GENETICS CH 23 - Genomics II: Functional Genomics, Proteomics and Bioinformatics

You have created a mouse that has a homozygous knockout for your gene of interest. What techniques could you use to show that the gene is no longer expressed?

- DNA microarray - RNA-Seq - 2-D gel electrophoresis - Antibody microarray Explanation: - Chromatin immunoprecipitation is used to find DNA sequences which are bound by certain proteins. - Both RNA-Seq and DNA microarrays could be used to determine if an RNA transcript was present from your knockout gene. - 2-D gel electrophoresis and an antibody microarray could be used to confirm that the protein of interest is not being made.

Describe the technique of chromatin immunoprecipitation (ChIP)

- a protein that is noncovalently bound to DNA can be more tightly attached to chromatin by addition of formaldehyde or some other agent that covalently crosslinks protein to the DNA - cells are lysed, DNA is broken by sonication into pieces approx 200 - 1000 bp long - antibody that is specific for the protein of interest is added - researcher must suspect that particular protein binds to DNA and have previously made or obtained an antibody that recognizes that protein - antibodies bind to DNA-protein complexes and cause the complexes to form a pellet following centrifugation - collect complexes in pellet, add chemical that breaks the crosslinks to remove the protein - (1) conduct PCR using primers to a specific DNA region OR (2) covalently attach DNA linkers to the ends of the DNA - (1) if PCR amplifies DNA the protein I'd interest was bound to the DNA region that is flanked by the primers - (2) conduct PCR using primers that are complementary to the linkers. incorporate fluorescently labeled nucleotides during PCR - denature DNA and hybridized to a microarray

Understand the methods of 2-D gel electrophoresis and what info it can give us

- a sample of cells is lysed, and the proteins are loaded onto the top of a tube gel that separates them according to their net charge at a given pH - protein migrates to the point in the gel where it's net charge is zero - after the tube gel run, it's laid horizontally on top of a polyacrylamide slab gel that contains sodium dodecyl sulfate (SDS) - SDS coats proteins with negative charges and denatures them - proteins in slab gel are separated according to molecular mass - proteins within the gel can be stained with a dye, collection of spots, each corresponds to. unique cellular protein - spots for proteins of larger mass remaining higher in the gel

Describe technique of RNA sequencing and explain what type of info RNA seq yields

- begins with isolation of RNA molecules from a sample of one or more types of cells - after desired population of RNAs has been obtained, next step is to produce cDNAs - the RNAs are fragmented into small pieces - one way to make cDNAs is to attach short segments of DNA, called linkers, to 5' and 3' ends of the RNA fragments - primers are added that are complementary to the linkers, and cDNAs are made via reverse transcriptase PCR - population of cDNAs is then subjected to next-generation DNA sequencing - cDNA sequences are aligned with the genomic DNA sequence - when a cDNA sequence aligns with a gene sequence within the genome, this result means that the gene was expressed, because each cDNA sequence is derived from RNA molecule

Recognize why the proteome is likely so much larger than the genome of organisms

- changes in pre-mRNAs may ultimately affect the resulting amino acid sequence of a protein - most important alteration that occurs commonly in eukaryotic species is alternative splicing

Explain the basic strategies that are used to analyze DNA sequences

- computer programs can be designed to scan very long sequences, such as those obtained from genome-sequencing projects, and locate meaningful features within them - sequence recognition: program has the information that a specific sequence of symbols has a specialized meaning - pattern recognition: in bioinformatics, this term refers to the ability of a computer program to recognize a pattern of symbols - located specialized sequences within a very long sequences - locate an organization of sequences - locate a pattern of symbols

Understand mass spectrometry and what info they can give us

- goal is to correlate a given spot on a 2D gel with a particular protein - peptides are mixed with organic acid and dried on a metal slide - laser beam ejects peptides from slides as ionized gas with peptides positively charged - charged peptides are accelerated via an electric field and fly toward a detector - the time they spend in flight is determined by their mass/charge ratio, provides accurate way determining the mass of a peptide

Explain how DNA microarray is used to study gene expression patterns

- mixture of mRNAs is isolated from a sample of cells is used to create cDNAs that are fluorescently labeled - cDNAs are applied to the microarray - after hybridization some spots become fluorescent and can be visualized using a laser scanner

Analyze a multiple sequence alignment to deduce important features of a gene or protein sequence (figure 23.10)

- multiple-sequence alignment has shown that a group of nine genes fall into two closely related subgroups - alignment identified particular amino acids within polypeptide sequences that are highly conserved

Describe what a DNA microarray is

- small slide dotted with many short DNA sequences corresponding to the sequence of a known gene that functions through hybridization

