1. What is the difference between constitutive and regulated transcription?
Constitutive transcription is genes being transcribed continuously with no regulatory control and regulated transcription is the need for agile and calibrated responses to changing environmental conditions
2. What is meant by "negative control" and "positive control"?
Negative control of transcription involves the binding of a repressor protein to a regulatory DNA sequence, consequence of preventing transcription of a gene or cluster of genes; Positive control of transcription involves the binding of an activator protein to regulatory DNA, result of initiating gene transcription
3. Which two functional domains are usually found in prokaryotic repressors and activators?
DNA binding domain-responsible for locating and binding operator DNA sequence or other target regulatory sequences; allosteric domain- binds a molecule or protein, in so doing, causes a change in the conformation of the DNA binding site
4. What are the common features of the DNA binding domains of regulatory proteins and the sites on DNA where they bind?
Common features: Inducer to bind to allosteric site in order to change repressors configuration, Corepressor-binding of molecule at the allosteric site to activate the DNA binding site, positive control is accomplished by activator proteins that bind to activator binding sites, allostertic effector compound, inhibitor that adds to activator protein
5. Describe the structural features of a helix-turn-helix motif. What is the function of this type of domain?
two alpha helical regions in each of two polypeptides in a homo dimer interact with inverted repeat regulatory sequences. one alpha helical region forms the recognition helix. turns to connect to a short string of amino acids known as the stabilizing helix that holds the two together
6. What is an operon? What kinds of genes are usually found in operons?
Operon is a cluster of genes undergoing coordinated transcriptional regulation by a shared regulatory region, often found in bacterial genomes, participate in the same metabolic or biosynthesis pathway
7. How is the sugar lactose metabolized in E. coli and what proteins are involved? How does the level of these proteins change when lactose is present versus when lactose is absent?
the enzyme B-galactosidase breaks the B-galactoside linkage to release glucose and galactose, small amount of allolactose in the break down of lactose to produce small amount of allolactose which plays a role as an inducer compound for the lac operon, level of proteins go down when glucose is up and lactose is absent
8. Describe the arrangement of genes and regulatory sequences in the lac operon. Include the following terms in your answer: structural genes, promoter, and operator.
Promoter (binds RNA polymerase), Operator (sequence that binds the lac repressor protein, and the third is the CAP-cAMP region---then structural genes: lacZ gene-enzyme B-galactodidase, lacY-encodes the enyzme permease, lacA encodes transacetylase
9. Explain how the lac repressor regulates transcription of the lac operon when lactose is present or absent. Which gene encodes the lac repressor and where is it located on the E. coli chromosome relative to the lac operon?
The lacI has its own promoter that is not regulated and drives consititutive transcription, when there is glucose it still constitvely transcribes
10. Describe the mechanism that prevents transcription of the lac operon when the sugar glucose is present.
There is no allolactose in the cell and the lac repressor protein binds to lacO, using its DNA binding domain, blocks RNA polymerase binding to lacP and prevents transcription initiation (it is silent)
11. The model of lac operon repression was originally based on the analysis of mutants that affect lac operon expression. Explain the phenotype that is associated with each of the following mutants: lacZ-, lacY-�, lacI-, lacIs.
lacZ-:No functional B-galactosidase, lacY-: no functional permease, lacI-:unable to bind to operator, lacls: Unable to bind the inducer (allolactose)
12. What are the two distinct binding domains of the repressor and what phenotype results from mutation of each domain?
DNA binding site which is lacI- when the repressor cannot bind to the lacO and the allosteric site, where the repressor cannot be induced by allolactose
13. What is a lacOc mutation and what is the phenotype of this mutant?
lacOc-fails to bind repressor protein, constitutively transcribes because there is no repressor to block transcriptase
14. What test did Jacob and Monod perform to distinguish between lacI- and lacOc mutants? What did they learn from this test?
postulated that lacI- is a mutation site for producing a regulatory protein, lacOc-the other is the target DNA binding site for the regulatory protein binding
15. How did molecular studies contribute to the understanding of lac operon regulation?
Observations: the repressor protein binding location at the lac operator overlaps with the promoter binding location of RNA polymerase, and three distinct segments of operator DNA sequence
1. Describe the organization of regulatory elements and structural genes of the trp operon. What is the function of the trp gene products?
