Exam 3 Objectives

What is conservative transposition?

"cut-and-paste"

Define and know the difference between inversion, translocation, duplication, and deletion.

Deletion: loss of chromosomal segment Duplication: duplication/retention of already present chromosomal segment Inversion: reversal of orientation of a given region Translocation: transfer of a chromosomal segment to another chromosome

What is the difference between forward and reverse genetics?

Forward genetics identifies heritable differences by their phenotypes and map locations and precedes the molecular analysis of the gene products. Reverse genetics starts with an identified protein or RNA and works toward mutating the gene that encodes it (and in the process, discovered the phenotype when the gene is mutated).

Intercalating agents produce mutations of which type? Give a simple explanation of their mechanism of action.

Intercalating agents are planar molecules that wedge between nitrogen bases in DNA. They cause INDEL and frameshift mutations.

How can transposable elements be utilized (or manipulated) to transfer genes in laboratory experiments?

P element carrying gene of interest is inserted into multinucleate embryo. P element has ends that bind transposase and allow transposition. P elements and gene make their way into gametes and genes are transferred to the next generation. Gerald Rubin and Allan Spradling showed that P-element DNA can be an effective vehicle for transferring donor genes into the germ line of a recipient fly. P elements mobilize only in germ-line cells.) Two types of DNA are injected into embryos of this type. The first is a bacterial plasmid carrying a defective P element into which the ry+ gene has been inserted. The defective P element resembles the maize Ds element in that it does not encode transposase but still has the ends that bind transposase and allow transposition. This deleted element is not able to transpose, and so, as mentioned earlier, a helper plasmid encoding transposase but without the terminal repeats (so it cannot transpose) also is injected. Flies developing from these embryos are phenotypically still rosy mutants, but their offspring include a large proportion of ry+ flies. In situ hybridization confirmed that the ry+ gene, together with the deleted P element, was inserted into one of several distinct chromosome locations. None appeared exactly at the normal locus of the rosy gene. These new ry+ genes are found to be inherited in a stable, Mendelian fashion.

What is a chromatin immunoprecipitation assay? How does it work? What does it enable the study of?

Tries to understand how proteins interact with DNA. What is the specific DNA sequence the protein binds to? 1. cross link protein to DNA 2. break chromatin into small pieces to isolate the DNA segment bound to the protein. 3. add antibody that recognizes the protein of interest - some sort of nanoparticle (glass or metal bead) 4. centrifuge - weighted DNA/protein/antibody pellet falls to the bottom 5. reverse cross link, amplify DNA sequence

Describe dosage compensation in Drosophila and humans.

X chromosome gene expression is the same for males as it is for females - males have half the amount of X genes but they don't have half the amount of gene product. Drosophila males have a hyperactive X gene. In mammals, a single X gene is inactivated in each female cell. This may explain why XXX and XXY is normal.

Polyploid

a cell having 3 or more sets of chromosomes when the normal is diploid. This is an abberant euploid.

In browsing through the chimpanzee genome, you find that it has three homologs of a particular gene, whereas humans have only two. a. What are two alternative explanations for this observation? b.How could you distinguish between these two possibilities?

a. It may be that the ancestor of both chimpanzee and humans had three copies of the gene and one was lost after the human ancestors diverged from chimpanzee, or conversely that the common ancestor had two and a duplication has occurred in the chimpanzee line. b. One would determine how many copies are present in the other great apes. This would allow one to determine when in the evolution of apes the change occurred.

Monoploid

an organism (in an otherwise diploid species) with a single chromosome set (n) ex: male wasps and bees

What are the two types of transposons in bacteria?

composite and simple

Aneuploid

organism whose chromosome # differs from the wild type by PART of a chromosome set 2 cases of coke + 1 single can

How do piRNAs and crRNAs function to repress/repel invading transposable element viruses?

piRNAs are short RNAs that interact with the Argonaute protein to degrade complementary mRNAs. piRNAs don't originate from the dsRNA pathway, they originate from pi-clusters (traps that ensnare active transposons). Invading virus genome captured by bacterial loci called CRISPER where they are transcribed into long RNAs that are processed to short crRNAs = guide bacterial protein complexes to degrade complementary RNAs from invading viral genome. Both of these mechanisms: new insertions of transposons or viral DNA elements result in permanent, genetic change in these loci (pi clusters/CRISPER) that is inherited by progeny.

What are the two possible types of transposon mobilization in bacteria?

replicative & conservative

What is a target site duplication?

