Genetics Exam 4

negative control in an inducible operon will be synthesized initially as an

active repressor

insertion mutation

addition of one or more nucleotides

acetylation of histone proteins

alters chromatin structure and permits some transcription factors to bind to DNA

intragenic mutation

an existing gene can be modified by changes in its DNA sequence, through various types of error that occur mainly in the process of DNA replication

frameshift mutation

mutation that shifts the "reading" frame of the genetic message by inserting or deleting a nucleotide

slicer-independent mRNA decay

not fully understand, but requires RISC

amino acid operon

(trp/arg) when amino acid is abundant, the operon should be turned off

what is catabolite repression? How does it allow a bacterial cell to use glucose in preference to other sugars?

In catabolite repression, the presence of glucose inhibits or represses the transcription of genes involved in the metabolism of other sugars. Because the gene expression necessary for utilizing other sugars is turned off, only enzymes involved in the metabolism of glucose will be synthesized. Operons that exhibit catabolite repression are under the positive control of catabolic activator protein (CAP). For CAP to be active, it must form a complex with cAMP. Glucose affects the level of cAMP. The levels of glucose and cAMP are inversely proportional—as glucose levels increase, the level of cAMP decreases. Thus, CAP is not activated.

How does bacterial gene regulation differ from eukaryotic gene regulation?

In eukaryotic cells, gene-coding regions are interrupted by introns, which are generally longer than exons. An individual intron may be much longer than the entire coding region. Gene expression requires the proper splicing of the pre-mRNA to remove these noncoding regions. In prokaryotic cells, gene-coding regions are usually not interrupted. In eukaryotic cells, chromatin structure plays a role in gene regulation. Chromatin that is condensed inhibits transcription. Therefore, for expression to occur, the chromatin must be altered to allow for changes in structure. Acetylation of histone proteins and DNA methylation are important in these changes. At the level of transcription initiation, the process is more complex in eukaryotic cells. In eukaryotes, initiation requires a complex machine involving RNA polymerase, general transcription factors, and transcriptional activators. Bacterial RNA polymerase is either blocked or stimulated by the actions of regulatory proteins. Finally, in eukaryotes the action of activator proteins binding to enhancers may take place at a great distance from the promoter and structural gene. These distant enhancers occur much less frequently in bacterial cells

What would be the most likely effect of deleting flowering locus D (FLD) in Arabidopsis thaliana? Explain how this related to the function of FLC.

It is likely that flowering will not occur if the flowering locus D is deleted. The protein encoded by FLD is a deacetylase enzyme. This deacetylase enzyme normally removes acetyl groups from histones surrounding the flowering locus C (FLC). Once the acetyl groups are removed, the chromatin structure within this region is restored to be transcriptionally repressed. The restored chromatin inhibits transcription from the FLC locus. FLC codes for a transcriptional activator whose expression activates other genes that suppress flowering. If FLC transcription is active due to a deletion in FLD, then flowering will not occur

Briefly explain how an antibiotic-resistance gene and the lacZ gene can be used to determine which cells contain a particular plasmid

Many plasmids designed as cloning vectors carry a gene for antibiotic resistance and the lacZ gene. The lacZ gene on the plasmid has been engineered to contain multiple unique restriction sites. Foreign DNAs are inserted into one of the unique restriction sites in the lacZ gene of plasmids and the plasmids are transformed into E. coli cells lacking a functional lacZ gene. Transformed cells are plated on a medium containing the appropriate antibiotic to select for cells that carry the plasmid. The medium also contains an inducer for the lac operon, so the cells express the lacZ gene, and X-gal, a substrate for beta-galactosidase that will turn blue when cleaved by β-galactosidase. The colonies that carry plasmid without foreign DNA inserts will have intact lacZ genes, make functional β-galactosidase, cleave X-gal, and turn blue. Colonies that carry plasmid with foreign DNA inserts will not make functional β-galactosidase because the lacZ gene is disrupted by the foreign DNA insert. They will remain white. Thus, cells carrying plasmids with inserts will form white colonies. This is known as blue/white selection

How do riboswitches differ from RNA mediated repression?