Promoter (trpP), Operator (trpO) and a leader region (trpL) that contains the attenuator region, five structural genes trpE, trpD, trpC, trpB, trpA, outside the operon, a sixth gene, trpR, encodes the repressor protein that is not activated until it pairs with tryptophan
2. Describe how repression of the trp operon works.
Tryptophan acts as a corepressor by binding to and activating the trp repressor protein that is not active without its bound corepressor, operon is activated in absence of tryphophan
3. What happens when trpR- mutants are grown in the presence of high versus low levels of tryptophan?
trpR- high levels of tryptophan: slightly more than usual, 33% and if there are low levels than a lot (mutant for repressor protein, it will show constitutive transcription of operon genes, regardless of whether tryphton is present)
4. What is the difference between an inducible operon and a repressible operon? Give an example of each.
inducible: lac operon, called upon when glucose is depleted and alternative sugar is available, repressible: involved in anablic pathways, can be regulated by negative feedback mechanisms that operate through activity of the end product of the pathway to block operon gene transcription
5. What is the name of the second mechanism that regulates trp operon expression? What is the purpose of this regulation?
Attenuation-the ability to fine tune transcription to match the momentary requirements of the cell to maintain a steady state concentration
6. Where are the sequences involved in attenuation control located?
in the leader region trpL
7. Describe the distinctive features of small polypeptide encoded by the trp leader region.
Four repeat DNA sequences, and the mRNA transcript contains complementary repeats as well as start codon and stop codon sequences and 14 amino acids
8. Describe the alternative secondary structures that are capable of forming.
1-2 stem loop temporarily pauses the progress of RNA polymerase , lasts only long enough for the ribosome to bind at the start codon in trpL and begin translation of the 14-amino acid polypeptide, ribosome and RNA polymerase begin their coupled progression
9. What is the 3-4 stem loop, and how is it related to a concept that we learned about earlier?
It is a termination stem loop, signals transcription termination, halts RNA polymerase progress along the DNA, terminating transcription in the leaders region before it reaches the structural genes of the operon, followed by a poly uracil sequence like the intrinsic termination of transcription in bacteria (on sequence 4)
10. Describe what happens during translation of the leader peptide when tryptophan is present at high versus low levels. What effect does this have on secondary structures formation?
More 3-4 stem loops and fewer 2-3 stem loops at higher levels of tryptophan concentration, lower levels: more 2-3 stem loops, fewer 3-4 loops
11. What is the outcome of attenuation control when tryptophan is present at high versus low levels?
steady concentrations of tryptophan by turning the synthesis of tryptophan synthesis up or down
12. What kinds of mutations affect attenuation?
there is an importance of back to back tryptophan, base pair substitions, missense
13. Compare and contrast attenuation control at the trp operon with other amino acid biosynthetic operons.
Helps with coupling of transcription and translation while lac operon does not depend solely on the amount of tryphophan but also glucose and lactose, and a catabolite control of glucose.
1. What is a bacteriophage? What are the requirements for successful phage infection?
viruses that infect bacterial cells, successful phage infections require that the genetic regulatory switches be controlled through phage gene expression to redirect the action of host genes and that phage gene expression initiate a sequence of events leading the bacterium to participate in the expression of phage genetic information
2. Phage ? is a temperate phage, meaning that it is capable of lytic and lysogenic growth. Briefly, how do the lytic and lysogenic cycles differ from each other?
lytic cycle: lambda phage ends with lysis of the host cell (phage heads, tails, and DNA assemble into progeny phages) lysogenic cycle: integration of the phage into the host chromosome, converting the host into a lysogen
3. What happens during the initial phase of ? infection?
immediate circularization inside the host cell that is accomplished by the joining of two single stranded cohesive ends, host DNA ligase seals the two gaps that are left when cos ends join
4. How is the ? genome organized after ? circularizes?
a series of operons
5. What are the early and late genes?
Immediate early transcription (red) starts at the rightward and leftward promoters (PR and PL) that flank the repressor gene (cI); transcription stops at the rho-dependent terminators (t) after the N and cro genes, ) Delayed early transcription (blue) begins at the same promoters, but bypasses the terminators by virtue of the N gene product, which is an antiterminator, Late transcription (green) begins at a new promoter (PR'); it would stop short at the terminator (t) without the Q gene product, another antiterminator.
