biol101 ch23

What is the primary goal of functional genomics?

To elucidate the roles and functions of genetic sequences within a species.

The term used to describe the entire collection of proteins an organism can produce is the _____.

Proteome

How does proteomics differ from genomics in its ultimate research aim?

It focuses on understanding the functional roles and interplay among the proteins of a species.

Define bioinformatics in the context of biological research.

The analysis of biological information using mathematical and computational approaches to extract meaning from genetic data.

What technological advancement allows researchers to monitor the expression of thousands of genes simultaneously?

DNA microarrays (also known as gene chips).

Describe the physical structure of a DNA microarray.

A small silica, glass, or plastic slide dotted with thousands of different synthetic DNA sequences acting as probes.

What method is used to generate the DNA fragments typically spotted onto a microarray slide?

They are either amplified via PCR or synthesized directly on the microarray surface.

Application: How are microarrays used in tumor profiling?

By revealing distinct gene expression profiles to subclassify morphologically indistinguishable cancer cells and improve clinical treatment.

In microarray technology, how is genetic variation detected between a mutant and wild-type allele?

Through differences in hybridization efficiency, as mutant alleles may not bind to probes as effectively as wild-type alleles.

What is the primary purpose of Chromatin Immunoprecipitation (ChIP)?

To determine where specific proteins bind to DNA regions within the chromatin of living cells.

In the ChIP protocol, what chemical agent is used to cross-link proteins to the DNA they are bound to?

Formaldehyde

Process: Identify the step in ChIP that follows cell lysis and DNA breakage.

A specific antibody is used to precipitate the protein of interest along with its bound DNA.

What is a 'ChIP-chip' assay?

A technique where the DNA fragments isolated via ChIP are identified by using them as probes on a DNA microarray.

Define the term 'transcriptome' as analyzed by RNA-Seq.

The complete set of all RNA molecules, including mRNAs and non-coding RNAs, transcribed in a cell or population.

Contrast alternative splicing and RNA editing in terms of their impact on the proteome.

Alternative splicing is the most important eukaryotic alteration producing multiple protein versions, while RNA editing is much less common and changes mRNA coding sequences.

Name three factors that determine the specific subset of the proteome a multicellular cell produces.

Cell type, stage of development, and environmental conditions.

Process: Describe the separation mechanism of the first dimension in two-dimensional gel electrophoresis.

Proteins are separated by $pH$ and charge interactions using an isoelectric focusing gel.

Process: Describe the separation mechanism of the second dimension in two-dimensional gel electrophoresis.

Proteins are separated according to their molecular mass using an SDS gel.

At what point do proteins stop migrating during isoelectric focusing?

When they reach the $pH$ where their net charge is zero.

Why are very abundant protein spots in a two-dimensional gel of special interest to researchers?

They may be critical for the specific structure or specialized function of that cell type.

What technique is used to reveal the amino acid sequence of a protein purified from a 2D gel spot?

Tandem mass spectrometry.

In tandem mass spectrometry, what is the specific role of the first spectrometer?

It measures the mass of a peptide generated from protein digestion.

In tandem mass spectrometry, what is the specific role of the second spectrometer?

It fragments the peptide from one end and analyzes the resulting smaller pieces to determine the sequence.

How are peptides ejected as ionized gas in a mass spectrometer?

The sample is subjected to a laser beam that ejects the peptides with one or more positive charges.

Logic: Once a short peptide sequence is obtained via mass spectrometry, how is the corresponding gene located?

Software uses possible codon sequences as query sequences to scan the entire genome sequence for matches.

How can mass spectrometry be used to identify protein phosphorylation?

By detecting an increase in the protein's mass equal to the mass of a phosphate group.

Identify two challenges in developing protein microarrays compared to DNA microarrays.

Proteins are more easily damaged by manipulations and are more time-consuming to synthesize and purify.

Contrast the utility of antibody microarrays versus functional microarrays.

Antibody microarrays assess protein expression levels, while functional microarrays probe protein activities, such as phosphorylation by kinases.

What logic does a computer program use when performing a 'search by signal' for gene prediction?

It seeks an organization of known sequence elements, such as promoters and start/stop codons, typically found within a gene.

What logic does a computer program use when performing a 'search by content' for gene prediction?

It identifies sequences where the distribution of codons differs from random due to codon bias in structural genes.

Define 'codon bias' in the context of genomics.

The phenomenon where certain codons are used more frequently than others to encode the same amino acid.

What defines an Open Reading Frame (ORF)?

A nucleotide sequence that does not contain any stop codons.

How many total translational reading frames are possible for a newly discovered DNA sequence?

Six (three forward and three reverse).

Why is locating ORFs more complex in eukaryotic genomes than in prokaryotic ones?

Eukaryotic chromosomal coding sequences are frequently interrupted by introns.

What is the E-value (Expect value) in a BLAST search?

A value representing the number of times a sequence match would be expected to occur by random chance in the database.

In a BLAST result, what does a very small E-value (e.g., $10^{-154}$) signify?

It indicates that the sequence similarity is highly unlikely to be due to chance, suggesting the sequences are homologous.

Name the database that serves as the primary repository for three-dimensional protein structures determined by X-ray crystallography.

PDB (Protein Data Bank).

Which database would a researcher use to find microarray or ChIP-chip gene expression data sets?

GEO (Gene Expression Omnibus).

What is a 'conserved site' in molecular evolution?

A genetic site that is identical or highly similar across multiple different species.

Concept: Sequence Motif

Definition: A genetic sequence with a particular known function, such as a promoter or splice site.