RNA-mediated repression occurs through the action of a ribozyme. In RNA-mediated repression, an RNA sequence within the 5' untranslated region can act as a ribozyme that when stimulated by the presence of a regulatory molecule can induce self-cleavage of the mRNA molecule, which prevents translation of the molecule. When bound by a regulatory molecule, RNA mediated repression results in the self-cleavage of the mRNA molecule. When bound by a regulatory molecule, riboswitch sequences stimulate changes in the secondary structure of the mRNA molecule that affect gene expression.

cutting and recombining DNA fragments in vitro

Restriction Enzymes from bacteria are used to cut DNA at specific sequences. These cuts often result in "sticky" cohesive ends that aid in recombination

What normal role do restriction enzymes play in bacteria? How do bacteria protect their own DNA from the action of restriction enzymes?

Restriction enzymes cut foreign DNA, such as viral DNA, into fragments. Bacteria protect their own DNA by modifying bases, usually by methylation, at the recognition sites

briefly describe the lac operon and how it controls the metabolism of lactose

The lac operon consists of three structural genes involved in lactose metabolism, the lacZ gene, the lacY gene, and the lacA gene. Each of these three genes has a different role in the metabolism of lactose. The lacZ gene codes for the enzyme β-galactosidase, which breaks the disaccharide lactose into galactose and glucose, and converts lactose into allolactose. The lacY gene, located downstream of the lacZ gene, codes for lactose permease. Permease is necessary for the passage of lactose through the E. coli cell membrane. The lacA gene, located downstream of lacY, encodes the enzyme thiogalactoside transacetylase whose function in lactose metabolism has not yet been determined. All of these genes share a common overlapping promoter and operator region. Upstream from the lactose operon is the lacI gene that encodes the lac operon repressor. The repressor binds at the operator region and inhibits transcription of the lac operon by preventing RNA polymerase from successfully initiating transcription. When lactose is present in the cell, the enzyme β-galactosidase converts some of it into allolactose. Allolactose binds to the lac repressor, altering its shape and reducing the repressor's affinity for the operator. Since this allolactose-bound repressor does not occupy the operator, RNA polymerase can initiate transcription of the lac structural genes from the lac promoter

what controls RNA stability in eukaryotic cells?

The presence of the 5ʹ cap, 3' poly(A) tail, the 5' UTR, 3' UTR, and the coding region in the mRNA molecule are features that can affect the stability of the mRNA molecule. Poly(A) binding proteins (PABP) bind at the 3' poly(A) tail. These proteins contribute to the stability of the tail and protect the 5' cap through direct interaction. Once a critical number of adenine nucleotides have been removed from the tail, the protection is lost and the 5' cap is removed. The removal of the 5' cap allows for 5' to 3' nucleases to degrade the mRNA. AU-rich sequence elements in the 3ʹ UTR can also increase degradation of the mRNA

what role does RNA stability play in gene regulation?

The total amount of protein synthesized is dependent on how much mRNA is available for translation. The amount of mRNA present is dependent on the rates of mRNA synthesis and degradation. Less-stable mRNAs will be degraded faster so there will be fewer copies available to serve as templates for translation

why are they called restriction enzymes

They are part of bacterial Restriction/Modification Systems for inhibiting foreign DNA in the cell. These systems modify the host (self) DNA, typically by methylation of specific bases. Endonucleases are unable to bind to modified DNA, but recognize un-modified sequences and cleave DNA at those sites

how does an enhancer affect the transcription of distant genes?

Transcription at a distant gene is affected when the DNA sequence located between the gene's promoter and the enhancer is looped out, allowing for the interaction of the enhancer-bound proteins with proteins needed at the promoter, which in turn stimulates transcription. Additionally, the transcription of short enhancer (e)RNA molecules from an enhancer template may be involved in transcriptional activation, but a precise mechanism for such activation has not been determined

briefly explain how transcriptional activator and repressor proteins affect the level of transcription of eukaryotic genes.

Transcriptional activator proteins stimulate transcription by binding DNA at specific base sequences such as an enhancer or regulatory promoter and attracting or stabilizing the basal transcription factor apparatus. Repressor proteins bind to silencer sequences or promoter regulator sequences. These proteins may inhibit transcription by either blocking access to the enhancer sequence by the activator protein, preventing the activator from interacting with the basal transcription apparatus, or preventing the basal transcription factor from being assembled

What are the mechanisms by which the attenuator forms when tryptophan levels are high and the antiterminator forms when tryptophan levels are low?