A short, direct-repeat DNA sequence (2 bp-10 bp) adjacent to the ends of a transposable element that was generated during the element's integration in to the host chromosome. Virtually all transposable elements (in both prokaryotes and eukaryotes) are flanked by a target-site duplication, indicating that all use a similar mechanism of insertion. It is important to keep in mind that the transposable elements have inverted repeats at their ends and that the inverted repeats are flanked by the target-site duplication—which is a direct repeat.

What is a composite transposon?

A type of bacterial transposable element containing a variety of genes that reside between two nearly identical insertion sequence (IS) elements.

What are some examples of eukaryotic class II TEs?

Ac, Ds, Tam-3, P elements

What is the yeast two-hybrid system? How does it work? What does it enable the study of?

Allows you to study whether or not two proteins interact physically. Gal-4 dimer with an active domain and a DNA binding domain

What is the difference between a bacterial IS element and a bacterial transposon?

An IS element is a mobile piece of bacterial DNA (several hundred nucleotide pairs in length) capable of inactivating a gene into which it inserts. Features common to all IS elements include: transposase gene and flanking inverted repeat (IR) sequences. IS elements are short mobile sequences that encode only those proteins necessary for their mobility. Composite transposons and simple transposons contain additional genes that confer new functions to bacterial cells.

Base analogs produce mutations of which type? Give a brief definition of a base analog and a simple explanation of its mechanism of action.

Base analogs are a chemical whose molecular structure mimics that of a DNA base. They cause mispairing.

Explain the approach that you would apply to sequencing the genome of a newly discovered bacterial species.

Because bacteria have relatively small genomes (roughly three megabase pairs) and essentially no repeating sequences, the whole genome shotgun approach would be used.

Understand how genomics has changed society and the sociology of biological research.

Characterizing whole genomes is fundamental to understanding the entire body of genetic information underlying the physiology and development of living organisms, and to the discovery of new genes such as those having roles in human genetic diseases.

What color are the kernels of a strain with c-m(Ds) and no Ac?

Colorless - Ds cannot be excised from the gene.

Which repair pathway recognizes DNA damage during transcription? What happens if the damage is not repaired?

DNA damage that stalls transcription is repaired by TC-NER (transcription-coupled nucleotide excision repair). Humans lacking this pathway suffer from Cockayne syndrome. A consequence of this defect is that a cell is much more likely to activate its apoptosis (cell suicide) pathway. Affected individuals are very sensitive to sunlight and have short stature, the appearance of premature aging, and a variety of developmental disorders.

There are big spots of color on a kernel when Ds leaves the C gene early/later in development.

Early. The spots are smaller if it leaves later in development.

A common transition mutation caused by the oxidative deamination of cytosine to uracil is C-G to T-A. Illustrate how such a mutation can be brought about in two rounds of DNA replication following the deamination event.

Normal: C-G Mutant mispairing: U-A After replication: T-A

Know how balanced rearrangements can lead to irregularities during meiosis and the production of irregular meiotic products.

Paracentric inversion: normal product (A-E), deletion product (A-D), deletion product (A), inversion product (ADCBE) Pericentric inversion: ABCD, ABCA, DBCD, DBCA Adjacent-1: T1 + N2, T2 + N1 = inviable Alternate: T1+T2, N1 + N2 = complete, viable

Terminal-sequencing reads of clone inserts are a routine part of genome sequencing. How is the central part of the clone insert ever obtained?

Terminal sequence reads of cloned inserts, such as those generated during whole genome shotgun sequencing, are assembled into a scaffold by matching homologous sequences shared by reads from overlapping clones. In essence, the central sequence of any single clone will be generated from the terminal sequences of overlapping clones.

Know and understand the difference between balanced and unbalanced rearrangements.

Unbalanced rearrangements result in a change in the amount of DNA. This includes: deletions and duplications. Balanced rearrangements result in no net change in the amount of DNA. This includes: inversions and translocations.

Explain how the properties of P elements in Drosophila make gene-transfer experiments possible in this organism.

Under certain conditions, they are highly mobile and can be used to generate new mutations by random insertion and gene knockout. P elements can also be manipulated and used to insert almost any DNA or gene in to the Drosophila genome. This requires inserting the DNA of interest between the inverted repeats necessary for P element transposition. A helper intact P element supplies transposase. Co-injected into very early embryos and progeny are screened for the gene of interest.

If you observed a dicentric bridge at meiosis, what rearrangement would you predict had taken place?

A crossover within a paracentric inversion heterozygote results in a dicentric bridge (and an acentric fragment).

Which, if any, of the following sex-chromosome aneuploids in humans are fertile: XXX, XXY, XYY, XO?

Both XYY (male) and XXX (female) would be fertile. XO (Turner syndrome) and XXY (Klinefelter syndrome) are known to be sterile.

