Chapter 6: Identifying Gene Function Flashcards
Module 5
Homologous genes
- share a common evolutionary ancestor, revealed by sequence similarities between the genes
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Orthologous genes
- homologs present in different organisms
- common ancestor predates the split between the species
- usually have the same or very similar functions
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Paralogous genes
- present in the same organism
- often as members of a recognized multigene family
- common ancestor may or may not predate the species in which the genes are now found
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A homology search can be conducted with a _____ sequence, but usually a tentative gene sequence is converted into an _____ _____ sequence before the search is carried out
- DNA
- amino acid
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Why in a homology search, is the sequence converted into an amino sequence?
- there are 20 different amino acids in proteins
- only four nucleotides in DNA
- genes that are unrelated usually appear to be more different from one another when their amino acid sequences are compared
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how is a homology search performed
- by making alignments between the query sequence and sequences from the databases
- For each alignment, a score is calculated
- two ways of generating the score
- simplest programs
- count the number of positions at which the same amino acid is present in both sequences
- when converted into a percentage, gives the degree of identity between two sequences
- uses chemical relatedness between nonidentical amino acids to assign a score
- higher score for identical or closely related amino acids
- lower score for less closely related amino acids
- To achieve the highest possible score, the algorithm introduces gaps at various positions in one or both sequences
- parallels processes thought to occur during the evolution of genes
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standard BLAST program is efficient at identifying homologous genes that have more than _____% sequence similarity but is less effective at recognizing evolutionary relationships if the similarity is _____
- 40
- lower
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PSI-BLAST (position-specific iterated BLAST)
identifies more distantly related sequences by combining the homologous sequences from a standard BLAST search into a profile, the features of which are used to identify additional homologous sequences that were not detected in the initial search.
Problems with BLAST
the presence in the databases of genes whose stated functions are incorrect.
may be possible to deduce at least some part of the function of the gene by searching the amino acid sequence for _____ that encode _____ _____ of known function
- motifs
- protein domains
Zinc fingers are _____-_____ structures, so identification in an unknown gene of an amino acid sequence that can encode a zinc finger indicates that the gene codes for
- DNA-binding
- a DNA-binding protein
searching for motifs called _____ _____, which direct proteins to organelles such as the nucleus or mitochondria or might specify that the protein is secreted from the cell
sorting sequences
The presence of a shared domain indicates that two proteins can perform a similar _____ _____, but that does not necessarily mean that the proteins have
- biochemical activity
- similar overall functions
Identification of a domain sequence in an unknown gene therefore identifies a specific ____ _____ to be identified, but on its own this does not enable the actual _____ of the gene to be assigned
- biochemical activity
- function
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If the starting point is the gene, rather than the phenotype, then one strategy is
- to mutate the gene and identify the phenotypic change that results
- basis of most of the techniques used to assign functions to unknown genes
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Gene Inactivation
The easiest way to inactivate a specific gene is to disrupt it with an unrelated segment of DNA. This can be achieved by _____ _____. The vector carries two segments of DNA that match the _____ of the gene to be inactivated. These end segments _____ with the chromosomal copy of the target gene. As a result, the target gene becomes disrupted.
- homologous recombination
- ends
- recombine
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Gene Inactivation w/Homologous Recombination
Deletion Cassette
- deletion cassette consists of
- promoter sequence
- followed by an antibiotic-resistance gene
- new segments of DNA are attached to either end
- they have identical parts of the yeast gene to be inactivated
- flanked by two restriction sites
- start and end segments of the target gene are inserted into the restriction sites, and the vector is introduced into yeast cells
- Recombination between the gene segments in the vector and the chromosomal copy of the target gene results in disruption of the latter
- Cells in which the disruption has occurred are identifiable because they now express the antibiotic-resistance gene and will grow agar medium containing geneticin
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Gene Inactivation w/Homologous Recombination
barcode deletion
- A high-throughput version of the gene inactivation method
- uses a modified version of the basic deletion cassette
- also includes two 20-nucleotide barcode sequences
- different for each deletion, that act as tags for that particular mutant
- Each barcode is flanked by the same pair of sequences and so can be amplified by a single PCR
- groups of mutated yeast strains, each with a different inactivated gene, can be mixed together and their phenotypes can be screened in a single experiment
- the relative abundance of each barcode indicates the abundance of each mutant after growth in glucoserich medium
- Barcodes that are absent or present only at low abundance indicate mutants whose inactivated genes were needed for growth under these conditions.
