", "collection": "thesis", "collection_id": "7334", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12132012-153519784", "primary_object_url": { "basename": "Jongeward_gd_1993.pdf", "content": "final", "filesize": 42474088, "license": "other", "mime_type": "application/pdf", "url": "/7334/1/Jongeward_gd_1993.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Negative regulators of the let-23 EGF receptor in Caenorhabditis elegans vulval differentiation", "author": [ { "family_name": "Jongeward", "given_name": "Gregg D.", "clpid": "Jongeward-G-D" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Lipshitz", "given_name": "Howard D.", "orcid": "0000-0002-7372-4419", "clpid": "Lipshitz-H-D" }, { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Vulval induction in C. elegans is an example of the use of an EGF\r\n(Epidermal Growth Factor) mediated signal transduction system. At least\r\nfive genes are involved in the negative regulation of vulval induction.
\r\n\r\nMutations at the silent locus sli-1 (suppressor of lineage defect) are\r\nsufficient to suppress all of the phenotypes associated with hypomorphic\r\nalleles of let-23. sli-1 functions to modify the activity of let-23 but muations\r\nat sli-1 do not bypass the requirement for let-23. Based on the phenotypes of\r\nanimals bearing mutations at sli-1 and other genes, sli-1 may function at or\r\nnear the let-23 or sem-5 step of vulval differentiation.
\r\n\r\nNull alleles of the pleiotropic locus unc-101 cause a number of mutant\r\nphenotypes including neural defects and suppression of the vulval defects\r\nassociated with some weak let-23 mutations. These unc-101 mutations\r\ninteract with mutations in other genes required for proper vulval\r\ndifferentiation but do not act as generalized suppressors. This locus has been\r\ncloned and encodes the C. elegans homolog of the Golgi-associated clathrin\r\nadaptor protein AP47.
\r\n\r\nAnimals mutant for both unc-101 and sli-1 display excessive vulval\r\ndifferentiation. Animals mutant at only one of these loci display no vulval\r\nabnormalities. This excessive vulval differentiation requires the inductive\r\nsignal and functionallet-23, suggesting that sli-1 and unc-101 function to\r\nnegatively regulate the response to the inductive signal, rather than the\r\nbasal activity of let-23.
\r\n\r\nRare mutant alleles at lin-2, lin-7, and let-23 result in excessive vulval\r\ndifferentiation. These alleles are genetically similar to more common alleles\r\nof these genes which result in the failure to differentiate vulval tissue. These\r\nthree genes apparently are required for the activation of both positive and negative regulators of vulval differentiation.
\r\n\r\nA number of negative regulators function to control the activity of let-23.\r\nAt least three pathaways of negative regulation have been genetically\r\nidentified. These negative regulators act to limit the response to a growth or\r\ndifferentiation factor.
\r\n", "doi": "10.7907/87ea-tr80", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:7334", "collection": "thesis", "collection_id": "7334", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12132012-153519784", "primary_object_url": { "basename": "Jongeward_gd_1993.pdf", "content": "final", "filesize": 42474088, "license": "other", "mime_type": "application/pdf", "url": "/7334/1/Jongeward_gd_1993.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Negative regulators of the let-23 EGF receptor in Caenorhabditis elegans vulval differentiation", "author": [ { "family_name": "Jongeward", "given_name": "Gregg D.", "clpid": "Jongeward-G-D" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Lipshitz", "given_name": "Howard D.", "orcid": "0000-0002-7372-4419", "clpid": "Lipshitz-H-D" }, { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Vulval induction in C. elegans is an example of the use of an EGF\r\n(Epidermal Growth Factor) mediated signal transduction system. At least\r\nfive genes are involved in the negative regulation of vulval induction.
\r\n\r\nMutations at the silent locus sli-1 (suppressor of lineage defect) are\r\nsufficient to suppress all of the phenotypes associated with hypomorphic\r\nalleles of let-23. sli-1 functions to modify the activity of let-23 but muations\r\nat sli-1 do not bypass the requirement for let-23. Based on the phenotypes of\r\nanimals bearing mutations at sli-1 and other genes, sli-1 may function at or\r\nnear the let-23 or sem-5 step of vulval differentiation.
\r\n\r\nNull alleles of the pleiotropic locus unc-101 cause a number of mutant\r\nphenotypes including neural defects and suppression of the vulval defects\r\nassociated with some weak let-23 mutations. These unc-101 mutations\r\ninteract with mutations in other genes required for proper vulval\r\ndifferentiation but do not act as generalized suppressors. This locus has been\r\ncloned and encodes the C. elegans homolog of the Golgi-associated clathrin\r\nadaptor protein AP47.
\r\n\r\nAnimals mutant for both unc-101 and sli-1 display excessive vulval\r\ndifferentiation. Animals mutant at only one of these loci display no vulval\r\nabnormalities. This excessive vulval differentiation requires the inductive\r\nsignal and functionallet-23, suggesting that sli-1 and unc-101 function to\r\nnegatively regulate the response to the inductive signal, rather than the\r\nbasal activity of let-23.
\r\n\r\nRare mutant alleles at lin-2, lin-7, and let-23 result in excessive vulval\r\ndifferentiation. These alleles are genetically similar to more common alleles\r\nof these genes which result in the failure to differentiate vulval tissue. These\r\nthree genes apparently are required for the activation of both positive and negative regulators of vulval differentiation.
\r\n\r\nA number of negative regulators function to control the activity of let-23.\r\nAt least three pathaways of negative regulation have been genetically\r\nidentified. These negative regulators act to limit the response to a growth or\r\ndifferentiation factor.
\r\n", "doi": "10.7907/87ea-tr80", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:6632", "collection": "thesis", "collection_id": "6632", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08302011-103945566", "primary_object_url": { "basename": "Funkhouser_wk_1992.pdf", "content": "final", "filesize": 47915613, "license": "other", "mime_type": "application/pdf", "url": "/6632/1/Funkhouser_wk_1992.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Demyelinating autoimmunity: murine T cell epitopes of MBP and primate T cell receptor V\u03b2 variation", "author": [ { "family_name": "Funkhouser", "given_name": "William Keith Jr.", "clpid": "Funkhouser-William-Keith-Jr" } ], "thesis_advisor": [ { "family_name": "Wise", "given_name": "Mark B.", "orcid": "0000-0002-9125-801X", "clpid": "Wise-M-B" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Wise", "given_name": "Mark B.", "orcid": "0000-0002-9125-801X", "clpid": "Wise-M-B" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Autoimmune diseases result from inappropriate self-reactivity by lymphocytes. The long-term goal is to generate specific therapies for autoimmune diseases of humans, the success of which hinges on the definition of specific therapeutic targets. Experimental allergic encephalomyelitis (EAE) is a good animal model for the human demyelinating autoimmune disease, multiple sclerosis (MS). Risk for these diseases stratifies by major\r\nhistocompatibility complex (MHC) allele, as well as by T\r\ncell receptor (TCR) locus RFLP, in the case of MS. These\r\ndata suggest that (TCR-self peptide-MHC) complexes are\r\nassociated, possibly causally, with pathogenesis. This work\r\nfocused on the self peptide and TCR components of this\r\ncomplex. One specific aim was to document and characterize\r\nthe T cell epitopes of the autoantigen, myelin basic protein\r\n(MBP), in the EAE-susceptible mouse strain, B10.PL. Inbred\r\nB10.PL mice which were immunized with self MBP in complete\r\nFreund's adjuvant activated lymphocytes specific for\r\nepitopes estimated by peptides MBP(NAc1-20), MBP(31-50), and\r\nMBP(121-140). These mice generated the bulk of their immune\r\nresponse to the MBP(NAc1-20) epitope. The responses to self\r\nMBP immunization of B10.PL wildtype and MBP null \"shiverer\"\r\nmice were compared, and it was found that MBP(12I-140) is\r\ntolerogenic in animals which express MBP. A similar result\r\nwas observed in BALB/c wildtype and shiverer mice. These\r\ndata demonstrate that MBP is not a sequestered antigen, that\r\nmultiple epitopes tolerize T cells independently, and that\r\nincomplete, rather than absent, tolerance is present in mice\r\nsusceptible to EAE. A second specific aim was to document\r\nthe degree of variation in the primate TCR V\u03b2 8 subfamily,\r\nthree members of which are adjacent to a BamHI RFLP\r\nrestriction site linked to multiple sclerosis (MS) disease\r\nrisk. V\u03b2 8.1 and 8.2 were compared in a number of primates.\r\nIt was found that the overall coding sequences, but not the\r\nCDR coding sequences, were conserved compared with adjacent\r\nnon-coding flanking sequences. CDR coding sequences were\r\nnot demonstrably positively selected compared with noncoding\r\nflanking sequences or with synonymous coding sequences. A comparison of unrelated normal humans failed to demonstrate any non-synonymous Substitutions within V\u03b2 8.1 and 8.2, and demonstrated a single non-synonymous Substitution in V\u03b2 8.3. These data demonstrate that germline V\u03b2 8 gene segments are conserved and minimally polymorphic, implying that final TCR protein diversity derives from other mechanisms. Occasional allelism has been demonstrated in other V\u03b2 subfamilies, and our data does not rule out that certain TCR V\u03b2 alleles may ultimately be found to contribute to autoimmunity disease risk.\r\n", "doi": "10.7907/kmkw-9619", "publication_date": "1992", "thesis_type": "phd", "thesis_year": "1992" }, { "id": "thesis:6632", "collection": "thesis", "collection_id": "6632", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08302011-103945566", "primary_object_url": { "basename": "Funkhouser_wk_1992.pdf", "content": "final", "filesize": 47915613, "license": "other", "mime_type": "application/pdf", "url": "/6632/1/Funkhouser_wk_1992.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Demyelinating autoimmunity: murine T cell epitopes of MBP and primate T cell receptor V\u03b2 variation", "author": [ { "family_name": "Funkhouser", "given_name": "William Keith Jr.", "clpid": "Funkhouser-William-Keith-Jr" } ], "thesis_advisor": [ { "family_name": "Wise", "given_name": "Mark B.", "orcid": "0000-0002-9125-801X", "clpid": "Wise-M-B" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Wise", "given_name": "Mark B.", "orcid": "0000-0002-9125-801X", "clpid": "Wise-M-B" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Autoimmune diseases result from inappropriate self-reactivity by lymphocytes. The long-term goal is to generate specific therapies for autoimmune diseases of humans, the success of which hinges on the definition of specific therapeutic targets. Experimental allergic encephalomyelitis (EAE) is a good animal model for the human demyelinating autoimmune disease, multiple sclerosis (MS). Risk for these diseases stratifies by major\r\nhistocompatibility complex (MHC) allele, as well as by T\r\ncell receptor (TCR) locus RFLP, in the case of MS. These\r\ndata suggest that (TCR-self peptide-MHC) complexes are\r\nassociated, possibly causally, with pathogenesis. This work\r\nfocused on the self peptide and TCR components of this\r\ncomplex. One specific aim was to document and characterize\r\nthe T cell epitopes of the autoantigen, myelin basic protein\r\n(MBP), in the EAE-susceptible mouse strain, B10.PL. Inbred\r\nB10.PL mice which were immunized with self MBP in complete\r\nFreund's adjuvant activated lymphocytes specific for\r\nepitopes estimated by peptides MBP(NAc1-20), MBP(31-50), and\r\nMBP(121-140). These mice generated the bulk of their immune\r\nresponse to the MBP(NAc1-20) epitope. The responses to self\r\nMBP immunization of B10.PL wildtype and MBP null \"shiverer\"\r\nmice were compared, and it was found that MBP(12I-140) is\r\ntolerogenic in animals which express MBP. A similar result\r\nwas observed in BALB/c wildtype and shiverer mice. These\r\ndata demonstrate that MBP is not a sequestered antigen, that\r\nmultiple epitopes tolerize T cells independently, and that\r\nincomplete, rather than absent, tolerance is present in mice\r\nsusceptible to EAE. A second specific aim was to document\r\nthe degree of variation in the primate TCR V\u03b2 8 subfamily,\r\nthree members of which are adjacent to a BamHI RFLP\r\nrestriction site linked to multiple sclerosis (MS) disease\r\nrisk. V\u03b2 8.1 and 8.2 were compared in a number of primates.\r\nIt was found that the overall coding sequences, but not the\r\nCDR coding sequences, were conserved compared with adjacent\r\nnon-coding flanking sequences. CDR coding sequences were\r\nnot demonstrably positively selected compared with noncoding\r\nflanking sequences or with synonymous coding sequences. A comparison of unrelated normal humans failed to demonstrate any non-synonymous Substitutions within V\u03b2 8.1 and 8.2, and demonstrated a single non-synonymous Substitution in V\u03b2 8.3. These data demonstrate that germline V\u03b2 8 gene segments are conserved and minimally polymorphic, implying that final TCR protein diversity derives from other mechanisms. Occasional allelism has been demonstrated in other V\u03b2 subfamilies, and our data does not rule out that certain TCR V\u03b2 alleles may ultimately be found to contribute to autoimmunity disease risk.\r\n", "doi": "10.7907/kmkw-9619", "publication_date": "1992", "thesis_type": "phd", "thesis_year": "1992" }, { "id": "thesis:6670", "collection": "thesis", "collection_id": "6670", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09142011-114200593", "primary_object_url": { "basename": "Aroian_rv_1992.pdf", "content": "final", "filesize": 34350162, "license": "other", "mime_type": "application/pdf", "url": "/6670/1/Aroian_rv_1992.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The let-23 gene of the nematode C. elegans : genetics and molecular biology of a member of the EGF receptor tyrosine kinase family", "author": [ { "family_name": "Aroian", "given_name": "Raffi V", "clpid": "Aroian-Raffi-V" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Lipshitz", "given_name": "Howard D.", "orcid": "0000-0002-7372-4419", "clpid": "Lipshitz-H-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Genetic studies indicate that the let-23 gene affects several developmental decisions in the nematode\r\nCaenorhabditis elegans. let-23 is required for the proper development of the hermaphrodite vulva, the male tail, and the posterior ectoderm. In addition, let-23 mutations can cause lethality and hermaphrodite sterility. These five let-23 functions can be independently mutated, suggesting that the let-23 protein encodes tissuespecific functions . Furthermore, let-23 controls two opposing pathways: one that stimulates and another that inhibits vulval\r\ndevelopment. These two pathways ensure that the proper level of vulval development occurs. Twenty let-23 alleles exist: 14 eliminate function (null), three reduce function in all tissues (hypomorphic), and three reduce function in certain tissues (tissue-specific). In addition, two of these alleles are defective in the inhibitory vulval pathway.