In a two-hybrid test, a certain gene A gave positive results with two clones, M and N. When M was used, it gave positives with three clones, A, S, and Q. Clone N gave only one positive (with A). Develop a tentative interpretation of these results.

The yeast two-hybrid test detects possible physical interactions between two proteins. These results indicate that gene A codes for a protein that interacts with proteins encoded by clones M and N, and further, that clone M encodes a protein that also interacts with proteins encoded by clones S and Q. For example:

Recognize the Next Generation DNA sequencing technologies have revolutionized the speed and cost of determining genome sequences. Compare and contrast traditional WGS from next gen WGS.

Traditional: (1) construct genomic libraries - fragments cloned in to plasmids, (2) amplify library clones by growth in bacteria, (3) sequence entire insert or paired ends of many clones, (4) computer algorithm finds sets of inserts with sequence overlap. This enable assembly of contigs. Next-gen: (1) uses new sequencing technologies (e.g. pyrosequencing), (2) DNA is not cloned into vectors or amplified in bacteria, but rather amplified by PCR, (3) millions of DNA fragments are sequenced in parallel = very high throughput, (4) MANY MANY SHORT sequences

T/F An allele of the Ac type can be transformed into an allele of the Ds type.

True. Ds is most likely an incomplete, mutated version of Ac itself.

What are all the transversions that can be made starting with the codon CGG? Which of these transversions will be missense? Can you be sure?

By transversion, CGG (arginine) can be become AGG (arginine), GGG (glycine), CCG (proline), CUG (leucine), CGC (arginine), or CGU (arginine).

The normal sequence of nine genes on a certain Drosophila chromosome is 123 · 456789, where the dot represents the centromere. Some fruit flies were found to have aberrant chromosomes with the following structures: a. 123 · 476589 b. 123 · 46789 c. 1654 · 32789 d. 123 · 4566789 Name each type of chromosomal rearrangement, and draw diagrams to show how each would synapse with the normal chromosome.

a. Paracentric inversion b.Deletion c. Pericentric inversion d. Duplication

Distinguish between gene mutations and chromosome mutations.

A chromosome mutation is any type of change in chromosome structure or number. Gene mutations affect a segment of protein coding DNA within a chromosome.

Is a trisomic an aneuploid or a polyploid?

Aneuploid. Trisomic refers to three copies of one chromosome. Triploid refers to three copies of all chromosomes.

To inactivate a gene by RNAi, what information do you need? Do you need the map position of the target gene?

Gene position is not a necessary requirement for using RNAi. What is needed is the sequence of the gene that you intend to inactivate, as you need to start with double-stranded RNA that is made with sequences that are homologous to part of the gene.

What are some examples of eukaryotic class I TEs?

LINEs, SINEs, LTR-retrotransposons

What is the C-value paradox? What's the connection between the C-value paradox and transposable elements?

The C-value paradox is the lack of correlation between the DNA content of an organism and its biological complexity. There are many transposable elements in genomes and repetitive sequences in higher organisms. In humans, for every 1 kb of DNA that is protein coding there are 20 kb of DNA containing TEs. In higher organisms, TEs constitute the majority of the genome.

Distinguish between the two subclasses of base substitution mutations: transition and transversions.

Transition: purine to purine or pyrimidine to pyrimidine Transversion: purine to pyrimidine, vice versa.

What are bypass polymerases? How do they differ from the replicative polymerases? How do their special features facilitate their role in DNA repair?

Translesion or bypass polymerases are able to replicate past damaged DNA that otherwise would stall replicative polymerases. They differ from replicative polymerases in that they can tolerate large adducts on the bases (as they have much larger active sites that can accommodate damaged bases), they are much more error-prone (as they lack the 3′ to 5′ proofreading function), and they can only add relatively few nucleotides before falling off. Their main function is to unblock the replication fork, not to synthesize long stretches of DNA that could contain many mismatches.

Euploid

a cell having any number of complete chromosome sets (having a whole number multiple of a haploid chromosome set) a normal diploid would have 2 sets with 12 chromosomes in each for a total of 24 chromosomes

What are some of the different approaches for conducting reverse genetics experiments? How do these approaches differ from one another? What are the advantages and disadvantages of each?

1. Introduce random mutations into the genome and hone in on the gene of interest by molecule identification of mutations in the gene. Con: must sift through all the mutations. Pro: well established mechanism. 2. Conduct a targeted mutagenesis that produces mutations directly in the gene of interest. Con: more labor intensive. Pro: more straightforward. 3. Create phenocopies: effects are comparable to mutant phenotypes (usually by treatment with agents that interfere with mRNA transcripts of the gene). Pro: very efficient, especially with libraries for some model organisms

What is a simple transposon?

