A mechanism for RNA–RNA splicing is proposed. A species of RNA (‘splicer’ RNA) hybridizes to precursor mRNA across the splice point. This hybridization can be with intron or exon sequences or both. The double-stranded RNA structure precisely indicates to the splicing enzymes the exact location for exon ligation.

A model for the control of gene expression is presented. The regulation of synthesis of different splicer RNAs will also control which precursor mRNA molecules are spliced. The removal of intervening sequences from a precursor mRNA molecule could be both a signal for that molecule to be transported to the cytoplasm and a means of allowing gene expression.

A mechanism for gene conversion is proposed which overcomes many of the difficulties that any copy choice model encounters. It is suggested that along with general genetic pairing of homologous genomes at meiosis, effective pairing over short regions of the genetic material occurs at the molecular level by the separation of the strands of the DNA double helices, followed by the annealing of strands from two homologous chromatids. If the annealed region happens to span a heterozygous site, mispairing of bases will occur. Such a situation may be analogous to that in DNA which is damaged by mutagens; the same or similar repair mechanisms may operate, and these, by adjusting the base sequences in order to restore normal base pairing, would bring about gene conversion in the absence of any genetic replication. The model indicates how precise breakage and rejoining of chromatids could occur in the vicinity of the conversion, so that conversion would frequently be accompanied by the recombination of outside markers. The model also proposes that the distance between two mutant sites on a fine structure map depends not so much on the frequency of a recombinational event occurring between them, but rather on the degree of inhibition of the processes of genetic pairing by the mutants themselves.

The model will explain almost all the data in a formal way, and it has the advantage over copy choice mechanisms for gene conversion in (1) being compatible with semi-conservative replication of DNA, (2) not invoking DNA synthesis during or after genetic pairing, (3) providing a molecular mechanism for close specific pairing, (4) making it unnecessary to postulate sister strand exchange or a process akin to this, (5) suggesting why rates of gene conversion in opposite directions are sometimes unequal and (6) providing an explanation of the clustering of mutant sites, a basis for map expansion and for the apparently capricious departure of fine structure maps from additivity. Although the model proposed is a general rather than a specific one, it suggests that the process of conversion and intragenic recombination is more complex than is usually believed, since it depends on several interacting factors. Nevertheless, it is hoped that the introduction of a model with this complexity will help to stimulate specific experiments, and that these will provide definitive information which would never be obtained if simpler models of conversion and intragenic recombination were believed to explain the genetic data sufficiently well.

In order to develop a system for the study of the mechanism of intragenic recombination in Ustilago, mutants lacking nitrate reductase activity were isolated, and five alleles were combined in pairs in ten vegetative heteroallelic diploids. The diploids have the mutant phenotype, i.e. inability to utilize nitrate as sole source of nitrogen, but they will recombine to produce wild-type cells much more frequently than the back-mutation rates of haploids or homoallelic diploids. The spontaneous rate of recombination can be enormously increased by low doses of UV light, particularly if treatment is during the period of DNA synthesis in the mitotic cycle. By means of half-tetrad analysis it has been shown that this process of intragenic recombination, as in other fungi, is due to gene conversion rather than reciprocal exchange. It has also been shown that the frequency of UV-induced conversion under standard conditions gives a rough measure of the distance between two mutant sites, since it was possible to use these frequencies to make a linear fine structure map of the gene. These results are discussed in relation to a hybrid DNA model for gene conversion slightly modified from that previously suggested for meiotic recombination.

A method is described which makes it possible to detect induced mutations in the pre-existing gene by determining whether both daughter cells of the first division after mutagenic treatment carry the mutation. The method depends on the use of diploid strains of Ustilago maydis which have biochemical markers in both homozygous and heterozygous condition. Cells which are induced to mutate at a homozygous locus will also occasionally undergo mitotic crossing-over. Since only mutant cells are selected, the reciprocal products of such a cross-over will not be detected if the mutation is transmitted to only one daughter cell after treatment. Mutations induced by UV light were examined in three different diploids, and with each, by use of the appropriate selective media and the replica plating technique, it was possible to detect mutant colonies which did consist of the reciprocal products of the exchange. It is deduced that a high proportion of the mutations must occur in the pre-existing gene; and a hypothesis is suggested which attempts to reconcile this conclusion with the current concept of the structure and replication of the gene.

