Screening of human gene promoter activities using transfected-cell arrays

Promoters are the best characterized transcriptional regulatory sequences in complex genomes because of their predictable location immediately upstream of transcription start sites. Despite a substantial body of literature describing transcriptional promoters, the identification of true start sites for all human transcripts is far from complete. The same is true of the key structural and functional elements responsible for promoter action in different cell types. In order to identify elements responsible for promoter activity, we applied transfected-cell array technology to functionally evaluate promoters for genes involved in inflammatory bowel disease. Seventy-four promoters were examined by reverse transfection of a promoter-fluorescent reporter constructs into a human embryonic kidney cell line (HEK293T). Sixteen (21.6%) promoters were found to be active in HEK293 T cells. Correlations between promoter activity and endogenous transcript level were calculated, and 75% of active promoters were found to be associated with transcriptional activity of their gene counterparts. These results provide experimental evidence of promoter activity, which may aid in understanding the regulation of gene expression. Moreover, this is the first large-scale functional study of regulatory sequences to use a high-throughput transfected-cell array technique

Wplyw preparatu blonnika pszennego na jakosc sensoryczna potraw miesnych

W pracy określono wpływ preparatu błonnika pszennego Vitacel na jakość sensoryczną potraw z mięsa wieprzowego - pulpetów i kotletów. Oznaczono również właściwości funkcjonalne preparatu Vitacel, tj. wodochłonność, absorpcję oleju oraz zdolność do wymiany kationów. Stwierdzono, że preparat Vitacel charakteryzował się wysoką wodochłonnością i absorpcją oleju oraz niską zdolnością do wymiany kationów. Wykazano zróżnicowany wpływ dodatku preparatu Vitacel na jakość sensoryczną pulpetów i kotletów. Kotlety z 2% dodatkiem Vitacel charakteryzowały się lepszą jakością sensoryczną w porównaniu z pulpetami. Natomiast większy dodatek preparatu (4%) pogorszył jakość zarówno pulpetów, jak i kotletów.In the research the effect of wheat dietary fiber Vitacel on sensory quality of dishes from pork meat (cooked meat balls and fried meat balls) was evaluated. The functional properties, that is water holding capacity, oil absorption and cation exchange capacity, were also determined. It was shown, Vitacel had high water holding capacity and oil absorption, but low cation exchange capacity. Vitacel had different influence on sensory quality of cooked meat balls and fried meat balls. Fried meat balls with 2% addition of Vitacel were better than cooked meat balls. However higher Vitacel addition (4%) decreased the sensory quality of cooked meat balls as well as fried meat balls

Oddzialywanie soli jodowanej na zmiany ilosciowe i jakosciowe tiaminy w wybranych potrawach miesnych

Celem pracy było ustalenie stopnia oddziaływania dodatku soli jodowanej jodkiem potasu na zmiany ilościowe i jakościowe tiaminy, w procesie gotowania i późniejszego przechowywania wybranych potraw mięsnych. W badaniach uwzględniono mięso o zachowanej strukturze histologicznej - tzw. „sztukę mięsa”, mięso rozdrobnione (pulpety) oraz mięsny farsz pierogowy. Przygotowane potrawy przechowywano w warunkach chłodniczych przez 7 dni oraz zamrażalniczych przez 30 dni. Przeprowadzone badania wykazały, że najmniejsze ubytki tiaminy ogólnej stanowiące 53% stwierdzono podczas gotowania mięsa w kawałku w obecności chlorku sodu. Mniejszą podatność na degradację termiczną wykazywała tiamina wolna niż związana. Większe straty tiaminy stwierdzono w mięsie rozdrobnionym niż w litym kawałku mięsa. Wprowadzenie soli jodowanej przyspieszało dynamikę rozpadu tiaminy wolnej i związanej. Przechowywanie badanych potraw w warunkach chłodniczych, jak i zamrażalniczych zwiększało straty zarówno tiaminy wolnej, jak i związanej.The aim of this work was to evaluate the influence of salt iodized with potassium iodide addition on quantitative and qualitative changes of thiamine in the process of cooking and storage of selected meat dishes. In experiments were taken into account meat demonstrating original histological structure (one piece of meat), minced meat balls (cooked) and meat - pie stuffing. All samples were kept under cooling conditions (temp. +4°C) for 7 days and frozen state (-18°C) for 30 days. The results indicated least losses equal to about 53% of total thiamine content in the heat cooked in one piece in presence of NaCl only. The free thiamine was less resistant to thermal degradation in compare with bound thiamine. The minced meat showed higher losses of thiamine in compare with the whole piece of meat. The addition of salt iodized with potassium iodide accelerated break-up dynamics of free thiamine as well as bound one. The storage of meat dishes both under conditions cooling and as well as freezing resulted in higher of free and bound thiamine

Functional analysis and identification of cis-regulatory elements of human chromosome 21 gene promoters

Given the inherent limitations of in silico studies relying solely on DNA sequence analysis, the functional character-ization of mammalian promoters and associated cis-regulatory elements requires experimental support, which demands cloning and analysis of putative promoter regions. Focusing on human chromosome 21, we cloned 182 gene promoters of 2500 bp in length and conducted reporter gene assays on transfected-cell arrays. We found 56 promoters that were active in HEK293 cells, while another 49 promoters could be activated by treatment of cells with Trichostatin A or depletion of serum. We observed high correlations between promoter activities and endogenous transcript levels, RNA polymerase II occupancy, CpG islands and core promoter elements. Truncation of a subset of 62 promoters to ∼500 bp revealed that truncation rarely resulted in loss of activity, but rather in loss of responses to external stimuli, suggesting the presence of cis-regulatory response elements within distal promoter regions. In these regions, we found a strong enrichment of transcription factor binding sites that could potentially activate gene expression in the presence of stimuli. This study illustrates the modular functional architecture of chromosome 21 promoters and helps to reveal the complex mechanisms governing transcriptional regulation

Scarcity of Recurrent Regulatory Driver Mutations in Colorectal Cancer Revealed by Targeted Deep Sequencing

Background: Genetic testing of cancer samples primarily focuses on protein-coding regions, despite most mutations arising in noncoding DNA. Noncoding mutations can be pathogenic if they disrupt gene regulation, but the benefits of assessing promoter mutations in driver genes by panel testing has not yet been established. This is especially the case in colorectal cancer, for which few putative driver variants at regulatory elements have been reported.Methods: We designed a unique target capture sequencing panel of 39 colorectal cancer driver genes and their promoters, together with more than 35 megabases of regulatory elements focusing on gene promoters. Using this panel, we sequenced 95 colorectal cancer and matched normal samples at high depth, averaging 170x and 82x coverage, respectively.Results: Our target capture sequencing design enabled improved coverage and variant detection across captured regions. We found cases with hereditary defects inmismatch and base excision repair due to deleterious germline coding variants, and we identified mutational spectra consistent with these repair deficiencies. Focusing on gene promoters and other regulatory regions, we found little evidence for base or region-specific recurrence of functional somatic mutations. Promoter elements, including TERT, harbored few mutations, with none showing strong functional evidence. Recurrent regulatory mutations were rare in our sequenced regions in colorectal cancer, though we highlight some candidate mutations for future functional studies.Conclusions: Our study supports recent findings that regulatory driver mutations are rare in many cancer types and suggests that the inclusion of promoter regions into cancer panel testing is currently likely to have limited clinical utility in colorectal cancer