Characterization of the transcription factor encoding gene, KlADR1: metabolic role in Kluyveromyces lactis and expression in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Adr1 is a zinc-finger transcription factor involved in the transcriptional activation of ADH2. Deletion of KlADR1, its putative ortholog in Kluyveromyces lactis, led to reduced growth in glycerol, oleate and yeast extract-peptone medium suggesting, as in S. cerevisiae, its requirement for glycerol, fatty acid and nitrogen utilization. Moreover, growth comparison on yeast extract and peptone plates showed in K. lactis a KlAdr1-dependent growth trait not present in S. cerevisiae, indicating different metabolic roles of the two factors in their environmental niches. KlADR1 is required for growth under respiratory and fermentative conditions like KlADH, alcohol dehydrogenase genes necessary for metabolic adaptation during the growth transition. Using in-gel native alcohol dehydrogenase assay, we showed that this factor affected the Adh pattern by altering the balance between these activities. Since the activity most affected by KlAdr1 is KlAdh3, a deletion analysis of the KlADH3 promoter allowed the isolation of a DNA fragment through which KlAdr1 modulated its expression. The expression of the KlADR1-GFP gene allowed the intracellular localization of the factor in K. lactis and S. cerevisiae, suggesting in the two yeasts a common mechanism of KlAdr1 translocation under fermentative and respiratory conditions. Finally, the chimeric Kl/ScADR1 gene encoding the zinc- finger domains of KlAdr1 fused to the transactivating domains of the S. cerevisiae factor activated in Scadr1D the transcription of ADH2 in a ScAdr1-dependent fashion

Transcription as a Threat to Genome Integrity

Genomes undergo different types of sporadic alterations, including DNA damage, point mutations, and genome rearrangements, that constitute the basis for evolution. However, these changes may occur at high levels as a result of cell pathology and trigger genome instability, a hallmark of cancer and a number of genetic diseases. In the last two decades, evidence has accumulated that transcription constitutes an important natural source of DNA metabolic errors that can compromise the integrity of the genome. Transcription can create the conditions for high levels of mutations and recombination by its ability to open the DNA structure and remodel chromatin, making it more accessible to DNA insulting agents, and by its ability to become a barrier to DNA replication. Here we review the molecular basis of such events from a mechanistic perspective with particular emphasis on the role of transcription as a genome instability determinant

A compendium of Caenorhabditis elegans regulatory transcription factors: a resource for mapping transcription regulatory networks

Background Transcription regulatory networks are composed of interactions between transcription factors and their target genes. Whereas unicellular networks have been studied extensively, metazoan transcription regulatory networks remain largely unexplored. Caenorhabditis elegans provides a powerful model to study such metazoan networks because its genome is completely sequenced and many functional genomic tools are available. While C. elegans gene predictions have undergone continuous refinement, this is not true for the annotation of functional transcription factors. The comprehensive identification of transcription factors is essential for the systematic mapping of transcription regulatory networks because it enables the creation of physical transcription factor resources that can be used in assays to map interactions between transcription factors and their target genes. Results By computational searches and extensive manual curation, we have identified a compendium of 934 transcription factor genes (referred to as wTF2.0). We find that manual curation drastically reduces the number of both false positive and false negative transcription factor predictions. We discuss how transcription factor splice variants and dimer formation may affect the total number of functional transcription factors. In contrast to mouse transcription factor genes, we find that C. elegans transcription factor genes do not undergo significantly more splicing than other genes. This difference may contribute to differences in organism complexity. We identify candidate redundant worm transcription factor genes and orthologous worm and human transcription factor pairs. Finally, we discuss how wTF2.0 can be used together with physical transcription factor clone resources to facilitate the systematic mapping of C. elegans transcription regulatory networks. Conclusion wTF2.0 provides a starting point to decipher the transcription regulatory networks that control metazoan development and function

Transcription enhancement of a digitised multi-lingual pamphlet collection: a case study and guide for similar projects

