Genetics Exam 2

Auxotrophs vs. Prototrophs

(1) Auxotrophs: lack one or more enzymes necessary for synthesizing essential molecules (2) Prototrophs: wild-type bacteria that uses simple ingredients to synthesize all the compounds that they need for growth and reproduction

How would you genetically engineer plants with insecticide? Bacillus thuringiensis (Bt Cry1 genes)

(1) Cry toxin gene isolated from Bt (2) Cloned into E. Coli (3) Multiple gene constructs fused to neo+ which confers kan(r)/neo(r) (4) insert gene constructs into an expression vector with poly(A) consensus sequence (5) Transform Agrobacterium with expression vector (6) Recombination transfers gene constructs with promoter and poly(A) to Ti plasmid

What are the three steps of Polymerase Chain Reaction?

(1) DNA is heated to 90-100 degrees celsius, H-bonds are BROKEN between strands (2) DNA solution is cooled between 30-65 celsius, PRIMERS attach to template strands (3) Solution is heated for a minute or less to 72 celsius, DNA polymerases synthesizes the new strands creating 2 new double stranded DNA molecules

Define forward genetics and reverse genetics.

(1) FORWARD genetics: approach that begins with a phenotype (mutant individual) and proceeds to a gene that encodes to the phenotype (2) REVERSE genetics: begins with a genotype (DNA) and proceeds to the phenotype by altering the sequence or inhibiting its expression

The DNA library is a collection of clones containing all the DNA fragments from one source. What are two types?

(1) Genomic DNA library: consists solely of chromosomal DNA (2) cDNA libraries: consist on of those DNA sequences transcribed into mRNA isolate poly(A) mRNA fraction >> reverse transcribe mRNAs >> RNAse RNA:DNA hybrid >> ssDNA acts as DNA pol template

Define what an(1) inversion is? what is a (2) Paracentric inversion? (3) Pericentric inversion?

(1) INVERSION: chromosome segment is turned 180 degrees (2) PARACENTRIC: do not include the centromere (3) PERICENTRIC: include the centromere

How diseases such as Crown gall tumor infect plants? Pathogen: Agrobacterium tumefaciens

(1) In natural HGT Agrobacterium invades plant at wound site (2) part of Ti transferred to plant cell (3) Integrates into plant chromosome in nucleus - Flanking sequences TL and TR required to transfer DNA from bacteria to plant cell

Assume that a mutation occurs in the gene that codes for each of the following RNA polymerases. Match the mutation w/ possible effects by placing letter in the blank (more than one effect for each mutated polymerase.) A Mutation in gene that codes for: (1) RNA Polymerase I (2) RNA Polymerase II (3) RNA Polymerase III Possible Effects: (a) tRNA not synthesized (b) some ribosomal RNA not synthesized (c) ribosomal RNA not processed (d) pre-mRNA not processed (e) some mRNA molecules not degraded (f) pre-mRNA not synthesized

(1) RNA Polymerase I: (b)some ribosomal RNA not synthesized, (c) ribosomal RNA not processed (2) RNA Polymerase II: (d) pre-mRNA not processed, (e) some mRNA molecules not degraded, (f) pre-mRNA not synthesized (3) RNA Polymerase III: (a) tRNA not synthesized, (b) some ribosomal RNA not synthesized, (c) ribosomal RNA not processed

Define the following spontaneous mutations (occur under normal conditions): (1) Tautomeric shift (2) Mispairing due to other structures

(1) TAUTOMERIC: positions of protons in the DNA bases change (2) MISPAIRING: can arise due to the wobble effect

How do you create a Genomic DNA library?

(1) isolate total genomic DNA (2) partial restriction digest to create overlapping fragments (3) LIGATE into cloning vector (4) Transform E.coli or yeast w/ vectors (plasmids) that have a selectable marker, and usually "screenable" phenotype (5) plate out cells (6) some clones contain entire gene of interest, others part, most none.

