Part I. Synchronization of the cell division cycle of HeLa cells in suspension cultures. Part II. Studies on chromosomal proteins of HeLa cells during the cell division cycle

Part I

These studies investigate the potential of single and double treatments with either 5-fluorodeoxyuridine of excess thymidine to induce cell division synchrony in suspension cultures of HeLa cells. The patterns of nucleic acid synthesis and cell proliferation have been analyzed in cultures thus synchronized. Several changes in cell population during long incubation with 5-fluorodeoxyuridine or excess thymidine are also described. These results are subjected to detailed evaluation in terms of the degree and quality of synchrony finally achieved.

Part II

Histones and non-histone proteins associated with interphase and metaphase chromosomes of HeLa cells have been qualitatively and quantitatively analyzed. Histones were fractionated by chromatography on Amberlite CG-50 and further characterized by analytical disc electrophoresis and amino acid analysis of each chromatographic fraction. It is concluded that histones of HeLa cells are comprised of only a small number of major components and that these components are homologous to those of other higher organisms. Of all the histones, arginine-rich histone III alone contains cysteine and can polymerize through formation of intermolecular disulfide bridges between histone III monomers.

A detailed comparison by chromatography and disc electrophoresis established that interphase and metaphase histones are made up of similar components. However, certain quantitative differences in proportions of different histones of interphase and metaphase cells are reported. Indirect evidence indicates that a certain proportion of metaphase histone III is polymerized through intermolecular disulfide links, whereas interphase histone III occurs mainly in the monomeric form.

Metaphase chromosomes are associated with an additional acid-soluble protein fraction which is absent from interphase chromosomes. All of these additional acid-soluble proteins of metaphase chromosomes are shown to be non-histones and it is concluded that the histone/DNA ratio is identical in interphase and metaphase chromosomes. The bulk of acid-soluble non-histone proteins of metaphase chromosomes were found to be polymerized through disulfide bridges; corresponding interphase non-histone proteins displayed no evidence of similar polymerization.

The factors responsible for the condensed configuration and metabolic inactivity of metaphase chromosomes are discussed in light of these findings.

The relationship between histone and DNA synthesis in nondividing differentiated chicken erythrocyte cells and in rapidly dividing undifferentiated HeLa cells is also investigated. Of all the histones, only arginine-rich histones are synthesized in mature erythrocytes. Histone synthesis in HeLa cells was studied in both unsynchronized and synchronized cultures. In HeLa cells, only part of the synthesis of all histone fractions is dependent on concurrent DNA synthesis, whereas all histones are synthesized in varying degrees even in the absence of DNA synthesis.

Part I. Approximate Hartree-Fock wavefunctions one-electron properties and electronic structure of the water molecule. Part II. Perturbation-variational calculation of the nuclear spin-spin isotope coupling constant in HD

Part I

Several approximate Hartree-Fock SCF wavefunctions for the ground electronic state of the water molecule have been obtained using an increasing number of multicenter s, p, and d Slater-type atomic orbitals as basis sets. The predicted charge distribution has been extensively tested at each stage by calculating the electric dipole moment, molecular quadrupole moment, diamagnetic shielding, Hellmann-Feynman forces, and electric field gradients at both the hydrogen and the oxygen nuclei. It was found that a carefully optimized minimal basis set suffices to describe the electronic charge distribution adequately except in the vicinity of the oxygen nucleus. Our calculations indicate, for example, that the correct prediction of the field gradient at this nucleus requires a more flexible linear combination of p-orbitals centered on this nucleus than that in the minimal basis set. Theoretical values for the molecular octopole moment components are also reported.

Part II

The perturbation-variational theory of R. M. Pitzer for nuclear spin-spin coupling constants is applied to the HD molecule. The zero-order molecular orbital is described in terms of a single 1s Slater-type basis function centered on each nucleus. The first-order molecular orbital is expressed in terms of these two functions plus one singular basis function each of the types e^-r/r and e^-r ln r centered on one of the nuclei. The new kinds of molecular integrals were evaluated to high accuracy using numerical and analytical means. The value of the HD spin-spin coupling constant calculated with this near-minimal set of basis functions is J_HD = +96.6 cps. This represents an improvement over the previous calculated value of +120 cps obtained without using the logarithmic basis function but is still considerably off in magnitude compared with the experimental measurement of JHD</sub> = +43 0 ± 0.5 cps.</sub>

I. The 3.7 Å crystal structure of horse heart ferricytochrome C. II. The application of the Karle-Hauptman tangent formula to protein phasing

I. The 3.7 Å Crystal Structure of Horse Heart Ferricytochrome C.

The crystal structure of horse heart ferricytochrome c has been determined to a resolution of 3.7 Å using the multiple isomorphous replacement technique. Two isomorphous derivatives were used in the analysis, leading to a map with a mean figure of merit of 0.458. The quality of the resulting map was extremely high, even though the derivative data did not appear to be of high quality.

