Lecture 21 Flashcards
the complete set of DNA within a single cell of an organism
genomes
is a discipline in genetics that applies recombinant DNA, DNA sequencing methods, and bioinformatics to sequence, assemble, and analyze the function and structure of genomes
genomics
Human genome project sequenced human genome, model system genomes, and –
EST (expressed sequence tags) = cDNAs
map the fragments, sequence the fragments, and piece it together = slow and careful (public)
clone-by-clone sequencing approach
Take a lot of copies of the genome
Break it up into little fragments
Sequenced all the little fragments
Computer puts it all together
shotgun sequencing
next generation sequencing: after each nucleotide is added, its identity is determined by –
fluorescence
Once a sequence is obtained, it must be –
annotated
annotating a sequence includes – the genes, promoters, exons/introns, and function of genes
locating
process of attaching biological functions to DNA sequences
- describes both structural and functional features of a gene
- goal = identify known genes, regulatory sequences, etc as well as to identify sequences that are likely to be genes through their function
annotation
– cDNAs allow designation of all introns and exons
complete
– cDNAs (referred to as expressed sequence tags (EST)) allow expressed genes to be identified
incomplete
same info from all cell types from the same individual
includes: cis-elements (promoter, enhancer, etc.)
introns, repetitive sequences, etc.
genomic DNA
– is expressed genes that is derived from mRNA contains coding regions (no introns or regulatory sequences)
cDNA
What type of library do you need to piece the genome together?
genomic
What type of library do you need to determine the exon/intron complete sequence and structure of a gene?
genomic and cDNA
expression levels are based on frequency of –
cDNA clones
exon/intron structure are based on
cDNA clones
alternative splicing are based on –
cDNA clones
databases contain info on function which is based on –
homology to known genes
Where experimental data is not available, – is used to predict gene structure from ORFs, splice sites, promoter sequences
bioinformatics
Bioinformatics: the use of – approaches to decipher DNA-sequence information
computational
sequences without a stop codon, appear to possibly code for polypeptides
open reading frames
DNA – analysis can analyze mRNA concentrations – a cheaper alternative to sequencing.
microarray
Sequence analysis of all living organisms in an environment
metagenomics
in metagenomics – are over represented
most common species
Addition of foreign DNA into the genome.
transgenic
Removal of a specific piece of DNA from the genome.
knock-out
Replacement of a specific piece of DNA from the genome with a different piece of DNA.
gene replacement
transgenes from different species
heterologous
human cells can properly translate bacterial genes because the genetic code is –
universal
– sequences are not universal: promoters, enhancers are species specific so need to use sequences specific for host
regulatory
– vectors are vectors that have been furnished with sequences capable of directing efficient transcription and translation of transgenes
expression
All vectors need
– and Selectable marker so that you can transform it and select for things
Origin of replication
Series of restriction sites into which the gene to be expressed is inserted in recombinant clones
MCS (multi-cloning site)
You can put a human gene in the MCS
If you have the bacterial promoter and transcription terminator you can express a human gene
But need to get rid of human introns since bacteria cannot splice introns therefore you need to use –
cDNA
Eukaryotic expression vector must contain –, polyadenylation signal, adn MCS
TATA box
different species may prefer different codons, reflected in levels of particular tRNA
codon bias
factors that affect heterologous protein synthesis:
codon bias, – and high levels of synthesis can lead to aggregation and precipitation of inactive protein
post-translational modifications (rarely occur in bacterial cells)
some proteins produced in E. coli for humans include insulin
human growth hormone
erythropoietin (induces red blood cell formation)
–
proteases used in detergents
– is also commonly used to express heterologous genes
Yeast
replicates in both bacteria and yeast
shuttle vector
With this shuttle vector, DNA sequences can be manipulated in – where manipulation is easier, after which the modified plasmids can be shuttled into yeast for heterologous protein expression
E. coli (bacteria)
an advantage of using S. cerevisiae as a protein factory is that it has the ability to splice introns and – modify proteins
post-translationally
S. cerevisiae can – proteins out of the cell
secrete
stable transgenic yeast can be made by integration of a plasmids without –
an origin of replication
stable transgenic yeast can be made by double crossover through -
circular or linear plasmid with homology to genome
integrates introduced DNA at a random, nonhomologous location
illegitimate recombination
integration of entire plasmid is useful for – or making transgenic yeast
gene disruption
double crossover is useful for gene disruption or –
gene replacement
In homologous recombination with circular DNA, a single crossover results in integration of introduced DNA without the – of target gene
replacement
In homologous recombination with circular DNA, a double crossover results in the – of target gene
replacement
In homologous recombination with linear DNA, a single crossover results in integration of introduced DNA and –
terminal deletion
In homologous recombination with linear DNA, a double crossover results in the – of target gene
replacement (or knockout)
linearized DNA molecules recombine at a – frequency than circular ones so in yeast a double crossover at linearized plasmid is the best choice
higher
Common technique used to determine the function of a gene is to disrupt it and examine the phenotype
gene knockout
Using a –, it is a simple process in yeast to disrupt any gene
selectable marker
Drosophila genome contains a predicted POU-domain gene, pdm-3 but nothing is known about its function.
How can you figure out where in the body pdm-3 is functioning?
transgenic fly
Advantages of transgenic animals: can examine the – of a gene of interest
expression pattern
Advantages of transgenic animals: can – particular cells or cell types with a reporter
label
Advantages of transgenic animals: can – a normal or mutant allele of a gene to look for associated phenotypes
overexpress
Disadvantages of transgenic animals: integration site is – so expression can vary depending on where the foreign DNA integrates
random
Disadvantages of transgenic animals: Random integration can sometimes disrupt genes and cause –.
unrelated mutant phenotypes
Disadvantages of transgenic animals: Often, multiple copies of the transgene are integrated, so the expression level cannot be controlled and is often much – than normal.
higher