Define these terms: (1) Duplications (2) Tandem Duplication

(1) part of the chromosome has been doubled (2) duplicated region is immediately adjacent to the original segment

(1) Alternative Processing (2) Multiple 3' cleavage sites

(1) pre-mRNA can be spliced in more that one way to yield multiple mRNAs that are translated into different AA sequences and thus different proteins (2) Two or more potential sites for cleavage and polyadenylation are present in the pre-mRNA

Process of Splicing (1) What is cut first? (2) What does this free? (3) What combines together? (4) What structure does this make? (5) What is this structure called? (6) Where is a cut made next?

(1) pre-mRNA is cut at the 5' splice site (2) frees EXON1 from the intron (3) 5' end of intron attaches to branch point (4) Intron FOLDS back on itself (5) LARIAT (6) Cut is made at the 3' SPLICE SITE and the 3' end of EXON1 becomes covalently attached to the 5' end of EXON2

Where are the different places that an organism can regulate the activity of a gene?

(1)By changing DNA in the genome (including rearrangements, somatic hypermutation) (2)Transcriptional regulation - Initiation, Cell-specificity, Splicing, mRNA Stability (3)Translation - Location or sequestration, Degradation (micro RNAs), Post-translational

Define these terms: (3) Displaced Duplication (4) Reverse Duplication

(3) DISPLACED: duplicated segment is located some distance from the original segment, either on the same chromosome or a different one (4)REVERSE: the duplication is inverted

Define the following spontaneous mutations (occur under normal conditions): (3) Incorporation errors (4) Replicated errors

(3) INCORPORATION: a mismatched base has been incorporated into a newly synthesized nucleotide chain (4) REPLICATED: incorporated error leads to this, which creates a permanent mutation because all the base pairings are correct and there is no mechanism for repair systems to detect the error

Genome Assembly. You are performing de novo genome assembly of an organism with 14 linear chromosomes. (A) what is the minimum number of contigs you can expect? (B) Please explain your rationale.

(A) 14 (B) In a perfect world, the number of contigs we see would perfectly match to the number of chromosomes we have. If the entire genome is represented in the reads and they are assembled correctly, we will see a 1:1 correlation between contigs and chromosomes.

Addition of Poly (A) Tail (A) What is added? (B) What are three functions?

(A) 50-250 adenine nucleotides are added to the 3' end (B) increases STABILITY of mRNA, facilitates attachment of RIBOSOMES to mRNA, aids in EXPORTING RNA to cytoplasm

Addition of 5' Cap (A) What is added? (B) What are three functions?

(A) 7' Methylguanine (B) Helps in TRANSLATION initiation, increases STABILITY of mRNA, influences splicing of INTRONS

Define the following base substitutions: (A) Transversion (B) Expanding nucleotide repeat

(A) A PURINE is replaced by a PYRIMIDINE and vice versa (B) Mutations in which the number of copies of a set of nucleotides increases **molecular change

Mobile Genetic Elements. You study a strain of E. coli carrying an R-plasmid (pKan) with kanamycin resistance. Upon recovering plasmid DNA from a culture of cells, tetracycline resistance was found associated with pKan. Analysis shows that this plasmid was now several kilobases larger in size than wild-type pKan. (A) How do you explain the origin of this plasmid derivative? (B) Imagine that tet-r became associated with a plasmid that had become 1 kb smaller than wild-type pKan. What would explain this finding?

(A) A transposon carrying tetracycline resistance integrated into pKan (B) Loss of an insertion sequence from pKan, which had been blocking tet-r expression, Also, pKanTet could have originated from a small pTet plasmid into which a Tn-bearing KanR sequence had recombined.

You decide to use microarrays to obtain an expression profile of your favorite organism (which must be yeast!) (A) In such a microarray experiment, exactly what is on the "chip?" (B) Of what consists the population of molecules being analyzed?

(A) Probe oligonucleotides that are homologous with, and therefore complementary to, some segment of a gene or intergenic DNA sequence, be it the organism under study (or one unrelated = negative control). (B) The population consists of cDNAs that have been reverse transcribed from a pool of poly(A) RNA transcripts. These transcripts are "tagged" typically with a photofluor that has characteristic excitation/emission wavelengths.

