Rat-liver chromatin has been separated into nuclease-sensitive\r\nand resistant fractions after mild digestion with DNAase II. The\r\nnuclease-sensitive material is further fractionated into Mg++-soluble\r\nand insoluble chromatin fractions. The kinetics of production of\r\nthese chromatin fractions have been investigated. After a brief\r\nenzyme treatment (5 min under standard conditions), 11% of the input\r\nchromatin DNA is found in the Mg++-soluble fraction. This DNA has a\r\nweight-average single strand length of about 400 nucleotides and, as\r\ndetermined by renaturation kinetics, comprises a subset of middle\r\nrepetitive and nonrepetitive DNA sequences of the rat genome. Cross-reassociation\r\nexperiments show that a fractionation of whole genomal\r\nDNA sequences has been achieved. Moreover, the Mg++-soluble fraction\r\nof liver chromatin is enriched in nonrepeated sequences coding for\r\nliver RNA but not for brain RNA. Fractionation does not depend on\r\nsome general property of chromatin but is specific with regard to the\r\ntemplate activity of the tissue from which the chromatin was obtained.\r\nThe Mg++-soluble, template-active fraction is enriched five-fold in\r\nDNA sequences complementary to RNA.
\r\n\r\nThe Mg++-soluble fraction is enriched in nonhistone chromosomal\r\nproteins and depleted in histone protein. Histone I (fl) is absent\r\nfrom the Mg++-soluble active fraction. About half of the DNA of both\r\nMg++-soluble and Mg++-insoluble fractions is resistant to prolonged\r\ndigestion with DNAase II or staphylococcal nuclease. The nuclease-\r\nresistant structures of inactive (Mg++-insoluble) chromatin are DNAhistone\r\ncomplexes which sediment at 11-13S. Two nuclease-resistant\r\nspecies are present in active chromatin. These particles sediment\r\nat 15 and 20S, respectively, and contain DNA, RNA, histone and nonhistone\r\nproteins. Thermal denaturation studies suggest that the\u00b7\r\nDNA of active chromatin is complexed primarily with nonhistone proteins.
", "doi": "10.7907/3ayy-cg55", "publication_date": "1976", "thesis_type": "phd", "thesis_year": "1976" }, { "id": "thesis:14363", "collection": "thesis", "collection_id": "14363", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09152021-165317789", "primary_object_url": { "basename": "Compton_JL_1975.pdf", "content": "final", "filesize": 38037918, "license": "other", "mime_type": "application/pdf", "url": "/14363/1/Compton_JL_1975.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Specific DNA Restriction Fragments as Internal Markers in the Electron Microscope: Superimposing the \u0278X174 Genetic Map on \u0278X174/S13 DNA Heteroduplexes", "author": [ { "family_name": "Compton", "given_name": "John Lee", "clpid": "Compton-John-Lee" } ], "thesis_advisor": [ { "family_name": "Sinsheimer", "given_name": "Robert L.", "clpid": "Sinsheimer-R-L" } ], "thesis_committee": [ { "family_name": "Sinsheimer", "given_name": "Robert L.", "clpid": "Sinsheimer-R-L" }, { "family_name": "Vinograd", "given_name": "Jerome", "clpid": "Vinograd-J" }, { "family_name": "Wood", "given_name": "William Barry", "clpid": "Wood-W-B" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Chapter I
\r\n\r\n\u0278X174 RF II* molecules were constructed in vitro by annealing circular \u0278X174 viral DNA and linear \u0278X174 complementary strand DNA. When a specific fragment of \u0278X174 RF purified from a digestion with the restriction endonuclease Hind from Hemophilus influenzae was included in the renaturation mixture \u0278X174 RF II molecules containing a loop corresponding in size to the fragment were seen. This was confirmed for three different DNA fragments.
\r\n\r\n\u0278X174 RF II molecules were constructed in the presence of two restriction fragments. In addition to molecules containing one loop corresponding in size to one of the fragments molecules containing two loops were seen. The two loops corresponded in size to the two restriction fragments and the distances between the two loops corresponded to the distances between the two fragments on the Hind cleavage map of \u0278X174 RF.
\r\n\r\nBy including two fragments of distinguishable size that map asymmetrically on the cleavage map in the renaturation mixture, \u0278X174 RF II molecules were constructed which could be oriented on the cleavage map. Using the two loops as internal markers the \u0278X174 genetic map was superimposed on the electron micrographs of these molecules.
\r\n\r\nChapter II
\r\n\r\n\u0278X174/S13 DNA heteroduplexes were constructed by annealing purified \u0278X174 complementary strand DHA and S13 viral DNA. The heteroduplexes were mounted for electron microscopy under a series of increasingly denaturing conditions and photographed. The micrographs were measured and oriented to form a map showing the progressive denaturation of various regions of the molecule.
