The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition

Background

MicroRNAs are modifiers of gene expression, acting to reduce translation through either translational repression or mRNA cleavage. Recently, it has been shown that some microRNAs can act to promote or suppress cell transformation, with miR-17-92 described as the first oncogenic microRNA. The association of miR-17-92 encoded microRNAs with a surprisingly broad range of cancers not only underlines the clinical significance of this locus, but also suggests that miR-17-92 may regulate fundamental biological processes, and for these reasons miR-17-92 has been considered as a therapeutic target.

Results

In this study, we show that miR-17-92 is a cell cycle regulated locus, and ectopic expression of a single microRNA (miR-17-5p) is sufficient to drive a proliferative signal in HEK293T cells. For the first time, we reveal the mechanism behind this response - miR-17-5p acts specifically at the G1/S-phase cell cycle boundary, by targeting more than 20 genes involved in the transition between these phases. While both pro- and anti-proliferative genes are targeted by miR-17-5p, pro-proliferative mRNAs are specifically up-regulated by secondary and/or tertiary effects in HEK293T cells.

Conclusion

The miR-17-5p microRNA is able to act as both an oncogene and a tumor suppressor in different cellular contexts; our model of competing positive and negative signals can explain both of these activities. The coordinated suppression of proliferation-inhibitors allows miR-17-5p to efficiently de-couple negative regulators of the MAPK (mitogen activated protein kinase) signaling cascade, promoting growth in HEK293T cells. Additionally, we have demonstrated the utility of a systems biology approach as a unique and rapid approach to uncover microRNA function.     

S-adenosylmethionine transport in Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the R. prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a K(T) of 2.3 micro M. Transport was inhibited by S-adenosylethionine and S-adenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of R. prowazekii and in Rickettsia typhi. By screening Escherichia coli clone banks for AdoMet transport, the R. prowazekii gene coding for a transporter, RP076 (sam), was identified. AdoMet transport in E. coli containing the R. prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.     

Norfolk Island Robins are a distinct endangered species: ancient DNA unlocks surprising relationships and phenotypic discordance within the Australo-Pacific Robins

Uncertain taxonomy hinders the effective prioritization of taxa for conservation. This problem is acute for understudied island populations in the southwest Pacific Ocean, which are increasingly threatened by habitat loss, predation and climate change. Here, we offer the first test of taxonomic limits and phylogenetic affinities of the iconic Pacific Robin radiation (Petroica multicolor) in order to prioritize the conservation of its nominotypical subspecies, the endangered Norfolk Island Robin (P. m. multicolor). We integrate phylogenetic analyses of ancient DNA and quantitative measures of plumage and morphometric variation to show that the Norfolk Island Robin should be recognized as a distinct species. Phenotypic and genetic datasets contradict the longstanding treatment of Pacific Robins (including Norfolk Island Robins) and Scarlet Robins (P. boodang) as a single species. Instead, we show that Norfolk Island Robins are deeply divergent from Scarlet Robins and have more genetic similarity to Red-capped Robins (P. goodenovii) than to other Pacific Robins. This finding is unrepresentative of the current taxonomic and conservation status of the Norfolk Island Robin, which we propose should be recognised as an endemic endangered species. Our study clearly shows that in the absence of contemporary tissues, ancient DNA approaches using historical museum specimens can address taxonomic questions that morphological traits are unable to resolve. Further, it highlights the need for similar studies of other threatened Norfolk fauna with uncertain taxonomic status in order to ensure appropriate conservation prioritization.     

Fluorescence Activated Cell Sorting of Rickettsia prowazekii-Infected Host Cells Based on Bacterial Burden and Early Detection of Fluorescent Rickettsial Transformants

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that replicates only within the cytosol of a eukaryotic host cell. Despite the barriers to genetic manipulation that such a life style creates, rickettsial mutants have been generated by transposon insertion as well as by homologous recombination mechanisms. However, progress is hampered by the length of time required to identify and isolate R. prowazekii transformants. To reduce the time required and variability associated with propagation and harvesting of rickettsiae for each transformation experiment, characterized frozen stocks were used to generate electrocompetent rickettsiae. Transformation experiments employing these rickettsiae established that fluorescent rickettsial populations could be identified using a fluorescence activated cell sorter within one week following electroporation. Early detection was improved with increasing amounts of transforming DNA. In addition, we demonstrate that heterogeneous populations of rickettsiae-infected cells can be sorted into distinct sub-populations based on the number of rickettsiae per cell. Together our data suggest the combination of fluorescent reporters and cell sorting represent an important technical advance that will facilitate isolation of distinct R. prowazekii mutants and allow for closer examination of the effects of infection on host cells at various infectious burdens.     

Quantitation of individual tocopherols in plasma, platelets, lipids, and livers by high-performance liquid chromatography

A rapid, sensitive procedure is described for the analysis of the tocopherols (α-, β-, γ-, and δ-) in plasma, platelets, lipids, and liver using high-performance liquid chromatography and fluorometric detection. Excellent recoveries of these tocopherols in plasma were obtained - greater than 90%. Separation and quantitation of the four tocopherols required as little as 0.2 ml plasma.     

