Cytoplasmic DNA: Differentiation by reassociation kinetics

Rat liver nuclear and cytoplasmic DNA samples were denatured and the kinetics of their reassociation was measured. About 85% of the soluble cytoplasmic (mitochondrial) DNA reannealed rapidly with a $\text{C}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.03}$ while 65% of the particulate (microsomal) DNA reassociated with a $\text{C}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.14}$ Both nucleic acids were clearly differentiated from nuclear DNA in their reassociation kinetics. The results indicate that both mitochondrial and microsomal DNA consist mainly of single components or closely related families with repetitive sequences.     

Developmental biochemistry of cotton seed embryogenesis and germination. VII. Characterization of the cotton genome.

The DNA of cotton, Gossypium hirsutum, has been characterized as to spectral characteristics, buoyant density in CsCl, base composition, and genetic complexity. The haploid genome size is found to bo 0.795 pg DNA/cell. However, the amount of DNA per cell in the cotyledons increases during embryogenesis to an average ploidy level of 12N in the mature seed cotyledons. Reassociation kinetics indicate that this increase is due to endoreduplication of the entire genome.Non-repetitive deoxynucleotide sequences account for approximately 60.5% of the cotton genome ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$pure⁵ = 437); highly repetitive sequences (> 10,000 repetition frequency) constitute about 7.7% of the genome. ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{pure}\text{= 4.6 × 10}{}^{\mathrm{?4}}$) and intermediately repetitive sequences constitute the remaining 27% of the genome ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{pure}\text{= 1.46}$). Hybridization of ¹²⁵I-labeled cytoplasmic ribosomal RNA to whole-cell DNA on filters and in solution indicate approximately 300 to 350 copies of the rRNA cistrons per haploid genome.The interspersion of repetitive sequences that reassociate between C₀t values of 0.1 and 50 with non-repetitive sequences of the cotton genome has been examined by determining the reassociation kinetics of DNA of varying fragment lengths and by the electron microscopy of reassociated molecules. About 60% of the genome consists of non-repetitive regions that average 1800 base-pairs interpersed with repetitive sequences that average 1250 base-pairs. Approximately 20% of the genome may be involved in a longer period interspersion pattern containing non-repetitive sequences of approximately 4000 base-pairs between repetitive sequences. Most of the individual sequences of the interspersed repetitive component are much smaller than the mass average size, containing between 200 and 800 base-pairs. Sequence divergence is evident among the members of this component.Highly repetitive sequence elements that are reassociated by a C₀t value of 0.1 average 2500 base-pairs in length, appear to have highly divergent regions and do not appear to be highly clustered. A portion of this highly repetitive component reassociates by C₀t = 10^?4, zero-time binding DNA, and accounts for less than 3% of the genome. At least a third of these sequences appear by electron microscopy to be intramolecular duplexes (palindromes) of 150 to 200 base-pairs and to occur in clusters.     

Synthesis and characterization of a DNA probe complementary to rat liver 28S ribosomal RNA.

Conditions for the production of a complementary DNA sequence for use in studies of ribosomal RNA are described. $\text{E}$. $\text{coli}$ DNA polymerase I is used to transcribe highly purified 28S ribosomal RNA from rat liver. The reaction is sensitive to the tertiary structure of the rRNA template-primer. The complementary DNA hybridizes to its rRNA template with a $\text{R}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.02. The hybrid formed between 28S ribosomal RNA and complementary DNA has a T_m of 73°C. The probe reacts with total rat nuclear RNA with a $\text{R}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.0.     

Optimizing enzyme assays with one or two coupling enzymes

The cost of assays using one or two coupling enzymes is optimized by using equations to calculate the minimum amount(s) of enzyme(s) which should be used to obtain a given time (t₉₉) in which 99% of the rate V₀ of the first reaction is obtained. Using two coupling enzymes and given a value of t₉₉, the induction period L = L₁ + L₂ fulfills the requirement $\text{t}{}_{99}\text{2}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{4.6}\text{≥ L ≥}\text{t}{}_{99}\text{4.6}$, allowing one to choose a cost lower than that derived from the until-now generally applied assumption of t₉₉ = 4.6L. Being $\text{α =}\text{L}{}_1\text{L}{}_2$, in optimized assays the values α, t₉₉, and L are related by $\text{T}{}_{99}\text{=4.6}\text{(1+α)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{1+α}\text{L}$, thus allowing (graphical) calculation of the amounts of coupling enzymes which will minimize the cost for every chosen t₉₉ or L. Maximum practical rates, allowed in some supposed interesting cases, have been calculated.     