Chromosome Rearrangement. A chromosome initially has the following segments: A B •C D E F G Draw the chromosome, identifying its segments that would result from each of the following mutations: (A) Tandem duplication of DEF (B) Displaced duplication of DEF (C) Deletion of FG

(A) Tandem duplication of DEF A B • C D E F D E F G (B) Displaced duplication of DEF A B • C D E F G D E F (or other arrangements where the duplicated DEF is not adjacent to the original DEF) (C) Deletion of FG A B • C D E

Chromosome Rearrangement. A chromosome initially has the following segments: A B •C D E F G Draw the chromosome, identifying its segments that would result from each of the following mutations: (D) Paracentric inversion that includes DEFG (E) Pericentric inversion of BCDE

(D) Paracentric inversion that includes DEFG (inversion in which the breakpoints are confined to one arm of a chromosome; the inverted segment does not span the centromere) A B • C G F E D (E) Pericentric inversion of BCDE (An inversion in which the breakpoints occur on both arms of a chromosome. The inverted segment spans the centromere.) A E D C • B F G

NextGen Sequencing. Draw and annotate three rounds of Illumina bridge amplification, beginning with a single fragment of DNA hybridized to a slide. After three rounds, how many copies of the original DNA fragment do you have?

***DRAW 3 rounds now Number of amplicons = 2n where n=number of rounds of amplification 3 rounds = 23 = 8 fragments

What is Taq polymerase?

- A DNA polymerase (derived from YNP) - Enzyme used in step 1 of PCR because it is stable at high temperatures and is not denatured in strand separation

What is under the hood of an E. Coli expression vector?

- Expression vectors contain operon sequences that allow inserted DNA to be transcribed and translated - they also include sequences that regulate-turn on or off- the desired gene

Gel electrophoresis separates DNA by size & charge, what are some of the experimental conditions you can vary?

- Gel composition - strength of current - run time - orientation of current switch time

Phylogenomics. Explain the difference between a supermatrix and a supertree. Given the data to resolve a phylogeny are different DNA sequences from several genes from N number of species.

- SUPERMATRIX results from the concatenation of these genes, tree building software can then be used on this data set and a best supported tree for the data is produced. - SUPERTREE involves building a phylogenetic tree for each gene in our data set; this will result in the same number of trees as we have genes. Some of these topologies may be the same, or they may all be different. We can then put together a most probable species tree based on the support of different nodes within different gene trees.

What is a SPLICEOSOME? What does it contain?

- Structure where splicing occurs - consists of five RNA molecules and ~300 proteins; contains snRNAs

In RNA interference what are some of the triggers inducing this processing?

- Transcription of INVERTED REPEATS into and RNA molecule that then base pair with itself to form double stranded RNA - SIMULTANEOUS transcription of two different RNA molecules that are complementary to one another and that pair forming double stranded RNA - infection by VIRUSES that make double stranded RNA

What are some of the effects of Chromosome duplications?

- alignment at Prophase I results in one of the chromosomes having to loop out in order for the two homologs to be the same length - unbalanced gene dosage

What are small interfering RNAs (siRNAs)?

- bind at the 3' UTR but also other places - CUT mRNA at the region they bind, causing mRNA decay; lack of transcription - complete complementarity

What are micro RNAs (miRNAs)?

- bind to the 3' UTR - blocks TRANSLATION directly - limited complementarity - Silence genes that are distinct from those which the miRNAs were transcribed

What are some possible effects of Translocations?

- can physically link genes that were formerly located on different chromosomes - chromosomal breaks that cause translocations may take place within a gene and disrupt its function - heterozygous individuals with a reciprocal translocation would possess one normal copy of each chromosome and one translocated copy

What are some of the effect of Inversions?