6. What is the molecular "tug of war" that occurs early after infection?
...
7. Describe the roles played by the early gene products N and cro.
First to be transcribed in PR and PL, expressed and produced shortly after circulation
8. What is the product of the cI gene, and what role does this protein play?
The product of the cI gene is lambda repressor and it helps lysogeny occur where there is the integration of phage and it replicates throughout the bacterial cell instead of cro protein leading to lysis, the repressor blocks that pathway
9. What determines whether ? enters the lytic or lysogenic cycle?
if the environment is nutrient rich or not, if it is, then the cro protein will be transcribed over cI because the cro protein covers the OR3 and cII will be degraded by protease, if it isnt then the cI will be transcribed and the lambda repressor will block operator 1 and 2 and transcribe more lambda
10. Which operator sequences are bound by cro and ? repressor? Describe the differences in affinity of these two proteins for operator sites.
the cro binds to the operator sequence O3, and lambda repressor O1 and O2, as for the different affinities: as cro protein concentration increases, it binds more to the O3 though it tends to have an equal affinity for O3 and O2 and not O1; binding affinity for O1 and O2 better for lambda repressor than O3
11. Which promoters are controlled by cro binding and how? What are the consequences of this regulation?
(PR) and PL sites
12. Which promoters are controlled by ? repressor and how? What are the consequences of this regulation?
(PRE)
13. What conditions bring lysogeny to an end and reinitiate the lytic cycle? What happens at the molecular level when a ? lysogen is induced?
UV induction: the process that brings lysogeny to an end and reinitates the lytic cycle, protease activity of RecA protein is also activated and targets the amino acid segment of lambda repressor monomers, allows production of cro protein, goes to lytic cycle
14. In summary, what is the crucial interaction that determines which way the genetic switch is flipped, i.e., toward lysogeny or lysis?
protein products of early genes cro and cI that compete for operator sequences
15. In summary, what levels of regulation control the expression of genes in the ? genome?
prevailance of cro or cI
1. What challenge does the size of eukaryotic genomes pose for the process transcription?
make complexes larger
2. In what ways are the processes that regulate gene expression more varied than the processes governing gene expression in bacteria?
Structural motifs-proteins are the primary sources of regulated gene expression in eukaryotes (protein secondary structures and alpha helices), functional domains-allow the proteins to identify and specific regulatory DNA sequences in the major groove or minor groove
3. In a broad sense, what are the similarities and differences in the mechanisms that control transcription in bacteria versus eukaryotes?
Similarities: there are activator proteins-bind regulatory sequences to stimulate transcription, repressor proteins-bind other sequences to stimulate transcription, differences: many larger complexes of them, there are structural motifs and functional domain
4. List four structural motifs that are found in DNA-binding regulatory proteins, and explain the function of each.
Helix-loop-helix: two alpha helices separated by a loop in each polypeptide. two polypeptides join to form a DNA-binding dimeric protein. one alpha helix from each dimer binds regulatory /DNA Leucine zipper-two alpha helices one containing multiple leucine amino acids, in each polypeptide. two polypeptides form a functional dimeric protein. The leucine-containing alpha helices face one another and interdigitate to form the "zipper" and the other alpha helix of each polypeptide binds DNA/ Homeodomain-three alpha helices form in single functional polypeptides, and the longest helix interacts with regulatory DNA sequences/ Zinc finger-protruding loops or fingers containing about 24 amino acids each contain a central molecule of zinc bound to two cysteine and two histidine amino acids. two or three zinc fingers form in each polypeptide, and each finger interacts with regulatory DNA
5. List the three types of regulatory sequences that are commonly associated with genes, explain where they are located and which proteins bind there.
enhancer sequences-great distances from the core promoter, occasionally found within genes/ cis-acting regulatory sequences- regulate transcription of genes located on the same chromosome as the sequences, not on other chromosomes
6. What is meant by cis-acting regulatory sequences and trans-acting regulatory proteins?
able to act only transcription of genes on the same chromosome, able to identify and bind to any core promoter region if the right transcriptiona factors are present
7. Which aspects of transcription are controlled by enhancers?
controls the timing and location of eukaryotic gene transcription to ensure the proper function and development of organisms; control the expression of genes in specific tissues or cell types, producing tissue-specifc patterns of polypeptide production
8. How many binding sites for regulatory proteins are found in the SV40 enhancer?
seven
9. What was learned from comparing the sequences of the ?-interferon enhancer of different mammals?
fewer than half of the conserved sequences bind proteins, those conserved sequences may have their diversitification constrained by the requirement that they be efficiently recognized by regulatory proteins.
