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Complementary sex determination (CSD) is a widespread sex determination mechanism in haplodiploid Hymenoptera. Under CSD, sex is determined by the allelic state of one or multiple CSD loci. Heterozygosity at one or more loci leads to female development, whereas hemizygosity of haploid eggs and homozygosity of diploid eggs results in male development. Sexual (arrhenotokous) reproduction normally yields haploid male and diploid female offspring. Under asexual reproduction (thelytoky), diploidized unfertilized eggs develop into females. Thelytoky is often induced by bacterial endosymbionts that achieve egg diploidization by gamete duplication. As gamete duplication leads to complete homozygosity, endosymbiont-induced thelytokous reproduction is presumed to be incompatible with CSD, which relies on heterozygosity for female development. Previously, we excluded CSD in four Asobara (Braconidae) species and proposed a two-step mechanism for Wolbachia-induced thelytoky in Asobara japonica. Here, we conclusively reject CSD in two cynipid wasp species, Leptopilina heterotoma and Leptopilina clavipes. We further show that thelytoky in L. clavipes depends on Wolbachia titer but that diploidization and feminization steps cannot be separated, unlike in A. japonica. We discuss what these results reveal about the sex determination mechanism of L. clavipes and the presumed incompatibility between CSD and endosymbiont-induced thelytoky in the Hymenoptera.

Sex-determination mechanisms evolve rapidly and vary between species. Occasionally, polymorphic systems are found, like in the housefly. Studying the dynamics and stability of such systems can provide a better understanding of the evolution of sex-determination systems. In the housefly, dominant male-determining loci (M) can lie not only on the Y chromosome (MY ), but also on autosomes (MA ) or the X chromosome (MX ). M enforces male development by inhibiting the female-determining gene transformer (tra). A mutant tra allele, traD , is insensitive to M and is a dominant female determiner. MY prevails at high latitudes and polymorphic M loci together with traD at low latitudes. To get more insight into the stability and frequencies of these sex determiners with mutually exclusive dominance, we investigated 5 regional Spanish populations. We found strong variation among populations. Two populations with hemizygous MIII were found, 2 contained homozygous MX with additional hemizygous MI and MII in 1 population. One population contained homozygous and hemizygous MX with additionally hemizygous MII . All females in populations with homozygous M, had traD , whereas no traD was found in populations without homozygous M. Our results indicate locally stable systems may either harbor a single hemizygous M and no traD , corresponding to a male heterogametic system, or homozygous and/or multiple M and heterozygous traD , reminiscent of a female heterogametic system. They support that M loci can accumulate in the presence of a dominant female determiner. Limited migration between populations may contribute to the stability of these systems.

Paternal factor specifies female wasps Not all animals have specialized sex chromosomes to determine their sex. In hymenopteran insects, for example, unfertilized eggs become males and fertilized eggs become females. Prior work showed that the paternal genome provides instruction for female development. Zou et al. identified a sex determination instructor gene, wasp overruler of masculinization , with parent-of-origin effect, in the parasitoid Nasonia vitripennis . It is only transcribed from the paternally provided genome in fertilized eggs to initiate female development. This discovery provides insights into the molecular basis and evolution of sex determination. Science , this issue p. 1115

Adaptation to heterogeneous sensory environments has been implicated as a key parameter in speciation. Cichlid fish are a textbook example of divergent visual adaptation, mediated by variation in the sequences and expression levels of cone opsin genes (encoding the protein component of visual pigments). In some vertebrates including fish, visual sensitivity is also tuned by the ratio of Vitamin A1/A2-derived chromophores (i.e. the light-sensitive component of the visual pigment, bound to the opsin protein), where higher proportions of A2 cause a more red-shifted wavelength absorbance. Here, we explore variation in chromophore ratios across multiple cichlid populations in Lake Victoria, using as a proxy the enzyme CYP27C1 that catalyses the conversion of Vitamin A1-into A2. We focus on sympatric Pundamilia cichlids, where species with blue or red male coloration co-occur at multiple islands, but occupy different depths and consequently different visual habitats. In the red species, we found higher cyp27c1 expression in populations from turbid-water than from clear-water locations, but there was no such pattern in the blue species. Across populations, differences between the sympatric species in cyp27c1 expression had a consistent relationship with species differences in opsin expression patterns, but the red/blue identity reversed between clear- and turbid-water locations. To assess the contribution of heritable versus environmental causes of variation, we tested whether light manipulations induce a change in cyp27c1 expression in the laboratory. We found that cyp27c1 expression was not influenced by experimental light conditions, suggesting that the observed variation in the wild is due to genetic differences. Establishing the biological importance of this variation requires testing the link between cyp27c1 expression and A1/A2 ratios in the eye, as well as its consequences for visual performance.

