J.R. Preer
Differences that persist when cells are cultured under constant environmental conditions are due to heredity, those that disappear are due to environment and those that vary in a cyclical fashion are due to development. Three ways to study hereditary differences are: uniparental genetics, molecular genetics and biparental genetics. Each is useful, but each has its limitations.
The first studies on protozoan genetics utilized only uniparental genetics. Uniparental genetics has been used primarily to study mutation. Mutation enables us to identify and determine the nature of the different elements controlling a given system. Uniparental genetics has played a major role in the study of viruses and bacteria, as well as those eukaryotes which are haploid or have a haploid stage in which characters can be identified. An example is the analysis of photosynthesis in the asexual alga Chlorella. In diploids or polyploids its use is severely limited because new mutations are generally recessive and hence do not change the phenotype.
The advent of molecular, genetics has made it possible to study the fine structure and transcription of individual genes in virtually all organisms, sexual or asexual. The numbers of genes of a given kind can be counted and similar ones compared. A transformation system is of great importance; if one is available it makes it possible to introduce variant genes and study their effects.
Nevertheless, there are many features of the genetic system that cannot be approached without the use of the sexual cycle or some other means of obtaining genetic recombination between distant elements. Crosses make it possible to obtain recombinants and learn about the overall structure of the genome––the numbers of chromosomes and linkage groups, the organization of genes and groups of genes into different regions of the chromosome. Much can also be learned about gene action by studying the ways that mutations are expressed when placed, into the genome in various combinations and determining whether they can complement or recombine. In many organisms where very large numbers of segregants can be examined (by plating them onto agar or by other means), it has been possible to probe genetic fine structure. Moreover, the study of mutations in diploid or polyploid organisms is made possible by taking advantage of the sexual cycle, and mutagenesis is the most powerful, technique available to explore the complete repertoire of elements controlling a given trait. Finally, mutation and recombination can serve as a partial substitute for transformation in molecular studies.
In bringing an organism under experimental control for genetic studies, however, one needs much more than the discovery of a mating system. Some of the essential elements are so trivial that their importance is easily overlooked. First, satisfactory means of culture must be found. Second, it must be possible to make large numbers of isolations and establish clonal cultures with ease so that rare mutants and rare recombinants can be found. Third, the life cycle must not only be established, but it must be brought under laboratory control so that the different stages can be induced at will. Fourth, a means of controlling promiscuous mating is of considerable advantage, so that the desired crosses can be obtained. Finally, if the peculiarities of the life cycle of particular organisms are discovered they can often be utilized to considerable advantage.
The establishment of Paramecium as a useful protozoan for genetic studies on surface antigens illustrates many of these principles. The alternative serotypes found in Paramecium persist under constant environmental conditions, but all cells in a culture can be induced to switch to another type by suitable treatments. Nevertheless, crosses with appropriate markers show that the differences in expression are determined by differences in the cytoplasm. Our ability to control and measure the amount of cytoplasm exchanged at conjugation has led to experiments that confirm this conclusion. Strains from different geographical locations usually show differences in serotype proteins and crosses reveal a series of independent genetic loci. The analysis was aided by using the process of autogamy which renders all loci homozygous at once, and whose induction is under control of the investigator. Molecular studies reveal that only one of the serotype genes is expressed at a time. The isolation of mutants has also contributed to our knowledge of the control of expression; in addition to the gene coding for the A protein, at least two other loci are necessary for expression. X-ray induced deletions of the A gene have produced no-A mutations, and further X-irradiation of them has produced no-A, no-B mutations. The lack of DNA sequences in the no-A mutants capable of hybridizing to isolated genomic DNA clones thought to be the A gene shows unambiguously that there is only one A gene in the genome and that it has been cloned and identified.
One group of deletions of the A and B genes show the genes in their micronuclei but have defective macronuclei. New macronuclei, forming under control of the defective macronuclei at conjugation and autogamy, not only lack the A (or B) gene(s), but, like the old macronuclei, are defective in their ability to support normal processing at the next conjugation or autogamy. These conclusions are supported by the experiments of Harumoto who transformed mutants permanently into wild type by transferring wild-type macronuclear material into mutant macronuclei. Similar mechanisms have been found for other traits by Sonneborn using heterokaryon formation induced by the process of macronuclear regeneration. Recently Doerder has obtained virtually identical results for certain mutations unable to produce the H serotype protein in Tetrahymena.
