Unit 3 (Week 11 Genetic Technologies and Genomics) Flashcards
1
Q
What is hemophilia A and why does it happen?
A
A blood clotting disorder that is inherited as an X-linked recessive trait.
2
Q
What protein is in hemophilia A which is needed in a pathway required for normal blood clotting?
A
Factor VIII
3
Q
How is factor VIII made if it is not made from humans?
A
Purified factor VIII is made by cells grown in a laboratory. They are genetically modified to synthesize factor VIII in large amounts.
4
Q
What do you call the use of laboratory techniques to bring together fragments of DNA from multiple sources?
A
Recombinant DNA technology
5
Q
What was some first successes in making recombinant DNA molecules?
A
In the early 1970s, independent groups at Stanford University: David Jackson, Robert Symons, and Paul Berg and another group, Peter Lobban and A. Dale Kaiser.
Both groups were able to isolate and purify pieces of DNA in a test tube and then covalently link two or more DNA fragments. Once inside a host cell, the recombinant molecules were replicated to produce many identical copies.
6
Q
What is the process of making multiple copies of a particular gene?
A
Gene cloning
7
Q
The use of laboratory techniques to isolate and manipulate fragments of DNA is known as _____ DNA technology.
A
Recombinant
8
Q
The introduction of recombinant DNA molecules into living cells where the molecules are replicated to produce many identical copies is a process known as ______.
Multiple choice question.
stem cell therapy
gene cloning
DNA sequencing
complementation
A
gene cloning
9
Q
The molecular analysis of the entire genome of a species is defined as _______.
A
Genomics
10
Q
A goal of gene __________ is to gather many copies of a gene of interest in order to study DNA directly or to use the DNA as a tool.
A
Cloning
11
Q
What is a good example as to why someone was to clone genes?
A
For example, geneticists may want to determine the sequence of a gene from a person with a disease to see if the gene carries a mutation.
12
Q
Why would researchers want to clone genes to gather large amount of gene product like a protein?
A
For example, biochemists use gene cloning to obtain large amounts of proteins to study their structure and function. In recent years, gene cloning has provided the foundation for critical technical advances in a variety of disciplines, including molecular biology, genetics, cell biology, biochemistry, and medicine.
13
Q
What is one way to carry out gene cloning that consists of making a carrier of the DNA segment that is to be cloned? This may carry a small segment of chromosomal DNA, perhaps only a single gene.
A
A vector or Vector DNA
By comparison, a chromosome carries a few thousand genes.
14
Q
Where were vectors used in gene cloning originally derived from in two natural sources?
A
Plasmids and Viruses
15
Q
What are small, circular pieces of DNA that are found naturally in many strains of bacteria and exist independently of the bacterial chromosome?
A
Plasmids.
Commercially available plasmids have been genetically engineered for effective use in cloning experiments. They contain unique sites into which geneticists can easily insert pieces of chromosomal DNA.
16
Q
What is derived from viruses, which can infect living cells and propagate themselves by taking control of the host cell’s metabolic machinery?
When a chromosomal gene is inserted into a ______ _______, the gene is replicated whenever the ______ DNA is replicated. Therefore, viruses can be used as vectors to carry other pieces of DNA.
A
Viral Vectors; viral
17
Q
What are some separation techniques used in gene cloning?
A
Chromatography and centrifugation.
18
Q
In the second step of gene cloning, what must you do next in the experiment?
A
Insertion of the gene of interest into the vector.
This step creates the recombinant vector.
19
Q
What is a restriction enzyme?
A
An enzyme that recognizes a particular DNA sequence and cleaves the DNA backbone at two sites.
20
Q
Who discovered the restriction enzymes which were found to be made naturally by many different species of bacteria as protection mechanism against invading viruses by degrading the viral DNA into small fragments?
A
Werner Arber, Hamilton Smith, and Daniel Nathans in the 1960s and 1970s.
Several hundred different restriction enzymes from various bacterial species have been identified and are commercially available to molecular biologists.
