On the origin and evolution of the genetic code. II. Origin of the genetic code as a primordial collector language. The pairing-release hypothesis.

The genetic code is treated as a language used by primordial “collector societies” of tRNA molecules (meaning: societies of RNA molecules specialized in the collection of amino acids and possibly other molecular objects), as a means to organize the delivery of collected material. Its origin is ascribed to the utilization of the complementarity between each tRNA and the genome segment from which it was originally copied, as a means to identify by annealing operations the tRNA molecules returning from their collection trips, and elicit the release of the amino acids they are carrying (the pairing release hypothesis).The gradual conversion of tRNA complements into codon-triplets in the regions of the primordial RNA genomes which specialized in the task of directing the delivery of amino acids by returning tRNA molecules, is ascribed to the removal of genetic redundancy in a gradual reorganization process.A reconstruction of the codon-triplets in one of the earliest genetic codes is attempted by the wobbling reintroduction procedure used in a preceding paper.     

Multilayer adsorption of lysozyme on a hydrophobic substrate.

Macromolecular adsorption is known to occur as a complex process, often in a series of steps. Several models are discussed in the literature which describe the microscopic structure of the adsorbate. In the present study we investigated the adsorption of hen egg white lysozyme on alkylated silicon oxide surfaces. A combination of fluorescence excitation in the evanescent field and fluorescence recovery after photobleaching allowed us to measure the amount of adsorbed fluorescent lysozyme and the equilibrium exchange kinetics with molecules in solution. We found that a model with at least three classes of adsorbed molecules is necessary to describe the experimental results. A first layer is formed by the molecules which adsorb within a short time after the beginning of the incubation. These molecules make up approximately 65% of the final coverage. They are quasi-irreversibly adsorbed and do not measurably exchange with bulk molecules within one day even at temperatures up to 55 degrees C. A second layer, which reaches equilibrium only after several hours of incubation, shows a pronounced exchange with bulk molecules. The on-off kinetics show a distinct temperature dependence from which an activation barrier of delta E approximately 22 kcal/mol is derived. A third layer of molecules that exchange rapidly with the bulk can be seen to comprise approximately 10% of the total coverage. The exchange rate is on the order of fractions of a second. The binding of the latter two classes of adsorbed molecules is exothermic. From the temperature dependence of the coverage, the binding enthalpy of the slowly exchanging layer was estimated to be delta Hads approximately 3.8 kcal/mol. The second and third class of molecules remain enzymatically active as a muramidase, which was tested by the lysis of the cell walls of Micrococcus lysodeiktikus. The molecules in the first layer, on the other hand, showed no enzymatic activity.     

Chemical markers of prebiotic chemistry in hydrothermal systems.

The goal of this chapter is to suggest some organic compounds which may be indicative of prebiotic processes in hydrothermal systems or laboratory simulations of them. While the exact processes which led to the origins of life are not known, studies of life's origins of the past forty years have uncovered a plethora of potential precursor molecules. Some of these same molecules were probably present in hydrothermal systems if chemical processes there had a role in the origins of life. The types of molecules formed in primitive Earth simulation experiments and observed in the interstellar medium, on comets and meteorites will be reviewed in Section 2 of this chapter. Some reactions involving these molecules which may have been important in prebiotic syntheses will be outlined. Since near- to supercritical water is found in hydrothermal systems, its properties and aspects of organic chemistry in supercritical water at high temperature and pressure will be discussed in Section 3. Fischer-Tropsch type (FTT) reactions, which are a potential source of the building blocks of biological molecules in hydrothermal systems, are discussed in Section 4. In the concluding section, Section 5, the possible formation in hydrothermal systems of organic molecules that are believed to have been important for the origins of life is discussed.     

