CYTOLOGICAL, ULTRASTRUCTURAL, AND BIOCHEMICAL STUDIES OF THE STRUCTURE OF PREMATURELY CONDENSED CHROMOSOMES

The phenomenon of premature chromosome condensation, resulting from fusion between mitotic and interphase cells, includes dissolution of the interphase nuclear framework, thus allowing a direct visualization of interphase chromosomes. Light microscope morphology of prematurely condensed chromosomes (PCC) from synchronized HeLa cells supports the model of an interphase "chromosome condensation cycle". PCC are increasingly attenuated as cells progress through G(,1). A maximum degree of decondensation is observed at active sites of DNA replication during S phase, and a condensed morphology is rapidly resumed following completion of replication of a chromosome segment.^ To permit ultrastructural and biochemical studies of PCC, a procedure was developed to induce premature chromosome condensation at high frequency. This was achieved by polyethylene glycol (PEG)-mediated fusion of a dense monolayer of mitotic and interphase cells induced by centrifugation onto lectin-coated culture dishes. Using this method, PCC induction frequencies of 60-90% are routinely obtained.^ Scanning electron microscope analysis of PCC spreads revealed that the extension of PCC during progression through G(,1) is accompanied by a transition of the basic 30 nm chromatin fiber from tightly packed looping fibers to extended longitudinal fibers. Sites of active DNA replication is S-PCC were indicated to be organized a single longitudinal fibers. Following replication of a chromosome segment, a rapid reorganization from the extended longitudinal fiber to packed looping fibers occurs. The postreplication maturation process appears to include the assembly of a chromosome core consisting of multiple longitudinal fibers.^ The role of histone H1 phosphorylation in PCC formation was investigated by acidurea polyacrylamide gel electrophoresis of total histone extracted from metaphase chromosomes and PCC following high frequency fusion. This investigation failed to demonstrate an extensive phosphorylation of H1 associated with PCC formation. However, significant dephosphorylation of superphosphorylated metaphase chromosome H1 was observed, indicating that interphase H1-phosphatase activity is dominant over metaphase H1 kinase activity. These observations provide evidence against models suggesting a role for H1 superphosphorylation in triggering mitotic condensation of chromosomes. ^

A systematic review of the animal and human literature on the use of vaccines to prevent dental caries

Streptococcus mutans has been identified as the primary etiological agent of human dental caries. Since its identification, there has been research focused on the development of a vaccine to prevent this disease. Preliminary research has been conducted to test both active and passive vaccines for Streptococcus mutans in animals and humans. Although a vaccine for dental caries caused by Streptococcus mutans would most likely be administered to children, no testing of any type of dental caries vaccines has been conducted on children as of yet. The public health imperative for the development of a vaccine is great. Not only will a vaccine reduce the various consequences, but it would also improve quality of life for many individuals. Among the many possible vaccine antigen candidates, researchers have also been focusing on protein antigens, GTFs, and Gbps as possible candidates for a vaccine. There are also many routes of administration under research, with topical, oral, and intranasal showing a lot of promise. This review will provide an overview on the current state of research, present key factors influencing prevalence of caries, and summarize and discuss the results of animal and human studies on caries vaccines against Streptococcus mutans. The progress and obstacles facing the development of a vaccine to fight dental caries will also be discussed. ^

Macromolecular interaction studies on FF1-3 of human CA150 and STAT1-importin alpha5 using biophysical and biochemical methods

