RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension.

The RNA world hypothesis regarding the early evolution of life relies on the premise that some RNA sequences can catalyze RNA replication. In support of this conjecture, we describe here an RNA molecule that catalyzes the type of polymerization needed for RNA replication. The ribozyme uses nucleoside triphosphates and the coding information of an RNA template to extend an RNA primer by the successive addition of up to 14 nucleotides-more than a complete turn of an RNA helix. Its polymerization activity is general in terms of the sequence and the length of the primer and template RNAs, provided that the 3' terminus of the primer pairs with the template. Its polymerization is also quite accurate: when primers extended by 11 nucleotides were cloned and sequenced, 1088 of 1100 sequenced nucleotides matched the template.

Constructing an RNA world.

A popular theory of life's origins states that the first biocatalysts were not made of protein but were made of RNA or a very similar polymer. Experiments are beginning to confirm that the catalytic abilities of RNA are compatible with this 'RNA world' hypothesis. For example, RNA can synthesize short fragments of RNA in a template-directed fashion and promote formation of peptide, ester and glycosidic linkages. However, no known activity fully represents one presumed by the 'RNA world' theory, and reactions such as oxidation and reduction have yet to be demonstrated. Filling these gaps would place the hypothesis on much firmer ground and provide components for building minimal forms of RNA-based cellular life.

RNA-catalysed nucleotide synthesis.

The 'RNA world' hypothesis proposes that early life developed by making use of RNA molecules, rather than proteins, to catalyse the synthesis of important biological molecules. It is thought, however, that the nucleotides constituting RNA were scarce on early Earth. RNA-based life must therefore have acquired the ability to synthesize RNA nucleotides from simpler and more readily available precursors, such as sugars and bases. Plausible prebiotic synthesis routes have been proposed for sugars, sugar phosphates and the four RNA bases, but the coupling of these molecules into nucleotides, specifically pyrimidine nucleotides, poses a challenge to the RNA world hypothesis. Here we report the application of in vitro selection to isolate RNA molecules that catalyse the synthesis of a pyrimidine nucleotide at their 3' terminus. The finding that RNA can catalyse this type of reaction, which is modelled after pyrimidine synthesis in contemporary metabolism, supports the idea of an RNA world that included nucleotide synthesis and other metabolic pathways mediated by ribozymes.

Cancer genes and risk assessment.

In the radiation protection approach to risk assessment, it is assumed that cancer induction follows low-level radiation exposure in a probabilistic way. The stochastic model underlying all present risk assessment methods derives risks from cancer incidence frequencies in exposed populations and associates disease outcomes totally with the level of exposure to an environmental source. Exposure is the risk factor that affects the probability of the disease outcome. But cancer risk also reflects pre-existing underlying genetic predisposition (genetic risk factors) in individuals who are exposed. The distribution of genetic risk factors in time and space is governed by the biological and social processes involved in reproduction (biological risk factors). To include both genetic and biological risk factors in cancer risk assessment, a genetic cancer risk factors model must be developed. We tested the plausibility of the genetic cancer risk factors model by surveying all genetic disorders associated with cancer in the Online Mendelian Inheritance in Man database, determined the gene map location, if known, and attached DNA sequence information if it was available. We found 641 genetic disorders associated with cancer, of which 495 have been mapped into about 120 clusters on the human genome, and of which DNA sequence data are at least partially available for 253. From the molecular variants of various cancer risk genes that have been described, and from the breeding patterns that determine carrier frequencies in the population, we deduce that significant numbers of members of the population may carry such genes. If such carriers differ in radiogenic cancer risk from non-carriers in the population, then their variability needs to be taken into account in risk assessment models.

Accessing rare activities from random RNA sequences: the importance of the length of molecules in the starting pool.

BACKGROUND: In the past few years numerous binding and catalytic motifs have been isolated from pools of random nucleic acid sequences. To extend the utility of this approach it is important to learn how to design random-sequence pools that provide maximal access to rare activities. In an effort to better define the relative merits of longer and shorter pools (i.e. pools with longer or shorter random-sequence segments), we have examined the inhibitory effect of excess arbitrary sequence on ribozyme activity and have evaluated whether this inhibition overshadows the calculated advantage of longer pools. RESULTS: The calculated advantage of longer sequences was highly dependent on the size and complexity of the desired motif. Small, simple motifs were not much more abundant in longer molecules. In contrast, larger motifs, particularly the most complex (highly modular) motifs, were much more likely to be present in longer molecules. The experimentally determined inhibition of activity by excess sequence was moderate, with bulk effects among four libraries ranging from no effect to 18-fold inhibition. The median effect among 60 clones was fivefold inhibition. CONCLUSIONS: For accessing simple motifs (e.g. motifs at least as small and simple as the hammerhead ribozyme motif), longer pools have little if any advantage. For more complex motifs, the inhibitory effect of excess sequence does not approach the calculated advantage of pools of longer molecules. Thus, when seeking to access rare activities, the length of typical random-sequence pools (< or = 70 random positions) is shorter than optimal. As this conclusion holds over a range of incubation conditions, it may also be relevant when considering the emergence of new functional motifs during early evolution.