Anomalies in sedimentation. I. Stability theory of sedimenting entanglements in the "tight-bending" limit

A simple model is introduced to investigate the stability of a sedimenting entanglement. The sedimenting entanglement is represented by a sedimenting sieve. Solvent can pass through it, but single-chain molecules that flow into it become entangled and their flow decreases or, if permanent entanglements form, ceases entirely. With this model we are able to find the conditions under which the mass of a sedimenting entanglement remains constant, grows or decays to a stable value, grows beyond limit, or decays to the mass of a single chain. The theory is applied to the sedimentation of small concentrations of large chain molecules in solutions of small chain molecules in solutions of small chain molecules for the case in which the entanglements are long-lived. Equations are derived which, (1) give the stable entanglement mass as a function of rotor speed and concentration and, (2) for a given concentration predict the rotor speed at which the entanglement mass grows without limit. Numerical results for small concentrations of T2 DNA sedimenting in solutions of T7 DNA are presented.     

Species-specific aggregation factor in sponges. VI. Aggregation receptor from the cell surface.

An aggregation receptor from the siliceous sponge Geodia cydonium has been isolated and purified in an almost pure form. It sediments at about 2-6s, has a buoyant density of 1-51 g/ml in CsCl and elutes from Sephadex G-50 at a Ve/V0 value of 1-311. Chemical analysis revealed that the receptor consists of 81% neutral carbohydrate and 7-5% protein. The activity of the receptor is rapidly destroyed by Na-periodate. The receptor is released from the cell surface after removal of Ca2+ from the medium or after incubation of the cells with trypsin. The depleted cells can be charged again with isolated receptor molecules. The binding of the receptor molecules on the cell surface is prevented in the presence of trypsin. For optimal binding, physiological salt concentrations with respect to NaCl (540 mM NaCl) and Ca2+ ions are necessary. The receptor whose isolation is described in this report, is involved in secondary aggregation processes, which are initiated by a soluble aggregation factor. The primary aggregation of the cells is not influenced by the receptor. Time-course studies with receptor-depleted cells revealed that new aggregation receptor molecules are formed during the aggregation process. By competition experiments it could be shown that high concentrations of soluble aggregation receptor molecules inhibit secondary aggregation. The soluble receptor molecules can complete with surface-bound receptor molecules only if these are not linked with the aggregation factor.     

In vitro synthesis of simian virus 40 DNA. I. Synthesis by a soluble extract from infected CV1 cells.

Simian Virus 40 (SV40) DNA replication was studied in vitro using cell free extracts prepared from SV40 infected CV1 cells. The cells were fractionated into a soluble cytoplasmic fraction and nuclei. The nuclei were lysed with high salt and used to prepare a soluble nuclear fraction. Both fractions displayed DNA polymerase activity as measured with activated calf thymus DNA. However, only the cytoplasmic fraction was active when SV40 DNA comonent I molecules were used as template. Under these conditions, the cytoplasmic extract was shown to catalyse the SV40 DNA dependent, in vitro incorporation of the four deoxyribonucleotides into DNA molecules which had, at both neutral and alkaline pH, the same sedimentation behavior as authentic SV40 DNA component I and component II molecules. Optimal Mg++ concentration was 5-8 mM. Incorporation of label into DNA component I molecules showed an initial lag of about 15 min., after which it was linear with time for up to 5 hrs at 32 degrees. Incorporation into DNA component II molecules proceeded without obvious lag and reached a plateau after approximately 2 hrs of incubation. It is concluded that the cytoplasmic extract supports the in vitro synthesis of SV40 DNA and that DNA component II molecules appear to be a precursor to DNA component I molecules in the reaction. Labeling of viral DNA molecules was highly dependent on ATP and on an ATP generating system. In the absence of ATP and of the energy generating system, incorporation occurred but both template and newly synthesized DNA molecules were extensively degraded.     

Mouse gamete adhesion molecules.