Cockroaches on a treadmill: Aerobic running

Five species of cockroach were tested on a miniature treadmill at three velocities as O₂ consumption () was measured: Gromphadorhina chopardi, Blaberus discoidalis, Eublaberus posticus, Byrsotria fumagata and Periplaneta americana. All cockroaches showed a classical aerobic response to running: increased rapidly from a resting rate to a steady-state on-response varied from under 30 s to 3 min. Recovery after exercise was rapid as well; $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ off-response varied from under 30 s to 6 min. These times are faster or similar to mammalian values. varied directly with velocity as in running mammals, birds and reptiles. during steady-state running was only 4–12 times higher than at rest. Running is energetically much less costly per unit time than flying, but the cost of transport per unit distance is much more expensive for pedestrians. The minimal cost of transport (M_run), the lowest necessary to transport a given mass a specific distance, is high in cockroaches due to their small size. The new data suggest that insects may be less economical than comparable sized vertebrates.     

Time lag in a model of a biochemical reaction sequence with end product inhibition

The model studied is that of Goodwin, in which all but one of the reactions obey linear kinetics, while the end-product inhibits the first reaction in a term of Michaelis-Menten form, with Hill coefficient ?:

$\text{z=}\text{∫}\text{?∞}\text{t}\text{x}{}_{\mathrm{n}}\text{(T)G(t?T)dt}$

The results obtained relate to time lag in the off diagonal terms in these equations. The time lag is taken in distributed form, for example replacing x_n in the first equation by

$\text{dx}{}_{\mathrm{t}}\text{dt}\text{=k}{}_1\text{x}{}_{\mathrm{t??}}\text{1?b}{}_1\text{x}{}_{\mathrm{t}}\text{, i=2, …n.}$

For any non-negative G, time lag in these terms can not destabilize the equilibrium point in the case ? = 1. For a particular class of functions G one can obtain some insight into the consequences of time lag by relating the model to that with a longer loop of reactions. Then known results can be used for general ? and n.     

Properties of bilayer membranes in the presence of dipicrylamine. A comparative study by optical absorption and electrical relaxation measurements

In order to test the question if a pool of lipophilic ions may exist in black lipid membranes which cannot be detected by electrical relaxation measurements we have performed simultaneously measurements of the optical absorption of a lipophilic ion. The absorbance of membrane-bound dipicrylamine at 410 nm was measured with a sensitive spectrophotometer which can detect absorbance changes $\text{? 4 · 10}{}^{\mathrm{?5}}$. A minimal concentration of about 6 · 10¹¹ dipicrylamine ions per cm² of the membrane could be detected with this instrument. The dipicrylamine concentration in the membrane obtained with the optical method $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ is compared with the concentrations $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$ obtained from simultaneous electrical relaxation measurements. $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ and $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$ agreed at low dipicrylamine concentrations (10^?8–10^?7 M in the aqueous phase) and showed saturation at higher concentrations (up to 5 · 10^?6 M). In the saturation range $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ was maximally four times higher than $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$. The significance of this difference is discussed together with general aspects of the saturation phenomenon.     

