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.

The generation of site-directed mutants within the actin binding domain of the EGF receptor modulates receptor function in internalization and ligand binding. In addition, truncation of the EGFr at residue 996 results in a loss of high affinity ligand binding, inhibited internalization and reduced signaling capacity. Mutation of tyrosine 992 to phenylalanine (Y992F) and glutamate 991 to glutamine (E991Q) increases the rate at which receptors are internalized. The presence of a phenylalanine residue eliminates EGFr-mediated phosphorylation at Tyr992 while the E991Q mutation might also eliminate phosphorylation at this position due to a disruption of the kinase recognition motif. Thus, phosphorylation of Tyr992 appears to function in the regulation of receptor internalization. The mutation of tyrosine 992 to a glutamate residue (Y992E) causes a three-fold increase in receptor affinity for its ligand and demonstrates the existence of novel third and potentially fourth affinity states for the EGFr. A very high affinity EGFr state with a K$\sb{\rm d}$ of approximately 10 pM has been identified as has an intermediate state of 1.5 nM. The deletion of the C-terminal 190 amino acids of the EGFr causes a complete abolition of the previously observed high affinity state of the EGFr and also causes a significant reduction in the affinity of the low affinity state of the EGFr. Phorbol ester treatment of wild type and mutant EGFr causes a loss of the high affinity receptors, and also a decrease in the overall affinity of the receptor for its ligand which is similar to the loss seen in the deletion mutant. This suggests that control of the affinity state of the EGFr is mediated through the C-terminal 190 amino acids of the receptor. In addition, the C-terminal 190 amino acids of the receptor have been identified as containing a domain which regulates the phorbol ester induced conversion of receptor affinity. The amino acid composition in the vicinity of tyrosine 992 has been shown to play a role in the internalization of the EGF receptor and in the regulation of receptor affinity for its ligand.

Isolates of Burkholderia cepacia differed in their ability to utilize D-serine as a carbon and energy source. Strain 17616 is one of the strains which ordinarily fails to utilize D-serine. D-serine also inhibited the growth of this strain on alternative carbon sources such as mannitol. D-serine resistant (Dsd$\sp+$) mutants of strain 17616 were isolated which formed high constitutive levels of a D-serine deaminase not present in the wild type. The majority of such mutants also utilized D-serine as sole carbon source. A 5.5-kb fragment of B. cepacia DNA containing the dsd gene was cloned into the DsdA$\sp-$ strain E. coli AC6082. Introduction of recombinant plasmids carrying the dsd gene into strains 17616 or AC6082 resulted in high levels of constitutive D-serine deaminase activity as well as expression of a new peptide with the predicted size (45 kDa) of the the dsd gene product. Efforts were made to define the mechanism of activation of the cryptic dsd gene in strain 17616. Comparison of PCR products of the region upstream of the dsd gene in the wild type and Dsd$\sp+$ strains indicated that dsd gene expression was not a consequence of insertion of tranposable-gene-activating elements upstream of the cryptic gene as had been observed for activation of foreign bla and lac genes in this strain. Furthermore, no other alterations were detected in this upstream region that would account for dsd gene activation. Analysis the region downstream of the dsd gene revealed the presence of a marR-like repressor gene and adjacent multi-drug resistance transporter gene. The orientation of the latter two genes was opposite to that of the dsd gene. We were interested in the possibility that the marR (multiple antibiotic resistance/multiple adaptive response) protein might control dsd gene expression. To explore this possibility, I compared the nucleotide sequences of the marR gene from the representative Dsd$\sp+$ strain 249-50 with the corresponding wild type gene. The wild type and mutant sequences were identical.