Finally, transformation has been shown to be a practical method of studying gene expression in Paramecium. Godiska and others have just discovered up to 257 transformation to serotype A in an A-deleted strain by microinjecting a cosmid with the A gene into the micronuclei of the deficient cells. The trait persists until the next autogamy. Expression of A in the transformants appears to be under normal cellular and environmental control.
During macronuclear regeneration if autogamy is induced in a heterozygote Aa, half of the progeny will be AA and half aa in the new micronucleus. However, all will be Aa for their old macronucleus. Any cell which arises from a fragment of the old macronucleus, i.e. from macronuclear regeneration, will be Aa and will show the dominant characteristic, whereas all of those which arise from the new macronucleus will be AA or aa. Half of these will show the recessive phenotype and one can follow cytologically to tell which was derived from which. Thus each fragment which arises from a previous macronucleus is just like the old macronucleus. This applies not just to this gene but to the entire macronuclear content. The ploidy level reaches about 1,000 and the number of fragments is about 30 to 40.
Although the chromosomes are difficult to work with, there may be about 45 chromosomes in the haploid condition in Paramecium. Tetrahymena has five chromosomes (like Drosophila) as a haploid number. Autosomal strains have been produced and genes have been mapped to individual arms of chromosomes. The hypotrich ciliates have polytene chromosomes like Drosophila. The DNA content of Tetrahymena is about 108 base pairs. Paramecium is about the same. Two reports of pulsed field electrophoresis have shown, as expected, that chromosomes are broken into fragments when the micronucleus is formed. There is extensive remodelling of the whole genome. The average size of the ‘chromosomes’ is of the order of 300 kb for Paramecium and greater than 600 kb in Tetrahymena.
In Paramecium and Tetrahymena the mating factors are insoluble surface proteins. However, this is not true of other ciliates where soluble factors are released, and these have been isolated in some cases. There are many mating types. In P. tetraurelia there are as many as 14 from different localities: 1 mates with 2, 3 with 4 etc. Selfing sometimes occurs. The mating type of the progeny can be affected in mating in a complex way. Sometimes it is simply genie, i.e. A and a. At other times, as in the d48 type, there is a cytoplasmic contribution and macronuclear differentiation occurs.
Despite the large aggregates that form during mating in Paramecium, there are only rarely other than biparental fusions, largely because the mating factors are insoluble. In hypotrichs, selling and multiple fusions are more common. Selfing occurs because both mating types are present transiently. It should be noted that the surface-antigen and mating-type factors are both surface molecules but separate from one another.
It is not known if there is loss of DNA during maturation of the micronucleus in Paramecium, but it is probably not very much. In Tetrahymena there is only about 10% loss, although in hypotrichs nearly 90% may be lost. Bruns has suggested that mating type is inherited through the presence of two loci within the micronucleus with mating type depending on whether one or other of these gets into the macronucleus. The d48 mutation does not affect mating-type determination and, like other mutations, is quite independent of mating type.
D. Walliker
Malaria parasites (Plasmodium spp.) undergo cycles of asexual division and gametocyte production in their vertebrate host, while fertilization of gametes takes place in the mosquito vector. Malaria parasites are analysed genetically by feeding mosquitoes on a mixture of gametocytes of two cloned lines differing in a number of genetically determined characters, to permit cross-fertilization of gametes of each clone. The sporozoites which develop are then used to infect a new host, and the resulting blood forms are examined for the presence of parasites exhibiting non-parental combinations of characters.
Early studies on the genetics of rodent malaria species, P. yoelii and P. chabaudi, made use of characters such as isoenzymes, drug-resistance and antigens as strain markers. These studies showed that (1) the malaria parasite appears to undergo a normal Mendelian pattern of inheritance of such characters, (2) the blood forces are haploid, meiosis probably occurring during early division of the zygote in mosquitoes, (3) resistance to drugs such as pyrimethamine and chloroquine is due to gene mutation, and (4) recombination occurs readily following cross-fertilization.