Sexually dimorphic traits in insects are subject to sexual selection, but our knowledge of the underlying molecular mechanisms is still scarce. Here we investigate how the highly conserved gene, Doublesex (Dsx), is involved in shaping sexual dimorphism in the model parasitoid wasp Nasonia vitripennis (Hymenoptera: Pteromalidae). First, we present the revised Dsx gene structure including an alternative transcription start, and two additional male NvDsx transcript isoforms. We show sex-specific NvDsx expression and splicing throughout development, and demonstrate that transient NvDsx silencing in different male developmental stages shifts two sexually dimorphic traits from male to female morphology, with the effect being dependent on the timing of silencing. In addition, we determined the effect of NvDsx on the development of reproductive organs. Transient silencing of NvDsx in early male larvae affects the growth and differentiation of the internal and external reproductive tissues. We did not observe phenotypic changes in females after NvDsx silencing. Our results indicate that male NvDsx is required to suppress female-specific traits and/or to promote male-specific traits during distinct developmental windows. This provides new insights into the regulatory activity of Dsx during male wasp development in the Hymenoptera.

The tremendous diversity of Hymenoptera is commonly attributed to the evolution of parasitoidism in the last common ancestor of parasitoid sawflies (Orussidae) and wasp-waisted Hymenoptera (Apocrita). However, Apocrita and Orussidae differ dramatically in their species richness, indicating that the diversification of Apocrita was promoted by additional traits. These traits have remained elusive due to a paucity of sawfly genome sequences, in particular those of parasitoid sawflies. Here, we present comparative analyses of draft genomes of the primarily phytophagous sawfly Athalia rosae and the parasitoid sawfly Orussus abietinus. Our analyses revealed that the ancestral hymenopteran genome exhibited traits that were previously considered unique to eusocial Apocrita (e.g., low transposable element content and activity) and a wider gene repertoire than previously thought (e.g., genes for CO2 detection). Moreover, we discovered that Apocrita evolved a significantly larger array of odorant receptors than sawflies, which could be relevant to the remarkable diversification of Apocrita by enabling efficient detection and reliable identification of hosts.

In insect sex determination a primary signal starts the genetic sex determination cascade that, in most insect orders, is subsequently transduced down the cascade by a transformer (tra) ortholog. Only a female-specifically spliced tra mRNA yields a functional TRA-protein that forms a complex with TRA2, encoded by a transformer-2 (tra2) ortholog, to act as a sex specific splicing regulator of the downstream transcription factors doublesex (dsx) and fruitless (fru). Here, we identify the tra2 ortholog of the haplodiploid parasitoid wasp N. vitripennis (Nv-tra2) and confirm its function in N. vitripennis sex determination. Knock down of Nv-tra2 by parental RNA interference (pRNAi) results in complete sex reversal of diploid offspring from female to male, indicating the requirement of Nv-tra2 for female sex determination. As Nv-tra2 pRNAi leads to frequent lethality in early developmental stages, maternal provision of Nv-tra2 transcripts is apparently also required for another, non-sex determining function during embryogenesis. In addition, lethality following Nv-tra2 pRNAi appears more pronounced in diploid than in haploid offspring. This diploid lethal effect was also observed following Nv-tra pRNAi, which served as a positive control in our experiments. As diploid embryos from fertilized eggs have a paternal chromosome set in addition to the maternal one, this suggests that either the presence of this paternal chromosome set or the dosage effect resulting from the diploid state is incompatible with the induced male development in N. vitripennis caused by either Nv-tra2 or Nv-tra pRNAi. The role of Nv-tra2 in activating the female sex determination pathway yields more insight into the sex determination mechanism of Nasonia. (C) 2017 The Authors. Published by Elsevier Ltd.