The polymorphic phase behaviour and miscibility properties of synthetic phosphatidylethanolamines

(1) The polymorphic phase behaviour of aqueous dispersions of various synthetic phosphatidylethanolamines, both singly and in mixtures, has been investigated by ³¹P-NMR. (2) $\text{14:0}\text{14:0}$ PE remains in the lamellar phase up to 90°C. $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibits a lamellar to hexagonal (H_II) transition between 60°C and 63°C. For $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the lamellar to hexagonal (H_II) transition occurs between 7 and 12°C, whereas for $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the hexagonal (H_II) phase is the preferred structure above ?15°C. (3) Mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit near-ideal miscibility behaviour. For mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{14:0}\text{14:0}$ PE there is evidence of fluid-solid immiscibility at temperatures below the gel-liquid crystalline transition temperature of the $\text{14:0}\text{14:0}$ PE component. Mixtures of $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit complex phase behaviour involving limited fluid-solid immiscibility at low temperatures and formation of a phase allowing isotropic motional averaging at higher temperatures. (4) ³¹P-NMR provides a graphic method for investigating the miscibility properties of mixed PE systems.     

Isolation and characterization of isocitrate lyase of castor endosperm

Isocitrate lyase (threo-D_S-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified to homogeneity from castor endosperm. The enzyme is a tetrameric protein (molecular weight about 140,000; gel filtration) made up of apparently identical monomers (subunit molecular weight about 35,000; gel electrophoresis in the presence of sodium dodecyl sulfate). Thermal inactivation of purified enzyme at 40 and 45 °C shows a fast and a slow phase, each accounting for half of the intitial activity, consistent with the equation: , where A₀ and A_t are activities at time zero at t, and k₁ and k₂ are first-order rate constants for the fast and slow phases, respectively. The enzyme shows optimum activity at pH 7.2–7.3. Effect of [S]on enzyme activity at different pH values (6.0–7.5) suggests that the proton behaves formally as an “uncompetitive inhibitor.” A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pK_a equal to 6.9. Successive dialysis against EDTA and phosphate buffer, pH 7.0, at 0 °C gives an enzymatically inactive protein. This protein shows kinetics of thermal inactivation identical to the untreated (native) enzyme. Full activity is restored on adding Mg²⁺ (5.0 mm) to a solution of this protein. Addition of Ba²⁺ or Mn²⁺ brings about partial recovery. Other metal ions are not effective.     

Cytoplasmic poly(A) polymerase from sea urchin eggs, merogons, and embryos

The presence of cytoplasmic poly(A) polymerase has been established in sea urchin eggs and four-cell embryos by subcellular fractionation and use of enucleate egg halves. ATP is the only ribonucleoside triphosphate incorporated. This incorporation is time dependent, contingent on input protein concentration, and immune to a variety of antimetabolites known to inhibit DNA-directed RNA synthesis. Both the unfertilized egg and the four-cell embryo cytoplasmic poly(A) polymerase activities display a preference for Mn²⁺. While oligo(A)₄ is inactive as a primer, addition of $\text{oligo(A)}{}_{\mathrm{<mtext>16</mtext>}}\text{,}\text{poly(A)}{}_{\mathrm{<mtext>45</mtext>}}$ and $\text{poly(A)}{}_{\mathrm{<mtext>90</mtext>}}$ stimulates ATP incorporation. On a unit per milligram protein basis, the endogenous activity associated with cytoplasmic fractions obtained from nucleate and enucleate egg halves is 36 and 83% that obtained with the cytoplasmic fraction prepared from the unfertilized egg. In the presence of $\text{oligo(A)}{}_{\mathrm{<mtext>16</mtext>}}$, both the nucleate and enucleate egg halves exhibit 81% of the activity associated with the unfertilized egg cytoplasmic fraction. The level of Mn²⁺ cytoplasmic poly(A) polymerase activity from the four-cell embryo is approximately 50% that of the unfertilized egg. This decrease does not appear to be due to either a postfertilization alteration in the subcellular localization of poly(A) polymerase or an increase in RNase activity. Supplementation with $\text{oligo(A)}{}_{\mathrm{<mtext>16</mtext>}}$ failed to restore the four-cell embryo cytoplasmic poly(A) polymerase potential to a level comparable to that of the unfertilized egg. Suppression of postfertilization protein synthesis by emetine, however, prevents this developmental decline in ATP incorporation thereby suggesting that postfertilization cytoplasmic poly(A) polymerase activity is subject to negative translational control.     