Two types of secondary structures can be formed by the mRNA 5ʹ UTR of the trp operon. If the 5ʹ UTR forms two hairpin structures from the base pairing of region 1 with region 2 and the pairing of region 3 with region 4, then transcription of the structural genes will not occur. The hairpin structure formed by the pairing of region 3 with region 4 results in a terminator being formed that stops transcription. When region 2 pairs with region 3, the resulting hairpin acts as an antiterminator allowing for transcription to proceed. Region 1 of the 5' UTR also encodes a small protein and has two adjacent tryptophan codons (UGG). Tryptophan levels affect transcription due to the coupling of translation with transcription in bacterial cells. When tryptophan levels are high, the ribosome quickly moves through region 1 and into region 2, thus preventing region 2 from pairing with region 3. Therefore, region 3 is available to form the attenuator hairpin structure with region 4, stopping transcription. When tryptophan levels are low, the ribosome stalls or stutters at the adjacent tryptophan codons in region 1. Region 2 now becomes available to base pair with region 3, forming the antiterminator hairpin. Transcription can now proceed through the structural genes.

expression vectors

a DNA vector, such as a plasmid, that carries a DNA sequence for the expression of an inserted gene into mRNA and protein in a host cell

what is a response element?

a regulatory DNA sequence consisting of short specific sequences located at various distances from the genes that they regulate

operon

a single transcriptional unit that includes a series of structural genes, a promoter, and an operator

list some important differences between bacterial and eukaryotic cells that affect the way in which genes are regulated

a. Bacterial genes are frequently organized into operons with coordinate regulation, and genes with operons can be transcribed as on a single long mRNA. Eukaryotic genes are not organized into operons and are singly transcribed from their own promoters. b. In eukaryotic cells, nucleosome structure of the DNA is remodeled prior to transcription occurring. Essentially, the chromatin must assume a more open configuration state, allowing for access by transcription-associated factors. c. Activator and repressor molecules function in both eukaryotic and bacterial cells. However, in eukaryotic cells activators appear to be more common than in bacterial cells. d. In bacteria, transcription and translation can occur concurrently. In eukaryotes, the nuclear membrane separates transcription from translation both physically and temporally. This separation results in a greater diversity of regulatory mechanisms that can occur at different points during gene expression

base excision repair

abnormal bases, modified bases, and pyrimidine dimers

name six different levels at which gene expression might be controlled

alteration or modification of the gene structure at the DNA level, transcription regulation, regulation at the level of mRNA processing, regulation of mRNA stability, regulation of translation, regulation by post translational modification of the synthesized protein.

What is antisense?

antisense RNA molecules are small RNA molecules that are complementary to other DNA or RNA sequences and that form RNA-protein complexes.

Fragile X chromosome

associated with a characteristic constriction on the long arm

activator proteins

bind to enhancer sites which may be thousands of bases away from the start site. these usually activate transcription non-specifically and are position and orientation independent

repressor proteins

bind to silencers. unlike bacterial repressors, eukaryotic repressors usually compete with activators for binding, or prevent activator interaction with the basal transcription apparatus

transcriptional activator proteins

bind to sites on DNA and stimulate transcription

insulator binding protein

blocks the action of an enhancer on a promoter when the insulator lies between the enhancer and the promoter

regulatory promoter sequences

can be mixed and matched to affect transcription differences at different promoters

ribozymes

catalytic RNA, mRNA sequences can form secondary structures that can autodegrade

induced mutation

chemicals, radiation

the pink circle causing the yellow repressor to become active is called (look at picture)

co-repressor

neutral mutation

codes for new amino acid but function doesn't change

deletion mutation

deletion of one or more nucleotides

missense mutation

different codon/amino acid

draw a picture of an operon and identify its parts

draw it.

why is gene regulation important for bacterial cells?

gene regulation allows for biochemical and internal flexibility while maintaining energy efficiency by the bacterial cells

mutation

heritable change in genetic information

what is a repressible operon?

in a repressible operon, transcription normally occurs. transcription is turned off either by the repressor becoming active in a negative repressible operon or by the activator becoming inactive in a positive repressible operon

regulator protein is a repressor of an inducible operon

in an inducible operon, a mutation at the operator site that blocks binding of the repressor would result in constitutive expression and transcription would occur all the time

How does antisense control gene expression?