Rapid, sequential changes in surface morphology of PC12 pheochromocytoma cells in response to nerve growth factor

The effect of nerve growth factor (NGF), a substance that promotes the differentiation and maintenance of certain neurons, was studied via scanning electron microscopy utilizing the PC12 clonal NGF-responsive pheochromocytoma cell line. After 2-4 d of exposure to NGF, these cells acquire many of the properties of normal sympathic neurons. However, by phase microscopy, no changes are discernible within the first 12-18 h. Since the primary NGF receptor appears to be a membrane receptor, it seemed likely that some of the initial responses to the factor may be surface related. PC12 cells maintained without NGF are round to ovoid and have numerous microvilli and small blebs. After the addition of NGF, there is a rapidly initiated sequential change in the cell surface. Ruffles appear over the dorsal surface of the cells with 1 min, become prominent by 3 min, and almost disappear by 7 min. Microvilli, conversely, disappear as the dorsal ruffles become prominent. Ruffles are seen at the the periphery of cell at 3 min, are prominent on most of the cells by 7 min and are gone by 15 min. The surface remains smooth from 15 min until 45 min when large blebs appear. The large blebs are present on most cells at 2 h and are gone by 4 h. The surface remains relatively smooth until 6-7 h of NGF treatment, when microvilli reappear as small knobs. These microvilli increase in both number and length to cover the cell surface by 10 h. These changes were not observed with other basic proteins, with α-bungarotoxin (which binds specifically to PC12 membranes), and were not affected by an RNA synthesis inhibitor that blocks initiation of neurite outgrowth. Changes in the cell surface architecture appear to be among the earlist NGF responses yet detected and may represent or reflect primary events in the mechanism of the factor’s action.     

PURIFICATION OF CHOLINE ACETYLTRANSFERASE FROM DROSOPHILA MELANOGASTER

Abstract— Purification of choline acetyltransferase (ChAc) from heads of Drosophila melanogaster , the richest known source of ChAc, has been accomplished. The stability of the enzyme was preserved by working with a concentration of protein above 0.1 mg/ml. The purification was carried out with ammonium sulfate fractionation and column chromatography on QAE-Sephadex, CoA-Sepharose, G-200 Sephadex, and PCMB-Sepharose. In a procedure using 100 g of Drosophila heads, the specific activity of the crude homogenate was 0.028 μmol/min/mg protein and that of the final product was 43 μmol/min/mg protein, representing a 1500 fold purification. A single protein band, containing all of the ChAc activity, was seen by polyacrylamide gel electrophoresis. A sharp pH optimum at 7.2 was observed. Apparent K_m's for acetyl CoA and choline were 90 μM and 47 μM , respectively. The molecular weight was determined to be 69,000. Isoelectric focusing of extracts of Drosophila heads showed only one peak of choline acetyltransferase activity with an apparent pi of 5.1.     

Unexpected molecular and morphological diversity of hemichordate larvae from the Neotropics

The diversity of tropical marine invertebrates is poorly documented, especially those groups for which collecting adults is difficult. We collected the planktonic tornaria larvae of hemichordates (acorn worms) to assess their hidden diversity in the Neotropics. Larvae were retrieved in plankton tows from waters of the Pacific and Caribbean coasts of Panama, followed by DNA barcoding of mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal DNA to estimate their diversity in the region. With moderate sampling efforts, we discovered six operational taxonomic units (OTUs) in the Bay of Panama on the Pacific coast, in contrast to the single species previously recorded for the entire Tropical Eastern Pacific. We found eight OTUs in Bocas del Toro province on the Caribbean coast, compared to seven species documented from adults in the entire Caribbean. All OTUs differed from each other and from named acorn worm sequences in GenBank by >10% pairwise distance in COI and >2% in 16S. Two of our OTUs matched 16S hemichordate sequences in GenBank: one was an unidentified or unnamed Balanoglossus from the Caribbean of Panama, and the other was an unidentified ptychoderid larva from the Bahamas. The species accumulation curves suggest that nearly all the species have been collected and only one more species might still remain undetected in the Pacific. In contrast, the Caribbean species accumulation curve suggests that further sampling could yield more than 10 additional OTUs. Tornaria from the 14 OTUs exhibited typical planktotrophic morphologies, and, in some cases, may be distinguished by differences in pigmentation and by the number of telotrochal ciliary bands, but in general, few diagnostic differences were detected.     

A multigene phylogeny examining evolutionary and ecological relationships in the Australo-papuan wrens of the subfamily Malurinae (Aves)

Nucleotide sequences from four mitochondrial genes and three nuclear introns were used to examine phylogenetic relationships within the Australo-papuan fairy-wrens (Passeriformes: Maluridae: Malurinae). A well-resolved and well-supported phylogenetic hypothesis of all species in the subfamily was generated. The tree contained three clades corresponding to groups with similar plumages previously identified in earlier studies: the "bi-color," "blue," and "chestnut-shouldered" groups. The genus Malurus was not monophyletic -Malurusgrayi formed a clade with two New Guinean genera Sipodotus and Clytomyias. We recommend M. grayi be reclassified into the genus Chenorhamphus Oustalet 1898. One other taxonomic change is recommended based on the large genetic distance between the two subspecies of Chenorhamphus grayi - the elevation of C. g.campbelli to specific status (= C. campbelli). Although the family Maluridae appears to have had its origins in Australia, the DNA data supports a New Guinean origin for the Malurini (Sipodotus, Clytomyias, Chenorhamphus, Malurus).