\r\n\r\nSuch heteroduplex molecules were totally denatured and mixed with a denatured specific fragment of \u0278X174 RF* purified from a digestion with the restriction endonuclease Hind of Hemophilus influenzae. After renaturation the molecules were mounted for electron microscopy under standard conditions. Each of three different \u0278X174 RF fragments could be seen in a different; specific region of the heteroduplex and could be oriented with respect to the others to correspond to the known \u0278X174 cleavage map.
\r\n\r\nThe \u0278X174 genetic map was thus superimposed on the heteroduplex denaturation map by using the included fragments as reference points. Under mildly denaturing conditions three regions of the heteroduplex are conserved as double-stranded: two regions of about 10% of the \u0278X174 genome each in genes A and H and a region of about 25% of the \u0278X174 genome covering gene E and most of gene F. Under increasingly denaturing conditions only two small duplex regions of 2 and 4% are conserved, one each in genes E and F.
", "doi": "10.7907/nr2r-8c58", "publication_date": "1975", "thesis_type": "phd", "thesis_year": "1975" }, { "id": "thesis:9639", "collection": "thesis", "collection_id": "9639", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04012016-120503899", "primary_object_url": { "basename": "Konopka_rj_1972.pdf", "content": "final", "filesize": 25109976, "license": "other", "mime_type": "application/pdf", "url": "/9639/1/Konopka_rj_1972.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Circadian Clock Mutants of Drosophila melanogaster", "author": [ { "family_name": "Konopka", "given_name": "Ronald Jerome", "clpid": "Konopka-Ronald-Jerome" } ], "thesis_advisor": [ { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" } ], "thesis_committee": [ { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" }, { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" }, { "family_name": "Vinograd", "given_name": "Jerome", "clpid": "Vinograd-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Three mutants of Drosophila melanogaster have been isolated in which the free-running period of the circadian eclosion rhythm and the adult locomotor activity rhythm is affected. One mutant is arrhythmic, another has a short period of 19 hours, and the third has a long period of 28 hours. The mutants retain their phenotypes over the temperature range 18\u00b0 to 25\u00b0 C. All three mutants map near the tip of the X chromosome (distal to the centromere). By deficiency mapping, the short-period mutation has been localized to the 3B1-2 region. Complementation tests show that all three mutations affect the same functional gene.
\r\n\r\nAnalysis of activity rhythms of individual mosaic flies indicates that the site of action of the short-period mutation is probably located in the head of the fly. A few activity patterns of split-head and mixed-head mosaics appear to possess both mutant and heterozygous components, suggesting that the fly head may contain\r\ntwo complete clocks capable of maintaining their periodicities independently.
\r\n\r\nThe short-period mutation affects both the duration of the light-insensitive part of the oscillation and the degree to which the clock can be reset during the light-sensitive part of the oscillation.
\r\n\r\nBoth the short-period and long-period mutant eclosion rhythms can be entrained to a period of 24 hours by a 12:12 light-dark cycle having a light intensity at least two orders of magnitude greater than that required to entrain the normal rhythm. The arrhythmic mutant does not entrain under these conditions. In the presence of a temperature cycle, however, the arrhythmic mutant does entrain, but its rhythm damps out when the temperature cycle is removed.
\r\n\r\nEvidence is presented that Pittendrigh's two-oscillator model for the clock in D. pseudoobscura applies to D. melanogaster as well. The three clock mutations primarily affect the light- sensitive driving oscillator. The arrhythmic mutation appears to have eliminated the driving oscillator while leaving the temperature-sensitive driven oscillator relatively intact.
