Genetics exam 3

In bacteria, the regulation of gene expression most commonly occurs at

the transcription level

An activator is a () that () transcription

regulatory transcription factor, enhances

Which of the following combinations will cause the rate of transcription to increase in bacteria.

a repressor plus an inducer

What is an operon

a group of genes under the control of a single promoter in bacteria, which are coordinately regulated for the production of related proteins.

The binding of () to lac repressor causes lac repressor to () to the operator site, thereby () transcription

allolactose, not bind, increasing

In the () of glucose, the binding of cAMP to CAP occurs, which causes CAP to bind to the CAP site, thereby () transcription.

absence, increasing

Without tryptophan binding, trp repressor () to the trp operator site and () transcription.

does not bind, allow

Leader peptide that is involved in the trp operon regulation

it is encoded in the trpL gene.

It contains two tryptophans that sense tryptophan levels.

It is involved in attenuation

In bacteria, the rate of translation can be regulated by

translational repressor or antisense RNAS.

Gene regulation

The level of gene expression can vary under different conditions.

The benefits of regulating genes is that coded proteins will be produced only when required.

Regulation can occur at any of the points on the pathway to gene expression.

Constitutive

genes that are unregulated

they have essentially constant levels of expression

frequently, constitutive genes code proteins that are continuously necessary for the survival of the organism

the lac operon is regulated by

lac repressor + CAP

The trp operon is regulated by

trp repressor + attenuation

Repressors

(Regulatory transcription factors (RTFS) )bind to DNA and inhibit transcription

Activators

(regulatory transcription factors (RTFs)) bind to DNA and increase transcription.

Negative control

regulation by repressor proteins

Positive control

regulation by activator proteins

Small effector molecules

affect transcription regulation, these bind to regulatory transcription factors but not to DNA directly.

Inducers

small effector molecules that increase transcription.

bind to activators and cause them to bind to DNA

Bind to repressors and prevent them from binding to DNA.

genes that are regulated in this manner are termed inducible.

Corepressors

bind to repressors, causing them to bind to DNA>

Inhibitors

bind to activators, preventing them from binding to DNA

Promoter

binds to RNA polymerase

Operator

binds the lac repressor protein

CAP site

binds the Catabolite activator protein (CAP)

Terminator

ends transcripition

lacZ

codes beta-galactosidase

enzymatically cleaves lactose and lactose analogs

also converts lactose to allolactose

lacY

codes lactose permease

membrane protein required for the transport of lactose and analogs

lacA

codes galactosidase transacetylase

prevents toxic buildup of nonmetabolizable lactose analogs

The lac operon can be transcriptionally regulated

1.) by a repressor protein (negative control)

it involves lac repressor, the inducer is allolactose (allolactose binds to lac repressor and prevents the repressor from binding to the DNA)

2.) by an activator protein ( positive control)

in involves catabolite activator protein (CAP)

the inducer is cyclic AMP (cAMP) (it is produced from ATP via the enzyme adenyly cyclase)

no lactose in the environment

in the absence of lactose ( no allolactose), the repressor protein is tightly bound to the operator site, thereby inhibiting the ability of RNA polymerase to transcribe the operon.

lactose present

when lactose is available, allolactose is produced. it binds to the repressor. this alters the conformation of the repressor protein, which prevents it from binding to the operator site. Therefore RNA polymerase can transcribe the operon.

lactose and glucose (low cAMP)

in the presence of glucose, the enzyme adenylyl cyclase is inhibited.

This decreases the levels of cAMP in the cell.

Therefore, cAMP is no longer available to bind CAP.

