BIO 325 Exam 3

Uses of expressing cloned genes in Research:

-Help explain its cellular function. -Aids in purification of large amounts of protein needed for biochemical or biophysical studies.

Uses of expressing cloned genes in Biotechnology:

-Introduced into bacteria to make pharmaceutical products such as insulin. -Introduced into plants & animals to make transgenic species with desirable traits.

PCR: 3 Steps

1. Denaturation 2. Annealing 3. Extension

Steps in Base Excision Repair

1. N-glycosylase recognizes an abnormal base and cleaves the bond between the base & sugar 2. If a purine is removed, an apurinic site is created. If a pyrimidine is removed, an apyrimidinic site is created. 3. The region is recognized by AP endonuclease, which makes a cut at the 5' side of the abnormal nucleotide. 4. DNA polymerase removes the abnormal region and fills it in with normal DNA 5. DNA ligase seals the repaired strand.

Steps in Nucleotide Excision Repair in E. coli:

1. UvrA/UvrB complex scans along DNA until encounter a thymine dimer 2. UvrA dimer is released 3. UvrC binds to UvrB 4. UvrC makes two cuts in the damaged DNA strand (one 8 nt 5' to the site, one 5 nt 3' from the site) 5. UvrD (a helicase) binds to the site and causes the damaged strand to be removed 6. UvrB and UvrC are then released 7. DNA Polymerase resynthesizes a complementary strand 8. DNA ligase makes the final connection

a small effector molecule that causes transcription to decrease can happen in what two ways?

1. corepressor (binds to a repressor & causes the protein to bind to DNA) 2. inhibitor (binds to activator protein & prevents it from binding to DNA)

Researches often measure the amount of ____ in a sample of DNA to determine the extent of oxidative stress

8-oxoG

Name the cause of this spontaneous mutation: Abnormal crossing over may cause deletions, duplications, translocations, and inversions

Aberrant recombination

Name the cause of this spontaneous mutation: Abnormal chromosomal segregation may cause aneuploidy or polyploidy

Aberrant segregation

A ___ protein can remove methyl or ethyl groups from guanine bases that have been mutagenized by alkylating agents.

Alkyltransferase

(BER/NER) is particularly important for the repair of oxidative DNA damage

BER

Name this DNA Repair System: An abnormal base or nucleotide is first recognized and removed from the DNA. A segment of DNA is excised, and then the complementary DNA strand is used as a template to synthesize normal DNA.

BER & NER (Base Excision Repair and Nucleotide Excision Repair)

the lacZ gene encodes the enzyme _____

Beta-galactosidase

Name 4 medical conditions that overaccumulation of ROS has been implicated.

Cardiovascular disease, Alzheimer disease, chronic fatigue syndrome, aging

(BER) Base excision repair involves _____ enzymes that recognize an abnormal base & cleave the bond beween it and the sugar backbone, creating an apurinic or apyrimidinic site.

DNA N-glycosylases

(Gel retardation assay/DNase I footprinting) can determine the regions of a DNA molecule that are bound by a protein

DNase I footprinting

Name the cause of this spontaneous mutation: Cytosine and 5-methylcytosine spontaneously deaminates to create uracil or thymine

Deamination

Name the cause of this spontaneous mutation: linkage between purines & deoxyribose spontaneously breaks. If not fixed, can lead to a mutation

Depurination

Name this DNA Repair System: An enzyme recognizes an incorrect alteration in DNA structure and directly converts it back to a correct structure

Direct repair

Name the cause of this spontaneous mutation: Mistake in DNA polymerase causing a point mutation

Errors in DNA replication

T/F ROS is always harmful

False (ROS are produced by the immune system as a means of killing pathogens and some ROS are used in cell signaling)

(Gel retardation assay/DNase I footprinting) can determine if a protein binds to a specific DNA fragment or RNA molecule because the binding of the protein slows down the movement of the DNA through a gel.

Gel retardation assay

Name the Technique of Gene Cloning that: Uses labeled DNA strandes from a cloned gene to identify similar or identical genes or RNA. Southern & Northern blot analysis methods are used. Used to localize genes within intact chromosomes and DNA fingerprinting.

Gene probes

Name the Technique of Gene Cloning that: Provides enough DNA to subject the gene to DNA sequencing . The sequence can reveal the gene's promoter, regulatory sequences, and coding sequence. Important in the identification of alleles that cause cancer and inherited human diseases.

