SOX2-Dependent Transcription in Clock Neurons Promotes the Robustness of the Central Circadian Pacemaker

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Direct interaction between Teashirt and Sex combs reduced proteins, via Tsh's acidic domain, is essential for specifying the identity of the prothorax in Drosophila

teashirt (tsh) encodes a zinc-finger protein that is thought to be part of a network that contributes to regionalization of the Drosophila embryo and establishes the domains of Hox protein function. tsh and the Hox gene Sex combs reduced (Scr) are essential to establish the identity of the first thoracic segment. We used the development of the first thoracic segment as a paradigm for Scr dependent regional morphological distinctions. In this specific context, we asked whether Tsh protein could have a direct influence on Scr activity. Here we present evidence that Tsh interacts directly with Scr and this interaction depends in part on the presence of a short domain located in the N-terminal half of Teashirt called "acidic domain". In vivo, expression of full length Tsh can rescue the tsh null phenotype throughout the trunk whereas Tsh lacking the Scr interacting domain rescues all the trunk defects except in the prothorax. We suggest this provides insights into the mechanism by which Tsh, in concert with Scr, specifies the prothoracic identity.     

Major histocompatibility complex heterozygosity reduces fitness in experimentally infected mice

It is often suggested that heterozygosity at major histocompatibility complex (MHC) loci confers enhanced resistance to infectious diseases (heterozygote advantage, HA, hypothesis), and overdominant selection should contribute to the evolution of these highly polymorphic genes. The evidence for the HA hypothesis is mixed and mainly from laboratory studies on inbred congenic mice, leaving the importance of MHC heterozygosity for natural populations unclear. We tested the HA hypothesis by infecting mice, produced by crossbreeding congenic C57BL/10 with wild ones, with different strains of Salmonella, both in laboratory and in large population enclosures. In the laboratory, we found that MHC influenced resistance, despite interacting wild-derived background loci. Surprisingly, resistance was mostly recessive rather than dominant, unlike in most inbred mouse strains, and it was never overdominant. In the enclosures, heterozygotes did not show better resistance, survival, or reproductive success compared to homozygotes. On the contrary, infected heterozygous females produced significantly fewer pups than homozygotes. Our results show that MHC effects are not masked on an outbred genetic background, and that MHC heterozygosity provides no immunological benefits when resistance is recessive, and can actually reduce fitness. These findings challenge the HA hypothesis and emphasize the need for studies on wild, genetically diverse species.     

Structural and functional studies of RegB, a new member of a family of sequence-specific ribonucleases involved in mRNA inactivation on the ribosome

The RegB endoribonuclease participates in the bacteriophage T4 life cycle by favoring early messenger RNA breakdown. RegB specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites. Its activity is very low but can be enhanced up to 100-fold by the ribosomal 30 S subunit or by ribosomal protein S1. RegB has no significant sequence homology to any known protein. Here we used NMR to solve the structure of RegB and map its interactions with two RNA substrates. We also generated a collection of mutants affected in RegB function. Our results show that, despite the absence of any sequence homology, RegB has structural similarities with two Escherichia coli ribonucleases involved in mRNA inactivation on translating ribosomes: YoeB and RelE. Although these ribonucleases have different catalytic sites, we propose that RegB is a new member of the RelE/YoeB structural and functional family of ribonucleases specialized in mRNA inactivation within the ribosome.     

A reliable general purpose method for extracting genomic DNA from Dictyostelium cells

In this protocol, we present a standard method for extracting DNA from cells of the social amoeba Dictyostelium discoideum. While this procedure is similar to other phenol:chloroform-based purification methods, it is modified to account for the high level of carbohydrate and nucleases found in Dictyostelium cells. Genomic DNA can be isolated from wild-type and genetically modified cells using the described protocol, allowing molecular genetic analyses to be performed. Following cell lysis, nucleic acid extraction, and precipitation, the isolated DNA is suitable for digestion by restriction enzymes, amplification by PCR and Southern blotting. This procedure takes approximately 3 h to complete.     

A general purpose method for extracting RNA from Dictyostelium cells

Here we present a protocol for the extraction of RNA from Dictyostelium discoideum. Dictyostelium is a social amoeba that undergoes a basic developmental program, and therefore analysis of RNA levels over a time course is a commonly used technique. This procedure is similar to other guanidine thiocyanate-based methods; however, it has been adjusted because of the large quantities of carbohydrate and nucleases found in Dictyostelium cells. After cell lysis and phenol:chloroform extraction, the resulting high-quality RNA isolated with the described protocol allows the molecular genetic analysis of wild-type and genetically modified cells. The purified RNA can be used for analyses such as northern blotting, RT-PCR and microarrays. This procedure requires approximately 2 h to complete.     