- could break the gene in two parts - position effect: most genes regulated in a position-dependent manner, if altered their expression may be altered - homozygous individuals will have no problems arise - heterozygous individuals, the gene order of two homologous differs, and the homologous sequences can align and pair only if the two chromosomes form an inversion loop

What is a Suppressor Mutation and how does it work in the body?

- genetic change that hides or suppresses the effect of another mutation - occurs at a site that is distinct from the site of the original mutation

Alu is a Transposable element common in humans. Alu sequences create short flanking direct repeats when they insert into DNA.

- have characteristics that suggest they have transposed through an RNA intermediate - Alu belongs to class of Short interspersed elements (SINEs) - most are copies of transposable elements, shortened at the 5' end

A deletion is defined as the loss of a chromosome segment. What are some effects of deletions?

- phenotypic effects depend on which genes are located in the duplicated region - many are lethal in the homozygous state - PSEUDODOMINANCE: expression of a normally recessive mutation - HAPLOINSUFFICIENT gene: some genes must be present in two copies for normal function this occurs when a single copy of gene is insufficient to produce a wild-type phenotype

What are some limitations of the Polymerase Chain Reaction?

-prior knowledge of target DNA - contamination - accuracy - amplified fragments usually less than <2 kb - but long-range PCR ~ 20 kb in complex templates

What mutation alters the amino acid sequence of the protein but does not significantly change its function?

A NEUTRAL mutation (type of missense mutation) - AA is replaced by one that is chemically similar, affected AA has little influence

What is a Missense mutation?

A base substitution that results in a different amino acid in the protein **effect on translation

(A) What are composite transposons? (B) What features cause them to be mobile DNA elements capable of capturing sets genes that encode such functions as antibiotic and heavy metal resistance?

A composite transposon contains two IS elements, each of which flanks gene(s) such as those encoding antibiotic resistance. Each of the two IS elements is composed of inverted repeats (IR) flanking a transposase gene that favors recombination with sequences homologous to the inverted repeat

Mutations can, of course, be detected by directly sequencing an organism's DNA. Briefly explain the roles of "sequencing primers" and "fluorescently-labeled dideoxynucleotide" the most commonly used direct sequencing method.

A sequencing primer complementary base pairs with a sequence adjacent to the region one wishes to sequence and initiates replication by DNA polymerase. - ATP, buffer, divalent cation and pool of nucleotides are provided for the sequencing reaction. - The POOL includes both deoxyribose nucleotides (dA, dT, dC, dG) and fluorescently-labeled dideoxynucleotides (ddA, ddT, ddC, ddG). - The LATTER CAUSE CHAIN TERMINATION of newly-synthesized DNAs, and fluorescently label the terminal position. - SIZE-SORTING of these fragments by electrophoresis, then reading which photofluor has labeled fragments N, N+1, N+2, N+3 . . . N+n enables one to deduce the DNA sequence downstream of the sequencing primer.

Which of the following modifications occurs during transcriptional silencing: A. histone methylation occurs B. histone acetylation occurs C. nucleosome repositioning takes place D. RNA polymerase II is replaced by RNA polymerase III E. global DNA demethylation occurs

A. HISTONE METHYLATION OCCURS

The term "chromatin remodeling" refers to A. alteration of chromatin structure in association with transcription. B. a process that only bacteria perform since they contain no nucleus. C. a process that is exclusively associated with transcription by reverse transcriptase in eukaryotes. D. alteration in chromatin structure

A. alteration of chromatin structure in association with transcription.

What is the purpose of Chromatin Remodeling Complexes?

Alter CHROMATIN structure without altering the CHEMICAL structure of the histones directly - they bind directly to sites on DNA and reposition on the nucleosomes, allowing transcription factors and RNA polymerase to bind to promoters

What is the function of the enzyme DICER? How is it connected to siRNA and miRNA?