Macromolecular interactions, such as protein-protein interactions and protein-DNA interactions, play important roles in executing biological functions in cells. However the complexity of such interactions often makes it very challenging to elucidate the structural details of these subjects. In this thesis, two different research strategies were applied on two different two macromolecular systems: X-ray crystallography on three tandem FF domains of transcription regulator CA150 and electron microscopy on STAT1-importin α5 complex. The results from these studies provide novel insights into the function-structure relationships of transcription coupled RNA splicing mediated by CA150 and the nuclear import process of the JAK-STAT signaling pathway. ^ The first project aimed at the protein-protein interaction module FF domain, which often occurs as tandem repeats. Crystallographic structure of the first three FF domains of human CA150 was determined to 2.7 Å resolution. This is the only crystal structure of an FF domain and the only structure on tandem FF domains to date. It revealed a striking connectivity between an FF domain and the next. Peptide binding assay with the potential binding ligand of FF domains was performed using fluorescence polarization. Furthermore, for the first time, FF domains were found to potentially interact with DNA. DNA binding assays were also performed and the results were supportive to this newly proposed functionality of an FF domain. ^ The second project aimed at understanding the molecular mechanism of the nuclear import process of transcription factor STAT1. The first structural model of pSTAT1-importin α5 complex in solution was built from the images of negative staining electron microscopy. Two STAT1 molecules were observed to interact with one molecule of importin α5 in an asymmetric manner. This seems to imply that STAT1 interacts with importin α5 with a novel mechanism that is different from canonical importin α-cargo interactions. Further in vitro binding assays were performed to obtain more details on the pSTAT1-importin α5 interaction. ^

Functional analysis of the Galpha protein, GNA -3, in the development of Neurospora crassa using biochemical and genetic studies

Extracellular signals regulate fungal development and, to sense and respond to these cues, fungi evolved signal transduction pathways similar to those in mammalian systems. In fungi, heterotrimeric G proteins, composed of α, β, and γ subunits, transduce many signals, such as pheromones and nutrients, intracellularly to alter adenylyl cyclase and MAPK cascades activity. ^ Previously, the Gα proteins GNA-1 and GNA-2 were characterized in regulating development in the fungus Neurospora crassa. R. A. Baasiri isolated a third Gα, gna-3, and P. S. Rowley generated Δgna-3 mutants. GNA-3 belongs to a fungal Gα family that regulates cAMP metabolism and virulence. The Δ gna-3 sexual cycle is defective in homozygous crosses, producing inviable spores. Δgna-3 mutants have reduced aerial hyphae formation and derepressed asexual sporulation (conidiation), causing accumulation of asexual spores (conidia). These defects are similar to an adenylyl cyclase mutant, cr-1; cAMP supplementation suppressed Δ gna-3 and cr-1. Inappropriate conidiation and expression of a conidiation gene, con-10, were higher in Δ gna-3 than cr-1 submerged cultures; peptone suppressed conidiation. Adenylyl cyclase activity and expression demonstrated that GNA-3 regulates enzyme levels. ^ A Δgna-1 cr-1 was analyzed with F. D. Ivey to differentiate GNA-1 roles in cAMP-dependent and -independent pathways. Δ gna-1 cr-1 defects were worse than cr-1 and refractory to cAMP, suggesting that GNA-1 is necessary for sensing extracellular CAMP. Submerged culture conidiation was highest in Δgna-1 cr-1, and only high cell density Δgna-1 cultures conidiated, which correlated with con-10 levels. Transcription of a putative heat shock cognate protein was highest in Δgna-1 cr-1. ^ Functional relationships between the three Gαs was analyzed by constructing Δgna-1 Δgna-2 Δ gna-3, Δgna-1 Δgna-3, and Δgna-2 Δgna-3 strains. Δ gna-2 Δgna-3 strains exhibited intensified Δ gna-3 phenotypes; Δgna-1 Δgna-2 Δgna-3 and Δgna-1 Δ gna-3 strains were identical to Δgna-1 cr-1 on plates and were non-responsive to cAMP. The highest levels of conidiation and con-10 were detected in submerged cultures of Δ gna-1 Δgna-2 Δgna-3 and Δgna-1 Δgna-3 mutants, which was partially suppressed by peptone supplementation. Stimulation of adenylyl cyclase is completely deficient in Δgna-1 Δ gna-2 Δgna-3 and Δgna-1 Δ gna-3 strains. Δgna-3 and Δ gna-1 Δgna-3 aerial hyphae and conidiation defects were suppressed by mutation of a PKA regulatory subunit. ^

Studies on the interaction of NADPH cytochrome P-450 reductase and cytochromes P-450