1. Many of the Ustilaginales, or smut fungi, appear to have the qualities necessary for the application of modern techniques of microbial genetics. Ustilago maydis is considered the most suitable species.

2. Investigations of the mating system confirm reports that the production of diploid brandspores in the host is controlled by alleles at two loci.

3. Genetic markers were obtained by inducing mutations in a wild-type strain with ultra-violet light. Of 100 biochemical mutants which were isolated, the growth requirements of 94 were identified. Thirty of these were used in genetic tests.

4. The compact growth of colonies on artificial media allowed new techniques to be developed by means of which large samples of progeny could be isolated and identified easily. The analysis of brandspore colonies consisting of the products of single meiotic divisions is the quickest method for detecting linkage, but its accurate measurement appears to be achieved by examining the individual members of tetrads.

5. Linkage was detected relatively rarely, but eight markers, including the a mating-type locus, were assigned to one or other of two linkage groups. Although recombination values were not always determined accurately owing to irregular basidiospore germination, the auxotrophic markers in each group could be mapped in a linear order. Since no indication of other linkage groups was obtained, the genetic evidence is so far consistent with cytological reports that the basic haploid chromosome number is two in the smut fungi.

6. Three linked markers were used to investigate chromatid interference by tetrad analysis. None was detected in a total of eighteen double exchanges.

1. Two different methods of selection were used to obtain stable prototrophic solopathogenic strains of Ustilago maydis from haploids with different biochemical requirements. The strains were shown to be heterozygous diploids.

2. Two diploids which were examined in detail had four markers in common, but they were in different coupling and repulsion phases. Rare spontaneous segregation was detected in one of the diploids, but a high frequency of segregation was obtained in both after treatment with ultra-violet light. The proportion of segregants amongst the survivors increased with the dose. These auxotrophic segregants were detected by means of the replica plating technique.

3. The types of segregant which were obtained from both diploids were consistent with the view that they arose as a result of mitotic crossing-over. After low doses of radiation the reciprocal products of crossing-over were often detected. There was no evidence from the phenotypes of the segregants that haploidization was occurring. The diploidy of a sample of the segregants was confirmed by mating-type tests, and by the fact that they showed further segregation after another dose of radiation.

4. A slow-growing unstable segregant recovered after a high dose of radiation proved to be a monosomic strain which consistently reverted to a stable diploid homozygous for one chromosome. It was possible to use this auxotrophic diploid together with a haploid with different biochemical requirements, to synthesize a prototrophic triploid strain. The triploid was much less stable than the diploid strains.

5. By means of pathogenicity tests with certain segregants it was possible to distinguish the function of the two loci which control the mating system. The a locus is responsible for the fusion of haploid sporidia and has no effect on the pathogenicity of the heterokaryon which is under the control of the b locus.

6. The effects of ultra-violet light on mitotic crossing-over do not seem to be easily compatible with a copy choice or similar mechanism of recombination.

A method is now available for synchronizing the division of populations of cells of the smut fungus Ustilago maydis. In two experiments carried out with a diploid strain heterozygous for several biochemical markers, samples of cells were removed at intervals through the synchronized division cycle and treated with a constant dose of ultra-violet light. Cell survival and the frequency of the various recombinants resulting from induced mitotic crossing-over were recorded. In addition the period of DNA synthesis in the unirradiated population has been measured. During this period the cells are very sensitive to ultra-violet light and those that survive contain the highest proportion of induced recombinants. In so far as the markers make it possible to locate the position of cross-overs, cells which are irradiated early in the period of genetic replication show most crossing-over towards the ends of the chromosome arms, whilst cells which are treated late in this period show crossing-over near the centromeres. The data are most easily interpreted by supposing that chromosome replication begins at the ends of the arms and proceeds to the centromere, and that the temporary interruption of this process by ultra-violet light can result in pairing and crossing-over in the vicinity of the points of interruption.

The transition from Palaeoindian to Archaic societies in North America is often viewed as a linear progression over a brief but time-transgressive period. New evidence from the Wilson-Leonard site in Texas suggests social experimentation by Palaeoindians over a 2500-year period eventually resulted in Archaic societies. The process was neither short nor linear, and the evidence shows that different but contemporaneous lifeways existed in a variety of locales in the south-central US in the Early Holocene.