New Research on Sexual Orientation and Gender Identity Discrimination: Effect of State Policy on Charges Filed at the EEOC.

In 2013, the Equal Employment Opportunity Commission (EEOC) began allowing anyone who believed that they experienced sexual orientation or gender identity (SOGI) discrimination to file charges of sex discrimination under Title VII of the Civil Rights Act. Very little is known about the impact of the EEOC's decision and whether it has enhanced protections for LGBT people. In this brief report, we present preliminary findings on trends and patterns in charge filing, paying particular attention to differences that emerge in charges filed in states with and without SOGI employment nondiscrimination laws. Differences in the characteristics of charging parties, allegations, and charge outcomes suggest that legal protections operating at the state level shape the experiences and disputing behaviors of LGBT individuals in pursuing Title VII remedies.

Same-sex legal marriage and psychological well-being: findings from the California Health Interview Survey.

OBJECTIVES: We examined whether same-sex marriage was associated with nonspecific psychological distress among self-identified lesbian, gay, and bisexual adults, and whether it had the potential to offset mental health disparities between lesbian, gay, and bisexual persons and heterosexuals. METHODS: Population-based data (weighted) were from the 2009 adult (aged 18-70 years) California Health Interview Survey. Within-group analysis of lesbian, gay, and bisexual persons included 1166 individuals (weighted proportion = 3.15%); within-group heterosexual analysis included 35 608 individuals (weighted proportion = 96.58%); and pooled analysis of lesbian, gay, and bisexual persons and heterosexuals included 36 774 individuals. RESULTS: Same-sex married lesbian, gay, and bisexual persons were significantly less distressed than lesbian, gay, and bisexual persons not in a legally recognized relationship; married heterosexuals were significantly less distressed than nonmarried heterosexuals. In adjusted pairwise comparisons, married heterosexuals had the lowest psychological distress, and lesbian, gay, and bisexual persons who were not in legalized relationships had the highest psychological distress (P < .001). Psychological distress was not significantly distinguishable among same-sex married lesbian, gay, and bisexual persons, lesbian, gay, and bisexual persons in registered domestic partnerships, and heterosexuals. CONCLUSIONS: Being in a legally recognized same-sex relationship, marriage in particular, appeared to diminish mental health differentials between heterosexuals and lesbian, gay, and bisexual persons. Researchers must continue to examine potential health benefits of same-sex marriage, which is at least in part a public health issue.

Jak-STAT regulation of cyst stem cell development in the Drosophila testis.

Establishment and maintenance of functional stem cells is critical for organ development and tissue homeostasis. Little is known about the mechanisms underlying stem establishment during organogenesis. Drosophila testes are among the most thoroughly characterized systems for studying stem cell behavior, with germline stem cells (GSCs) and somatic cyst stem cells (CySCs) cohabiting a discrete stem cell niche at the testis apex. GSCs and CySCs are arrayed around hub cells that also comprise the niche and communication between hub cells, GSCs, and CySCs regulates the balance between stem cell maintenance and differentiation. Recent data has shown that functional, asymmetrically dividing GSCs are first established at ∼23 h after egg laying during Drosophila testis morphogenesis (Sheng et al., 2009). This process correlates with coalescence of the hub, but development of CySCs from somatic gonadal precursors (SGPs) was not examined. Here, we show that functional CySCs are present at the time of GSC establishment, and that Jak-STAT signaling is necessary and sufficient for CySC maintenance shortly thereafter. Furthermore, hyper-activation of Jak in CySCs promotes expansion of the GSC population, while ectopic Jak activation in the germline induces GSC gene expression in GSC daughter cells but does not prevent spermatogenic differentiation. Together, these observations indicate that, similar to adult testes, Jak-STAT signaling from the hub acts on both GSCs and CySC to regulate their development and differentiation, and that additional signaling from CySCs to the GSCs play a dominant role in controlling GSC maintenance during niche formation.

Gene order constrains adaptation in bacteriophage T7.

The order of genes in the genome is commonly thought to have functional significance for gene regulation and fitness but has not heretofore been tested experimentally. We adapted a bacteriophage T7 variant harboring an ectopically positioned RNA polymerase gene to determine whether it could regain the fitness of the wild type. Two replicate lines maintained the starting gene order and showed only modest recovery of fitness, despite the accumulation of over a dozen mutations. In both lines, a mutation in the early terminator signal is responsible for the majority of the fitness recovery. In a third line, the phage evolved a new gene order, restoring the wild-type position of the RNA polymerase gene but also displacing several other genes to ectopic locations. Due to the recombination, the fitness of this replicate was the highest obtained but it falls short of the wild type adapted to the same growth conditions. The large benefits afforded by the terminator mutation and the recombination are explicable in terms of T7 biology, whereas several mutations with lesser benefits are not easily accounted for. These results support the premise that gene order is important to fitness and that wild-type fitness is not rapidly re-evolved in reorganized genomes.