Gene sequencing

Name this DNA Repair System: Occurs at double-strand breaks or when DNA damage causes a gap in synthesis during DNA replication. The strands of a normal chromatid are used to repair a damaged chromatid.

Homologous recombination repair

Name this DNA Repair System: Similar to an excision repair except the DNA defect is a base pair mismatch, not an abnormal nucleotide. The mismatch is recognized, and a segment of DNA is removed. The parental strand is used to synthesize a normal daughter strand of DNA.

Mismatch repair

The number of mutant genes divided by the total number of genes within a population

Mutation frequency

The likelihood that a gene will be altered by a new mutation. (expressed as the # of new mutations in a given gene per cell generation)

Mutation rate

In (NER/BER) several nucleotides in the damaged strand are removed from the DNA--and the intact strand is used as a template for re-synthesis of a normal compelementary strand.

NER

Name this DNA Repair System: Occurs at double-strand breaks. The broken ends are recognized by proteins that keep the ends together; the broken ends are eventually rejoined.

Nonhomologous end joining

(Southern/Northern/Western) blotting uses a labeled DNA probe to detect a specific RNA within a mixture of many different RNAs

Northern

____ refers to an imbalance between the production of ROS and an organism's ability to break them down

Oxidative stress

UV light is a Physical Mutagen that can cause what effect on DNA structure?

Promotes pyrimidine dimer formation, such as thymine dimers

______ ____, also called ___ _____, cut chromosomal DNA and vector DNA to produce sticky ends that will hydrogen bond with each other. _____ ____ is needed to make a covalent link in the DNA backbone.

Restriction enzymes; restriction endonucleases; DNA ligase

cDNA can be made from mRNA via _____ ____

Reverse Transcriptase

Name the Technique of Gene Cloning that: Can be manipulated to change its DNA sequence. Mutations within genes can help to identify gene sequences such as promoters & regulatory elements. This study can also help to explain its normal function and how its expression may affect the roles of other genes. Can reveal which amino acids are important for a protein's structure and function.

Site-directed mutagenesis

(Southern/Northern/Western) blotting uses a labeled DNA probe to detect the presence of a particular gene sequence within a mixture of many gene sequences. At low stringency, gene families may be detected.

Southern

Name the cause of this spontaneous mutation: Spontaneous shifts in base structure causing mutations if they occur immediately prior to DNA replication

Tautomeric shifts

Name the cause of this spontaneous mutation: The products of normal metabolic processes, such as reactive oxygen species (ROS) may be chemically reactive agents taht can alter teh structure of DNA

Toxic metabolic products

Name the cause of this spontaneous mutation: elements inserting themselves into the sequence of a gene

Transposable elements

Name the cause of this induced mutation: physical phenomena that can damage the DNA

UV light & X-rays

Uses of expressing cloned genes in Clinical Trials:

Used in clinical trials involving gene therapy

In Prokaryotes, the NER system requires four key proteins:

UvrA, UvrB, UvrC, and UvrD (& help of DNA polymerase & DNA ligase)

(Southern/Northern/Western) blotting uses antibodies to detect a specific protein within a mixture of many different proteins

Western

How do tautomers interconvert?

a chemical reaction involving the migration of a hydrogen atom and a switch of a single bond and an adjacent double bond.

the lac operon is regulated by a (repressor/activator)

activator

positive control occurs with (repressors/activators)

activators

The common, stable form of adenine and cytosine is the (amino/imino) form

amino

Deamination involves removal of an ____ from the ___ base, producing ___

amino group; cytosine; uracil

what does an effector molecule bind to?

an activator or repressor

Some TNRE disorders have the unusual fature of a progressively worsening severity in future generations, called _____ or ____ _____

anticipation; dynamic mutation

X-rays are Physical Mutagens that can cause what 4 effects on DNA structure?

base deletions, single-stranded nicks in DNA, cross-linking, chromosomal breaks

tautomers are ___ which exist in ___ and ___ or ___ and ___ forms.