A type of bacterial transposable element containing a variety of genes that reside between short inverted repeat sequences.

Distinguish between base altering compounds, such as EMS, and base damaging agents, such as aflotoxin, which creates an apurinic site. What is an apurinic site?

Aflotoxin forms a bulky adduct & breaks the bonds between sugar and base. This results in a apurinic site and stalls replication. Apurinic site is a place where DNA has lost a purine residue. EMS adds an ethyl group (C2H5) to bases, which causes mispairing.

Understand the difference between autopolyploidy and allopolyploidy.

Autopolyploidy: chromosome sets from 1 species only. These are homologous chromosomes. Can arise spontaneously from unreduced gametes or cross between 4n x 2n. Examples: seedless watermelons and bananas (3n) = sterile. Allopolyploidy: chromosome sets from different species. These are homelogous chromosomes. Can be used to combine desirable traits in nature/create new species.

How did Ron Plasterk's lab learn that components of the RNAi pathway also regulated transposable element activity in C. elegans?

C. elegans with mutant unc 22 gene + TC1 transposable element inserted into it had a twitching motion. If they inactivated the genes that were repressing TC1 mobility, it would excise and C. elegans would have smooth movement again. Products of the genes that were silenced were integral components of RNAi pathway, including proteins found in DICER and RISC. Tc1 has inverted repeats at ends so when transcribed it forms dsRNA. siRNA bound to RISC targets all transposase transcripts to be degraded.

How do composite transposons and simple transposons differ?

Composite transposons contain a variety of genes that reside between two nearly identical IS elements that are oriented in opposite direction (Figure 15-6a) and, as such, form what is called an inverted repeat sequence. Transposase encoded by one of the two IS elements is necessary to catalyze the movement of the entire transposon. Simple transposons also consist of bacterial genes flanked by inverted repeat sequences, but these sequences are short (< 50 bp) and do not encode the transposase enzyme that is necessary for transposition. Thus, their mobility is not due to an association with IS elements. Instead, simple transposons encode their own transposase in the region between the inverted repeat sequences in addition to carrying bacterial genes.

What genetic factor was located at the site of the break in chromosome 9 of maize?

Ds

What is replicative transposition?

generates a single additional copy. uses a cointegrate.

What is the relationship between bacterial transposons and the spread of antibiotic resistance?

A transposon can jump from a plasmid to a bacterial chromosome or from one plasmid to another plasmid. In this manner, multiple-drug-resistant plasmids are generated. An R plasmid may contain several transposons carrying resistance genes.

What is meant by autonomous v. non-autonomous? How do non-autonomous transposable elements mobilize?

Autonomous: transposable element that encodes the proteins necessary for its own transposition and the transposition of non-autonomous elements in the same family. Ex: transposase and transcriptase

What observations in maize led to the initial discovery of transposable elements?

Barbara McClintock noticed that chromosomal breakage was common for chromosome #9 at one particular site & that breaks gave rise to strains with odd kernel color patterns and unstable mutant phenotypes that revert to wild type. McClintock began to suspect that these were mobile genetic elements when she found it impossible to map Ac. Two required genetic factors: Ac and Ds. Ds = disassociation at the site of the break Ac = activator, unlinked and unmapable

What is functional genomics? What is reverse genetics? How does reverse genetics differ from forward genetics?

Functional genomics is the study of global patterns of expression and interaction of gene products, often in the context of environmental factors.

In what system were transposable elements first isolated at the DNA level?

Insertion sequences in E. coli in the 1960s. Gal- mutants with large insertions in the gal operon. Same insertion sequence found in independently isolated mutants.

A large plant arose in a natural population. Qualitatively, it looked just the same as the others, except much larger. Is it more likely to be an allopolyploid or an autopolyploid? How would you test that it was a polyploid and not just growing in rich soil?

It would be more likely an autopolyploid. To make sure it was polyploid, you would need to microscopically examine stained chromosomes from mitotically dividing cells and count the chromosome number.

Define the term mutagen. Describe at least four broad classes of mutagenic agent or events that result in mutation.

Mutagens incur mutations. Induced mutations occur via replacement, alteration, or damage. 1. base analogs 2. alkylating agents 3. intercalating agents 4. damage (UV, aflotoxin)

Understand the implications of the Luria-Delbruck fluctuation test on our understanding of the random nature of mutations.

Mutation is a random process. Any allele in any cell may mutate at any time. Mutations occurred during the growth of bacterial cultures and were only shown once plated with the T1 phage.

Under what circumstances could nonhomologous end joining be said to be error prone?