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Gene Inactivation w/Homologous Recombination
model organism, knockout
- engineered embryonic stem cell is injected into a mouse embryo
- mouse embryo develops and gives rise to a chimera (mouse whose cells are a mixture of mutant ones), derived from the engineered ES cells embryonic site and nonmutant ones, derived from all the other cells in the embryo
- This is still not quite what we want, so the chimeric mice are allowed to mate with one another
- Some of the offspring result from fusion of two mutant gametes producing knockout mice
- works well for many gene inactivations, but some are lethal and so cannot be studied in a homozygous knockout mouse
- Instead, a heterozygous mouse is obtained in the hope that the phenotypic effect of the gene inactivation will be apparent even though the mouse still has one correct copy of the gene being studied
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Gene Inactivation w/out Homologous Recombination
RNA interference, or RNAi
- natural processes by which short RNA molecules influence gene expression in living cells
- provides a means of silencing the expression of a target gene
- doesn’t disrupt gene itself but destroys its mRNA
- short double-stranded RNA molecules, whose sequences match that of the mRNA being targeted are introduced into the cell
- double-stranded RNAs are broken down into shorter molecules, which induce degradation of the mRNA
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Gene Inactivation w/out Homologous Recombination
programmable nuclease
- a nuclease that can be directed to a specific site in a genome
- can be programmed to make a double-stranded cut in a selected gene
- stimulates nonhomologous end-joining
- results in a short insertion or deletion which will inactivate the gene
- creates a true knockout
- example: Cas9 endonuclease
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Gene Inactivation w/out Homologous Recombination
RNA interference, or RNAi issues
- does not always result in complete silencing of the target gene
- Often the silencing is incomplete and is referred to as knockdown rather than knockout
- so short that offtarget effects are possible
- interfering RNAs bind to mRNAs other than the targets, resulting in silencing of more than one gene
- In mammals it often results in activation of signaling proteins called interferons, which stimulate an antiviral defense resulting in phenotypic changes that can mask the specific change occurring due to silencing of the target gene.
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Gene overexpression
- used to assess function
- organism is engineered w/test gene much more active than normal (gain of function) to determine what changes, if any, this has on the phenotype
- a multicopy vector is used, that multiplies inside the host organism to 40–200 copies per cell and also contain a highly active promoter sequence
- must be treated w/caution because gene product may synthesized in excessive amounts, possibly in tissues in which the gene is normally inactive making it difficult to distinguish a phenotype change that is due to the specific function of an overexpressed gene
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The critical aspect of a gene inactivation or overexpression experiment is the need to identify a phenotypic change. This can be much more difficult than it sounds. Why?
- the range of phenotypes that must be examined is immense
- effect of gene inactivation can be very subtle and may not be recognized
- many gene inactivations appear to give no discernible phenotypic change
- effects of these genes only become apparent, if at all, when the cells are grown under a range of different conditions or when groups of genes that contribute to the same phenotype are co-inactivated
- In the human genome, there appears to be a subset of several hundred genes that are nonessential, both copies of which can be inactivated, due to natural mutation, without any discernible effect on the health of the individual.
- These observations suggest that a complete functional annotation of the genomes of many species will not be achievable by approaches that are based solely on gene inactivation or overexpression.
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Additional insights into gene function can be obtained by identifying in which tissues, and at what times, a gene is expressed and by direct examination of the
protein coded by the gene.
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Reporter Genes
- used to determine the pattern of gene expression within an organism
- cells that express the reporter gene may become blue, fluoresce, or give off some other visible signal
- must be subject to the same regulatory signals as the test gene, so ORF of the test gene is replaced w/the ORF of the reporter gene
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Reporter Genes
- It is important to know two things: which cells a gene is expressed and
- Reporter genes cannot help here because the DNA sequence upstream of the gene—the sequence to which the reporter gene is attached—is not involved in targeting the protein product to its correct intracellular location. Instead, the _____ ______ ______ of the protein itself contains the targeting information.