Genome properties and the limits of adaptation in bacteriophages.

Eight bacteriophages were adapted for rapid growth under similar conditions to compare their evolved, endpoint fitnesses. Four pairs of related phages were used, including two RNA phages with small genomes (MS2 and Qbeta) two single-stranded DNA phages with small genomes (phiX174 and G4), two T-odd phages with medium-sized, double-stranded DNA genomes (T7 and T3), and two T-even phages with large, double-stranded DNA genomes (T6 and RB69). Fitness was measured as absolute growth rate per hour under the same conditions used for adaptation. T7 and T3 achieved the highest fitnesses, able to increase by 13 billionfold and three-quarters billionfold per hour, respectively. In contrast, the RNA phages achieved low fitness maxima, with growth rates approximately 400-fold and 4000-fold per hour. The highest fitness limits were not attributable to high mutation rates or small genome size, even though both traits are expected to enhance adaptation for fast growth. We suggest that major differences in fitness limits stem from different "global" constraints, determined by the organization and composition of the phage genome affecting whether and how it overcomes potentially rate-limiting host processes, such as transcription, translation, and replication. Adsorption rates were also measured on the evolved phages. No consistent pattern of adsorption rate and fitness was observed across the four different types of phages, but within each pair of related phages, higher adsorption was associated with higher fitness. Different adsorption rate limits within pairs may stem from "local" constraints-sequence differences leading to different local optima in the sequence space.

Experimental evolution yields hundreds of mutations in a functional viral genome.

Two lines of the bacteriophage T7 were grown to fix mutations indiscriminately, using a combination of population bottlenecks and mutagenesis. Complete genome sequences revealed 404 and 299 base substitutions in the two lines, the largest number characterized in functional microbial genomes so far. Missense substitutions outnumbered silent substitutions. Silent substitutions occurred at similar rates between essential and nonessential genes, but missense substitutions occurred at a higher rate in nonessential genes than in essential genes, as expected if they were less deleterious in the nonessential genes. Viral fitness declined during this protocol, and subsequent passaging of each mutated line in large population sizes restored some of the lost fitness. Substitution levels during these recoveries were less than 6% of those during the bottleneck phase, and only two changes during recovery were reversions of the original mutations. Exchanges of genomic fragments between the two recovered lines revealed that fitness effects of some substitutions were not additive-that interactions were accumulating which could lead to incompatibility between the diverged genomes. Based on these results, unprecedented high rates of nucleotide and functional divergence in viral genomes should be attainable experimentally by using repeated population bottlenecks at a high mutation rate interspersed with recovery.

Experimental genomic evolution: extensive compensation for loss of DNA ligase activity in a virus.

Deletion of the viral ligase gene drastically reduced the fitness of bacteriophage T7 on a ligase-deficient host. Viral evolution recovered much of this fitness during long-term passage, but the final fitness remained below that of the intact virus. Compensatory changes occurred chiefly in genes involved in DNA metabolism: the viral endonuclease, helicase, and DNA polymerase. Two other compensatory changes of unknown function also occurred. Using a method to distinguish compensatory mutations from other beneficial mutations, five additional substitutions from the recovery were shown to enhance adaptation to culture conditions and were not compensatory for the deletion. In contrast to the few previous studies of viral recovery from deletions, the compensatory changes in T7 did not restore the deletion or duplicate major regions of the genome. The ability of this deleted genome to recover much of the lost fitness via mutations in its remaining genes reveals a considerable evolutionary potential to modify the interactions of its elements in maintaining an essential set of functions.

A general mechanism for viral resistance to suicide gene expression.