Explain what BLAST program does

- starts with a particular genetic sequence and then locates homologous sequences within a large database - determines which sequences in this database are the closest matches to the amino acid sequence of human phenylalanine hydroxylase

Explain why gene knockout collections are useful

- understand how the protein products of genes participate in a particular cellular pathway or contribute to a complex trait - study of inherited human diseases

Assay levels of protein

2-D gel electrophoresis

Compare DNA and Protein sequences

BLAST

Knockout mice can also be created using _________ technology

CRISPR-Cas

Find DNA sequences bound by protein

ChIP chip assay

A __________ is a slide with many DNA sequences spotted on it

DNA microarray

Determine changes in RNA expression

DNA microarray

You are carrying out a chromatin immunopreciptation experiment. At the step where you are supposed to add the antibody to your protein of interest, you accidentally add a mixture of antibodies that someone was storing in the lab. What will your results show?

DNA will still be isolated, but it will correspond to DNA that was bound by any protein recognized by the antibody mixture. Explanation: - The ChIP assay will still isolate DNA, but instead of being DNA bound by just one protein, it will contain DNA bound by any of the proteins to which there was an antibody in the antibody mixture.

The value that represents the number of times that the match or a better match would be expected to be found by random chance in an entire database is known as the _______

E value

Study protein function

Functional protein microarray

Two or more paralogs in a single organism

Gene family

Determine identify of a protein

Mass spectrometry

You are studying a transcription factor. You have identified the DNA sequence to which it binds through a ChIP-chip experiment. You now wish to find other genes whose transcription is controlled by this transcription factor. What approach should you take?

Use a computer program to search by signal Explanation: - Since you have already identified the DNA sequence to which the transcription factor binds, it makes sense to take a search by signal approach. - In this approach, you would use a computer program to search for genes that are flanked by the DNA sequence to which the transcription factor binds. - This sequence is likely to be the promoter

Define database

a large number of computer data files, such as those containing genetic sequences, collected and stored in a single location

Open reading frame

a region in a genetic sequence that does not contain stop codons

RNA sequencing (RNA-Seq)

a technology for determining the sequences of RNA molecules isolated from a sample of cells

Search by content

an approach in which a computer program predicts the location of a gene based on the fact that the nucleotide content of a particular region differs significantly (due to codon bias) from a random distribution

Search by signal

an approach in which a computer program relies on known sequences such as promoters, start and stop codons, and splice sites to help predict whether or not a DNA sequence contains a protein-encoding gene

If the files in the database include additional information such as the name of the organism from which the sequence was obtained the database is said to be _________

annotated

In an antibody microarray___________ to specific proteins are spotted onto the chip

antibodies

A protein microarray that can determine protein expression levels is the

antibody microarray

To use a microarray, mRNA from cells of interest are first converted to __________.

cDNA

When mRNA is used to direct the synthesis of DNA, the resulting DNA is called _______

cDNA

Chromatin immunoprecipitation involves

chemical cross linking of DNA to protein

A protein microarray that can demonstrate protein-to-protein interactions is the

functional protein microarray

DNA microarray can be used to look for insertions or deletions if labeled __________ is used

genomic DNA

The BLAST program starts with a protein or DNA sequence and then locates ___________ sequences within a database

homologous

Genes derived from the same ancestral gene

homologous genes

cDNA from two cell types can be differentially labeled and ________ to the microarray

hybridized

Gene Knockout

in the case of diploid species, the condition in which both copies of a gene have been altered to an inactive form

A gene knockout means that the gene has been altered in a way that _______ it's function

inactivates

Gene knockouts may be created by using transposable elements to ______ into a gene

insert

The goal of the Knockout Mouse Project and other large-scale mouse knockout efforts is to create at least one _________ mutation in each of the protein encoding genes in the mouse genome

loss-of-function

The better the match the _______ the E value

lower

To compare gene expression between two cells ________ is isolated from each cell type

mRNA

Gene knockouts are useful as many disease and syndromes are the result of _________ that inactivate genes

mutations

Two homologous genes found in different species

orthologs

Two or more homologous genes found in the same organism

paralogs

In a functional protein array the ________ whose function is to be tested are spotted onto the chip

proteins

Define homology

similarity resulting from common ancestry

DNA microarrays contain

single stranded DNA

In the microarray procedure, what molecule is labeled with a fluorescent tag?

single-stranded cDNA

One reason the proteome is larger than the genome is

some mRNAs are alternatively spliced Explanation: - There are more types of proteins in the cell than genes because mRNA can produce variant proteins through alternative splicing and RNA editing. - Also proteins can be modified after translation.

Transcriptome

the set of all RNA molecules, including mRNAs and non-coding RNAs, that are transcribed in one cell or in a population of cells

If there is a 100% match between the sequence used for searching and a sequence in the database the E value would be

zero