10. Describe the organization and function of yeast genes that are required for galactose metabolism. How is the expression of these genes regulated?
GAL genes regulated by Gal4, which is a transcription activator protein that binds to an enhancer like element (UPS), binding to Gal80 helps block transcription, binding of Gal3 to Gal80 helps Gal4 bind to UAS to aide transcription of GAL genes
11. What is the function of the locus control region (LCR) that is associated with the ?-globin gene complex?
highly specialized enhancer elements that regulate the transcription of multiple genes packaged in complexes of closely related genes, orchestrate the sequential developmental expression of the B-globin complex genes
12. How do eukaryotic repressors, like the one at the yeast GAL1 gene, work?
They block transcription by directly preventing enhancer mediated transcription
13. How can the same sequence act as an enhancer and a silencer?
depends on the regulatory proteins that bind the sequence , sometimes DNA loops formation can bring even very distant sequences together
14. Explain how transcription factors and enhancers regulate the expression of the SHH (Sonic hedgehog) gene.
The brain specific enhancer is bound by brain specific transcription factors and activates SHH transcription in brain cells; different, limb specific enhancer binds different, limb specific transcription factors to express SHH differently in limb cells
15. Describe a mechanism that prevents enhancers from inappropriately activating nearby genes.
insulators-cis-acting sequences located between enhancers and promoters of genes that are to be insulated from the effects of the enhancer
16. What explains the fact that some thalassemia patients that mutations in either the ?-globin or ?-globin genes?
due to deletion or chromosome rearranement mutations that alter the LCR of one of the globin gene complexes , result in enhancer mutations that alter the level of transcription of affected genes, lead to an imbalance of polypeptide production
17. What kinds of mutations can disrupt enhancer function?
base substituion mutations on enhancers, also frameshift
18. Considering the fact that eukaryotic DNA is packaged into chromatin, how do transcription factors gain access to the DNA? Describe three mechanisms.
regulatory sequences are not tightly bound by histones, which thus allow more or less direct entry to the regulatory DNA, proteins called chromatin remodelers can enzymatically change the distribution or composition of histone octamers (nucleosomes), chromatin modifiers can enymatically modify histones by adding or removing methyl or acetyl groups at specific amino acid residues, most commonly lysine
19. What is the expression pattern of genes with open promoters? Why are these promoters called "open"?
cause genes to be consecutively transcribed, have nucleosome depleted regions, constrain few nucleosomes that lies immediately upstream of the start of transcription, do not contain a TATA box, histone 2A protein is readily modified for removal from the transcription start site at transcription initation, allowing transcription to start
20. What is the expression pattern of genes with covered promoters? What has to happen for transcription to occur at this type of gene?
characterize genes whose transcription is regulated, transcription is blocked until nucleosomes are displaced of removed from the promoter sequence, to allow ACT to bind
21. What experimental technique can be used to determine if chromatin is open or closed? What result is observed with closed versus open chromatin?
Assessing the sensitivity of the region to the DNA digesting enzyme DNase I, this enzyme randomly cuts DNA in open chromatin regions but is not able to do so where chromatin is closed; regions of open chromatin, sensitive to DNase I digestion are known as DNase I hypersensitive sites
22. Generally speaking, how do chromatin remodelers affect chromatin structure?
the protein complexes that carry out chromatin remodeling by moving nucleosomes in two principal ways: slide nucleosomes along DNA (spool around nucleosomes until promoter or enhancer sequences are free from their connection. And they can relocated from one DNA molecule to another.