21
Q
What, in gene cloning experiments, can bind to a specific base sequence and then cleave the DNA backbone at two defined locations, one in each strand?
A
Restriction enzymes
22
Q
How does restriction enzymes digest DNA?
A
Into fragments with single-stranded ends (termed “sticky” ends) that hydrogen-bond to other DNA fragments that are cut with the same enzyme and thus have complimentary sequences.
23
Q
What is the process called when vector DNA and chromosomal DNA pieces are hydrogen-bonded together at complimentary bases to form a recombinant vector?
A
Annealing
24
Q
What must be completed after annealing to stabilize the vector and create a permanent connection?
A
The sugar-phosphate backbones of DNA strands must be covalently linked, or LIGATED.
This linkage is catalyzed by DNA ligase which fills the gaps in the backbone.
25
What are the general properties of plasmids?
Plasmids are small, circular DNA molecules that exist independently of the bacterial chromosome. They have their own origin of replication. Many plasmids carry genes that convey some type of selective advantage to the host cell, such as antibiotic resistance.
26
What happens when the two ends of the vector simply ligate back together restoring it to its circular structure?
Forms a recircularized vector.
27
[Review] What is a vector containing a piece of chromosomal DNA?
Recombinant vector
28
What is the third step of gene cloning?
The actual cloning of the gene of interest.
The hope is for bacteria to take up the recombinant vector carrying the gene of interest which have been rendered permeable to DNA molecules using agents.
29
What do you call it when a bacteria has been rendered permeable by agents to take up recombinant vectors?
A process called transformation.
30
What do we call bacterial cells with the ability to take up DNA?
Competent
The term used to describe bacterial strains that have the ability to take up DNA from the environment.
31
In this cloning experiment, what is the purpose of having the lacZ gene in the vector?
The insertion of chromosomal DNA into the vector disrupts the lacZ gene, thereby preventing the expression of β-galactosidase. The functionality of lacZ can be determined by providing the growth medium with a colorless compound, X-Gal, which is cleaved by β-galactosidase into a blue dye. Bacterial colonies containing recircularized vectors form blue colonies, whereas colonies containing recombinant vectors carrying a segment of chromosomal DNA will be white.
32
The bacterial cells were originally sensitive to ampicillin, yet, the vector which is a plasmid, carries an antibiotic-resistance gene called the ampR gene. What is the purpose of this gene in a cloning experiment?
This gene is called a SELECTABLE MARKER - because the presence of the antibiotic in the medium selects for growth of cells expressing the ampR gene.
Definition - A gene whose presence can allow organisms (such as bacteria) to grow under a certain set of conditions. For example, an antibiotic-resistance gene is a selectable marker that allows bacteria to grow in the presence of the antibiotic.
33
What does the ampR gene encode?
It encodes an enzyme known as B-lactamase that degrades the antibiotic amplicillin, which normally kills bacteria.
As such, bacteria that have not taken up a plasmid are killed by the antibiotic.
In contrast, any bacterium that has taken up a plasmid carrying the ampR gene grows and divides many times to form a visible bacterial colony containing tens of millions of cells.
34
How is the functionality of lacZ gene determined?
By adding to the growth medium a colorless compound, X-Gal, which is cleaved by B-galactosidase into blue dye.
Bacteria grown in the presence of X-Gal form blue colonies if they produce a functional β-galactosidase enzyme, and white colonies if they do not.
The lacZ gene is active = blue colonies
The lacZ gene is inactive = white colonies
35
How often do bacterial cells divide?
Every 20 minutes and cells can have more than one copy of recombinant vector.
36
The use of what can actually produce many different types of DNA fragments?
Restriction enzymes.
37
What is created after all of the fragments of DNA are ligated individually to vectors and now the researchers has a collection of many recombinant vectors, with each vector containing a particular fragment of chromosomal DNA of a given organism?
DNA library
A collection of recombinant vectors, each containing a particular fragment of DNA from a given organism.
38
What is a type of DNA library in which the inserts are derived from chromosomal DNA?