Transcription rate decreases.

lactose, no glucose (high cAMP)

in the absence of glucose, cyclic AMP (cAMP) is produced from ATP via the enzyme adenylyl cyclase,

the cAMP- CAP complex binds to the CAP site near the lac promoter and increases transcription (an example of inducible, positive control)

trp operon

involved in the biosynthesis of the amino acid tryptophan.

the genes trpE, trpD, trpC, trpB and trpA code enzymes involved in tryptophan biosyntesis.

trpR

codes the trp repressor proteins, functions in repression

trpL

codes a short peptide called the leader peptide, which functions in attenuation

Low tryptophan levels

transcription of the entire trp operon occurs. When tryptophan levels are low, trp repressor is inactive ( no tryptophan binding). Under these conditions, RNA polymerase can transcribe the operon, leading to an increase in tryptophan biosynthesis

high tryptophan levels

repression occurs, when tryptophan levels are high, tryptophan acts as a corepressor that binds to trp repressor. The tryptophan-trp reprssor complex the binds to the operator site to inhibit transcription

Attenuation

another mechanism of regulation. When attenuation occurs, the RNA is transcribed only to the attenuator sequence and then transcription is terminated (before trpE/D/C/B/A). No production of tryptophan (attenuation can occur in bacteria because transcription and translation are coupled)

low tryptophan levels, 2 to 3 stem loop forms Attenuation does not occur. transcriptions occurs

high tryptophan levels, 3 to 4 stem loop forms. attenuation occurs. terminated transcription

A translational repressor inhibits translation in one of two ways.

1.) binding next to the Shine-Dalgarno sequence and/or the start codon to hinder the ribosome from initiating translation.

2.) binding outside the Shine-Dalgarno/started codon region, which stabilizes an mRNA secondary structure, preventing initiation

Also found in eukaryotes.

Antisense RNA

The second way to regulate translation is via the synthesis of antisense RNA (an RNA strand that is complementary to mRNA)

Posttranslational regulation

A common mechanism to regulate the activity of metabolic enzymes is feedback inhibition.

the final product in a pathway often can inhibit an enzyme that acts early in the pathway

if the concentration of the final product becomes high, it will bind to enzyme 1, thereby inhibiting its abilityty to convert substrate 1 into intermediate 1

Combinatorial control refers to the phenomenon that

The combination of many factors determines the expression of any given gene

Binding of () to () increases the rate of transcription

an activator an enhancer

How enhancers function in gene regulation in eukaryotes?

Many enhancers are typically located at a distance from the core promoter of a gene, and DNA must form to bring the enhancer (when bound by activators) and the core promoter close together to activate gene transcription.

Regulatory transcription factors can be modulated by

covalent modifications, protein-protein interactions, and the binding of small effector molecules.

Characteristics are typical of a eukaryotic gene that can be transcribes

its core promoter and terminator are found in nucleosome-free regions

acetylation of histone tails by () would () the interaction between histone octamers and DNA

histone acetyltrasnferasne, loosen

By binding to an enhancer, an activator can bring () to remodel nucleosomes near the core promoter of a eukaryotic gene to activate transcription.

ATP- dependent remodeling complex(es)

One way for methylation at a CpG island to affect transcription is that

it may inhibit the binding of an activator to an enhancer, thereby decrasing transcrition

a key difference between microRNA (miRNA) and small-interfering RNA (siRNA) is that

miRNA usually inhibits mRNA translation, whereas siRNA causes mRNA degradation

Combinatorial control

most eukaryotic genes are regulated by many factors.

1.) one or more activator proteins may stimulate transcription

2.) one or more repressor proteins may inhibit transcrption

3.) activators and repressor may by modulated by binding of small effector molecules, protein-protein interactions and covalent modifications.

4.) regulatory proteins may alter nucleosomes near the promoter

5.) DNA methylation mya inhibit transcription

6.) heterochromatin formation may inhibit transcrption

General transcription factors

required for the binding of the RNA pol to the core promoter and its progression to the elongation stage. Necessary for basal transcription

Regulatory transcription factors (activators/repressors)

serves to regulate the rate of transcription of target genes.

They influence the ability of RNA polymerase to begin transcription of a particular gene.

Regulatory elements

bidirectional

Some are located within a few hundred nucleotides upstream of the promoter; however, often found several thousands nucleotides away, downstream from the promoter, even within introns.

closed conformation (heterochromatin)

chromatin may be tightly packed,

transcription may be difficult or impossible

open conformation (euchromatin)

chromatin is accessible to transcription factors

transcription can take place

ATP -dependent chromatin remodeling

refers to dynamic changes in chromatin structure, which ranges from a few nucleosomes to large scale changes. Carried out by diverse multiprotein machines that reposition and restructure nucleosomes. make the DNA more or less amenable to transcription.

al remodeling complexes have a catalytic ATPase subunit called DNA translocases, energy of ATP hydrolysis is used to drive change in location and/or composition of nucleosomes.