Although it was impossible to fit the known amino acid sequence to the calculated structure in an unambiguous way, many important features of the molecule could still be determined from the 3.7 Å electron density map. Among these was the fact that cytochrome c contains little or no α-helix. The polypeptide chain appears to be wound about the heme group in such a way as to form a loosely packed hydrophobic core in the molecule.

The heme group is located in a cleft on the molecule with one edge exposed to the solvent. The fifth coordinating ligand is His 18 and the sixth coordinating ligand is probably neither His 26 nor His 33.

The high resolution analysis of cytochrome c is now in progress and should be completed within the next year.

II. The Application of the Karle-Hauptman Tangent Formula to Protein Phasing.

The Karle-Hauptman tangent formula has been shown to be applicable to the refinement of previously determined protein phases. Tests were made with both the cytochrome c data from Part I and a theoretical structure based on the myoglobin molecule. The refinement process was found to be highly dependent upon the manner in which the tangent formula was applied. Iterative procedures did not work well, at least at low resolution.

The tangent formula worked very well in selecting the true phase from the two possible phase choices resulting from a single isomorphous replacement phase analysis. The only restriction on this application is that the heavy atoms form a non-centric cluster in the unit cell.

Pages 156 through 284 in this Thesis consist of previously published papers relating to the above two sections. References to these papers can be found on page 155.

Part I. Regulation of development in the cellular slime mold Dictyostelium discoideum. Part II. Polysomes and RNA synthesis during early development of the surf clam Spisula solidissima

Part I. The cellular slime mold Dictyostelium discoideum is a simple eukaryote which undergoes a multi-cellular developmental process. Single cell myxamoebae divide vegetatively in the presence of a food source. When the food is depleted or removed, the cells aggregate, forming a migrating pseudoplasmodium which differentiates into a fruiting body containing stalk and spore cells. I have shown that during the developmental cycle glycogen phosphorylase, aminopeptidase, and alanine transaminase are developmentally regulated, that is their specific activities increased at a specific time in the developmental cycle. Phosphorylase activity is undetectable in developing cells until mid-aggregation whereupon it increases and reaches a maximum at mid-culmination. Thereafter the enzyme disappears. Actinomycin D and cycloheximide studies as well as studies with morphologically aberrant and temporally deranged mutants indicate that prior RNA and concomitant protein synthesis are necessary for the rise and decrease in activity and support the view that the appearance of the enzyme is regulated at the transcriptional level. Aminopeptidase and alanine transaminase increase 3 fold starting at starvation and reach maximum activity at 18 and 5 hours respectively.

The cellular DNA s of D. discoideum were characterized by CsC1 buoyant density gradient centrifugation and by renaturation kinetics. Whole cell DNA exhibits three bands in CsCl: ρ = 1.676 g/cc (nuclear main band), 1.687 (nuclear satellite), and 1.682 (mitochondrial). Reassociation kinetics at a criterion of Tm -23°C indicates that the nuclear reiterated sequences make up 30% of the genome (Cot_1/2 (pure) 0.28) and the single-copy DNA 70% (Cot_1/2(pure) 70). The complexity of the nuclear genome is 30 x 10⁹ daltons and that of the mitochondrial DNA is 35-40 x 10⁶ daltons (Cot_1/2 0.15). rRNA cistrons constitute 2.2% of nuclear DNA and have a ρ = 1.682.

RNA extracted from 4 stages during developmental cycle of Dictyostelium was hybridized with purified single-copy nuclear DNA. The hybrids had properties indicative of single-copy DNA-RNA hybrids. These studies indicate that there are, during development, qualitative and quantitative changes in the portion of the single-copy of the genome transcribed. Overall, 56% of the genome is represented by transcripts between the amoeba and mid-culmination stages. Some 19% are sequences which are represented at all stages while 37% of the genome consists of stage specific sequences.

Part II. RNA and protein synthesis and polysome formation were studied during early development of the surf clam Spisula solidissima embryos. The oocyte has a small number of polysomes and a low but measurable rate of protein synthesis (leucine-³H incorporation). After fertilization, there is a continual increase in the percentage of ribosomes sedimenting in the polysome region. Newly synthesized RNA (uridine-5-³H incorporation) was found in polysomes as early as the 2-cell stage. During cleavage, the newly formed RNA is associated mainly with the light polysomes.