The parasitoid wasp genus Nasonia has rapidly become a genetic model system for developmental and evolutionary biology. The release of its genome sequence led to the development of high-resolution genomic tools, for both interspecific and intraspecific research, which has resulted in great advances in understanding Nasonia biology. To further advance the utility of Nasonia vitripennis as a genetic model system and to be able to fully exploit the advantages of its fully sequenced and annotated genome, we developed a genetically variable and well-characterized experimental population. In this study, we describe the establishment of the genetically diverse HVRx laboratory population from strains collected from the field in the Netherlands. We established a maintenance method that retains genetic variation over generations of culturing in the laboratory. As a characterization of its genetic composition, we provide data on the standing genetic variation and estimate the effective population size (N-e) by microsatellite analysis. A genome-wide description of polymorphism is provided through pooled resequencing, which yielded 417331 high-quality SNPs spanning all five Nasonia chromosomes. The HVRx population and its characterization are freely available as a community resource for investigators seeking to elucidate the genetic basis of complex trait variation using the Nasonia model system.

Across species, animals have diverse sex determination pathways, each consisting of a hierarchical cascade of genes and its associated regulatory mechanism. Houseflies have a distinctive polymorphic sex determination system in which a dominant male determiner, the M-factor, can reside on any of the chromosomes. We identified a gene

International audience; Male fitness depends on the number of lifetime progeny of their mates and could be constrained by the chance of finding a mate, lifespan and temporal patterns of sperm production and allocation. Here, we used the parasitic wasp Nasonia vitripennis with a two-week lifespan and a gregarious lifestyle, to analyze how the reproductive system is organized to allocate spermatozoa over consecutive matings. Results show that spermatogenesis is synchronized and completed one day before emergence so that males emerge with a full sperm complement. We also found a regulation of spermatozoa transfer between testis and seminal vesicles that allows males to partition small ejaculates over multiple matings. Overall, this study shows that for N. vitripennis, male fertilization potential is determined (1) at the pupal stage, when spermatogenesis takes place to generate a complete life-long stock, (2) on emergence, when transport of spermatozoa from testes to seminal vesicles is initiated and (3) in adulthood, during which spermatozoa are partitioned over successive copulations. Such life history-traits are consistent with the gregarious lifestyle of N. vitripennis.

SNP genotyping was supported by the Natural Environment Research Council (NERC), UK, and performed at NERC Bio-molecular Analysis Facility (NBAF) at the University of Sheffield with project number NBAF653. This study was funded by Marie Curie Initial Training Network “Understanding the evolutionary origin of biological diversity” (ITN-2008-213780 SPECIATION). F.J. acknowledges support from the Technische Universität München – Institute for Advanced Study, funded by the German Excellence Initiative and the European Union Seventh Framework Program under grant agreement no. 291763. A major focus in speciation genetics is to identify the chromosomal regions and genes that reduce hybridization and gene flow. We investigated the genetic architecture of mating behavior in the parasitoid wasp species pair Nasonia giraulti and Nasonia oneida that exhibit strong prezygotic isolation. Behavioral analysis showed that N. oneida females had consistently higher latency times, and broke off the mating sequence more often in the mounting stage when confronted with N. giraulti males compared with males of their own species. N. oneida males produce a lower quantity of the long-range male sex pheromone (4R,5S)-5-hydroxy-4-decanolide (RS-HDL). Crosses between the two species yielded hybrid males with various pheromone quantities, and these males were used in mating trials with females of either species to measure female mate discrimination rates. A quantitative trait locus (QTL) analysis involving 475 recombinant hybrid males (F2), 2148 reciprocally backcrossed females (F3), and a linkage map of 52 equally spaced neutral single nucleotide polymorphism (SNP) markers plus SNPs in 40 candidate mating behavior genes revealed four QTL for male pheromone amount, depending on partner species. Our results demonstrate that the RS-HDL pheromone plays a role in the mating system of N. giraulti and N. oneida, but also that additional communication cues are involved in mate choice. No QTL were found for female mate discrimination, which points at a polygenic architecture of female choice with strong environmental influences. Publisher PDF

Insects exhibit a variety of sex determining mechanisms including male or female heterogamety and haplodiploidy. The primary signal that starts sex determination is processed by a cascade of genes ending with the conserved switch doublesex that controls sexual differentiation. Transformer is the doublesex splicing regulator and has been found in all examined insects, indicating its ancestral function as a sex-determining gene. Despite this conserved function, the variation in transformer nucleotide sequence, amino acid composition and protein structure can accommodate a multitude of upstream sex determining signals. Transformer regulation of doublesex and its taxonomic distribution indicate that the doublesex-transformer axis is conserved among all insects and that transformer is the key gene around which variation in sex determining mechanisms has evolved.