\r\n\r\nThe let-23 primary structure resembles that of the mammalian epidermal growth factor receptor (EGFR). The let-23 protein possesses putative ligand binding,\r\ntransmembrane, and tyrosine kinase domains, as well as cysteine-rich regions, all with the characteristics of the EGFR family. Like let-23, mammalian EGFR is multifunctional, encodes tissuespecific functions, and functions in stimulatory and inhibitory pathways. let-23 may be the receptor in the vulva for the anchor-cell\r\ninductive signal. Furthermore, genetic data indicate let-23 acts upstream of the let-60 ras gene, supporting mammalian studies that suggest a link between EGFR and ras.
\r\n\r\nTo investigate how EGFR primary structure relates to function, mutations in eight let-23 alleles have been\r\nsequenced. Five null alleles alter sequences in both the kinase and the extracellular domains. These alterations suggest that let-23 has kinase activity and that the extra cysteine domain found only in invertebrate EGFRs is important. A strong hypomorphic allele mutates one of the conserved extracellular cysteines close to the ligand binding domain. A tissue-specific allele mutates an\r\nintronlexon boundary in the C-terminus. This mutation suggests that the C-terminus can provide tissue-specific information. Finally, a hypomorphic allele that is defective in the let\u202223 inhibitory vulval pathway alters a different intron/exon boundary in the C-terminus. This mutation results in numerous, unexpected transcripts. Models are suggested to account for the behavior of this allele.
\r\n", "doi": "10.7907/q10c-b807", "publication_date": "1992", "thesis_type": "phd", "thesis_year": "1992" }, { "id": "thesis:6670", "collection": "thesis", "collection_id": "6670", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09142011-114200593", "primary_object_url": { "basename": "Aroian_rv_1992.pdf", "content": "final", "filesize": 34350162, "license": "other", "mime_type": "application/pdf", "url": "/6670/1/Aroian_rv_1992.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The let-23 gene of the nematode C. elegans : genetics and molecular biology of a member of the EGF receptor tyrosine kinase family", "author": [ { "family_name": "Aroian", "given_name": "Raffi V", "clpid": "Aroian-Raffi-V" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Lipshitz", "given_name": "Howard D.", "orcid": "0000-0002-7372-4419", "clpid": "Lipshitz-H-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Genetic studies indicate that the let-23 gene affects several developmental decisions in the nematode\r\nCaenorhabditis elegans. let-23 is required for the proper development of the hermaphrodite vulva, the male tail, and the posterior ectoderm. In addition, let-23 mutations can cause lethality and hermaphrodite sterility. These five let-23 functions can be independently mutated, suggesting that the let-23 protein encodes tissuespecific functions . Furthermore, let-23 controls two opposing pathways: one that stimulates and another that inhibits vulval\r\ndevelopment. These two pathways ensure that the proper level of vulval development occurs. Twenty let-23 alleles exist: 14 eliminate function (null), three reduce function in all tissues (hypomorphic), and three reduce function in certain tissues (tissue-specific). In addition, two of these alleles are defective in the inhibitory vulval pathway.
\r\n\r\nThe let-23 primary structure resembles that of the mammalian epidermal growth factor receptor (EGFR). The let-23 protein possesses putative ligand binding,\r\ntransmembrane, and tyrosine kinase domains, as well as cysteine-rich regions, all with the characteristics of the EGFR family. Like let-23, mammalian EGFR is multifunctional, encodes tissuespecific functions, and functions in stimulatory and inhibitory pathways. let-23 may be the receptor in the vulva for the anchor-cell\r\ninductive signal. Furthermore, genetic data indicate let-23 acts upstream of the let-60 ras gene, supporting mammalian studies that suggest a link between EGFR and ras.
\r\n\r\nTo investigate how EGFR primary structure relates to function, mutations in eight let-23 alleles have been\r\nsequenced. Five null alleles alter sequences in both the kinase and the extracellular domains. These alterations suggest that let-23 has kinase activity and that the extra cysteine domain found only in invertebrate EGFRs is important. A strong hypomorphic allele mutates one of the conserved extracellular cysteines close to the ligand binding domain. A tissue-specific allele mutates an\r\nintronlexon boundary in the C-terminus. This mutation suggests that the C-terminus can provide tissue-specific information. Finally, a hypomorphic allele that is defective in the let\u202223 inhibitory vulval pathway alters a different intron/exon boundary in the C-terminus. This mutation results in numerous, unexpected transcripts. Models are suggested to account for the behavior of this allele.
\r\n", "doi": "10.7907/q10c-b807", "publication_date": "1992", "thesis_type": "phd", "thesis_year": "1992" }, { "id": "thesis:2673", "collection": "thesis", "collection_id": "2673", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06212007-075739", "primary_object_url": { "basename": "Hoh_jh_1991.pdf", "content": "final", "filesize": 16024320, "license": "other", "mime_type": "application/pdf", "url": "/2673/1/Hoh_jh_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Studies on the structure and molecular diversity of the gap junction", "author": [ { "family_name": "Hoh", "given_name": "Jan Hakan", "clpid": "Hoh-Jan-Hakan" } ], "thesis_advisor": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "thesis_committee": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Lipshitz", "given_name": "Howard D.", "orcid": "0000-0002-7372-4419", "clpid": "Lipshitz-H-D" }, { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "An improved method for the isolation of hepatic gap junctions that substantially shortens preparation time and improves the yield of previous methods is described. The topology of the 28 kD protein component (connexin-32, Cx32) of gap junctions isolated with this method is examined using proteases and antibodies against specific peptides. These experiments are consistent with the current model for the organization of the protein in the membrane, but reveal that an unexpectedly large part of the carboxy-terminus is protected from proteolytic attack. Together with data from comparisons of the Cx32 protein sequence with other channel proteins, a modified topological model is proposed.\r\n\r\nThe structure of the gap junction is further studied by atomic force microscopy. Using this new technology, high resolution images of a gap junction in phosphate buffered saline are obtained, and after \"force dissection,\" which removes half the plaque, the extracellular domains of individual connexons in a hexagonal array with lattice constant of 9.1 nm are revealed. These are the first images of an ion channel by atomic force microscopy, and the observations open the door for a variety of new experiments not previously possible.\r\n\r\nLow stringency screening of a rat genomic library produced genomic clones for Cx32 and a new member of the gene family, connexin-31 (Cx31) or [beta]3. Cx31 has a unique distribution and is found in the eye, Harderian gland, skin, and placenta. Comparison of the Cx31 with the other known connexins, reveals unique and conserved domains in the protein sequences. This comparison is extended to a phylogenetic analysis of the entire gene family that shows two major branches of connexins that diverged 1.3-1.9 billion years ago. Comparison with other ion channels reveals a short sequence similarity between the connexins and channels such as the voltage activated K+ channel. In K+ channels the sequence has been shown to line the aqueous pore, and the model for connexin organization is modified to account for this possibility. The similarity also suggests that gap junctions are part of a superfamily of ion channels.", "doi": "10.7907/8n7d-2t98", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:2673", "collection": "thesis", "collection_id": "2673", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06212007-075739", "primary_object_url": { "basename": "Hoh_jh_1991.pdf", "content": "final", "filesize": 16024320, "license": "other", "mime_type": "application/pdf", "url": "/2673/1/Hoh_jh_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Studies on the structure and molecular diversity of the gap junction", "author": [ { "family_name": "Hoh", "given_name": "Jan Hakan", "clpid": "Hoh-Jan-Hakan" } ], "thesis_advisor": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "thesis_committee": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Lipshitz", "given_name": "Howard D.", "orcid": "0000-0002-7372-4419", "clpid": "Lipshitz-H-D" }, { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "An improved method for the isolation of hepatic gap junctions that substantially shortens preparation time and improves the yield of previous methods is described. The topology of the 28 kD protein component (connexin-32, Cx32) of gap junctions isolated with this method is examined using proteases and antibodies against specific peptides. These experiments are consistent with the current model for the organization of the protein in the membrane, but reveal that an unexpectedly large part of the carboxy-terminus is protected from proteolytic attack. Together with data from comparisons of the Cx32 protein sequence with other channel proteins, a modified topological model is proposed.\r\n\r\nThe structure of the gap junction is further studied by atomic force microscopy. Using this new technology, high resolution images of a gap junction in phosphate buffered saline are obtained, and after \"force dissection,\" which removes half the plaque, the extracellular domains of individual connexons in a hexagonal array with lattice constant of 9.1 nm are revealed. These are the first images of an ion channel by atomic force microscopy, and the observations open the door for a variety of new experiments not previously possible.\r\n\r\nLow stringency screening of a rat genomic library produced genomic clones for Cx32 and a new member of the gene family, connexin-31 (Cx31) or [beta]3. Cx31 has a unique distribution and is found in the eye, Harderian gland, skin, and placenta. Comparison of the Cx31 with the other known connexins, reveals unique and conserved domains in the protein sequences. This comparison is extended to a phylogenetic analysis of the entire gene family that shows two major branches of connexins that diverged 1.3-1.9 billion years ago. Comparison with other ion channels reveals a short sequence similarity between the connexins and channels such as the voltage activated K+ channel. In K+ channels the sequence has been shown to line the aqueous pore, and the model for connexin organization is modified to account for this possibility. The similarity also suggests that gap junctions are part of a superfamily of ion channels.", "doi": "10.7907/8n7d-2t98", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:2675", "collection": "thesis", "collection_id": "2675", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06212007-131544", "primary_object_url": { "basename": "Bowman_jl_1991.pdf", "content": "final", "filesize": 73373417, "license": "other", "mime_type": "application/pdf", "url": "/2675/1/Bowman_jl_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Molecular genetics of flower development in Arabidopsis thaliana", "author": [ { "family_name": "Bowman", "given_name": "John L.", "clpid": "Bowman-J-L" } ], "thesis_advisor": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" } ], "thesis_committee": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Flowers of Arabidopsis thaliana consist of a precise pattern of organs arranged in four concentric whorls, with each whorl containing a different type of floral organ in characteristic positions and numbers. Arabidopsis flowers begin their development as small outgrowths of cells on the flank of the inflorescence meristem. Cells within each flower primordium must somehow assess their position relative to others and subsequently differentiate accordingly. Homeotic mutations at four loci (AGAMOUS, APETALA2, APETALA3, PISTILLATA) identified in Arabidopsis appear to cause cells in two adjacent whorls of the developing flower primordium to misinterpret their position and differentiate inappropriately. The development of wild-type flowers and that of several alleles of each locus is analyzed as well as the development of double and triple mutant combinations between the homeotic mutations. Based on this genetic data, a model is proposed for how this limited number of floral homeotic genes, by being expressed in overlapping fields of cells in the meristem of the developing flower primordium, and acting alone and in combination, could specify the identity of each of the whorls. AGAMOUS and APETALA2 are proposed to negatively regulate each other's activity with the result that they are expressed in mutually exclusive domains, APETALA2 in the outer two whorls of the flower and AGAMOUS in the inner two whorls. The activities of APETALA3 and PISTILLATA are proposed to be localized to the second and third whorls, with another gene, SUPERMAN, negatively regulating their activities in the fourth whorl. By protein sequence homology to known transcriptions factors, SRF of humans and MCM1 of yeast, AGAMOUS encodes a putative transcription factor. In support of the proposed model, RNA tissue in situ hybridizations to developing flowers show that AGAMOUS RNA is spatially localized to the inner two whorls in developing floral buds. Furthermore, in apetala2 mutant flowers, AGAMOUS RNA is detected in all floral whorls suggesting that APETALA2 negatively regulates AGAMOUS expression in the outer two whorls at the trancriptional