The explicit analysis of consecutive pseudo-first-order reactions: Application to kinetics of thiolysis of dithiasuccinoyl (Dts) amino acids

An explicit set of general methods for the experimental determination of the rates k₁ and k₂ of consecutive pseudo-first-order reactions is described and discussed. These rely on the direct simultaneous analytical quantitation of the starting material, intermediate, and product of the reaction, and thus differ from present techniques based on measurement of coreactant consumption or coproduct appearance. The quantity $\text{k}{}^{\mathrm{<mtext>env</mtext>}}\text{=}\text{k}{}_1\text{k}{}_2\text{(k}{}_1\text{+ k}{}_2\text{)}$ is shown to define a good “envelope” approximation to product formation according to the simple law 100% [1 ? exp(?k^envt)]. The theory of envelopes is useful for comparing overall rates of reactions with widely differing values of $\text{κ =}\text{k}{}_2\text{k}{}_1$. The kinetic pattern of thiolysis of dithiasuccinoyl amino acids to carbamoyl disulfide intermediates to product free amino acids is analyzed and shown to agree quantitatively with theory.     

Stereoselective disposition of methadone in man.

Stable isotope labeled methadone (pentadeuteromethadone) has been used in conjunction with gas chromatography-chemical ionization mass spectrometry to study plasma disappearance rates and urinary excretion of pharmacologically active R-(?)-methadone (l-methadone) and inactive S-(+)-methadone (d-methadone) in three adult methadone maintenance patients. In all three cases, the analgesically active enantiomeric form of the drug had a significantly longer elimination half-life ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\beta </mtext>}}$) when studied in a steady state than did the inactive form ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\beta </mtext>}}$ for active R-(?)-methadone, 51.7 to 61.8 hours; $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\beta </mtext>}}$ for inactive S-(+)-methadone, 31.8 to 37.0 hours). The ratio of drug elimination half-lives } R-(?)-/S-(+)- ranged between 1.40 and 1.94. In the two cases so studied, slower plasma disappearance of active R-(?)-enatiomer than the inactive S-(+)-enantiomer was also observed ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2\beta </mtext>}}$ R-(?)-, 42.8 and 52.5 hours; $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2\beta </mtext>}}$ S-(+)-, 38.3 and 41.3 hours).     

The asymptotic normality of a simple batch epidemic process

A group of n susceptible individuals exposed to a contagious disease isconsidered. It is assumed that at each point in time one or more susceptible individuals can contract the disease. The progress of this simple batch epidemic is modeled by a stochastic process X_n(t), t∈[0, ∞), representing the number of infectiveindividuals at time t. In this paper our analysis is restricted to simple batch epidemics with transition rates given by $\text{[α}{}^2\text{X}{}_{\mathrm{n}}\text{(t){n ?X}{}_{\mathrm{n}}\text{(t) +X}{}_{\mathrm{n}}\text{(0)}]}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{, t∈[0, ∞), α∈(0, ∞)}$. This class of simple batch epidemics generalizes a model used and motivated by McNeil (1972) to describe simple epidemic situations. It is shown for this class of simple batch epidemics, that X_n(t), with suitable standardization, converges in distribution as n→∞ to a normal random variable for all t∈(0, t₀), and t₀ is evaluated.     

Catalytic function of thymidylate synthase is confined to S phase due to its association with replitase

Catalytic activity of thymidylate synthase, as measured $\text{in}\text{,}\text{vivo}$, is tightly linked to S phase of the cell cycle in Chinese hamster embryo fibroblast cells. This activity, as measured $\text{in}\text{,}\text{vitro}$, is found in all parts of the cell cycle. Thymidylate synthase activity in nuclear (karyoplast) extracts increased as the cells progressed from $\text{G}{}_0\text{G}{}_1$ to S phase. This enzymatic activity in the nuclei of S phase cells is associated with the multienzyme complex (replitase) that also contained DNA polymerase and other enzymes of DNA replication and precursor synthesis. The degree of association of thymidylate synthase with replitase, which increased co-ordinately as the cells progressed from $\text{G}{}_0\text{G}{}_1$ phase to S phase, coincided strongly with the level of $\text{in}\text{,}\text{vivo}$ activity of the enzyme.     