UCL Library Services holds an extensive collection of over 9,000 Jewish pamphlets, many of these extremely rare. Over the past five years, UCL has embarked on a project to widen access to this collection through an extensive programme of cataloguing, conservation and digitisation. With the cataloguing complete and the most fragile items conserved, the focus is now on making these texts available to global audiences via UCL Digital Collections website. The pamphlets were ranked for rarity, significance and fragility and the highest-scoring selected for digitisation. Unique identifiers allocated at the point of cataloguing were used to track individual pamphlets through the stages of the project. This guide details the text-enhancement methods used, highlighting particular issues relating to Hebrew scripts and early-printed texts. Initial attempts to enable images of these pamphlets to be searched digitally relied on the Optical Character Recognition (OCR) embedded within the software used to create the PDF files. Whilst satisfactory for texts chiefly in Roman script, it provided no reliable means to search the extensive corpus of texts in Hebrew. Generous advice offered by the National Library of Israel led to our adoption of ABBYY FineReader software as a means of enhancing the transcriptions embedded within the PDF files. Following image capture, JPEG files were used to create multi-page PDF files of each pamphlet. Pre-processing in ABBYY FineReader consisted of: setting the language and colour mode; detecting page orientation; selecting and refining areas of the text to be read; reading the text to produce a transcription. The resultant files were stored in folders according to language of text. The software highlighted spelling errors and doubtful readings. A verification tool allowed transcribers to correct these as required. However, some erroneous or doubtful readings were nevertheless genuine words and not highlighted; it was therefore essential to proofread the text, particularly for early-printed scripts. Transcribers maintained logs of common errors; additionally, problems with Hebrew vocalisations, cursive and Gothic scripts were noted. During initial quality checks of the transcriptions, many text searches were unsuccessful due to previously unidentified spacings occurring within words. This was generally linked to the font size being too small. Maintaining logs of font sizes used led to the adoption of a minimum of Arial 8 or Times New Roman 10 in transcribed text. The methodology was revised to include the preliminary quality-checking of one page. We concluded that it was difficult to develop a standardised procedure applicable to all texts given the variance in language, script and typography. However, we concluded that the font Arial gave the most successful accuracy ratings for Hebrew script, minimum text size 17, minimum title size 25. ABBYY file preparation took a minimum of 1.5 hours per pamphlet; transcription correction took an average of 10.4 minutes per page; the final quality check took 30 minutes per pamphlet. On average, the work on each pamphlet took a minimum of 6 hours to complete. As a result of the project, average accuracy ratings improved from 60% to 89%, the greatest improvement being for pre-1800 and Hebrew script publications. We are therefore inclined to focus future transcription-enhancement activity on these types of publication for the remainder of our Jewish Pamphlet Collections

Automated tone transcription

In this paper I report on an investigation into the problem of assigning tones to pitch contours. The proposed model is intended to serve as a tool for phonologists working on instrumentally obtained pitch data from tone languages. Motivation and exemplification for the model is provided by data taken from my fieldwork on Bamileke Dschang (Cameroon). Following recent work by Liberman and others, I provide a parametrised F_0 prediction function P which generates F_0 values from a tone sequence, and I explore the asymptotic behaviour of downstep. Next, I observe that transcribing a sequence X of pitch (i.e. F_0) values amounts to finding a tone sequence T such that P(T) {}~= X. This is a combinatorial optimisation problem, for which two non-deterministic search techniques are provided: a genetic algorithm and a simulated annealing algorithm. Finally, two implementations---one for each technique---are described and then compared using both artificial and real data for sequences of up to 20 tones. These programs can be adapted to other tone languages by adjusting the F_0 prediction function.Comment: 12 pages, 4 postscript figures, uses examples.sty, newapa.sty, latex-acl.sty, ipamacs.st

In Vitro Analysis of the Thyroid Hormone Receptor in Mitochondrial Transcription

The central dogma theory relates how DNA is transcribed into messenger RNA (mRNAs) and then translated into proteins. Since the nucleus contains the majority of the DNA in cells, research related to transcription and translation focuses on these processes within the nucleus and cytosol; however, these processes are also taking place within the mitochondrial organelle. Mitochondria are most widely known for their essential role in producing energy for the cell, but the organelle also contains its own small, circular genome. Transcription of mitochondrial DNA (mtDNA) follows similar mechanisms as does transcription of nuclear DNA. During this essential process, specific mitochondrial transcription factors, such as TFAM and TFB2M, regulate the attachment of the mitochondrial RNA polymerase (POLRMT) to the promoter and initiation of transcription. With a fully functioning mitochondrial RNA polymerase, transcription is properly conducted, and transcripts can be translated to protein by the mitochondrial ribosome. Mitochondrial transcription is a major regulatory process within the organelle, and determining transcription factors involved in this control point is important for understanding mitochondrial function and many diseases relating to mitochondrial dysfunction. Numerous transcription factors are found both in the nucleus as well as in the mitochondria where their function is not well understood. One such transcription factor is the thyroid hormone receptor. Previous research suggests that when the hormone triiodothyronine (T3) is present and taken up in cells, mitochondrial transcription increases. The mechanism behind the T3 stimulation of transcription is thought to be a coordinated effect by interacting with both the mitochondrial and nuclear thyroid hormone receptor. Our aim is to analyze the level of interaction that the mitochondrial thyroid hormone receptor (mt-TRalpha1) has with the mitochondrial DNA and other core mitochondrial transcription factors in the presence and absence of the T3 hormone. With this information, we further understand another component of mitochondrial transcription that could have implications in mitochondrial dysfunction and disease