RNA extracted from polysomes labeled at the 4-cell stage is polydisperse, nonribosomal, and non-4 S. Actinomycin D causes a reduction of about 30% of the polysomes formed between fertilization and the 16-cell stage.

In the early cleavage stages the light polysomes are mostly affected by actinomycin.

Proton magnetic resonance studies of ribonucleic acid complexes. I. Complexes of biological bases and oligonucleotides with RNA. II. Template recognition and the degeneracy of the genetic code

Part I. Complexes of Biological Bases and Oligonucleotides with RNA

The physical nature of complexes of several biological bases and oligonucleotides with single-stranded ribonucleic acids have been studied by high resolution proton magnetic resonance spectroscopy. The importance of various forces in the stabilization of these complexes is also discussed.

Previous work has shown that purine forms an intercalated complex with single-stranded nucleic acids. This complex formation led to severe and stereospecific broadening of the purine resonances. From the field dependence of the linewidths, T₁ measurements of the purine protons and nuclear Overhauser enhancement experiments, the mechanism for the line broadening was ascertained to be dipole-dipole interactions between the purine protons and the ribose protons of the nucleic acid.

The interactions of ethidium bromide (EB) with several RNA residues have been studied. EB forms vertically stacked aggregates with itself as well as with uridine, 3'-uridine monophosphate and 5'-uridine monophosphate and forms an intercalated complex with uridylyl (3' → 5') uridine and polyuridylic acid (poly U). The geometry of EB in the intercalated complex has also been determined.

The effect of chain length of oligo-A-nucleotides on their mode of interaction with poly U in D<sub>20 at neutral pD have also been studied. Below room temperatures, ApA and ApApA form a rigid triple-stranded complex involving a stoichiometry of one adenine to two uracil bases, presumably via specific adenine-uracil base pairing and cooperative base stacking of the adenine bases. While no evidence was obtained for the interaction of ApA with poly U above room temperature, ApApA exhibited complex formation of a 1:1 nature with poly U by forming Watson-Crick base pairs. The thermodynamics of these systems are discussed.

Part II. Template Recognition and the Degeneracy of the Genetic Code

The interaction of ApApG and poly U was studied as a model system for the codon-anticodon interaction of tRNA and mRNA in vivo. ApApG was shown to interact with poly U below ~20°C. The interaction was of a 1:1 nature which exhibited the Hoogsteen bonding scheme. The three bases of ApApG are in an anti conformation and the guanosine base appears to be in the lactim tautomeric form in the complex.

Due to the inadequacies of previous models for the degeneracy of the genetic code in explaining the observed interactions of ApApG with poly U, the "tautomeric doublet" model is proposed as a possible explanation of the degenerate interactions of tRNA with mRNA during protein synthesis in vivo.

Part I: Total yield measurements for the ^(21)Ne(α,n)^)(24)Mg reaction. Part II: A study of the ^(12)C(He,p)^(14)N reaction

PART I

The total cross-section for the reaction ²¹Ne(α, n)²⁴Mg has been measured in the energy range 1.49 Mev ≤ E_cm ≤ 2.6 Mev. The cross-section factor, S(O), for this reaction has been determined, by means of an optical model calculation, to be in the range 1.52 x 10¹² mb-Mev to 2.67 x 10¹² mb-Mev, for interaction radii in the range 5.0 fm to 6.6 fm. With S(O) ≈ 2 x 10¹² mb-Mev, the reaction ²¹Ne(α, n)²⁴Mg can produce a large enough neutron flux to be a significant astrophysical source of neutrons.

PART II

The reaction¹²C(³He, p)¹⁴N has been studied over the energy range 12 Mev ≤ E_lab ≤ 18 Mev. Angular distributions of the proton groups leading to the lowest seven levels in ¹⁴N were obtained.

Distorted wave calculations, based on two-nucleon transfer theory, were performed, and were found to be reliable for obtaining the value of the orbital angular momentum transferred. The present work shows that such calculations do not yield unambiguous values for the spectroscopic factors.