Bacteriophage T7 was challenged with either of two toxic genes expressed from plasmids. Each plasmid contained a different gene downstream of a T7 promoter; cells harboring each plasmid caused an infection by wild-type T7 to abort. T7 evolved resistance to both inhibitors by avoidance of the plasmid expression system rather than by blocking or bypassing the effects of the specific toxic gene product. Resistance was due to a combination of mutations in the T7 RNA polymerase and other genes expressed at the same time as the polymerase. Mutations mapped to sites that are unlikely to alter polymerase specificity for its cognate promoter but the basis for discrimination between phage and plasmid promoters in vivo was not resolved. A reporter assay indicated that, relative to wild-type phage, gene expression from the plasmid was diminished several-fold in cells infected by the evolved phages. A recombinant phage, derived from the original mutant but lacking a mutation in the gene for RNA polymerase, exhibited intermediate activity in the reporter assay and intermediate resistance to the toxic gene cassettes. Alterations in both RNA polymerase and a second gene are thus responsible for resistance. These findings have broad evolutionary parallels to other systems in which viral inhibition is activated by viral regulatory signals such as defective-interfering particles, and they may have mechanistic parallels to the general phenomena of position effects and gene silencing.

Big-benefit mutations in a bacteriophage inhibited with heat.

High temperature inhibits the growth of the wild-type bacteriophage phiX174. Three different point mutations were identified that each recovered growth at high temperature. Two affected the major capsid protein (residues F188 and F242), and one affected the internal scaffolding protein (B114). One of the major capsid mutations (F242) is located in a beta strand that contacts B114 in the procapsid during viral maturation, whereas the other capsid mutation (F188) is involved in subunit interactions at the threefold axis of symmetry. Selective coefficients of these mutations ranged from 13.9 to 3.8 in the inhibitory, hot environment, but all mutations reduced fitness at normal temperature. The selective effect of one of the mutations (F242) was evaluated at high temperature in four different genetic backgrounds and exhibited epistasis of diminishing returns: as log fitness of the background genotype increased from -0.1 to 4.1, the fitness boost provided by the F242 mutation decreased from 3.9 to 0. 8. These results support a model in which viral fitness is bounded by an upper limit and the benefit of a mutation is scaled according to the remaining opportunity for fitness improvement in the genome.

Different trajectories of parallel evolution during viral adaptation.

The molecular basis of adaptation is a major focus of evolutionary biology, yet the dynamic process of adaptation has been explored only piecemeal. Experimental evolution of two bacteriophage lines under strong selection led to over a dozen nucleotide changes genomewide in each replicate. At least 96 percent of the amino acid substitutions appeared to be adaptive, and half the changes in one line also occurred in the other. However, the order of these changes differed between replicates, and parallel substitutions did not reflect the changes with the largest beneficial effects or indicate a common trajectory of adaptation.

Viral escape from antisense RNA.

RNA coliphage SP was propagated for several generations on a host expressing an inhibitory antisense RNA complementary to bases 31-270 of the positive-stranded genome. Phages evolved that escaped inhibition. Typically, these escape mutants contained 3-4 base substitutions, but different sequences were observed among different isolates. The mutations were located within three different types of structural features within the predicted secondary structure of SP genomic RNA: (i) hairpin loops; (ii) hairpin stems; and (iii) the 5' region of the phage genome complementary to the antisense molecule. Computer modelling of the mutant genomic RNAs showed that all of the substitutions within hairpin stems improved the Watson-Crick pairing of the stem. No major structural rearrangements were predicted for any of the mutant genomes, and most substitutions in coding regions did not alter the amino acid sequence. Although the evolved phage populations were polymorphic for substitutions, many substitutions appeared independently in two selected lines. The creation of a new, perfect, antisense RNA against an escape mutant resulted in the inhibition of that mutant but not of other escape mutants nor of the ancestral, unevolved phage. Thus, at least in this system, a population of viruses that evolved to escape from a single antisense RNA would require a cocktail of several antisense RNAs for inhibition.

Exceptional convergent evolution in a virus.

Replicate lineages of the bacteriophage phiX 174 adapted to growth at high temperature on either of two hosts exhibited high rates of identical, independent substitutions. Typically, a dozen or more substitutions accumulated in the 5.4-kilobase genome during propagation. Across the entire data set of nine lineages, 119 independent substitutions occurred at 68 nucleotide sites. Over half of these substitutions, accounting for one third of the sites, were identical with substitutions in other lineages. Some convergent substitutions were specific to the host used for phage propagation, but others occurred across both hosts. Continued adaptation of an evolved phage at high temperature, but on the other host, led to additional changes that included reversions of previous substitutions. Phylogenetic reconstruction using the complete genome sequence not only failed to recover the correct evolutionary history because of these convergent changes, but the true history was rejected as being a significantly inferior fit to the data. Replicate lineages subjected to similar environmental challenges showed similar rates of substitution and similar rates of fitness improvement across corresponding times of adaptation. Substitution rates and fitness improvements were higher during the initial period of adaptation than during a later period, except when the host was changed.