NHEJ (nonhomologous end joining) is error prone as some sequence may be lost in the repair process. The consequences of imperfect repair may be far less harmful than leaving the lesion unrepaired. Presumably, this repair pathway evolved because, unless repaired, the broken ends can degrade further, leading to loss of more genetic information. Also, these lesions can initiate potentially harmful chromosomal rearrangements that could lead to cell death.

What are orthologs? What are paralogs? How do they arise?

Orthologs: genes in different species that evolved from a common ancestral gene by speciation Paralogs: genes that are related by gene duplication within a genome

What were the circumstances leading to the discovery of P elements in Drosophila?

P elements were discovered by Margaret Kidwell, who was studying hybrid dysgenesis—a phenomenon that occurs when females from laboratory strains of D. melanogaster are mated with males derived from natural populations. In such crosses, the laboratory stocks are said to possess an M cytotype (cell type), and the natural stocks are said to possess a P cytotype. In a cross of M (female) × P (male), the progeny show a range of surprising phenotypes that are manifested in the germ line, including sterility, a high mutation rate, and a high frequency of chromosomal aberration and nondisjunction (Figure 15-17). These hybrid progeny are dysgenic, or biologically deficient (hence, the expression hybrid dysgenesis). Interestingly, the reciprocal cross, P (female) × M (male), produces no dysgenic offspring. An important observation is that a large percentage of the dysgenically induced mutations are unstable; that is, they revert to wild type or to other mutant alleles at very high frequencies.

What color are the kernels of a strain with c-m(Ds) and Ac is present?

Spotted kernels because Ac activates Ds to leave the C gene in some cells.

A disomic product of meiosis is obtained. What is its likely origin? What other genotypes would you expect among the products of that meiosis under your hypothesis?

The likely origin of a disomic (n + 1) gamete is nondisjunction during meiosis. Depending whether the nondisjunction took place during the first or second division, you would expect one nullosomic (n - 1) gamete, or two nullosomic gametes and another disomic gamete, respectively.

In browsing through the human genome sequence, you identify a gene that has an apparently long coding region, but there is a two-base-pair deletion that disrupts the reading frame. a. How would you determine whether the deletion was correct or an error in the sequencing? b. You find that the exact same deletion exists in the chimpanzee homolog of the gene but that the gorilla gene-reading frame is intact. Given the phylogeny of great apes below, what can you conclude about when in ape evolution the mutation occurred?

(a) To determine whether the 2-bp deletion was a sequencing error, you would simply resequence that region of the genome, or a cDNA copy of the gene. (b) You would conclude that the 2-bp deletion occurred in the common ancestor of both chimpanzee and human after the divergence from the ancestor of gorillas. cDNA = DNA molecule that's complementary to an mRNA sequence. They provide direct evidence that a given segment of the genome is expressed and may thus encode a gene. cDNA is complementary to the mature RNA and the introns have already been removed.

Understand the differences between comparative genomics and functional genomics and the role of bioinformatics in each.

Comparative genomics: comparison of genomes of related species for evolutionary and functional insights Functional genomics: uses a variety of techniques to understand the function of all the genes in the genome Bioinformatics: analysis of the information content of entire genomes

What are microarrays? How do they allow one to examine gene expression? What is the transcriptome?

Microarray = a set of DNAs containing all or most genes in a genome deposited on a small glass chip. It allows you to examine what genes are active in a cell under certain conditions (RNA transcripts in a cell can tell us what genes are active). Transcriptome = sequence and expression patters of all RNA transcripts (which kinds, where in the tissues, how much, when they are expressed)

Recognize that the SOS system can be activated with excess damage, allowing introduction of random bases, and that this can lead to mutations (but might prevent cell death).

SOS (repair) system: an error-prone process in E. coli whereby a bypass polymerase replicates past DNA damage at a stalled replication fork by inserting non-specific bases. This is a form of damage tolerance that allows the cell to trade death for a certain level of mutagenesis.

The insertion of transposable elements into genes can alter the normal patterns of expression. In the following situations, describe the possible consequences on gene expression. (a) A LINE inserts into an enhancer of a human gene. (b) A transposable element contains a binding site for a transcriptional repressor and inserts adjacent to a promoter. (c) An Alu element inserts into the 3' splice site (AG) of an intron in a human gene. (d) A Ds element that was inserted into the exon of a maize gene excises imperfectly and leaves three base pairs behind in the exon. (e) Another excision by that same Ds element leaves two base pairs behind in the exon. (f) A Ds element that was inserted into the middle of an intron excises imperfectly and leaves five base pairs behind in the exon.