level. Expression patterns of AGAMOUS late during flower development suggest that AGAMOUS may also play a role in cell fate specification during cellular differentiation of stamens and carpels.", "doi": "10.7907/VJCE-Z966", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:2675", "collection": "thesis", "collection_id": "2675", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06212007-131544", "primary_object_url": { "basename": "Bowman_jl_1991.pdf", "content": "final", "filesize": 73373417, "license": "other", "mime_type": "application/pdf", "url": "/2675/1/Bowman_jl_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Molecular genetics of flower development in Arabidopsis thaliana", "author": [ { "family_name": "Bowman", "given_name": "John L.", "clpid": "Bowman-J-L" } ], "thesis_advisor": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" } ], "thesis_committee": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Flowers of Arabidopsis thaliana consist of a precise pattern of organs arranged in four concentric whorls, with each whorl containing a different type of floral organ in characteristic positions and numbers. Arabidopsis flowers begin their development as small outgrowths of cells on the flank of the inflorescence meristem. Cells within each flower primordium must somehow assess their position relative to others and subsequently differentiate accordingly. Homeotic mutations at four loci (AGAMOUS, APETALA2, APETALA3, PISTILLATA) identified in Arabidopsis appear to cause cells in two adjacent whorls of the developing flower primordium to misinterpret their position and differentiate inappropriately. The development of wild-type flowers and that of several alleles of each locus is analyzed as well as the development of double and triple mutant combinations between the homeotic mutations. Based on this genetic data, a model is proposed for how this limited number of floral homeotic genes, by being expressed in overlapping fields of cells in the meristem of the developing flower primordium, and acting alone and in combination, could specify the identity of each of the whorls. AGAMOUS and APETALA2 are proposed to negatively regulate each other's activity with the result that they are expressed in mutually exclusive domains, APETALA2 in the outer two whorls of the flower and AGAMOUS in the inner two whorls. The activities of APETALA3 and PISTILLATA are proposed to be localized to the second and third whorls, with another gene, SUPERMAN, negatively regulating their activities in the fourth whorl. By protein sequence homology to known transcriptions factors, SRF of humans and MCM1 of yeast, AGAMOUS encodes a putative transcription factor. In support of the proposed model, RNA tissue in situ hybridizations to developing flowers show that AGAMOUS RNA is spatially localized to the inner two whorls in developing floral buds. Furthermore, in apetala2 mutant flowers, AGAMOUS RNA is detected in all floral whorls suggesting that APETALA2 negatively regulates AGAMOUS expression in the outer two whorls at the trancriptional level. Expression patterns of AGAMOUS late during flower development suggest that AGAMOUS may also play a role in cell fate specification during cellular differentiation of stamens and carpels.", "doi": "10.7907/VJCE-Z966", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:6290", "collection": "thesis", "collection_id": "6290", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04112011-112805564", "type": "thesis", "title": "Genetic, Molecular and Biochemical Studies of Vacuole Biogenesis and Maintenance in the Yeast Saccharomyces cerevisiae", "author": [ { "family_name": "Robinson", "given_name": "Jane Suzanna", "clpid": "Robinson-Jane-Suzanna" } ], "thesis_advisor": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "thesis_committee": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Using a selection for spontaneous mutants that mislocalize a vacuolar CPY-Inv\r\nfusion protein to the cell surface, 505 Saccharomyces cerevisiae mutants with defects in\r\nprotein sorting were identified. Seventeen of these mutants were dominant; the others\r\ndefined 25 new vps (for vacuolar protein sorting) complementation groups. Alleles of\r\neach vps complementation group exhibit defects in the targeting and final processing of\r\nsoluble vacuolar enzymes (CPY, PrA and PrB). Two of the genes, VPS17 and VPS15,\r\nmap to ChXV and ChII, respectively. The vpsll, vps16, vps18 and vps33 mutants\r\nexhibit morphological defects, their cells containing debris of a membranous nature and\r\nhighly abnormal vacuole remnants. Intercrosses with other vacuole function-defective\r\nmutants (vpl, pep, sip and end), revealed genetic overlaps. It is evident that more than\r\n50 gene products are involved in biogenesis and maintenance of the yeast vacuole.\r\nAlleles of 7 of the vps complementation groups are temperature-sensitive for vegetative\r\ncell growth at 37\u00b0C, and this recessive, Ts phenotype cosegregated with the vps defect\r\nin each case. This easily complemented phenotype has facilitated cloning of six of the\r\ngenes.\r\nThe VPS18 gene was chosen for further studies. A plasmid complementing the\r\nTs growth defect of vps18-1 was isolated and shown by integrative mapping to carry\r\nDNA from the VPS18 locus. Yeast strains with a deletion of the entire VPS18 coding\r\nregion (\u0394vps18), are viable and exhibit the same phenotypes as vps18-1. \u0394vps18, \u03b1 strains have smaller \u03b1-factor halos on sst2,a lawns than do VPS18, \u03b1 strains.\r\nImmunoprecipitation of \u03b1-factor indicated that it is secreted from the \u0394vps18 mutant in\r\nprecursor form. Several of the other severely defective vps mutants also show this \u03b1-factor\r\nprocessing defect. DNA sequencing of VPs18 showed an open reading frame\r\nencoding a 918aa protein, hydrophilic in nature. The protein sequence revealed a zinc finger like, cysteine-rich motif in its C-terminal region. A synthetic mutant with a\r\ncysteine to serine alteration has a temperature-conditional CPY sorting defect with very\r\nrapid onset. Therefore, Vps18p may be a zinc-binding protein, directly necessary for\r\ncorrect vacuolar protein sorting and proper functioning of the Golgi compartment that\r\ncontains Kex2p.\r\n", "doi": "10.7907/zfzc-0035", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:6299", "collection": "thesis", "collection_id": "6299", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04112011-142633680", "primary_object_url": { "basename": "Wang_ks_1991.pdf", "content": "final", "filesize": 2963696, "license": "other", "mime_type": "application/pdf", "url": "/6299/1/Wang_ks_1991.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Molecular characterization of a receptor for the Togavirus Sindbis virus", "author": [ { "family_name": "Wang", "given_name": "Kang-Sheng", "clpid": "Wang-Kang-Sheng" } ], "thesis_advisor": [ { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Strauss", "given_name": "Ellen G.", "clpid": "Strauss-E-G" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The first step in any virus entry process is binding to the plasma membrane of the host cell. The nature of this obligatory step depends upon both the viral and cellular components and may be quite diverse among viruses. The entry of Sindbis virus into a host cell is reported to occur via receptor-mediated endocytosis. We have used several approaches to isolate and characterize the receptor(s) for Sindbis virus. In one such approach, we searched for monoclonal antibodies (mAb) that could interfere with Sindbis virus attachment to and infection of baby hamster kidney (BHK) cells. Mice were immunized with multiple injections of whole BHK cells or with BHK cell membranes. Hybridomas were prepared and supernatants from approximately 3600 hybridoma clones were screened by a plaque reduction assay for their ability to interfere with virus infection. One IgM mAb from a mouse immunized with whole BHK cells inhibited Sindbis virus attachment to BHK cells by 80% at 20 \u00b5g/ml, and immunoprecipitated a 68 kDal membrane protein from BHK cells. This mAb also inhibits virus attachment to two other mammalian cell lines tested, Vero cells (monkey) and SW13 cells (human), and also immunoprecipitates a 68 Kd protein from these cells. The mAb does not interfere with virus infection of chicken cells but did immunoprecipitate a 71 kDal protein from chicken cells. This mAb was used to screen 10^6 plaques from a \u03bbgt11 cDNA library from BHK cells, and 15 reactive phages were found. Six of the fifteen were shown by sequence analysis to react with overlapping regions of a protein that was identical in sequence to the mouse high affinity laminin receptor. By rescreening with a probe from one of these reactive phages, other lambda phages containing the remaining regions of the gene were found. The complete sequence of this protein was deduced by sequence analysis of the cDNA clones and was identical to that of the mouse laminin receptor and 99% identical to the human laminin receptor. A full length cDNA clone of the gene was constructed and inserted into a high efficiency expression vector. BHK cell lines stably transfected with vector expressing the plus sense BHK laminin receptor cDNA are 3-5 fold more susceptible to infection by Sindbis virus as measured by plaque assay, and overexpress the receptor protein on their surface as assayed by flow cytometry analysis. Conversely, cell lines transfected with vector expressing antisense laminin receptor cDNA are only about one half as susceptible to infection by Sindbis virus as the nontransformed BHK cells, and expression of laminin receptor on the cell surface is reduced as measured by flow cytometry analysis. In a second study we looked for Sindbis virus receptors on the surface of chicken cells, using specific molecular mimicry to identify receptor molecules. It has been postulated that viral receptors may share structural features (idiotypes) with antibodies directed against the cell attachment protein of virus. Using antiidiotypic antibodies directed against Sindbis-specific neutralization antibodies, we have demonstrated that an antiidiotypic antibody to a neutralizing mAb reactive with the E2 glycoprotein of Sindbis virus specifically interferes with the binding of wild type Sindbis virus to chicken cells. This antiidiotypic antibody also immunoprecipitates a 63 kDal protein from chicken cells and binds to the surface of these cells. This 63 kDal protein is presumably a receptor for Sindbis virus in chicken cells. The relationship between this protein and the laminin receptor used as a Sindbis receptor in mammalian cells remains to be determined. We also wished to determine the domains of the virus envelope proteins that are responsible for attachment to the cell membrane. The Sindbis virus envelope contains two species of integral membrane glycoproteins, El and E2, which assemble into heterodimers. Each spike on the surface of the virion is a trimer of these dimeric units. We attempted to map the neutralization epitopes on the surface of the virus, including epitopes implicated in virus binding to cells by the antiidiotypic antibody results described above. A \u03bbgt11 expression library was constructed containing cDNA inserts 100-300 nucleotides in length obtained by randomly primed synthesis on Sindbis genomic RNA. This library was probed with several neutralizing monoclonal antibodies specific for E2 and one neutralizing antibody specific for El. Four positive clones, all of which contained inserts from the region of the Sindbis genome that encodes amino acids 173 to 220 of glycoprotein E2, were found from the screening with mAb 23. No reactive clones could be identified using any of the other antibodies. We hypothesize that this domain of E2 centered at residue 200 forms part of the virus binding site for attachment to the cell to initiate infection.", "doi": "10.7907/qdr9-p250", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:6299", "collection": "thesis", "collection_id": "6299", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04112011-142633680", "primary_object_url": { "basename": "Wang_ks_1991.pdf", "content": "final", "filesize": 2963696, "license": "other", "mime_type": "application/pdf", "url": "/6299/1/Wang_ks_1991.