bases; keto and enol or amino and imino

inducible operons typically encode (catabolic/anabolic) enzymes, whereas repressible operons often encode (catabolic/anabolic) enzymes

catabolic; anabolic

the lac operon can be regulated by (catabolite/anabolite) repression and the trp operon can be regulated by (catabolite/anabolite) repression

catabolite; anabolite

If ROS accumulate, they can damage _____ including ___, ___ and ___

cellular molecules including DNA, proteins and lipids

Name the cause of this induced mutation: substances that may cause changes in the structure of DNA

chemical agents

what controls the switch during the lytic cycle?

cro protein

Nitroous acid is a Chemical Mutagen that ______

deaminates bases

Most common type of chemical change that occurs naturally:

depurination

mutation of a regulatory element/operator site may ___ the ability of the gene to be properly regulated

disrupt

an inducer is a small effector molecule that causes transcription to _____

increase

mutation of a promoter sequence may ______ the rate of transcription

increase or decrease

3. Primer Extension: temp & function

incubate at slightly higher temp, allows DNA synthesis to occur

_____ mutations are caused by environmental agents

induced

lac operon and ara operon are (inducible/repressible) systems

inducible

trp operon is a (inducible/repressible) system

inducible

Allolactose binds to the repressor and prevents repressor from binding to operator, (inhibiting/inducing) transcription

inhibiting

lac repressor protein binds to lac operator and (inhibits/induces) transcription

inhibits

Reverse transcriptase PCR (is/is not) extraordinarily sensitive and (can/cannot) detect the expression of small amounts of RNA from a single cell.

is; can

The common, stable form of guanine and thymine is the (keto/enol) form

keto

the ara operon is similar to the (lac/trp) operon

lac

what controls the switch during the lysogenic cycle?

lambda repressor

2. Primer Annealing: temp & function

low temp allows primers to bind to template DNA

the expression of cII protein favors the (lytic/lysogenic) cycle. The expression of cro protein favors the (lytic/lysogenic cycle)

lysogenic; lytic

Reverse transcriptase PCR begins with ___ to study gene expression

mRNA

mutatino of the 5'-UTR/3'-UTR may alter the ability of the ___ to be translated and may alter ___ stability

mRNA; mRNA

In eukaryotes, ROS are _____ produced as (wanted/unwanted) by-products of energy production in mitochondria

naturally; unwanted

when tryptophan levels are high, tryptophan acts as a corepressor that binds to the trp repressor protein and binds to the operator site, turning (on/off) the operon.

off

(ROS) Reactive oxygen species (such as hydrogen peroxide, superoxide, and hydroxyl radical) are products of ____ metabolism in all ____ organisms.

oxygen; aerobic

Cloning vectors can be derived from ___ or ___

plasmids; viruses

feedback inhibition & covalent modification are forms of (transcriptiona/translational/posttranslational) regulation

posttranslational

mutation of the splice recognition sequence may alter the ability of ____ to be properly spliced

pre-mRNA

what do constitutive genes frequently code for?

proteins that are continuously needed for the survival of the bacterium

Depurination involves removal of a ___ from the DNA, creating an ___ site

purine; apurinic site

the genes that corepressors & inhibitors regulate are (inducible/repressible) genes

repressible

the trp operon is regulated by a (repressor/activator)

repressor

negative control occurs with (repressors/activators)

repressors

1. Denaturation: function & temp

separates DNA strands; high temp

_______ mutations are changes in DNA structure that result form abnormalities in biological processes

spontaneous

What enzymes do cells use to prevent buildup of ROS?

superoxide dimutase and catalase (antioxidants like Vitamin C)

intergenic suppressor

suppressor mutation in a different gene from the first mutation (find info about proteins that have similar or redundant functional roles)

deamination of 5-methylcytosine prouduces ____

thymine

Nucleotide Excision Repair (NER) can repair what 4 types of mutations?

thymine dimers, chemically modified bases, missing bases, certain types of crosslinks

What is the purpose of vector DNA

to act as a carrier of the DNA segment that is to be cloned

antisense RNA is form of (transcriptiona/translational/posttranslational) regulation

translational

constitutive genes are (unregulated/regulated) genes

unregulated

deamination of cytosine produces ____

uracil

A ___ is a small DNA molecule that can replicate independently of host cell chromosomal DNA and produce many identical copies of an inserted gene.

vector

intragenic supressor

when the second mutant site is within the same gene as the first (change in protein structure that compensates for an abnormality in protein structure caused by the first mutation)