An enzyme that CLEAVES and processes double-stranded RNA to produce single stranded siRNAs or miRNAs that pair with proteins to form the RNA-induced silencing complex (RISC)

A mutant microorganism unable to synthesize an essential compound but able to grow if that compound is supplied exogenously is called a(n)_______________________. A. Phototroph B. Auxotroph C. Heterotroph D. Prototroph E. Heliotroph

B. AUXOTROPH

The process in which recipient bacterial cells can acquire genes from free DNA molecules in the surrounding medium is called A. Specialized transduction B. Transformation C. Generalized transduction D. Conjugation E. Recombination

B. TRANSFORMATION

Most transcriptional activator proteins affect transcription by interacting with A. introns B. the basal transcription apparatus C. DNA polymerase D. nucleosomes

B. the basal transcription apparatus

What are base substitutions? (A) define transition

BASE substitutions: alteration of a single nucleotide in the DNA (A) a PURINE is replaced by a different purine, or PYRIMIDINE w/ another pyrimidine

Transformation

Bacterial cells take up DNA from the external environment - occurs when the plasmid is introduced into bacterial cells - some cells undergo transformation naturally and others need to be treated chemically or physically

What modification neutralizes the charges on histones and loosens up the interactions between histones and DNA? A. phosphorylation B. methylation C. acetylation D. polyadenylation

C. ACETYLATION

Two types of post-transcriptional modifications that take place in mRNA of eukaryotes are A. addition of a poly T sequence at the 5' end of the gene and addition of a poly U tail at the 3' end. B. addition of a poly A sequence at the 5' end and addition of a "cap" at the 3' end of the RNA transcript. C. addition of a cap at the 5' end of the transcript and addition of a poly A sequence at the 3' end of the message. D. excision of the introns and addition of a cap to the 3' end.

C. ADDITION of a CAP at the 5' end of the transcript and ADDITION of a POLY A SEQUENCE at the 3' end of the message.

Pulsed Field Gel electrophoresis would be best suited for separating A. PCR products B. Plasmid clones C. Intact yeast or human chromosomes D. Genomic DNA cut with eight-base restriction endonucleases E. Genomic DNA cut with four-base restriction endonucleases F. Ribosomes

C. INTACT YEAST OR HUMAN CHROMOSOMES

Base substitutions in coding regions that result in changed amino acids are called A. Conditional mutations B. Transversions C. Missense mutations D. Nonsense mutations E. Silent mutations

C. Missense mutations

What is the 3' Consensus sequence?

CAG

Describe the process of Histone methylation?

Can cause ACTIVATION or REPRESSION of transcription - Histone methyltransferases add methyl groups to specific AA's of histone proteins - Histone DEMETHYLASES remove methyl groups

What is a Nonsense mutation?

Changes a SENSE codon (one that specifies an amino acid) into a NONSENSE codon (one that terminates translation) "stop codon" **effect on translation

Define a silent mutation.

Changes a codon to a synonymous codon that specifies the SAME amino acid

What are Plasmid Vectors?

Circular DNA molecules naturally existing in bacteria; commonly used for cloning DNA fragments in bacteria

Transposons. Discuss the differences between Class I and Class II transposons. Which class is more common in prokaryotes? Eukaryotes?

ClASS I: "retrotransposons" utilize an RNA intermediate, the transposable element is transcribed to RNA then back to DNA via reverse transcriptase. This results in two copies of the transposon, thus a 'copy/paste' mechanism, and are more common in eukaryotes, there are no known retrotransposons in bacteria CLASS II: or "DNA transposons" do not use an RNA intermediate and are more common in bacteria. They can be replicative or conservative

Protein Coding Region

Comprises the codons that specify the AMINO ACID sequence of the protein - begins with START codon and ends with STOP codon

What role do consensus sequences play?

