The messenger RNA’s that encode the Proteins rat serum albumin (RSA) and rat alphafetoprotein (RAFP) have been purified to virtual homogeneity by a combination of immunoprecipitation of polysomes and other physical isolation methods. Radioactive cDNA copies of these mRNA’s have been prepared and used to monitor the changes in abundance of RSA- and RAFP-synthesizing polysomes in neonatal rat liver and in Morris hepatoms 7777, a rat liver tumor. These cDNA probes have also been used to determine the stability and reiteration frequency of their respective genes in various rat tissues.
The RSA mRNA sequence has been converted into a series of bacterial plasmids by recombinant DNA methodology, and the RSA gene has been isolated from a library of recombinant bateriophage. Restriction endonuclease site mapping, R-loop mapping, “Southern” blot and extensive nucleotide sequence determination have been employed to elucidate the organization of the cloned sequences.
The rat serum albumin gene has been found to be interrupted at fourteen locations by introns. The fifteen exons are spread over approximately 15,000 nucleotides of contiguous chromosomal DNA. The evolutionary history of albumin has been deduced by analysis of the patterns of internal periodic homology in this gene. Albumin apparently evolved by a series of at least three intragenic duplications followed by accumulation of many point mutations and small deletions. These events probably occurred over 300 million years ago. The rat alphafetoprotein gene has been shown to be related to the rat serum albumin gene by a common ancestor gene, and thus two (or possibly more) highly complex genes with many exons and many protein domains have evolved by duplication mechanisms. That this might be an important general source of the complexity of eukaryotic genomes is discussed.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/9V3M-QH52, author = {Stumph, William Edward}, title = {Gene enrichment using antibodies to DNA/RNA hybrids : mapping the ribosomal DNA of slime mold and rat}, school = {California Institute of Technology}, year = {1979}, doi = {10.7907/9V3M-QH52}, url = {https://resolver.caltech.edu/CaltechTHESIS:02182014-134518249}, abstract = {A novel method for gene enrichment has been developed and applied to mapping the rRNA genes of two eucaryotic organisms. The method makes use of antibodies to DNA/RNA hybrids prepared by injecting rabbits with the synthetic hybrid poly(rA)•poly(dT). Antibodies which cross-react with non-hybrid nucleic acids were removed from the purified IgG fraction by adsorption on columns of DNA-Sepharose, oligo(dT)-cellulose, and poly(rA)-Sepharose. Subsequent purification of the specific DNA/RNA hybrid antibody was carried out on a column of oligo(dT)-cellulose to which poly(rA) was hybridized. Attachment of these antibodies to CNBr-activated Sepharose produced an affinity resin which specifically binds DNA/RNA hybrids.
In order to map the rDNA of the slime mold Dictyostelium discoideum, R-loops were formed using unsheared nuclear DNA and the 178 and 268 rRNAs of this organism. This mixture was passed through a column containing the affinity resin, and bound molecules containing R- loops were eluted by high salt. This purified rDN A was observed directly in the electron microscope. Evidence was obtained that there is a physical end to Dictyostelium rDN A molecules approximately 10 kilobase pairs (kbp) from the region which codes for the 268 rRNA. This finding is consistent with reports of other investigators that the rRNA genes exist as inverse repeats on extra-chromosomal molecules of DNA unattached to the remainder of the nuclear DNA in this organism.
The same general procedure was used to map the rRNA genes of the rat. Molecules of DNA which contained R-loops formed with the 188 and 288 rRNAs were enriched approximately 150- fold from total genomal rat DNA by two cycles of purification on the affinity column. Electron microscopic measurements of these molecules enabled the construction of an R-loop map of rat rDNA. Eleven of the observed molecules contained three or four R-loops or else two R-loops separated by a long spacer. These observations indicated that the rat rRNA genes are arranged as tandem repeats. The mean length of the repeating units was 37.2 kbp with a standard deviation of 1.3 kbp. These eleven molecules may represent repeating units of exactly the same length within the errors of the measurements, although a certain degree of length heterogeneity cannot be ruled out. If significantly shorter or longer repeating units exist, they are probably much less common than the 37.2 kbp unit.
The last section of the thesis describes the production of antibodies to non-histone chromosomal proteins which have been exposed to the ionic detergent sodium dodecyl sulfate (SDS). The presence of low concentrations of SDS did not seem to affect either production of antibodies or their general specificity. Also, a technique is described for the in situ immunofluorescent detection of protein antigens in polyacrylamide gels.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/18PT-FD16, author = {Chong, Ming Ta}, title = {Investigations of chromatin bound enzymes}, school = {California Institute of Technology}, year = {1974}, doi = {10.7907/18PT-FD16}, url = {https://resolver.caltech.edu/CaltechETD:etd-11152005-155825}, abstract = {NOTE: Text or symbols not renderable in plain ASCII are indicated by […]. Abstract is included in .pdf document.
Part I. Rat liver chromatin contains a neutral protease with a marked preference for chromosomal proteins as substrates. The enzyme has been purified 705-fold from chromatin by salt extraction, chromatography on Bio-Rex 70, Sepharose 6B, calcium phosphate gel and QAE Sephadex. The enzyme has a molecular weight of 200,000 with two identical subunits of molecular weight 100,000. It attacks rat liver histones, NHC proteins and L-poly-lysine preferentially, is essentially inactive with rat liver cytosol proteins, and slowly degrades caseine, L-poly-arginine and protamines. The […] for histones is 0.5 mg/ml; for NHC proteins […] is 1 mg/ml. The enzyme is quite stable when stored at -20[degrees] for 4 months. Activity is diminished to 50% by heating to 62[degrees] for 15 min and totally destroyed at 70[degrees]. The enzyme has an optimal pH at 7.0 and a half maximal activity at pH 6.0, suggesting that a histidine residue is involved in catalysis. It is inhibited by DFP and PMSF, suggesting that a serine residue is involved. Hg++ inhibits enzyme activity, suggesting sulfhydryl group is important for enzyme activity. High salt (above 1M NaCl) inhibits enzyme activity completely but reversibly. The enzyme needs divalent ions as activators; especially potent is Mn++ (6-8 mM) which stimulates activity about 2 fold. The isolated enzyme appears to be similar to that responsible for the endogeneous degradation of histones in chromatin. The susceptibility of the five histone fractions to proteolysis is critically dependent upon whether or not the histones are complexed with DNA. In the intact nucleohistone four major histones are rather resistant to proteolytic attack, while histone I is rapidly attacked. If histones are freed from DNA all the histone molecules are attacked at about the same rate except histone I, which is relatively resistant.
Part II. Rat liver chromatin also contains a nonspecific esterase which cleaves the artificial substrate–[…]-tosyl arginine methyl ester (TAME). The enzyme has been purified to 510 fold from chromatin by salt extraction, chromatography on Bio-Rex 70, Sephadex G-200, calcium phosphate gel and SE Sephadex. The molecular weight of the purified enzyme is estimated to be about 15,000. The enzyme has an optimal pH at 8.2 and half maximal activities at 6.9 and 10.5, suggesting that histidine and lysine residue might be involved in catalysis. It is inhibited by DFP and PMSF, suggesting that a serine residue is involved. At high substrate concentrations inhibition is noted. The […] for TAME is 0.16 mM.
Part III. Ferritin tagged c-RNA molecules were hybridized to rat ascites nuclear DNA in an effort to map the arrangement of these sequences in the rat genome by electron microscopy. The ferritin acts as an electron dense marker. The interferritin distance is also the inter RNA distance and c-RNA hybridizes specifically to middle repetitive DNA sequences. Thus measuring the distances between ferritin molecules attached to c-RNA reveals that middle repetitive DNA sequences (about 300 nucleotides in length) were arranged either singly or in tandem and immediately followed by a unique sequence about 500-1500 nucleotides in length.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/XDYW-S154, author = {Holmes, David Salway}, title = {Studies on Nuclear RNA}, school = {California Institute of Technology}, year = {1973}, doi = {10.7907/XDYW-S154}, url = {https://resolver.caltech.edu/CaltechTHESIS:07192018-114123511}, abstract = {The isolation of giant nuclear RNA (HnRNA) from rat ascites cells is described. By the criteria of sedimentation through sucrose, formaldehyde and dimethyl sulfoxide, it is estimated that the majority of the radioactivity of giant HnRNA after a 30 minute pulse of 3H-uridine is associated with molecules in the range 5-10 x 106 daltons. In the electron microscope, under denaturing conditions, 84% (mass %) of giant HnRNA has a contour length of 4-9µ corresponding to a molecular weight of about 5-10 x 106 daltons.
Giant HnRNA has a “DNA-like” base composition (G+C = 46-54%) and has considerable secondary structure (ca. 60% helix conformation) as judged by its melting profile and reactivity with formaldehyde.
Rat nuclear DNA is characterized by its reassociation profile ((Na+) = 0.18 at 62°, Tm - 23°) as judged by chromatography on hydroxyapatite. Single-copy DNA (Cot 1/2 observed = 1.5 x 103) comprises 65% of the genome and 19% of the genome consists of sequences repeated an average 1,800 times (middle repetitive DNA, Cot 1/2 observed = 1.0). 9% of the genome (highly repetitive DNA) reassociates faster than is measured in these experiments (Cot 1/2 observed < 2 x 10-2).
Middle repetitive and single-copy DNA are isolated and characterized with respect to their reassociation kinetics and melting profiles. They reassociate with kinetics similar to the kinetics describing these components when they are present in total DNA. The reassociated single-copy DNA has a high thermal stability indicative of fidelity of base pairing; the reassociated middle repetitive DNA has a lower thermal stability which is probably attributable, in part, to base-pair mismatch.
Rat giant nuclear RNA (HnRNA, 5-10 x 106 daltons) is hybridized to isolated single copy or middle repetitive DNA ((Na+) = 0.18 at 62°) HnRNA hydbridizes to about 4.5% of the single-copy and 9.4% of the middle repetitive DNA. The Tms of single-copy and middle repetitive hybrids are 1-2° lower than those of the reassociated single-copy and middle repetitive DNA respectively. The DNA isolated from the single-copy or middle repetitive hybrids reassociates with kinetics similar to the input single-copy or middle repetitive DNA respectively. HnRNA is hybridized to total genomic DNA present in excess. 37% of the HnRNA hybridizes with kinetics (Cot 1/2 = 2.0 x 103) similar to single-copy DNa and 12% hybridizes with kinetics (Cot 1/2 = 5.6), a little more slowly than the major reassociating component of middle repetitive DNA.