23. Briefly describe the functions of three chromatin remodeling complexes.
SWI/SNF Complex: pronounced "switch-sniff
24. Generally speaking, how do chromatin modifiers affect chromatin structure?
chemically modify histone proteins in the nucelosomes by adding or removing specific chemical groups strength of association between nucleosomes and DNA
25. Briefly describe the functions of three chromatin modifiers, including information about their amino acid targets. Then explain how each type of modification affects chromatin structure.
histone acetyltransferases-most common chemical modification associated with the opening of chromosome structure is the addition of acetyl groups (COCH3), add acetyl groups to specific positively charged amino acids in N terminal tails of histones; histone deacetylases-a modification associated with the closing of chromatin structure; histone methyltransferase-addition of methyl (CH3) (lysine main target)
26. Explain how expression of the yeast PHO5 gene is regulated.
transcription is activated by phosphate starvation, repressed when phosphate level is high
27. What does the term "epigenetic" mean? How can epigenetic states be maintained through cell division?
resulting from the distinct chromatin states affecting gene transcription in specific types of cells
28. In what situations is it important to maintain epigenetic states and when do epigenetic programs need to be reset?
...
29. What is genomic imprinting? Describe how this mechanism controls expression of H19 and IGF2. What is unusual about the expression pattern of these genes?
kind of resetting of genetic patterns in meiosis,
30. Which nucleotide is commonly methylated in mammals? At what sites does this occur? How does this affect chromatin structure and transcription?
CpG dinucleotides, clustered at promoters, unmethylatred-allows access by transcription factors and RNA polymerase, and vice versa for methylation
1. Define the term genomics.
the scientific study of biological processes from the perspective of the whole genome, originated in the human genome project
2. Briefly compare and contrast the strategy that is used in clone-by-clone versus whole-genome shotgun (WGS) sequencing.
Clone-by-clone sequencing-each chromosome is first broken into overlapping clones that are then arranged in linear order to produce a physical map of the genome, each clone in the map is sequenced seperately (relies on availability of specific genetic resources) while Whole genome shotgun sequencing (WGS) involves DNA representing the entire genome fragmented into smaller pieces, and a larger number of fragments are chosen at random and sequenced
3. What is the purpose of sequencing DNA libraries with three different sizes of inserts in WGS sequencing?
Provides information on whether two particular sequences are physically linked and the approximate distance between two sequences, even if repetitive DNA occurs between the paired end sequences, they can still be linked into a scaffold. dispersed repetitive DNA in the genome often consist of simple, short ends or transposable element sequences (repetitive sequences longer than the largest available clones cannot be spanned, cause gaps between contigs)
4. What does the process of genome annotation involve?
the process of attaching biological functions to DNA sequences, identifies the location of genes and other functional sequences within the genome sequence-biochemical, cellular, and biological function ( use clones cDNA representing all the genes from an organism) partial cDNA sequnce reads are called expressed sequence tags (ESTs)
5. How were cDNA clones and bioinformatics approaches useful in genome annotation?
cDNA clones are useful because they are compared with the genomic sequences to identify the parts of the genome that undergo transcription leading to the production of RNA molecules and bioinformatics is the use of computational approaches to decipher DNA sequence information, predict gene structure by identifying open reading frames-sequences that have the potential to code for polypeptides, search for ORFs larger than 50 amino acids
6. Why can it be difficult to identify genes in large eukaryotic genomes?
Because exons are often small relative to introns and are dispersed over large distances
7. What approaches are used to annotate the biological functions of genes?
Genes that are similar to each other in sequence are assumed to encode gene products with similar biochemical function
8. What is the correlation between gene density and organismal complexity?
a high gene density is attributable to the lack of introns, the more compact size of regulatory sequences, and the generally less complex structures of most encoded proteins, and encode fewer genes than those of multicellular organism, species that have evolved to be obligate parasites often experience genome contraction (lose genes they no longer need)
