Lecture 1 - Exam 2 Flashcards
What is the generation of a recombinant DNA molecule?
Start with human DNA and a plasmid vector. Cut both DNA strands with restriction enzyme like EcoRI. The fragment of human DNA is inserted into the plasmid DNA vector? The resulting recombinant molecule is then introduced into E. coli, where it replicated along with the bacteria to yield a population of bacteria carrying plasmids with the human DNA insert.
During the generation of a recombinant DNA molecule, there is the process of joining DNA molecules. What does that look like?
Insert and vector DNAs are digested with a restriction endonuclease (such as EcoRI) which cleaves at staggered sites leaving overhanging single-stranded tails. Insert and vector DNAs can then associate by complementary base pairing, and covalent joining of the DNA strands by DNA ligase yields a recombinant molecule.
What is the process of cDNA cloning?
Start with an mRNA molecule. A reverse transcriptase is used to generate a cDNA copy of a mRNA molecule. Then, oligonucleotides linkers containing restriction endonuclease cleavage sites are added to the ends of the cDNA. cDNA is ligated to an appropriate vector.
What is the process of cloning in plasmid vectors?
Start with human cDNA fragments and a plasmid vector that uses EcoRI to digest a section of the vector. The cDNA is inserted and EcoRI helps ligate the fragment to the vector. Transform E. coli with recombinant plasmids. Then, plate bacteria on medium containing ampicillin. The ampicillin colonies will persist and grow, and then isolate a colony. Now, you have an E. coli containing E. coli DNA and the recombinant plasmid.
Does genome size of many eukaryotes correlate to genomic complexity?
No. The number of genes of eukaryotic genomes is not simply related to either genome size or genomic complexity.
The number of genes in eukaryotic genomes is not simply related to either genome size or genomic complexity. So, what’s up?
Genomes of most eukaryotic cells contain protein-coding sequences as well as large amounts of DNA that does not code for proteins.
Genomes of most eukaryotic cells contain protein-coding sequences as well as large amounts of DNA that does not code for proteins. What do we call this?
We call this non-coding DNA, and many noncoding sequences play critical roles in our cells.
The gene encoding the adenovirus hexon (a major structural protein of the viral particle) consists of how many exons and introns?
Consists of four exons, interrupted by three introns.
There is an illustration on the powerpoint that shows a hypothetical hybrid between hexon mRNA and a portion of adenovirus DNA. The exons are seen as regions of what? What are they separated by?
The exons are seen as regions of RNA-DNA hybrid, which are separated by single-stranded DNA loops corresponding to the introns.
The structure of eukaryotic genes: Most eukaryotic genes contain segments of?
Protein-coding sequences (exons) interrupted by noncoding sequences (introns).
The structure of eukaryotic genes: What is the process of chromosomal DNA becoming mRNA?
Starts with two DNA strands with exons and introns. Transcription of exons and introns will occur, resulting in one strand that is the primary RNA transcript (in the 5’ to 3’ direction). The introns are then removed by splicing to form the mature mRNA.
The structure of eukaryotic genes, roles of introns:
Where are nested genes contained?
A nested gene is contained within an intron of a larger host gene.
When nested genes are present, what is the yield of transcription?
Transcription will yield primary transcripts of both the host and nested genes, which are spliced to yield host gene and nested gene mRNAs.
These account for more then 5% of genes in Drosophila, but less than 1% in humans.
What are the roles of introns?
Transcriptional regulatory elements that are commonly called cis-regulatory elements.
All cells contain the ______ genes, but only a subset of genes is expressed in _____ cell. How?
Same ; each
Noncoding regulatory elements control transcription of mRNAs in each cell.
Regulatory elements that control transcription of a gene may be located up to?
Hundreds of kilobases away from the gene, immediately upstream of the gene, within noncoding exons or within introns.
Where are most regulatory elements within genes?
In the introns or in the 5’ untranslated region (5’ UTR) encoded by the first exon.
If a gene has 6 exons and is separated by 5 introns, what does alternative splicing allow?
Alternative splicing allows these exons to be joined in different combinations, resulting in the formation of three distinct mRNAs and proteins from the single primary transcript.
_____ of the human genome is transcribed, which is much more than what?
75%.
Much more than protein coding genes.
What’s up with miRNA?
miRNA genes are transcribed to yield primary transcripts (pri-miRNAs) that contain hairpin structures.
What are miRNAs?
Noncoding micro RNAs.
After miRNA genes are transcribed to yield primary transcripts (pri-miRNAs) that contain hairpin structures, what happens?
Pre-miRNAs are sequentially cleaved by the nucleases Drosha and Dicer to yield double-stranded miRNAs of approximately 22 nucleotides.
After Pre-miRNAs are sequentially cleaved by the nucleases Drosha and Dicer to yield double-stranded miRNAs of approximately 22 nucleotides, what happens?
miRNAs associate with the RISC complex in which the two strands of the miRNA are unwound. The miRNA then targets RISC to the 3’ untranslated region (3’ UTR) of an mRNA, leading to repression of translation and mRNA degradation.
What are IncRNAs?
Noncoding RNAs that are greater than 200 BPs. There are more than 50,000 IncRNAs in the human genome, that’s more than the number of protein coding genes!!!
Importantly, expression of the IncRNAs is mostly _______ specific. What does this suggest?
Tissue.
This suggests that many have specific functions depending on the cell type.