A chromatin-associated RNA (cRNA) prepared from rat ascites cells hybridizes to about 16% of isolated middle repetitive and 1% of isolated single copy rat DNA. In a hybridization reaction to total DNA, present in excess, at least 50% of the cRNA hybridizes at an average rate similar to the major component of the middle repetitive DNA. These experiments indicate that the majority of cRNA consists of repetitive transcripts. Under conditions which assay essentially only repetitive transcripts cRNA hybridizes to about 4.7% and giant nuclear RNA (HnRNA) hybridizes to about 4.6% of total nuclear rat DNA immobilized on filters. The Tm of cRNA hybrids (73.5°) and HnRNA hybrids (75.5°) are considerably lower than the Tm of native rat DNA (85.5°). This lowering of Tm is probably attributable, at least in part, to base-pair mismatch. Under the same conditions of hybridization there is some hybridization competition for complementary DNA sites between cRNA and HnRNA, presumably between repetitive transcripts. Due to probable base-pair mismatch it is possible to infer only that there is a similarity between HnRNA and cRNA transcripts and not necessarily an identity.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/M8Q4-DB66, author = {Froehner, Stanley Charles}, title = {The Isolation, Purification and Characterization of Three RNA Polymerases from Novikoff Hepatoma Ascites Tumor}, school = {California Institute of Technology}, year = {1973}, doi = {10.7907/M8Q4-DB66}, url = {https://resolver.caltech.edu/CaltechTHESIS:07062018-115304742}, abstract = {DNA-dependent RNA polymerase has been isolated from nuclei of Novikoff hepatoma ascites tumor cells and resolved into three activities, designated Ia, Ib, and II, by a combination of phosphocellulose and DEAE cellulose chromatography. Ia and Ib have been further purified by sucrose density centrifugation. Both gradient profiles exhibit coincidence of the polymerase activity and protein peaks, suggesting that the two may be homogeneous enzymes. Ia migrates as a single species on non-denaturing polyacrylamide gel electrophoresis. SDS polyacrylamide gel electrophoresis indicates that Ia contains subunits of 170,000, 125,000, 69,000, 49,000, 44,000 and 37,000 molecular weights in equimolar ratios except for the 69,000 and 37,000 dalton subunits which may be present in two copies per enzyme molecule. A molecular weight of 600,000 for the enzyme calculated from the molecular weights of the subunits is in good agreement with that determined by exclusion chromatography. The probable molecular structure of Ib is subunits of 190,000 and 135,000 daltons, each present twice per enzyme molecule. The enzymological characterization of these three enzymes suggests that Ia and Ib are the nucleolar polymerases while II is nucleoplasmic. Ia and Ib are most active at low ionic strength with Mg++ on native DNA and are insensitive to α-amanitin. II prefers Mn++, high ionic strength, a denatured template and is inhibited by low concentrations of α-amanitin. A factor present in the material which does not bind to the DEAE cellulose column used in the purification scheme, stimulates the activity of all three of the enzymes. Ia and Ib are inactive at low enzyme concentrations in the absence of this factor. The active agent in the factor is probably a protein, since it is heat sensitive, and may be a subunit of the enzyme.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/6VGH-RZ17, author = {Elgin, Sarah Carlisle Roberts}, title = {Investigations of nonhistone chromosomal proteins}, school = {California Institute of Technology}, year = {1972}, doi = {10.7907/6VGH-RZ17}, url = {https://resolver.caltech.edu/CaltechTHESIS:04182016-080802139}, abstract = {
The major nonhistone chromosomal proteins (NHC proteins) are a group of 14-20 acidic proteins associated with DNA in eukaryotic chromatin. In comparisons by SDS gel electrophoresis (molecular weight sieving) one observes a high degree of homology among the NHC protein fractions of different tissues from a given species. Tissue-specific protein bands are also observed. The appearance of a new NHC protein, A, in the NHC proteins of rat liver stimulated to divide by partial hepatectomy and of rat ascites cells suggests that this protein may play a role in preparing the cell for division. The NHC proteins of the same tissue from different species are also very similar. Quantitative but not qualitative changes in the NHC proteins of rat uterus are observed on stimulation (in vivo) with estrogen. These observations suggest that the major NHC proteins play a general role in chromatin structure and the regulation of genome expression; several may be enzymes of nucleic acid and histone metabolism and/or structural proteins analogous to histones. One such enzyme, a protease which readily and preferentially degrades histones, can be extracted from chromatin with 0.7 N NaCl.
Although the NHC proteins readily aggregate, they can be separated from histone and fractionated by ion exchange chromatography on Sephadex SE C-25 resin in 10 M urea-25% formic acid (pH 2.5). Following further purification, four fractions of NHC protein are obtained; two of these are single purified proteins, and the other two contain 4-6 and 4-7 different proteins. These NHC proteins show a ratio of acidic to basic amino acids from 2.7 to 1.2 and isoelectric points from apparently less than 3.7 to 8.0. These isolated fractions appear more soluble and easier to work with than any whole NHC protein preparation.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/G15P-H973, author = {Firtel, Richard Alan}, title = {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}, school = {California Institute of Technology}, year = {1972}, doi = {10.7907/G15P-H973}, url = {https://resolver.caltech.edu/CaltechTHESIS:04112016-144248288}, abstract = {
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 (Cot1/2 (pure) 0.28) and the single-copy DNA 70% (Cot1/2(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 (Cot1/2 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.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/D6DK-F035, author = {McConnell, David John}, title = {Investigations on (i) Chromosomal Ribonucleic Acid of Ascites Tumour, (ii) RNA Polymerase of E. Coli}, school = {California Institute of Technology}, year = {1971}, doi = {10.7907/D6DK-F035}, url = {https://resolver.caltech.edu/CaltechTHESIS:05252018-095226235}, abstract = {
PART I: When chromatin isolated from rat ascites cells is dissociated in the presence of high salt and the chromosomal proteins separated from the DNA by buoyant density centrifugation, a portion of the RNA contained in the chromatin remains associated with the chromosomal proteins. This RNA (chromosomal RNA) is characterized by its small size, s20,w = 3.3S, its high content of dihydroribothymidine and its ability to form hybrid with about 4% of the nuclear DNA. It appears to have no sequences in common with ascites transfer, ribosomal, or messenger RNA.
A class of RNA (cytoplasmic 3S RNA) with similar properties but associated with the cytoplasmic proteins has also been isolated. This RNA hybridizes to about 2% of the nuclear DNA and contains very few, if any, sequences not also contained in chromosomal RNA. This fraction of RNA is however unable to compete with about 50% of the sequence present in chromosomal RNA indicating that a large portion of chromosomal RNA is confined to the chromatin. A further class of RNA associated with the nuclear sap proteins appears to be identical to the RNA associated with cytoplasmic proteins.
A further class of RNA (nuclear 3S RNA) with hybridization properties similar to the cytoplasmic 3S RNA has been isolated from the nuclear sap. It hybridises to a lower extent than chromosomal RNA and is strongly competed by cytoplasmic 3S RNA.
PART II. RNA polymerase, more than 95% pure, has been prepared from E. coli D-10 and B. It is free from ribonuclease and phosphatase. It carries out poly A synthesis on E. coli or ascites tumour DNA but not on T7 DNA. Phosphate incorporation (TCA precipitable) from the γ phosphate of ATP in the absence of primer may be caused by a slight contamination with polyphosphate kinase. This can be controlled. Initiation of synthesis on T7, E. coli and ascites tumour DNA differ in the response to the σ sub-unit. RNA synthesized on T7 DNA can initiate with A or G. A salt- or rifam- picin-stable complex between T7 DNA can be formed when a single nucleoside triphosphate (commercial reagent) is present, but requires a small amount of propagation. After purification of the nucleotides the efficiency of complex foration is greatly reduced.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/RTXG-PQ59, author = {Griffith, Jack Denney}, title = {High resolution electron microscope studies of chromosomal fibers}, school = {California Institute of Technology}, year = {1970}, doi = {10.7907/RTXG-PQ59}, url = {https://resolver.caltech.edu/CaltechTHESIS:08042015-105247195}, abstract = {
Techniques are described for mounting and visualizing biological macromolecules for high resolution electron microscopy. Standard techniques are included in a discussion of new methods designed to provide the highest structural resolution. Methods are also discussed for handling samples on the grid, for making accurate size measurements at the 20 Å level, and for photographically enhancing image contrast.
The application of these techniques to the study of the binding of DNA polymerase to DNA is described. It is shown that the electron micrographs of this material are in agreement with the model proposed by Dr. Arthur Kornberg. A model is described which locates several active sites on the enzyme.
The chromosomal material of the protozoan tetrahymena has been isolated and characterized by biochemical techniques and by electron microscopy. This material is shown to be typical of chromatin of higher creatures.
Comparison with other chromatins discloses that the genome of tetrahymena is highly template active and has a relatively simple genetic construction.
High resolution electron microscope procedures developed in this work have been combined with standard biochemical techniques to give a comprehensive picture of the structure of interphase chromosome fibers. The distribution of the chromosomal proteins along its DNA is discussed.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/K05B-3W74, author = {Lee, Chong Sung}, title = {I. Studies on bacteriophage DNA’s and minicircular DNA’s in M. lysodeikticus and E. coli 15. II. Flow dichroism of DNA solutions}, school = {California Institute of Technology}, year = {1970}, doi = {10.7907/K05B-3W74}, url = {https://resolver.caltech.edu/CaltechTHESIS:08102015-160156690}, abstract = {
Part I
Chapter 1…..A physicochemical study of the DNA molecules from the three bacteriophages, N1, N5, and N6, which infect the bacterium, M. lysodeikticus, has been made. The molecular weights, as measured by both electron microscopy and sedimentation velocity, are 23 x 106 for N5 DNA and 31 x 106 for N1 and N6 DNA’s. All three DNA’s are capable of thermally reversible cyclization. N1 and N6 DNA’s have identical or very similar base sequences as judged by membrane filter hybridization and by electron microscope heteroduplex studies. They have identical or similar cohesive ends. These results are in accord with the close biological relation between N1 and N6 phages. N5 DNA is not closely related to N1 or N6 DNA. The denaturation Tm of all three DNA’s is the same and corresponds to a (GC) content of 70%. However, the buoyant densities in CsCl of Nl and N6 DNA’s are lower than expected, corresponding to predicted GC contents of 64 and 67%. The buoyant densities in Cs2SO4 are also somewhat anomalous. The buoyant density anomalies are probably due to the presence of odd bases. However, direct base composition analysis of N1 DNA by anion exchange chromatography confirms a GC content of 70%, and, in the elution system used, no peaks due to odd bases are present.
Chapter 2…..A covalently closed circular DNA form has been observed as an intracellular form during both productive and abortive infection processes in M. lysodeikticus. This species has been isolated by the method of CsC1-ethidium bromide centrifugation and examined with an electron microscope.
Chapter 3…..A minute circular DNA has been discovered as a homogeneous population in M. lysodeikticus. Its length and molecular weight as determined by electron microscopy are 0.445 μ and 0.88 x 106 daltons respectively. There is about one minicircle per bacterium.
Chapter 4…..Several strains of E. coli 15 harbor a prophage. Viral growth can be induced by exposing the host to mitomycin C or to uv irradiation. The coliphage 15 particles from E. coli 15 and E, coli 15 T- appear as normal phage with head and tail structure; the particles from E. coli 15 TAU are tailless. The complete particles exert a colicinogenic activity on E.coli 15 and 15 T-, the tailless particles do not. No host for a productive viral infection has been found and the phage may be defective. The properties of the DNA of the virus have been studied, mainly by electron microscopy. After induction but before lysis, a closed circular DNA with a contour length of about 11.9 μ is found in the bacterium; the mature phage DNA is a linear duplex and 7.5% longer than the intracellular circular form. This suggests the hypothesis that the mature phage DNA is terminally repetitious and circularly permuted. The hypothesis was confirmed by observing that denaturation and renaturation of the mature phage DNA produce circular duplexes with two single-stranded branches corresponding to the terminal repetition. The contour length of the mature phage DNA was measured relative to φX RFII DNA and λ DNA; the calculated molecular weight is 27 x 106. The length of the single-stranded terminal repetition was compared to the length of φX 174 DNA under conditions where single-stranded DNA is seen in an extended form in electron micrographs. The length of the terminal repetition is found to be 7.4% of the length of the nonrepetitious part of the coliphage 15 DNA. The number of base pairs in the terminal repetition is variable in different molecules, with a fractional standard deviation of 0.18 of the average number in the terminal repetition. A new phenomenon termed “branch migration” has been discovered in renatured circular molecules; it results in forked branches, with two emerging single strands, at the position of the terminal repetition. The distribution of branch separations between the two terminal repetitions in the population of renatured circular molecules was studied. The observed distribution suggests that there is an excluded volume effect in the renaturation of a population of circularly permuted molecules such that strands with close beginning points preferentially renature with each other. This selective renaturation and the phenomenon of branch migration both affect the distribution of branch separations; the observed distribution does not contradict the hypothesis of a random distribution of beginning points around the chromosome.