Chromatin remodeling complexes change chromatin structure in one of 3 ways: change in the position of nucleosomes, eviction of histone octamers and change in the composition of nucleosomes.

Histone modifications: acetylation

attachment of the acetyl group (-COCH3) disrupts the electrostatic attraction between the histone protein and the negatively charged DNA back bone

DNA methylation

is the covalent attachment of methyl group (-CH3).

carried out by DNA methyltransferase.

DNA methylation usually inhibits eukaryotic gene transcription.

CpG islands

in vertebrates and plants, many geners contain CpG islands near their promoters, CpG islands contain high number of CpG sites.

in housing keeping genes, the CpG islands ae unmethylated. Genes tend to be expressed in most cell types.

in tissue-specific genes, the expression of these genes may be silences by the methylation of CpG islands

Methylation may influence the binding of transcription factors.

Methyl-CpG binding proteins proteins may recruit factors that lead to compaction of the chromatin

gene activation

series of events that allow a gene to be transcribed to produce an RNA molecule.

For eukaryotic genes controlled by regulatory transcription factors:

1.) one or more regulatory transcription factors (activators) bind to an enhancer

2.) The activators recruit coactivators (chromatin remodeling complexes and histone-modifying enzymes)

3.) RNA polymerase binds to the core promoter to form a preinitiation complex

4.) RNA polymerase proceeds to the elongation phase and makes an RNA transcript.

microRNAs (miRNAs)

transcribed from endogenous eukaryotic genes.

regulate gene expression, commonly a single type of miRA inhibits the translation of several different mRNAs through partial complementaruty.

As many as 60% of humans genes may be regulated by miRNAs

Small interfering RNAs (siRNAs)

ncRNAs that usually originate from exogenous sources (not normally made by cells)

can be from viruses or experimentally injected by researchers.

A typical plasmid vector contains

an origin of replication, an antibiotic gene, and a cloning site (unique restriction site)

() is a restriction enzyme that is used in gene-cloning experiments to () DNA molecules at specific DNA sequences.

EcoRI, cut

Which is the proper order of the following steps in a gene-cloning experiment involving vectors?

1.) Add DNA ligase

2.) Incubate the chromosomal DNA and the vector DNA with a restriction enzyme.

3.) Introduce the DNA into living cells

4.) Mix the digested chromosomal DNA and vector DNA together.

2,4,1,3

The function of reverse transcriptase is to

use RNA as template to make cDNA

Which of the following parts of sgRNA binds to the complementary region of a target gene in gene editing?

the spacer region

During Western blotting, the primary antibody recognizes

the protein of interest

Chromosomal DNA

serves as the source of the DNA segment of interest. Extract and purify DNA from cellular tissue/cells using a variety of biochemical techniques.

Vector DNA

serves as the carrier for the ENA segment that is to clones. Can be replicated independently of the host chromosomal DNA

The vectors commonly used in gene cloning 1.) plasmids, many have selectable markers (genes conferring antibiotic resistance to the host cell) and origin or replication

Restriction endonucleases

binds to specific DNA sequences and then cleaves the DNA at two defined locations, one in each strand.

Recognition sequences are typically palindromic. The sequence is identical when read in the opposite direction in the complementary strand.

amp^R

confers antibiotic resistance to the host cell, identifies cells that have taken up the vector.

lacZ

codes for beta-galactosidase, providing a means to identify bacteria that have a vector with an inserted DNA fragment.

Steps in gene cloning

1.) cut/digest human DNA and plasmid vector with the same enzyme in separated tubes

2.) combine digested human DNA and palsid vector DNA

3.) one of human DNA fragments can be inserted into the vector DNA (not always)

4.) cells that can take up DNA are called competent cells

5.) Transformation means uptake of plasmid vectors by a bacterial cell

6.) cells that did not take up a vector cannot grow on this plate that contains ampicillin

Polymerase Chain Reaction (PCR)

can copy DNA without the aid of vectors and host cells, must know enough about the gene of interest to have the sequence of short primers.

