Jacques R. Fresco

Protein fingerprinting by SDS-gel electrophoresis after partial fragmentation with CNBr.

Abstract The CNBr fragmentation reaction was adapted for the degradation of microgram quantities of protein in sodium dodecyl sulfate gel bands. These bands are then electrophoresed so as to generate easily distinguishable protein fingerprints. The method was tested on three proteins of known sequence and shown to generate nearly the full range of possible peptide fragments. It was also employed to compare the structures of several yeast aminoacyl tRNA synthetases and a contaminating crystallizable protein.

Polynucleotides: VII. Spectrophotometric study of the kinetics of formation of the two-stranded helical complex resulting from the interaction of polyriboadenylate and polyribouridylate

The kinetics of interaction of equimolar mixtures of poly A and poly U in low and moderate [Na+] at several temperatures has been followed at three wavelengths: 259·0 mμ, where the hypochromic change per U residue incorporated into helix is the same for poly (A + U) and poly (A + U + U); 280·0 mμ, where the hypochromic change is due only to the formation of poly (A + U + U); and 283·5 mμ. where the hyperchromic change is due only to the formation of poly (A + U). The results indicate that, under conditions that do not allow formation of poly (A + U + U) (0·01 M -Na+ at 25°C), poly (A + U) is formed directly and at a rate that can be described by a standard second-order kinetic expression. By contrast, under conditions that do allow poly (A + U + U) formation (0·01 M -Na+ at M -Na+ at 25°C), the complete formation of poly (A + U) does not proceed directly. In addition to rapid formation of poly (A + U), substantial amounts of poly (A + U + U) appear with an induction period. This formation of incidental poly (A + U + U) reaches a maximum shortly before all the poly U is consumed. Thereafter, the formation of poly (A + U) is notably slower and proceeds at a rate that is indistinguishable from the rate of disappearance of a half-equivalent of poly (A + U + U). Complete formation in 0·2 M -Na+ at 25°C is not approached until approximately 72 hours. Four rate expressions derived from standard kinetic laws usually applied to reactions between small molecules are proposed as a quantitative description of the entire sequence of events.

Increased Frequency of Cysteine, Tyrosine, and Phenylalanine Residues Since the Last Universal Ancestor

Analysis of extant proteomes has the potential of revealing how amino acid frequencies within proteins have evolved over biological time. Evidence is presented here that cysteine, tyrosine, and phenylalanine residues have substantially increased in frequency since the three primary lineages diverged more than three billion years ago. This inference was derived from a comparison of amino acid frequencies within conserved and non-conserved residues of a set of proteins dating to the last universal ancestor in the face of empirical knowledge of the relative mutability of these amino acids. The under-representation of these amino acids within last universal ancestor proteins relative to their modern descendants suggests their late introduction into the genetic code. Thus, it appears that extant ancient proteins contain evidence pertaining to early events in the formation of biological systems.

Structural and energetic consequences of noncomplementary base oppositions in nucleic acid helices.

Publisher Summary This chapter describes that secondary structure in nucleic acids generally consists of double-stranded helical regions that are stabilized by a variety of interbase forces. Bases in the opposing strands are regularly oriented with respect to the long axis of the helix by at least two hydrogen bonds, and only certain oppositions seem to be allowed, usually a purine and a pyrimidine. In the chapter, the variety of codon-anticodon interactions observed in protein synthesis suggests that there can be some flexibility in these requirements, such that pairing need not be restricted to base oppositions that are precisely accommodated by the one-dimensional lattice of a long double helix. The conformational possibilities of base oppositions that fall outside these complementary categories also merit definition, particularly in view of several recent indications that noncomplementary residues may have important functional roles in naturally occurring nucleic acids. The conformational alternatives available to noncomplementary residues within helical regions logically fall into two general categories, one being intrahelical and the other extrahelical or “looped-out’’. The conformational class that is adopted in a particular case depends upon the interplay of some factors, such as: (1) the degree of local stereochemical distortion of the helix when the non-complementary residue is left intrahelical, (2) conformatianal entropy, (3) solvation, (4) charge effects, and (5) the relative stacking tendency of the noncomplementary base

Laboratory culturing of a thermophilic alga at high temperature.

Conditions for laboratory growth of the thermophilic alga Cyanidium caldarium at elevated temperatures have been developed. Growth characteristics of the organisms are described.

A novel method for estimating ancestral amino acid composition and its application to proteins of the Last Universal Ancestor

MOTIVATION Knowledge of how proteomic amino acid composition has changed over time is important for constructing realistic models of protein evolution and increasing our understanding of molecular evolutionary history. The proteomic amino acid composition of the Last Universal Ancestor (LUA) of life is of particular interest, since that might provide insight into the early evolution of proteins and the nature of the LUA itself. RESULTS We introduce a method to estimate ancestral amino acid composition that is based on expectation-maximization. On simulated data, the approach was found to be very effective in estimating ancestral amino acid composition, with accuracy improving as the number of residues in the dataset was increased. The method was then used to infer the amino acid composition of a set of proteins in the LUA. In general, as compared with the modern protein set, LUA proteins were found to be richer in amino acids that are believed to have been most abundant in the prebiotic environment and poorer in those believed to have been unavailable or scarce. Additionally, we found the inferred amino acid composition of this protein set in the LUA to be more similar to the observed composition of the same set in extant thermophilic species than in extant mesophilic species, supporting the idea that the LUA lived in a thermophilic environment. AVAILABILITY The program is available at http://compbio.cs.princeton.edu/ancestralaa