EXPERIMENTAL MOLECULAR EVOLUTION OF BACTERIOPHAGE T7.

We present an analysis of molecular evolution in a laboratory-generated phylogeny of the bacteriophage T7, a virus of 40 kilo-base pairs of double-stranded DNA. The known biology of T7 is used in concert with observed changes in restriction sites and in DNA sequences to produce a model of restriction-site convergence and divergence in the experimental lineages. During laboratory propagation in the presence of a mutagen, the phage lineages changed an estimated 0.5%-1.5% in base pairs; most change appears to have been G → A or C → T, presumably because of the mutagen employed. Some classes of restriction-site losses can be explained adequately as simple outcomes of random processes, given the mutation rate and the bias in mutation spectrum. However, some other classes of sites appear to have undergone accelerated rates of loss, as though the losses were selectively favored. Overall, the wealth of knowledge available for T7 biology contributes only modestly to these explanations of restriction-site evolution, but rates of restriction-site gains remain poorly explained, perhaps requiring an even deeper understanding of T7 genetics than was employed here. Having measured these properties of molecular evolution, we programmed computer simulations with the parameter estimates and pseudo-replicated the empirical study, thereby providing a data base for statistical evaluation of phylogeny reconstruction methods. By these criteria, replicates of the experimental phylogeny would be correctly reconstructed over 97% of the time for the three methods tested, but the methods differed significantly both in their ability to recover the correct topology and in their ability to predict branch lengths. More generally, the study illustrates how analyses of experimental evolution in bacteriophage can be exploited to reveal relationships between the basics of molecular evolution and abstract models of evolutionary processes.

Experimental phylogenetics: generation of a known phylogeny.

Although methods of phylogenetic estimation are used routinely in comparative biology, direct tests of these methods are hampered by the lack of known phylogenies. Here a system based on serial propagation of bacteriophage T7 in the presence of a mutagen was used to create the first completely known phylogeny. Restriction-site maps of the terminal lineages were used to infer the evolutionary history of the experimental lines for comparison to the known history and actual ancestors. The five methods used to reconstruct branching pattern all predicted the correct topology but varied in their predictions of branch lengths; one method also predicts ancestral restriction maps and was found to be greater than 98 percent accurate.

Identification of a cytoskeletal protein localized in the myoplasm of ascidian eggs: localization is modified during anural development.

The myoplasm of ascidian eggs is a localized cytoskeletal domain that is segregated to presumptive larval tail muscle cells during embryonic development. We have identified a cytoskeletal protein recognized by a vertebrate neurofilament monoclonal antibody (NN18) which is concentrated in the myoplasm in eggs and embryos of a variety of ascidian species. The NN18 antigen is localized in the periphery of unfertilized eggs, segregates with the myoplasm after fertilization, and enters the larval tail muscle cells during embryonic development. Western blots of one-dimensional and two-dimensional gels showed that the major component recognized by NN18 antibody is a 58 x 10(3) Mr protein (p58), which exists in at least three different isoforms. The enrichment of p58 in the Triton X-100-insoluble fraction of eggs and its reticular staining pattern in eggs and embryos suggests that it is a cytoskeletal protein. In subsequent experiments, p58 was used as a marker to determine whether changes in the myoplasm occur in eggs of anural ascidian species, i.e. those exhibiting a life cycle lacking tadpole larvae with differentiated muscle cells. Although p58 was localized in the myoplasm in eggs of four urodele ascidian species that develop into swimming tadpole larvae, this protein was distributed uniformly in eggs of three anural ascidian species. The eggs of two of these anural species contained the actin lamina, another component of the myoplasm, whereas the third anural species lacked the actin lamina. There was no detectible localization of p58 after fertilization or segregation into muscle lineage cells during cleavage of anural eggs. NN18 antigen was uniformly distributed in pre-vitellogenic oocytes and then localized in the perinuclear zone during vitellogenesis of urodele and anural ascidians. Subsequently, NN18 antigen was concentrated in the peripheral cytoplasm of post-vitellogenic oocytes and mature eggs of urodele, but not anural, ascidians. It is concluded that the myoplasm of ascidian eggs contains an intermediate filament-like cytoskeletal network which is missing in anural species that have modified or eliminated the tadpole larva.