9. How many genes are found in the human genome, and how does this compare to the number of genes in Drosophila?
20,000-25,000 and for Drosophila 14,000
10. Explain how BLAST can be used to search for homologous sequences.
searches for homologous sequences (derived from common ancestral genes) searches that can be performed: nucleotide blast: a nucleotide query sequence is compared to nucleotide sequences in the database; tblastn: a protein query sequence is compared with the nucleotide databases, hypothetically translated into all six potential reading frames, tblastx-a nucleotide query sequence is translated into all six possible reading frames and compared againist the nucleotide sequences in the database that have also been translated into all six possible reading frames
11. Compare the human genome with other primates.
humans have 46 chromosomes while other primates have 48 chromosomes, difference of a pair of chromosomes or segments fused to form a single chromosome, there is still synteny
12. What types of nucleotide sequence polymorphisms are the main sources of human genetic diversity?
SNPS (single nucleotide polymorphisms)
13. How does the level of genetic diversity in humans compare to other species? What is this interpreted to mean?
genetic diversity among humans is relatively low compared to other species, homo sapiens is a comparatively young species and that populations have not had much time to diverge from one another
14. What is a microarray?
Consist of collections of synthesized DNA fragments, attached to a solid support and representing sequences present in a genome, specific DNA sequences are chemically synthesized on a silicon substrate, called a chip, at a high density, all located on a different spot.
15. Compare and contrast the information obtained using expression microarrays, high throughput cDNA sequencing, and Northern blotting.
Northern blotting is used to analyze gene expression, expression array-carries unique sequences from every annotated gene of the genome, only limited by the degree to which mRNA can be extracted from specific cells or tissues/ high throughput cDNA sequencing-Rna is isolated from the cells of interest and converted into cDNA, which is fragmented and sequenced
1. What is the definition of recombinant DNA technology? What is the major challenge of this approach?
The set of techniques developed for amplifying, maintaining, and manipulating specific DNA sequences in vitro as well as in vivo, the major challanges are the identification of specific DNA sequences and their manipulation in vitro
2. Describe the common features of restriction enzyme cleavage sites. What types of DNA fragment ends are typically generated upon cleavage?
Each restriction enzyme recognizes a specific restriction sequence at which it cuts both strands of the sugar-phosphate backbone of the DNA, cleaving the restriction sequence in the same way each time it is encountered. Sticky ends-single stranded segments at the ends of each fragment that stick to a complementary base pair sequence by hydrogen bonding (5' overhangs or with 3' overhangs)
Blunt ends-lack a single stranded segment
3. What is the purpose of using a cloning vector?
it is a carrier fragment of DNA that will allow amplification in a biological system, can amplify DNA making many identical copies called DNA clones, can be analyzed by a variety of techniques
4. What are the three basic steps in molecular cloning?
1. The joining together of the cloning vector and a donor DNA fragment to produce a recombinant clone
2.Selection of organism containing copies of the cloned DNA segment of interest
3. Amplification of the recombinant clone in a biological system
5. What is the difference between recombinant and nonrecombinant vectors?
recombinant DNA molecules have a single piece of source DNA is combined with a single cloning vector molecule while non-recombinant vectors is where the ends of vectors rejoin with each other rather than incorporating a donor insert
6. Name three features of plasmids that facilitate cloning.
Directional cloning-use two restriction enzymes with different compatible ends, inserts DNA fragments possessing the two different compatible ends will be efficiently cloned into the vector
linkers-makes blunt ends into sticky ends by ligation of short oligonucleotides
multiple cloning site (MCS)-a site that has several different restriction enzyme sites into which DNA can be cloned
7. How are recombinant DNA molecules amplified?
DNA is mixed with E.coli in a test tube, the bacteria are chemically treated with divalent cations (Ca+) or an electrical shock to open pores in their membranes, thus making the bacteria competent to take up exogenous DNA by transformation
8. Describe several other types of vectors that can be used in cloning besides plasmids. What are the advantages of these vectors?
