Chapter 24

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

0

paralogs

2 homologous genes found in a single organism

orthologs

2 homologous genes found in different species

gene family

2 or more copies of homologous genes within genome of single organism

tandem mass spectrometry

2 spectrometers used; first measures mass of given peptide generated from protein digestion; second analyzes peptide after digestion into smaller fragments

assay levels of protein

2D gel electrophoresis

number of reading frames possible in a newly discovered sequence

6

Compare DNA and Protein sequences

BLAST

find DNA sequences bound by protein

ChIP chip assay

involves chemical cross linking of DNA to protein

Chromatin immunoprecipitation

Determine changes in RNA expression

DNA microarray

slide with many DNA sequences spotted on it

DNA microarray

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

DNA microarrays

The better the match the lower the

E value

to identify the DNA bound by the protein, researchers can use

PCR, DNA microarray

any given cell of a multicellular organism will produce only

a subset of proteins in its proteome

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

annotated

protein microarray that can determine protein expression levels

antibody microarray

2 common approaches to protein microarray analysis

antibody microarrays and functional microarrays

homology-based modeling

approach to predicting protein structure based on one protein's similarity to another protein with a known structure

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

cDNA

resulting DNA when mRNA is used to direct the synthesis of DNA

cDNA

chromatin immunoprecipitation

can determine if proteins can bind to a particular region of DNA in chromatin of living cells

subset of proteins produced depends primarily on

cell type, stage of development, environmental conditions

spots present in only given circumstances

cells exposed to a hormone versus those that are not

ORF in eukaryotes

chromosomal coding sequences may be interrupted by introns

database

collection of computer files stored in one place

Computer programs have been developed to predict RNA and protein structures based on

comparison to RNA and protein molecules of similar sequence and function

antibody microarrays

consist of a collection of antibodies that recognize short peptide sequences; used to asses level of protein expression

functional microarrays

consist of many different cellular proteins; used to probe function of proteins

steps of tandem mass spectrometry

digest protein to fragments with protease; determine mass of these fragments with mass spectrometer; analyze fragment individually with second spectrometer

RNA-seq involves directly sequencing the RNA

false

SDS coats the proteins to give them a net positive charge

false

Two dimensional gel electrophoresis separates proteins based on size alone

false

2D gel electrophoresis involves 2 different gel electrophoresis experiments

first separates by pH/charge interactions; second separates by size

Programs that are used to predict protein structure frequently rely on

frequency of amino acids found within structures that have already been crystallized

protein microarray that can demonstrate protein-to-protein interactions

functional protein microarray

DNA microarray

gene chip/DNA chip/biochip; collection of microscopic DNA spots attached to a solid surface

Two or more paralogs in a single organism

gene family

gene knockout

gene has been altered in a way that inactivates its function

may replace microarrays

high-throughput RNA sequencing

will provide whole genome sequence information between cancer/normal cells of the same and different individuals

high-throughput RNA sequencing

Genes derived from the same ancestral gene

homologous genes

In mice many knockouts are created by

homologous recombination

The BLAST program starts with a protein or DNA sequence and then locates

homologous sequences within a database

To fully study what the consequences of a knockout the animal should be

homozygous for knock out

cDNA from two cell types can be differentially labeled and

hybridized to the microarray

examine translational reading frames

in a DNA sequence, the reading of codons could begin with the first, second, or third nucleotides; reading frame 1, 2, and 3

Gene knockouts may be created by using transposable elements to

insert into a gene

RNA-Seq technique

isolate RNA from a sample of cells; break the RNAs into small fragments; attach short oligonucleotide linkers to the ends of the RNAs; synthesize cDNAs via reverse transcriptase PCR, using the RNAs as templates; PCR primers are complementary to linkers; sequence cDNAs using a next-gen sequencing technology; using computer technology, align the cDNA sequences along the genomic sequence

ORF in prokaryotes

long ORFs are contained within the chromosomal gene sequences

Some programs that predict RNA structure may also use calculations to determine the form with

lowest energy state

to compare gene expression between two cells,

mRNA is isolated from each cell type

proteins which are very abundant in a cell type

may be important for that cells structure or function

DNA microarrays can be used to

measure expression levels of large numbers of genes simultaneously or genotype multiple regions of genome

Gene knockouts are useful as many disease and syndromes are the result of

mutations that inactivate genes

Genbank

nucleotide database

open reading frame

nucleotide sequence that does not contain any stop codons

Two homologous genes found in different species

orthologs

Two or more homologous genes found in the same organism

paralogs

sequence recognition searches require a researcher to input a sequence of interest but

pattern recognition search does not

sample subjected to laser beam

peptides become ejected as an ionized gas in which peptide contains one or more positive charges

tandem mass spectrometry protocol

peptides mixed with organic acid and dried onto metal slide; sample subjected to laser beam; charged peptides accelerated via electric field and fly toward detector

gene prediction

process of identifying regions of genomic DNA that encode genes; protein-encoding genes; genes for non-coding RNAs

search by content

program identifies sequences that differ significantly from random distribution due to codon bias within structural genes; some codons used more frequently than others for same amino acid

search by signal

program tries to locate an organization of known sequence elements normally found within a gene (promoter, start/stop codons)

application of protein microarrays

protein expression, protein function, protein=protein interactions, pharmacology

technology to make DNA microarrays is being applied to make

protein microarrays

Prosite

protein motif database

development of protein microarrays is more challenging

proteins are much more easily damaged by manipulations that occur during microarray formation; synthesis and purification of proteins is more time-consuming than DNA

ChiP protocol

proteins in living cells cross-linked to DNA they are bound to with formaldehyde; cells lysed and DNA fragmented; antibody used to precipitate protein of interest; DNA chemically freed from cross-links; DNA PCR amplified; sequence of DNA identified directly or by using it as a probe on microarray (ChIP-chip assay)

protein microarrays

proteins rather than DNA are spotted onto a slide

specific spots may be of special interest

proteins which are very abundant in a cell type; spots present in only given circumstances; spots present only in abnormal cells

computer programs can employ different strategies to locate genes

search by signal; search by content; examine translational reading frames

2-dimensional gel electrophoresis

separation technique that can distinguish hundreds or even thousands of different proteins in a cell extract

DNA microarrays contain

single-stranded DNA

what molecule is labeled with a fluorescent tag in a microarray

single-stranded cDNA

readon proteome is larger than genome

some mRNAs are alternatively spliced

how to correlate a given spot on a 2D gel with a protein

spot is cut out from gel; protein purified from it

In a functional protein array the proteins whose function is to be tested are

spotted onto the chip

In an antibody microarray antibodies to specific proteins are

spotted onto the chip

in mass spectrometry, the amino acid sequence of the protein is revealed via

tandem mass spectrometry

charged peptides accelerated via an electric field and fly toward a detector

time they spend in flight is determined by mass and net charge and reveals mass of peptide

DNA microarrays are used to study patterns of gene expression

true

DNA microarrays may be used to study how a cell responds to specific environmental conditions

true

During isoelectric focusing proteins are separated by the pH at which they have a net neutral charge

true

SDS allows proteins to separate based on molecular mass

true

Two dimensional electrophoresis allows proteins that differ by one charged amino acid to be separated

true

common technique in field of proteomics is

two-dimensional gel electrophoresis

E value

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

spots present only in abnormal cells

very common in cancer cells