The repetitive sequence structure of component alpha DNA and its relationship to the nucleosomes of the African green monkey.

An analysis of the repeat structure of the highly repetitive sequence, component α DNA of the African green monkey, shows that the DNA contains restriction sites for EcoRI, $\text{Eco}\text{RI}{}^1$, HindIII and HaeIII. All four restriction enzyme activities indicate a basic repeat length of 176 ± 4 base-pairs. In addition to primary $\text{Eco}\text{RI}{}^1$ and HindIII sites, about 59% of the repeat sequences contain secondary $\text{Eco}\text{RI}{}^1$ sites and about 36% of the repeat sequences contain secondary HindIII sites. The secondary sites are located less than 176 base-pairs from the primary sites and their cleavage yields several complex series of minor, intermediate segments in gels of the partial $\text{Eco}\text{RI}{}^1$ or HindIII digests. Cleavage at the secondary sites yields segments shorter than the unit monomer in the limit digests. The sites for EcoRI, $\text{Eco}\text{RI}{}^1$, HindIII and HaeIII have been mapped within the repeat unit.Treatment of the monkey nuclei with micrococcal nuclease at 2 °C and in the presence of 80 mm-NaCl reveals two distinct populations of nucleosomes. One population contains bulk DNA sequences, and after cleavage with micrococcal nuclease this population yields heterogeneous segments of DNA spanning 180 to 200 base-pairs in length. The other population contains component α sequences and after cleavage with micrococcal nuclease yields homogeneous segments of component α DNA that are exact multiples of the basic sequence repeat unit of 176 base-pairs. Thus, the cleavage by micrococcal nuclease of nucleosomal arrays containing component α sequences is as regular and precise as the cleavage of the purified DNA by the restriction enzymes. The resolution of the two distinct subsets of nucleosomes in the monkey nuclei is dependent upon the conditions of ionic strength and temperature employed during the nuclear isolation and the micrococcal nuclease digestion.These observations are consistent with a phase relation between the component α repeat sequences and the associated nucleosomal proteins (Musich et al., 1977b). They are also in accord with the hypothesis that the subunit structure of constitutive heterochromatin modulates or determines the repeat sequence structure and hence, the evolution of many highly repetitive mammalian DNAs (Maio et al., 1977).     

The effect of prostaglandins E1 and E2 on the human erythrocyte as monitored by spin labels

The effects of the prostaglandins PGE₁ and PGE₂ on the deformability of the human erythrocyte were studied using spin-labeled erythrocytes. Two magnetic resonance parameters were measured: (1) The orientation relaxation time, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, for the erythrocyte, and (2) the order parameter, S, for a fatty acid spin label bound to the membrane. Prostaglandins PGE₁ and PGE₂ exhibited opposite effects on both $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S. PGE₂ made the cell less deformable (increases of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S) and PGE₁ made the erythrocyte more deformable (decrease of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S).     

A unified approach to ion transport through membranes. I. Membrane-soluble ions.

Theoretical membrane potential transient produced by applying a current step to nerve cells has been derived based on the compartment neuron model and also on the equivalent cylinder model developed by W. Rall. It is expressed as a sum of exponential functions as

$\text{∑}\text{i=0}\text{n?1}\text{E}{}_{\mathrm{i}}\text{[1?}\text{exp}\text{(}\text{t}\text{τ}{}_{\mathrm{i}}\text{)]}$

where n is the number of compartments. The ratio of the amplitudes of the first and the second largest exponential functions, ($\text{E}{}_1\text{E}{}_0$), was found to be proportional to that of their respective time constants, ($\text{τ}{}_1\text{τ}{}_0$), in these neuron models. The constant of proportionality is given in a form that depends on the number of compartments as $\text{E}{}_1\text{E}{}_0\text{= (1 +}\text{cos}\text{π}\text{n}\text{)}\text{τ}{}_1\text{τ}{}_0$. This theoretical result is discussed in the light of recent experimental results in cat red nucleus neurons.