Transcription-associated mutation promotes RNA complexity in highly expressed genes - a major new source of selectable variation

Alternatively spliced transcript isoforms are thought to play a critical role for functional diversity. However, the mechanism generating the enormous diversity of spliced transcript isoforms remains unknown, and its biological significance remains unclear. We analyzed transcriptomes in saker falcons, chickens, and mice to show that alternative splicing occurs more frequently, yielding more isoforms, in highly expressed genes. We focused on hemoglobin in the falcon, the most abundantly expressed genes in blood, finding that alternative splicing produces 10-fold more isoforms than expected from the number of splice junctions in the genome. These isoforms were produced mainly by alternative use of de novo splice sites generated by transcription-associated mutation (TAM), not by the RNA editing mechanism normally invoked. We found that high expression of globin genes increases mutation frequencies during transcription, especially on nontranscribed DNA strands. After DNA replication, transcribed strands inherit these somatic mutations, creating de novo splice sites, and generating multiple distinct isoforms in the cell clone. Bisulfate sequencing revealed that DNA methylation may counteract this process by suppressing TAM, suggesting DNA methylation can spatially regulate RNA complexity. RNA profiling showed that falcons living on the high Qinghai–Tibetan Plateau possess greater global gene expression levels and higher diversity of mean to high abundance isoforms (reads per kilobases per million mapped reads ≥18) than their low-altitude counterparts, and we speculate that this may enhance their oxygen transport capacity under low-oxygen environments. Thus, TAM-induced RNA diversity may be physiologically significant, providing an alternative strategy in lifestyle evolution

In vivo delivery of transcription factors with multifunctional oligonucleotides.

Therapeutics based on transcription factors have the potential to revolutionize medicine but have had limited clinical success as a consequence of delivery problems. The delivery of transcription factors is challenging because it requires the development of a delivery vehicle that can complex transcription factors, target cells and stimulate endosomal disruption, with minimal toxicity. Here, we present a multifunctional oligonucleotide, termed DARTs (DNA assembled recombinant transcription factors), which can deliver transcription factors with high efficiency in vivo. DARTs are composed of an oligonucleotide that contains a transcription-factor-binding sequence and hydrophobic membrane-disruptive chains that are masked by acid-cleavable galactose residues. DARTs have a unique molecular architecture, which allows them to bind transcription factors, trigger endocytosis in hepatocytes, and stimulate endosomal disruption. The DARTs have enhanced uptake in hepatocytes as a result of their galactose residues and can disrupt endosomes efficiently with minimal toxicity, because unmasking of their hydrophobic domains selectively occurs in the acidic environment of the endosome. We show that DARTs can deliver the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) to the liver, catalyse the transcription of Nrf2 downstream genes, and rescue mice from acetaminophen-induced liver injury

Transcriptional Regulation: a Genomic Overview

The availability of the Arabidopsis thaliana genome sequence allows a comprehensive analysis of transcriptional regulation in plants using novel genomic approaches and methodologies. Such a genomic view of transcription first necessitates the compilation of lists of elements. Transcription factors are the most numerous of the different types of proteins involved in transcription in eukaryotes, and the Arabidopsis genome codes for more than 1,500 of them, or approximately 6% of its total number of genes. A genome-wide comparison of transcription factors across the three eukaryotic kingdoms reveals the evolutionary generation of diversity in the components of the regulatory machinery of transcription. However, as illustrated by Arabidopsis, transcription in plants follows similar basic principles and logic to those in animals and fungi. A global view and understanding of transcription at a cellular and organismal level requires the characterization of the Arabidopsis transcriptome and promoterome, as well as of the interactome, the localizome, and the phenome of the proteins involved in transcription