Genomic Library
39
What is used after isolating mRNA to make DNA molecules when this isolated mRNA is used as a starting material?
Enzyme reverse transcriptase
40
What is the library called when reverse transcriptase enzyme is used to take mRNA, as a starting material, and convert it to DNA?
Complementary DNA, cDNA, or cDNA library.
41
What is the advantage of cDNA libraries?
It lacks introns.
Introns are intervening sequences that do not translate into proteins. Because they are so large, it is much simpler for researchers to insert cDNAs into vectors rather than chromosomal DNA segments if they want to focus their attention on the coding sequence of a gene.
42
What is a second advantage of creating a cDNA library over a genomic library?
Since bacteria do not splice out introns, using cDNAs provides an advantage if researchers want to express the gene of interest in bacteria.
43
What is a technique used to separate macromolecules by applying an electric field that causes them to migrate through a gel matrix?
Gel electrophoresis.
This method is often used to evaluate the results of a cloning experiment. For example, gel electrophoresis is used to determine the sizes of DNA fragments that have been inserted into recombinant vectors.
44
How does gel electrophoresis separate macromolecules such as proteins or DNA?
Based on their charge, size/length, and mass.
45
[Focus Electrophoresis]
Explain the process when separating different fragments of DNA based on mass.
The flat slab of semisolid gel has depressions at the top called wells, where samples are added. Electrodes are located at each end of the gel. An electric current is applied to the gel, which causes charged molecules, either proteins or nucleic acids, to migrate from the top of the gel toward the bottom—a process called electrophoresis.
DNA is negatively charged and moves toward the positively charged electrode, which is at the bottom in this figure. As gel electrophoresis occurs, the DNA fragments are separated into distinct bands within the gel. Smaller DNA fragments move more quickly through the gel than larger ones in a given amount of time and therefore are located closer to the bottom of the gel than the larger ones. The fragments in each band can then be stained with a dye for identification.
46
One DNA fragment contains 600 bp and another has 1,300 bp. Following electrophoresis, which will be closer to the bottom of the gel?
The 600-bp fragment will be closer to the bottom. Smaller pieces travel faster through the gel.
47
What is another technique in cloning that DOES NOT use vectors and host cells to copy DNA that was developed by Kary Mullins in 1985?
Polymerase Chain Reaction (PCR)
A technique to make many copies of a gene in vitro; primers are used that flank the region of DNA to be amplified.
48
What is the goal of PCR?
To make many copies of DNA in a defined region, perhaps encompassing a gene or part of a gene.
Several reagents are required for the synthesis of DNA.
49
What are the two primers needed in PCR that are complementary to sequences at each end of the DNA region to be amplified and are about 20 nucleotides long?
A forward primer and a reverse primer.
50
What else does PCR need which is a heat stable form of DNA polymerase and is isolated from the bacterium Thermus aquatics, which lives in hot springs and can tolerate temperatures up to 95 degrees Celsius?
Taq polymerase. T= THermus.... aq = aquatics
Remember: Archaea live in hot springs.
A heat-stable form of DNA polymerase is necessary because PCR is conducted at high temperatures that would inactivate DNA polymerase from most other bacteria.
51
Other than the two primers and Taq polymerase, what is the last thing PCR needs?
All four deoxynucleoside triphosphates (dNTPs)
52
Why do the primers used in PCR bind specifically to the primer-annealing sites?
The primers are complementary to sequences at each end of the DNA region to be amplified.
53
[Information] 3-Steps of PCR
To make copies of a DNA region, the following three steps occur:
A sample of chromosomal DNA, called the template DNA, is heated to separate (denature) the DNA into single-stranded molecules.
The primers bind to the DNA as the temperature is lowered. The binding of the primers to the specific sites in the template DNA is called primer annealing.
After the primers have annealed, the temperature is slightly raised and Taq polymerase uses dNTPs to catalyze the synthesis of complementary DNA strands, thereby doubling the amount of DNA in the region that is flanked by the primers. This step is called primer extension because the length of the primers is extended by the synthesis of DNA.