Chapter 5….Some physicochemical studies on the minicircular DNA species in E. coli 15 (0.670 μ, 1.47 x 106 daltons) have been made. Electron microscopic observations showed multimeric forms of the minicircle which amount to 5% of total DNA species and also showed presumably replicating forms of the minicircle. A renaturation kinetic study showed that the minicircle is a unique DNA species in its size and base sequence. A study on the minicircle replication has been made under condition in which host DNA synthesis is synchronized. Despite experimental uncertainties involved, it seems that the minicircle replication is random and the number of the minicircles increases continuously throughout a generation of the host, regardless of host DNA synchronization.
Part II
The flow dichroism of dilute DNA solutions (A260≈0.1) has been studied in a Couette-type apparatus with the outer cylinder rotating and with the light path parallel to the cylinder axis. Shear gradients in the range of 5-160 sec.-1 were studied. The DNA samples were whole, “half,” and “quarter” molecules of T4 bacteriophage DNA, and linear and circular λb2b5c DNA. For the linear molecules, the fractional flow dichroism is a linear function of molecular weight. The dichroism for linear A DNA is about 1.8 that of the circular molecule. For a given DNA, the dichroism is an approximately linear function of shear gradient, but with a slight upward curvature at low values of G, and some trend toward saturation at larger values of G. The fractional dichroism increases as the supporting electrolyte concentration decreases.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Davidson, Norman R.}, } @phdthesis{10.7907/DQN1-VN19, author = {Smart, John Edward}, title = {Studies on the role of histones in the structure and function of chromatin}, school = {California Institute of Technology}, year = {1970}, doi = {10.7907/DQN1-VN19}, url = {https://resolver.caltech.edu/CaltechTHESIS:09032015-162652124}, abstract = {Studies on the dissociation of histones from chromatin by increasing concentrations of sodium deoxycholate (DOC) have shown that histrone II is removed at lowest concentrations of DOC, while slightly higher concentrations remove histones III and IV. Still higher concentrations remove histone I.
The complete separation of chromatin and 14C-DOC by sucrose sedimentation indicated that the binding of DOC to chromatin is readily and completely reversible.
The dissociation of histones from chromatin by increasing concentrations of related cholanic acids and some of their conjugated derivatives were studied. The results suggested that the driving force for the interaction between the cholanic acid anion and histones is the lowering of the activity coefficient of the cholanic acid anion which occurs when it is partially removed from solution by interaction with hydrophobic regions of the positively charged histones.
The role of histones in the structure of chromatin has been studied by comparing the effects of selective removal of histones from chromatin by increasing concentrations of DOC with those caused by NaCl (removes histone I at lowest concentrations, while higher concentrations remove histones II, III, and IV). Properties studied included thermal denaturation, sedimentation velocity, flow dichroism, relaxation times of molecules oriented in a flow field, and the irreversible disruption of a 130 S, cross-linked component of sheared chromatin. The data indicated that none of the structural or chemical parameters with which these properties are correlated show a dependence on the presence of one particular histone fraction.
The template activity (ability to prime a 0.2 M KC1 DNA-dependent RNA synthesis system catalyzed by E. coli RNA polymerase) increases from that of native chromatin (approximately 25 per cent of that pure DNA) to that of pure DNA in a fashion which shows a nearly linear relationship to the amount of histone coverage of the template. The precipitability of partially dehistonized chromatin samples in 0.15 M NaCl shows a large dependence on the presence of histone I.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/HQQY-2J93, author = {Shih, Thomas Yu-tzong}, title = {I. Chromosomal RNA of Calf Thymus Chromatin. II. The Template Properties of DNA-Polypeptide Complexes. III. Studies on DNA Complexes with Purified Histone Fractions}, school = {California Institute of Technology}, year = {1969}, doi = {10.7907/HQQY-2J93}, url = {https://resolver.caltech.edu/CaltechTHESIS:03312017-130624894}, abstract = {
Part I. Chromosomal RNA of Calf Thymus Chromatin
Calf thymus chromatin is shown to contain an associated chromosomal RNA as do the chromatins of other species. The chromosomal RNA of calf thymus chromatin is present in an amount of 1% of that of DNA. The purified material eluted from DEAE-Sephadex column at a NaCl concentration of 0.56 M as do the chromosomal RNA’s of other organisms. Its average chain length by end-group assay is approximately 40 nucleotides and it contains approximately 3-4 dihydrouridylic acid residues per chain. Calf thumus chromosomal RNA is associated with chromosomal protein in a form not dissociable by high salt concentration.
Part II. The Template Properties of DNA-Polypeptide Complexes
DNA complexes with poly-L-lysine, poly-L-arginine and protamine were prepared by a salt gradient dialysis. The complexes possess the stoichiometry of one lysine or arginine residue per nucleotide residue as determined from the biphasic melting profiles. The template activity of a complex in support of RNA synthesis in the presence of excess RNA polymerase and required substrates is proportional to its fractional content of free DNA segments. The complexed DNA region is quantitatively blocked and does not act as template. Kinetic analysis of the template behavior reveals two different modes of inhibition by the polypeptides. If the template is in a finely dispersed state, it is available to the enzyme as shown by the fact that the equal template concentrations of complex and of pure DNA are required for half saturation of a given amount of enzyme (K). Inhibition of RNA synthesis is, we propose, due to interference with local untwisting of DNA. If the template is in a highly aggregated state, K is drastically increased and it is unavailable to the enzyme. The several species of histone molecules normally complexed with DNA in the eucaryotic organisms differ among themselves in content of lysine and arginine. The present studies show that the arginine residues are as effective as the lysine residues in abolishing DNA template activity.
Part III. Studies on DNA Complexes with Purified Histone Fractions
Well-defined DNA complexes with calf thymus histones Ia, Ib, IIb and IV have been prepared by a salt gradient dialysis in the presence of 5 M urea. The complexes with subequivalent histone/DNA ratio exhibit biphasic melting profiles. Tm,1 is the melting of free DNA segments, and Tm,2 that of the histone-complexed regions. Tm,2 is characteristic for each DNA-basic protein complex. Tm,2(°C) of, the complexes in 2.5 x 10-4 M sodium EDTA, pH 8.0 is as follows: DNA, 47.2; chromatin, 74.3; DNA-histone Ia, 75.4; DNA-histone Ib, 76.3; DNA-histone IIb, 81.5; DNA-histone IV, 83.7; DNA-protamine, 92.5; DNA-polyarginine, 98.0; and DNA-polylysine, 99.5. The stoichiometric ratio (histone lysine plus arginine to nucleotide) of the equivalent complexes as detennined from the biphasic melting profiles is DNA-histone Ia and Ib, 0.8; DNA-histone IIb, 1.2 and DNA-histone IV, 1.5. The general shape of UV spectrum of DNA is not changed by complexing with various histone species. DNA-histone IV complex is inactive in priming RNA synthesis in E.coli RNA polymerase system. Possible structures of the DNA-binding parts of histone molecules have been discussed and illustrated with CPK molecular models in the case of histone IV (pp. 157, 158).
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/APSP-5851, author = {Dahmus, Michael Edward}, title = {I. Studies on Chromosomal RNA. II. Effect of Hydrocortisone on the Template Activity of Liver Chromatin}, school = {California Institute of Technology}, year = {1968}, doi = {10.7907/APSP-5851}, url = {https://resolver.caltech.edu/CaltechETD:etd-10032002-102109}, abstract = {Chapter I.
Chapter I of this thesis is concerned with the isolation and characterization of ascites chromosomal RNA. The isolation of rat ascites chromosomal RNA as well as some of its physical and chemical properties are described in Section 1. Chromosomal RNA is characterized by its small size, lack of amino acid acceptor activity, and, relative to transfer RNA, low content of methylated bases. It has a base composition similar to that of ascites ribosomal RNA and is not labeled when the cells are exposed to a short pulse of ³²P. An RNA (3S cytoplasmic RNA), with properties similar to those of chromosomal RNA but contained in the cytoplasm, has also been isolated.
Section 2 is concerned with the hybridization properties of ascites chromosomal RNA to denatured ascites nuclear DNA. Chromosomal RNA hybridizes to about 4% of ascites nuclear DNA and has no sites in common with ascites messenger RNA. 3S cytoplasmic RNA hybridizes to about 20 of ascites nuclear DNA and contains no sequences not also contained in chromosomal RNA. The 3S RNA contained in the nuclear sap is homologous to 3S cytoplasmic RNA. Therefore, it appears that a fraction of chromosomal RNA is confined to the chromatin while the remainder is homologous to an RNA contained in both the cytoplasm and nuclear sap.
Chapter II.
This chapter is concerned with the effect of hydrocortisone on the template activity of liver chromatin. Hydrocortisone administered to an adrenalectomized rat causes a two- to threefold increase in the rate of RNA synthesis in the liver. Chromatin isolated from the liver of hydrocortisone-treated rats possesses a 30 % greater template activity for DNA-dependent RNA synthesis than does chromatin isolated from control rats. The difference in template activity is abolished by removal of the proteins associated with the DNA. Hydrocortisone, administered to isolated purified chromatin, does not alter its template activity.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/5GTY-NC55, author = {Sadgopal, Anil}, title = {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}, school = {California Institute of Technology}, year = {1968}, doi = {10.7907/5GTY-NC55}, url = {https://resolver.caltech.edu/CaltechTHESIS:01042016-083704592}, abstract = {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.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/C95J-PM40, author = {Fambrough, Douglas McIntosh, Jr.}, title = {Studies on Plant and Animal Histones}, school = {California Institute of Technology}, year = {1968}, doi = {10.7907/C95J-PM40}, url = {https://resolver.caltech.edu/CaltechTHESIS:04262018-155935430}, abstract = {
The histones of the pea plant, Pisum sativum L., and of calf thymus have been fractionated and further characterized in order to determine the extent of heterogeneity and the main chemical features of these basic nuclear proteins. Histones were fractionated by chromatography on Amberlite CG-50 and by preparative disc electrophoresis. The resulting highly purified histone fractions were further characterized by analytical disc electrophoresis, amino acid analysis, N-terminal and C-terminal analyses, and the preparation of tryptic peptide maps. Calf thymus histones Ia, Ib, IIb1, III and IV (f1, f1, f2a2, f3, and f2a1 in the nomenclature of Johns, Phillips and Butler) and pea bud histones Ib, IIb, III and IV were obtained as electrophoretically pure components and each appears to be a single molecular species on the basis of N-terminal and C-terminal analysis and the number of tryptic peptides. The total number of major histones in calf thymus appears to be six, in pea bud, eight. The apparent heterogeneity of calf thymus histones demonstrated by disc electrophoresis is largely due to the formation of histone III complexes by disulfide bridges between histone III monomers. While calf thymus histone III contains two cysteines per molecule pea bud histone III contains but one and thus can form only dimers.