- the only way to determine where the protein is located is to
- what position within the cell where the protein coded by the gene is found is important. ie. mitochondria, in the nucleus, or on the cell surface
- amino acid sequence
- search for it directly
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immunocytochemistry
- used to locate the position within a cell where the protein can be found
- uses a labeled antibody that is specific for the protein of interest
- antibody binds only to this protein
- label allows protein to be visualized via fluorescent labeling and confocal microscopy or electron microscopy
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site-directed or in vitro mutagenesis
Oligonucleotide-directed mutagenesis
- an oligonucleotide containing a single base-pair mismatch, corresponding to the desired mutation, is annealed to a single-stranded version of the relevant gene
- it primes a strand-synthesis reaction that continues all the way around the circular template molecule
- DNA replication produces numerous copies of this recombinant DNA molecule
- Half of these are copies of the original strand of DNA, and half are copies of the strand that contains the mutated sequence
- mutant ones are identified by hybridization probing with the original oligonucleotide
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site-directed or in vitro mutagenesis
PCR
- only one mutation can be created per experiment
- one normal primer: forms a fully base-paired hybrid with the template DNA
- one mutagenic primer: contains a single basepair mismatch
- mutation is initially present in two PCR products, each corresponding to one half of the starting DNA molecule
- The two PCR products are then mixed together and a final PCR cycle is carried out to construct the full-length, mutated DNA molecule.
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site-directed or in vitro mutagenesis
- The problem w/a site-directed mutagenesis experiment is
- The answer is to use a more complex, two-step gene replacement. explain.
- that we must be sure that any change in the activity of the gene being studied is the result of the specific mutation that was introduced into the gene, rather than the indirect result of changing its environment in the genome by inserting a marker gene next to it
- two-step gene replacement:
- target gene is replaced with the marker gene
- cells where recombination takes place are identified by selecting for the marker gene phenotype
- the marker gene in these cells is replaced by the mutated gene
- we now look for for cells that have lost the marker gene phenotype
Module 6
Serial Analysis of Gene Expression (SAGE)
Process
- oligo(dT) strands are attached to cellulose beads
- mRNA is immobilized in a chromatography column by annealing the poly(A) tails present at the 3’ ends to the beads
- The mRNA is converted into doublestranded cDNA via restriction enzyme
- treated with a restriction enzyme AluI that recognizes a 4 bp target site (5’–AGCT–3’.) and so cuts frequently in each cDNA
- 44 cut is every 256 bp
- leaves a blunt cut
- terminal restriction fragment of each cDNA remains attached to the cellulose beads
- all the other fragments to be eluted and discarded
- short ds oligonucleotide containing a recognition sequence for BsmFI, is attached to the free end of each cDNA
- BsmFI is restriction enzyme that rather than cutting within its recognition sequence, it cuts 10–14 nucleotides downstream
- BsmFI therefore removes a fragment with an average length of 12 bp from the end of each cDNA
- fragments are collected, ligated head-to-tail to produce a concatamer, and sequenced
- the concatamer that is formed is made up partly of sequences derived from the BsmFI oligonucleotides
- to avoid this the oligonucleotide can be designed so that the end that ligates to the cDNA contains the recognition sequence for a third restriction enzyme
- Treatment with this enzyme cleaves the oligonucleotide from the cDNA fragment
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issues with microarray or chip to study one or more transcriptomes
Further complications arise if the objective is to compare two or more transcriptomes. name an alternative method.
- design the experiment so that the two transcriptomes can be directly compared, in a single analysis using a single array
- done by labeling the cDNA preparations with different fluorescent probes
- then scanning the array at the appropriate wavelengths to determine the relative intensities of the two fluorescent signals at each position
- determines the differences between the mRNA contents of the two transcriptomes
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issues with microarray or chip to study one or more transcriptomes
Further complications arise if the objective is to compare two or more transcriptomes. why?
- For comparisons to be valid, differences between the hybridization intensities for the same gene with two different microarrays or chips must represent genuine differences in mRNA amount
- data analysis must include normalization procedures that enable results from different array experiments to be accurately compared
- data analysis include negative controls so that the background can be determined in each experiment, as well as positive controls that should always give identical signals
- actin gene is often used as a positive control as its expression level tends to be fairly constant in a particular tissue
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issues with microarray or chip to study one or more transcriptomes
with all but the simplest transcriptomes, hybridization analysis will have insufficient specificity to distinguish between every mRNA that is present. why?
