Proc. Natl. Acad. Sci. USA Vol. 77, No. 6, pp. 3539-3543, June 1980 Genetics Predicting coding function from nucleotide sequence or survival of "fitness" of tRNA (DNA/mRNA/sequence constraints/genotypic selection/bonded discontinuous ultrathin acrylamide gel) GEORGE PIECZENIK Department of Biochemistry, Bureau of Biological Research, Rutgers University-Busch Campus, New Brunswick, New Jersey 08903 Communicated by Rollin D. Hotchkiss, December 26, 1979 ABSTRACT The sequence of a nucleotide region of fI Presented are two oligonucleotide sequences unselected bacteriophage was determined on a bonded ultrathin acryl- except in the respect that they were determined by me and that amide gel with a discontinuous buffer system by using the di- they offer little in the form of initiator or terminator codons. deoxy-DNA sequencing method. This sequence and one other were analyzed for maximal base pairing with tRNAs. The results When they are first determined, such short sequences give little allow a prediction of the direction and phase of possible coding or no clue as to coding functions. The two sequences will be functions. The implication of sequence constraints on mRNA analyzed as they were first viewed in the light of the mRNA codon frequency, tRNA structure, the origin of protein synthesis, constraint, without reference to any collateral information. The and triplet reading are discussed in terms of neutral, Darwinian, results will be compared with later proposals for coding func- and genotypic selectionist perspectives of evolution. The model of F. H. C. Crick, S. Brenner, A. Klug, and G. Pieczenik [(1976) tion. Oriins ofLife 7,389-397] for the origin of the genetic code is used to interpret contemporary adaptive and functional nucleic METHODS acid sequences. Determining a Nucleotide Sequence. Bacteriophage fi is Pieczenik et al. (1) have tested the assumption that mRNA is a single-stranded DNA bacteriophage, parts of which have been directly determined by its complementary sequence. In this sequenced (6, 11, 12). Single-stranded viral DNA, (+)strand, paper I wish to question the assumption that triplet codon was prepared from plaque-purified fI bacteriophage as de- translation necessarily implies that the tRNA-mRNA interac- scribed (13) and made up to a concentration of 0.6 mg/ml. tion is a three-base-pair interaction. It is widely believed that Covalently closed, duplex, supercoiled replication form (RF) DNA sequences evolve or are conserved in evolution as the I of fI DNA was prepared by the published method (6) with the result of selective pressures exerted on organisms carrying their modification of eliminating the sucrose gradient. Escherichia functional end products. The view that nucleic acid sequences coli K-38 was the host strain. themselves may be subject to direct biochemical selection, Hae III restriction fragments were prepared by digesting 100 which imposes evolutionary constraints on nucleotide order (2, ,Ag of RF I DNA with Hae III (obtained from New England 3), was in part illustrated by a recent proposal for the primordial BioLabs) for 15 min at 1 unit/tg of DNA for 2 hr in 50 Ul of 10 mechanism of protein synthesis. In this proposal, Crick et al. mM Tris-HCl, pH 7.8/66 mM MgCl2/60 mM 2-mercaptoeth- (4) suggested that in the primitive protein-synthesizing system, anol. Fragments were separated on a modified version of an the tRNAs used five base pair interactions with the mRNAs. Ornstein-Davis discontinuous acrylamide gel system (2, 14, 15). From this, a comma-free, triplet, partially overlapping code The stacking gel was 2.75% acrylamide (1:10 bisacrylamide/ could easily develop. In the present paper, the possibility of such acrylamide) at pH 6.7. The separating gel was composed of 5% overlapping interactions will be examined for existing con- acrylamide and 10% acrylamide at pH 8.9 at a 1:40 bisacryla- temporary nucleotide sequences and known tRNAs. mide/acrylamide ratio. The running buffer was Tris glycine Such interactions would impose constraints upon mRNA at pH 8.3 (15). Electrophoresis was at 0°C at 100-200 V until sequences and these would be independent of, but compatible the bromophenol blue marker reached the bottom of a 20-cm with, the internal (out-of-phase) terminator constraint for de- gel. Eight of the nine recognized restriction fragments were termining whether a sequence is a message sequence (5, 6). separated. The seventh band from the origin was eluted elec- Because the universality of the UGA terminator codon has now trophoretically into a dialysis bag, acidified with sodium acetate been questioned (7), an additional method for determining (pH 5.0), and precipitated with 2.5 vol of 95% (vol/vol) ethanol phase-of-reading becomes more