The Wasps and the Bees Sex development in bees and wasps depends on whether or not they develop from a haploid unfertilized egg (resulting in males) or diploid fertilized egg (resulting in females). Although these ploidy-level developmental processes are conserved among bees and wasps, the mechanisms that direct development down a male or female pathway differ significantly. By examining the sex-determining genes in the parasitoid wasp Nasonia , Verhulst et al. (p. 620 ) have shown that a maternal messenger RNA (mRNA) is necessary to initiate the female pathway. The mRNA operates in combination with conserved maternal and paternal genes to produce females, which explains why females are diploids. In unfertilized eggs, maternal provisioning of the gene transcript is too low to initiate the female pathway.

Sex determining mechanisms are highly diverse. Like all Hymenoptera, the parasitic wasp Nasonia vitripennis reproduces by haplodiploidy: males are haploid and females are diploid. Sex in Nasonia is not determined by complementary alleles at sex loci. Evidence for several alternative models is considered. Recent studies on a polyploid and a gynandromorphic mutant strain point to a maternal product that is balanced against the number of chromosomal complements in the zygote and a parent-specific (imprinting) effect. Research is now focused on the molecular details of sex determination in Nasonia. (c) 2007 Elsevier Ltd. All rights reserved.

Background Haplodiploidy, where females develop from diploid, fertilized eggs and males from haploid, unfertilized eggs, is abundant in some insect lineages. Some species in these lineages reproduce by thelytoky that is caused by infection with endosymbionts: infected females lay haploid eggs that undergo diploidization and develop into females, while males are very rare or absent. It is generally assumed that in thelytokous wasps, endosymbionts merely diploidize the unfertilized eggs, which would then trigger female development. Results We found that females in the parasitoid wasp Asobara japonica infected with thelytoky-inducing Wolbachia produce 0.7–1.2 % male offspring. Seven to 39 % of these males are diploid, indicating that diploidization and female development can be uncoupled in A. japonica. Wolbachia titer in adults was correlated with their ploidy and sex: diploids carried much higher Wolbachia titers than haploids, and diploid females carried more Wolbachia than diploid males. Data from introgression lines indicated that the development of diploid individuals into males instead of females is not caused by malfunction-mutations in the host genome but that diploid males are most likely produced when the endosymbiont fails to activate the female sex determination pathway. Our data therefore support a two-step mechanism by which endosymbionts induce thelytoky in A. japonica: diploidization of the unfertilized egg is followed by feminization, whereby each step correlates with a threshold of endosymbiont titer during wasp development. Conclusions Our new model of endosymbiont-induced thelytoky overthrows the view that certain sex determination mechanisms constrain the evolution of endosymbiont-induced thelytoky in hymenopteran insects. Endosymbionts can cause parthenogenesis through feminization, even in groups in which endosymbiont-diploidized eggs would develop into males following the hosts’ sex determination mechanism. In addition, our model broadens our understanding of the mechanisms by which endosymbionts induce thelytoky to enhance their transmission to the next generation. Importantly, it also provides a novel window to study the yet-poorly known haplodiploid sex determination mechanisms in haplodiploid insects. Electronic supplementary material The online version of this article (doi:10.1186/s12862-015-0370-9) contains supplementary material, which is available to authorized users.

In recent years, our knowledge of the conserved master-switch gene doublesex (dsx) and its function in regulating the development of dimorphic traits in insects has deepened considerably. Here, a comprehensive overview is given on the properties of the male- and female-specific dsx transcripts yielding DSX(F) and DSX(M) proteins in Drosophila melanogaster, and the many downstream targets that they regulate. As insects have cell-autonomous sex determination, it was assumed that dsx would be expressed in every somatic cell, but recent research showed that dsx is expressed only when a cell is required to show its sexual identity through function or morphology. This spatiotemporal regulation of dsx expression has not only been established in D. melanogaster but in all insect species studied. Gradually, it has been appreciated that dsx could no longer be viewed as the master-switch gene orchestrating sexual development and behaviour in each cell, but instead should be viewed as the interpreter for the sexual identity of the cell, expressing this identity only on request, making dsx the central nexus of insect sex determination.