Chromosome content and ultrastructure of radiation-induced micronuclei.

Unrepaired or misrepaired radiation damage in mammalian chromosomes can result in micronucleus formation at the first cell division. This represents loss of genomic information which may cause cell death. To improve our understanding of the mechanism of radiation-induced micronucleus formation, we characterized micronucleus ultrastructure and identified the origin of micronucleus DNA. Immunofluorescence microscopy showed that micronuclei were structurally similar to main nuclei since they contained nuclear lamins A and C and were encapsulated by a network of vimentin intermediate filaments. The contents of radiation-induced micronuclei were characterized using fluorescence in situ hybridization to probe for DNA originating from chromosomes 2, 7, 11 and 16. We postulated that if incorporation of DNA into micronuclei were random, then the probability of chromosomal DNA in micronuclei would be related to the target, i.e. chromosome size. Our results demonstrated that incorporation of DNA from smaller chromosomes (11 and 16) was not different from expected values but incorporation of DNA from the larger chromosomes (2 and 7) was significantly greater than expected. Not all chromosomes in the human genome, therefore, were equally susceptible to genomic loss by micronucleus encapsulation. In conclusion, radiation-induced micronuclei have similar structural characteristics to main nuclei, chromosome damage and/or repair after ionizing radiation may be non-random, and micronucleus formation may reflect this variability.

The REC1 gene of Ustilago maydis, which encodes a 3'-->5' exonuclease, couples DNA repair and completion of DNA synthesis to a mitotic checkpoint.

Mutation in the REC1 gene of Ustilago maydis results in extreme sensitivity to killing by ultraviolet light. The lethality of the rec1-1 mutant was found to be partially suppressed if irradiated cells were held artificially in G2-phase by addition of a microtubule inhibitor. This mutant was also found to be sensitive to killing when DNA synthesis was inhibited by external means through addition of hydroxyurea or by genetic control in a temperature-sensitive mutant strain defective in DNA synthesis. Flow cytometric analysis of exponentially growing cultures indicated that wild-type cells accumulated in G2 after UV irradiation, while rec1-1 cells appeared to exit from G2 and accumulate in G1/S. Analysis of mRNA levels in synchronized cells indicated that the REC1 gene is periodically expressed with the cell cycle and reaches maximal levels at G1/S. The results are interpreted to mean that a G2-M checkpoint is disabled in the rec1-1 mutant. It is proposed that the REC1 gene product functions in a surveillance system operating during S-phase and G2 to find and repair stretches of DNA with compromised integrity and to communicate with the cell cycle apparatus.

Can tetraplex recombination models explain observations in induced mitotic gene conversion?

Induced mitotic gene conversion studies on the CYC1 gene of yeast have shown that the actual base pair changes, the types of changes (base substitution, deletion or addition) and the distances between mutations all affect gene conversion yields. In crosses between mutations less than four bases apart, gene conversion rates are as low as back mutation rates. The same mutants crossed to alleles more than five bases away may recombine 50-fold more. In crosses between mutations five or more base pairs apart, recombination rates varying by up to ten-fold are observed when comparing mutations at the same codon sites. The actual mutations in crosses affect recombination rates at these distances. The data rules out models in which mutants are repaired independently. Models with large gaps at the initiation site are ruled out if the mutants are within the gap. Recombination models are favoured in which both mutations can interact at a distance to affect the probability of recombination; such interactions may reflect the geometry of the recombinational junctions. The specific interactions proposed are that the actual mutations, and residual mismatches arising on excision resynthesis, affect both the further migration of the recombinational junction, and the probability that excision-repair will detect and correct residual mismatches. Junction models in which interactions are expected include those composed of base tetraplexes. The data is interpreted in terms of these models. Meiotic recombination data is consistent with these models.

Non-cloning amplification of specific DNA fragments from whole genomic DNA digests using DNA 'indexers'.

A highly systematic, non-cloning method of distinguishing and isolating every fragment in a class-IIS or interrupted palindrome restriction digest has been developed in our laboratory. These enzymes produce informative, non-identical cohesive ends which can be selectively modified by ligation to individual synthetic oligodeoxyribonucleotides with the corresponding complementary ends. In this way, polymerase chain reaction and sequencing primer sites and labels can be introduced specifically into a single fragment in a total genomic digest. Known and unknown fragments from genomes of the complexity of Escherichia coli can be isolated directly in sequencable form without the necessity of synthesizing unique primers. Human DNA has also been assessed in this way. Problems intrinsic to cloning (selective fragment loss, mutation and sequence rearrangement) are avoided. Systematic characterization of DNA fragments by their cohesive ends and length provides tremendous power and flexibility for analysis of any DNA molecule without specific clones, probes or libraries. We report proof of principle of this remarkable system and indicate potential applications in DNA sequence tagged site and restriction mapping, sequencing, restriction-fragment-length polymorphism analysis and DNA diagnostics.