- because two different mRNAs might have similar sequences
- when two or more paralogous genes are active in the same tissue
- when two or more different mRNAs are derived from the same because of of alternative splicing
- exons from a pre-mRNA are assembled in different combinations to give a series of related but different mRNAs
- The array must be designed very carefully if all of these variants are to be detected and accurately quantified
Module 6
Using a microarray or chip to study one or more transcriptomes
When a transcriptome is analyzed, there are two key objectives. Name them and how are they addressed w/a microarray and a chip
- identify the genes whose mRNAs are present
- demands that every relevant gene be represented by at least one probe in the array
- W/a microarray it’s achieved by using PCR products or cDNAs that are derived from the genes of interest
- W/a DNA chip it’s achieved by synthesizing at each position a mixture of oligonucleotides which match different positions in the relevant gene
- determine the relative amounts of these various mRNAs
- met because the microarray or chip contains up to 109 copies of the probe molecules
- higher than the anticipated copy number for any mRNA
- no position ever becomes saturated (i.e., so much hybridization that every probe molecule is base-paired to a target molecule)
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microarray or chip analysis
hierarchical clustering
- identifies genes that display similar expression profiles that are likely to be ones with related functions
- involves comparing the expression levels of every pair of genes in every transcriptome that has been analyzed, and assigning a value that indicates the degree of relatedness between those expression levels.
- data can be expressed as a dendrogram, in which genes with related expression profiles are clustered together
- The dendrogram gives a clear visual indication of the functional relationships between genes.
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phage display
- methods for studying protein–protein interactions
- cloning vector used for phage display is a bacteriophage genome with a unique restriction site located within a gene for a coat protein
- technique was originally carried out with the gene III coat protein of the filamentous phage called M13, but has now been extended to other phages including λ
- DNA sequence coding for the test protein is ligated into the restriction site so that a fused reading frame is produced—one in which the series of codons continues unbroken from the test gene into the coat protein gene.
- After transformation of Escherichia coli, this recombinant molecule directs synthesis of a hybrid protein made up of the test protein fused to the coat protein
- Phage particles produced by these transformed bacteria display the test protein in their coats.
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phage display library
- The test protein is immobilized within a well of a microtiter tray
- phage display library added
- After washing, the phages that are retained in the well are those displaying a protein that interacts with the test protein
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activators
- responsible for controlling the expression of genes in eukaryotes
- activator must bind to a DNA sequence upstream of a gene and stimulate the RNA polymerase enzyme that copies the gene into RNA
- DNA-binding and polymerase activation—are specified by different parts of the activator
- some activators will work even after cleavage into two segments, one segment containing the DNA-binding domain and one containing the activation domain. In the cell, the two segments interact to form the functional activator
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two-hybrid system
- methods for studying protein–protein interactions
- uses Saccharomyces cerevisiae strain that lacks an activator for a reporter gene → gene is switched off
- Hibryd I
- artificial gene coding for the activator DNA-binding domain ONLY is ligated to another gene coding the protein of interest
- gene of interest can come from any organism
- Hibryd I is introduced into yeast
- recombinant yeast strain cannot express the reporter gene because it’s missing the activation domain for Hibryd I
- it cannot influence the RNA polymerase, it can only bind to DNA
- Hibryd II
- artificial gene encoding the activation domain ONLY fused to a DNA fragment that specifies a protein able to interact with the protein from Hibrid I
- gene is ligated with a mixture of DNA fragments so that many different constructs are made
- Hibryd II is introduced into recombinant yeast strain
- cells are plated out
- A colony in which the reporter gene is expressed contains fusion proteins whose human segments interact, thereby bringing the DNA-binding and activation domains into proximity and stimulating the RNA polymerase
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affinity chromatography
- works with intact proteins
- test protein is attached to a chromatography resin and placed in a column
- cell extract is passed through the column in a low-salt buffer, which allows formation of the hydrogen bonds that hold proteins together in a complex
- proteins that interact with the bound test protein are retained in the column & others are washed away
- interacting proteins are then eluted with a high-salt buffer
- disadvantage: need to purify protein, which is time-consuming and difficult
- identities of the purified proteins are determined by mass spectrometry
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tandem-affinity purification (TAP)
- gene for the test protein is modified so that the protein, when synthesized, has a C–terminal extension that binds to a second protein called calmodulin
- cell extract prepared under gentle conditions so multiprotein complexes do not break down
- extract is passed through an affinity chromatography column packed with a resin containing calmodulin molecules
- results in immobilization both of the test protein and others with which it is associated
- identities of the purified proteins are determined by mass spectrometry
interactome networks
- Protein interaction maps
- display all of the interactions that occur between the components of a proteome
The proteome is part of the final link that connects the genome with the _____ of the cell.
- biochemistry