in bacterial cells, antisense RNA molecules can bind to a complementary region in the 5' UTR of a mRNA molecule, blocking the attachment of the ribosome to the mRNA and stopping translation or they pair with specific regions of the mRNA and cleave the mRNA stopping translation

negative control in a repressible operon will be synthesized initially as an

inactive repressor

intercalating agents

increase the likelihood of insertions and deletions in DNA during subsequent replication

Increases in trinucleotide copy number are often correlated to

increases in the severity of the disease or increases in the possibility of further expansion of copy number

the pink circle causing the purple repressor to become inactive is called (look at picture)

inducer

allolactase

induces expression of the lac operon

the lacZ gene is

interrupted and inactivated when DNA is clones into the restriction sites located within a gene

what are some of the limitations of PCR?

it is error prone (Taq poly fidelity; lack of proof reading), need to know sequence and provide primer

wobble base pairing

leads to a replicated error

strand slippage

leads to either insertions or deletions

unequal crossing over

leads to insertions and deletions

RNA silencing

leads to the degradation of mRNA, or the inhibition of translation or transcription

transposable elements

may cause mutations by inserting into genes to disrupt them or disrupt regulatory sites. may cause mutations by leading increased recombination, resulting in rearrangements

inhibition of translation

mediated by miRNA-associated RISC

base modification

methylation of DNA at CpG islands correlates with lack of transcription

how do base analogs lead to mutations?

mismatched at higher frequencies

methylases

modify the DNA at the recognition sequences, preventing the endonucleases from acting

RNA cleavage

mostly by siRNA-associated RISC

Give three important characteristics of cloning vectors.

must be small in size, must be self-replicating inside host cell, must possess a restriction site

incorporated errors

occur when a mismatched base is incorporated into a newly synthesized strand

replicated errors

occur when the incorporated error is replicated so the mutation exists on both strands

intergenic mutation

occurs in a gene other than the one bearing the original mutation

reactive forms of oxygen

often lead to oxidative damage to the DNA

expression vectors contain

operon sequences that allow inserted DNA to be transcribed and translated

three important properties of cloning vectors

origin of replication for maintenance in new host, marker (gene) to enable selection of bacteria that contain the vector, and restriction sites where genes of interest can be inserted

why do you think the motif of the DNA-binding proteins shown is called a zinc-finger motif?

part of the protein containing zinc extends into the major groove of DNA like a finger

what is the difference between positive and negative control?

positive transcriptional control requires an activator protein to stimulate transcription at the operon. in negative control, a repressor protein inhibits or turns off transcription at the operon

direct repair

pyrimidine dimers; other specific types of alterations - changes nucleotides back into their original structures

restriction endonucleases

recognize specific sequences of DNA and break phosphodiester bonds between adjacent nucleotides

Recombinant DNA technology

recombining DNA from one place to another; finding the target gene in the source genome, separation of the target from the rest of the genome, stabilization of the target for maintenance in the new host, identification and isolation of host cells that have received the target DNA

expression vectors enable

regulated transcription of cloned genes

What are riboswitches?

regulatory sequences in RNA molecules

mismatch repair

replication errors, including mispaired bases and strand slippage

spontaneous mutation

replication errors, may be due to tautomeric base forms, may be due to flexibility of the DNA structure, spontaneous chemical changes

base analogs

resemble normal bases but are mismatched at higher frequency, may become incorporated and then replicated errors

pyrimidine dimers

result from ultraviolet light

the lacZ gene can be used to

screen bacteria containing recombinant plasmids

briefly explain how the polymerase chain reaction is used to amplify a specific DNA sequence.

separate DNA strand (denaturation) -> annealing (primers attach) -> extension (DNA synthesis) and repeat many times over to get tons of product that hopefully is the same fragment. Reading the fragments from shorter to longer, an automated DNA sequencer can determine the sequence of the template DNA molecule

expression vectors include

sequences that either turn on or turn off the desired gene

Riboswitches

sequences within the mRNA that can fold to prevent ribosome binding

transcriptional silencing

siRNAs bind with RNA-induced transcriptional silencing proteins and can bind to DNA and direct chromatin structure changes

supressor mutation

suppresses the effect of an earlier mutation at a different site

What conclusion would you make if the number of bacterial colonies in Figure 18.22 were the same on the control plate and the treatment plate? Explain your reasoning (look at picture)

the chemical tested is not mutagenic and likely not carcinogenic. The number of colonies on each plate represents the number of bacterial cells that underwent a mutation. Because the number of cells undergoing mutation on the control plate, without the tested chemical, was the same as the number undergoing mutation on the plate treated with the chemical, there is no evidence that the chemical elevates the mutation rate or is potentially carcinogenic

what is anticipation?