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Molecular characterization of a receptor for the Togavirus Sindbis virus", "author": [ { "family_name": "Wang", "given_name": "Kang-Sheng", "clpid": "Wang-Kang-Sheng" } ], "thesis_advisor": [ { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Strauss", "given_name": "Ellen G.", "clpid": "Strauss-E-G" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The first step in any virus entry process is binding to the plasma membrane of the host cell. The nature of this obligatory step depends upon both the viral and cellular components and may be quite diverse among viruses. The entry of Sindbis virus into a host cell is reported to occur via receptor-mediated endocytosis. We have used several approaches to isolate and characterize the receptor(s) for Sindbis virus. In one such approach, we searched for monoclonal antibodies (mAb) that could interfere with Sindbis virus attachment to and infection of baby hamster kidney (BHK) cells. Mice were immunized with multiple injections of whole BHK cells or with BHK cell membranes. Hybridomas were prepared and supernatants from approximately 3600 hybridoma clones were screened by a plaque reduction assay for their ability to interfere with virus infection. One IgM mAb from a mouse immunized with whole BHK cells inhibited Sindbis virus attachment to BHK cells by 80% at 20 \u00b5g/ml, and immunoprecipitated a 68 kDal membrane protein from BHK cells. This mAb also inhibits virus attachment to two other mammalian cell lines tested, Vero cells (monkey) and SW13 cells (human), and also immunoprecipitates a 68 Kd protein from these cells. The mAb does not interfere with virus infection of chicken cells but did immunoprecipitate a 71 kDal protein from chicken cells. This mAb was used to screen 10^6 plaques from a \u03bbgt11 cDNA library from BHK cells, and 15 reactive phages were found. Six of the fifteen were shown by sequence analysis to react with overlapping regions of a protein that was identical in sequence to the mouse high affinity laminin receptor. By rescreening with a probe from one of these reactive phages, other lambda phages containing the remaining regions of the gene were found. The complete sequence of this protein was deduced by sequence analysis of the cDNA clones and was identical to that of the mouse laminin receptor and 99% identical to the human laminin receptor. A full length cDNA clone of the gene was constructed and inserted into a high efficiency expression vector. BHK cell lines stably transfected with vector expressing the plus sense BHK laminin receptor cDNA are 3-5 fold more susceptible to infection by Sindbis virus as measured by plaque assay, and overexpress the receptor protein on their surface as assayed by flow cytometry analysis. Conversely, cell lines transfected with vector expressing antisense laminin receptor cDNA are only about one half as susceptible to infection by Sindbis virus as the nontransformed BHK cells, and expression of laminin receptor on the cell surface is reduced as measured by flow cytometry analysis. In a second study we looked for Sindbis virus receptors on the surface of chicken cells, using specific molecular mimicry to identify receptor molecules. It has been postulated that viral receptors may share structural features (idiotypes) with antibodies directed against the cell attachment protein of virus. Using antiidiotypic antibodies directed against Sindbis-specific neutralization antibodies, we have demonstrated that an antiidiotypic antibody to a neutralizing mAb reactive with the E2 glycoprotein of Sindbis virus specifically interferes with the binding of wild type Sindbis virus to chicken cells. This antiidiotypic antibody also immunoprecipitates a 63 kDal protein from chicken cells and binds to the surface of these cells. This 63 kDal protein is presumably a receptor for Sindbis virus in chicken cells. The relationship between this protein and the laminin receptor used as a Sindbis receptor in mammalian cells remains to be determined. We also wished to determine the domains of the virus envelope proteins that are responsible for attachment to the cell membrane. The Sindbis virus envelope contains two species of integral membrane glycoproteins, El and E2, which assemble into heterodimers. Each spike on the surface of the virion is a trimer of these dimeric units. We attempted to map the neutralization epitopes on the surface of the virus, including epitopes implicated in virus binding to cells by the antiidiotypic antibody results described above. A \u03bbgt11 expression library was constructed containing cDNA inserts 100-300 nucleotides in length obtained by randomly primed synthesis on Sindbis genomic RNA. This library was probed with several neutralizing monoclonal antibodies specific for E2 and one neutralizing antibody specific for El. Four positive clones, all of which contained inserts from the region of the Sindbis genome that encodes amino acids 173 to 220 of glycoprotein E2, were found from the screening with mAb 23. No reactive clones could be identified using any of the other antibodies. We hypothesize that this domain of E2 centered at residue 200 forms part of the virus binding site for attachment to the cell to initiate infection.", "doi": "10.7907/qdr9-p250", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:2612", "collection": "thesis", "collection_id": "2612", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06152007-080238", "primary_object_url": { "basename": "Altman_e_1991.pdf", "content": "final", "filesize": 5721050, "license": "other", "mime_type": "application/pdf", "url": "/2612/1/Altman_e_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Characterization of the SecB Protein, a Chaperone that Facilitates Protein Secretion in Escherichia coli", "author": [ { "family_name": "Altman", "given_name": "Elliot Charles", "orcid": "0000-0002-0721-0022", "clpid": "Altman-Elliot-Charles" } ], "thesis_advisor": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "thesis_committee": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Campbell", "given_name": "Judith L.", "orcid": "0000-0001-8291-5551", "clpid": "Campbell-J-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "It has become increasingly clear that in Escherichia coli, most exported proteins are translocated either posttranslationally or late in their synthesis, and that a component of the export apparatus, SecB, facilitates the export of a subset of the secreted proteins by maintaining them in an export-competent, unfolded form. In an effort to understand how SecB functions as an antifolding factor, we mapped and characterized the sites of SecB interaction in the outer membrane protein LamB. We found that the interaction of SecB with LamB was dependent on the LamB signal sequence as well as on a region in the mature LamB protein. The simplest interpretation of these findings is that SecB binds to both the LamB signal sequence and a mature region in LamB, and that this interaction promotes the antifolding activity of SecB.
\r\n\r\nGiven the fact that several heat-shock proteins have also been shown to function as antifolding factors, we wanted to investigate whether heat-shock proteins might act in a manner analogous to SecB in facilitating the export process. We found that induction of the heat-shock response could substitute for SecB function (SecB is not a heat-shock protein), and that a basal level of heat-shock proteins was necessary for the cell to survive in the absence of SecB protein. These results suggested that heat-shock proteins might indeed be involved in the secretory process and function in a manner similar to that of SecB.
\r\n\r\nIn an attempt to identify these proteins, suppressors of a secB null mutation were isolated and characterized. Not unexpectedly, most of these suppressors mapped to the rpoH locus. Since rpoH encodes \u03c3\u00b3\u00b2, the heat-shock transcription factor, it is likely that these suppressors affect the synthesis levels of heat-shock proteins, which can substitute for SecB function. The remaining suppressors did not map to any known heat-shock or export genes, and potentially represent unidentified heat-shock proteins or export factors that act in a manner similar to SecB in facilitating the export process in E. coli.
", "doi": "10.7907/tq2t-3k47", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:2656", "collection": "thesis", "collection_id": "2656", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06202007-083735", "primary_object_url": { "basename": "Herman_pk_1991.pdf", "content": "final", "filesize": 13913514, "license": "other", "mime_type": "application/pdf", "url": "/2656/1/Herman_pk_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Protein Sorting in the Eukaryotic Secretory Pathway: An Essential Role for a Novel Yeast Protein Kinase", "author": [ { "family_name": "Herman", "given_name": "Paul Kenneth", "clpid": "Herman-Paul-Kenneth" } ], "thesis_advisor": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "thesis_committee": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Dunphy", "given_name": "William G.", "orcid": "0000-0001-7598-8939", "clpid": "Dunphy-W-G" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The yeast vps mutants are defective for the intracellular sorting of proteins to the vacuolar compartment. Mutants from two particular vps complementation groups, vpsl5 and vps34, share a common set of phenotypes that suggested that the VPS15 and VPS34 gene products might be functioning at a similar step of the vacuolar protein sorting pathway. vpsl5 and vps34 mutants exhibit specific defects in the sorting of soluble hydrolases to the vacuolar compartment. Whereas soluble hydrolases such as carboxypeptidase Y are almost quantitatively mislocalized to the cell surface, vacuolar membrane proteins appear to be properly localized to the vacuole.\r\n\r\nThe wild-type VPS15 and VPS34 genes were both cloned from yeast genomic DNA libraries by complementation of temperature-sensitive growth defects associated with mutations in these genes. Haploid yeast strains carrying a disruption of either locus were viable but exhibited a severe ts growth defect indicating that both genes are essential for vegetative growth at elevated temperatures. The vps34 null mutant was also found to exhibit a defect in the segregation of the vacuolar compartment upon cell division.\r\n\r\nThe predicted sequence of the VPS15 gene product exhibits significant similarity to the catalytic domains of the serine/threonine family of protein kinases. Point mutations altering specific amino acid residues of Vpsl5p that are highly conserved in all protein kinases result in the biological inactivation of Vpsl5p. The kinase domain mutants exhibit severe vacuolar protein sorting and ts growth defects. In addition, Vpsl5p is phosphorylated in vivo in a reaction that requires a wild-type Vpsl5p kinase domain. Subcellular fractionation experiments indicate that Vpsl5p is peripherally associated with the cytoplasmic face of a late Golgi or vesicle compartment. A vpsl5 mutant that encodes a protein lacking 30 carboxy-terminal amino acids exhibits a severe ts defect in vacuolar protein delivery. At the restrictive temperature, carboxpeptidase Y accumulates in a specific intracellular compartment that may represent a normal transport intermediate between the Golgi and vacuolar compartments. The vacuolar delivery defect in this mutant has an extremely rapid rate of onset suggesting that Vps15p is directly involved in the sorting of soluble proteins to the vacuole. Altogether, these data suggest that Vpsl5p regulates specific protein phosphorylation reactions in vivo that are required for the delivery of soluble hydrolases to the vacuole.", "doi": "10.7907/38ba-0w26", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:3266", "collection": "thesis", "collection_id": "3266", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08292007-085427", "primary_object_url": { "basename": "Topol_j_1990.pdf", "content": "final", "filesize": 11212571, "license": "other", "mime_type": "application/pdf", "url": "/3266/1/Topol_j_1990.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Transcriptional control of the drosophila segmentation gene fushi tarazu", "author": [ { "family_name": "Topol", "given_name": "Joanne", "clpid": "Topol-J" } ], "thesis_advisor": [ { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" } ], "thesis_committee": [ { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The Drosophila segmentation gene fushi tarazu (ftz) is expressed in a characteristic pattern of seven stripes during early embryogenesis. The promoter sequences sufficient to direct this stripe pattern are located within the 670 base pairs (bp) proximal to the ftz transcriptional start site. When we extract nuclear proteins from 0-12 hour Drosophila embryos, we find sequence-specific DNA binding proteins that recognize multiple sites within the 670 by zebra stripe promoter. This observation suggests that the control of ftz zebra stripe expression may require a complex array of transcriptional regulators. The results of our P element-mediated transformation experiments using ftz promoter/ [beta]-galactosidase fusion genes confirm this notion. They demonstrate that the zebra stripe promoter contains multiple, distinct activator and repressor recognition elements responsible for the formation of ftz stripes. The transformation studies also reveal that a pattern of general activation, that is, a continuous band of gene expression throughout the germ band, can be generated when repressor recognition sites are deleted from the fusion gene promoter and activator sites are retained. This result strongly supports a mechanism for ftz stripe formation involving general transcriptional activation and localized repression.\n\nStudies with constructs in which individual protein binding sites have been deleted or added to the ftz promoter correlate protein recognition elements with regulatory functions. We characterized two distinct interband repressor sites and two distinct general activator sites with this approach. One activator site recognizes the product of the homeobox gene caudal (cad); the other contains a DNA sequence motif found in the cis-activators of a number of Drosophila genes. As would be expected for general activators, both these sites are able to mediate expression throughout most of the germ band. We also demonstrate that when multiple copies of two distinct repressor recognition sites are independently ligated to a portion of the ftz promoter, they transform the continuous band of gene expression generated by a group of endogenous cis-activators into the characteristic seven stripe pattern of ftz expression. Finally, we find that multiple copies of these repressor elements are more capable of mediating repression than single copies.\n", "doi": "10.7907/mvhc-0a34", "publication_date": "1990", "thesis_type": "phd", "thesis_year": "1990" }, { "id": "thesis:2519", "collection": "thesis", "collection_id": "2519", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06082007-084319", "primary_object_url": { "basename": "Hardy_wr_1990.pdf", "content": "final", "filesize": 13015288, "license": "other", "mime_type": "application/pdf", "url": "/2519/1/Hardy_wr_1990.