For each calf thymus histone there appears to be an homologous pea bud histone. It is proposed that the homologous pea and calf histones are related by evolution and perform identical biological functions. This hypothesis is based upon remarkable similarities in chromatographic and electrophoretic behavior, amino acid compositions, terminal amino acids, and in some cases even peptide maps of corresponding pea and calf histones. Peptide maps of the arginine-rich histone III contain 29 soluble peptides of which 26 are common to calf and pea; maps of histone IV contain 32 peptides of which 27 are common to calf and pea.
By chromatography and electrophoresis the histones of various pea tissues are qualitatively identical to those of pea bud. There are, however, quantitative differences and these have been accurately measured by a method of quantitative analytical disc electrophoresis. Young pea cotyledons contain only about a third as much lysine-rich histone as do mature cotyledons. Exploratory experiments on the synthesis of histone in pea cotyledons as a function of development and in relation to other macromolecular parameters are described in an appendix.
The dissociation of histones from pea bud nucleohistone by NaCl was studied, employing quantitative disc electrophoresis. Histone I (lysine-rich) is selectively dissociated by 0.5 M NaCl and the remaining histones are non-selectively dissociated primarily over the range 0.5 - 1.5 M NaCl. These data are compared with data for the dissociation of calf thymus histones from nucleohistone by NaCl and the general similarities are noted.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/X7X3-QH34, author = {Tuan, Dorothy Y.H.}, title = {Part I. Interaction of DNA and histone in nucleohistone; Part II. Dormancy associated with repression of genetic activity}, school = {California Institute of Technology}, year = {1967}, doi = {10.7907/X7X3-QH34}, url = {https://resolver.caltech.edu/CaltechETD:etd-10042002-162144}, abstract = {
PART I. INTERACTION OF DNA AND HISTONE IN NUCLEOHISTONE
Chapter 1. SELECTIVE DISSOCIATION OF HISTONE FROM NUCLEOHISTONE
With increasing concentration of NaCl solution, an increasing amount of histone is dissociated from dissolved nucleohistone. The dissociated histone fractions are identified by gel electrophoresis. The lysine rich histone fraction I is dissociated from nucleohistone in the range 0.3-0.5 F NaCl; slightly lysine rich histone II in the range 0.8-1.6 F NaCl; arginine rich histone III+IV in the range 0.9-1.6 F NaCl. The results suggest that both electrostatic and non-electrostatic interactions contribute to the strength of binding between DNA and histones.
Chapter 2. OPTICAL ROTATORY DISPERSION STUDIES ON HISTONES
The optical rotatory dispersion spectra of histones free and in reconstituted nucleohistone (in which histone is complexed to DNA) are recorded. By the criterion of optical rotatory dispersion at wavelengths below 220 mu, histone I is the least helical of the histones, histone II the most helical. The helicity of DNA-bound histones in reconstituted nucleohistone is greater than that of free histones, but the order, histone II most helical and histone I least, is still preserved.
Chapter 3. OPTICAL ROTATORY DISPERSION STUDIES ON THE DNA OF NATIVE NUCLEOHISTONE AND OF PARTIALLY DEHISTONIZED NUCLEOHISTONES
The conformation of DNA in native nucleohistone is altered by the DNA-histone interaction. The dissociation of histone I does not produce significant conformational change in DNA of nucleohistone but removal of histone II and of histone III+IV bring about changes which cause the conformation of DNA in nucleohistone to resume virtually that characteristic of free DNA. The possibility of DNA supercoiling in nucleohistone is discussed.
PART II. DORMANCY ASSOCIATED WITH REPRESSION OF GENETIC ACTIVITY
Chapter 1. THE DORMANCY OF POTATO BUDS
Chromatin of the buds of dormant potato tubers is almost totally incapable of the support of DNA-dependent RNA synthesis in the presence of added exogenous RNA polymerase. The chromatin of non-dormant buds of potato tubers (in which dormancy has been broken by treatment with ethylene chlorohydrin) is highly effective in the support of DNA-dependent RNA synthesis by added exogenous RNA polymerase. It is therefore concluded that the genetic material of the buds of dormant potato tubers is largely in a repressed state, and that the breaking of dormancy is accompanied by derepression of the genetic material.
Chapter 2. THE DORMANCY OF ONION BULBS
The chromosomal material of non-growing and non-dividing onion buds possesses template activity in support of in vitro DNA-dependent RNA synthesis. If we define dormancy not only by the absence of visible growth and mitotic division, but also by the lack of ability to direct in vitro DNA-dependent RNA synthesis, potato buds are then dormant but onion buds are not. And the block to onion bud growth must lie somewhere else than in the repression of genetic material.
Chapter 3. ISOLATION OF GLADIOLUS CHROMATIN
Gladiolus corms contain very little isolatable chromatin material and the isolated chromatin is highly contaminated by the presence of starchy material.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/1276-DQ97, author = {Widholm, Jack Milton}, title = {Physical and biological studies of crab deoxyribonucleic acid}, school = {California Institute of Technology}, year = {1966}, doi = {10.7907/1276-DQ97}, url = {https://resolver.caltech.edu/CaltechETD:etd-09232002-114928}, abstract = {NOTE: Text or symbols not renderable in plain ASCII are indicated by […]. Abstract is included in .pdf document.
The native dAT components of C. antennarius and of C. borealis DNA have been separated in pure form by a new mercury binding technique.
Two further DNA species, not heretofore described, are apparently also present in C. antennarius DNA. They have melting points and buoyant densities intermediate between those of dAT and of the major DNA component.
A series of tests using optical density melting electrophoresis, buoyant density, electron microscopic, band sedimentation, exonuclease I susceptibility and actinomycin D inhibition techniques are employed to show that melted and renatured C. antennarius dAT remains partially denatured. After strand separation and recombination approximately 7% of the base pairs do not reform. These unpaired bases are predominantly guanine and cytosine (G and C) since exonuclease I hydrolysis assays and RNA synthesis inhibition by actinomycin D give the results which would be expected on this basis. The C. antennarius dAT contains 3.5% G and C bases. C. borealis dAT with 2.5% GC behaves similarly to C. antennarius dAT, except that the denatured structure is less evident, apparently because of the lower GC content.
When the aforementioned tests are applied to synthetic dAT, which contains no G or C bases, the results indicate that this alternating dAT copolymer is fully hydrogen bonded both before and after melting.
dAT is present to the extent of 10-11% in another Cancer species, C. anthonyi.
dAT is found in all C. antennarius tissues and is localized in the cell nucleus.
Studies with the E. coli RNA polymerase system show that dAT is copied at a rate 2.6 times as fast as is the main DNA component. dAT also has a higher affinity for the enzyme.
Actinomycin D inhibits the transcription of native primers more than it does that of melted ones. Incorporation of GC into RNA is preferentially inhibited.
Protamine, bistones Ib, IIb, III and IV, listed in order of decreasing effectiveness, inhibit the RNA synthesis supported by DNA. These basic proteins inhibit incorporation of A and U preferentially indicating preferential binding of the inhibitors to AT rich regions of the template.
The RNA synthesized from the dAT template by E. coli RNA polymerase is double-stranded and melts at 67[degrees]C. This rAU has an S value of 5-6 after ribonuclease T1 treatment. This result is due to the low G content of the dAT, 1.8%.
Actinomycin D inhibition studies with crab hepatopancreas tissue indicate that rAU is not synthesized at an appreciable rate in vivo if at all.
The RNA from this tissue also contains no rAU as shown by melting and by T1 treatment. The rAU if present would melt sharply and be detectable and would show up as 5-6 S material in sucrose density gradient centrifugation of the hepatopancreas RNA after T1 treatment.
Chromatin is less active as template for RNA synthesis than is whole crab DNA and the chromatin dAT component is particularly repressed.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/HRVF-V763, author = {Filner, Philip}, title = {Studies on exponential cultures of plant cells}, school = {California Institute of Technology}, year = {1965}, doi = {10.7907/HRVF-V763}, url = {https://resolver.caltech.edu/CaltechETD:etd-04082003-112136}, abstract = {The properties of tobacco cells growing exponentially in a chemically defined liquid medium were investigated for the purpose of characterizing the exponential plant cell culture and to explore its potential as a developmental model system.
The cells multiply exponentially with a generation time of 2 days, and exponential multiplication persists for 4.5 generations. The replication of DNA was studied by means of N[superscript 15] density labeling. All the DNA in the cells replicates in about one generation, and replication proceeds by a semiconservative mechanism. The average cell properties of the culture are not constant during the exponential phase, indicating that the population is not in a steady state condition. The number of cells per group, the water content per cell, the soluble protein content, and the rate of RNA synthesis vary. Enzyme levels of the soluble protein fraction do not vary in parallel with the protein content, but rather they vary in their own characteristic ways.
The changes in cell properties are related to changes in the chemical environment which the cells themselves bring about. The cells deplete the medium of phosphorus after three generations, and they deplete it of potassium and nitrogen by the end of the exponential phase. Nitrogen is the growth limiting nutrient, and nitrogen may be supplied either as nitrate or as a complete amino acid mixture. The nitrate reductase (NR) level in the culture falls as the nitrate supply is depleted, so that the enzyme must be induced before the cells can grow when subcultured into a medium in which nitrate is the sole nitrogen source. There is a lag in the onset of mitosis which correlates with the time necessary for NR induction. The initial and terminal events of the exponential phase have been tentatively identified as the induction of NR and the depletion of nitrate, respectively.
The regulation of NR involves both substrate induction and end-product repression. NR is induced by nitrate and repressed by a complete amino acid mixture in proportion to the ability of the mixture to meet the nitrogen requirement of the cells. If amino acids are available at a sub-optimal level, and nitrate is also present, sufficient NR is induced and nitrate reduced to bring the nitrogen supply up to the optimal level.
The amino acids may be classed as repressors or derepressors, according to their action in the regulation of NR. The derepressors are arginine, lysine, cysteine and isoleucine. A single repressor inhibits the growth of the cells on a nitrate medium as a consequence of the condition of nitrogen starvation which results from NR repression. In the presence of a repressor and an appropriate derepressor, NR is induced and the cells grow. Since the growth of the cells can be controlled through the NR regulatory mechanism, it has been suggested that this mechanism may be used by the whole organism to regulate the growth of its parts.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/G6WY-AS68, author = {Morré, D. James F.}, title = {Biochemistry of cell extension}, school = {California Institute of Technology}, year = {1963}, doi = {10.7907/G6WY-AS68}, url = {https://resolver.caltech.edu/CaltechTHESIS:11022012-144309062}, abstract = {By subjecting various plant tissues to conditions which influence growth, such as treatment with the growth hormone, 3-indoleacetic acid (IAA), changes in certain cytoplasmic constituents were observed which parallel simultaneous changes in the mechanical properties of the cell wall.
Treatment of maize roots for one hour in the presence of concentrations of IAA that normally promote shoot growth and, therefore, inhibit root growth followed by measurement of the deformability of the rapidly elongating region under artificially imposed load, demonstrated a concentration-dependent, IAA-induced plasticity component, not observable in untreated roots. The response is transient; half-maximal plasticity is induced by ca. 5 x 10^(-7) M IAA, one hour after treatment (a concentration comparable to that of Avena sections). Increased deformability is paralleled by increased growth of root sections, also transient, with maximum IAA-induced increase in growth rate coinciding with the maximum for increased wall plasticization. The initial response of maize roots to IAA, therefore, resembles that of Avena coleoptiles both qualitatively and quantitatively. The end result, however, is to effectively shorten the period over which the root can elongate.