Consensus sequences HAVE to be present when splicing begins

Which of the following statements about basal transcription factors is TRUE? A. they are essential for transcription B. they cannot increase the rate of transcription by themselves C. they can decrease the rate of transcription by themselves D. A and B E. A, B, and C

D. A and B A. they are essential for transcription B. they cannot increase the rate of transcription by themselves

DNA methylation may be a significant mode of genetic regulation in eukaryotes. Methylation refers to: A. altering RNA polymerase activity by methylation of RNA polymerase B. altering translational activity especially of highly methylated tRNAs. C. alteration of DNA polymerase activity by addition of methyl groups to glycine residues. D. addition of methyl groups to the cytosine of CG doublets

D. addition of methyl groups to the cytosine of CG doublets

Two graduate students are discussing how to determine if the basic Helix-loop-helix transcription factor they are working on binds a specific 10 base-pair DNA sequence located distal to the stranded at second gene in Drosophila. (A) What assay could these perplexed students use to verify protein-DNA interactions? (B) Explain how the assay works

DNase Protection Assay or DNase Footprinting Assay: - This assay works by exposing radioactively end-labeled DNA to deoxyribonuclease (DNase) which cuts the DNA. - The product is then run on a gel (gel electrophoresis) for DNA sequencing. Areas of the DNA that are contacted or protected by the bHLH protein (i.e at a promoter or enhancer regions) would be protected from DNase enzymatic cleavage. - A sample done without the presence of the protein expected to bind is compared with a sample(s) in the presence of protein (or increasing concentrations). - Areas that were protected by the protein will appear clear (without bands) and thus reveal were the protein bound to the DNA and give you a promoter sequence.

Regulation of Gene Expression. Explain how the following process complicates the concept of Colinearity. [colinearity = concept that there is a direct correspondence between the nucleotide sequence of a gene and the continuous sequence of amino acids in a protein] (B) Alternative splicing

Different mature mRNAs from a single gene can be produced by alternative splicing. Different arrangements of the gene's exons can occur in the mature mRNAs. Thus, different proteins can be encoded within the same gene as opposed to one gene corresponding to one protein as is predicted by the concept of colinearity.

Transposons. What do we mean when we are talking about a transposon's 'footprint'? What feature of a transposon serves as a footprint?

Direct repeats serve as a transposon's footprint. When these repeats are found in other areas of the genome, the transposable element is able to move in. Site-specific endonuclease makes staggered cuts at a target site, since there is complementary sequence in the DNA and attached to the transposon it is able to move in

What is the 5' Consensus sequence?

GU(A/G) AGU: 5' splice site

Define Gene Cloning? (A) what is a Cloning Vector? (B) what does a cloning vector contain?

Gene cloning = a fragment of DNA is placed in a bacterial cell where it is replicated producing identical copies of original piece of DNA (A) Stable, replicating DNA molecule to which a foreign DNA fragment can attach for introduction into a cell (B) Origin of Replication - selectable markers that enable any cells containing the vector to be selected or identified - one or more unique restriction sites into which a DNA fragment can be inserted

Regulation of Gene Expression. Compare the roles of general transcription factors and transcriptional activator proteins.

General transcription factors form the basal transcription apparatus together with RNA polymerase are needed to initiate minimal levels of transcription. Transcriptional activator proteins bring about higher levels of transcription by stimulating the assembly of the basal transcriptional apparatus at the start site

HindIII restriction enzyme recognizes this sequence: 5'- AAGCTT -3' 3'- TTCGAA -5' How would the new strands be cut?

HindIII cuts the sugar-phosphate backbone of each strand, generating short fragments with sticky "cohesive" ends 5'- A AGCTT -3' 3'- TTCGA A -5' - Cohesive ends are complementary to each other and can spontaneously pair to connect fragments

Phylogenomics. Describe a phenomena that can lead to different topologies between a supermatrix and supertree when attempting to resolve the phylogeny of a group of organisms using several homologous genes as your dataset.

Horizontal gene transfer, Introgression and Incomplete lineage sorting. All three of these examples could lead to gene trees with inconsistent phylogenies when compared to a 'truer' species phylogeny. If we take a supertree approach and have genes with more straight-forward evolutionary histories, we can catch potential mistakes in our final topology. However, if we use a supermatrix approach, genes with these sorts of histories may confound our final topology, giving us erroneous results.

