Incorporation of aryl bromide functionality into proteins was achieved via engineering the bacterial biosynthetic apparatus. A phenylalanine auxotrophic E. coli host was equipped with a phenylalanyl-tRNA synthetase (PheRS) variant that has a broadened substrate specificity. The mutant pheS gene (pheS*), which codes for the α-subunit of the enzyme PheRS and confers relaxed substrate specificity, was encoded on a multiple-copy plasmid that also bears the target gene dihydrofolate reductase (DHFR). Constitutive over-expression of pheS* and subsequent expression of the target gene in the presence of phenylatanine analog, para-bromophenylalanine (p-Br-phe), allowed over 85% replacements of phe residues by p-Br-phe in DHFR. The level of bromination can be controlled by varying the relative amounts of phe and p-Br-phe in the culture medium. Introduction of aryl bromide functionality into proteins offers great potential for selective chemical modification of proteins via transition metal-catalyzed reactions, which are orthogonal to existing protein chemistry. Moreover, bromination may be useful in X-ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique. The utility of the aryl bromide as a unique functionality was investigated in collaboration with Isaac Carrico. Artificial extracellular matrix (ECM) proteins were synthesized using the principles of recombinant DNA technology. These proteins were designed for eventual application in vascular grafts. The engineered ECM proteins contained alternating blocks of cell-binding domains derived from CS1or CS5 regions of human fibronectin for endothelial cell attachment, and elastin-like repeats for mechanical integrity. One phe residue per elastin block [(VPGVG)2VPGFG(VPGVG)2] was designed, which could be replaced with p-Br-phe and subsequently used for chemical cross-linking of the proteins. Protein expression yields of 75–90 mg/L were obtained with 50–60% substitution of phe by p-Br-phe. Preliminary exploration of Pd(0)-catalyzed Heck and Sonagashira couplings with p-Br-phe demonstrate feasibility of these reactions under mild conditions required for protein modification as well as compatibility with side chains of all natural amino acids (except cysteine). Site-specific incorporation of p-Br-phe was tested in an E. coli strain equipped with a yeast PheRS/tRNA Phe amber supressor pair. While p-F-phe can be site-specifically incorporated using this system, attempts at p-Br-phe incorporation were unsuccessful, probably due to unfavorable interaction of p-Br-phe with the bulky and polar tyrosine residue in the binding pocket of yeast PheRS.

U14 small nucleolar RNA (snoRNA) is required for processing of 18S ribosomal RNA. It was hypothesized that U14 might base pair with 18S RNA through two highly conserved U14 sequence elements known as domains A and B. Using Saccharomyces cerevisiae as the experimental system, I showed that: (1) the domain A and B elements are functionally interdependent, and (2) single-point mutations in domain A combined with complete substitution of domain B causes lethality while either mutation alone does not. Direct interaction of U14 with 18S RNA was shown by demonstrating that a lethal mutation in U14 domain A can be suppressed with a mutation which restores complementarity in the corresponding region of 18S RNA. Y-domain in yeast U14 was postulated to serve as a recognition element for vital intermolecular or intramolecular interactions. Consistent with this assumption, mutations in several conserved nucleotides of the loop cause growth defects. In contrast, alterations to the stem have little or no effect. Using a lethal mutation in the loop, three different intragenic suppressor mutations were mapped to three positions adjacent to the primary mutation, and are predicted to influence the structure of the loop. An extragenic suppressors (UF1) able to rescue a cold-sensitive mutation in the loop encodes an essential putative ATP-dependent RNA helicase. Loss of UF1 gene expression caused a reduction in 18S rRNA production, without affecting accumulation of 25S rRNA or U14 snoRNA. Pulse-chase analysis showed that depletion of UF1 protein impaired pre-18S rRNA processing. Finally, an effort was made to define minimum pre-rRNA substrates that can be used to produce functional 18S and 25S rRNAs in vivo. The rDNA operon was split either between the 18S RNA and 5.8S/25S coding units, or between the 18S/5.8S RNA and 25S RNA coding units. The test fragments were expressed from GAL7 promoters. The results showed that functional rRNAs could be produced in trans, but only when the operon was divided between the 18S RNA and 5.8S/25S RNA coding sequence.