None
54
Why is this method using Taq polymerase called a chain reaction?
This method is called a chain reaction because the products of each step are used as reactants in subsequent steps. A device that controls the temperature and automates the timing of each step, known as a thermocycler, is used to carry out PCR. The PCR technique can amplify a sample of DNA by a staggering amount. After 30 cycles of denaturation, primer annealing, and primer extension, a DNA sample will have increased by 230, approximately a billionfold, in a few hours!
55
A vector could also be referred to as a
Multiple choice question.
DNA carrier
retrovirus
piece of cloned DNA
DNA carrier
56
Recombinant DNA technology is the use of laboratory techniques to bring together fragments of __________ from multiple sources.
DNA
57
In cloning experiments, a(n) ___________
| may carry a small segment of chromosomal DNA, perhaps only a single gene.
vector or plasmid
58
The process of making multiple copies of a particular gene is best described as gene ________
cloning
59
Select all that apply
Select all of the following that are vectors commonly used in gene cloning experiments.
Multiple select question.
viruses
mouse cells
restriction enzymes
plasmids
viruses
plasmids
60
The second step in a gene cloning experiment is the insertion of the gene of interest into a(n) ________-.
vector or plasmid
61
Gene cloning allows you to produce a lot of copies of a gene and also to obtain a large amount of the gene product, such as a specific _______.
mRNA, protein, or RNA
62
Restriction enzymes are most commonly produced in nature by species of which of the following?
Multiple choice question.
Archaea
Animals
Bacteria
Plants
Bacteria
63
A carrier of a DNA segment to be cloned is termed a(n) _______ .
vector
64
Bacteria use restriction enzymes to protect themselves from _______.
viruses, bacteriophages, phages, foreign DNA, virus, bacteriophage, or phage
65
A limitation of vectors is that they ______.
Multiple choice question.
can only carry DNA into prokaryotic cells
can only carry small segments of DNA
cannot replicate within host cells
can only carry small segments of DNA
66
Restriction enzymes work by _________
to a specific base sequence and then __________
the DNA backbone in two locations, one on each strand.
binding, annealing, or attaching
cleaving, cutting, cleave, breaking, or severing
67
The third step of gene cloning involves treatment of host cells with agents to render them ______ to DNA so that they may incorporate the recombinant plasmid. This process is called ______.
Multiple choice question.
permeable ; transduction
permeable ; transformation
impermeable ; transduction
impermeable ; transformation
permeable ; transformation
68
In gene cloning, what is the next step after the vector and chromosomal DNA are cut with a restriction enzyme?
Multiple choice question.
Separating the DNA using gel electrophoresis
Determining colonies that contain recombinant vectors
Inserting the gene of interest into the vector
Allowing the recombinant vector to propagate
Inserting the gene of interest into the vector
69
In cloning experiments, there is a need to identify cells that have taken in a specific plasmid. That is why plasmids are designed to contain ______ _________, such as those for antibiotic resistance.
Blank 1: selectable
| Blank 2: markers or marker
70
The ampicillin-resistance gene is an example of a(n)
__________ marker that can be used to distinguish between cells that contain plasmid DNA and cells that do not contain plasmid DNA.
Selectable
71
The antibiotic-resistance gene ampR is used as a selectable marker. Cells with the ampR gene can grow in the presence of ampicillin, which means that they ______.
Multiple choice question.
have successfully taken up the plasmid vector
can synthesize ampicillin
mutated to resist ampicillin
have ampicillin resistance genes in their genome
have successfully taken up the plasmid vector
72
Why is ampicillin included in the media used to grow bacterial cells that have been exposed to vectors?
Multiple choice question.
To keep the media free of other bacteria
To determine if the bacterial cell took up the vector or not
To discourage the growth of the transformed cells
To prevent the bacteria from breaking down the vector
To determine if the bacterial cell took up the vector or not
73
Why are complementary DNA libraries simpler for researchers to use?