Complementary adhesion molecules are located on the surface of mouse eggs and sperm. These molecules support species-specific interactions between sperm and eggs that lead to gamete fusion (fertilization). Modification of these molecules shortly after gamete fusion assists in prevention of polyspermic fertilization. mZP3, an 83,000-Mr glycoprotein located in the egg extracellular coat, or zona pellucida, serves as primary sperm receptor. Gamete adhesion in mice is carbohydrate-mediated, since sperm recognize and bind to certain mZP3 serine/threonine- (O-) linked oligosaccharides. As a consequence of binding to mZP3, sperm undergo the acrosome reaction, which enables them to penetrate the zona pellucida and fertilize the egg. A 56,000-Mr protein called sp56, which is located in plasma membrane surrounding acrosome-intact mouse sperm heads, is a putative primary egg-binding protein. It is suggested that sp56 recognizes and binds to certain mZP3 O-linked oligosaccharides. Acrosome-reacted sperm remain bound to eggs by interacting with mZP2, a 120,000-Mr zona pellicida glycoprotein. Thus, mZP2 serves as secondary sperm receptor. Perhaps a sperm protease associated with inner acrosomal membrane, possibly (pro)acrosin, serves as secondary egg-binding protein. These and, perhaps, other egg and sperm surface molecules regulate fertilization in mice. Homologous molecules apparently regulate fertilization in other mammals.     

Heavy chain variable region allotypic sub-specificities of rabbit immunoglobulins. I. Identification of three subpopulations of a1 IgG molecules.

Four anti-al Ab subpopulations were isolated from an anti-al antiserum by sequential immunoadsorption chromatography. These four anti-al Ab subpopulations were differentially bound by two "limited heterogeneity" Abs having different components of the al allotypic specificity. Each of the four anti-al Ab subpopulations reacted with al IgG molecules obtained from a2 and a3 rabbits. A subpopulation designated anti-al Ab reacted with 100% of al IgG molecules. Thus, the anti-al-A Ab recognizes al determinants common to all al IgG molecules. Each of the other three subpopulations, designated anti-al-B Ab, anti-al-C Ab, and anti-al-D Ab, reacted with only a fraction of the al IgG molecules but the sum of the percentages of al IgG molecules which reacted with each of these three anti-al Ab subpopulations approximated 100% of the al IgG molecules. Thus each of the anti-al-B Ab, anti-al-C Ab, and anti-al-D Ab recognizes non-common determinants distinct for each of three subpopulations of al IgG molecules. Although 65 to 90% of IgG molecules in al homozygous rabbits have the al allotypic specificity, these IgG molecules are heterogeneous with respect to their antigenic determinants comprising the al allotype; at least three kinds of al IgG molecules are identified. This heterogeneity probably reflects variation in the amino acid sequence of the Vh region of al IgG molecules and, therefore, poses a similar argument which had led to the hypothesis of two genes for one polypeptide chain and to the theory of episomal insertions for the genetic control of immunoglobulin synthesis.     

Binding of D-glucose to insulin.

Binding of D-glucose to insulin has been studied by equilibrium dialysis. The binding is not very specific and probably takes place in two steps. The average amount of glucose molecules bound per insulin molecule is eight, two molecules in the first and six during the second step of binding. The intrinsic binding constants for both steps are almost the same (6-10-2 M-minus 1 and 1-10-3 M-minus 1) which can be explained by assuming: (1) that after binding of the first two molecules a conformational change of insulin occurs which facilitates the binding of the next six molecules of D-glucose; or (2) that in the second step of binding the glucose binds to hydrophobic regions which are unmasked by dissociation of the insulin dimer. Using a three-dimensional model of the insulin molecule areas of the protein molecule where binding of glucose can occur were selected. The glucose-binding site very probably involves the area at the insulin surface where most of the invariant and modification-selective residues are present.     

Radiation-induced DNA damage as a function of hydration. I. Release of unaltered bases.

