Possible ecological risks of transgenic organism release when transgenes affect mating success: Sexual selection and the Trojan gene hypothesis

Widespread interest in producing transgenic organisms is balanced by concern over ecological hazards, such as species extinction if such organisms were to be released into nature. An ecological risk associated with the introduction of a transgenic organism is that the transgene, though rare, can spread in a natural population. An increase in transgene frequency is often assumed to be unlikely because transgenic organisms typically have some viability disadvantage. Reduced viability is assumed to be common because transgenic individuals are best viewed as macromutants that lack any history of selection that could reduce negative fitness effects. However, these arguments ignore the potential advantageous effects of transgenes on some aspect of fitness such as mating success. Here, we examine the risk to a natural population after release of a few transgenic individuals when the transgene trait simultaneously increases transgenic male mating success and lowers the viability of transgenic offspring. We obtained relevant life history data by using the small cyprinodont fish, Japanese medaka (Oryzias latipes) as a model. Our deterministic equations predict that a transgene introduced into a natural population by a small number of transgenic fish will spread as a result of enhanced mating advantage, but the reduced viability of offspring will cause eventual local extinction of both populations. Such risks should be evaluated with each new transgenic animal before release.

A gene–culture coevolutionary model for brother–sister mating

We present a gene–culture coevolutionary model for brother–sister mating in the human. It is shown that cultural—as opposed to innate—determination of mate preference may evolve, provided the inbreeding depression is sufficiently high. At this coevolutionary equilibrium, sib mating is avoided because of cultural pressures.

The mating-type and pathogenicity locus of the fungus Ustilago hordei spans a 500-kb region

The fungal pathogen Ustilago hordei causes the covered smut disease of barley and oats. Mating and pathogenicity in this fungus are controlled by the MAT locus, which contains two distinct gene complexes, a and b. In this study, we tagged the a and b regions with the recognition sequence for the restriction enzyme I-SceI and determined that the distance between the complexes is 500 kb in a MAT-1 strain and 430 kb in a MAT-2 strain. Characterization of the organization of the known genes within the a and b gene complexes provided evidence for nonhomology and sequence inversion between MAT-1 and MAT-2. Antibiotic-resistance markers also were used to tag the a gene complex in MAT-1 strains (phleomycin) and the b gene complex in MAT-2 strains (hygromycin). Crosses were performed with these strains and progeny resistant to both antibiotics were recovered at a very low frequency, suggesting that recombination is suppressed within the MAT region. Overall, the chromosome homologues carrying the MAT locus of U. hordei share features with primitive sex chromosomes, with the added twist that the MAT locus also controls pathogenicity.

A serotonin transporter gene intron 2 polymorphic region, correlated with affective disorders, has allele-dependent differential enhancer-like properties in the mouse embryo

Polymorphic regions consisting of a variable number of tandem repeats within intron 2 of the gene coding for the serotonin transporter protein 5-HTT have been associated with susceptibility to affective disorders. We have cloned two of these intronic polymorphisms, Stin2.10 and Stin2.12, into an expression vector containing a heterologous minimal promoter and the bacterial LacZ reporter gene. These constructs were then used to produce transgenic mice. In embryonic day 10.5 embryos, both Stin2.10 and Stin2.12 produced consistent β-galactosidase expression in the embryonic midbrain, hindbrain, and spinal cord floor plate. However, we observed that the levels of β-galactosidase expression produced by both the Stin2.10 and Stin2.12 within the rostral hindbrain differed significantly at embryonic day 10.5. Our data suggest that these polymorphic variable number of tandem repeats regions act as transcriptional regulators and have allele-dependent differential enhancer-like properties within an area of the hindbrain where the 5-HTT gene is known to be transcribed at this stage of development.

Is sexual selection and species recognition a continuum? Mating behavior of the stalk-eyed fly Drosophila heteroneura

If behavioral isolation between species can evolve as a consequence of sexual selection within a species, then traits that are both sexually selected and used as a criterion of species recognition by females should be identifiable. The broad male head of the Hawaiian picture-winged fly Drosophila heteroneura is a novel sexual dimorphism that may be sexually selected and involved in behavioral isolation from D. silvestris. We found that males with broad heads are more successful in sexual selection, both through female mate choice and through aggressive interactions. However, female D. heteroneura do not discriminate against hybrids on the basis of their head width. Thus, this novel trait is sexually selected but is not a major contributor to species recognition. Our methods should be applicable to other species in which behavioral isolation is a factor.