Architecturally well-defined polymeric materials with precisely controlled chain length, sequence, stereochemistry and interchain interactions can be produced using the fidelity of biological protein synthesis. A set of periodic protein polymers of repeating unit sequence (AlaGly)$\sb3$-X-Gly, where X is Asn, Phe, Ser, Val, or Tyr, has been produced to examine the relation between amino acid residue volume and crystalline unit cell dimensions. The proteins were overexpressed in Escherichia coli and purified on the basis of acid/ethanol precipitation or insolubility in aqueous sodium dodecyl sulfate. The monodisperse populations of purified polypeptides were processed in the form of oriented crystalline mats by precipitation from formic acid under mechanical shear. Analysis by infrared spectroscopy and x-ray diffraction showed that the artificial proteins adopt a chain-folded lamellar structure comprised of anti-parallel $\beta$-sheets with polar orientation and three-residue folds at the lamellar surface; as seen for ((AlaGly)$\rm\sb3GluGly\rbrack\sb{36}$ (Krejchi, 1997). The x-ray diffraction signals for each of the (AlaGly)$\sb3$-X-Gly polymers were indexed on an orthorhombic unit cell with invariant a (hydrogen bond direction) and c (chain direction) axes. However, the b-axis (sheet stacking direction) spacing increased linearly with the volume of the substituted amino acid, indicating a linear relationship between the average intersheet stacking dimension and the volume of the residue at position X. Analysis of the Phe variant utilizing proton spin diffusion in solid state NMR spectroscopy, provided direct evidence for the confinement of Phe residues at the lamellar surface. The chain-folded lamellar architecture adopted by this family of periodic polypeptides accommodates a wide range of residues differing in charge, steric bulk, and hydrophobicity. These results provide a new approach to the controlled engineering of intermolecular interactions in polymeric solids.

Genetic screens which assay behavior have been successfully used to identify genes required for neural function. This thesis is the analysis of midline uncoordinated (muc), a mutation identified for its effect on grooming behavior. This mutation was caused by a single P (lac W) insertion at position 28A. A number of additional muc alleles have been generated by excision of the P element. Using markers for two types of femoral chordotonal neurons we have been able to show that muc disrupts the axon trajectories of these cells. In addition to grooming behavior and neuroanatomy, many muc alleles also affect midline parting of the thoracic microchaetae, flightlessness, lethality and male sterility. Genetic analysis of the various muc mutations suggest that they form a unique complementation group. Three transcripts were found near the area of the muc mutation. The most likely gene affected by muc is the Drosophila homolog of dihydrolipoamide acetyltransferase, component E$\sb2$ of the mitochondrial pyruvate dehydrogenase complex. The P (lac W) element sits in an intron of this gene. We have found that the most severe grooming alleles retain all or almost of all of the P element used to cause the original mutation. In addition to severe grooming behavior, these alleles also have severe axon projection defects. Revertant alleles which have cleanly excised the P element have wild type grooming behavior and normal axon projection patterns.

This dissertation focuses on dioecious angiosperms with a genetic system of sex determination based on a pair of heteromorphic sex chromosomes. Silene latifolia and S. dioica (Caryophyllaceae), with their X/Y mechanism and heterogametic males, have become model organisms for the study of genetic sex determination in angiosperms. Random amplified polymorphic DNA (RAPD) markers provide a valuable tool for the study of the genetic nature of the male determining Y chromosome. We first embarked on a search for Y chromosome RAPD markers using the breeding technique of bulked segregant analysis, obtaining 101 Y chromosome markers which together, were shown to characterize the two species. Genetic mapping placed one of these Y chromosome molecular markers in the pseudoautosomal region of the sex chromosomes. This finding provides a tool for the study of recombination rates among sex chromosomes and relative rates of evolution of X and Y chromosomes. Finally, we investigated the molecular nature of a highly conserved Y chromosome marker and looked for homologous sequences in other related dioecious and hermaphroditic species. The marker of choice had been found to be conserved across males from four species of Silene, section Elisanthe. It was cloned, sequenced and analyzed by Southern hybridization. This Y chromosome marker is a high copy sequence that shares homology to female DNA. Forward and reverse primers were designed to amplify the male specific band only. The amplification pattern of the resulting sequence characterized amplified region (SCAR) in related taxa provide evidence for: (1) a common ancestry of the Y chromosomes of dioecious Silene, section Elisanthe, (2) a different branch of the evolution of dioecy in section Otites, (3) the distant relationship of the hermaphrodite S. noctiflora to the dioecious members of section Elisanthe and (4) homology between the Y chromosome of dioecious Silene dioica and autosomes of hermaphroditic S. flos-cuculi.

