DNA rearrangements and mating-type determination in Paramecium tetraurelia

Ciliated protozoa have separate germline and somatic nuclei, yet unlike larger organisms, both nuclei reside in the same cytoplasm. The micronuclei contain the germline and the macronucleus is the somatic nucleus. Thousands of DNA elements are normally removed from the micronuclear genome as it forms a new macronucleus during each sexual cycle. A recent study directly links the excision of these internal eliminated sequences (IESs) to mating type determination by showing that a pleiotropic mutation affecting mating type also prevents the excision of an IES from a surface protein gene⁽¹⁾. Remarkably, once the IES is present in the old macronucleus it prevents excision of that specific IES during formation of the next macronucleus.     

The developmental fate of fission yeast cells is determined by the pattern of inheritance of parental and grandparental DNA strands.

A key feature for development consists of producing sister cells that differ in their potential for cellular differentiation. Following two cell divisions, a haploid Schizosaccharomyces pombe cell produces one cell in four 'granddaughters' with a changed mating cell type, implying nonequivalence of sister cells in each of two consecutive cell divisions. The observed pattern of switching is analogous to the mammalian 'stem cell' lineage by which a cell produces one daughter like itself while the other daughter is advanced in its developmental program. It is tested here whether sisters differ because of unequal distribution of cytoplasmic and/or nuclear components to them or due to inheriting a specific parental DNA chain at the mating type locus. Only the DNA strand-segregation model predicts that those cells engineered to contain an inverted tandem duplication of the mating type locus should produce equivalent sisters. Consequently, two 'cousins' in four related granddaughter cells should switch. The results verified the prediction, thus establishing that all cells otherwise fully possess the potential to switch. Therefore, the program of cell type change in S.pombe cell lineages is determined by the pattern of DNA strand inheritance at the mating type locus. A specific DNA sequence present at the mating type locus is postulated to be the cause of developmental asymmetry between sister cells. A general model for cellular differentiation is proposed in which the act of DNA replication itself is hypothesized to produce developmentally nonequivalent sister genomes.     

A Mendelian Mutation Affecting Mating-Type Determination Also Affects Developmental Genomic Rearrangements in Paramecium Tetraurelia

In Paramecium tetraurelia, mating type is determined during the differentiation of the somatic macronucleus from a zygotic nucleus genetically competent for both types, O and E. Determination of the developing macronucleus is controlled by the parental macronucleus through an unknown mechanism resulting in the maternal inheritance of mating types. The pleiotropic mutation mtF(E) affects macronuclear differentiation. Determination for E is constitutive in mutant homozygotes; a number of unrelated mutant characters are also acquired during development. We have examined the possibility that the mutation causes a defect in the developmental rearrangements of the germ-line genome. We show that the excision of an IES (internal eliminated sequence) interrupting the coding sequence of a surface antigen gene is impaired in the mutant, resulting in an alternative macronuclear version of the gene. Once established, the excision defect is indefinitely transmitted across sexual generations in the cytoplasmic lineage, even in a wild-type genetic context. Thus, the processes of mating-type determination and excision of this IES, in addition to their common sensitivity to the mtF(E) mutation, show a similar maternal inheritance of developmental alternatives in wild-type cells, suggesting a molecular model for mating-type determination.     

Genetic Mapping and Non-Mendelian Segregation of Mating Type Loci in the Oomycete, Phytophthora Infestans

DNA markers linked to the determinants of mating type in the oomycete, Phytophthora infestans, were identified and used to address the genetic basis of heterothallism in this normally diploid fungus. Thirteen loci linked to the A1 and A2 mating types were initially identified by bulked segregant analysis using random amplified polymorphic DNA markers (RAPDs) and subsequently scored in three crosses as RAPD markers, restriction fragment length polymorphisms (RFLPs), single-strand conformational polymorphisms (SSCP), cleaved amplified polymorphisms (CAPS), or allele-specific polymerase chain reaction markers (AS-PCR). All DNA markers mapped to a single region, consistent with a single locus determining both mating types. Long-range restriction mapping also demonstrated the linkage of the markers to one region and delimited the mating type locus to a 100-kb region. The interval containing the mating type locus displayed non-Mendelian segregation as only two of the four expected genotypes were detected in progeny. This is consistent with a system of balanced lethal loci near the mating type locus. A model for mating type determination is presented in which the balanced lethals exclude from progeny those with potentially conflicting combinations of mating type alleles, such as those simultaneously expressing A1 and A2 functions.     