\r\n", "doi": "10.7907/R04B-3425", "publication_date": "1972", "thesis_type": "phd", "thesis_year": "1972" }, { "id": "thesis:3050", "collection": "thesis", "collection_id": "3050", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08082006-142417", "primary_object_url": { "basename": "Burke_pv_1971.pdf", "content": "final", "filesize": 25454241, "license": "other", "mime_type": "application/pdf", "url": "/3050/1/Burke_pv_1971.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Freezing Phycomyces Sporangiophores in Superfluid Helium for Ultrastructure Studies", "author": [ { "family_name": "Burke", "given_name": "Patricia Virginia", "clpid": "Burke-Patricia-Virginia" } ], "thesis_advisor": [ { "family_name": "Delbruck", "given_name": "Max", "clpid": "Delbr\u00fcck-M" } ], "thesis_committee": [ { "family_name": "Delbruck", "given_name": "Max", "clpid": "Delbr\u00fcck-M" }, { "family_name": "Goodstein", "given_name": "David L.", "clpid": "Goodstein-D-L" }, { "family_name": "Liepmann", "given_name": "Hans Wolfgang", "clpid": "Liepmann-H-W" }, { "family_name": "Vinograd", "given_name": "Jerome", "clpid": "Vinograd-J" }, { "family_name": "Van Harreveld", "given_name": "Anthonie", "clpid": "Van-Harreveld-A" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Knowledge of the ultrastructure of the growing zone of Phycomyces sporangiophores is a desirable adjunct to studies of their sensory physiology. This includes discovery of the type of organelles present and their spatial arrangement. Chemical fixation preserves some of the cell's organelles satisfactorily, but considerable disruption and dislocation occur. Physical fixation by freezing should preserve the spatial distribution of organelles within the cell if the freezing is rapid enough to prevent ice crystal formation. Ice crystal inhibitors do not penetrate the cell satisfactorily, so that crystal size is determined only by the freezing rate.
\r\n\r\nLiquid He II exhibits a quantum mechanical mechanism of heat transfer which is much more efficient than the normal, classical heat conduction in fluid. The heat transfer rate is qualitatively described by the Landau equations for liquid He II with additional terms (Gorter-Mellink mutual friction) to account for frictional forces within the liquid. For the case of a cylindrical heater, the maximum steady state heat transfer rate before film boiling occurs depends on the depth of the heater below the liquid surface (hydrostatic head), the pressure of the gas above the liquid, and the liquid temperature. For a gas pressure of 20 Torr or more in excess of the equilibrium vapor pressure, the appearance of the film boiling changes from a uniform gas film to a fine haze, presumably of tiny gas bubbles. If He4 gas is used to pressurize the liquid, the temperature of the liquid He bath rises to about 2 K and a layer of liquid He I forms at the gas-liquid interface. This layer of the He I grows at the expense of the bulk He II as heat is supplied by the warm gas. The phase boundary between the two liquid phases moves down through the liquid at about 5 cm/min. The conditions for optimal heat transfer occur immediately after the pressure increase. These conditions are a pressure excess of 20 Torr or more and a bath temperature of 1.9 - 2 K.
\r\n\r\nSporangiophores are suspended by an iron filing from an electromagnet in a special chamber (room temperature) above a helium cryostat. As soon as the pressure excess in the cryostat exceeds 20-60 Torr they are released and fall freely into the superfluid helium through a tube of heated gas. The sporangiophores are collected in a plastic beaker at the bottom of the cryostat and transferred to a liquid nitrogen storage dewar. Frozen sporangiophores are prepared for electron microscopy using freeze-substitution and freeze-fracture techniques.
\r\n\r\nThe thin sections and the freeze-fracture replicas show extensive ice crystal damage to stage IV sporangiophores which have a large central vacuole. Ice crystal damage is considerably less in stage I sporangiophores which have a much smaller vacuole. Presumably, the vacuole is responsible for the poor preservation of the ultra-structure.
\r\n", "doi": "10.7907/PY8S-BH09", "publication_date": "1971", "thesis_type": "phd", "thesis_year": "1971" }, { "id": "thesis:3051", "collection": "thesis", "collection_id": "3051", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08082006-151750", "primary_object_url": { "basename": "Young_et_1967.pdf", "content": "final", "filesize": 8998735, "license": "other", "mime_type": "application/pdf", "url": "/3051/1/Young_et_1967.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Structure and Synthesis of Bacteriophage Lambda DNA", "author": [ { "family_name": "Young", "given_name": "Elton Theodore, II", "clpid": "Young-Elton-Theodore" } ], "thesis_advisor": [ { "family_name": "Sinsheimer", "given_name": "Robert L.", "clpid": "Sinsheimer-R-L" } ], "thesis_committee": [ { "family_name": "Sinsheimer", "given_name": "Robert L.", "clpid": "Sinsheimer-R-L" }, { "family_name": "Weigle", "given_name": "Jean J.", "clpid": "Weigle-J-J" }, { "family_name": "Vinograd", "given_name": "Jerome", "clpid": "Vinograd-J" }, { "family_name": "Delbruck", "given_name": "Max", "clpid": "Delbr\u00fcck-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The research in this thesis represents an attempt to study the replication of bacteriophage lambda in terms of the structure of the vegetative DNA. A spheroplast assay for lambda DNA is described in Part I and several parameters affecting the efficiency of the assay are investigated. The biological activity of intracellular lambda DNA is then studied using this assay. In contrast to the results obtained with the transformation or helper assay, no eclipse of infectivity of vegetative lambda DNA occurs in the spheroplast assay. The DNA species responsible for most of the infectivity in the spheroplast assay of DNA extracted from infected, immune bacteria, is the fast-sedimenting \"super-coiled\" form of lambda DNA.