interesting. in 100 The existence of such an mRNA constraint would also bring at -20°C. The fragment was pelleted and resuspended under question the assumption that synonymous codon usage ,ul of water. This restriction fragment, called Hae III H frag- is selectively neutral (8, 9). It also argues that selection may not ment, was used to prime the fi (+)strand for generating a se- be exclusively at the total organism level and that nucleotide quence. sequences are a simple record of the successful and adaptive The DNA sequence was determined with chain-terminating historical accidents a species has encountered in its survival in dideoxy inhibitors by the procedure of Sanger et al. (16), with nature (10). An mRNA constraint based on tRNA interaction the modification that all four terminators were 2',3'-dideoxy- can be a historical vestige of the original protein-synthesizing triphosphates and at a concentration 100 times the respective mechanism based on the Darwinian principle of descent from deoxytriphosphate concentrations. The dideoxytriphosphates a common ancestor (4), or it may have arisen as a consequence were purchased from P-L Biochemicals. Reaction mixtures of continuous selection at the nucleotide level [i.e., genotypic were prepared for [a-32P]dATP as the labeling triphosphate selection (3)], or it may be a consequence of another, as yet, as well as for [ct-32P]dCTP. The specific activities were in the unspecified limitation. range of 300-500 Ci/mmol (1 Ci = 3.7 X 1010 becquerels). In addition to the dATP "chase" for the [32P]dATP-labeled re- reaction mixture was chased with its The publication costs of this article were defrayed in part by page action, each respective charge payment. This article must therefore be hereby marked "ad- unlabeled triphosphate in 1 Al of 0.5 mM dNTP. The primer vertisement" in accordance with 18 U. S. C. §1734 solely to indicate was recleaved with Hae III (4 units per reaction mixture for 10 this fact. min at 37°C). Reactions were terminated with EDTA and then 3539 Downloaded by guest on September 23, 2021 3540 Genetics: Pieczenik Proc. Nati. Acad. Sci. USA 77 (1980) EDTA/dye in formamide as described. Nucleotides were 3 fractionated on two gel systems. One was a continuous system r G A T C G A T ) of Tris borate/7 M urea/12% acrylamide (1:20 bisacrylam- COiC.,, A ide/acrylamide) at pH 8.3 with a thickness of 1 mm (17). The 2l A other was a discontinuous buffer system and the gel was cova- co G a -0s.NA q" lently bonded to its backing, allowing an 0.18-mm gel. C Ultrathin Discontinuous Bonded Acrylamide Gel System. ,C 4IS.: By covalently bonding the gel to its glass backing one solves two T problems of resolution. One can make an ultrathin gel and shrink the gel, without distortion, immediately after electro- phoresis. This limits bandwidth as a consequence of gel thick- ness and diffusion. The question of gel thickness has also already G been addressed by Sanger and Coulson (18). The strategy of C developing a thin starting zone was originally devised by C Ornstein and Davis (14, 15), using a moving boundary. They A set up a discontinuous pH system to generate moving -T T boundaries; I have set up a system with cations of different _mIA mobilities. C4,WT The gel was a 12% Tris borate/urea gel at pH 8.9; the upper A (and lower) reservoir buffer was Tris glycine at pH 8.3. The concentrations were as given for Tris glycine buffer (15) and TB/TB TG/TB D Tris borate buffer and Tris borate/urea 12% gels (17). The gel FIG. 1. DNA sequence of bacteriophage fi with Hae III restric- was bonded to its glass plate with y-methacryloxypropyltri- tion fragment used to prime the viral DNA (+)strand and dideoxy methoxysilane (19). Runners for the vertical ultrathin gel were sequencing method of Sanger et al. (16). TB/1TB refers to continuous made from cut x-ray film with the emulsion removed. Slot Tris borate buffer gel electrophoresis system. TG/TB refers to Tris formers of the same material were 1 cm wide X 1 cm high X glycine buffer and Tris borate gel buffer and bonded acrylamide gel 0.18 mm thick with a 2-mm space between the slots. The gel electrophoresis. was prerun with Tris borate buffer for 1/2 hr prior to layering internal terminators or symmetry elements 3'-ward to it to of sample and change of buffer system. This thin gel runs at 2 satisfy sequence constraints for initiator regions (6). The single kV and 10-14 mA, but can be run as low as 1 kV and 5 mA. The terminator codon UAA in phase 3 might eliminate one of the gel was dehydrated in 95% ethanol immediately after elec- possible six phases; on the other hand it might represent an trophoresis, which removes both the urea and water and shrinks actual carboxyl terminus in this phase.