Genetic recombination of homologous plasmids catalysed by cell-free extracts of topo-isomerase mutant strains of Saccharomyces cerevisiae.

Cell-free extracts of the yeast Saccharomyces cerevisiae can be used to catalyse the recombination of bacterial plasmids in vitro. Recombination between homologous plasmids containing different mutations in the gene encoding tetracycline resistance is detectable by the appearance of tetracycline-resistance following transformation of the recombinant plasmid DNA into Escherichia coli DH5. This in vitro recombination system was used to determine the involvement of eukaryotic topo-isomerases in genetic recombination. Cell-free extracts prepared from a temperature-sensitive topo-isomerase II mutant (top2-1) of S. cerevisiae yielded tetracycline-resistant recombinants, when the recombination assays were performed at both a non-restrictive temperature (30°C) and the restrictive temperature (37°C). This result was obtained whether or not ATP was present in the recombination buffer. Extracts from a non-conditional topo-isomerase I mutant (top1-1) of S. cerevisiae yielded tetracycline-resistant recombinants, as did a temperature-sensitive double mutant (top2-1/top1-8) at the restrictive temperature. The results of this study indicate that neither topo-isomerase I nor topo-isomerase II was involved in the recombinational activity examined.

The RBE of neutrons for induced mitotic gene conversion in "error-prone repair" defective yeast.

Mitotic gene conversion was induced in the diploid yeast strain D7.rad6 which lacks "error-prone repair" and thus does not mutate. Neutrons (14.5 MeV), 60Co gamma rays, and 150 kVp X rays delivered under oxic or anoxic conditions were compared for their ability to induce gene conversion. Doses were chosen to minimize cell killing. A lack of induced mutation in this strain at the ilv1-92 allele was confirmed. Gene conversion of the trp5-27/trp5-12 alleles was induced with a linear dose response, and the yield of convertants per gray was significantly enhanced over yields reported previously for a wild-type stain. The relative biological effectiveness (RBE) of neutrons relative to low-LET radiations was found to be about 2.2 for either oxic or anoxic radiation in contrast to wild-type where the oxic RBE was 1.7 and the anoxic RBE 2.7. Absence of the rad6 function was therefore associated with an altered RBE for the conversional end point. The oxygen enhancement ratio (OER) for gene conversion was found to be about 1.7 for all radiations in contrast to the wild type where the OER for neutrons was 1.7, but for low-LET radiations it was 2.7. As repair of ionizing damage in the rad6 strain did not lead to mutation, owing to the loss of "error-prone repair," the changes in yield, RBE, and OER were consistent with the hypothesis that some of the lesions processed by wild type to generate mutations could, in the rad6 strain, lead instead to gene conversion.

The relative biological effectiveness of 14.5-MeV neutrons for the induction of gene conversion and mutation in yeast.

The relative biological effectiveness (RBE) and oxygen enhancement ratio (OER) were determined in the yeast Saccharomyces cerevisiae for the induction of gene conversion (the product of recombinational repair) and mutation (the product of error prone repair) by 14.5-MeV neutrons in comparison with 60Co gamma rays and 150 KVp X rays. Neutron irradiation in oxic or anoxic conditions induced significantly higher yields of convertants and mutants than sparsely ionizing radiations under the same conditions. RBEs for both gene conversion and mutation under anoxia were significantly higher than under oxic conditions. RBEs for mutant induction under anoxia were lower than the RBEs for gene conversion under the same conditions. The data support the hypothesis that the production of lesions leading to the genetic consequences of gene conversion and mutation differ in their dependence upon LET and the presence of oxygen during irradiation, and therefore the two DNA repair processes which produce these end points recognize, at least in part, different classes of damage.

Nucleic acid-binding glycoproteins which solubilize nucleic acids in dilute acid. Re-examination of the Ustilago maydis glycoproteins.

Holloman ((1975) J. Biol. Chem. 250, 2993-3000) reported the isolation from Ustilago maydis of a glycoprotein which prevented the precipitation of nucleic acids in cold 5% trichloroacetic acid. Two glycoprotein fractions from U. maydis with this nucleic acid-solubilizing activity were isolated in our laboratory using improved purification procedures. The activity was not due to nuclease contamination. The glycoproteins are distinguished by: their ability to bind to concanavalin A-Sepharose; their differential binding to double- and single-stranded deoxyribonucleic acid, and to ribonucleic acid; their molecular weights (46,000 and 69,000); and the relative amounts present in growing versus nongrowing cells. Both fractions required sulfhydryl-reducing conditions for optimal yields, specific activity, and stability. Nucleic acid binding was cooperative, the minimum number of glycoproteins required to make a native T7 DNA molecule soluble in dilute acid being estimated at 2 and 15, respectively.