Associated with increased plasticity and growth rate of maize roots is rapid formation (or maintenance) of a protein-bound carbohydrate fraction. Disruption of the complex by such agents as extremes of pH or organic solvents results in increased turbidity of aqueous solutions and acquisition of solutility properties characteristic of lipides. Results of preliminary characterization studies also suggest that material is of lipoprotein origin.
Evidence for a similar fraction, increased in amount by treatment of the tissue with IAA, has been extended to include Avena coleoptiles, pea epicotyls and pea embryo axes. In addition to lipide-soluble components, these fractions contain approximately equimolar quantities of carbohydrate (including hexose) and of esterified phosphate. The material, therefore, has been designated as a protein-bound glycolipide (PGL).
A cytoplasmic origin of PGL is suggested. Also associated with the 2- to 4-fold increase in amount of protein-bound PGL is a decrease in the heat coagulability of cytoplasmic proteins. Similarily, a portion of an apparent IAA-induced increase in acid phosphatase activity may be attributable to increased stabilization as well. An electron microscopic survey of IAA effects on the fine structure of subcellular organelles revealed no major structural changes, however, the number of vesicles associated with the central Golgi structure of both maize roots and Avena sections may be increased by IAA treatment.
A study of cell wall pectic constituents has revealed that although versene-soluble pectin does represent a solubility class distinct from hot water-soluble pectin, the conditions for extraction do not correspond to those of the classical residual pectin fraction. The existence of pectin in the cold buffer-soluble, 70% ethanol-insoluble fraction of Avena coleoptiles (cold water-soluble pectin) seems doubtful, however.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/GV9J-HV27, author = {Studier, Frederick William}, title = {Studies on the DNA of bacteriophage T7}, school = {California Institute of Technology}, year = {1963}, doi = {10.7907/GV9J-HV27}, url = {https://resolver.caltech.edu/CaltechTHESIS:11092012-141743346}, abstract = {
The molecular weight of T7 DNA was determined to be 15-17 x 10^6 by the method of sedimentation equilibrium in a CsCl density gradient. Possible sources of error in the optical reproduction of the concentration distribution in the cell were considered and evaluated. Comparison of the above molecular weight with the weight of the total DNA content of T7 phage determined by Davison and Freifelder (22) suggests: 1) that the entire DNA content of T7 bacteriophage can be released into solution as a single molecule and 2) that the density gradient method for determining molecular weights of homogeneous DNA samples is accurate within 10-20%.
The denaturation and renaturation of T7 DNA was studied using formamide as the denaturing agent. The complete cycle of denaturation and renaturation can thus be studied at room temperature without degradation of the primary structure of DNA.
Kinetics of denaturation were found to be essentially independent of concentration but were not first order, the rate falling off with time. Irreversible denaturation does not commence until the DNA has reached 80-90% of its full hyperchromicity. Samples of DNA isolated from denaturing conditions appear to be mixtures of fully denatured molecules and molecules which exhibit native characteristics to a high degree. Stirring solutions of DNA under denaturing conditions markedly accelerates the rate of denaturation. It seems possible that the molecular configuration of DNA molecules in intermediate states of denaturation may be a factor in stability to denaturation.
Optimally renatured T7 DNA has 85-90% of the native hyperchromic effect and bands at a slightly denser position than native DNA in a CsCl density gradient. Preliminary studies indicate second order kinetics for the renaturation reaction.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/ZQ00-1H14, author = {McCalla, Dennis Robert}, title = {I. Chemical study of necrotic corn mutants. II. Metabolism of a kinin. III. The chemical nature of an insect gall growth factor}, school = {California Institute of Technology}, year = {1961}, doi = {10.7907/ZQ00-1H14}, url = {https://resolver.caltech.edu/CaltechETD:etd-04102006-135936}, abstract = {
Part I:
The leaves of nec rd (a single gene necrotic maize mutant) become necrotic when they are exposed to light for a few days. Extensive analysis has failed to reveal any difference in chemical composition between leaves of normal plants and of homozygous nec rd plants before any necrotic symptoms are visible (prenecrotic leaves). Treatment of nec rd seedlings with various metabolites (B vitamins, purines, pyrimidines, amino acids, etc.) did not prevent the appearance of necrosis. The rate of photosynthesis of prenecrotic leaves is normal at low light intensities but 20 to 50% of normal at saturating light intensity. C14O2 feeding experiments indicate that the carbon fixing reactions function normally in the mutant. Hill reaction rates are also similar in mutant and normal plants, as is the metabolism of labelled inorganic phosphate. CMU, which specifically inhibits photosynthesis to the extent of about 90%, delays the onset of visible necrotic damage and reduces the severity of subsequent necrotic symptoms. It is suggested that the nec rd lesion is in some reaction associated with photosynthesis and that it causes the accumulation of one or more toxic substances. These lower rate of photosynthesis and damage cell membranes. The necrotic phenotype would appear to be the result of the breakdown of cell compartmentalization.
Part II:
The kinin, 6-benzylaminopurine (benzyladenine), is converted to a number of low molecular weight materials by senescing leaves of Xanthium pensylvanicum Wall.(cocklebur). A major product is the riboside, benzyladenosine, which has been identified by comparison of its properties with those of benzyladenylic synthesized enzymatically using E. coli nucleoside phosphorylase and by degradative studies. The ribotide, benzyladenylic acid, also appears to be present. Labelled adenylic, guanylic and inosinic acids are produced, as are small amounts of adenine and guanine. Substantial amounts of label are also found in urea and in a ureide. Small amounts of labelled adenylic and guanylic acids are found in the RNA of the leaf, but benzyladenylic acid itself does not appear to be incorporated into RNA in measurable amounts.
Part III:
Low molecular weight material obtained from excised accessory glands of Pontania pacifica (gall-wasp) promotes the growth of Pontania galls on Salix alba (willow). Paper chromatographic analysis has indicated that uridine, uric acid and two unidentified adenine containing compounds are prominent constituents of this mixture. Uric acid and the two adenine containing compounds substantially increase the growth rate of small galls from which the larva has been removed while uridine has slight growth promoting activity. Various related compounds (e.g. adenosine, adenine and guanosine) also have growth-promoting activity. It appears likely that such compounds play an important role in the growth and development of Pontania galls.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/1A8H-5F84, author = {Davern, Cedric Inglis}, title = {I. Chemotherapy of High Temperature Inhibition of Plant Growth. II. Studies of the Relationship between Tobacco Mosaic Virus Infection and the DNA Metabolism of Tobacco Leaves. III. The Conservation of Microsomal RNA in Escherichia coli}, school = {California Institute of Technology}, year = {1959}, doi = {10.7907/1A8H-5F84}, url = {https://resolver.caltech.edu/CaltechETD:etd-01272006-132352}, abstract = {Part I. Chemotherapy of High Temperature Inhibition of Plant Growth: The effects of biochemical supplements on the growth of five subterranean clover varieties grown under supra-optimal temperature conditions were studied. Some evidence of a chemotherapeutic effect of the high temperature growth inhibition was obtained. Part II. Studies on the Relationship between Tobacco Mosaic Virus Infection and the DNA Metabolism of Tobacco Leaves: A comparison of the DNA metabolisms of uninfected and TMV infected excised tobacco leaves, using p(32)-orthophosphate incorporation into the DNA as a measure of its metabolism, indicated that the DNA metabolism is not affected by TMV infection. This result was corroborated by the results of studies on the effect of 5-fluorouracil, a specific inhibitor of DNA synthesis, on the multiplication of TMV in tobacco-leaf discs. Although partial inhibition of TMV multiplication was observed, the absence of inhibition reversal thymidine, indicated that the mechanism of TMV inhibition probably did not involve a specific block of DNA synthesis. Finally unsuccessful attempts were made to see if intact host DNA was necessary for TMV infection by treating tobacco-leaf discs with DNAase. Part III. The Conservation of Microsomal RNA in Escherichia coli: A uniformly C13 N15 labeled exponentially growing culture of E. coli was transformed to light isotope substrates, and the metabolic fate of the pre-transfer synthesized heavy isotope microsomal RNA molecules followed by means of equilibrium sedimentation analysis of the RNA molecules in a density gradient. The results demonstrated complete conservation of the heavy isotopes by the pre-transfer RNA molecules remaining intact after transfer.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/K6BH-7R13, author = {Albersheim, Peter}, title = {Metabolism of the Pectic Substances}, school = {California Institute of Technology}, year = {1959}, doi = {10.7907/K6BH-7R13}, url = {https://resolver.caltech.edu/CaltechETD:etd-01312006-131728}, abstract = {This thesis examines pectic metabolism by oat coleoptile sections. Pectin accounts for 5 per cent of the dry weight of such sections. Approximately 5 per cent of the pectin is cold water soluble–70 per cent alcohol insoluble. Approximately 50 per cent of the carboxyl groups of this fraction are methyl esterified. Hot water solubilizes from the cell wall a highly esterified pectin fraction which represents 15 per cent of the total. In the remaining hot water insoluble pectin, only 30 per cent of the pectic carboxyl groups are combined as esters.
Pectins, once formed, are metabolized very slowly. There is little or no mixing of the various pectic fractions during a 15-hour period.
The methyl ester group of pectin is supplied by the methyl group of methionine. Oat coleoptile sections, either intact or as homogenates, form S-methylmethionine and methionine sulfoxide from methionine. Both of these compounds are also active as methyl donors for the formation of pectic esters.
Indoleacetic acid accelerates both incorporation of the methyl of methionine into methyl ester moieties and the incorporation of glucose into galacturonic acid residues of water soluble pectins. This increase is a measure of an accelerated rate of pectin synthesis. Indoleacetic acid does not increase the rate of synthesis of the water insoluble pectins.
Evidence is presented which favors the supposition that the methyl ester groups are formed before polymerization of the galacturonic acid residues.