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The characterization and processing of the nonstructural proteins of Sindbis virus", "author": [ { "family_name": "Hardy", "given_name": "Winters Reef", "clpid": "Hardy-W-R" } ], "thesis_advisor": [ { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "thesis_committee": [ { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\n\nSHORT ABSTRACT:\n\nThe nonstructural proteins of Sindbis virus, the type alphavirus of the family Togaviridae, are produced by proteolytic cleavage of two polyprotein precursors. One precursor (P123) is 1,896 amino acids in length and contains the sequences of nsP1, nsP2, and nsP3, while the second (P1234) of 2,513 amino acids is produced by readthrough of an opal termination codon and contains a fourth nonstructural protein, nsP4.\n\nIn order to study the kinetics of processing of these polyproteins, monospecific antisera were produced in rabbits to fusion protein antigens containing the N-terminal two-thirds of the trpE protein of E. coli fused to a large part of the sequence within each of the nonstructural proteins of Sindbis virus. Using these antisera the following details of processing were elucidated: i) most nonstructural proteins arise from the processing of the completed precursor P123 and its cleavage product, P12; ii) in the P123 precursor, the primary cleavage occurs in trans, between nsP2 and nsP3 to generate P12 and nsP3 with a half-life of [...]19 min. in vivo, followed by processing of P12 to produce nsP1 and nsP2, either intramolecularly or in trans, at a rate which exceeds that of the first cleavage; iii) processing of the 3-4 site is complex; and iv) nsP3 was found to be phosphorylated during posttranslational modification.\n\nIn vitro, processing could be inhibited with antibodies to nsP2, but not with antisera to the other three nonstructural proteins, suggesting that the nonstructural proteinase is in nsP2. Deletion-mapping studies were performed which further localized the proteinase to a region of approximately 334 amino acids at the C terminus of nsP2. From a comparison of the deduced sequences of several alphaviruses in this part of nsP2 with the sequences of cellular proteinases, a hypothesis was presented that the proteinase is a thiol protease related to papain. Finally, the examination of several temperature-sensitive mutants of Sindbis virus has confirmed the importance of this region as only mutants that possessed a mutation in the C-terminal domain of nsP2 produced aberrant processing patterns at the nonpermissive temperature.\n", "doi": "10.7907/gk9d-5371", "publication_date": "1990", "thesis_type": "phd", "thesis_year": "1990" }, { "id": "thesis:2549", "collection": "thesis", "collection_id": "2549", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06112007-104332", "type": "thesis", "title": "Vacuolar Protein Sorting in Yeast: Characterization of Mutants and Identification of a Protein Required for Vacuole Biogenesis", "author": [ { "family_name": "Banta", "given_name": "Lois Margaret", "clpid": "Banta-Lois-Margaret" } ], "thesis_advisor": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "thesis_committee": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The lysosome-like vacuole of the yeast Saccharomyces cerevisiae is an acidic compartment containing a number of hydrolytic glycoproteins including carboxypeptidase Y (CPY), proteinase A (PrA) and proteinase B (PrB). A gene fusion-based selection scheme was utilized to isolate ~600 mutants defective in the localization and processing of vacuolar proteins. These vacuolar protein sorting (vps) mutants define >33 complementation groups and exhibit hybrid protein-independent defects in the sorting of CPY, PrA, and PrB. Light and electron microscopic analyses of the vacuole morphology revealed three distinct classes of vps mutants. The class A mutants (26 complementation groups) contain 1-3 large vacuoles that resemble those of the parental strain. One class A mutant is sensitive to low pH and exhibits a defect in vacuole acidification. Consistent with a role for vacuolar pH in protein sorting, perturbation of vacuole acidification resulted in the missorting and secretion of CPY and PrA in wild-type cells. Mutants in the three class B complementation groups exhibit a fragmented vacuole morphology. The class C vps mutants (four complementation groups) lack any compartment resembling a wild-type vacuole, but accumulate vesicles and other membranous structures. Many class C strains exhibit genetically linked defects including temperature-sensitivity and sensitivity to osmotic stress. Unlike other vps mutants, these mutants secrete up to 50% of a vacuolar membrane marker enzyme. The gene defined by one class C mutant, vps33, has been cloned. The predicted VPS33 gene product is hydrophilic and shares sequence similarity with a family of ATP-binding proteins. Disruption of VPS33 is not lethal but results in temperature-sensitive growth. Vps33p-specific antisera recognize a cytosolic protein of ~75 kD. One temperature-sensitive vps33 mutant carrying a missense mutation contains apparently normal vacuoles at the permissive temperature, but lacks vacuoles specifically in the bud at the nonpermissive temperature. We propose that the abnormalities in vacuole morphology and inheritance in vps33 mutants are a consequence of a primary defect in Golgi-to-vacuole protein delivery. A second VPS gene, VPS28, has also ben cloned. Our data suggest that the VPS28 gene product only indirectly affects vacuole protein sorting, but may function in a late protein modification process.
", "doi": "10.7907/TZHF-3J73", "publication_date": "1990", "thesis_type": "phd", "thesis_year": "1990" }, { "id": "thesis:2500", "collection": "thesis", "collection_id": "2500", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06072007-082551", "type": "thesis", "title": "Analysis of the Structure, Expression and Evolution of the Shark Myelin Proteins and Genes", "author": [ { "family_name": "Fors", "given_name": "Lance", "clpid": "Fors-Lance" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Myelin is a compacted multilamellar membrane which encases axons to provide electrical insulation and facilitates the rapid transmission of nerve impulses. The major myelin structural proteins produced by oligodendrocyctes in the central nervous system (CNS) of mammals are proteolipid protein (PLP) and myelin basic protein (MBP). In contrast, the major myelin structural proteins produced by the Schwann cells in the peripheral nervous system (PNS) of mammals are protein zero (Po) and MBP. Sharks (class Chondrichthyes) are the oldest living vertebrates that have a concentric multilamellar \"mammalian-like\" myelin structure around axons. In addition to this structural similarity, the shark and mammalian myelin proteins appeared to be distantly related biochemically and immunologically even though they diverged from each other about 400 million years ago. Logically those regions in the shared proteins, genes and promoters which are most similar between sharks and mammals are likely to be functionally important to both. Therefore by analyzing these elements in shark myelin and comparing them to what is already known about mammalian myelin we could learn about shark myelin, its evolution and what regions are essential for the proper function and expression of mammalian myelin. This thesis contains an analysis of the structure, expression and evolution of the shark myelin proteins and genes.
\r\n\r\nThe first chapter (Saavedra, R., Fors, L., Aebersold, R., Arden, B., Horvath, S., Sanders, J., and Hood, L. J. Mol. Evol. 29:149) describes the isolation and sequencing of the two major shark CNS proteins Po and MBP and their corresponding cDNAs. This study shows that the myelin proteins of the shark brain are similar to the myelin proteins of the mammalian peripheral nervous system in both primary and secondary structures.
\r\n\r\nThe second chapter (Fors, L., Saavedra, R., and Hood, L. Nuc. Acids Res., Submitted) contains a novel genomic walking technique that was developed to clone the shark Po and MBP promoters. Using this technique it was possible to clone approximately 400 nucleotides immediately upstream of the shark Po and MBP transcription initiation sites. This genomic walking technique will be generally useful for cloning promoters or other sequences of interest without the need for constructing or screening genomic libraries.
\r\n\r\nThe third chapter presents and discusses the similarity between these shark Po and MBP promoters, the JC virus enhancer (which directs tissue-specific expression in oligodendrocytes), and the mouse Po and MBP promoters. The implications of these findings on nervous system specific and CNS vs. PNS specific gene expression are discussed.
\r\n\r\nLastly, the appendix describes the current status of Shiverer transgenic mouse experiments in which constructs bearing the shark MBP gene are injected into mouse eggs. These transgenic experiments are testing if the structural similarity between shark and mammalian MBPs translates into any measurable functional similarity in vivo.
", "doi": "10.7907/840h-0p67", "publication_date": "1990", "thesis_type": "phd", "thesis_year": "1990" }, { "id": "thesis:2500", "collection": "thesis", "collection_id": "2500", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06072007-082551", "type": "thesis", "title": "Analysis of the Structure, Expression and Evolution of the Shark Myelin Proteins and Genes", "author": [ { "family_name": "Fors", "given_name": "Lance", "clpid": "Fors-Lance" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Myelin is a compacted multilamellar membrane which encases axons to provide electrical insulation and facilitates the rapid transmission of nerve impulses. The major myelin structural proteins produced by oligodendrocyctes in the central nervous system (CNS) of mammals are proteolipid protein (PLP) and myelin basic protein (MBP). In contrast, the major myelin structural proteins produced by the Schwann cells in the peripheral nervous system (PNS) of mammals are protein zero (Po) and MBP. Sharks (class Chondrichthyes) are the oldest living vertebrates that have a concentric multilamellar \"mammalian-like\" myelin structure around axons. In addition to this structural similarity, the shark and mammalian myelin proteins appeared to be distantly related biochemically and immunologically even though they diverged from each other about 400 million years ago. Logically those regions in the shared proteins, genes and promoters which are most similar between sharks and mammals are likely to be functionally important to both. Therefore by analyzing these elements in shark myelin and comparing them to what is already known about mammalian myelin we could learn about shark myelin, its evolution and what regions are essential for the proper function and expression of mammalian myelin. This thesis contains an analysis of the structure, expression and evolution of the shark myelin proteins and genes.
\r\n\r\nThe first chapter (Saavedra, R., Fors, L., Aebersold, R., Arden, B., Horvath, S., Sanders, J., and Hood, L. J. Mol. Evol. 29:149) describes the isolation and sequencing of the two major shark CNS proteins Po and MBP and their corresponding cDNAs. This study shows that the myelin proteins of the shark brain are similar to the myelin proteins of the mammalian peripheral nervous system in both primary and secondary structures.
\r\n\r\nThe second chapter (Fors, L., Saavedra, R., and Hood, L. Nuc. Acids Res., Submitted) contains a novel genomic walking technique that was developed to clone the shark Po and MBP promoters. Using this technique it was possible to clone approximately 400 nucleotides immediately upstream of the shark Po and MBP transcription initiation sites. This genomic walking technique will be generally useful for cloning promoters or other sequences of interest without the need for constructing or screening genomic libraries.
\r\n\r\nThe third chapter presents and discusses the similarity between these shark Po and MBP promoters, the JC virus enhancer (which directs tissue-specific expression in oligodendrocytes), and the mouse Po and MBP promoters. The implications of these findings on nervous system specific and CNS vs. PNS specific gene expression are discussed.
\r\n\r\nLastly, the appendix describes the current status of Shiverer transgenic mouse experiments in which constructs bearing the shark MBP gene are injected into mouse eggs. These transgenic experiments are testing if the structural similarity between shark and mammalian MBPs translates into any measurable functional similarity in vivo.
", "doi": "10.7907/840h-0p67", "publication_date": "1990", "thesis_type": "phd", "thesis_year": "1990" }, { "id": "thesis:713", "collection": "thesis", "collection_id": "713", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-02222007-113422", "type": "thesis", "title": "Kinetics of RNA Polymerase \u03b2 Subunit Synthesis and Acid End Product Transport in Escherichia coli", "author": [ { "family_name": "Axe", "given_name": "Douglas D.", "clpid": "Axe-Douglas-D" } ], "thesis_advisor": [ { "family_name": "Bailey", "given_name": "James E.", "clpid": "Bailey-J-E" } ], "thesis_committee": [ { "family_name": "Bailey", "given_name": "James E.", "clpid": "Bailey-J-E" }, { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "An approach to modeling the regulation of synthesis of crucial bacterial proteins has been developed. The unique features of this approach are that it focuses on maintenance of a steady state rather than on transitions between steady states, and that stochastic fluctuations in the number of transcripts per cell are treated as the perturbation. It has been used to investigate various models of translational regulation of RNA polymerase \u03b2 subunit synthesis. The simplest autogenous regulatory mechanism, binding of a single RNA polymerase molecule to the rpoBC mRNA, appears to provide inadequate control. A more sophisticated mechanism, sequential binding of multiple polymerase molecules in a cooperative manner, was shown to dramatically improve the control characteristics.
