Eukaryotic genome complexity is enriched by embedded introns which expand the number of proteins produced by alternative splicing, provide unique environments to embed genes and regulatory elements, and create opportunities for new gene assembly through recombination and intron evolution. Such benefits come at a cost, however, as approximately 15% of human genetic disorders result from splicing errors associated with cis- (splicing substrate) mutations that alter gene-delimited splice patterns or trans- (spliceosome subunit) mutations that may impair general pre-mRNA splicing. A better understanding of spliceosome composition and the molecular basis of splice site selection will facilitate the diagnosis and, ultimately, the treatment or correction of splicing-related disorders. The contribution of spliceosome assembly to the mechanism of pre-mRNA splicing is the focus of our work.

Metazoan genes may contain dozens of intron/exon borders, some of which are used only in response to specific developmental or environmental cues. For many genes it is the stable recruitment of the U2 snRNP to the branchpoint region of the pre-mRNA that modulates splice site choice. Pre-mRNA branchpoint recognition is complex and even in the case unregulated transcripts, progresses through the sequential association of multiple splicing factors (e.g., SF1/BBP – U2AF65/Mud2p) and snRNPs (U1, U2, and U6).

While the basic pathway of spliceosome assembly is well conserved through evolution, Saccharomyces cerevisiae (henceforth yeast) lacks canonical SR splicing factor regulators found in metazoa and relies on more rigidly conserved pre-mRNA consensus elements to direct splice site choice. Yeast gene structure is also simpler, with few genes containing more than a single intron. Accordingly, yeast offers an excellent model to investigate the assembly and function of the basal splicing apparatus in the absence of complications resulting from complex gene organization and splicing. Currently, we using genetic and proteomic approaches to investigate the dynamics of pre-mRNA branchpoint selection in vitro and in living cells.   

Rymond, BC., Going my way?  A tale of enzyme recruitment and activation 2016, Atlas of Science,

Rymond, B.C. The use of Saccharomyces cerevisiae to study the mechanism of pre-mRNA splicing. 2012,  Alternative Pre-mRNA Splicing: Theory and Protocols, Wiley-Blackwell,  (Eds., Stefan Stamm, Chris Smith, Reinhard Luhrmann)

Course Materials

2017 LAB MANUAL, SYLLABUS and HOMEWORK (updated)

2016 Final Exam

2016 Final Exam Answer Key

 

 

FINAL EXAM ANSWER KEY 2015

 

• Friday Lecture Slides (subject to change)
• Extra in-lab discussion slides (subject to change)
• Notebook Guidelines
• Participation Sheet
• 2016 Radiation Safety Lecture
• Example Quiz
• Study Guide - first half of semester
• Study Guide - final exam
• Old First Exam (questions only)
• Old First Exam with answer key
• Old Final Exam
• Old Final Exam with answer key
• 2014 Exam I answer key
• 2015 Exam I answer key
• 2016 Midterm exam
• 2016 Midterm answer key

Scientific Resources, Maps, Tools and Course Reading Assignments (reference in manual)

• NIH Guidelines for Recombinant DNA Use
• UK Radiation Training Manual
• Original 1974 Paul Berg et al. call for caution with recombinant DNA
• Paul Berg & Maxine Singer reflection 20 years later
• pTZ19u Map
• pL4440 Map
• 1 kbp DNA Ladder (NEB)
• Qiagen QIAquick Gel Extraction Kit
• Ethidium Bromide Information
• SYBR Safe
• Semi-Log Paper
• Stratalinker
• Riboprobe Manual
• Epicentre MasterPure DNA Purification Kit
• BioRad BioSpin P6 Column
• Cloning PRP38 by Complementation
• Vincent et al Paper Illustrating Use of Plasmid Shuffle
• James et al., Paper on the Yeast Two Hybrid Approach
• pACT and pAS2 Two Hybrid Plasmid Descriptions
• Biological Buffers - CalBiochem
• Use of RNAi to Score Gene Function in C. elegans
• Growth and propagation of C. elegans
• Typhoon 8600 Phosphoimager User Manual
• Typhoon 8600 Phosphoimager Specifications
• NEB Impact Protein Fusion System
• B-PER Bacterial Lysis Reagent
• iCycer Realtime PCR Manual
• Realtime PCR for Measuring mRNA Levels (Review)
• ProtoScript RT-PCR Manual
• Nanosting Technology
• Overview of DNA Microarray Technology
• NEB Broad-range Protein Marker
• Ion Torrent Paper
• Next Generation DNA Sequencing Review 1
• Next Generation DNA Sequencing Review 2
• C. elegans life cycle and anatomy
• PAP antibody
• Luminol detection of Horseradish peroxidse
• Forward genetics - ts mutant screen
• Dosage suppression
• Extragenic suppression and proteomics
• Bioinformatics - guilt by similarity
• Synthetic lethality paper
• C. elegans GFP/RFP alternative splicing screen
• E. coli genotype features review
• Overview of proteomics
• Gateway technology
• CRISPR-Cas9 in yeast
• NEB 2X MasterMix

 

Class Data (links active when class data becomes available)