The release of unaltered bases from irradiated DNA, hydrated between 2.5 and 32.7 mol of water per mole of nucleotide (gamma), was investigated using HPLC. The objective of this study was to elucidate the yield of the four DNA bases as a function of dose, extent of hydration, and the presence or absence of oxygen. The increase in the yield of radiation-induced free bases was linear with dose up to 90 kGy, except for the DNA with gamma = 2.5, for which the increase was linear only to 10 kGy. The yield of free bases as a function of gamma was not constant in either the absence or the presence of oxygen over the range of hydration examined. For DNA with gamma between 2.5 and 15, the yield of free bases was nearly constant under nitrogen, but decreased under oxygen. However, for DNA with gamma greater than 15, the yield increased rapidly under both nitrogen and oxygen. The yield of free bases was described by a model that depended on two factors: 1) a change in the DNA conformation from a mixture of the A and C conformers in vacuum-dried DNA to predominantly the B conformer in the fully hydrated DNA, and 2) the proximity of the water molecules to the DNA. Irradiation of the inner water molecules (gamma less than 15) was less efficient than irradiation of the outer water molecules (gamma greater than 15), by a factor of approximately 3.3, in forming DNA lesions that resulted in the release of an unaltered base. This factor is similar to the previously published relative efficiency of 2.8 with which hydroxyl radicals and base cations induce DNA strand breaks. Our irradiation results are consistent with the hypothesis that the G value for the first 12-15 water molecules of the DNA hydration layer is the same as the G value for the form of DNA to which it is bound (i.e., the pseudo-C or the B form). Thus we suggest that the release of bases originating from irradiation of the hydration water is obtained predominantly: (1) by charge transfer from the direct ionization of the first 12-15 water molecules of the primary hydration layer and (2) by the attack of hydroxyl radicals generated in the outer, more loosely bound water molecules.     

In vitro lymphocyte cytotoxicity. II. Unstable lymphotoxins (beta-LT) secreted and inactivated by mitogen-stimulated human lymphocytes.

Supernatants from phytohemagglutinin (PHA)-activated human lymphocytes contain two major classes of cytotoxins (α-LT and β-LT). While α-LT appears to be stable, and the major component in 5-day culture supernatants, the majority of cytolytic activity at earlier intervals in these cultures is due to a “family” of highly unstable cytotoxins which are both secreted and destroyed at a rapid rate. The inactivation of the unstable LT molecules appears to be due to: (a) inherent instability o β-LT molecules, and (b) a lymphocyte-mediated inactivation mechanism(s) which involves serum.     

Phospholipid interactions in model membrane systems. I. Experiments on monolayers.

We study the lateral headgroup interactions among phosphatidylcholine (PC) molecules and among phosphatidylethanolamine (PE) molecules in monolayers and extend our previous models. In this paper, we present an extensive set of pressure-area isotherms and surface potential experiments on monolayers of phospholipids ranging from 14 to 22 carbons in length at the n-heptane/water interface, over a wide range of temperature, salt concentration, and pH on the acid side. The pressure data presented here are a considerable extension of previous data (1) to higher surface densities, comprehensively checked for monolayer loss, and include new data on PE molecules. We explore surface densities ranging from extremely low to intermediate, near to the main phase transition, in which range the surface pressures and potentials are found to be independent of the chain length. Thus, these data bear directly on the headgroup interactions. These interactions are observed to be independent of ionic strength. PC and PE molecules differ strongly in two respects: (a) the lateral repulsion among PC molecules is much stronger than for PE, and (b) the lateral repulsion among PC molecules increases strongly with temperature whereas PE interactions are almost independent of temperature. Similarly, the surface potential for PC is found to increase with temperature whereas for PE it does not. In this and the following paper we show that these data from dilute to semidilute monolayers are consistent with a theoretical model that predicts that, independent of coverage, for PC the P-N+ dipole is oriented slightly into the oil phase because of the hydrophobicity of the methyl groups, increasingly so with temperature, whereas for PE the P-N+ dipole is directed into the water phase.     