\r\n\r\nThe interaction between RNA polymerase and rpoBC mRNA was examined experimentally. RNA polymerase was incubated with RNA that is identical to a small portion of the native rpoBC message. Gel mobility-shift assays were performed to detect complexes. The relative amount of RNA in different complexes was determined by radiolabeling the transcript. Data obtained in this way indicate that cooperative binding is occuring.
\r\n\r\n\u00b3\u00b9P NMR studies of intact E. coli cells suggested a difference in membrane function between a plasmid-containing strain and the plasmid-free host. Similar \u00b3\u00b9P NMR experiments were complemented with \u00b9\u00b3C NMR experiments to examine the transport of lactate and acetate through the cytoplasmic membrane during anaerobic glycolysis. Methods were developed to measure cytoplasmic and extracytoplasmic solution volumes and intra- and extracellular acid concentrations using \u00b9\u00b3C NMR. The results demonstrated significant differences in the transport of the two acids. Acetate was determined to permeate the membrane at comparable rates in the dissociated and undissociated forms. The mode of lactate transport in cells that are actively glycolyzing was found to be different from that of cells that have exhausted their supply of glucose. Lactate thus appears to be transported by a system that is sensitive to some indicator of glycolytic activity. It also appears to diffuse across the membrane in both forms.
\r\n\r\nA kinetic approach was used to deduce constraints on the unidirectional fluxes for a general protein-mediated, ATP-independent transport process. The conclusion was that under certain circumstances, the Ussing-Teorell flux ratio equation applies to protein-mediated transport. From the analysis, an expression was derived for the driving force of such a transport process, and the relationship between this and the net flux is discussed.
", "doi": "10.7907/ejy5-sm51", "publication_date": "1990", "thesis_type": "phd", "thesis_year": "1990" }, { "id": "thesis:503", "collection": "thesis", "collection_id": "503", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-02052007-153354", "type": "thesis", "title": "Dissimilative Fe(III) Reduction by Alteromonas putrefaciens Strain 200", "author": [ { "family_name": "DiChristina", "given_name": "Thomas J.", "clpid": "DiChristina-Thomas-J" } ], "thesis_advisor": [ { "family_name": "Lidstrom", "given_name": "Mary E.", "clpid": "Lidstrom-M-E" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Lidstrom", "given_name": "Mary E.", "clpid": "Lidstrom-M-E" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" }, { "family_name": "Morgan", "given_name": "James J.", "clpid": "Morgan-J-J" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" }, { "family_name": "Gunsalus", "given_name": "Robert P.", "clpid": "Gunsalus-Robert-P" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Complementary genetic and biochemical analyses have been used to study the (dissimilative) iron reduction system of Alteromonas putrefaciens strain 200. Preliminary kinetic data suggested that A. putrefaciens 200 possessed a ferri-reductase that was physiologically distinct from either cytochrome oxidase or nitrate reductase. A suite of iron-reduction-deficient mutants was generated via chemical (EMS) and transposon (Tn5) mutagenesis procedures. A newly developed screening technique was subsequently used to identify mutants deficient in both high-rate and low-rate iron reduction activity. A conjugal gene transfer system was developed for mobilization of IncP1-based cloning vectors to A. putrefaciens 200. The broad host range (IncPl) cosmid cloning vector pVK100 was used to construct A. putrefaciens 200 gene clone banks in E. coli strains HB101 and S17-1 (mobilizing strain). Both three-way and two-way mating (conjugation) procedures were used to mobilize the gene clone banks into the suite of iron-reduction-deficient mutants during genetic (complementation) analysis. Two iron reduction clones (designated S4-E-2 and S18-F-4) were identified by their ability to restore iron reduction activity to several of the iron-reduction-deficient mutants. Preliminary biochemical characterization of selected mutant strains has indicated that cytochrome content may play an important role in the iron reduction process. Based on the results of the complementary genetic and biochemical studies, the iron reduction mutants have been placed into four classes: Class I (deficient in both high-rate and low-rate iron reduction activity, complemented by clones S4-E-2 and S18-F-4, possible b- or c-type cytochrome mutants), Class II (deficient in both high-rate and low-rate iron reduction activity, complemented by clone S4-E-2 but not by S18-F-4), Class III (proficient in high-rate iron reduction activity, but deficient in low-rate iron reduction activity, not complemented by either clone S4-E-2 or S18-F-4), and Class IV (deficient in both high-rate and low-rate iron reduction activity, but not complemented by either clone S4-E-2 or S18-F-4, possible d-type cytochrome or anaerobic regulatory mutant).
", "doi": "10.7907/temn-0p39", "publication_date": "1989", "thesis_type": "phd", "thesis_year": "1989" }, { "id": "thesis:2497", "collection": "thesis", "collection_id": "2497", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06072007-075259", "type": "thesis", "title": "Functional Analysis of Viral Nonstructural and Structural Proteins", "author": [ { "family_name": "Hahn", "given_name": "Young Shin Lim", "clpid": "Hahn-Young-Shin-Lim" } ], "thesis_advisor": [ { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" } ], "thesis_committee": [ { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The genera Alphavirus and Flavivirus contain enveloped RNA viruses which are similar in size, morphology, and RNA content, and were once considered to belong to the same family, the Togaviridae. Recently the flaviviruses were reclassified as a separate family, the Flaviviridae, because they differ markedly from alphaviruses in replication strategy and mode of assembly as well as genome organization. In this thesis, representatives of both groups have been studied in order to understand the functions of the various virus-encoded proteins in replication and pathogenesis. Part I describes the mapping of temperature-sensitive (ts) RNA- mutants of the alphavirus Sindbis virus, to elucidate the function of each nonstructural protein during RNA replication. Part II includes the determination of the complete nucleotide sequence of the flavivirus dengue 2, comparative analysis of conserved elements in the 3' untranslated region of various flaviviruses, and expression of dengue 2 structural proteins in a recombinant vaccinia virus.
\r\n\r\nDuring alphavirus replication, the parental 49S plus-strand RNA is transcribed into a complementary minus-strand RNA which serves as the template for the synthesis of both plus-strand 49S genomic RNA and 26S subgenomic RNA. The nonstructural proteins, which are involved in viral RNA replication, are translated from the genomic 49S RNA as two polyprotein precursors that are processed by cotranslational or posttranslational cleavage into four final polypeptide products.
\r\n\r\nts RNA- mutants of Sindbis virus have been isolated previously and grouped by complementation into four groups (A, B, F, G); these mutants fail to make RNA after infection at a nonpermissive temperature and were presumed to contain ts lesions in the nonstructural proteins. Over a number of years, work in our own and other laboratories has established the details of the RNA- phenotypes of these mutants, which include defects in RNA chain elongation, in initiation of genomic and 26S RNA synthesis, in regulation of minus-strand template synthesis, and in processing of polyprotein precursors. However, it is only with the mapping described here that it has been possible to unambiguously assign these functions to particular nonstructural polypeptides. The mutations responsible for the is phenotype of Sindbis complementation group F mutants ts6, tsl10, and tsl18, have been determined. ts6 and ts110 have a single base substitution in nsP4 resulting in a replacement of Gly by Glu at position 153 or position 324, respectively. It is of interest that nsP4 contains the Gly-Asp-Asp motif characteristic of a number of viral replicases, and this together with the fact that all RNA synthesis in ts6-infected cells is shut off upon shift-up from the permissive to the nonpermissive temperature suggests that nsP4 is the viral RNA polymerase or elongation enzyme. tsl18 is a double mutant where one mutation is in nsP2 (the Val at residue 425 is changed to Ala). This mutation alone causes the formation of minute plaques at the nonpermissive condition without a reduction in the plaque number. The second change (Gln-93 of nsP4 changed to Arg) has little apparent phenotype on its own, but in combination with the change in nsP2 it leads to a temperature-sensitive phenotype. This suggests that nsP2 and nsP4 interact with one another in a complex.
\r\n\r\nWe also have mapped representatives of RNA- complementation groups A, B, and G. Mutants belonging to groups A and G have been found to map in nsP2, suggesting that this protein is required for initiation of 26S RNA synthesis, proteolytic processing of the nonstructural precursor, and shut off of minus-strand synthesis. tsl1, the only member of group B, has a mutation in nsP1, indicating that this protein is responsible for initiation of minus-strand synthesis.
\r\n\r\nTo understand the role of viral structural proteins in the pathogenesis of flaviviruses, we studied one of the dengue viruses, which constitute a worldwide health problem of increasing dimensions, and determined the complete nucleotide sequence of the PRl59-S1 strain of dengue 2 virus except for l5 nucleotides at the 5' end. There is one long open reading frame which is translated to give the structural proteins, capsid (C), membrane-like protein (M), and envelope protein (E), followed by nonstructural proteins, NS1, NS2, NS3, NS4, and NS5. The individual proteins appear to be produced by posttranslational cleavage of a precursor polyprotein. There are nucleotide sequences in the 5' terminal region (in the coding region for the capsid protein) and in the 3' terminal region (in the 3' untranslated sequence) that are invariant among flaviviruses examined to date and that may be involved in cyclization of the RNA. These sequences are presumed to be important for viral replication. In addition, the 3' terminal 79 nucleotides are capable of forming a hairpin structure.
\r\n\t\r\nWe have expressed the structural proteins of dengue 2 using a recombinant vaccinia virus to study the role of these proteins in the immunological response. The vaccinia recombinant containing a cDNA copy of the 5' region of the dengue genome virus expressed dengue structural proteins which are correctly cleaved and modified. This suggests that the sequences encoding the structural proteins specify all the necessary catalytic activities or recognition signals required to ensure the proper synthesis and maturation of the polypeptides. And also, this recombinant can generate dengue-specific antibodies in mice which neutralize viral infectivity.
", "doi": "10.7907/3trk-k698", "publication_date": "1989", "thesis_type": "phd", "thesis_year": "1989" }, { "id": "thesis:7997", "collection": "thesis", "collection_id": "7997", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10172013-115606044", "primary_object_url": { "basename": "Vijayraghavan 1989.pdf", "content": "final", "filesize": 28447380, "license": "other", "mime_type": "application/pdf", "url": "/7997/1/Vijayraghavan 1989.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "A Genetic and Biochemical Analysis of pre-mRNA Splicing in Saccharomyces cerevisiae", "author": [ { "family_name": "Vijayraghavan", "given_name": "Usha", "clpid": "Vijayraghavan-Usha" } ], "thesis_advisor": [ { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" } ], "thesis_committee": [ { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Pre-mRNA splicing requires interaction of cis- acting intron sequences with trans -acting factors: proteins and small nuclear ribonucleoproteins (snRNPs). The assembly of these factors into a large complex, the spliceosome, is essential for the subsequent two step splicing reaction. First, the 5' splice site is cleaved and free exon 1 and a lariat intermediate (intron- exon2) form. In the second reaction the 3' splice site is cleaved the exons ligated and lariat intron released. A combination of genetic and biochemical techniques have been used here to study pre-mRNA splicing in yeast.
\r\n\r\nYeast introns have three highly conserved elements. We made point mutations within these elements and found that most of them affect splicing efficiency in vivo and in vitro, usually by inhibiting spliceosome assembly.