Mating type differentiation in tetrahymena thermophila: Characterization of the delayed refeeding effect and its implications concerning intranuclear coordination

Mating type determination in Tetrahymena thermophila involves developmentally programmed, heritable alterations of the macronucleus, localized to the mtd locus. This determination can be predictably controlled by the environmental conditions during macronuclear development, eg, temperature and time of refeeding. In this article we have further characterized the effects of delayed refeeding on mating type determination, as revealed by the frequency of mating types among the progeny of a cross. Our results show that 1) the magnitude of this starvation effect decreases with temperature of conjugation and becomes undetectable at 18°C; 2) starvation during the interval 14 to 22 hr (after conjugation is induced at 30°C) is a necessary and sufficient condition for the induction of starvation effects; 3) relative mating type frequencies vary monotonically with nutrient concentration present during this critical period; and 4) sister macronuclei, developing under starvation conditions in the same cytoplasm, differentiate majority mating types characteristic of early or late refeeding; sister macronuclei show no apparent correlation with each other. On the basis of our observations on early and late refed cells, we propose that the composition of the newly developed macronucleus is the outcome of two key events: 1) mating type determination at the mtd locus and 2) differential molecular cloning of generally one or two autonomously replicating fragments (ARFs) of the macronuclear DNA bearing the mtd locus.     

Directed Replacement of Mt a by Mt a-1 Effects a Mating Type Switch in Neurospora Crassa

To test the functions of a mating type genes, we developed an efficient strategy to select transformants of Neurospora crassa in which resident A mating type DNA was replaced by cloned DNA from the mt a idiomorph. Cloned a idiomorphic DNA could specify all functions, including fertility, of a mating type, but only when it replaced A DNA at the mating type locus. Only the mt a-1 region of the a idiomorph was necessary in order to specify a mating type. Gene replacement events involved the homologous sequences flanking the unique mating type idiomorphic DNA, resulting in apparently isogenic a and A strains. These isogenic strains were fertile when crossed with one another, indicating that no determinants outside the transforming DNA are necessary for fertility as a and that no host sequences of A strains interfere with fertility as a. One a replacement strain bore a duplication of the transforming mt a-1 and hph DNA. The duplication strain had unexpected properties. Although mating type segregated 1:1 in crosses of this strain to A, the duplicated regions were efficiently altered during the sexual process to generate a single copy in the progeny. No progeny were recovered that had undergone RIP (repeat induced point mutation) sufficient to inactivate the mt a-1 gene. We infer that the mt a-1 gene is necessary and sufficient to specify a mating type identity in all vegetative and sexual activities. Mt a-1 may also play an essential role in ascosporogenesis after fertilization.     

香菇自然群体中个体间的空间分布及其遗传联系*

应用体细胞不亲和性反应、交配型因子分析和基因组ＤＮＡ的ＲＡＰＤ分析,研究了一个分布于方圆约１ｋｍ的６根倒木上的１８个香菇野生菌株间的遗传差异。结果表明,该群体大多数菌株间配对（８０．４％）体细胞不亲和,而同一倒木菌株间配对的体细胞亲和率达 ６２．５％。不同倒木菌株间未发现体细胞亲和的配对。该群体存在 １１个特异的Ａ因子和７个特异的Ｂ因子。同一倒木的菌株有的交配型因子相同,有的则不同。不同倒木的菌株大多数交配型因子不同,未发现交配型因子完全相同的菌株。ＲＡＰＤ分析显示,体细胞亲和的菌株,交配型因子完全相同的菌株,在基于ＤＮＡ相似系数的遗传相关聚类中,首先聚为小类。总起来看,在自然群体中,香菇个体间的遗传差异与其空间分布之间存在一定的联系,随着空间距离的增大,菌株间的异质性相应增高。     

Doubling of the cytoplasm volume improves the developmental competence of porcine oocytes injected with freeze-dried somatic cells

In the present study using pig cells, we examined the effect of the cryoprotectant trehalose on the DNA integrity of freeze-dried cells. We then investigated whether donor cell types and storage duration had impact on DNA integrity in freeze-dried cells or developmental competence of oocytes injected with freeze-dried somatic cells. We also examined whether double cytoplasm nuclear transfer (DCNT) would improve developmental competence of such oocytes. Furthermore, using a PCR-based method for sex identification, we determined whether the blastocysts obtained had actually been generated from the freeze-dried cells. It was found that, for a short storage duration at low temperature, trehalose had no beneficial effect on protection from DNA damage, and that donor cell type had no effect on the DNA integrity of freeze-dried somatic cells or the developmental competence of oocytes injected with them. We also confirmed that all of the blastocysts obtained following nuclear transfer were of freeze-dried somatic cell origin. Storage of freeze-dried somatic cells for up to 1 year at low temperature did not degrade DNA integrity in comparison with storage for 1 month, 1 week or 1 day. Following injection of freeze-dried cells, the proportion of oocytes that developed to blastocysts after storage for up to 1 year was similar to that after storage for 1 month, 1 week or 1 day. Moreover, DCNT significantly improved the developmental competence of oocytes treated in this way. In summary, using DCNT, we have demonstrated that freeze-dried porcine somatic cells subjected to long-term storage at 4 °C have nearly the same potential to develop to blastocysts as non-freeze-dried cells.     