• EtBr vs Sybr Safe Gel Image
• Impact of buffer concentration on DNA resolution during gel electrophoresis image
• In vitro transcription - radiolable incorporation results
• Northern blot to detect yeast RNA and determine direction of transcription
• Inverse PCR Results
• DNA sequencing results for use in Blast search
• Realtime PCR Results
• Class Group Picture 1 - 2016
• Class Group Picture 2 - 2016
• Spp382(1-121) protein purification image (NEB Impact system)
• Illumina sequencing of yeast RNA and examples of alterate splicing
• rtPCR confirmation of alternative mRNA processing
• Nitrous Acid Mutagenesis Results
• Ponceau red stain of TAP-tagged protein western transfer
• TAP-tagged protein western blot - AP development
• empty vector & Sup-12 RNAi GFP/RFP

 

 

 

 

 

 

 

CLASS SYLLABUS for BCH/BIO/MI/PLS/PPA 601 Special Topics in Molecular and Cellular Genetics 

 

ORIENTATION OVERVIEW POWERPOINT (when available)

                                

                                      2017 VISITOR INFORMATION

 

 

Dr. Jeffrey Wilusz, Ph.D., Professor, Department of Microboligy, Immunology & Pathology; College of Veterinary Medicine & Biomedical Sciences, Colorado State University, Fort Collins, Colorado http://csu-cvmbs.colostate.edu/academics/mip/Pages/Jeffrey-Wilusz.aspx

Topic: Regulation of mRNA metabolism in mammalian cells & disease

Pre-meeting: March 3, 2017 (Friday), 4:00-5:30 PM 109 TH Morgan Building

Student Lecture: March 6, 2017 (Monday 8:00-8:50 AM, 109 TH Morgan Building

Student Lunch: March 6, 2017 (Monday), 12:30-1:30 PM, 305 TH Morgan Building

Seminar: March 6, 2017 (Monday) 4:00 PM, 116 TH Morgan Building     

            Title: TBA

Host: Brian Rymond, rymond@uky.edu

Reading list for Dr. Wilusz

XRN1 Stalling in the 5’ UTR of Hepatitis C Virus and Bovine Viral Diarrhea Virus Is Associated with Dysregulated Host mRNA Stability (2015) Moon SL, Blackinton JG, Anderson JR, Dozier MK, Dodd BJ, Keene JD, Wilusz CJ, Bradrick SS, Wilusz J. PLOS Pathogens | DOI:10.1371/journal.ppat.1004708

Sindbis Virus Usurps the Cellular HuR Protein to Stabilize Its Transcripts and Promote Productive Infections in Mammalian and Mosquito Cells (2010) Kevin J. Sokoloski, Alexa M. Dickson, Emily L. Chaskey, Nicole L. Garneau, Carol J. Wilusz, and Jeffrey Wilusz,  Cell Host & Microbe, 8: 2, 196–207

Cytoplasmic Viruses: Rage against the (Cellular RNA Decay) Machine (2013, review) Moon SL, Wilusz J. PLoS Pathog. 9(12):e1003762.

Diverse Strategies Used by Picornaviruses to Escape Host RNA Decay Pathways, Ullmer, W, Semler (2016, review) Viruses 8:E335

 

John Dueber, Ph.D., Professor, Department of Bioengineering, University of California, Berkeley, California 94720 http://dueberlab.berkeley.edu/about/     

Topic: Synthetic biology, strategies for introducing designable, modular control in living cells

Pre-meeting: March 24, 2017, 4:00-5:30 PM, Cameron Williams Lecture Hall (Plant Sciences Building)

Student Lecture: March 27, 2017 (Monday), 8:00-8:50 AM, Cameron Williams Lecture Hall (Plant Sciences Building)

Student Lunch: March 27, 2017 (Monday), 12:30-1:30 PM, Room 460 Plant Sciences Building

Seminar: March 27 (Monday) 4:00 PM, Cameron Williams Lecture Hall (Plant Sciences Building)

            Title: TBA

Host: Seth DeBolt, sdebo2@email.uky

Reading list for Dr. Dueber (coming soon)

 

James Patton, Ph.D., Stevenson Professor, Department of Biological Sciences, Vanderbilt University Medical Center, Nashville, TN, https://medschool.vanderbilt.edu/patton-lab/

Topic: The role of miRNAs play in vertebrate development & regeneration; extracellular RNA communication

Pre-meeting: April 7, 2017 (Friday), 4:00-5:30 PM 109 TH Morgan Building

Student Lecture: April 10, 2017 (Monday 8:00-8:50 AM, 109 TH Morgan Building

Student Lunch: April 10, 2017 (Monday), 12:30-1:30 PM, 305 TH Morgan Building

Seminar: April 10, 2017 (Monday) 4:00 PM, 116 TH Morgan Building     

            Title:Regulation of Retina Regeneration by miRNAs, GABA Signaling, and Exosomes

Host: Brian Rymond, rymond@uky.edu

Reading list for Dr. Patton: Posted on Canvas

 

Alejandro Sánchez Alvarado, Ph.D.  Howard Hughes Medical Institute Investigator, Stowers Institute for Medical Research http://www.stowers.org/faculty/s%C3%A1nchez-lab

NOTE: changes in days/times

Topic: The molecular basis for regeneration

Pre-meeting: March 29, 2017 (Wednesday), 4:00-5:30 PM 116 TH Morgan Building (tentative, depending on room availability)

Student Lecture: None

Student Lunch: March 30, 2017 (Thursday), 305 TH Morgan Building times TBA  

Seminar: March 30, 2017 (Thursday) 4:00 PM, 116 TH Morgan Building 

            Title: TBA

Host: Dr. Jeramiah Smith

Reading list for Dr. Alvarado is posted on the 601 Canvas site.