Genetic studies on the human parasite P. falciparum have been started recently. In the first cross, two clones were chosen which differed in pyrimethamine resistance, in electrophoretic forms of adenosine deaminase (ADA), in epitopes of two blood-form antigens detected by monoclonal antibodies, in certain proteins detected by two-dimensional polyacrylamide gel electrophoresis (PAGE), in patterns of hybridization of a repetitive DNA probe, and in chromosome sizes as revealed by pulse field electrophoresis. Gametocytes of each alone were grown in culture, then mixed together and fed to mosquitoes (Anopheles freeborni). Sporozoites were used to infect a chimpanzee, and the resulting blood forms were established in culture. Clones were isolated from the progeny of the cross and examined for each parental line marker. The principal findings of this study were:
1. Recombination between each parent-line character was detected. The inheritance patterns of enzyme, antigen and pyrimethamine-resistance markers confirmed the haploid nature of the blood forms.
2. The chromosome studies showed that considerable genome rearrangements take place following cross-fertilization. The two parent lines differed in the size of chromosomes 3 and 4. Certain progeny clones exhibited a chromosome 4 of intermediate size between that of each parent. In other clones, chromosome 2 was larger than in either parent. Changes in some other chromosomes were also apparent.
3. Recombination appeared to occur at a remarkably high frequency, suggesting that cross-fertilization was favoured over self fertilization. Among 14 clones examined, 12 were recombinants; whereas if fertilization had occurred randomly between gametes, it would have been predicted that at least 50% of the progeny would be parental types.
In view of the fact that malaria patients are frequently infected with more than one genetically distinct parasite, these results show that novel genotypes are likely to arise frequently following mosquito transmission. This finding has important implications for measures designed to control P. falciparum by chemotherpy or vaccination.
During presentation of the paper, the importance of the timing of cloning to determine recombinant progeny was suggested to the speaker and readily agreed. Because of experimental constraints, the cloning step had been carried out only once in the reported experiments on P. falciparum. However, cloning proved relatively easy by the method of limiting dilution although occasional mixtures of progeny types had resulted.
The apparent non-appearance of parental types bearing the histidine rich protein (HRP)-negative phenotype was difficult to explain unless cross-fertilization was favoured. Speculatively, if the HRP-negative phenotype is associated with loss of the knobbed phenotype, it might then find difficulty in establishing in the mammalian host (chimpanzee). However, the speaker felt that published work on the disappearance and reacquisition of the knobbed phenotype was unconvincing.
In the case of rodent malarias, it is relatively easy to achieve mating between strains, though occasionally it is not possible between geographically separated isolates. In the case of P. falciparum, it was only achieved once. Although the existence of mating types remains a possibility, recombination was obtained between virtually every pair of strains tested except for the HRP types.
Evidence of recombination in the bloodstream has not been sought in P. falciparum. In rodents, workers deliberately mixed sporozoites or blood forms, infected mice and looked for recombination. Although only two drug markers were used, no recombination was found.
If a phenomenon occurs in malaria similar to parasex in fungi, in addition to the sexual cycle, an excess of recombinants might arise. However, such a situation is not known to exist in malaria.
J. Glassberg
Mutants of Crithidia fasciculata were isolated, following mutagenesis, and fell into the following classes: auxotrophic, drug-resistant and colony-morphology. The single auxotrophic mutant found required cysteine for growth. This requirement was satisfied by cystathione but not by homocysteinethiolactone. Drug-resistant mutants were found resistant to actinomycin D, azauracil, azeuridine and 5-fluorouracil. These mutants showed no cross-resistance. Azauracil and fluororacil mutants were uptake mutants.
Crossing an actinomycin D-resistant strain with an azauracil-resistant strain resulted in the appearance of doubly drug-resistant colonies. These colonies remained resistant to both drugs even after growth on non-selective plates.
Crossing an actinomycin D-resistant, azauridine-resistant strain with an azauracil-resistant, fluorouracil-resistant strain resulted in a complex assortment of colonies exhibiting both parental and recombinant phenotypes. Most of the recombinants were unstable but some stable wild-type and azauridine-resistant clones were found. These data suggest that Crithidia undergoes some type of genetic recombination and must be diploid at some time during the process.
in the case of resistance to azauridine, repeated ‘picking’ showed the same phenotype, but it is not known how long this stability would be maintained. However, each colony contains many generations. The hypothesis concerning the existence of unstable diploids which resolve into stable haploids was questioned on the ground that the wild-type chromosomes seemed to survive the drug pressure.