Bacteriophage vectors-cos sites in the lambda genome will interact with lambda genomes into discrete phage particles in vitro. DNA can be replicated as a plasmid in the bacterium, it is the replication of the phage within the bacterium that amplifies the recombinant DNA molecule
Artificial chromosomes-YACS-first artifical chromosomes developed and are used as cloning vectors in S.cerevisiae, contains sequences corresponding to a centromere, telomeres, a selectable marker, and a cloning site (accepts 200kb to 2 megabases)
BACs-have a smaller insert capactiy, but can be used in E.coli rather than yeast as a host, contain an origin of replication, a selectable marker gene, and an MCS, derived from the F-factor plasmid
9. What is the advantage of using molecular cloning instead of PCR to amplify DNA? What are the advantages of using PCR?
Molecular cloning does not involve prior knowledge for a specific DNA sequence to be cloned, but PCR is fast and inexpensive enzymatic method for amplifying specific DNA sequences, but you need prior knowledge of the DNA sequence being amplified
10. Compare and contrast genomic libraries and cDNA libraries.
Genomic libraries-derived from the genomic DNA of an organism,exons, introns of genes, the regulatory sequences controlling gene expression, intergenic sequences (noncoding sequences between genes) and repetitive sequences such as centromeres, telomeres, ribosomal DNA,transposons, retroelements). cDNA (complementary DNA libraries)-derived from mRNA, represent the DNA sequences that are transcribed in the tissue from which the mRNA is derived
11. Describe the steps involved in making a genomic library. What is the purpose of partially digesting DNA with a restriction enzyme?
Steps in making a genomic library: a single individual is isolated and fragmented into smaller pieces that are then ligated into cloning vectors. The recombinant vectors are transformed into bacteria or used to infect bacteria that grow into colonies or plaques that collectively contain clones representing the entire genome
Purpose of partially digesting DNA with a restriction enzyme: leads to random fragmentation, use of less restriction enzyme than would be needed to cut the DNA at every restriction sequence, generates random, large genomic DNA fragments with sticky ends
12. Describe the steps involved in making a cDNA library. Why does the composition of a cDNA library vary, depending on the tissue from which the mRNA was extracted?
cloning of mRNA sequences can be accomplished by synthesizing a double stranded cDNA copy of the mRNA through reverse transcriptase and then ligating the cDNA into a vector.
Genes that are highly expressed are represented in the mRNA at a higher frequency than genes expressed at a lower level, and genes not expressed in the tissue of origin are not represented
13. What sequences are missing from cDNA libraries?
lack intronic and intergenic sequences
14. Describe the process used in screening DNA libraries to identify specific clones. Compare and contrast this process to Southern blotting.
In southern blotting, single stranded DNA fixed to a solid support membrane is probed with a labeled, single stranded DNA. , similarly, a labeled probe is mixed with cloned DNA fragments from a library-a membrance is laid on top of the bacterial colonies grown on a petri dish, membrance bound bacteria are lysed and DNA is denatured.
15. What is a transgenic organism? What challenges are associated with creating a transgenic organism?
the introduction of a gene from one organism into the genome of another organism, the challenges: the need to introduce DNA into a cell in such a way that the DNA integrates into the genome, the need to provide appropriate regulatory sequences so that the transgene will be properly expressed
16. What is an expression vector?
vectors that have been furnished with sequences capable of directing efficient transcription and translation of transgenes
17. Which regulatory sequences must be present in order for a transgene to be properly expressed in bacteria?
promoter sequences that bind RNA polymerase upstream of the MCS, shine dalgarno sequence
18. How can the expression of a heterologous gene be regulated in bacteria?
by the addition or removal of inducer compounds
19. Describe two factors that complicate the efficient production of transgenic products.
efficiency of translation, use of specific codons,post translational modification many proteins must undergo in order to function
20. What features must be present on eukaryotic expression vectors?
sequences for the regulation of transcription (TATA Box), enhancer sequences for qualitative and quantitative control of transcription, and polyadenylation and transcription termination signals
21. Describe the strategy that was used to produce insulin in bacteria.
synthetic genes were cloned into separate plasmid vectors
22. Give several examples of other proteins used in industrial processes that are produced in bacteria.
HGH extracted from the pituitary glands of human cadevars and insulin extractions, proteases in laundry detergent
23. Briefly describe the strategy that is used to create genetically modified plants. Which traits are commonly engineered into transgenic crops? What type of genetic engineering is being done with rice?
Reengineering of Ti plasmid seperates sequences responsible for transfer of T-DNA from the T-DNA itself, disarmed plasmid contains genes required for virulence and conjugative transfer, genes on disarmed plasmid produce conjugative and virulence proteins that act in trans on T-DNA border sequences of transformation vector to effect transfer of T-DNA into plant cells, grown into transgentic plants