Multiple select question.
Isolating mRNA is easier than DNA.
The fragments lack introns.
The vectors are easier to express in bacteria.
The vectors are easier to insert into bacteria.
The fragments lack introns.
The vectors are easier to express in bacteria.
The vectors are easier to insert into bacteria.
74
If a recombinant vector is introduced successfully into a host cell, the function of the lacZ gene will be disrupted and the cell will lose the ability to degrade ______ to a blue color.
Multiple choice question.
X-Gal
Y-Glu
Y-Gal
Z-Lac
X-Glu
X-Gal
75
Select all that apply
Gel electrophoresis is used to separate which of the following items on a gel?
Multiple select question.
proteins
carbohydrates
lipids
DNA
proteins
DNA
76
By comparing the movement of a molecule of known size with the movement of a molecule of unknown size during gel electrophoresis it is possible to ______.
Multiple choice question.
isolate the molecule from the gel
duplicate the unknown molecule many times
determine the size of the unknown molecule
combine the two molecules
determine the size of the unknown molecule
77
This technique is used to copy DNA without the aid of a vector:
Multiple choice question.
gel electrophoresis
cell culture
polymerase chain reaction
polymerase chain reaction
78
Complementary DNA (cDNA) libraries are preferred by researchers when studying the expression of the gene of interest because these libraries contain gene sequences that lack _________.
introns, intervening sequences, intron, or intervening sequence
79
What are genomic methods aimed at studying the expression of a genome?
Functional genomics
80
What is the procedure used to determine the base sequence of DNA?
DNA sequencing
81
What is one type of DNA sequencing, developed by Frederick Sanger in 1977, that is based off of our knowledge of DNA replication?
Dideoxy chain-termination method or more simply, dideoxy sequencing.
82
Explain how dideoxy chain-termination method or dideoxy sequencing works.
1. Typically, DNA polymerase connects adjacent deoxynucleoside triphosphates (dNTPs) by catalyzing a covalent linkage between the phosphate group at the 5′ position on an incoming nucleotide and the —OH group at the 3′ position on the growing strand.
2. Chemists create a different dNTP called a ddNTP. This ddNTP has a H instead of -OH which prevents the strand from growing longer hence chain-termination.
ddNTP = dideoxynucleoside triphosphate
83
What happens when a ddNTP is incorporated into a growing DNA strand?
If a ddNTP is added to a growing DNA strand, the strand can no longer grow because the 3′ —OH group, the site of attachment for the next nucleotide, is missing.
84
What is a technology used to monitor the expression of thousands of genes simultaneously?
DNA microarray
85
With the DNA microarray, what is typically on the small silica, glass, or plastic slide?
Dotted with many different sequences of single-stranded DNA, each corresponding to a short sequence within a known gene.
86
T/F Each spot on a microarray contains multiple copies of a known DNA sequence.
True.
For example, one spot in a microarray may correspond to a sequence within the β-globin gene; another might correspond to a different gene, such as a gene that encodes a glucose transporter. A single slide contains tens of thousands of different spots in an area the size of a postage stamp. These microarrays are typically produced using a technology that “prints” spots of DNA sequences onto a slide, similar to the way that an inkjet printer deposits ink on paper.
87
What is the purpose of using a DNA microarray?
the goal is to determine which genes are transcribed into mRNA from a particular sample of cells. In other words, which genes in the genome of these cells are expressed?
88
If a fluorescent spot appears on a microarray, what information does this provide regarding gene expression?
A fluorescent spot identifies a cDNA that is complementary to a particular DNA sequence. Because the cDNA was generated from mRNA, the fluorescence identifies a gene that has been transcribed in a particular cell type under a given set of conditions.
89
[Information of Application of DNA Microarrays]
Cell-specific gene expression
A comparison of microarray data using cDNAs derived from mRNAs of different cell types can identify genes that are expressed in a cell-specific manner.