In vitro incorporation of the methyl of methionine into methyl ester groups of pectin has not been achieved. Limited success has been obtained with the in vitro incorporation of glucose into galacturonic acid residues of water soluble pectins.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/SXP9-4527, author = {Thompson, Guy Allen}, title = {The Biosynthesis of Carotenes in the Tomato}, school = {California Institute of Technology}, year = {1959}, doi = {10.7907/SXP9-4527}, url = {https://resolver.caltech.edu/CaltechETD:etd-02232006-084220}, abstract = {NOTE: Text or symbols not renderable in plain ASCII are indicated by […]. Abstract is included in .pdf document. Ripening tomatoes were injected with 1C(14)-acetic acid or 2C(14)-mevalonic(3,5-dihydroxy-3-methylvaleric) acid and, after various incubation times, the carotenes were extracted and purified. The pattern of labeling among the carotenes leads to the conclusion that the more unsaturated pigments are not formed by the dehydrogenation of less unsaturated carotenes, but, rather, that most carotenes are synthesized by independent pathways, although from common precursors. 2C(14)-Mevalonic acid was found to be incorporated into the carotene fraction with high efficiency. Most of the radioactivity was found by chromatographic separation of the polyenes to be associated with the phytoene fraction. Purification of this fraction yielded phytoene of little radioactivity and an unknown compound of specific activity higher than that of any purified carotene. This compound, designated as fraction II, has been shown to be a 20-carbon-atom isoprenoid hydrocarbon containing a pair of conjugated double bonds. Its probable structure is given below. […] Fraction II is rapidly synthesized from mevalonic acid in both ripening and immature tomatoes. A complete pathway for the formation of the carotenes from mevalonic acid is proposed. It is postulated that fraction II is formed throuhghout the life of the fruit and is continuously converted to an as yet uncharacterized polyene, which yields carotenes at the time of ripening. The incorporation into carotenes of C(14)O(2), 1C(14)-glucose, uniformly C(14)-labeled-glucose, and uniformly C(14)-labeled leucine is described, and the patterns of labeling are compared with those obtained using mevalonate or acetate.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/CJQ9-P816, author = {Park, Roderic Bruce}, title = {I. The Biosynthesis of Open Chain Terpenes in Plants. II. Fractionation of the Stable Carbon Isotopes in Plants}, school = {California Institute of Technology}, year = {1958}, doi = {10.7907/CJQ9-P816}, url = {https://resolver.caltech.edu/CaltechETD:etd-01252006-084039}, abstract = {I: Open chain terpene synthesis in plants was studied by measurement of the incorporation of potential intermediates into the rubber of the rubber producing plants Taraxacum kok saghyz and Hevea brasiliensis. Intact plants incorporate 1-C14-acetate and 2-C14-acetate into rubber without randomization of the label. [Beta]-Methylcrotonic acid was found to be an ineffective rubber precursor in intact plants. Enzymatic experiments were performed using Hevea latex as a source of enzyme. C14-acetate is rapidly incorporated into a volatile, non-acidic, non-polar substance in this system under anaerobic conditions. C14-acetate is not incorporated into rubber. Mevalonic acid is rapidly incorporated into rubber in this system. Partial degradation of the rubber indicates that no randomization occurs during incorporation. This result suggests that mevalonic acid is on the pathway of terpene synthesis in plants. II: The two stable carbon isotopes, C12 and C13, occur in nature in the ratio of about ninety to one. Various workers have shown that this ratio is not fixed, but may vary by as much as 5%. Interestingly enough, this variation is not random. Carbon reservoirs such as limestone, atmospheric CO2, land plants, algae and coal all exhibit characteristic C13/C12 ratios. This section of the dissertation is concerned with the differences between the C13/C12 ratios of plants and those of the carbon sources from which such plants have grown. Both algae and terrestrial plants have smaller C13/C12 ratios than those of dissolved carbonates and atmospheric CO2 respectively. The magnitude of this fractionation was determined for tomato plants by growing the plants from seed in CO2 of known isotopic composition. Separation of the plant material into its component chemical constituents showed that only the lipid fraction differed markedly in C13/C12 ratio from that of the plant as a whole. The lipid fraction is enriched in C12 and possesses a C13/C12 ratio similar to that of petroleums derived from land plants. A similar relation was found to exist between marine algae, their lipids, and petroleums of marine origin. The CO2 evolved by plant respiration is slightly enriched in C13 as compared to the plant. This process apparently closely related to the C12 enrichment in lipid fractions. A possible mechanism for fractionation of C13 and C12 in photosynthesis is suggested. This suggestion is supported by observations of the C13/C12 ratio of CO2 dissolved in higher plants and by determination of the fractionation which occurs during fixation of CO2 by the photosynthetic carboxylation enzyme.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick and Epstein, Samuel}, } @phdthesis{10.7907/GVR0-N928, author = {Clark, John Magruder}, title = {Studies on Amino Acid Activation and Protein Synthesis}, school = {California Institute of Technology}, year = {1958}, doi = {10.7907/GVR0-N928}, url = {https://resolver.caltech.edu/CaltechETD:etd-10072004-105753}, abstract = {This thesis concerns the overall subject of protein synthesis in plants. Several different experimental approaches to the problem are described. The initial studies concern the incorporation of amino acids into tissue sections and homogenates. Tissue sections show a constant rate of incorporation of labeled amino acids into protein for periods of time up to two hours. The rate of incorporation then decreases with time. Conversely, tissue homogenates show ever increasing rates of incorporation of labeled amino acids into protein. This incorporation is indicative of incorporation of labeled amino acids into bacteria within the homogenate rather than incorporation into plant protein. Sterile homogenate preparations do not show the kinetics of incorporation shown by other homogenate preparations. The incorporation of labeled amino acids into acid insoluble material obtained from sterile tissue homogenates is a function of the washing procedure used in the assay. Amino acid activation has been studied by a hydroxylamine trapping reaction and by pyrophosphate exchange. Plant tissues contain amino acid activating enzymes which may be detected by either method of assay. The pyrophosphate exchange assay is however more sensitive. The hydroxamate assay suffers from some added complications common to plant systems. The specific nature of amino acid activation in plant preparations has been studied with enzymes isolated from spinach leaves. The activation reaction requires ATP and amino acids, the products being pyrophosphate and probably a mixed anhydride between the 5’ phosphate of AMP and the carboxyl group of the amino acid. The enzymes occur in all plant tissues tested and appear to be a part of the soluble fraction of cells. Purification procedures have been developed which make it possible to remove the free amino acids from the preparations. The purified preparation is capable of activating all of the naturally occurring (in protein) amino acids except serine. The possible activation of serine is-not excluded. Preliminary evidence is presented which indicates that each amino acid is activated by its own specific amino acid activating enzyme. The possibility of linking the process of amino acid activation to protein synthesis is considered. Crude particulate containing preparations of spinach exhibit an ATP and RNA dependent incorporation of leucine. Attempts to obtain an amino acid dependent exchange of AMP into ATP with spinach preparations were unsuccessful. Even so it has been possible to demonstrate an ATP dependent RNAase sensitive incorporation of labeled leucine into acid and alcohol washed material.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/SFDB-W284, author = {Burrows, Vernon Douglas}, title = {Studies on Translocation}, school = {California Institute of Technology}, year = {1958}, doi = {10.7907/SFDB-W284}, url = {https://resolver.caltech.edu/CaltechETD:etd-10082004-111812}, abstract = {Studies have been made on the translocation of C14-labeled solutes (2,4-D, 2,4,6-T and sugar(s)) and labeled solvent (THO or H2O18) in the red kidney bean. Transport of 2,4-D can be controlled by regulating the supply of carbohydrate in the leaves. For the first six hours following treatment of leaves with 2,4-D, the amount of 2,4-D transported to the epicotyl increases linearly with time. Over short time intervals the amount of 2,4-D transported is linearly related to the concentration applied. Over longer time intervals high concentrations of 2,4-D tend to depress transport somewhat. Transport of 2,4-D does not however saturate at concentrations which saturate the growth process of the plant. Essentially the same amount of 2,4-D is transported to the epicotyl of plants grown under 1000 and 2000 f.c. of light. Growth of the epicotyls induced by equivalent amounts of 2,4-D is two to four times larger in plants grown under 2000 than in those grown under 1000 f.c. of light, however. The compounds 2,4-D and 2,4,6-T are equally well absorbed by bean leaves and travel at the same speed in the phloem. The amount of 2,4,6-T which enters the phloem of the leaf, per unit time, is less than the amount of 2,4-D which so enters. TIBA applied as a pre-treatment to petioles inhibits the transport of C14-labeled 2,4-D, 2,4,6-T and sugars (predominantly sucrose). The inhibition of sugar movement may be used to interpret the inhibitory effect of TIBA on 2,4-D and 2,4,6-T transport. Foliarly applied tritium labeled (THO) and O18-labeled (H2O18) water are transported downward in bean seedlings. The carbohydrate status of the leaf does not govern the transport of labeled water in the same manner as it governs 2,4-D transport. The transport of THO takes place equally well or better in girdled as in normal plants. Movement of tritium apparently takes place in the xylem rather than in the phloem.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/3BNE-T071, author = {Labouriau, Luiz Fernando Gouvêa}, title = {Studies on the Initiation of Sporangia in Ferns}, school = {California Institute of Technology}, year = {1958}, doi = {10.7907/3BNE-T071}, url = {https://resolver.caltech.edu/CaltechETD:etd-10082004-105932}, abstract = {
A survey of available data in the literature shows that initiation of sporangia in ferns is not strictly localized, but is possible in many areas of the fern organism, such as the gametophytes, the first leaves and several areas of the leaf of the adult plant, which do not have to be contiguous.
In the usual sites of initiation, sporangia show correlations with the leaf veins and they may be replaced by a variety of alternative differentiations, such as cell proliferations, vegetative buds and aposporous prothalli.
Initiation and differentiation of sporangia may be arrested in several stages, both in natural conditions and experimentally. Some of the experimental procedures that produce this effect use changes in environmental factors.
Experimental results recorded in the literature indicate a day-neutral behavior in several species and a qualitative short-day behavior in one species (Salvinia natans).
The results of experiments reported in this thesis show that Asplenium bulbiferum is a quantitative long-day plant with a critical night of 23 hours at 20°C. In this species adventitious buds are capable of producing sporangia on their leaves, while attached to the adult leaf, but this capacity is lost upon isolation of the buds.
In Osmunda claytoniana determination of the sporophylls was found to be caused by processes that are independent of those that determine the cataphylls and to be enhanced by long days and high temperatures.
Salvinia rotundifolia was found to behave as a short-day plant at temperatures above 20°C and as a long-day plant at 17°C. Initiation of sporangia in this species responds also to daily thermoperiodicity.
Regnellidium diphyllum was found to develop sporocarps as a response to seasonal and to daily thermoperiodicity. This response is quantitatively modified by photoperiodism.