Aneuploidy correlated 100% with chemical transformation of Chinese hamster cells

Aneuploidy or chromosome imbalance is the most massive genetic abnormality of cancer cells. It used to be considered the cause of cancer when it was discovered more than 100 years ago. Since the discovery of the gene, the aneuploidy hypothesis has lost ground to the hypothesis that mutation of cellular genes causes cancer. According to this hypothesis, cancers are diploid and aneuploidy is secondary or nonessential. Here we reexamine the aneuploidy hypothesis in view of the fact that nearly all solid cancers are aneuploid, that many carcinogens are nongenotoxic, and that mutated genes from cancer cells do not transform diploid human or animal cells. By regrouping the gene pool—as in speciation—aneuploidy inevitably will alter many genetic programs. This genetic revolution can explain the numerous unique properties of cancer cells, such as invasiveness, dedifferentiation, distinct morphology, and specific surface antigens, much better than gene mutation, which is limited by the conservation of the existing chromosome structure. To determine whether aneuploidy is a cause or a consequence of transformation, we have analyzed the chromosomes of Chinese hamster embryo (CHE) cells transformed in vitro. This system allows (i) detection of transformation within 2 months and thus about 5 months sooner than carcinogenesis and (ii) the generation of many more transformants per cost than carcinogenesis. To minimize mutation of cellular genes, we have used nongenotoxic carcinogens. It was found that 44 out of 44 colonies of CHE cells transformed by benz[a]pyrene, methylcholanthrene, dimethylbenzanthracene, and colcemid, or spontaneously were between 50 and 100% aneuploid. Thus, aneuploidy originated with transformation. Two of two chemically transformed colonies tested were tumorigenic 2 months after inoculation into hamsters. The cells of transformed colonies were heterogeneous in chromosome number, consistent with the hypothesis that aneuploidy can perpetually destabilize the chromosome number because it unbalances the elements of the mitotic apparatus. Considering that all 44 transformed colonies analyzed were aneuploid, and the early association between aneuploidy, transformation, and tumorigenicity, we conclude that aneuploidy is the cause rather than a consequence of transformation.

A Modulatory Role for Clathrin Light Chain Phosphorylation in Golgi Membrane Protein Localization during Vegetative Growth and during the Mating Response of Saccharomyces cerevisiae

The role of clathrin light chain phosphorylation in regulating clathrin function has been examined in Saccharomyces cerevisiae. The phosphorylation state of yeast clathrin light chain (Clc1p) in vivo was monitored by [32P]phosphate labeling and immunoprecipitation. Clc1p was phosphorylated in growing cells and also hyperphosphorylated upon activation of the mating response signal transduction pathway. Mating pheromone-stimulated hyperphosphorylation of Clc1p was dependent on the mating response signal transduction pathway MAP kinase Fus3p. Both basal and stimulated phosphorylation occurred exclusively on serines. Mutagenesis of Clc1p was used to map major phosphorylation sites to serines 52 and 112, but conversion of all 14 serines in Clc1p to alanines [S(all)A] was necessary to eliminate phosphorylation. Cells expressing the S(all)A mutant Clc1p displayed no defects in Clc1p binding to clathrin heavy chain, clathrin trimer stability, sorting of a soluble vacuolar protein, or receptor-mediated endocytosis of mating pheromone. However, the trans-Golgi network membrane protein Kex2p was not optimally localized in mutant cells. Furthermore, pheromone treatment exacerbated the Kex2p localization defect and caused a corresponding defect in Kex2p-mediated maturation of the α-factor precursor. The results reveal a novel requirement for clathrin during the mating response and suggest that phosphorylation of the light chain subunit modulates the activity of clathrin at the trans-Golgi network.