Chemical modification of cytochrome P-450 reductase was used to determine the involvement of charged amino acids in the interaction between the reductase and two forms of cytochrome P-450. Acetylation of 11 lysine residues of the reductase with acetic anhydride yielded a 20-40% decrease in the K$\sb{\rm m}$ of the reductase for cytochrome P-450b or cytochrome P-450c. Modification of carboxyl groups on the reductase with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and methylamine, glycine methyl ester, or taurine as nucleophiles inhibited the interaction with the cytochromes P-450. We were able to modify 4.0, 7.9, and 5.9 carboxyl groups using methylamine, glycine methyl ester, and taurine, respectively. The apparent K$\sb{\rm m}$ for cytochrome P-450c or cytochrome P-450b was increased 1.3 to 5.2 fold. There were varied effects on the V$\sb{\rm max}$. There was no significant change in the conformation of the reductase upon chemical modification. These results strongly suggest that electrostatic interactions as well as steric constraints play a role in the binding and electron transfer step(s) between the reductase and cytochrome P-450. Cytochrome P-450 protected 0.8 moles of carboxyl residues on the reductase from being modified with EDC. These protected amino acids on the reductase are presumably involved in binding to cytochrome P-450. The specific peptide containing these amino acids has been identified. ^

Studies on the {\it in vivo} function of a phosphatidylinositol transfer protein from {\it Saccharomyces cerevisiae}

Phosphatidylinositol transfer proteins (PI-TP's) catalyze the transfer of phosphatidylinositol and phosphatidylcholine between membranes in vitro. However the in vivo function of these proteins is unknown. In this thesis we have used a combined biochemical and genetic approach to determine the importance of PI-TP in vivo. An oligonucleotide based on the amino terminal sequence of the PI-TP from Saccharomyces cerevisiae, was used to screen a yeast genomic library for the gene encoding PI-TP (PIT1 gene). Yeast strains transformed with the positive clones showed overproduction of transfer activities and transfer protein in the 100,000 x g supernatants. The 5$\sp\prime$ terminus of the PIT1 gene correlates with the predicted codons for residues 3-30 of the determined protein sequence. Tetrad analysis of a heterozygous diploid (PIT1/pit1::LEU2) revealed that the PIT1 gene is essential for cell growth. Non-viable spores could be rescued by transformation of the above diploid prior to sporulation, with a plasmid borne copy of the wild type gene. Sequencing of the entire PIT1 gene has revealed that the PIT1 gene is identical to the SEC14 gene. The sec14 ts mutant which exhibits conditional defects at the Golgi stage of protein secretion, is also temperature sensitive for PI-TP activity in vitro. These findings represent the first instance in which a physiological function has been assigned to any phospholipid transfer protein. ^

Functional studies on DNA double-strand break repair proteins (MmRad51, Ku80) in mice

RecA in Escherichia coli and it's homologue, ScRad51 in Saccharomyces cerevisiae, play important roles in recombinational repair. ScRad51 homologues have been discovered in a wide range of organisms including Schizosaccharomyces pombe, lily, chicken, mouse and human. To date there is no direct evidence to describe that mouse Rad51(MmRad51) is involved in DNA double-strand break repair. In order to elucidate the role of MmRad51 in vivo, it was mutated by the embryonic stem (ES) cell/gene targeting technology in mice. The mutant embryos arrested in development shortly after implantation. There was a decrease in cell proliferation followed by programmed cell death, and trophectoderm-derived cells were sensitive to $\gamma$-radiation. Severe chromosome loss was observed in most mitotically dividing cells. The mutant embryos lived longer and developed further in a p53 mutant background; however, double-mutant embryonic fibroblasts failed to proliferate in tissue culture, reflecting the embryos limited life span. Based on these data, MmRad51 repairs DNA damage induced by $\gamma$-radiation, is needed to maintain euplody, and plays an important role in proliferating cells.^ Ku is a heterodimer of 70 and 80 kDs subunit, which binds to DNA ends and other altered DNA structures such as hairpins, nicks, and gaps. In addition, Ku is required for DNA-PK activity through a direct association. Although the biochemical properties of Ku and DNA-PKcs have been characterized in cells, their physiological functions are not clear. In order to understand the function of Ku in vivo, we generated mice homozygous for a mutation of the Ku80 gene. Ku80-deficient mice, like scid mice, showed severe immunodeficiency due to a impairment of V(D)J recombination. Mutant mice were semiviable and runted, cells derived from mutant embryos displayed hypersensitivity to $\gamma$-radiation, a decreased growth rate, a slow entry into S phase, altered colony size distributions, and a short life span. Based on these results, mutant cells and mice appeared to prematurely age. ^

