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Alcohol Dehydrogenase (ADH)
Enzyme involved in ethanol metabolism
ADH Expression
More produced in ethanol, males head
ADH Lesson
Gene expression can be influenced by factors in the environment, gender, and body part
ADH Organism
Drosophila melanogaster
ADH Methods
Cellulose acetate zymogram
ADH Male vs. Female
M: smaller, fuse bands; F: bigger, separate bands
RAPD PCR Abbreviation
Random Amplified Polymorphic DNA; Polymerase Chain Reaction
RAPD PCR Use
PCR technique used to amplify random unknown sequences
RAPD PCR Primer
One nonspecific primer
RAPD PCR Results
multiple bands in gel electrophoresis results because primer binds to multiple sequences
RAPD PCR Methods
PCR, gel electrophoresis
RAPD PCR Results Interpretation
Comparing bands in different lanes can indicate whether they are genetically similar
Traditional PCR
Uses forward and reverse primers to amplify known DNA sequence and results in one band on gel electrophoresis
Central Dogma
DNA transcribed into RNA (transcriptome). Its then translated into a protein.
Module 4: Splicing
Process in which introns are removed from pre-mRNA via the spliceosome to produce mature mRNA molecules; Mature mRNA is the template for protein synthesis
Transcriptome
all RNAs in a cell (noncoding & coding)
RNA-Seq Analysis
Extracted mRNA is reverse transcribed into cDNA
Fragments of cDNA are then sequenced and mapped back to the genome
RNA-Seq Reason
Analyzing mRNA molecules allows us to analyze transcription regulation/gene expression
Histograms
Y-axis: # of reads that map to the position on genome; X-axis: position of genome
Histogram Introns
Gaps of gene expression because they're spliced out from mature mRNA that's sequenced.
Histogram Exons
Higher number of reads
Isoforms
Difference in peak height can indicate alternative splicing effects
Male vs. Female Tracks Identification
Differential gene expression, sex-specific isoforms, Exon-Intron boundaries counts, Exon usage/Splice site variations
Viewing exon-intron boundaries
Zoom in
Set Screen to 15-20 nts
View nts & positions shown in window
Making a picture
Draw exons and introns
Label boundaries with the nucleotide positions from genome browser
Label splice donor/ acceptor sequences
Boxes
Exons; sequenced mRNA
Line
Introns
Exon junction
RNA fragment sequence matches that of DNA downstream. Why? Bases in between must have been removed
Non-randomized Splicing
Machinery recognizes specific sequences at the beginning and end of introns.
Splice donor: 1st two nucleotides of intron (GT/GU)- Splice acceptor: last two nucleotides of intron (AG)
Module 5: Translation
Process of converting mRNA molecule sequence to an amino acid sequence that encodes for protein via the ribosome
Reading Frame: Gene Transcription direction (- or +)
6 possibilities:
(-), Forward; (-), Reverse; (+), Forward; (+), Reverse; Direction (+); Direction (-)
Reading Frame: Start codon where?; Stop codons in exons?
Exons can have different reading frames; Stop codons in UTRs are okay
Coding DNA Sequence
Coding portion of exons
Phases: codon cut off by splice site?
1 base long partial codon at the end of the exon- Couldn’t be complete
2 base long partial codon at the end of the exon- May be compensated for
Complete codon before splice site- Normal translation
Module 6: Alternative Splicing
Exons of the same gene are joined in different combinations to produce different proteins
Alternative Splicing Reason
mRNA allows us to be adaptable to environment and through development. We need diverse proteomes to meet our needs
RNAi (RNA interference)
Small RNA molecules guide endonuclease activity to degrade mRNA transcripts. Degraded transcripts cannot be translated therefore their proteins are not expressed. This results in observable phenotype
RNAi Organism
Caenorhabditis elegans- roundworm or nematode
RNAi Experiment
C. elegans is fed E. coli with DPY( dumpy determines fat/ short phenotype) gene which results in phenotypic change of shorter C. elegans