Biology and assessment of the painted sweetlips (Diagramma pictum (Thunberg, 1792)) and the spangled emperor (Lethrinus nebulosus (Forsskål, 1775)) in the southern Arabian Gulf

The population biology and status of the painted sweeplips (Diagramma pictum) and spangled emperor (Lethrinus nebulosus) in the southern Arabian Gulf were established by using a combination of size-frequency, biological, and size-at-age data. Transverse sections of sagittal otoliths were characterized by alternating translucent and opaque bands that were validated as annuli. Comparisons of growth characteristics showed that there were no significant differences (P>0.05) between sexes. There were well defined peaks in the reproductive cycle, spawning occurred from April to May for both species, and the mean size at which females attained sexual maturity was 31.8 cm fork length (LF) for D. pictum and 27.6 cm (LF) for L. nebulosus. The mean sizes at first capture (21.1 cm LF for D. pictum and 26.4 cm LF for L. nebulosus) were smaller than the sizes for both at first sexual maturity and those at which yield per recruit would be maximized. The range of fishing-induced mortality rates for D. pictum (0.37−0.62/yr) was substantially greater than the target (Fopt=0.07/yr) and limit (Flimit=0.09/ yr) estimates. The range of fishing-induced mortality rates for L. nebulosus (0.15/yr to 0.57/yr) was also in excess of biological reference points (Fopt=0.10/yr and Flimit=0.13/yr). In addition to growth overfishing, the stocks were considered to be recruitment overfished because the biomass per recruit was less than 20% of the unexploited levels for both species. The results of the study are important to fisheries management authorities in the region because they indicate that both a reduction in fishing effort and mesh-size regulations are required for the demersal trap fishery.

Land-sea characterization of the St. Croix East End Marine Park, U.S. Virgin Islands

The St. Croix East End Marine Park (STXEEMP) was established in 2003 as the first multi-use marine park managed by the U.S. Virgin Islands Department of Planning and Natural Resources. It encompasses an area of approximately 155 km2 and is entirely within Territorial waters which extend up to 3 nautical miles from shore. As stated in the 2002 management plan, the original goals were to: protect and maintain the biological diversity and other natural values of the area; promote sound management practices for sustainable production purposes; protect the natural resource base from being alienated for other land use purposes that would be detrimental to the area’s biological diversity; and to contribute to regional and national development (The Nature Conservancy, 2002). At the time of its establishment, there were substantial data gaps in knowledge about living marine resources in the St. Croix, and existing data were inadequate for establishing baselines from which to measure the future performance of the various management zones within the park. In response to these data gaps, National Centers for Coastal Ocean Science (NCCOS), Center for Coastal Monitoring and Assessment, Biogeography Branch (CCMA-BB) worked with territorial partners to characterize and assess the status of the marine environment in and around the STXEEMP and land-based stressors that affect them. This project collected and analyzed data on the distribution, diversity and landscape condition of marine communities across the STXEEMP. Specifically, this project characterized (1) landscape and adjacent seascape condition relevant to threats to coral reef ecosystem health, and (2) the marine communities within STXEEMP zones to increase local knowledge of resources exposed to different regulations and stressors.

An ecological characterization of the marine resources of Vieques, Puerto Rico. Part I: Historical data synthesis

From the 1940s until 2003, portions of the island of Vieques, a municipality within the Commonwealth of Puerto Rico, were used by the US Navy as a base and training facility, resulting in development and zoning history that differ in comparison to other Caribbean islands. The majority of former Navy lands are now under the jurisdiction of the Department of the Interior’s Fish and Wildlife Service as a National Wildlife Refuge, while a smaller percentage of land was transferred to the Vieques municipality and the Puerto Rico Conservation Trust. An analysis of the distribution and status of the marine resources is timely in light of the recent land transfer, increases in development and tourism, and potential changes in marine zoning around the island. To meet this need, NOAA’s Biogeography Branch, in cooperation with the Office of Response and Restoration and other local and regional partners, conducted Part I of an ecological characterization to integrate historical data and research into a synthesis report. The overall objective of this report is to provide resource managers and residents a comprehensive characterization of the marine resources of Vieques to support research, monitoring, and management. For example, knowledge of the spatial distribution of physical features, habitats, and biological communities is necessary to make an informed decision of the establishment and placement of a marine protected area (MPA). The report is divided into chapters based on the physical environment (e.g., climate, geology, bathymetry), habitat types (e.g., reefs and hardbottom, seagrasses, mangroves) and major faunal groups (e.g. fish, turtles, birds). Each section includes five subsections: an overview, description of the relevant literature, methods of analysis, information on the distribution, status and trends of the particular resource, and a discussion of ecological linkages with other components of the Vieques marine ecosystem and surrounding environment. The physical environment of Vieques is similar to other islands within the Greater Antilles chain, with some distinctions. The warm, tropical climate of Vieques, mediated by the northeasterly trade winds, is characterized by a dry season (December-April) and a rainy season (May-November), the latter of which is characterized by the occasional passage of tropical cyclones. Compared to mainland Puerto Rico, Vieques is characterized by lower elevation, less annual precipitation, and higher average temperatures. The amount of annual precipitation also varies spatially within Vieques, with the western portion of the island receiving higher amounts of rainfall than further east. While the North Equatorial Current dominates the circulation pattern in the Greater Antilles region, small scale current patterns specific to Vieques are not as well characterized. These physical processes are important factors mitigating the distribution and composition of marine benthic habitats around Vieques. In general, the topography of Vieques is characterized by rolling hills. Mt. Pirata, the tallest point at 301 m, is located near the southwest coast. In the absence of island wide sedimentation measurements, information on land cover, slope, precipitation, and soil type were used to estimate relative erosion potential and sediment delivery for each watershed. While slope and precipitation amount are the primary driving factors controlling runoff, land use practices such as urban development, military activity, road construction, and agriculture can increase the delivery of pollution and sediments to coastal waters. Due to the recent land transfer, increased development and tourism is expected, which may result in changes in the input of sediments to the coastal environment.