Population and molecular evolution studies on the Melanocortin 1 receptor locus implicate its role in human pigmentation variation

Human pigmentation is a complex trait with the observed variation caused by the varied production of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) by the melanocytes. The melanocortin 1 receptor (MC1R), a G protein-coupled receptor expressed in the melanocytes, is a regulator eu- and phaeomelanin synthesis, and MC1R mutations causing skin and coat color changes are known in many mammals. To understand the role of MC1R in human pigmentation variation, I have sequenced the MC1R gene in 121 individuals sampled from world populations. In addition, I have sequenced the MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon to study the evolution of MC1R and to infer the ancestral human MC1R sequence. The ancestral MC1R sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. To further evaluate the role of MC1R variants in human pigmentation variation, I have combined these molecular evolution and population studies with functional assays on MC1R variants and primate MC1Rs. ^

STUDIES ON THE PATTERNS OF NUCLEOTIDE AND AMINO ACID SUBSTITUTION (ELECTROPHORESIS, MUTATION RATE, SELECTION, BASE, COMPOSITION)

Theoretical and empirical studies were conducted on the pattern of nucleotide and amino acid substitution in evolution, taking into account the effects of mutation at the nucleotide level and purifying selection at the amino acid level. A theoretical model for predicting the evolutionary change in electrophoretic mobility of a protein was also developed by using information on the pattern of amino acid substitution. The specific problems studied and the main results obtained are as follows: (1) Estimation of the pattern of nucleotide substitution in DNA nuclear genomes. The pattern of point mutations and nucleotide substitutions among the four different nucleotides are inferred from the evolutionary changes of pseudogenes and functional genes, respectively. Both patterns are non-random, the rate of change varying considerably with nucleotide pair, and that in both cases transitions occur somewhat more frequently than transversions. In protein evolution, substitution occurs more often between amino acids with similar physico-chemical properties than between dissimilar amino acids. (2) Estimation of the pattern of nucleotide substitution in RNA genomes. The majority of mutations in retroviruses accumulate at the reverse transcription stage. Selection at the amino acid level is very weak, and almost non-existent between synonymous codons. The pattern of mutation is very different from that in DNA genomes. Nevertheless, the pattern of purifying selection at the amino acid level is similar to that in DNA genomes, although selection intensity is much weaker. (3) Evaluation of the determinants of molecular evolutionary rates in protein-coding genes. Based on rates of nucleotide substitution for mammalian genes, the rate of amino acid substitution of a protein is determined by its amino acid composition. The content of glycine is shown to correlate strongly and negatively with the rate of substitution. Empirical formulae, called indices of mutability, are developed in order to predict the rate of molecular evolution of a protein from data on its amino acid sequence. (4) Studies on the evolutionary patterns of electrophoretic mobility of proteins. A theoretical model was constructed that predicts the electric charge of a protein at any given pH and its isoelectric point from data on its primary and quaternary structures. Using this model, the evolutionary change in electrophoretic mobilities of different proteins and the expected amount of electrophoretically hidden genetic variation were studied. In the absence of selection for the pI value, proteins will on the average evolve toward a mildly basic pI. (Abstract shortened with permission of author.) ^

STUDIES ON CHOLINERGIC FUNCTION AND MEMORY IN MICE (CHOLINOTOXINS, WORKING, REFERENCE MEMORY, RADIAL ARM MAZE)