\r\n\r\nTo study trans -acting splicing factors we generated and screened a bank of temperature-sensitive (ts) mutants. Eleven new complementation groups (prp17 to prp27) were isolated. The four phenotypic classes obtained affect different steps in splicing and accumulate either: 1) pre-mRNA, 2) lariat intermediate, 3) excised intron or 4) both pre-mRNA and intron. The latter three classes represent novel phenotypes. The excised intron observed in one mutant: prp26 is stabilized due to protection in a snRNP containing particle. Extracts from another mutant: prpl8 are heat labile and accumulate lariat intermediate and exon 1. This is especially interesting as it allows analysis of the second splicing reaction. In vitro complementation of inactivated prp18 extracts does not require intact snRNPs. These studies have also shown the mutation to be in a previously unknown splicing protein. A specific requirement for A TP is also observed for the second step of splicing. The PRP18 gene has been cloned and its polyadenylated transcript identified.
", "doi": "10.7907/cxmk-nj42", "publication_date": "1989", "thesis_type": "phd", "thesis_year": "1989" }, { "id": "thesis:7936", "collection": "thesis", "collection_id": "7936", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08232013-141734040", "primary_object_url": { "basename": "Sweder 1989.pdf", "content": "final", "filesize": 26958277, "license": "other", "mime_type": "application/pdf", "url": "/7936/1/Sweder 1989.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Isolation and Characterization of Proteins that Bind to Yeast Origins of DNA Replication", "author": [ { "family_name": "Sweder", "given_name": "Kevin Scot", "clpid": "Sweder-Kevin-Scot" } ], "thesis_advisor": [ { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" } ], "thesis_committee": [ { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Yeast chromosomes contain sequences called ARSs which function as origins of replication in vitro and in vivo. We have carried out a systematic deletion analysis of ARS1, allowing us to define three functionally distinct domains, designated A, B, and C. Domain A is a sequence of 11 to 19bp, containing the core consensus element that is required for replication. The core consensus sequence, A/TTTTATPuTTTA/T, is conserved at all ARSs sequenced to date. A fragment containing only element A and 8 flanking nucleotides enables autonomous replication of centromeric plasmids. These plasmids replicate very inefficiently, suggesting that flanking sequences must be important for ARS function. Domain B also provides important sequences needed for efficient replication. Deletion of domain B drastically increases the doubling times of transformants and reduces plasmid stability. Domain B contains a potential consensus sequence conserved at some ARSs which overlaps a region of bent DNA. Mutational analysis suggests this bent DNA may be important for ARS function. Deletion of domain C has only a slight effect on replication of plasmids carrying those deletions.
\r\n\r\nWe have identified a protein called ARS binding factor I (ABF-I) that binds to the HMR-E ARS and ARS1. We have purified this protein to homogeneity using conventional and oligonucleotide affinity chromatography. The protein has an apparent molecular weight of 135kDa and is present at about 700 molecules per diploid cell, based on the yield of purified protein and in situ antibody staining. DNaseI footprinting reveals that ABF-I binds sequence-specifically to an approximately 24bp sequence that overlaps element Bat ARS1. This same protein binds to and protects a similar size region at the HMR-E ARS.
\r\n\r\nWe also find evidence for another ARS binding protein, ABF-III, based on DN asei footprint analysis and gel retardation assays. The protein protects approximately 22bp adjacent to the ABF-I site. There appears to be no interaction between ABF-I and ABF-III despite the proximity of their binding sites.
\r\n\r\nTo address the function of ABF-I in DNA replication, we have cloned the ABF-I gene using rabbit polyclonal anti-sera and murine monoclonal antibodies against ABF-I to screen a \u03bbgt11 expression library. Four EcoRI restriction fragments were isolated which encoded proteins that were recognized by both polyclonal and monoclonal antibodies. A gene disruption can now be constructed to determine the in vivo function of ABF-I.
", "doi": "10.7907/d0tj-k332", "publication_date": "1989", "thesis_type": "phd", "thesis_year": "1989" }, { "id": "thesis:2312", "collection": "thesis", "collection_id": "2312", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05302007-153631", "type": "thesis", "title": "Characterization of the SEC18 Gene of S. cerevisiae: Identification of a Protein Involved in Yeast Secretion", "author": [ { "family_name": "Eakle", "given_name": "Kurt Andrew", "clpid": "Eakle-Kurt-Andrew" } ], "thesis_advisor": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "thesis_committee": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum and the Golgi complex. We have cloned the SEC18 gene by complementation of the secl8-1 mutation. Deletion/disruption of this gene has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2271 by open reading frame which would code for a protein of 83.9 kd. The predicted protein sequence showed no significant homology to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kd. Antisera raised against a Sec18-?-galactosidase fusion protein, detects two proteins from in vivo 35S labeled yeast cells identical in size to those seen by in vitro translation. Although potential sites for N-linked glycosylation are present in the Sec 18p sequence, the sizes of the in vivo SEC18 gene products are unaffected by the drug tunicamycin. Hydrophobicity analysis indicated that the protein is hydrophilic in nature and lacks any region that would be predicted to serve as a signal sequence or transmembrane anchor. These results suggest that the Secl8p resides in the cell cytoplasm. Pulse-chase experiments indicate that the two forms of Sec 18 protein are not the result of post-translational processing. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec 18 protein do not arise from mRNAs with different 5' ends. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of the Sec 18 protein. While cell fractionation studies show that the Sec 18p are not associated with ER or Golgi compartments, association with a 100,000 x g pellet fraction has been observed suggesting that Sec 18p may bind transiently to small vesicles such as those presumed to participate in ER to Golgi transport.
", "doi": "10.7907/6aqb-xd83", "publication_date": "1989", "thesis_type": "phd", "thesis_year": "1989" }, { "id": "thesis:7826", "collection": "thesis", "collection_id": "7826", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06032013-145407392", "primary_object_url": { "basename": "Normanly_j_1989.pdf", "content": "final", "filesize": 31581280, "license": "other", "mime_type": "application/pdf", "url": "/7826/1/Normanly_j_1989.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "An in vivo Approach to tRNA Identity", "author": [ { "family_name": "Normanly", "given_name": "Jennifer", "clpid": "Normanly-Jennifer" } ], "thesis_advisor": [ { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" } ], "thesis_committee": [ { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A leucine-inserting tRNA has been transformed into a serine-inserting tRNA by changing 12 nucleotides. Only 8 of the 12 changes are required to effect the conversion of the leucine tRNA to serine tRNA identity. The 8 essential changes reside in basepair 11-24 in the D stem, basepairs 3-70, 2-71 and nucleotides 72 and 73, all of the acceptor stem.
\r\n\r\nFunctional amber suppressor tRNA genes were generated for 14 species of tRNA in E. coli, and their amino acid specificities determined. The suppressors can be classified into three groups, based upon their specificities. Class I suppressors, tRNAAla2CUA, tRNAGlyUCUA, tRNAHisACUA, tRNALysCUA, and tRNAProHCUA, inserted the predicted amino acid. The Class II suppressors, tRNAGluACUA, tRNAGlyTCUA, and tRNAIle1CUA were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase (AAS). The Class III suppressors, tRNAArgCUA, tRNAAspMCUA, tRNAIle2CUA, tRNAThr2CUA, tRNAMet(m)CUA and tRNAValCUA inserted predominantly lysine.
", "doi": "10.7907/s9xt-6f20", "publication_date": "1989", "thesis_type": "phd", "thesis_year": "1989" }, { "id": "thesis:7481", "collection": "thesis", "collection_id": "7481", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02152013-142530976", "primary_object_url": { "basename": "Martin 1988.pdf", "content": "final", "filesize": 20542579, "license": "other", "mime_type": "application/pdf", "url": "/7481/1/Martin 1988.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Evolution and Expression at the 68C Glue Gene Cluster of Drosophila", "author": [ { "family_name": "Martin", "given_name": "Christopher Hayes", "clpid": "Martin-Christopher-Hayes" } ], "thesis_advisor": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" } ], "thesis_committee": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "This thesis describes investigations on the evolution of a region containing a cluster of three glue genes located at chromosomal site 68C in the Drosophila melanogaster genome. These studies have used a set of five closely related Drosophila species, all members of the melanogaster species subgroup. The first chapter serves as an introduction and summarizes this work. The second chapter describes the initial characterization of the glue gene clusters and the surrounding regions in the five Drosophila species. The third chapter describes the characterization at the sequence level of the boundary that was found between adjacent blocks of rapidly and slowly evolving sequences located at the 68C glue gene cluster. The fourth chapter describes the evolution of the largest of the three glue genes in the 68C glue gene cluster: Sgs-3. Together, these studies reveal that this region of the genome is evolving as a mosaic, with adjacent regions evolving at different rates and in very different ways.
", "doi": "10.7907/p2n6-q185", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:999", "collection": "thesis", "collection_id": "999", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03182008-132957", "primary_object_url": { "basename": "Arnold_rg_1987.pdf", "content": "final", "filesize": 15463718, "license": "other", "mime_type": "application/pdf", "url": "/999/1/Arnold_rg_1987.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Dissimilative Fe(III) Reduction by Pseudomonas sp. 200", "author": [ { "family_name": "Arnold", "given_name": "Robert Glenn", "clpid": "Arnold-Robert-Glenn" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "clpid": "Hoffmann-M-R" }, { "family_name": "Morgan", "given_name": "James J.", "clpid": "Morgan-J-J" }, { "family_name": "Bailey", "given_name": "James E.", "clpid": "Bailey-J-E" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" }, { "family_name": "North", "given_name": "Wheeler J.", "clpid": "North-W-J" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The feasibilities of (i) liberating free energy from dissimilative iron reduction and (ii) coupling oxidative phosphorylation to electron transport to Fe(III) are sensitive to the aqueous chemistry of iron. The addition of ligands, such as nitrilotriacetic acid (NTA), to solution significantly impacts both the overall thermodynamics and kinetics of dissimilative iron reduction. The overall free-energy change due to electron transfer from glucose or lactate ion to Fe(III) is negative, but when Fe(III) is presented as an iron oxide there may be insufficient free energy in the transformations to permit coupled ATP generation. A systematic investigation of iron-reduction kinetics as a function of Fe(III) speciation indicated that in Pseudomonas sp. 200 (i) iron-reduction rate was functionally related to the concentrations of individual iron species and (ii) direct contact between Fe(III) and the electron-transport chain (ferrireductase) was required for electron transfer. Iron reduction in the absence of microbial activity was negligible. The addition of equimolar quantities of NTA enormously accelerated the initial rate of microbial iron reduction, and the calculated concentration of Fe(NTA)(OH)22- correlated strongly with measured iron-reduction rates.
\r\n\r\nWhen Fe(III) was provided as an iron oxide, the overall reduction rate was much slower, though still dependent upon the concentration of NTA added to solution. Primary factors controlling mineral dissolution and Fe(III) reduction were mineral surface area (or concentration of high-energy surface sites), ligand concentration, and cell number. Saturation kinetics were evident, as indicated by the following relationship governing reductive dissolution of hematite:
\r\n\r\nd[Fe(II)] / dt = Vmax(I)Km(NTA)Vmax(II)[NTA] / Km(NTA) + [NTA] \u2022 [Fe(III)] / Km(Fe) + [Fe(III)]
\r\n\r\nwhere Vmax(I) = 2.8 x 10-5 M\u2022hr-1
\r\n \r\nVmax(II) = 6.3 x 10-4 M\u2022hr-1
\r\n\r\nKmax(NTA) = 1.2 x 10-3 M
\r\n\r\nKmax(Fe) = 1.0 x 10-1 M (as Fe)
\r\n\r\nNTA = nitrilotriacetic acid
\r\n\r\n[Fe(III)] = volume concentration of hematite (as Fe).
\r\n\r\nExperiments involving oxide/microorganism separation indicated that cell/mineral contact was essential to reductive dissolution of goethite.
\r\n\r\nSpecific respiratory inhibitors were utilized to identify elements of electron transport chains involved in reduction of molecular oxygen and Fe(III) and to compare transport-chain compositions of cells grown under high- versus limited-O2 conditions. Pseudomonas sp. 200 expressed both a constitutive (cytochrome o) and an inducible (cytochrome d) cytochrome oxidase. Induction of the alternate transport pathway resulted from growth at low oxygen tension (<0.01 atm.). Induced cells were capable of O2 utilization at moderately increased rates. Pseudomonas sp. 200 also expressed a constitutive and an inducible ferrireductase. Growth at low oxygen tension resulted in acceleration of the overall rate of dissimilative iron reduction by a factor of 6 to 8, but iron reduction appeared to be uncoupled from oxidative phosphorylation. Maximum rates of electron transfer in induced cells were independent of the identity of the electron acceptor indicating a common rate-limiting step. Dissimilative iron reduction occurred via an abbreviated electron transport chain in both the induced and uninduced cases. Electron-transport-chain compositions for the induced and uninduced cases are postulated.