\r\n\r\nIn Part II the structure and replication of the fast-sedimenting species of vegetative lambda DNA is examined physically and biologically. Its physical properties are those of a twisted, circular molecule each of whose two strands is closed upon itself. Using a mitomycin C technique to inhibit host DNA synthesis, the accumulation and semi-conservative replication of closed-circular lambda DNA specifically early in the infection is demonstrated with radio-isotopes. Biological analysis using the spheroplast assay verifies this conclusion. Chloramphenicol inhibits the synthesis of viral DNA at a lower-concentration than that needed to inhibit the synthesis of closed-circular DNA. Chloramphenicol does not prevent the conversion from viral to closed-circular DNA.\r\n\r\nThe data presented in Part III suggest that open-circular lambda DNA may act as a precursor to, both closed-circular and viral DNA. The closed-circular species is not a major precursor to viral DNA. Sedimentation analysis also reveals the existence of as yet unidentified intermediates in the replication of viral DNA.\r\n\r\nPart IV describes several methods for purifying intracellular lambda DNA and examines the biological activity of the purified DNA in the spheroplast assay. The circular DNA is infective before and after denaturation, whereas denaturation inactivates viral DNA. The increased infectivity of open-circular DNA after denaturation (relative to the native state) appears to be due to the appearance of biologicallyactive single-stranded rings of lambda DNA. The closed-circular DNA usually has the same infectivity before and after denaturation.", "doi": "10.7907/MHCY-S381", "publication_date": "1967", "thesis_type": "phd", "thesis_year": "1967" }, { "id": "thesis:3318", "collection": "thesis", "collection_id": "3318", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09032002-103837", "type": "thesis", "title": "Part I. Properties of Helical Polycytidylic Acid. Part II. Interactions of Purine with Proteins and Amino Acids. Part III. Binding of Basic Proteins to DNA", "author": [ { "family_name": "Akinrimisi", "given_name": "E. Olabisi", "clpid": "Akinrimisi-E-Olabisi" } ], "thesis_advisor": [ { "family_name": "Bonner", "given_name": "James Frederick", "clpid": "Bonner-J-F" } ], "thesis_committee": [ { "family_name": "Bonner", "given_name": "James Frederick", "clpid": "Bonner-J-F" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Horowitz", "given_name": "Norman", "clpid": "Horowitz-N" }, { "family_name": "Ts'o", "given_name": "Paul O. P.", "clpid": "Ts'o-Paul-O-P" }, { "family_name": "Vinograd", "given_name": "Jerome", "clpid": "Vinograd-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "This thesis is divided into three sections. The first part deals with the secondary structure of poly C in acid solution, as revealed by several of its physical-chemical properties in solution. The interpretation of the data was based on previous knowledge of the general properties of polynucleotides in solution and on the properties of poly C monomer.
\r\n\r\nThe second part of the thesis deals with the interaction of purine with the proteins. Conformation changes in the proteins are easily measurable in terms of changes in optical rotation even at the visible wavelength regions. Full use was made of this fact to study the nature of interaction of purine with the proteins. The most significant aspect of the studies is not the theoretical speculation on the mechanism of the interaction, but rather the possible practical applications of the findings. This is briefly considered in the discussion. The mechanism of urea. interaction with the proteins cannot be regarded as solved in spite of the voluminous literature on the subject. Similarly, the mechanism of purine interaction with proteins cannot be regarded as solved. The speculations presented at length on the mechanism of the interactions of purine or urea with the proteins, therefore, merely represent a simplified deduction from available data -- a deduction intended to stimulate more experiments and perhaps a modified interpretation of the nature of the interactions.
\r\n\r\nThe third part of the thesis presents initial findings on a very complex problem -- the relationship of polyvalent polymer-polymer interactions to some of their other physical-chemical and biochemical properties. The conclusions drawn from the data are not new or peculiar to current thinking about the nature of interaction of the histones or protamines to DNA. However, certain satisfactions are derived from the fact that it has been possible to carry out reliable physical-chemical measurements on the nucleohistone and nucleoprotamine systems by means of simple standard techniques. Such data are rare in the nucleohistone or nucleoprotamine literature. No doubt, the confirmation of more complex findings on these very important systems will often require these simple physical-chemical data.
\r\n", "doi": "10.7907/EFG9-2C60", "publication_date": "1964", "thesis_type": "phd", "thesis_year": "1964" } ]