Multiple Sex Pheromones and Receptors of a Mushroom-producing Fungus Elicit Mating in Yeast

The mushroom-producing fungus Schizophyllum commune has thousands of mating types defined, in part, by numerous lipopeptide pheromones and their G protein-linked receptors. Compatible combinations of pheromones and receptors encoded by different mating types regulate a pathway of sexual development leading to mushroom formation and meiosis. A complex set of pheromone–receptor interactions maximizes the likelihood of outbreeding; for example, a single pheromone can activate more than one receptor and a single receptor can be activated by more than one pheromone. The current study demonstrates that the sex pheromones and receptors of Schizophyllum, when expressed in Saccharomyces cerevisiae, can substitute for endogenous pheromone and receptor and induce the yeast pheromone response pathway through the yeast G protein. Secretion of active Schizophyllum pheromone requires some, but not all, of the biosynthetic machinery used by the yeast lipopeptide pheromone a-factor. The specificity of interaction among pheromone–receptor pairs in Schizophyllum was reproduced in yeast, thus providing a powerful system for exploring molecular aspects of pheromone–receptor interactions for a class of seven-transmembrane-domain receptors common to a wide range of organisms.

The shape of female mating preferences

The “shape” of a female mating preference is the relationship between a male trait and the probability of acceptance as a mating partner. The shape of preferences is important in many models of sexual selection, mate recognition, communication, and speciation, yet it has rarely been measured precisely. Here I examine preference shape for male calling song in a bushcricket (katydid). Preferences change dramatically between races of a species, from strongly directional to broadly stabilizing (but with a net directional effect). Preference shape generally matches the distribution of the male trait. This is compatible with a coevolutionary model of signal-preference evolution, although it does not rule out an alternative model, sensory exploitation. Preference shapes are shown to be genetic in origin.

Frequency of migrants and migratory activity are genetically correlated in a bird population: Evolutionary implications

Most migratory bird populations are composed of individuals that migrate and individuals that remain resident. While the role of ecological factors in maintaining this behavioral dimorphism has received much attention, the importance of genetic constraints on the evolution of avian migration has not yet been considered. Drawing on the recorded migratory activities of 775 blackcaps (Sylvia atricapilla) from a partially migratory population in southern France, we tested two alternative genetic models about the relationship between incidence and amount of migratory activity. The amount of migratory activity could be the continuous variable “underlying” the phenotypic expression of migratory urge, or, alternatively, the expression of both traits could be controlled by two separate genetic systems. The distributions of migratory activities in five different cohorts and the inheritance pattern derived from selective breeding experiments both indicate that incidence and amount of migratory activity are two aspects of one trait. Thus, all birds without measurable activity have activity levels at the low end of a continuous distribution, below the limit of expression or detection. The phenotypic dichotomy “migrant–nonmigrant” is caused by a threshold which may not be fixed but influenced both genetically and environmentally. This finding has profound implications for the evolution of migration: the transition from migratoriness to residency should not only be driven by selection favoring resident birds but also by selection for lower migratory activity. This potential for selection on two aspects, residency and migration distance, of the same trait may enable extremely rapid evolutionary changes to occur in migratory behavior.

Multiple benefits of multiple mating in guppies

The rewards of promiscuity for males are undisputed. But why should a female mate promiscuously, particularly when her partners offer no resources other than sperm and increase her chances of succumbing to predation or disease? This question has been hotly debated but at present remains largely unresolved [Jennions, M. D. & Petrie, M. (2000) Biol. Rev. 75, 21–64]. One possibility is that females exploit postcopulatory mechanisms, such as sperm competition, to increase both the quality and quantity of their offspring. In this paper, we use the Trinidadian guppy, a species with a resource-free mating system, to test the hypothesis that females gain multiple benefits from multiple mating. Our results indicate that multiply mated females secure substantive advantages: They have shorter gestation times and larger broods, and they produce offspring with better developed schooling abilities and escape responses than their singly mated counterparts.

Regulation of the pAD1 sex pheromone response of Enterococcus faecalis by direct interaction between the cAD1 peptide mating signal and the negatively regulating, DNA-binding TraA protein

The Enterococcus faecalis conjugative plasmid pAD1 (60 kb) encodes a mating response to the recipient-produced peptide sex pheromone cAD1. The response involves two key plasmid-encoded regulatory proteins: TraE1, which positively regulates all or most structural genes relating to conjugation, and TraA, which binds DNA and negatively regulates expression of traE1. In vitro studies that included development of a DNA-associated protein-tag affinity chromatography technique showed that TraA (37.9 kDa) binds directly to cAD1 near its carboxyl-terminal end and, as a consequence, loses its affinity for DNA. Analyses of genetically modified TraA proteins indicated that truncations within the carboxyl-terminal 9 residues significantly affected the specificity of peptide-directed association/dissociation of DNA. The data support earlier observations that transposon insertions near the 3′ end of traA eliminated the ability of cells to respond to cAD1.