CSE1, an essential Saccharomyces cerevisiae gene was initially isolated in a screen for genes important for accurate chromosome segregation. cse1 mutants have pleiotropic phenotypes including defects in the ubiquitin-mediated degradation of mitotic cyclins and in cell cycle progression. SRP1, encodes a nuclear localization signal (NLS) receptor protein involved in nuclear protein import that is an allele specific dosage suppressor of cse1-1. CSE1 can rescue certain srp1 mutant phenotypes, indicating that Cse1p and Srp1p are functionally related or have roles in similar pathways. New mutant alleles of CSE1 were generated by linker insertion mutagenesis, including a temperature sensitive allele, cse1-2, that causes arrest in G2/M, chromosome missegregation and defective mitotic cyclin degradation. Analysis of CSE1 mRNA and Cse1p indicate that their levels do not change significantly during the cell cycle and that Cse1p is not phosphorylated. Cse1p is located in the nucleus and concentrated at the nuclear periphery, probably in association with nuclear pores. Current evidence supports the model that Cse1p is required for the export of Srp1p from the nucleus. Srp1p improperly accumulates in the nuclei of both cse1-1 and cse1-2 cells. Reporter proteins that contain NLS sequences accumulate in the cytoplasm of cse1-1 and cse1-2 cells indicating that Cse1p function is also necessary for nuclear protein import. Cse1p binds the nuclear transport protein Ran/Gsp1p-GTP via a conserved amino-terminal motif. In addition, the human protein CAS, which is an export factor for importin-$\alpha,$ is 60% similar to Cse1p. Although a physical interaction between Srp1p and Cse1p has not been shown, a two-hybrid screen identified two potential Cse1p-interacting proteins, Scj1p and Yma5p. Scj1p is a DnaJ homologue involved in protein translocation, folding, and complex assembly. Yma5p is a novel non-essential protein with an as yet unknown role in these important nuclear processes.

We utilized the powerful molecular and genetic tools available for the analysis of neural development in Drosophila to characterize a mutation for its effects on imaginal sensory axons. Previous analysis demonstrated that loss of function alleles caused defects in axon anatomy. Molecular analysis revealed that these axon defects were due to a disruption in the 10.4 kDa cytoplasmic dynein light chain gene (Cdlc1). This molecular analysis involved the recovery of both genomic and cDNA clones, characterization of the transcription unit in both wild type and mutants, and sequence analysis. As verification that mutations in the cytoplasmic dynein light chain gene caused the axon defects, we generated transgenic flies in which expression of the gene was targeted to specific sensory neurons. When this targeting system was introduced into a Cdlc1$\rm\sp{null}$ genetic background the axon phenotype of the targeted neurons was rescued. Further, the rescuing effects of the targeted expression was restricted to the Cdlc1-positive neurons. Other sensory neurons that were not expressing the transgene in this system retained mutant axon phenotypes. The specificity of the rescue demonstrated that Cdlc1 function is cell autonomous, and that sensory neurons require Cdlc1 function for proper development of their axon projections. The dynein light chain has been shown to associate with the cytoplasmic dynein complex, myosin V and nitric oxide synthase. All of these molecules have neural function so it is important to determine whether any of these partners were involved in producing the axon defects we observed in Cdlc1 mutants. In Drosophila, mutant alleles are only available for genes encoding components of the cytoplasmic dynein complex. We used these alleles in a double mutant analysis to determine whether the axon phenotype was influenced by genetic interactions between mutations affecting the light chain and the dynein heavy chain and p150$\rm\sp{Glued}.$ Our data showed that loss of function mutations in the heavy chain gene acted as dominant suppressors of the Cdlc1 axon phenotype, while loss of function mutations in Glued acted as dominant enhancers of the phenotype. These results support a model in which the light chain functions with the cytoplasmic dynein complex during axon development.

Homosporous ferns such as Ceratopteris richardii produce only one type of spore; irrespective of the genotype, spores can develop into either ameristic males, or meristic, hermaphroditic gametophytes. Determination of sexual identity occurs after spore germination by the action of the hormone antheridiogen, which promotes male development and is secreted by the hermaphrodite. Meristem development antagonizes the antheridiogen response, whereas activation of maleness by antheridiogen involves the specific repression of female-associated traits, such as the meristem and the archegonia. To elucidate the possible relationship between the meristem and the antheridiogen sex determination pathway, I cloned a Ceratopteris gene (CKNOX-A or CK) homologous to the homeobox-containing, meristem-specific KNOTTED1 or KN1 gene. CK encodes a putative protein that contains an 88-aa region 76% identical to the KN1 homeodomain. Expression studies performed by RT-PCR and in situ hybridization showed that CK is most strongly expressed in tissues that contain a meristem, such as hermaphroditic gametophytes, and sporophyte apices and leaves. However, CK RNA was also detected in antheridial cells in males. Surprisingly, the cellular localization of the CK hybridization signal differed depending on the regions of the CK gene used as a probe. Using the CK 3$\sp\prime$ UTR probe, the hybridization signal localized to the nuclei, in contrast to the cytoplasmic localization with a homeobox-containing probe. Functional homology of the CK gene to KN1 was assessed by overexpressing CK in Arabidopsis plants (ecotype Columbia). Transformant phenotypes ranged between two classes: in one, leaf size was severely reduced and plants formed petite rosettes. In the other, plants displayed a loss of apical dominance, by overproducing rosette leaves that developed without a visible phyllotactic pattern, or by forming several inflorescences simultaneously. Contrary to the KN overexpressing phenotypes in Arabidopsis, leaf shape was not altered by lobe formation in CK transformants. In conclusion, CK can be recognized by the molecules that control the initiation and maintenance of the Arabidopsis meristem, which suggests that Ceratopteris CK may play a similar role to the KNOTTED gene of maize.