Developmental choices in mating-type interconversion in fission yeast.

Fission yeast cells follow a specific pattern of mating (cell) type switching in single cell pedigrees. Asymmetric cell divisions producing sisters of different developmental fates result from inheritance of specific parental DNA strands according to the classical model of semiconservative replication and segregation.     

Escherichia coli K-12 F? mutants that form mating aggregates but form transconjugants with low frequencies

Summary We have isolated Escherichia coli F^– mutants which, when mated with either Hfr or F, can form stable mating aggregates well but produce transconjugants with reduced frequencies. Selection procedure and other tests rule out the possibility that they are Rec^– strains. These mutants can be classified into two types: type I mutants can induce conjugal DNA replication in the donor, yet form transconjugants poorly; whereas, type II mutants induce conjugal DNA replication with poor efficiencies in the donor. Further tests indicate that type I mutants are very sensitive to lethal zygosis and their membranes, both inner and outer, show alterations in protein composition, whereas type II mutants are insensitive to lethal zygosis, and have an obvious alteration in the protein composition of their outer membrane. These results suggest that type I is defective in transconjugant formation primarily due to a change in the inner membrane, whereas type II is defective in generating a mating signal, which induces donor conjugal DNA replication, due to an alteration in the outer membrane.     

When are good genes good? Variable outcomes of female choice in wax moths

Female lesser wax moths (Achroia grisella) choose males based on characters of their ultrasonic advertisement signals. Because a female''s opportunity to obtain increased somatic benefits by mating with a particular male is limited, we investigated whether females obtain genetic benefits for their offspring via mate choice. Controlled breeding experiments conducted under favourable food and temperature conditions showed that developmental characters are heritable, that sire attractiveness and offspring survivorship are unrelated, but that females mating with attractive signallers produce offspring who mature faster than the offspring of females mating with non-attractive signallers. However, under some unfavourable food or temperature conditions, it is the offspring of females mating with non-attractive males who mature faster; these offspring are heavier as well. Thus, the relationship between male attractiveness and offspring development is not environmentally robust, and support for a good genes model of mate choice in A. grisella is dependent on conditions. These findings suggest genotype–environment interactions and emphasize the necessity of testing sexual selection models under a range of natural environments.     

Perturbance analysis of nuclear determination in Tetrahymena I: background, rationale, and illustrative example, employing temperature responses

A model based on comparative considerations, is presented for the nuclear determination of mating type in Tetrahymena thermophila. The model proposes a system of three binary control elements, each capable of stable persistence in one of two states. A general method is proposed for evaluating the model and for assigning particular mating types to particular compound states. Preliminary assignments of mating types are made from the responses of nuclei to temperature differentials.     

Mating type and mating strategies in Neurospora

In the heterothallic species Neurospora crassa, strains of opposite mating type, A and a, must interact to give the series of events resulting in fruiting body formation, meiosis, and the generation of dormant ascospores. The mating type of a strain is specified by the DNA sequence it carries in the mating type region; strains that are otherwise isogenic can mate and produce ascospores. The DNA of the A and a regions have completely dissimilar sequences. Probing DNA from strains of each mating type with labelled sequences from the A and the a regions has shown that, unlike in Saccharomyces cerevisiae, only a single copy of a mating type sequence is present in a haploid genome. The failure to switch is explainable by the physical absence of DNA sequences characteristic of the opposite mating type. While the mating type sequences must be of the opposite kind for mating to occur in the sexual cycle, two strains of opposite mating type cannot form a stable heterokaryon during vegetative growth; instead, they fuse abortively to give a heterokaryon incompatibility reaction, which results in death of the cells along the fusion line. The DNA sequences responsible for this reaction are coextensive with those sequences in the A and a regions which are necessary to initiate fruiting body formation. The genus Neurospora also includes homothallic species--ones in which a single haploid nucleus carries all the information necessary to form fruiting bodies, undergo meiosis, and produce new haploid spores. One such species, N. terricola, contains one copy each of the A and the a sequences within each haploid genome.(ABSTRACT TRUNCATED AT 250 WORDS)     