Quantitative assessment of enzyme specificity in vivo: P2 recognition by Kex2 protease defined in a genetic system

The specificity of the yeast proprotein-processing Kex2 protease was examined in vivo by using a sensitive, quantitative assay. A truncated prepro-α-factor gene encoding an α-factor precursor with a single α-factor repeat was constructed with restriction sites for cassette mutagenesis flanking the single Kex2 cleavage site (-SLDKR↓EAEA-). All of the 19 substitutions for the Lys (P2) residue in the cleavage site were made. The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prepro-α-factor. Cleavage of the 20 sites by Kex2, expressed at the wild-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude. All substitutions for Lys at P2 decreased mating, from 2-fold for Arg to >106-fold for Trp. Eviction of the Kex2-encoding plasmid indicated that cleavage of mutant sites by other cellular proteases was not a complicating factor. Mating efficiencies of strains expressing the mutant precursors correlated well with the specificity (kcat/KM) of purified Kex2 for comparable model peptide substrates, validating the in vivo approach as a quantitative method. The results support the conclusion that KM, which is heavily influenced by the nature of the P2 residue, is a major determinant of cleavage efficiency in vivo. P2 preference followed the rank order: Lys > Arg > Thr > Pro > Glu > Ile > Ser > Ala > Asn > Val > Cys > AsP > Gln > Gly > His > Met > Leu > Tyr > Phe > Trp.

A nonspecific fatty acid within the bumblebee mating plug prevents females from remating

The best mating strategy for males differs from that of females, because females gain from mating with several males (polyandry), but males gain from monopolizing the females. As a consequence, males have evolved a variety of methods, such as the transfer of inhibitory substances from their accessory glands, to ensure exclusive paternity of the female's offspring, generally with detrimental effects on female fitness. Inhibitory substances have been identified as peptides or other specific molecules. Unfortunately, in social insects male-mating traits are investigated only poorly, although male social insects might have the same fundamental influence on female-mating behavior as found in other species. A recently developed technique for the artificial insemination of bumblebee queens allowed us to investigate which chemical compound in the mating plug of male bumblebees, Bombus terrestris L., prevents females (queens) from further mating. Surprisingly, we found that the active substance is linoleic acid, a ubiquitous and rather unspecific fatty acid. Contrary to mating plugs in other insect species, the bumblebee mating plug is highly efficient and allows the males to determine queen-mating frequencies.

Ozone Sensitivity in Hybrid Poplar Is Correlated with a Lack of Defense-Gene Activation1

Ozone is a major gaseous pollutant thought to contribute to forest decline. Although the physiological and morphological responses of forest trees to ozone have been well characterized, little is known about the molecular basis for these responses. Our studies compared the response to ozone of ozone-sensitive and ozone-tolerant clones of hybrid poplar (Populus maximowizii × Populus trichocarpa) at the physiological and molecular levels. Gas-exchange analyses demonstrated clear differences between the ozone-sensitive clone 388 and the ozone-tolerant clone 245. Although ozone induced a decrease in photosynthetic rate and stomatal conductance in both clones, the magnitude of the decrease in stomatal conductance was significantly greater in the ozone-tolerant clone. RNA-blot analysis established that ozone-induced mRNA levels for phenylalanine ammonia-lyase, O-methyltransferase, a pathogenesis-related protein, and a wound-inducible gene were significantly higher in the ozone-tolerant than in the ozone-sensitive plants. Wound- and pathogen-induced levels of these mRNAs were also higher in the ozone-tolerant compared with the ozone-sensitive plants. The different physiological and molecular responses to ozone exposure exhibited by clones 245 and 388 suggest that ozone tolerance involves the activation of salicylic-acid- and jasmonic-acid-mediated signaling pathways, which may be important in triggering defense responses against oxidative stress.