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.     

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.     

Accommodation of pyrimidine dimers during replication of UV-damaged simian virus 40 DNA.

UV irradiation of simian virus 40-infected cells at fluences between 20 and 60 J/m2, which yield one to three pyrimidine dimers per simian virus 40 genome, leads to a fluence-dependent progressive decrease in simian virus 40 DNA replication as assayed by incorporation of [3H]deoxyribosylthymine into viral DNA. We used a variety of biochemical and biophysical techniques to show that this decrease is due to a block in the progression of replicative-intermediate molecules to completed form I molecules, with a concomitant decrease in the entry of molecules into the replicating pool. Despite this UV-induced inhibition of replication, some pyrimidine dimer-containing molecules become fully replicated after UV irradiation. The fraction of completed molecules containing dimers goes up with time such that by 3 h after a UV fluence of 40 J/m2, more than 50% of completed molecules contain pyrimidine dimers. We postulate that the cellular replication machinery can accommodate limited amounts of UV-induced damage and that the progressive decrease in simian virus 40 DNA synthesis after UV irradiation is due to the accumulation in the replication pool of blocked molecules containing levels of damage greater than that which can be tolerated.     

Detection of non-homology-containing heteroduplex molecules.

Heteroduplex DNA molecules which contain both the wild-type and mutant sequences of a deletion nonhomology possess a characteristic electrophoretic mobility in agarose and can be readily separated from both the wild-type and deletion-containing parental homoduplex fragments. Because of the partial single stranded character of these deletion-containing heteroduplex molecules, they are selectively bound to nitrocellulose filters, and once bound, can be selectively detected by hybridization with radioactively labeled single-stranded DNA which is homologous to the sequences absent in the deletion mutation.     

Molecular interactions in myosin assembly. Role of the 28-residue charge repeat in the rod.

We have used internal deletions of multiples of seven residues to change the phase of the 28-residue charge repeat in a light meromyosin cDNA construct expressed in Escherichia coli. The solubility behaviour of these mutants was similar to that of the wild-type material, but the molecular packing in the aggregates formed at low ionic strength was different. Whereas wild-type material formed paracrystals in which molecules were in close contact over most of their length, molecules in the paracrystals formed by the mutants were in close contact for only a short distance, which was just short enough to exclude the deletion from the overlap. These data indicate that, although the 28-residue charge periodicity is important in myosin molecular interactions, it is probably not the major driving force for myosin assembly and instead influences the detailed axial stagger of the interacting molecules.     

Folding of ribonuclease T1. 1. Existence of multiple unfolded states created by proline isomerization

It is our aim to elucidate molecular aspects of the mechanism of protein folding. We use ribonuclease T1 as a model protein, because it is a small single-domain protein with a well-defined secondary and tertiary structure, which is stable in the presence and absence of disulfide bonds. Also, an efficient mutagenesis system is available to produce protein molecules with defined sequence variations. Here we present a preliminary characterization of the folding kinetics of ribonuclease T1. Its unfolding and refolding reactions are reversible, which is shown by the quantitative recovery of the catalytic activity after an unfolding/refolding cycle. Refolding is a complex process, where native protein is formed on three distinguishable pathways. There are 3.5% fast-folding molecules, which refold within the millisecond time range, and 96.5% slow-folding species, which regain the native state in the time range of minutes to hours. These slow-folding molecules give rise to two major, parallel refolding reactions. The mixture of fast- and slow-folding molecules is produced slowly after unfolding by chain equilibration reactions that show properties of proline isomerization. We conclude that part of the kinetic complexity of RNase T1 folding can be explained on the basis of the proline model for protein folding. This is supported by the finding that the slow refolding reactions of this protein are accelerated in the presence of the enzyme prolyl isomerase. However, several properties of ribonuclease T1 refolding, such as the dependence of the relative amplitudes on the probes, used to follow folding, are not readily explained by a simple proline model.