Gene regulation
Because environmental conditions play an important role in gene regulation, a comparison of microarray data using cDNAs derived from mRNAs from cells exposed to two different environmental conditions may reveal genes that are induced under one set of conditions and repressed under another set.
Elucidation of metabolic pathways
Genes that encode proteins that participate in a common metabolic pathway are often expressed together and can be revealed from a microarray analysis.
Tumor profiling
Different types of cancer cells exhibit striking differences in their gene expression profiles, which can be revealed by a DNA microarray analysis. This approach is gaining use as a tool to classify tumors that are sometimes morphologically indistinguishable.
Genetic variation
A mutant allele may not hybridize to a spot on a microarray as well as a wild-type allele. Therefore, microarrays are gaining use as a tool for detecting genetic variation. This application has been used to identify disease-causing alleles in humans and to identify mutations that contribute to quantitative traits in plants and other species.
Microbial strain identification
Microarrays can distinguish between closely related bacterial species and subspecies.
None
90
What system has the components to be used to alter genes, an approach called gene editing?
CRISPR-Cas
Typically provides bacteria with a defense against bacteriophages.
91
What is an experimental technique to introduce mutations into genes?
CRISPR-Cas technology
Researchers have made a modification to the natural system to make it efficient for gene editing.
They create a single RNA in which tracrRNA and crRNA are linked to each other. This is called the single guide RNA (sgRNA).
92
What is the spacer region of the sgRNA designed to be?
Complementary to one of the DNA strands of a target gene that a researcher wants to edit.
93
What does the sgRNA bind to?
Cas9 which guides the sgRNA to the target gene.
Cas9 then makes a double-strand break in the gene.
94
How is the sgRNA different from certain components of the bacterial defense system described in Chapter 13 (refer back to Figure 13.8)?
The sgRNA is composed of two different components of the bacterial defense system, crRNA and tracrRNA, which have been linked together.
95
DNA microarrays allow researchers to ______.
Multiple choice question.
make sure that all genes are expressed
determine what gene is expressing the most protein
measure how many proteins one gene makes
monitor the expression of many genes
monitor the expression of many genes
96
The primers used in PCR are usually about ______ in length.
Multiple choice question.
20 nucleotides
5 nucleotides
200 nucleotides
100 nucleotides
50 nucleotides
20 nucleotides
97
Each spot in a(n) ________ slide contains multiple copies of a known DNA sequence.
microarray
98
CRISPR-Cas technology is used to
Multiple choice question.
mutate genes.
clone genes.
sequence genes.
separate genes.
mutate genes.
99
This technique is used to copy DNA without the aid of a vector:
Multiple choice question.
cell culture
polymerase chain reaction
gel electrophoresis
polymerase chain reaction
100
In the CRISPR-Cas technique, the target gene is recognized by ______.
Multiple choice question.
leucine zippers
telomeric RNA
restriction enzymes
Taq DNA polymerase
single guide RNA
single guide RNA
101
Why are researchers interested in the genomes of bacteria and archaea? (4)
Bacteria cause many different diseases that affect humans as well as other animals and plants. Studying the genomes of bacteria reveals important clues about the process of infection, which may also help us find ways to combat bacterial infections.
The knowledge that is obtained by studying bacterial and archaeal genomes often applies to larger and more complex organisms.
A third reason is evolution. The origin of the first eukaryotic cell probably involved a union between an archaeal cell and a bacterial cell. The study of bacterial and archaeal genomes helps us understand how all living species evolved.
Bacteria are often used as tools in research. A better understanding of their genomes can make them more effective tools.
102
What is equivalent to 1 million base pairs to genome researchers?
1 million bp = 1 megabase pair, abbreviated 1 Mb.
103
T/F Most bacteria and archaea contain a single type of chromosome, though multiple copies may be present in a single cell. However, some bacteria are known to have more than one type of chromosome.
True.
104
What disease does the bacteria, Vibrio cholerae, cause and what are the sizes of its two different chromosomes?
Diarrheal disease cholera, and it has two different chromosomes in each cell, one has 2.9 Mb and the other 1.1 Mb.