三种致病疫霉交配型分子检测方法的比较

致病疫霉Phytophthora infestans属于异宗配合卵菌,当A1、A2两种交配型同时存在时,可以进行有性生殖,产生卵孢子。检测疫霉菌交配型的传统方法是采用对峙培养,这种方法耗时长并且需要标准的A1、A2交配型菌株作为参照。因此,人们希望开发出更加简便和快捷的可直接基于核苷酸序列差异的分子检测方法。目前,已报道了3个与致病疫霉交配型紧密连锁的分子标记可用于交配型的检测。本研究用64株致病疫霉菌比较了3种基于交配型分子标记的检测方法与传统方法检测的结果。结果显示,依据分子标记的3种分子检测方法与传统对峙培养方法测定的交配型结果一致率为61%-73%,而且3种分子检测方法都不能检测出自育菌株。因此,致病疫霉交配型的分子检测方法还有待进一步研究。     

Linkage Mapping Identifies the Sex Determining Region as a Single Locus in the Pennate Diatom Seminavis robusta

The pennate diatom Seminavis robusta, characterized by an archetypical diatom life cycle including a heterothallic mating system, is emerging as a model system for studying the molecular regulation of the diatom cell and life cycle. One of its main advantages compared with other diatom model systems is that sexual crosses can be made routinely, offering unprecedented possibilities for forward genetics. To date, nothing is known about the genetic basis of sex determination in diatoms. Here, we report on the construction of mating type-specific linkage maps for S. robusta, and use them to identify a single locus sex determination system in this diatom. We identified 13 mating type plus and 15 mating type minus linkage groups obtained from the analysis of 463 AFLP markers segregating in a full-sib family, covering 963.7 and 972.2 cM, respectively. Five linkage group pairs could be identified as putative homologues. The mating type phenotype mapped as a monogenic trait, disclosing the mating type plus as the heterogametic sex. This study provides the first evidence for a genetic sex determining mechanism in a diatom.     

Transposable DNA elements and life history traits: II. Transposition of P DNA elements in somatic cells reduces fitness, mating activity, and locomotion of Drosophila melanogaster

Some transposable DNA elements in higher organisms are active in somatic cells, as well as in germinal cells. What effect does the movement of DNA elements in somatic cells have on life history traits? It has previously been reported that somatically active P and mariner elements in Drosophila induce genetic damage and significantly reduce lifespan. In this study, we report that the movement of P elements in somatic cells also significantly reduces fitness, mating activity, and locomotion of Drosophila melanogaster. If other elements cause similar changes in life history traits, it is doubtful if transposable DNA elements remain active for long in somatic cells in natural populations.     

DNA methyltransferase is developmentally expressed in replicating and non-replicating male germ cells.

Genomic methylation patterns are established during maturation of primordial germ cells and during gametogenesis. While methylation is linked to DNA replication in somatic cells, active de novo methylation and demethylation occur in post-replicative spermatocytes during meiotic prophase (1). We have examined differentiating male germ cells for alternative forms of DNA (cytosine-5)-methyltransferase (DNA MTase) and have found a 6.2 kb DNA MTase mRNA that is present in appreciable quantities only in testis; in post-replicative pachytene spermatocytes it is the predominant form of DNA MTase mRNA. The 5.2 kb DNA MTase mRNA, characteristic of all somatic cells, was detected in isolated type A and B spermatogonia and haploid round spermatids. Immunobolt analysis detected a protein in spermatogenic cells with a relative mass of 180,000-200,000, which is close to the known size of the somatic form of mammalian DNA MTase. The demonstration of the differential developmental expression of DNA MTase in male germ cells argues for a role for testicular DNA methylation events, not only during replication in premeiotic cells, but also during meiotic prophase and postmeiotic development.     

DNA damage induced mating type switching in Saccharomyces cerevisiae.

Haploid cells of the yeast Saccharomyces cerevisiae are able to undergo a differentiation-like process: they can switch their mating type between the a and the alpha state. The molecular mechanism of this interconversion of mating types is intrachromosomal gene conversion. It has been shown in a variety of studies that mating type switching in heterothallic strains can be induced by DNA damaging agents, and that different DNA damaging agents differ in the length of incubation after treatment required for induction. Because X-rays induce switching immediately after irradiation and because the DNA double-strand break repair pathway is required for switching, the event initiating heterothallic mating type switching is likely to be a DNA double-strand break. Therefore the assay for heterothallic mating type switching may screen for the induction of DNA double-strand breaks. Several aspects indicating a relationship of mating type switching to mechanisms associated with carcinogenesis are discussed.