A review of the fisheries of the Uganda waters of Lake Albert East Africa, 1928-1965/66, with catch data mainly from 1953

An historical account is given of the development of the Lake Albert fisheries since Worthington's survey in 1928. It is noted that the development of the fisheries was related to, and dependent upon, improvements in the type of gear and canoes, an incFease in the number of canoes and outboard engines in use, improved marketing facilities and better road communications. Summarized data, collected mainly since 1954, has been analysed and tabulated to show annual exports to the Congo, total annual catches 'and annual catches of individual species. A change in the relative abundance of the various species in the annual catches is described. It is noted that this change was caused by a change-over from large to small mesh size gill-nets, and that it was associated with an increased demand within Uganda for the smaller species of fish, such as Aleste's baremose and Hydrocynus forskahlii. A brief description of fish processing and marketing in the Lake Albert region is given, which emphasizes the suitability of salt-cured fish to the social and physical environment of the area. Finally, a summary of a recent survey of the potential fish resources of the lake is given in the discussion, and estimates of the 1965 catch at the north and south ends of the lake are compared with the findings of the survey. This showed that there is little danger of overfishing the Alestes baremose stocks of the Wanseko area at the 1965 rate of exploitation of the species, and that the total catch for 1965 at the south end of the lake was well below the estimated annual sustainable yield from the area.

The preservation of some East African freshwater fish

Quality changes during storage were investigated for several commercially important East African freshwater fish. Lates, Bagrus, Protopterus, Tilapia esculenta and T. nilotica were examined during storage in ice and at ambient temperature (250•C). After 24 hours at ambient temperature Lates and Bagrus were completely spoilt but Protopterus was still edible. In iced storage most fish were acceptable for at least 20 days. Organoleptic examination showed that T. nilotica was acceptable after 22 days storage in ice and that gutting was only marginally beneficial. Changes in physical appearance, which could form the basis of a fish inspection system, were recorded during storage. Possible chemical quality control indices were also investigated. It was found that total volatile bases and hypoxanthine are unlikely to be useful quality indices for the species studied with the possible exception of Lates. The bacterial counts of the flesh and skin of T. esculenta and T. niloticus were found to be low (a maximum of 10 organisms per sq cm of skin or per g of flesh) after 22 days storage in ice.

Measurement of the Interaction Between Recombinant I-domain from Integrin alpha 2 beta 1 and a Triple Helical Collagen Peptide with the GFOGER Binding Motif Using Molecular Force Spectroscopy.

The role of the collagen-platelet interaction is of crucial importance to the haemostatic response during both injury and pathogenesis of the blood vessel wall. Of particular interest is the high affinity interaction of the platelet transmembrane receptor, alpha 2 beta 1, responsible for firm attachment of platelets to collagen at and around injury sites. We employ single molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) to study the interaction of the I-domain from integrin alpha 2 beta 1 with a synthetic collagen related triple-helical peptide containing the high-affinity integrin-binding GFOGER motif, and a control peptide lacking this sequence, referred to as GPP. By utilising synthetic peptides in this manner we are able to study at the molecular level subtleties that would otherwise be lost when considering cell-to-collagen matrix interactions using ensemble techniques. We demonstrate for the first time the complexity of this interaction as illustrated by the complex multi-peaked force spectra and confirm specificity using control blocking experiments. In addition we observe specific interaction of the GPP peptide sequence with the I-domain. We propose a model to explain these observations.