Stabilization of the native tertiary structure of yeast tRNALeu3 by cationic metal complexes

Complexes of inert (slowly exchanging) multivalent transition metal cations and neutral ligands were found to effectively replace Mg 2+ as stabilizers of the biologically active “native” form of yeast tRNA Leu 3 . From an analysis of the sigmoidal dependence of the denatured native conformational equilibrium on complex cation concentration at 25°C in 0·15 m -KCl at pH 7·4, it is calculated that about five complex cations must be tightly bound to stabilize the native conformer. Since this number is larger than the three binding sites previously observed for alkaline earth metal ions and polyamines, there must be sites of high negative charge on the tRNA accessible to Mg 2+ and spermidine 3+ but not to the more bulky complex cations. In that case, the complex cations must occupy additional sites in order to stabilize the native conformer. The relative effectiveness with which the cations stabilize the native conformation is: Co(NH 3 ) 6 3+ ≳ Pt(en) 2 (en-H) 3+ > Co(en) 3 3+ > Ir(NH 3 ) 5 Cl 2+ ⋍ Pt(NH 3 ) 4 2+ . This order is consistent with general electrostatic considerations. Hydrogen bonding of the co-ordinated amines to tRNA phosphates could enhance the affinity of Co(NH 3 ) 6 3+ . Since the complex cation-tRNA association constants are even larger than those of the cobalt complexes with multivalent inorganic anions, a cluster of at least three phosphates could constitute the binding site of each tightly bound cation in tRNA.

THIRD-STRAND IN SITU HYBRIDIZATION (TISH) TO NON-DENATURED METAPHASE SPREADS AND INTERPHASE NUCLEI

Abstract. A methodology has been developed for binding oligodeoxyribonucleotide ’third strands’ to duplex DNA targets in fixed but not additionally denatured metaphase spreads and interphase nuclei under conditions found to be optimal in solution. Third-strand in situ hybridization (TISH) at pH 6.0 of a psoralen- and biotin-modified 16-nucleotide homopyrimidine third strand to a unique multicopy target sequence in human chromosome 17 α-satellite (D17Z1 locus) is described. UVA-photofixed third strands, rendered fluorescent by fluorescein isothiocyanate-labeled avidin, are reproducibly centromere-specific for chromosome 17, and visible without amplification of the signal in lymphocyte and somatic cell hybrid spreads and interphase nuclei. Two third-strand-specific D17Z1 haplotypes were identified. TISH has potential diagnostic, biochemical, and flow cytometric applicability to native metaphase and interphase chromatin.

Oligonucleotide binding to the native and denatured conformers of yeast transfer RNA3Leu

Abstract The equilibrium binding patterns of complementary oligonucleotides to the native and denatured conformers of yeast transfer RNA 3 Leu have been determined. The pattern of binding to the native conformer follows that observed previously with other tRNAs. The results indicate that the anticodon loop and 3′ terminus are free in solution, and that all stems of the cloverleaf appear intact, although the dihydrouracil and “extra arm” stems are sufficiently weak to be subject to competitive binding by the probe oligomers. The T ΨC loop is also inaccessible to oligomer binding, while the dihydrouracil loop shows a low level of binding suggestive of oligomer competition with existing RNA structure. By contrast, in the denatured conformer the dihydrouracil loop and stem show strong oligomer binding characteristics of random RNA segments, whereas the anticodon loop no longer binds complementary oligomers. Binding to other regions remains unchanged, suggesting that the three major cloverleaf stems are intact. These observations are used as a basis for consideration of models for the two conformers.

Repairing the Sickle Cell Mutation I. SPECIFIC COVALENT BINDING OF A PHOTOREACTIVE THIRD STRAND TO THE MUTATED BASE PAIR

A DNA third strand with a 3′-psoralen substituent was designed to form a triplex with the sequence downstream of the T·A mutant base pair of the human sickle cell β-globin gene. Triplex-mediated psoralen modification of the mutant T residue was sought as an approach to gene repair. The 24-nucleotide purine-rich target sequence switches from one strand to the other and has four pyrimidine interruptions. Therefore, a third strand sequence favorable to two triplex motifs was used, one parallel and the other antiparallel to it. To cope with the pyrimidine interruptions, which weaken third strand binding, 5-methylcytosine and 5-propynyluracil were used in the third strand. Further, a six residue “hook” complementary to an overhang of a linear duplex target was added to the 5′-end of the third strand via a T4 linker. In binding to the overhang by Watson-Crick pairing, the hook facilitates triplex formation. This third strand also binds specifically to the target within a supercoiled plasmid. The psoralen moiety at the 3′-end of the third strand forms photoadducts to the targeted T with high efficiency. Such monoadducts are known to preferentially trigger reversion of the mutation by DNA repair enzymes.