the increase in the severity of a disease over subsequent generations. in diseases influenced by expansion of polynucleotide repeats, the number of repeats increase in subsequent generations, leading to increases in the severity of the diseases in those progenies

Depurination

the loss of a purine base from a nucleotide, produces an apurinic site

in the lac operon

the operator overlaps the promoter and the 5' end of the first structural gene

Detection of Sequences via Hybridization

the probe is an oligonucleotide (typically synthetic), the label may be radioactive, fluorescent, or enzymatic, hybrid molecules indicate sequence similarity between probe and the nucleic acid to which it is bound

a mutation at the operator prevents

the regulator protein from binding

regulator protein is a repressor of a repressible operon

the regulator protein-corepressor complex would normally bind to the operator and inhibit transcription. if a mutation prevented the repressor protein from binding at the operator, then the operon would never be turned off and transcription would occur all the time

what is attenuation?

the termination of transcription prior to the structural genes of an operon. it is the result of the formation of a termination hairpin structure or attenuator in the mRNA

how are his-bacteria used in the ames test?

they are plated so only the mutants that revert the change to be his+ can grow (no his on plate)

southern blotting

this procedure enables identification of DNA fragments in the original digest that have specific sequences of interest, based on their complementation to the labeled probe

chromatin structure regulation

tightly condensed DNA in chromatin is less available to the transcriptional apparatus. the availability can be affected in various ways, histone modification, chromatin remodeling, DNA modification

lacl (the lactose repressor) binds to lacO (the lactose operator)

to prevent transcription when lactose levels are low

what is the purpose of the ames test?

to test mutagenesis or carcinogenicity of compounds

formation of hairpins facilitates

trinucleotide repeat expansion

pUC19 plasmid

typical cloning vector

ionizing radiation

x-rays and gamma rays damage DNA by dislodging electrons from atoms; these electrons then break phosphodiester bonds and alter the structure of bases

Briefly list some of the ways in which siRNAs and miRNAs regulate genes.

(1) Through cleavage of mRNA sequences through "slicer activity": The binding of RISCs containing either siRNA or miRNA to complementary sequences in mRNA molecules stimulates cleavage of the mRNA through "slicer activity." This is followed by further degradation of the cleaved mRNA. (2) Through binding of complementary regions with the mRNA molecule by miRNAs to prevent translation: The miRNAs as part of RISC bind to complementary mRNA sequences preventing either translation initiation or elongation, which results in premature termination. (3) Through transcriptional silencing due to methylation of either histone proteins or DNA sequences: The siRNA binds to complementary DNA sequences within the nucleus and stimulates methylation of histone proteins. Methylated histones bind DNA more tightly preventing transcription factors from binding the DNA. The miRNA molecules bind to complementary DNA sequences and stimulate DNA methylases to directly methylate the DNA sequences, which results in transcriptional silencing. (4) Through slicer-independent mRNA degradation stimulated by miRNA binding to complementary regions in the 3' UTR of the mRNA: A miRNA binds to the AU rich element in the 3' UTR of the mRNA stimulating degradation using RISC and dicer

sugar operon

(lac) when the sugar is present, you want to digest it. therefore the operon should be turned ON when sugar is present to transcribe code for the enzyme to digest.

How is CRISPR-Cas used to introduce specific changed into DNA sequences?

A single guide RNA (sgRNA) is engineered that combines a tracrRNA with a crRNA and contains approximately 20 nucleotides complementary to the target DNA sequence. The sgRNA binds to a Cas protein (such as Cas9)

how do riboswitches control gene expression?

At riboswitches, regulatory molecules bind and influence gene expression by affecting the formation of secondary structures within the mRNA molecule. The binding of the regulatory molecule to a riboswitch sequence may result in repression or induction. Some regulatory molecules bind the riboswitch sequence and stabilize a terminator structure in the mRNA, which results in premature termination of the mRNA molecule. Other regulatory molecules bind riboswitch sequences resulting in the formation of secondary structures that block the ribosome binding sites of the mRNA molecules, thus preventing translation initiation. In induction, the regulatory molecule acts as an inducer, stimulating the formation of a secondary structure in the mRNA that allows for transcription or translation to occur.