Although sex determination is a universal process in sexually reproducing organisms, sex determination pathways are among the most highly variable genetic systems found in nature. Nevertheless, general principles can be identified among the diversity, like the central role of transformer (tra) in insects. When a functional TRA protein is produced in early embryogenesis, the female sex determining route is activated, while prevention of TRA production leads to male development. In dipterans, male development is achieved by prevention of female-specific splicing of tra mRNA, either mediated by X-chromosome dose or masculinizing factors. In Hymenoptera, which have haplodiploid sex determination, complementary sex determination and maternal imprinting have been identified to regulate timely TRA production. In the parasitoid Nasonia, zygotic transformer (Nvtra) expression and splicing is regulated by a combination of maternal provision of Nvtra mRNA and silencing of Nvtra expression in unfertilized eggs. It is unclear, however, if this silencing is directly on the tra locus or whether it is mediated through maternal silencing of a trans-acting factor. Here we show that in Nasonia, female sex determination is dependent on zygotic activation of Nvtra expression by an as yet unknown factor. This factor, which we propose to term womanizer (wom), is maternally silenced during oogenesis to ensure male development in unfertilized eggs. This finding implicates the upstream recruitment of a novel gene in the Nasonia sex determining cascade and supports the notion that sex determining cascades can rapidly change by adding new components on top of existing regulators.

Although the role of DNA methylation in insect development is still poorly understood, the number and role of DNA methyltransferases in insects vary strongly between species. DNA methylation appears to be widely present among the social hymenoptera and functional studies in Apis have suggested a crucial role for de novo methylation in a wide variety of developmental processes. The sequencing of three parasitoid Nasonia genomes revealed the presence of three Dnmt1 (Dnmt1a, Dnmt1b and Dnmt1c) genes and one Dnmt2 and Dnmt3 gene, suggesting a role of DNA methylation in Nasonia development. In the present study we show that in Nasonia vitripennis all Dnmt1 messenger RNAs (mRNAs) and Dnmt3 mRNA are maternally provided to the embryo and, of these, Dnmt1a is essential during early embryogenesis. Lowering of maternal Dnmt1a mRNA results in embryonic lethality during the onset of gastrulation. This dependence on maternal Dnmt1a during embryogenesis in an organismal group outside the vertebrates, suggests evolutionary conservation of the function of Dnmt1 during embryogenesis.

The parasitoid wasp Nasonia vitripennis reproduces by haplodiploidy; males are haploid and females are diploid. Sex determination in Nasonia is not governed by complementary alleles at one or more sex loci. As in most other insects, the sex-determining pathway consists of the basal switch doublesex that is sex-specifically regulated by transformer. Analysis of a polyploid and a mutant gynandromorphic strain, suggested a parent-specific effect (imprinting) on sex determination in Nasonia. Zygotic activity of transformer is autoregulated and depends on a combination of maternal provision of tra mRNA and a paternal genome set. This constitutes a novel way of transformer control in insect sex determination implying maternal imprinting. The nature of the maternal imprint is not yet known and it remains to be determined how broadly the Nasonia sex-determining mechanism applies to other haplodiploids.

The transcription factor Fruitless exerts a broad range of functions during Drosophila development, the most apparent of which is the determination of sexual behavior in males. Although fruitless sequences are found in other insect orders, little is known about fruitless structure and function outside Diptera. We have performed a thorough analysis of fruitless transcripts in the haplo-diploid wasp Nasonia vitripennis and found both sex-specific and non-sex-specific transcripts similar to those found in Drosophila. In Nasonia, however, a novel, large fruitless transcript is present in females only. Putative binding sites for sex-specific splicing factors found in Nasonia fruitless and doublesex as well as Apis mellifera doublesex transcripts were sufficient to identify a corresponding female-specific fruitless exon in A. mellifera, suggesting that similar factors in both hymenopteran species could be responsible for sex-specific splicing of both genes. Furthermore, new C(2)H(2) zinc finger domains found in Nasonia fruitless transcripts were also identified in the fruitless locus of major holometabolous insect species but not in drosophilids. Conservation of important domains and sex-specific splicing in Diptera and Hymenoptera support the hypothesis that fruitless is an ancient gene and has conserved functions in insects. Considerable divergences in other parts of the gene are expected to underlie species-specific differences and may help to explain diversity observed in insect sexual behaviors.

Sex ratio theory offers excellent opportunities to examine the extent to which individuals adaptively adjust their behavior in response to local conditions. Hamilton's theory of local mate com- petition, which predicts female-biased sex ratios in structured pop- ulations, has been extended in numerous directions to predict in- dividual behavior in response to factors such as relative fecundity, time of oviposition, and relatedness between cofoundresses and be- tween mates. These extended models assume that foundresses use different sources of information, and they have generally been un- tested or have only been tested in the laboratory. We use microsatellite markers to describe the wild oviposition behavior of individual foun- dresses in natural populations of the parasitoid wasp Nasonia vitri- pennis, and we use the data collected to test these various models. The offspring sex ratio produced by a foundress on a particular host reflected the number of eggs that were laid on that host relative to the number of eggs that were laid on that host by other foundresses. In contrast, the offspring sex ratio was not directly influenced by other potentially important factors, such as the number of foun- dresses laying eggs on that patch, relative fecundity at the patch level, or relatedness to either a mate or other foundresses on the patch.