The regulation of genes involved in proliferation and cell cycle control plays a critical role in normal development and differentiation. Aberrant expression of genes promoting proliferation (oncogenes) or loss of genes involved in restraining cell growth (tumor suppressors) can result in cancer. The p53 tumor suppressor protein has been demonstrated to play a critical role in both tumorigenesis and normal developmental processes. The mdm2 proto-oncogene can regulate the activity and stability of p53 protein. This suggested that mdm2 functions in development and tumorigenesis through p53-dependent mechanisms. However, mdm2 has also been shown to interact with factors involved in the regulation of cell cycle control, transcription and ribosome biosynthesis. The aim of this dissertation was to examine whether mdm2 expression during breast tumorigenesis and during normal mammary gland development in the mouse was regulated by p53-dependent or -independent mechanisms. The first segment of this work involved the analysis of alternative splicing of mdm2 mRNA during breast tumorigenesis in mice and humans. The second element of this dissertation examined the role of p53 in regulating the expression of mdm2 mRNA in adult tissues from the mouse and during normal mammary gland development in the mouse. Results from these experiments demonstrated that truncated mdm2 mRNA are expressed in mouse and human breast tissues. These truncated transcripts are predicted to code for mdm2 proteins that have lost C-terminal sequences involved in regulating proteolytic cleavage of mdm2 itself and in targeting p53 for ubiquitin mediated proteosomal degradation.

To study polyhydroxyalkanoate (PHA) accumulation and PHA regulation in Bacillus megaterium, transposon mutagenesis with Tn917-LTV1 was carried out and seventy two mutants that produce more or less PHA than normal were isolated and partially characterized. The chromosomal regions of B. megaterium flanking the Tn917-LTV1 insertions were cloned and sequenced from two PHA over-producing and six leaky mutants. The results showed that Tn917-LTV1 was less than ideal for generating a mutant bank due to the unavoidable occurrence of sibling transposants. Furthermore, the sequence data revealed that chromosomal deletions mediated by Tn917-LTV1 insertions were common. An alternative method was developed using a direct plating procedure and it was shown to circumvent these two problems. One PHA leaky mutant and one overproducing mutant were studied in more detail. The PHA leaky mutant, T4, had significantly reduced levels of PHA accumulation in all media tested and could not form spores. Chromosomal DNA, contiguous with the transposon was cloned and sequenced. Analysis of the sequence data showed Tn917-LTV1 inserted 24-bp upstream of an operon encoding a putative RNA helicase (deaD) gene and a nuclease (nucP) gene. A chromosomal target repeat 5$\sp\prime$-TATTT-3$\sp\prime$ was found on both sides of the insertion which indicated that no deletion was involved in the Tn917-LTV1 transposition. A plasmid carrying the intact RNA helicase operon was constructed and transformed into mutant T4. The transformant recovered the ability to form spores and accumulate wild-type levels of PHA in minimal media. Deletions in either DeaD or NucP did not complement the T4 mutant to the phenotype of VT1660. The results show that both DeaD and NucP are involved in PHA accumulation and are required for spore formation. The DeaD protein of B. megaterium is the first protein in the DEAD-box helicase family that is not essential for the organism. The fact that the growth rate of mutant T4 and VT1660 were similar in both LB and minimal media indicates that this DeaD protein may target specific mRNA, such as those of pha and spo genes. B001S, a PHA over-producing mutant, unlike its parental strain VT1660, produced large quantities of PHA in rich media. The chromosomal DNA flanking the transposon was cloned from both sides of the insertion. The 8.4 kilobase pairs of chromosomal sequence from left end (IR-L side) of Tn917-LTV1 coded for sixteen open reading frames (ORFs). Ten putative products of the sixteen ORFs shared sequence homology with known gas vesicle proteins (Gvp). The 8.4-kb fragment and its deletion derivatives were cloned into pBluescriptIISK and the plasmids were transformed into E. coli. Gas vesicles were formed and observed by phase contrast microscope, differential interference contrast microscopy and electron microscopy. The deletion analysis and sequence comparison with known gas vesicle proteins suggested 14 out of the 16 ORFs formed the gvp operon of B. megaterium VT1660. The deletion analysis showed that 11 genes are the maximum required for the gas vesicle formation in E. coli. The E. coli cells containing gas vesicles showed increased buoyancy. This is the first time that a functional organelle has been transferred to E. coli.