In Silico Identification of Novel Biomarkers and Development of New Rapid Diagnostic Tests for the Filarial Parasites Mansonella perstans and Mansonella ozzardi.

Mansonelliasis is a widespread yet neglected tropical infection of humans in Africa and South America caused by the filarial nematodes, Mansonella perstans, M. ozzardi, M. rodhaini and M. streptocerca. Clinical symptoms are non-distinct and diagnosis mainly relies on the detection of microfilariae in skin or blood. Species-specific DNA repeat sequences have been used as highly sensitive biomarkers for filarial nematodes. We have developed a bioinformatic pipeline to mine Illumina reads obtained from sequencing M. perstans and M. ozzardi genomic DNA for new repeat biomarker candidates which were used to develop loop-mediated isothermal amplification (LAMP) diagnostic tests. The M. perstans assay based on the Mp419 repeat has a limit of detection of 0.1 pg, equivalent of 1/1000th of a microfilaria, while the M. ozzardi assay based on the Mo2 repeat can detect as little as 0.01 pg. Both LAMP tests possess remarkable species-specificity as they did not amplify non-target DNAs from closely related filarial species, human or vectors. We show that both assays perform successfully on infected human samples. Additionally, we demonstrate the suitability of Mp419 to detect M. perstans infection in Culicoides midges. These new tools are field deployable and suitable for the surveillance of these understudied filarial infections.

Broad, but not universal, transcriptional requirement for yTAFII17, a histone H3-like TAFII present in TFIID and SAGA.

The RNA polymerase II general transcription factor TFIID is a multisubunit complex comprising TATA box-binding protein (TBP) and associated factors (TAFIIs). Experiments in yeast have shown that although most TAFIIs are required for viability, many genes are transcribed normally upon inactivation of individual and even multiple yTAFIIs. Here we analyze yTAFII17, recently found to be present in both the SAGA HAT complex as well as TFIID. Functional inactivation of yTAFII17 by temperature-sensitive mutation or depletion results in loss of transcription of many, but not all, genes. The upstream activating sequence (UAS), which contains the activator binding sites, is the region that renders a gene yTAFII17 dependent. In conjunction with previous studies, our results reveal that different TAFIIs have remarkably distinct properties.

Yeast TAF(II)145 required for transcription of G1/S cyclin genes and regulated by the cellular growth state.

TFIID comprises the TATA box-binding protein and a set of highly conserved associated factors (TAF(II)s). yTAF(II)145, the core subunit of the yeast TAF(II) complex, is dispensable for transcription of most yeast genes but specifically required for progression through G1/S. Here we show that transcription of G1 and certain B-type cyclin genes is dependent upon yTAF(II)145. At high cell density or following nutrient deprivation, yeast cells cease division, enter a G0-like state, and terminate transcription of most genes. In this stationary phase, we find that the levels of yTAF(II)145, several other yTAF(II)s, and TBP are drastically reduced. Collectively, our results indicate that yTAF(II)145 and other TFIID components have a specialized role in transcriptional regulation of cell cycle progression and growth control.

Yeast TAF(II)90 is required for cell-cycle progression through G2/M but not for general transcription activation.

The RNA polymerase II general transcription factor TFIID is a multisubunit complex comprising TATA-box binding protein and associated factors (TAFIIs). In vitro experiments have suggested that TAFIIs are essential coactivators required for RNA polymerase II-directed transcription activation. Here, for the first time, we analyze systematically the in vivo function of a specific TAFII, yeast TAFII90 (yTAFII90). We show that functional inactivation of yTAFII90 by temperature-sensitive mutations or depletion leads to arrest at the G2/M phase of the cell cycle. Unexpectedly, in the absence of functional yTAFII90, a variety of endogenous yeast genes were all transcribed normally, including those driven by well-characterized activators. Taken together, our results indicate that yTAFII90 is not required for transcription activation in general, and reveal linkages between TAF function and cell-cycle progression.

Transcription activation in cells lacking TAFIIS.

The general transcription factor TFIID is composed of the TATA-box-binding protein (TBP) and a set of TBP-associated factors (TAFIIs). In vitro, TAFIIs are required for activated transcription, and have been proposed to be obligatory targets of transcriptional activator proteins (activators)2. The function of TAFIIs has not been investigated systematically in vivo. A Saccharomyces cerevisiae TAFII complex (yTAFII complex) has been identified that shares functional and structural similarities with higher eukaryotic TFIID. In particular, most yTAFIIs are the homologue of a higher eukaryotic TAFII. Here we report that inactivation or depletion of six different yTAFIIs, including the core yTAFII, that contacts TBP, does not compromise transcriptional activation. We conclude that in vivo, activated transcription of many genes can occur in the absence of functional yTAFIIS, and that in these instances another transcription component(s) must be the target of the activator.

Yeast TAFIIS in a multisubunit complex required for activated transcription.

In higher eukaryotes the RNA polymerase II transcription factor TFIID is composed of a TATA-box-binding protein (TBP) and a set of tightly bound polypeptides, designated TBP-associated factors (TAFIIS). One or more TAFIIS are coactivators that are required for activated but not basal transcription. The eukaryotic transcription machinery is highly conserved and it is therefore puzzling that TAFIIS have not been identified in yeast. Here we use TBP as a protein-affinity ligand to isolate from yeast a multisubunit complex that is required specifically for activated transcription by RNA polymerase II. Microsequence analysis and cloning of two subunits of this complex reveal that they are the homologues of known mammalian and Drosophila TAFIIS. The genes encoding these two yeast TAFIIS are essential, suggesting that activated transcription is required for viability of Saccharomyces cerevisiae.

The MyoD family of myogenic factors is regulated by electrical activity: isolation and characterization of a mouse Myf-5 cDNA.

A full-length cDNA coding for a homolog of the human Myf-5 was isolated from a BC3H-1 mouse library and characterized. The clone codes for a protein of 255 amino acids that is 89%, 88% and 68% identical to the human, bovine and Xenopus myf-5, respectively. The mouse Myf-5 cDNA (mmyf-5), as well as sequences coding for MyoD, myogenin and Mrf-4, were used to probe Northern blots to analyze the effects of innervation on the expression of the MyoD family of myogenic factors. Mouse myf-5, MyoD and myogenin mRNAs levels were found to decline in hind limb muscles of mice between embryonic day 15 (E15) and the first postnatal week, a period that coincides with innervation. In contrast, Mrf-4 transcripts increase during this period and reach steady-state levels by 1-week after birth. To distinguish if the changes in myogenic factor expression are due to a developmental program or to innervation, mRNA levels were analyzed at different times after muscle denervation. Mmyf-5 transcripts begin to accumulate 2 days postdenervation; after 1 week levels are 7-fold higher than in innervated muscle. Mrf-4, MyoD and myogenin transcripts begin to accumulate as soon as 8h after denervation, and attain levels that are 8-, 15- and 40-fold higher than found in innervated skeletal muscle, respectively. The accumulation of these three mRNAs precedes the increase of nicotinic acetylcholine receptor alpha subunit transcripts, a gene that is transcriptionally regulated by MyoD-related factors in vitro. Using extracellular electrodes to directly stimulate in situ the soleus muscle of rats, we found that 'electrical activity' per se, in absence of the nerve, represses the increases of myogenic factor mRNAs associated with denervation.