These results are discussed in relation to the available data on the differentiation of vascular tissues, on heteroblastic leaf development, photoperiodism and thermoperiodicity.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/2PSQ-9548, author = {Cleland, Robert Erskine}, title = {The Hormonal Control of Cell Wall Properties}, school = {California Institute of Technology}, year = {1957}, doi = {10.7907/2PSQ-9548}, url = {https://resolver.caltech.edu/CaltechETD:etd-07012004-102944}, abstract = {A technique has been developed for the separation of auxin action and cell elongation. Avena coleoptile sections are treated with auxin under conditions of nonexpansion. This is followed by expansion of the sections in water containing an inhibitor which blocks all auxin action. Under aerobic conditions, auxin initiates a loosening of the cell wall. The loosening can then persist over short periods of non-expansion under anaerobic conditions. The loosening can be abolished by general metabolic inhibitors such as KCN or counteracted by an auxin-independent stiffening process. The loosening is due to an increase in the plasticity of the cell wall. Ethionine administered during either phase of the process inhibits the residual auxin effect, suggesting that transmethylation is involved in the increase in plasticity. The metabolism of the cell wall components has been investigated by the use of labeled glucose and labeled methionine. The only effect of auxin which was found was an increased rate of incorporation of label from both glucose and methionine into the methyl ester groups of pectin. This increase is abolished by the same inhibitors which abolish the auxin-induced cell wall loosening. It is proposed that the increase in rate of pectin methyl ester incorporation is an integral part of the cell wall loosening process. Incorporation of label from methionine C-14 into the pectin fraction has also been achieved with homogenates of Avena coleoptile sections although the rate is markedly decreased by homogenization. A procedure has been developed for obtaining reproducible results with such homogenates. Dialysis of the homogenate supernatant after solubilization of the pectin by boiling has proven satisfactory. About 85 percent of the label from methionine C-14 incorporated into the pectin of intact tissues is in the form of esterified methyl groups. Upon homogenization of the tissues, however, only 20 percent of the label is located in the methyl groups.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/17XT-6E12, author = {Ts’o, Paul On Pong}, title = {On Plant Myosin}, school = {California Institute of Technology}, year = {1956}, doi = {10.7907/17XT-6E12}, url = {https://resolver.caltech.edu/CaltechETD:etd-07152004-154314}, abstract = {A systematic study has been carried out both on the cellular and the molecular level of the mechanism of protoplasmic streaming in the slime mold, Physarum polycephalum. This study is based on the hypothesis that ATP-protein interaction is an important feature of the mechanism. ATP has been found to have a liquefying effect on the gel structure of the streaming plasmodium. Furthermore, when ATP is allowed to react with protoplasmic material extracted from plasmodia under proper conditions, the viscosity of the solution decreases immediately. This abrupt decrease in viscosity of the protoplasmic solution is followed by a slow recovery. The active protein which is responsible for ATP induced viscosity lowering reaction has been identified by studies of viscosity, electrophoresis and ultracentrifuge. It has been purified to 75% by salt precipitation and differential centrifugation. It is proposed that this protein be termed myxomyosin because of its similarity to actomyosin. Myxomyosin is found to complex with RNA to ca. 9% of its own weight. The RNA does not appear to be essential for the ATP-protein interaction process. RNA does, however, exert a great influence on the physical states of myxomyosin in solution. Myxomyosin preparations possess an ATPase activity. The turnover number of this enzymatic activity is small, namely, 30. A refractory state of myxomyosin has been found. Immediately after myxomyosin reacts with a large excess of ATP, the system enters into a refractory state which persists during the recovery of the viscosity level. In this state, the protein is not sensitive to ATP. Viscosity, sedimentation, flow birefringence and electron microscopy studies reveal that myxomyosin is a rod-like molecule with a weight average molecular weight of 6 millions [plus or minus] 10% and with a range of 4.9 to 10 millions. The molecule possesses a diameter of 50 A [plus or minus] 5 A and a most frequent length of 4000 A with a range of 3000 to 6000 A. The effect of ATP on myxomyosin has been investigated by viscosity, sedimentation, flow birefringence, electron microscopy and electrophoresis studies. There is no indication that myxomyosin breaks down into small units or suffers an extensive change in shape when it reacts with ATP. The present data are best understood on the basis that in the absence of ATP, myxomyosin aggregates in a concentration-dependent manner. The binding of ATP to the myxomyosin moiety reduces the aggregation of the monomers. A proposed mechanism of protoplasmic streaming, suggested by the above observations is presented.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/VV4M-DG13, author = {Salisbury, Frank Boyer}, title = {Kinetic Studies on the Physiology of Flowering}, school = {California Institute of Technology}, year = {1955}, doi = {10.7907/VV4M-DG13}, url = {https://resolver.caltech.edu/CaltechETD:etd-01202004-162406}, abstract = {Xanthium pennsylvanicum is induced to flower by exposure to a single uninterrupted dark period equal to or exceeding some minimum duration (ca. 9 hours). A system of stages of floral bud development is described, which gives a quantitative measurement of the degree of induction caused by various treatments. A number of factors which may affect the degree of induction are investigated, and it is concluded that age of the leaf and light intensity before and after induction strongly influence the effects of a given inductive dark period, while other factors are less important in this respect. Methods in the use of auxin are also described.
Auxin is shown to inhibit induction in the leaf, but to promote floral bud development after induction is complete. Applied auxin will replace a requirement for the presence of active buds after and just before induction.
The induced state is discussed, and it is proposed that this condition consists of a given concentration of florigen in the plant which is maintained at the level brought about by the act of induction.
Concentration curves indicate that auxin acts in floral inhibition such as it does in other auxin-induced phenomena. Applied auxin has little effect upon the critical night length, but inhibits the rate of florigen synthesis which follows. Auxin is most effective when applied two to three hours after the beginning of the dark period, and its effectiveness decreases up until translocation of florigen out of the leaf is complete. Light interruption of the dark period is most effective after 8 hours. Light interruption and auxin are additive in their inhibitory effect.
It is suggested that the act of induction consists of at least three phases: transformation of photo-receptor pigment, preparatory reaction, and hormone synthesis. A possible mechanism of auxin action through destruction of florigen is discussed.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/NB9G-Q853, author = {Rogers, Bruce Joseph}, title = {Studies on the Amino Acid Metabolism of Higher Plants}, school = {California Institute of Technology}, year = {1955}, doi = {10.7907/NB9G-Q853}, url = {https://resolver.caltech.edu/CaltechETD:etd-01202004-155710}, abstract = {It has been known for some time that the protein level of detached leaves decreases as a result of the excision. The possibility that detached leaves are unable to form certain amino acids has been advanced as a cause for the decrease in the protein level. This hypothesis was tested in the present work. The incorporation with time of carbon-14 from C14-carboxyl-labeled acetate and C14-uniformly-labeled sucrose into the free and protein-bound amino acids of excised organs of red kidney bean (Phaseolus vulgaris) was studied. Of sixteen amino acids investigated, all were found to have incorporated carbon atoms from both acetate and sucrose in the excised leaves, stems and roots. On the basis of this work, it is felt that the decrease in protein level in detached leaves is not due to an inability of leaves to synthesize amino acids. The incorporation of radioactive amino acids (produced in vivo) into leaf, stem and root protein of red kidney bean was studied. It was found that radioactive amino acids are incorporated into root protein much more rapidly than into leaf or stem protein. The enzymatic decarboxylation and oxidation of a number of amino acids was studied in a water extract prepared from acorn squash. Great variation in the relative rates of reaction for the amino acids was demonstrated for both the decarboxylative and oxidative reactions. The partial N-terminal amino acid sequence of pancreatic trypsin inhibitor was found to be arginyl-phenylalanine. It is suggested that the system used would be applicable for the determination of the sequence of small peptides such as those discovered in the work with red kidney bean.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick and Went, Frits W. and Borsook, Henry}, } @phdthesis{10.7907/CPFB-ZF49, author = {Bernhard, Richard Allan}, title = {Studies on the Mode and Mechanism of Action of alpha-Chymotrypsin}, school = {California Institute of Technology}, year = {1955}, doi = {10.7907/CPFB-ZF49}, url = {https://resolver.caltech.edu/CaltechETD:etd-11212003-114338}, abstract = {NOTE: Text or symbols not renderable in plain ASCII are indicated by […]. Abstract is included in .pdf document. The enzyme-inhibitor dissociation constants, i. e. , K[subscript I] values, for alpha-chymotrypsin and two competitive inhibitors, indole-2-carboxylate and cinchoninamide, have been determined at pH 7. 9 and 25[degrees]. A new colorimetric procedure for the determination of proteolytic activity employing the reaction of ninhydrin with ammonia has been developed. A study has been made of the effect of buffer species and ions upon the course of alpha-chymotrypsin catalyzed hydrolyses. It has been demonstrated for the case at hand that K[subscript S] is essentially independent of buffer species and ionic strength. The discrepancies between values of k[subscript 3], evaluated in the presence of THAM-HCl buffers and in the presence of phosphate buffers, appear to be due principally to the effects of the increased ionic strength of the phosphate buffers. A number of enzyme-inhibitor dissociation constants have been evaluated in different buffer systems. During the evaluation of K[Subscript I] values for anionic, bifunctional, competitive inhibitors of alpha-chymotrypsin, it was determined that the presence of phosphate buffers apparently increased the affinity of the enzyme for the inhibitor. A possible mechanism for this phenomenon has been proposed and has been supported by experimental observations. It has been demonstrated that in a typical alpha-chymotrypsin catalyzed hydrolysis, the reaction proceeds in solution insofar as can be experimentally determined, and that wall effects are unimportant within the limits of experimental error. An investigation has been made of the possible use of dilatometry as a means of following the course of enzyme catalyzed hydrolyses. A number of instruments have been developed and discussed. Some typical data have been experimentally determined and analyzed in the usual manner.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Niemann, Carl G.}, } @phdthesis{10.7907/G9FF-M178, author = {Goldacre, Peter Lionel}, title = {Destruction of Indole-3-Acetic Acid by Plant Tissues}, school = {California Institute of Technology}, year = {1953}, doi = {10.7907/G9FF-M178}, url = {https://resolver.caltech.edu/CaltechETD:etd-05012003-110235}, abstract = {This paper concerns those systems present in the epicotyls of eliolated pea seedlings which inactivate the plant hormone indole-3-acetic acid (I.A.A.). It consists of two sections which deal with (a) the enzyme system collectively known as I.A.A. oxidase and (b) a group of dialyzable substances which sensitize the photodestruction of I.A.A. I.A.A. oxidase, which behaves as a flavoprotein coupled to a peroxidase is shown to have a partial cofactor requirement. Two alternative systems are postulated. The two systems are differentiated on the basis of their response to Mn++ and to 2,4-dichlorophenol (D.C.P.) and by their change in relative concentrations upon exposure of the seedlings to red light. D.C.P. is shown to increase the activity of I.A.A. oxidase at low concentrations. The mechanism of the effect is studied in detail. Although D.C.P. is a powerful inhibitor of catalase, which inhibits I.A.A. oxidase, it is concluded that the enhancement of I.A.A. oxidase by D.C.P. is not due to its inhibition of catalase. Probably D.C.P. is acting similarly to the native cofactor. D.C.P. increases the in vivo destruction of I.A.A. by some tissues, not by others. It is suggested as a useful tool for studying altered I.A.A. level in a tissue. The dialyzate from epicotyl brei contains at least four components which sensitize the inactivation of I.A.A. in the light but not in the dark. Blue light is the most effective. The kinetics of the action of the dialyzate has been studied. The active material resides principally in the buds. Preliminary methods of purification have been explored. Some possible physiological roles of the I.A.A. oxidase and the dialyzate have been discussed}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/H6ZH-WM47, author = {Boroughs, Howard James}, title = {I. Studies on the Acid Phosphatases of Green Leaves. II. Studies on the Role of Indoleacetic Acid in Cell Elongation}, school = {California Institute of Technology}, year = {1953}, doi = {10.7907/H6ZH-WM47}, url = {https://resolver.caltech.edu/CaltechETD:etd-04212003-115806}, abstract = {The bulk protein of green leaves has been shown to be dissociable and probably distinct from the acid phosphatase previously associated with it. This conclusion is based on the differential sedimentation rates in the analytical ultracentrifuge of the bulk protein and of phosphatase, on the relative amount of enzyme present in the bulk protein prepared by salt-precipitation or by differential centrifugation, and on the comparative stability of the two proteins toward heat and acid. Leaf phosphatase is further shown to be a mixture of isodynamic enzymes with different pH optima and Michaelis constants. A separation was accomplished by adsorption, dialysis, and by antigen-antibody reactions. The phosphatases are shown to lose enzymatic activity during dialysis, but to be capable of reactivation by certain metal ions. Copper ions are the most effective activators. Comparison of the spectrographic analysis of the ash of dialyzed phosphatase with enzymatic activity, however, disclosed a direct relation between activity and the amount of iron and manganese, as well as copper. Various comparative aspects of the enzymes were also studied. The influence of auxin on metabolic pathways is studied by a new method, in which the rate of incorporation of isotopically labeled intermediates into varied components of living tissue is studied as a function of the auxin supplied. It is found that auxin is without effect on the total protein amino nitrogen, or on the rate of incorporation of the C[superscript 14] label from glycine or leucine into tissue proteins of Avena and corn coleoptiles. Hence, auxin has no effect on protein metabolism during cell elongation. Similarly, auxin exerted only small effects on the rate of incorporation of C[superscript 14] from acetate into the lipid constituents of Avena coleoptiles. Added auxin induced no important changes in the incorporation of the C[superscript 14] of acetate or sucrose into the cell wall components of Avena. It was shown, however, that absence of auxin favors incorporation of the isotopic label of acetate or sucrose into the pectates and soluble polyuronide hemicelluloses, while the presence of auxin favors incorporation into the non-cellulosic polysaccharides. These effects are of small magnitude in relation to gross increase in cell length.