Starting materials for PCR

1.) template DNA- contains the region that needs to be amplified

2.) oligonucleotide primers- complementary to sequences at the ends of the END fragment to be amplified (20 nucleotides long)

3.) Deoxynucleoside triphosphates (dNTPs)- provide the precursors for DNA synthesis

4.) Taq polymerase, DNA polymerase isolated from the bacterium Thermus aquaticus. This thermostable enzyme is necessary because PCR involves heating steps that inactivate most other DNA polymerases.

3 steps of PCR cycle

1.) denaturation- DNA strands are separated (>95C)

2.) primer annealing- oligonucleotide primers bind to the DNA strands (~55C)

3.) primer extension- nucleotides are added to the primers, thereby extending their lengths (72 °C)

Reverse transcriptase

used to synthesize DNA from RNA. (uses RNA as a template to make a complementary DNA (cDNA)

cDNA

lacks introns.

Has two important uses

1.) allows researchers to focus their attention on the coding sequence of a gene

2.) it allows the expression of the encoded protein

Reverse transcriptase PCD (RT-PCR)

is used to detect and quantitate the amount of an RNA in living cells

Nonhomologous end joining

small deletion that inactivates the target gene

Homologous recombination repair (HRR)

in HRR is desired, donor DNA hmologous to the target region that also carries the desired mutation is added ( or fix a pre-existing mutation as way of gene theraoy)

Northen Blot

used to detect mRNA. mRNA are separated by gel electrophoresis, then transferred to a nitrocellulose or nylon filter for the detection of a specific mRNA with probe

Western blot

used to detect proteins that use antibiotics

To express a cloned gene, an active promoter must be located adjacent to the coding sequence of the cloned gene. In a typical expression vector, such a promoter can be found upstream from ()

a unique restriction (cloning) site

What is the key reason why A and B chains of insulin are made as a fusion proteins with beta-galactosidase

to prevent their degradation

a vaccine that contains a purified component from a pathogen, such as a protein or a polysaccharide, is a () vaccine

subunit

During mammalian reproductive cloning, () is fused with ()

a somatic cell, an enucleated egg cell

Which of the following stem cells are most practical and useful in treating a variety of human diseases?

iPS cells

You plan to produce human insulin growth factor (IGF) in cow milk by generating transgenic livestock. IGF is primarily produced in the liver and is needed for diabetic patients. AS a first step, you need to clone the gene coding this protein. For this purpose, which of the following materials would you use to obtain/amplify the coding sequences of this gene? Also, which technique would you use?

mRNA from liver cells, RT-PCR

Once you obatined the coding sequences of the insulin-like growth factor (IGF) gene, you placed it downstream from () in an expression vector, and the inject the DNA into hundreds of () to prodcue transgenic livestock.

beta- lactoglobulin promoter, cow oocytes

You screened hundreds of newborn calves for the presence/absence of the IGF transgene (). you then tested the expression of the transgene by () in idetified transgenic animals. However, you found that only 30% of them had detectable levels of transgene expression in milk. This is probably because ()

PCR, western blot, the transgene might have been integrated into heterochromatic regions.

Transgenic

an organism that has integrated recombinant DNA into its genome from a different species

Insulin

is a hormone composed of two polypeptide chains, called the A and B chains. Insulin regulates several physiological processes, particularly the upatke of glucose into fat and muscle cells.

it is produced by the beta cells of the pancreas. People with insulin-dependent diabetes have a defect in their beta cells. Sources of insulin used to include cows/humans cadvers. But not, patient most commonly use insulin made by recombinant bacteria.

Whole pathogen vaccines

consist of entire pathogens that have been completely inactivated of weakened

inactivated vaccines

contains a treated pathogen that cannot cause an infection ( that is influenza, hepatitis A and rabies). provide short term immunity.

Attenuated vaccines

created by reducing virulence of a pathogen but keeping it viable (MMR combined vaccine), chickenpox and yellow fever. Promote a long -lasting and strong immune response.

Subunit vaccines

contains only certain components (proteins/polusaccharides) that best stimuale the immune system (only a portion of a disease-casuing agent), often require the addition of adjuvants or substances that support immune response; antigens alone are not sufficient to induce adequate immunity; booster shots may be needed