(a) The consequences will be different for different genes and different insertions. In the simplest scenario, the insertion prevents the binding of transcriptional activators that are required for the ultimate binding of RNA polymerase to the promoter. In this case, the gene will not be expressed (no mRNA will be synthesized). In more complex scenarios, the gene may be regulated by many enhancers (as is the case for most human genes). For example, one enhancer might be required for transcription in the liver, one required for transcription in muscle cells, etc. In this instance, insertion of the LINE into the liver enhancer may prevent transcription of the gene in the liver but not interfere with its transcription in muscle. (b) Again, the consequences will differ depending on the gene that has sustained the insertion. In the simplest scenario, the presence of the transposable element will provide a binding site for the transcriptional repressor to bind near the promoter and prevent the binding of RNA polymerase II. (c) The Alu element will be transcribed into RNA with the rest of the gene sequences and will prevent the splicing of the intron that it has inserted into. The insertion will almost certainly result in a null allele as the Alu sequence and the intron will now be translated. The intron, Alu,or both probably will contain stop codons. (d) The insertion and excision will result in a 3-bp indel in the exon and slightly alter the amino acid sequence of the protein, but it will not produce a frameshift mutation. The minor change in the amino acid sequence may or may not affect the function of the encoded protein. (e) This insertion and excision will produce a frameshift mutation and is more likely than the situation in part d to impair protein function. (f) Chances are that this mutation will not affect gene expression, as the intron will probably still be spliced correctly.

Know and understand mechanisms of different DNA repair pathways. What types of mutations are associated with each repair pahtway?

1. direct reversal of DNA damage: photodimer, CPD enzyme 2. base-excision repair (BER): minor base damage such as deamination, or damage from reactive oxygen species 3. nucleotide excision repair (NER): activated when bulky adducts or multiple damaged bases are recognized 4. post replication: mismatch repair. Corrects DNA replication errors that the proofreading function fails to fix. 5. error-prone repair (translesion DNA synthesis): bypass lesions at the stalled replication fork to prevent cell death and leaves the mutation where it is. 6. double stranded break repair: error prone nonhomologous end joining (NHEJ) which stabilizes breaks and trims the ends & error free synthesis dependent strand annealing (SDSA) which uses sister chromatids as a template for repair and results in conservative replication.

What percent of the human genome encodes exons of mRNAs? What are psuedogenes? How are they thought to have arisen?

< 3% of the human genome are exons (protein-coding genes) psuedogenes are a mutationally inactive gene for which no functional counterpart exists in the wild type populations these are ORFs or partial ORFs that may at first appear to be genes but are not due to their manner of origin or to mutations. processed psuedogenes are DNA sequences that may have been reverse-transcribed from RNA and randomly inserted into the genome. Ninety percent or so of human pseudogenes appear to be of this type. About 900 pseudogenes appear to be conventional genes that have acquired one or more ORF-disrupting mutations in the course of evolution.

What is the goal of bioinformatics? How does one employ bioinformatics to learn about an organism's genome? Why are bioinformatics approaches necessary? What is annotation? Why are regulatory regions more difficult to identify than coding regions?

Bioinformatics is the study of the information content of genomes. Information in the genome can be though of as the sum of all the sequences that encode proteins and RNAs, plus the binding sites that govern the time and place of their action. Annotations = the identification of all the functional elements of the particular genome. Introns that are spliced out of mRNA make it difficult to identify regulatory sequences. There are also 6 total possible reading frames (3 on each strand)

Briefly describe the experiment that demonstrates that the transposition of the Ty1 element in yeast takes place through an RNA intermediate.

Boeke and Fink: constructed a plasmid using a Ty element that had a promoter that could be activated by galactose, and an intron inserted into its coding region. First, the frequency of transposition was greatly increased by adding galactose, indicating that an increase in transcript (and production of RNA) was correlated to rates of transposition. The newly transposed Ty DNA lacked the intron sequence. Because intron splicing occurs only during RNA processing, there must have been an RNA intermediate.

What is the distinction between eukaryotic class I and class II transposable elements? How do they differ structurally? How do they differ with regard to transposition?

Class I = retrotransposons. Similar in structure to retroviruses. Several genes flanked on either side by long terminal repeat sequences (LTR). Encode genes similar to the retrovirus gag and pol, which play a role in the maturation of the RNA genome and encode reverse transcriptase, respectively. Retrotransposons are transposed through an RNA intermediate. This is "copy and paste" mobilization. Class II = DNA transposons. Similar in structure to transposons found in bacteria, with flanking inverted repeats (IR) and interior transposase gene. DNA transposons are mobilized through a DNA intermediate. This is "cut and paste" transposition.