Identify the consensus sequence for the polyadenylation signal found in DNA.

$AATAAA$

Which eukaryotic core promoter element is characterized by sequences like the CAAT box or TATA box?

The core promoter.

What pattern of sequences is characteristic of transposable elements?

Direct repeats flanking inverted repeats.

Irreversible PTM: Give an example of a permanent covalent modification required for a functional protein.

Proteolytic processing or the attachment of prosthetic groups, sugars, or lipids.

Reversible PTM: Name three transient covalent modifications that affect protein function.

Phosphorylation, acetylation, and methylation.

In 2D gel electrophoresis, how does the SDS gel separate proteins?

It separates them strictly by molecular mass, with smaller proteins migrating further down the gel.

What is the primary advantage of RNA-Seq over older methods of studying gene expression?

It allows for the identification of all transcribed molecules and provides detailed information on alternative splicing and RNA editing.

What factor determines the 'time in flight' for a charged peptide in a mass spectrometer?

The peptide's mass and its net charge.

Why is homology among protein sequences easier to identify than DNA sequence homology?

The 20-amino acid alphabet provides more distinct information and higher conservation signals than the 4-nucleotide alphabet.

Formula/Logic: If a protein spot on a 2D gel is found only in cancer cells, what is its likely biological significance?

It may represent a protein uniquely involved in the oncogenic process or a biomarker for that specific tumor type.

The technique of _____ alignment is used to compare more than two genetic sequences to identify conserved regions.

Multiple sequence

Identify the $-10$ and $-35$ promoter sequences typically found in $E. coli$.

$TATAAT$ ($-10$) and $TTGACA$ ($-35$).

Which database collects amino acid sequence motifs characteristic of protein families or domain structures?

Prosite

The DNA fragments in a microarray are referred to as 'probes' because they _____.

Identify specific transcribed genes through complementary hybridization.

What information does a laser scanner provide after a microarray hybridization experiment?

It detects fluorescent signals at specific spots to quantify the level of gene expression.

Concept: Search by Organization

Definition: A bioinformatics strategy that seeks a specific arrangement of multiple sequence elements within a gene.

Application: How can functional microarrays identify kinase substrates?

By exposing a slide of cellular proteins to kinases and identifying which spots become phosphorylated.

What is the role of 'reverse transcriptase' in preparing samples for DNA microarrays?

It converts isolated mRNA into fluorescently labeled cDNA for hybridization.

In mass spectrometry, what is the role of the 'organic acid' added to the peptides?

It helps the peptides dry onto the metal slide and facilitates ionization by the laser beam.

How can microarrays determine metabolic pathways?

By identifying groups of genes that are expressed in a parallel manner under specific conditions.

The first computer approach to compare sequence similarity between whale myoglobin and human $\beta$-hemoglobin was proposed by _____.

Saul Needleman and Christian Wunsch (1970).

What does a 'Match 1' percentage of $100\%$ in a BLAST search usually indicate?

The query sequence is identical to a sequence already present in the database (often from the same species).

How does 2D gel electrophoresis distinguish between two proteins of the same molecular weight?

They will likely have different isoelectric points ($pI$) and thus separate at different $pH$ positions in the first dimension.

In the context of proteomics, what is the 'digestion' step before mass spectrometry?

The treatment of a purified protein with a protease to break it into smaller peptide fragments.

What is the significance of the 'postage stamp' size of a microarray slide?

It allows tens of thousands of different spots to be analyzed in a very compact area.

Which database would contain peptide sequences translated from the GenBank database?

Genpept

Bioinformatics Logic: Why is a computer program more efficient than a human at translating DNA?

It can instantly scan all six reading frames across millions of base pairs without manual error.

What is 'proteolytic processing' in protein maturation?

The irreversible removal of a portion of a polypeptide chain to activate or modify the protein.

Why might a mutant allele show lower fluorescence on a microarray spot compared to a wild-type allele?

Mismatches in the sequence reduce the strength of the hybridization bond between the cDNA and the probe.

Application: How are microarrays used in microbial strain identification?

By distinguishing between closely related bacterial species and subspecies based on unique genomic sequences.

What specific sequence element allows for the initiation of translation and is a key signal for gene prediction?

The start codon ($ATG$).

Describe the relationship between sequence homology and gene function.

There is a strong correlation, meaning homologous sequences across species often perform the same or similar functions.

In mass spectrometry, the peptide fragmentation in the second spectrometer occurs _____.

From one end of the peptide chain.

What is the primary role of the poly-dT primer in cDNA synthesis for microarrays?

It anneals to the polyA tail of all mRNAs to initiate reverse transcription.

In a 2D gel, what is the likely identity of a spot present after hormone exposure but absent in control cells?

A protein whose expression is induced by that specific hormone.

The goal of _____ is to analyze biological information using a computational approach.

Bioinformatics

Process: How is DNA 'chemically freed' in the ChIP protocol?

By adding a chemical that breaks the formaldehyde cross-links between the protein and the DNA.

Why must DNA be 'denatured' before it can hybridize to a microarray?

To ensure the DNA is single-stranded so it can bind to the complementary single-stranded probes on the slide.

What information does a 'multiple sequence alignment' of the globin gene family provide?

It identifies conserved sites that are likely functionally important for oxygen binding or protein structure.

What is the E-value for an orangutan phenylalanine hydroxylase when compared to the human version in BLAST?

$0$ (indicating essentially zero probability that the match is due to random chance).

Name the database of protein families that uses multiple amino acid sequence alignments.

Pfam