How are bacterial gene regulation and eukaryotic gene regulation similar?

Bacterial and eukaryotic gene regulation involves the action of protein repressors and protein activators. Cascades of gene regulation in which the activation of one set of genes affects another set of genes take place in both eukaryotes and bacteria. Regulation of gene expression at the transcriptional level is also common in both types of cells. Both have coordinated expression, although, through different mechanisms. Bacterial genes are often clustered in operons and are coordinately expressed through the synthesis of a single polygenic mRNA. Eukaryotic genes are typically separate, with each containing its own promoter and transcribed on individual mRNAs. Coordinate expression of multiple genes is accomplished through the presence of common [shared] response elements. Genes sharing the same response element will be regulated by the same regulatory factors

list the four different types of DNA repair and briefly explain how each is carried out

Base excision - remove base, create AP site, remove sugar and phosphate, DNA poly/ligase Direct - revert changed nucleotides back to normal structure; photolyase

transition/transversion mutation

Base substitution in which a purine replaces a purine or a pyrimidine replaces a pyrimidine

how many restriction sites are found within a genome?

By knowing the GC ratio of the target DNA, the chance of a specific base at a particular site can be determined. Then number of bases in the recognition site factors into the determination of how many sites may be found. Longer recognition sequences will occur less frequently. The size of the genome will also factor into the estimation of the number of a restriction sites present. Bigger genomes will likely have more sites

a mutation prevents the catabolite activator protein (CAP) from binding to the promoter in the lac operon. what will the effect of this mutation be on the transcription of the operon?

Catabolite activator protein binds the CAP site of the lac operon and stimulates RNA polymerase to bind the lac promoter, thus resulting in increased levels of transcription from the lac operon. If a mutation prevents CAP from binding to the site, then RNA polymerase will bind the lac promoter poorly. This will result in significantly lower levels of transcription of the lac structural genes

what changes take place in chromatin structure, and what role do these changes play in eukaryotic gene regulation?

Changes in chromatin structure can result in repression or stimulation of gene expression. As genes become more transcriptionally active, DNA shows increased sensitivity to DNase I digestion, suggesting that the chromatin structure is more open. Acetylation of histone proteins by acteyltransferase proteins results in the destabilization of the nucleosome structure and increases transcription as well as hypersensitivity to DNase I. The reverse reaction by deacetylases stabilizes nucleosome structure and lessens DNase I sensitivity. Other transcription factors and regulatory proteins, called chromatin remodeling complexes, bind directly to the DNA-altering chromatin structure without acetylating histone proteins. The chromatin remodeling complexes allow for transcription to be initiated by increasing accessibility to the promoters by transcription factors. DNA methylation is also associated with decreased transcription. Methylated DNA sequences stimulate histone deacetylases to remove acetyl groups from the histone proteins, thus stabilizing the nucleosome and repressing transcription. Demethylation of DNA sequences is often followed by increased transcription, which may be related to the deacetylation of the histone proteins

forward mutation

Changes the wild-type phenotype to a mutant phenotype

nucleotide excision repair

DNA damage that distorts the double helix, including abnormal bases, modified bases, and pyrimidine dimers

After DNA fragments have been separated by gel electrophoresis, how can they be visualized?

DNA molecules can be visualized by staining with a fluorescent dye, such as ethidium bromide, that intercalates between the stacked bases of the DNA double helix, and the dye-DNA complex fluoresces when irradiated with an ultraviolet light source.

what is an enhancer?

DNA sequences that are binding sites of transcriptional activator proteins

Why should the bacteria not be exposed to light?

Exposure of DNA to UV light results in the formation of pyrimidine dimers in the DNA molecule. Often the repair of these dimers leads to mutations. Because the SOS repair system is error-prone and leads to an increased accumulation of mutations, UV light produces more mutations in bacteria when the SOS repair system is activated to repair the damage caused by the UV light. However, many species of bacteria have a direct DNA repair system that can repair pyrimidine dimers by breaking the covalent linkages between the pyrimidines that form the dimer. The enzyme that repairs the DNA is called photolyase and is activated and energized by light. The photolyase is a very efficient repair enzyme and typically makes accurate repairs of the damage. If the bacteria in the UV radiation experiment are exposed to light, then the photolyase will be activated to repair the damage, resulting in fewer mutations in the irradiated bacteria

factors that affect mutation rate calculation

Frequency of change of the DNA, Rate of repair, Probability of detection

what is a germ line cell and what are its consequences of mutations

Germ line mutations- arise in cells that ultimately produce gametes. A germ line mutation can be passed to future generations producing offspring that carry the mutation in there somatic and germ line cells

how do repressors that bind to silencers in eukaryotes differ from repressors that bind to operators in bacteria?