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/9JPG-AE41, author = {Jansen, Leonard Leroy}, title = {Studies in Fruit Growth and in Vernalization}, school = {California Institute of Technology}, year = {1953}, doi = {10.7907/9JPG-AE41}, url = {https://resolver.caltech.edu/CaltechETD:etd-04232003-161523}, abstract = {Growth and development of flowers and fruits of red currant tomato (Lycopersicon pimpinellifolium) have been studied both on the intact plant and with the excised flower cultivated in vitro. A new interpretation of development of the inflorescence has been presented. Two types of growth response of ovaries of pollinated flowers have been identified both on the plant and in culture. In one type growth is linear and in the other it is exponential. The latter type, on the plant, produces seeded fruits, and size of fruits is correlated with number of seeds. Development on the plant is apparently optimal at a night temperature of 17[degrees]C. Smog is detrimental to development. Growth of the pollinated ovary in vitro can be supported on a minimal medium of mineral salts and sucrose. The two response types differ in carbohydrate requirements. A pollination-fertilization factor is postulated as the primary difference between them. Growth of the ovary in vitro can be influenced by temperature, auxin, casein hydrolysate, and concentration of sucrose. The temperature optimum for development in culture differs from that for development on the plant. Mechanisms of the responses have been discussed. The cold-requiring processes which promote flower initiation in Petkus winter rye have been analyzed more critically for their oxygen and sugar requirements by vernalizing the excised embryos under atmospheres of air or nitrogen on media with or without sugar. The first partial process of vernalization requires sugar but does not require oxygen. The second process does not require sugar but is dependent upon oxygen. Separation of the partial processes now becomes possible.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/R2GS-R439, author = {McRae, Dougal Harold}, title = {Studies on the Kinetics of Auxin-Induced Growth}, school = {California Institute of Technology}, year = {1953}, doi = {10.7907/R2GS-R439}, url = {https://resolver.caltech.edu/CaltechETD:etd-05082003-094406}, abstract = {The auxin-induced growth reaction of the Avena coleoptile has been treated by methods of classical enzyme kinetics. The kinetic treatment makes it possible to characterize the growth promoting activity of an auxin by two parameters, K[subscript s] and V[subscript max]. These express respectively the affinity of the auxin for the auxin-receptive site within the plant and the ability of the complex thus established to promote growth. Treatment of Avena section growth by the methods of enzyme kinetics has made it possible to determine rigorously whether or not inhibitors of auxin-induced growth are true antiauxins and act by competing with auxin for the auxin-receptive site within the plant. Certain auxin-inactive compounds have been shown to possess antiauxin activity. Among such substances are 2,4-dichloroanisole, 4,-chloro- and 2,4-dichlophenoxyisobutyric acids, and 2,6-dichloro-and 2,4,6-trichlorophenoxyacetic acids. Each of these substances can be considered as derived from an active auxin (2,4-dichlorophenoxyacetic acid) by elimination of one of the structural features essential to auxin activity and thereby capable of combining at one point of the auxin-receptive site but incapable of consumating the two-point attachment requisite for auxin activity. It is shown that chemically different auxins compete with one another for the same receptive sites within the plant. Auxins of low v[subscript max] are capable of inhibiting or augmenting the activity of auxins of greater V[subscript max]. Whether inhibition or promotion result depend on the concentrations of the two substances and the differential in V[subscript max] exhibits apparent antiauxin activity. This activity is shown to be different from competitive inhibition of true antiauxins. The relationship between Avena section growth rate and auxin concentration has been demonstrated to be predictable on the basis of a requirement for two-point attachment of the auxin molecule to some receptive entity within the plant. The growth inhibition resulting from high auxin concentrations is not alleviated by antiauxins but rather the auxin inhibition is augmented by the presence of sufficiently high antiauxin concentrations. A necessary corollary to the single point attachment antiauxin concert and the bimolecular complex formation concept for inhibitory auxin concentrations is therefore confirmed. A preliminary investigation concerning herbicidal activity of mixtures of an antiauxin and an auxin on bean plants is presented. The data obtained do not unequivocally establish that inactive bimolecular auxin receptor complex formation at high auxin concentrations is a factor contributing to herbicidal action of 2,4-D, but the possibility that this is in fact so is considered. A cultural technique for obtaining isolated flax root clones is described and data for some experiments with an isolated flax root clone are presented. The inhibitory action of certain antimetabolites on the growth of young tomato plants is described.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/MQ5F-JT09, author = {Eggman, William Luther}, title = {The Cytoplasmic Protein Component of Green Leaves}, school = {California Institute of Technology}, year = {1953}, doi = {10.7907/MQ5F-JT09}, url = {https://resolver.caltech.edu/CaltechETD:etd-05082003-154241}, abstract = {NOTE: Text or symbols not renderable in plain ASCII are indicated by […]. Abstract is included in .pdf document. The soluble cytoplasmic proteins of the leaves of dicotyledonous plant species have been characterized as to their chemical and physical chemical properties. Elephoretically, the proteins migrate as a single major component and from one to six minor components, the number depending upon the species. The principal component has a mobility of ca. ?5.0 to ?5.5 x 10[superscript ?5] [. . .] K-maleate buffer at pH 7.0, and constitutes ca. 50 to 80 per cent of the total protein. In the analytical ultracentrifuge, the proteins are resolved into two components; a high-molecular weight, apparently homogeneous component with a corrected sedimentation, constant of 19S which constitutes ca. 30 to 50 per cent of the total protein, and a low-molecular weight heterogeneous, fraction which contains the enzymatic activity. Chemically, the cytoplasmic proteins contain 13 to 15 per cent nitrogen and from 0.1 to 0.8 per cent phosphorus. This protein-bound [phosphorus] was found to be associated with the 19S component of cytoplasm in the form of ribonucleic acid. The principal component of cytoplasm is, therefore, a nucleoprotein. The nucleotide composition of the ribonucleic acid in the cytoplasms of leaves of different plant species and in leaves of different physiological ages but from the same plant species were studied. The composition varies with the plant species but not with physiological age. The effects of pH, of temperatures from 0[degrees] C. to 37[degrees] C., of dialysis and of storage at ?20[degrees] C. upon the stability of whole cytoplasm preparations was studied by chemical and ultracentrifugal analysis. Acidity greater than pH 6.5 and storage at ?20[degrees] C. for extended periods preferentially denatures the protein moiety of the nucleoprotein component, while dialysis or incubation at room temperatures for short periods of time causes the loss, apparently by enzymatic degradation, of the ribonucleic acid moiety. No method of entirely preventing the loss of nucleic acid was found, although maintenance of a low temperature partially suppresses it. A differential sedimentation procedure capable of preparing high-molecular weight fractions of cytoplasm (Fraction I protein preparations) containing only 5 to 10 per cent of low-molecular weight contaminants was developed when classical methods of chemical fractionation proved unsuitable. Such preparations were used to determine the physical properties of the nucleoprotein component. The nucleoprotein components of spinach and tobacco have been shown to be closely similar, although certain physical properties of the nucleoprotein, such as partial specific volume, sedimentation constant and molecular weight, are dependent on the ribonucleic acid content. The molecular weight of the nucleoprotein component in a particular preparation containing 11 per cent nucleic acid is estimated to be 360,000, based on determination of the partial specific volume and the sedimentation constant of this preparation together with an estimation of the frictional coefficient from a deduced shape factor and an assumed hydration value. Fraction I protein preparations always contain high-molecular weight components that are not initially present in whole cytoplasm. It is shown that these components probably have their origin in aggregation of the nucleoprotein component.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Wildman, Samuel G. and Bonner, James Frederick}, } @phdthesis{10.7907/RPCV-Y418, author = {Kurtz, Edwin B.}, title = {Studies on the Metabolism of Lipids in Plants}, school = {California Institute of Technology}, year = {1952}, doi = {10.7907/RPCV-Y418}, url = {https://resolver.caltech.edu/CaltechTHESIS:02062018-154306942}, abstract = {This work is concerned with some physiological and biochemical studies of the lipids of higher plants, a subject in which only an extremely limited number of studies have previously been made.
From a study of plants grown under controlled conditions, it was found that both character and amount of fat and wax produced by a plant may be affected by a factor of two or three by day and night temperatures and soil moisture. The effect of increased day or night temperature on the yield of fat was different for different species. Plants generally responded to increased temperature by producing less wax. The fats and waxes from plants at high temperatures were of a higher melting point than those from plants at low temperatures. Water stress plants also produced large amounts of fat, or in Larrea, resin. Although the wax content was only slightly affected by low soil moisture, in Nicotiana glauca an abundant formation of cuticle occurred under this condition. These and other effects of climate on lipids were discussed.
Whereas changes in climate effect up to three-fold changes in lipid yield, a series of recessive genes in corn was found to control ten-fold changes in wax yield. The genetic factors also affect the character and yield of the fats.
A system was obtained for studying the synthesis of fats in a higher plant. Preliminary results show that short chain compounds (ethyl alcohol, acetate, acetone, acetoacetate) may be rapidly utilized in the synthesis of fat. These substrates are readily used only when an energy source such as sugar and the vitamin biotin are supplied. The effects of other substrates and vitamins on fat synthesis were also studied and found to be small or altogether absent.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/6970-QP16, author = {Liverman, James Leslie}, title = {The Physiology and Biochemistry of Flowering}, school = {California Institute of Technology}, year = {1952}, doi = {10.7907/6970-QP16}, url = {https://resolver.caltech.edu/CaltechTHESIS:09272017-092747800}, abstract = {With the two SDP, Xanthium canadense and Chenopodium Amaranticolor, it has been possible to separate more clearly the partial reactions of the photoperiodic response of SDP and in some degree to associate these processes with particular biochemical or physiological processes of the plant. Thus it has been shown that sugars and Krebs cycle acids are able to replace the high intensity light process. Further investigations have shown that substances formed during an inductive dark process are still susceptible to auxin inactivation even after exposure to several hours of high intensity light immediately following the dark period. It has been shown that the effect of a flash of light in inhibiting the dark process can be reversed by anti-auxins. Further experiments have confirmed earlier work which show that LD leaves on SD plants inhibit flowering. Hypotheses have been advanced 1) to explain the nature of this inhibition and 2) to explain the kinetics of the dark process.
Experiments with a newly discovered LDP, Silene Armeria, have shown that its critical day length is reduced by increasing temperature. Studies on the auxin relations in the flowering of Silene and Hyoscyamus niger indicate that auxin causes flowering of these LDP under conditions in which the controls remain vegetative.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Bonner, James Frederick}, } @phdthesis{10.7907/TBXH-5W50, author = {Gottlieb, Sidney}, title = {Studies on a Growth Inhibitor in Guayule}, school = {California Institute of Technology}, year = {1943}, doi = {10.7907/TBXH-5W50}, url = {https://resolver.caltech.edu/CaltechTHESIS:01242017-111743193}, abstract = {