Invasive and adherent bacterial pathogens co-Opt host clathrin for infection

Infection by the bacterium Listeria monocytogenes depends on host cell clathrin. To determine whether this requirement is widespread, we analyzed infection models using diverse bacteria. We demonstrated that bacteria that enter cells following binding to cellular receptors (termed "zippering" bacteria) invade in a clathrin-dependent manner. In contrast, bacteria that inject effector proteins into host cells in order to gain entry (termed "triggering" bacteria) invade in a clathrin-independent manner. Strikingly, enteropathogenic Escherichia coli (EPEC) required clathrin to form actin-rich pedestals in host cells beneath adhering bacteria, even though this pathogen remains extracellular. Furthermore, clathrin accumulation preceded the actin rearrangements necessary for Listeria entry. These data provide evidence for a clathrin-based entry pathway allowing internalization of large objects (bacteria and ligand-coated beads) and used by "zippering" bacteria as part of a general mechanism to invade host mammalian cells. We also revealed a nonendocytic role for clathrin required for extracellular EPEC infections.     

Stress impacts telomere dynamics

Telomeres are DNA-protein complexes at the ends of chromosomes that control genomic integrity but appear to become shorter with age and stress. To test whether stress causes telomere attrition, we exposed the offspring of wild-caught house mice (Mus musculus) to stressful conditions and examined the changes in telomere length over six months. We found that females exposed to males and reproductive stress (either with or without crowding) had significantly shorter telomeres than controls, and males exposed to crowding stress had shorter telomeres than males that were not crowded. Our results indicate that stress alters telomere dynamics, causing attrition and hindering restoration, and these effects are sex dependent. Telomeres may thus provide a biomarker for assessing an individual's cumulative exposure or ability to cope with stressful conditions.     

Effects of ionizing radiation on the activity of the major hepatic enzymes implicated in bile acid biosynthesis in the rat

In the days following high-dose radiation exposure, damage to small intestinal mucosa is aggravated by changes in the bile acid pool reaching the gut. Intestinal bile acid malabsorption, as described classically, may be associated with altered hepatic bile acid biosynthesis, which was the objective of this work. The activity of the main rate-limiting enzymes implicated in the bile acid biosynthesis were evaluated in the days following an 8-Gy gamma(60)Co total body irradiation of rats, with concomitant determination of biliary bile acid profiles and intestinal bile acid content. Modifications of biliary bile acid profiles, observed as early as the first post-irradiation day, were most marked at the third and fourth day, and resulted in an increased hydrophobicity index. In parallel, the intestinal bile acids' content was enhanced and hepatic enzymatic activities leading to bile acids were changed. A marked increase of sterol 12 alpha-hydroxylase and decrease of oxysterol 7 alpha-hydroxylase activity was observed at day 3, whereas both cholesterol 7 alpha-hydroxylase and oxysterol 7 alpha-hydroxylase activities were decreased at day 4 after irradiation. These results show, for the first time, radiation-induced modifications of hepatic enzymatic activities implicated in bile acid biosynthesis and suggest that they are mainly a consequence of radiation-altered intestinal absorption, which induces a physiological response of the enterohepatic bile acid recirculation.     

A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs

Double muscling is a trait previously described in several mammalian species including cattle and sheep and is caused by mutations in the myostatin (MSTN) gene (previously referred to as GDF8). Here we describe a new mutation in MSTN found in the whippet dog breed that results in a double-muscled phenotype known as the “bully” whippet. Individuals with this phenotype carry two copies of a two-base-pair deletion in the third exon of MSTN leading to a premature stop codon at amino acid 313. Individuals carrying only one copy of the mutation are, on average, more muscular than wild-type individuals (p = 7.43 × 10⁻⁶; Kruskal-Wallis Test) and are significantly faster than individuals carrying the wild-type genotype in competitive racing events (Kendall's nonparametric measure, τ = 0.3619; p ≈ 0.00028). These results highlight the utility of performance-enhancing polymorphisms, marking the first time a mutation in MSTN has been quantitatively linked to increased athletic performance.