Considerable evidence suggests that central cholinergic neurons participate in either acquisition, storage or retrieval of information. Experiments were designed to evaluate information processing in mice following either reversible or irreversible impairment in central cholinergic activity. The cholinergic receptor antagonists, atropine and methylatropine were used to reversibly inhibit cholinergic transmission. Irreversible impairment in central cholinergic function was achieved by central administration of the cholinergic-specific neurotoxins, N-ethyl-choline aziridinium (ECA) and N-ethyl-acetylcholine aziridinium (EACA).^ ECA and EACA appear to act by irreversible inhibition of high affinity choline uptake (proposed rate-limiting step in acetylcholine synthesis). Intraventricular administration of ECA or EACA produced persistent reduction in hippocampal choline acetyltransferase activity. Other neuronal systems and brain regions showed no evidence of toxicity.^ Mice treated with either ECA or EACA showed behavioral deficits associated with cholinergic dysfunction. Passive avoidance behavior was significantly impaired by cholinotoxin treatment. Radial arm maze performance was also significantly impaired in cholinotoxin-treated animals. Deficits in radial arm maze performance were transient, however, such that rapid and apparent complete behavioral recovery was seen during retention testing. The centrally active cholinergic receptor antagonist atropine also caused significant impairment in radial arm maze behavior, while equivalent doses of methylatropine were without effect.^ The relative effects of cholinotoxin and receptor antagonist treatment on short-term (working) memory and long-term (reference) memory in radial arm maze behavior were examined. Maze rotation studies indicated that there were at least two different response strategies which could result in accurate maze performance. One strategy involved the use of response algorithms and was considered to be a function of reference memory. Another strategy appeared to be primarily dependent on spatial working memory. However, all behavioral paradigms with multiple trails have reference memory requirements (i.e. information useful over all trials). Performance was similarly affected following either cholinotoxin or anticholinergic treatment, regardless of the response strategy utilized. In addition, rates of behavioral recovery following cholinotoxin treatment were similar between response groups. It was concluded that both cholinotoxin and anticholinergic treatment primarily resulted in impaired reference memory processes. ^

The use of the computerized and revised vital record in conducting etiologic studies on neural tube defects

The purpose of this study was to evaluate the adequacy of computerized vital records in Texas for conducting etiologic studies on neural tube defects (NTDs), using the revised and expanded National Centers for Health Statistics vital record forms introduced in Texas in 1989.^ Cases of NTDs (anencephaly and spina bifida) among Harris County (Houston) residents were identified from the computerized birth and death records for 1989-1991. The validity of the system was then measured against cases ascertained independently through medical records and death certificates. The computerized system performed poorly in its identification of NTDs, particularly for anencephaly, where the false positive rate was 80% with little or no improvement over the 3-year period. For both NTDs the sensitivity and predictive value positive of the tapes were somewhat higher for Hispanic than non-Hispanic mothers.^ Case control studies were conducted utilizing the tape set and the independently verified data set, using controls selected from the live birth tapes. Findings varied widely between the data sets. For example, the anencephaly odds ratio for Hispanic mothers (vs. non-Hispanic) was 1.91 (CI = 1.38-2.65) for the tape file, but 3.18 (CI = 1.81-5.58) for verified records. The odds ratio for diabetes was elevated for the tape set (OR = 3.33, CI = 1.67-6.66) but not for verified cases (OR = 1.09, CI = 0.24-4.96), among whom few mothers were diabetic. It was concluded that computerized tapes should not be solely relied on for NTD studies.^ Using the verified cases, Hispanic mother was associated with spina bifida, and Hispanic mother, teen mother, and previous pregnancy terminations were associated with anencephaly. Mother's birthplace, education, parity, and diabetes were not significant for either NTD.^ Stratified analyses revealed several notable examples of statistical interaction. For anencephaly, strong interaction was observed between Hispanic origin and trimester of first prenatal care.^ The prevalence was 3.8 per 10,000 live births for anencephaly and 2.0 for spina bifida (5.8 per 10,000 births for the combined categories). ^