What type of mutation is show in the following sequence? Wild type ....5′ AAUCCUUACGGA 3′.... Mutant ....5′ AAUCCUACGGA 3′....

The mutant has a deletion of one base, and this will result in a frameshift (-1) mutation.

Differentiate between the elements of the following pairs: a. Transitions and transversions b. Synonymous and neutral mutations c. Missense and nonsense mutations d. Frameshift and nonsense mutations

a. A transition mutation is the substitution of a purine for a purine or the substitution of a pyrimidine for a pyrimidine. A transversion mutation is the substitution of a purine for a pyrimidine, or vice versa. b. Both are base-pair substitutions. A synonymous mutation is one that does not alter the amino acid sequence of the protein product from the gene because the new codon codes for the same amino acid as did the nonmutant codon. A neutral mutation results in a different amino acid that is functionally equivalent, and the mutation therefore has no known adaptive significance. c. A missense mutation results in a different amino acid in the protein product of the gene. A nonsense mutation causes premature termination of translation, resulting in a shortened protein. d.Frameshift mutations arise from addition or deletion of one or more bases in other than multiples of three, thus altering the reading frame for translation. Therefore, the amino acid sequence from the site of the mutation to the end of the protein product of the gene will be altered. Frameshift mutations can and often do result in premature stop codons in the new reading frame, leading to shortened protein products. A nonsense mutation causes premature termination of translation in the original reading frame, resulting in a shortened protein.

What are the consequences of meiosis in individuals heterozygous for a(n): inversion, translocation, duplication, deletion?

1. Deletion: deletion loops form during meiosis. Can uncover recessive alleles on a homolog - 'psuedodominance' 2. Duplication: gain of an additional copy for a segment of DNA. New source of genes/gene familes = evolutionary effect. 3 kinds: tandem, insertional, segmental. 3. Inversion: paracentric or pericentric. Inversion loop formed at meiosis I. For paracentric inversions, crossing over results in dicentric bridge and one acentric fragment. Meiotic deletion products result. For pericentric inversion, the result is 1 normal, 2 deletion products, and 1 inversion product. These are meiotic deletions and duplications. Both paracentric and pericentric inversions result in the number of viable gametes being reduced. 4. Translocation: exchange between 2 nonhomologous chromosomes. Cross shaped configuration at meiosis I. Adjacent 1 segregation meiotic products are often inviable. Alternate segregation products are complete and viable.

Define a point mutation and distinguish between its two major subclasses, base substitution and base addition/deletion (indel) mutation. Distinguish between an induced mutation and a spontaneous mutation.

A point mutation is the alteration of a single (or a few base pairs) of DNA. Base substitutions can be either a transition or a transversion. An INDEL is the insertion or deletion of a base pair. Spontaneous mutations occur during DNA replication or as a result of DNA lesions. Induced mutations are a result of exposure to mutagens (base analogs, alkylating agents, etc.)

Although DNA transposons are abundant in the genomes of multicellular eukaryotes, class I elements usually make up the largest fraction of very large genomes such as those from humans (~2500 kb), maize (~2500 Mb), and barley (~5000 Mb). Given what you know about class I and class II elements, what is it about their distinct mechanisms of transposition that would account for this consistent difference in abundance?

A single class I element can give rise to many transcripts, whereas class II elements insert by "cut-and-paste". Mechanisms have evolved to repress class I copying. In situations where it's not repressed, the host controls the conversion of RNA transcript into ds DNA.

What is comparative genomics? What is being compared? In comparing orthologous regions of two different species' genomes, why are some regions more conserved than others?

Comparative genomics is the analysis of the relations of the genome sequences of two or more species Non-coding sequences that are highly conserved across large evolutionary distances are strong candidates for having important regulatory functions. Many are situated in genes near development.

In mismatch repair in E. coli, only a mismatch in the newly synthesized strand is corrected. How is E. coli able to recognize the newly synthesized strand? Why does this ability make biological sense?

DNA in E. coli is methylated. To distinguish the old template strand from the newly synthesized strand, the mismatch repair mechanism takes advantage of a delay in the methylation of the new strand. This makes sense as replication errors produce mismatches only on the newly synthesized strand, so the mismatch repair system replaces the "wrong" base on that strand.

Provide examples of human monosomies and trisomies and describe their consequences.

Monosomy = (2n-1) -Turner syndrome (X0) -lethal for all autosomes in humans -some viability for sex chromosomes -Y0 isn't viable Trisomy = (2n+1) -Klinfelter's syndrome (XXY) = sterile males -Down syndrome (trisomy 21) is a result of nondisjunction in meiosis I -XXX = normal female -XYY = normal male -most autosomal trisomies are lethal

What is the difference between a draft sequence and a finished sequence (besides on that is done and other is not)? Explain the features of a draft sequence and how they might be finished. What is the role of paired-end reads in this process?