Two graduate students are discussing how to determine if the basic Helix-loop-helix transcription factor they are working on binds a specific 10 base-pair DNA sequence located distal to the stranded at second gene in Drosophila. (A) What assay could these perplexed students use to verify protein-DNA interactions? (B) Explain how the assay works

IN VIVO REPORTER GENE ANALYSIS: - The 10-base pair DNA sequence could be placed in front of a reporter gene (lacZ or GFP as examples) and a transgenic Drosophila line created. - If lacZ or GFP expression is observed than this enhancer region is sufficient to drive expression. - A second step would involve crossing the transgenic 10bp enhancer-lacZ line into a fly that was mutant for the bHLH transcription factor gene and determine if the lacZ expression is subsequently abolished.

Intragenic suppressor mutations vs. Intergenic suppressor mutations

INTRAGENIC: occurs in the same gene containing mutation being suppressed INTERGENIC: occurs in a gene other than the one bearing the original mutation

Regulation of Gene Expression. Explain how the following process complicates the concept of Colinearity. [colinearity = concept that there is a direct correspondence between the nucleotide sequence of a gene and the continuous sequence of amino acids in a protein] (C) RNA editing

In RNA editing, genetic information is added to the pre-mRNA after it is transcribed. In other words, the mature mRNA will contain information that was not part of the DNA from which it was transcribed. The result is that the nucleotide sequence of the gene does not correspond to the amino acid sequence of the protein—a clear violation of the concept of colinearity.

Regulation of Gene Expression. Explain how the following process complicates the concept of Colinearity. [colinearity = concept that there is a direct correspondence between the nucleotide sequence of a gene and the continuous sequence of amino acids in a protein] (A) Trans-splicing

In trans-splicing, exons from different mRNAs are spliced together during RNA processing events. Essentially, the mature mRNA product is not produced by DNA sequences that are contiguous or even necessarily on the same chromosome. This results in an amino acid sequence of the translated protein from trans-spliced mRNA being encoded by two or more different genes. According to the principle of colinearity, we would have expected the DNA sequence of a single gene to correspond to the amino acid sequence of the protein.

Nuclear pre-mRNA Introns

Located in the protein-encoding genes of the eukaryotic nucleus NOT self-splicing; require snRNAs and other proteins

What vector reports transcription factor binding?

Luciferase reporter vector: (1) transfect cell lines of interest (2) If TF binds to enhancer, luciferase expression occurs (3) harvest cell lines (4) incubate extracts with luciferase substrate >> light emission

What is Histone Modification [Histone code]?

Modifications to histones; they encode information that affects how genes are expressed

Transposons. Why are transposons so widely used for mutagenesis? (HINT: Provide at least two useful attributes.)

Most transposons insert randomly into the genome - serve as a tool to induce mutation throughout the genome for researchers which helps in determining gene function - the sequence of a TE used in mutagenesis is known, they also serve as a tag and/or a PCR primer for finding genes whose function has been altered by the insertion EXAMPLE (pg. 513) of your text outlines an experiment where a certain TE was able to proliferate in mice; occasionally it inserted into genes that were protective against cancer. When this occurred, the function of the gene was destroyed and it helped researchers to identify genes protective against cancer.

Translocations

Movement of genetic material between non-homologous chromosomes or within the same chromosome

Mutations. Explain, mechanistically and providing specific examples, why most mutations are neutral, but most non-synonymous substitutions are deleterious.

Mutations can be neutral when they are invisible to selection, either because they occur in noncoding regions such as introns or intergenic regions in Eukaryotes. They can also be neutral because the changes they cause in coding sequences fail to result in a phenotype that diminishes organismal performance, such as may be the case when the change is synonymous, or when mutation results in an AA sequence change to a similar AA or occurs in a part of the protein that preserves its structure function. Non-synonymous mutations (missense) mutations, however, tend on average to be more disruptive, diminishing activity or regulation of activity, and therefore deleterious

Non-Reciprocal translations vs. Reciprocal translations

Non-Reciprocal: genetic material moves from one chromosome to another without a reciprocal change Reciprocal: two-way exchange of segments between the chromosomes

Proximal promoter region: is the region located immediately upstream of the core promoter

One example feature that occurs in this region in many eukaryotic promoters is the CCAAT box. The CCAAT box can be recognized by multiple transcription factors, including the ubiquitous NF-Y

What is the RNA-Induced Silencing Complex (RISC)?