In bacteria, repressors that bind to the operator block RNA polymerase from binding to the promoter, and thus, directly block transcription. On the other hand, repressors that bind to silencers in eukaryotes block transcriptional activator proteins from binding at an activator site, thus eliminating transcriptional activation.

compare and contrast the use of PCR and gene cloning for amplifying DNA fragments

PCR: makes a ton of whatever DNA (from even just one double strand initially) you want but need to know sequence and add a primer, contamination means all product is messed up, Taq poly lacks proof reading. Gene cloning: need to mess with vectors, you're giving bacteria ampicillin resistance which theoretically could be a problem (if using ampR as a selective marker), but at least bacteria are pretty easy to make a farm out of and don't take up much space.

antisense RNA

RNA that can form a complement to the mRNA that is inhibitory to translation

outline the role of alternative splicing in the control of sex differentiation in drosophila

Sex development in fruit flies depends on alternative splicing as well as a cascade of genetic regulation. Early in the development of female fruit flies, a female-specific promoter is activated stimulating transcription of the sex-lethal (Sxl) gene. Splicing of the pre-mRNA of the transformer (tra) gene is regulated by the Sxl protein. The mature mRNA produces the Tra protein. In conjunction with another protein, the Tra protein stimulates splicing of the pre-mRNA from the doublesex (dsx) gene. The resulting Dsx protein is required for the embryo to develop female characteristics. Male fruit flies do not produce the Sxl protein, which results in the tra pre-mRNA in male fruit flies being spliced at an alternate location. The alternate Tra protein is not functional, resulting in the dsx pre-mRNA splicing at a different location as well. Protein synthesis from this mRNA produces a male-specific doublesex protein, which causes development of male-specific traits

Explain how gel electrophoresis is used to separate DNA fragments of different lengths

Smaller fragments move faster, and therefore further, than larger fragments as they snake through the gel

what is a somatic cell and what are its consequences of mutations

Somatic arise in somatic tissues which do not produce gametes. When a somatic cell with a mutation divides by mitosis, the mutation is passed on to the daughter cells leading to a population of genetically identical cells

Through evolutionary time, the proportion of AT base pairs in the DNA of these organisms increases. Can you suggest a possible mechanism for this increase?

Spontaneous deamination of 5-methylcytosine produces thymine. If the subsequent repair of the GT mispairing is repaired incorrectly or, more likely, not repaired at all because the thymine is a normal base, then a GC to AT transition will result. Over time, the incorrect repairs will lead to an increase in the number of AT base pairs

How do insertions and deletions arise?

Strand slippage - formation of small loops: new -> insertion on new; template -> deletion on newUnequal crossing over - deletion in one, insertion on other

Southern Blotting Technique

The procedure used to isolate and identify DNA fragments from a complex mixture. The isolated denatured fragments are transferred from an agarose gel to a nylon filter and identified by hybridization with probes

restriction digestion

The process of cutting DNA molecules into smaller pieces using restriction enzymes

how do response elements bring about the coordinated expression of eukaryotic genes?

Under conditions of stress, a transcription activator protein binds to the response element and stimulates transcription. If the same response element sequence is located in the control regions of different genes, then these genes will be activated by the same stimuli, thus producing a coordinated response

what is an inducible operon?

an inducible operon normally is not transcribed. it requires an inducer molecule to stimulate transcription either by inactivating a repressor protein in a negative inducible operon or by stimulating the activator protein in a positive inducible operon

What is an insulator?

an insulator or boundary element is a sequence of DNA that inhibits the action of regulatory elements called enhancers in a position dependent manner

reverse mutation

changes a mutant phenotype back to the wild-type phenotype

silent mutation

changes but same result/amino acid

base substitution

changes the base of a single DNA nucleotide

nonsense mutation

changes to stop codon