Pared-end reads help close the gaps in genomes with large repetitive fractions. Helps put together sequence contigs in their correct order and orientation. -large #s of repeats pose a problem for draft of a genome -challenge: how to glue together contigs in correct orientation and order when tandem repeats are longer than the length of the sequence read -solution: pairs of sequence reads from opposite ends of genomic inserts in the same clone -first: the unique sequence of overlaps between sequence reads are used to build contigs. paired-end reads are then used to span gaps and to order and orient the contigs into larger units called a scaffold

Given the highly mutagenic nature of transposable elements, how do species' genomes tolerate transposable elements?

Presumably, transposable elements insert into both exons and introns, but only the insertions into introns will remain in the population because they are less likely to cause a deleterious mutation. Insertions into exons are said to be subjected to negative selection. Second, humans, as well as all other multicellular organisms, can survive with so much mobile DNA in the genome because the vast majority is inactive and cannot move or increase in copy number. Most transposable-element sequences in a genome are relics that have accumulated inactivating mutations through evolutionary time. Others are still capable of movement but are rendered inactive by host regulatory mechanisms (RNAi machinery, piRNA defense).

At the structural (protein) level, point mutations can be expressed as synonymous, missense, conservative substitution, nonconservative substitution, nonsense, and frameshift mutations. Define each of these and explain their meanings.

Synonymous: altered codon specifies the same amino acid Missense (conservative): altered codon specifies a chemically similar amino acid Missense (non-conservative): altered codon specifies a chemically dissimilar amino acid Nonsense: altered codon signals a chain termination Frameshift: insertion or deletion of a nucleotide

A segment of cloned DNA containing a protein-encoding gene is radioactively labeled and used in an in situ hybridization to chromosomes. Radioactivity was observed over five regions on different chromosomes. How is this result possible?

The clone may contain DNA that hybridizes to a small family of repetitive DNA that is adjacent to the gene being studied or within one of its introns. Alternatively, the clone may share enough homology with members of a small gene family to hybridize to all or there may be four pseudogenes that are descendants of the unique gene being studied.

Before the integration of a transposon, its transposase makes a staggered cut in the host target DNA. If the staggered cut is at the sites of the arrows below, draw what the sequence of the host DNA will be after the transposon has been inserted. Represent the transposon as a rectangle. AATTTGGCC - TAGTACTAATTGGTTGG TTAAACCGGATCATGATT - AACCAACC

The staggered cut will lead to a nine-base-pair target site duplication that flanks the inserted transposon. See book.

What are the consequences of meiosis in tetraploids? In triploids?

Triploids are typically sterile -- chromosomes pair but segregation produces unbalances gametes (it's hard to divide 3 things in half) Tetraploids can (but not always) produce viable gametes. Pairing possibilities at meiosis include: two bivalents (2n) = fertile, one quadrivalent (2n) = fertile, and a univalent + trivalent (2n-1) or (2n+1) = sterile.

A woman with Turner syndrome is found to be color-blind (an X-linked recessive phenotype). Both her mother and her father have normal vision. a. Explain the simultaneous origin of Turner syndrome and color blindness by the abnormal behavior of chromosomes at meiosis. b. Can your explanation distinguish whether the abnormal chromosome behavior occurred in the father or the mother? c. Can your explanation distinguish whether the abnormal chromosome behavior occurred at the first or second division of meiosis? d.Now assume that a color-blind Klinefelter man has parents with normal vision, and answer parts a, b, and c.

a., b., and c. One of the parents of the woman with Turner syndrome (XO) must have been a carrier for color blindness, an X-linked recessive disorder. Because her father has normal vision, she could not have obtained her only X from him. Therefore, nondisjunction occurred in her father. A sperm lacking a sex chromosome fertilized an egg with the X chromosome carrying the color-blindness allele. The nondisjunctive event could have occurred during either meiotic division. d. If the color-blind patient had Klinefelter syndrome (XXY), then both X's must carry the allele for color blindness. Therefore, nondisjunction had to occur in the mother. Remember that during meiosis I, given no crossover between the gene and the centromere, allelic alternatives separate from each other. During meiosis II, identical alleles on sister chromatids separate. Therefore, assuming there have been no crossovers between the color-blind allele and the centromere, the nondisjunctive event had to occur during meiosis II because both alleles are identical. If the gene is far from the centromere, it would be difficult to determine if nondisjunction happened at MI or MII without molecular studies on haplotypes near the centromere.