An attractive way to improve our understanding of sex determination evolution is to study the underlying mechanisms in closely related species and in a phylogenetic perspective. Hymenopterans are well suited owing to the diverse sex determination mechanisms, including different types of Complementary Sex Determination (CSD) and maternal control sex determination. We investigated different types of CSD in four species within the braconid wasp genus Asobara that exhibit diverse life-history traits. Nine to thirteen generations of inbreeding were monitored for diploid male production, brood size, offspring sex ratio, and pupal mortality as indicators for CSD. In addition, simulation models were developed to compare these observations to predicted patterns for multilocus CSD with up to ten loci. The inbreeding regime did not result in diploid male production, decreased brood sizes, substantially increased offspring sex ratios nor in increased pupal mortality. The simulations further allowed us to reject CSD with up to ten loci, which is a strong refutation of the multilocus CSD model. We discuss how the absence of CSD can be reconciled with the variation in life-history traits among Asobara species, and the ramifications for the phylogenetic distribution of sex determination mechanisms in the Hymenoptera.

The ichneumonid wasp Venturia canescens (Hymenoptera) has been studied extensively for foraging behaviour and population dynamics of sexually (arrhenotokous) and parthenogenetically (thelytokous) reproducing individuals. Here we report the development of a set of microsatellite markers for V.canescens and use them to show that arrhenotokous individuals have more genetic variability than thelytokous ones, which are even homozygous for all tested loci. Crosses between arrhenotokous individuals suggested one marker, Vcan071, to be linked with the Complementary Sex Determiner (CSD) locus and one, Vcan109, with the Virus Like Protein (vlp-p40) locus. The genome size of V. canescens was estimated to be 274–279 Mb. We discuss how both reproductive modes can give rise to the observed genetic variability and how the new markers can be used for future genetic studies of V. canescens. Electronic supplementary material The online version of this article (doi:10.1007/s10709-012-9657-6) contains supplementary material, which is available to authorized users.

The enzyme ornithine decarboxylase (ODC; EC 4.1.1.17) catalyses the first and rate-limiting step in polyamine biosynthesis. Its activity is markedly increased in rapidly growing or regenerating tissue and is subject to regulation by a variety of trophic and mitogenic stimuli. ODC is therefore believed to play an essential role in the onset of cellular proliferation. In a molecular-biological approach to investigate ODC regulation upon induction by tumor promoters in rat liver we isolated an almost full-length rat ODC cDNA clone of 2.4 kb (designated pODC.ElO) from a cDNA library of testosterone-induced rat kidney poly(A)+ RNA. Characterization by restriction-endonuclease mapping and sequence analysis showed strong homology to mouse ODC cDNA sequences previously published [Gupta and Coffino, J. Biol. Chem. 260 (1985) 2941–2944; Kahana and Nathans, Proc. Natl. Acad. Sci. USA 82 (1985) 1673–1677; Hickok et al., Proc. Natl. Acad. Sci. USA 83 (1986) 594–598]. This homology is most pronounced in the 461-aa-spanning coding region, amounting to 94% and 97% at the DNA and protein levels, respectively. In the 423-nt 5′ leader the rat-mouse homology (approx. 75%) is most pronounced in a region of about 175 nt directly upstream from the translational start site. The leader sequence also contains a perfect inverted repeat of 54 nt and ten additional upstream ATG triplets, which are all followed by nonsense codons before the initiating ATG. In the 633-nt 3′ trailer region of pODC.ElO an additional polyadenylation signal is observed more than 300 nt upstream from the 3′ end. Rat-mouse homology is about 80% up to this first polyadenylation signal and is considerably less thereafter. The presence of two alternate polyadenylation sites most likely accounts for the 3′ size heterogeneity observed in the two ODC mRNAs of 2.1 and 2.6 kb, respectively. In rat liver both mRNAs are coordinately induced by different tumor promoters. Finally, Southern blot analysis of normal rat liver and rat hepatoma DNA revealed that rat ODC, as in other rodents, belongs to a multigene family.