Molecular Nutrition:
Application of Bioinformatics to the analysis of gene expression by 5’ deletion analysis of promoter regions
Bioinformatics exercise:
Intro session 9am Mon 17/02/2025 (A07 SB-Gateway). Mon, 24/02/2025 (14:00 – 17:00) and Mon 03/03/2025 (14:00 – 17:00), A07 SB-Gateway.
HAND IN DATE: WEEK 27. 27/03/2025 by 3pm by electronic submission only via Moodle.
This coursework can be completed individually or as a pair.
For a pair, one piece of work should be submitted. On the work should be clearly stated the identity of the 2 individuals that are part of the pair.
For those submitting work as pair, one mark will be given, which will be used as the mark for each member of the pair
This coursework can be completed individually or as a pair.
For a pair, one piece of work should be submitted. On the work should be clearly stated the identity of the 2 individuals that are part of the pair.
For those submitting work as pair, one mark will be given, which will be used as the mark for each member of the pair
This schedule contains the instructions for the coursework along with the associated questions at the end of this document. All sequence data needed to complete this work are located at the end of this document.
A separate submission sheet document is available on Moodle. This document contains only the questions, and should be completed then submitted electronically. Details of the electronic submission procedure will follow at a later date.
Aims:
1. To gain an appreciation of the experimental procedures that are employed to assess the effects of nutrients on gene expression.
2. To introduce the material that is freely available via the internet which gives explanations and resources explaining disorders that result from genetic variability.
3. Apply some of these programs to analyze and then interpret hypothetical data from a nutrient-gene experiment.
4. From interpreted data suggest further experiments to characterize the identified responding gene.
ONLY SECTION B CONTRIBUTES TO THE FINAL MARK FOR THIS PIECE OF COURSEWORK ENTITLED, 5’DELETION ANALYSIS AND PROMOTER INTEROGATION.
Introduction
Experiment Hypothesis: The expression of genes in skeletal muscle is directly regulated by glucose, independently of circulating endocrine factors that respond to glucose.
Introduction
An experiment was devised to try to determine the effects of glucose on gene expression in skeletal muscle. These experiments were designed to test the hypothesis that there are a group of genes in skeletal muscle whose expression is directly regulated by glucose. The aim of the experiment was to characterize the effects of glucose on skeletal muscle gene expression. To achieve this experimental aim, individuals were subjected to low insulinaemic with either a hyperglycaemic or a euglycaemic clamp. After 6 hours on the clamp, skeletal muscle biopsies were taken, analysed to determine whether insulin signalling had been activated, then the muscle samples prepared for gene expression analysis by using a transcriptome microarray to identify genes that were affected by glucose.
Glycaemic clamps and sampling
Twelve healthy men (age 22±1 yr, body mass 78±3 kg, BMI 24±1 kg/m2) were recruited from for this study. Six subjects experienced a low insulinaemic-euglycaemic (LIEu) clamp (target insulin level 60 pmol/l; target glucose level 5 mmol/l), whilst six subjects were exposed to a low insulinaemic-hyperglycaemic (LIHyp) clamp (insulin 60 pmol/l; glucose 10 mmol/l) for 6 hours. On the morning of the trial after an overnight fast, a catheter was placed into the antecubital vein for infusion of insulin, somatostatin, glucagon, and glucose. Another catheter was inserted retrogradely into a contralateral hand vein and the hand kept at 60°C in a thermoregulated box for sampling of arterialized venous blood. Blood glucose and insulin were measured before the start of the infusions (-1hr). At the beginning of the trial (time -10 minutes) somatostatin (250 microg/h; Somatostatine-ucb; UCB Pharma) and glucagon (1 ng/kg/min; Glucagen; NovoNordisk) were infused. At time 0hr infusions of insulin (Actrapid; Novo Nordisk) at a rate of 6 mU/m2 body surface area/ min and 10 or 20% (w/v) glucose at a variable rate to obtain eu- or hyperglycemia, respectively, were started. Calibrated syringe pumps administered all of the infusions. To clamp glucose at 5 or 10 mmol/l (eu- or hyperglycemic) from 0hr to 6hr, bedside plasma glucose concentration was measured on a Beckman Glucose Analyzer 2 every 30 min (3 samples over 10 minutes), likewise for determination of the concentration of plasma insulin. All skeletal muscle biopsies were obtained from the vastus lateralis using the percutaneous needle biopsy technique 6 hr after the beginning of the infusions, frozen in liquid nitrogen then stored at -70oC until analysed.
Total RNA extraction from Skeletal Muscle
Total RNA was isolated from skeletal muscle biopsy samples. The mRNA transcripts in the total RNA were copied into cDNA so that the two groups' cDNAs were labeled with different coloured fluorophores. A microarray was generated by using oligonucleotide probes which were micro-dotted onto slides. The sequences used for the array were cDNA sequences originating from a human skeletal muscle and liver cDNA libraries (transcriptome). After incubation of the array with the fluorescent labeled cDNAs from the two groups several dots indicated an increase in expression of specific genes in glucose exposed skeletal muscle (a significant increase in mRNA steady-state for various gene products). A responsive dot contained a probe that was generated from a calpain 10 cDNA sequence (accession number AF089088), this encoded for a mRNA which was an isoform. of the calpain 10 gene (CAPN10). The experiment indicated that there was statistically significant increase in expression of this mRNA transcript. in skeletal muscle under the influence of glucose (P<0.001). The expression of this transcript. in the high glucose group (low insulinaemic-hyperglycaemic (LIHyp) clamp) was increased by 223% relative to the control group (low insulinaemic-euglycaemic (LIEu) clamp) Further information about this gene (CAPN10) and the disease state that variations in it’s sequence is associated with can be found at the Online Mendelian Inheritance in Man® (OMIM) web site (https://www.omim.org/entry/605286?search=CAPN10&highlight=capn10) OMIM is a useful site with lots of information about the gene product and its function, it also gives information about genetic disorders associated with variants in this gene.
Your objectives in this coursework:
1. Identify DNA sequence elements involved in regulating transcription from the potential promoter.
2. Interpret data from an experiment devised to characterize the potential promoter of the gene.
3. Summarize your interpretation of the experimental data in an abstract.