\r\n", "doi": "10.7907/xsas-dd69", "publication_date": "1987", "thesis_type": "phd", "thesis_year": "1987" }, { "id": "thesis:920", "collection": "thesis", "collection_id": "920", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03102008-130217", "primary_object_url": { "basename": "Peretti_sw_1987.pdf", "content": "final", "filesize": 7336055, "license": "other", "mime_type": "application/pdf", "url": "/920/1/Peretti_sw_1987.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Theoretical Modeling and Experimental Investigation of Host-Plasmid Interactions in Recombinant Escherichia coli", "author": [ { "family_name": "Peretti", "given_name": "Steven William", "clpid": "Peretti-Steven-William" } ], "thesis_advisor": [ { "family_name": "Bailey", "given_name": "James E.", "clpid": "Bailey-J-E" } ], "thesis_committee": [ { "family_name": "Bailey", "given_name": "James E.", "clpid": "Bailey-J-E" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "clpid": "Hoffmann-M-R" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" }, { "family_name": "Bertani", "given_name": "Giuseppe", "clpid": "Bertani-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Microbial metabolism has been mathematically represented with sufficient mechanistic rigor to allow consideration of the ramifications of introducing recombinant vectors on host cell metabolic activity. The model was first verified using details of transcription and translation sufficiently stringent to test the simulation of RNA polymerase equilibrium distribution on promoter regions and the activation of ribosomes by binding of mRNA.
\r\n\r\nPlasmids were added to the formulation, and the dependence of productivity and growth rate was simulated. The results show good agreement with those obtained by other researchers. This model structure was then used to simulate potentially beneficial metabolic engineering scenarios in an attempt to identify those processes limiting productivity for recombinant systems. Simulation results indicate that transcription is the metabolic bottleneck that limits product synthesis. In addition, the most efficient strategy for enhancing plasmid product synthesis was shown to be increasing the efficiency of plasmid mRNA translation.
\r\n\r\nAn asynchronous population was simulated in order to study the transient behavior of the cell model. Based on plasmid-free simulation results, the model accurately reflects the predominant metabolic structure for control of macromolecular synthesis. Transient response considerations for plasmid-containing populations suggest that dynamic reactor operation, in the form of induction of plasmid promoter activity, leads to transient accumulation of product which exceeds that attained during the subsequent balanced growth.
\r\n\r\nFinally, the effect of different plasmid copy numbers and of the concommitant expression of a constitutive plasmid-borne gene were investigated experimentally. Radio-labeling techniques combined with filter hybridizations were used to study the transcription rate from the \u03b2-lactamase promoter as well as the level of corresponding mRNA present in the cell. Coupled with product activity measurements, the stability of the plasmid-derived message, as well as the efficiency of its translation into protein are reduced significantly as copy number increases.
", "doi": "10.7907/M1F6-R062", "publication_date": "1987", "thesis_type": "phd", "thesis_year": "1987" }, { "id": "thesis:11478", "collection": "thesis", "collection_id": "11478", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04172019-094731539", "type": "thesis", "title": "The Control of RNA Synthesis in Isolated HeLa Cell Mitochondria", "author": [ { "family_name": "Gaines", "given_name": "George Loweree, III", "clpid": "Gaines-George-Loweree-III" } ], "thesis_advisor": [ { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "thesis_committee": [ { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The transcription of DNA and processing of RNA in mitochondria was investigated using isolated HeLa cell mitochondria. The intact organelles transcribe their DNA and process their RNA both qualitatively and quantitatively similar to the in vivo situation. Changing the conditions for the transcription reactions allowed the identification of a processing pathway for the small ribosomal RNA species, and an RNA species from the region surrounding the origin of light-strand replication with novel electrophoretic properties. Removal of the mitochondria from the cellular environment simplified the investigations of the nuclear-cytoplasmic influences upon the mitochondrial transcription. Differential sensitivities of all three transcription events, synthesizing the rRNAs, mRNAs, and light-strand RNAs, was shown to exist to a small molecular weight factor(s) present in the cytoplasm, and the availability of energetic substrates. These sensitivities indicate a link between cytoplasmic and respiratory/oxidative phosphorylative control of mitochondrial transcription.
", "doi": "10.7907/1hj5-em11", "publication_date": "1986", "thesis_type": "phd", "thesis_year": "1986" }, { "id": "thesis:11279", "collection": "thesis", "collection_id": "11279", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11212018-100547724", "primary_object_url": { "basename": "Kuo_C-L_1984.pdf", "content": "final", "filesize": 28474761, "license": "other", "mime_type": "application/pdf", "url": "/11279/1/Kuo_C-L_1984.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Use of Temperature Sensitive Mutants to Study Yeast DNA Replication", "author": [ { "family_name": "Kuo", "given_name": "Chia-lam", "clpid": "Kuo-Chia-lam" } ], "thesis_advisor": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Campbell", "given_name": "Judith L.", "orcid": "0000-0001-8291-5551", "clpid": "Campbell-J-L" }, { "family_name": "Raftery", "given_name": "Michael A.", "clpid": "Raftery-M-A" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "An improved in vitro DNA replication system in Brij-treated Saccharomyces cerevisiae has been used to screen a random population of temperature-sensitive strains for mutants specifically defective in DNA synthesis. Twenty mutants defective in in vitro DNA synthesis have been isolated. Seven of them fall into three complementation groups -- cdc2, cdc8, and cdc16 -- involved in the control of the cell-division cycle. Because synthesis in vitro represents propagation of replication forks active in in vivo at the time of permeabilization, our findings that cdc2 and cdc16 mutants can incorporate dTMP into DNA in such permeabilized cells at 23\u00b0c but not at 37\u00b0c supports the conclusion that these two mutations directly affect DNA synthesis. Such an involvement was previously suggested by in vivo analysis for CDC2 but was less clear for CDC16. The usefulness of our screening procedure is further demonstrated by the isolation of replication mutants in previously undescribed complementation groups. One strain shows a serious defect in in vivo DNA synthesis but normal RNA synthesis.
\r\n\r\nThe in vitro system has also been used to purify the CDC8 protein. cdc8 mutant strains are temperature-sensitive for DNA chain elongation and the CDC8 gene product is required for DNA synthesis in vitro in permeabilized yeast cells. Extracts of wild-type A364a yeast restore DNA synthesis in Brij-treated cdc8 mutant. A small, heat-stable protein responsible for this complementation has been partially purified from wild-type cells.
\r\n\r\nThe CDC8 gene has been isolated on recombinant plasmids. The yeast-E. coli shuttle vector YCp50 was used to prepare a recombinant plasmid pool containing the entire yeast genome. Plasmids capable of complementing the temperature-sensitive cdc8-1 mutation were isolated by transformation of a cdc8-1 mutant and selection for clones able to grow at the nonpermissive temperature. The entire complementing activity is carried on a 0.75-kilobase fragment, as revealed by deletion mapping and DNA sequencing. This fragment lies 1 kilobase downstream from the well characterized sup4 gene, a gene known to be genetically linked to CDC8 thus confirming the cloned gene corresponds to the chromosomal CDC8 gene. Two additional recombinant plasmids that complement the cdc8-1 mutation but that do not contain the 0.75-kilobase fragment or any flanking DNA were also identified in this study. These plasmids may contain genes that compensate for the cdc8-1 mutation.
\r\n\r\nBy the following criteria, we have shown that thymidylate kinase, which catalyzes the phosphorylation of thymidine-5'-monophosphate to thymidine-5'-diphosphate in the pathway of synthesis of dTTP from dTMP, is the product of the CDC8 gene. First, transformed strains carrying the CDC8 gene on a stable high-copy-number plasmid express higher levels of both the gene transcript and the kinase activity than does wild type. Secondly, extracts of strains bearing different alleles of cdc8 show no detectable thymidylate kinase activity. Third, the DNA sequence of CDC8 gene reveals an open reading frame that encodes a protein of 216 amino acids with the same amino terminal sequence as thymidylate kinase purified from yeast.
", "doi": "10.7907/gekb-yq20", "publication_date": "1984", "thesis_type": "phd", "thesis_year": "1984" }, { "id": "thesis:11279", "collection": "thesis", "collection_id": "11279", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11212018-100547724", "primary_object_url": { "basename": "Kuo_C-L_1984.pdf", "content": "final", "filesize": 28474761, "license": "other", "mime_type": "application/pdf", "url": "/11279/1/Kuo_C-L_1984.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Use of Temperature Sensitive Mutants to Study Yeast DNA Replication", "author": [ { "family_name": "Kuo", "given_name": "Chia-lam", "clpid": "Kuo-Chia-lam" } ], "thesis_advisor": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Campbell", "given_name": "Judith L.", "orcid": "0000-0001-8291-5551", "clpid": "Campbell-J-L" }, { "family_name": "Raftery", "given_name": "Michael A.", "clpid": "Raftery-M-A" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "An improved in vitro DNA replication system in Brij-treated Saccharomyces cerevisiae has been used to screen a random population of temperature-sensitive strains for mutants specifically defective in DNA synthesis. Twenty mutants defective in in vitro DNA synthesis have been isolated. Seven of them fall into three complementation groups -- cdc2, cdc8, and cdc16 -- involved in the control of the cell-division cycle. Because synthesis in vitro represents propagation of replication forks active in in vivo at the time of permeabilization, our findings that cdc2 and cdc16 mutants can incorporate dTMP into DNA in such permeabilized cells at 23\u00b0c but not at 37\u00b0c supports the conclusion that these two mutations directly affect DNA synthesis. Such an involvement was previously suggested by in vivo analysis for CDC2 but was less clear for CDC16. The usefulness of our screening procedure is further demonstrated by the isolation of replication mutants in previously undescribed complementation groups. One strain shows a serious defect in in vivo DNA synthesis but normal RNA synthesis.
\r\n\r\nThe in vitro system has also been used to purify the CDC8 protein. cdc8 mutant strains are temperature-sensitive for DNA chain elongation and the CDC8 gene product is required for DNA synthesis in vitro in permeabilized yeast cells. Extracts of wild-type A364a yeast restore DNA synthesis in Brij-treated cdc8 mutant. A small, heat-stable protein responsible for this complementation has been partially purified from wild-type cells.
\r\n\r\nThe CDC8 gene has been isolated on recombinant plasmids. The yeast-E. coli shuttle vector YCp50 was used to prepare a recombinant plasmid pool containing the entire yeast genome. Plasmids capable of complementing the temperature-sensitive cdc8-1 mutation were isolated by transformation of a cdc8-1 mutant and selection for clones able to grow at the nonpermissive temperature. The entire complementing activity is carried on a 0.75-kilobase fragment, as revealed by deletion mapping and DNA sequencing. This fragment lies 1 kilobase downstream from the well characterized sup4 gene, a gene known to be genetically linked to CDC8 thus confirming the cloned gene corresponds to the chromosomal CDC8 gene. Two additional recombinant plasmids that complement the cdc8-1 mutation but that do not contain the 0.75-kilobase fragment or any flanking DNA were also identified in this study. These plasmids may contain genes that compensate for the cdc8-1 mutation.
\r\n\r\nBy the following criteria, we have shown that thymidylate kinase, which catalyzes the phosphorylation of thymidine-5'-monophosphate to thymidine-5'-diphosphate in the pathway of synthesis of dTTP from dTMP, is the product of the CDC8 gene. First, transformed strains carrying the CDC8 gene on a stable high-copy-number plasmid express higher levels of both the gene transcript and the kinase activity than does wild type. Secondly, extracts of strains bearing different alleles of cdc8 show no detectable thymidylate kinase activity. Third, the DNA sequence of CDC8 gene reveals an open reading frame that encodes a protein of 216 amino acids with the same amino terminal sequence as thymidylate kinase purified from yeast.
", "doi": "10.7907/gekb-yq20", "publication_date": "1984", "thesis_type": "phd", "thesis_year": "1984" } ]