PAIRS with mRNA molecule that possesses a sequence complementary to its siRNA or miRNA component and CLEAVES the mRNA leading to DEGRADATION of the mRNA or repressed translation

PvuII restriction enzyme recognizes this sequence: 5'- AAGCTT -3' 3'- TTCGAA -5' How would the new strands be cut?

PvuII cuts in the middle of its recognition site and the cuts on the two strands are directly opposite one other, producing "blunt" end fragments 5'- AAG CTT -3' 3'- TTC GAA -5'

What is RNA Interference (RNAi)?

RNAi = mechanism used by eukaryotic cells to limit the invasion of foreign genes and to censor the expression of their own genes

What are Restriction enzymes?

Recognize and make double-stranded cuts in DNA at specific sequences

Mutations. In many eukaryotic organisms, a significant proportion of cytosine bases are naturally methylated to 5-methylcytosine. 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.

Define the base substitution of Insertion or Deletion. (include frameshifts and their affect on the individual)

The ADDITION or REMOVAL of one or more nucleotide pairs - may lead to frameshift mutations (change in gene reading frame) FRAMESHIFT: generally have drastic effects on the phenotype - many code for premature stop codons (Phase variation in bacterial pathogens, Neisseria, Mycoplasma) **molecular change

(A) What is the difference between an enhancer and a promoter? (B) How does each contribute to the regulation of gene expression? [Hint: Be sure to indicate how and where transcriptional activators work.]

Transcriptional activators bind enhancer elements. The activation domain of the txn factor will interact with the basal txn machinery including RNA polymerase and increase the amount of txn from a promoter. Enhancers work upstream or downstream of the promoter, in either orientation, and from far away. Promoters are binding sites of general txn factors (basal txn machinery) including TFIID which includes the TATA-box binding factor. A promoter without enhancement by txn factors will have low levels of transcription as RNA Pol II is attracted at low levels. Promoters don't work in either orientation and set the start site and direction of transcription

What are the characteristics and modes of activity of type I and IV restriction enzymes?

Type I: Multi subunit complex; cut DNA at random away from recognition site Type IV: Require Mg2+ for activity; cuts modified DNA (normally methylated)

Transfer RNA Introns

found in tRNA; splicing relies on enzymes

Phylogenomics. INCOMPLETE LINEAGE SORTING is a phenomena that can lead to different topologies between a supermatrix and supertree when attempting to resolve the phylogeny of a group of organisms using several homologous genes as your dataset. How?

if time between speciation events is short, it is possible that certain genes or alleles may have a deeper and more complex history than species divergence.

Phylogenomics. INTROGRESSION is a phenomena that can lead to different topologies between a supermatrix and supertree when attempting to resolve the phylogeny of a group of organisms using several homologous genes as your dataset. How?

it is common that among organisms that readily hybridize, introgression can occur. EXAMPLE: using mtDNA to resolve phylogeny of bears. Using this as our data, it appears that polar bears diverged from brown bears only 150,000 ya.

Phylogenomics. HORIZONTAL GENE TRANSFER is a phenomena that can lead to different topologies between a supermatrix and supertree when attempting to resolve the phylogeny of a group of organisms using several homologous genes as your dataset. How?

it is more common in microorganisms, genes from a distantly related organism can become incorporated into a species' genome. EXAMPLE: the eukaryotic acidophiles in Yellowstone. If we use their arsenic reducing gene as data for tree building, they group within the bacteria.

3'UTR

sequence of nucleotides at the 3' end of the mRNA that are not translated into the protein - affects the STABILITY of the mRNA