These chromosomes in bacteria and archaea are typically circular.
105
How many genes within bacteria and archaea are found for every megabase pair (1 million base pairs)?
Roughly 1,000 genes.
106
What is some differences between bacterial and archaeal genomes compared to eukaryotic genomes?
Bacterial and archaeal genomes are less complex, lack centromeres and telomeres and have a single origin of replication.
Additionally, the chromosomes in these prokaryotes have little repetitive DNA, which is very repetitive in eukaryotic DNA.
107
What is the strategy for sequencing an entire genome by randomly sequencing many different DNA fragments?
Shotgun DNA sequencing
108
Why are researchers interested in the genomes of bacteria and archaea?
Multiple select question.
The information obtained can be applied to organisms with more complex genomes, including humans.
All human genes are also found in bacteria and archaea.
Bacteria and archaea may provide insight as to how life has evolved since the first common ancestor.
Bacteria are used as tools in research and biotechnology.
The information obtained can be applied to organisms with more complex genomes, including humans.
Bacteria and archaea may provide insight as to how life has evolved since the first common ancestor.
Bacteria are used as tools in research and biotechnology.
109
True or false: The DNA sequence of each spot in a microarray is already known.
Multiple choice question.
false
true
true
110
The first eukaryotic cell probably involved a union between a(n) _________ cell and a(n) _________ cell.
Blank 1: bacterial or bacteria
| Blank 2: archaeal, archaea, archaean, or archaeon
111
What technique can be used to identify which genes are transcribed by a specific cell?
Multiple choice question.
microarray
electrophoresis
sequencing
PCR
microarray
112
Bacterial chromosomes are usually ______.
Multiple choice question.
linear and single-stranded
circular and single-stranded
linear and double-stranded
circular and double-stranded
circular and double-stranded
113
Genes in living cells can be altered using an approach called _______-Cas technology.
CRISPR or C R I S P R
114
Researchers have made a modification to the natural CRISPR-Cas system. They have created a(n) ______-_________ RNA that binds to both the target gene and to the protein Cas9.
Blank 1: single
| Blank 2: guide
115
Eukaryotic genomes are typically ______ than those of their bacterial and archaeal counterparts.
Multiple choice question.
smaller but more complex
larger but less complex
smaller and less complex
larger and more complex
larger and more complex
116
Studying the genomes of bacteria provides insight into which of the following?
Multiple select question.
Evolution
Basic genetic mechanisms
Ways to combat bacterial infections
The structure and function of eukaryotic cells
Evolution
Basic genetic mechanisms
Ways to combat bacterial infections
117
What does eukaryotes involve?
Protists, fungi, animals, and plants.
Their genomes are larger and more complex than those of their bacterial and archaeal counterparts.
118
[Eukaryotic genomes comes from four main sources]
The availability of genome sequences makes it easier for researchers to identify and characterize the genes of model organisms. This was the impetus for genome projects involving baker’s yeast (Saccharomyces cerevisiae), the fruit fly (Drosophila melanogaster), a nematode worm (Caenorhabditis elegans), the flowering plant called thale cress (Arabidopsis thaliana), and the mouse (Mus musculus).
Studying eukaryotic genomes enables researchers to gather more information for identifying and treating human diseases. Researchers hope that knowing the DNA sequence of the human genome will help to identify genes in which mutation plays a role in disease.
Sequencing the genomes of agriculturally important species can lead to development of new strains of livestock and plant species with improved traits.
Biologists are increasingly relying on genome sequences as a way to establish evolutionary relationships.
None
119
The nucleus of eukaryotic species contains the genome which is found in sets of linear chromosomes. In humans, how many sets are there?
One set consists of 23 linear chromosomes
22 autosomes and 1 sex chromosome, X or Y.
Remember: Mitochondrial and Chloroplast DNA is separate from the nuclear genome.
120
What are two reasons that the groups of species shown in part (a) vary in their total amount of DNA?
One reason is that more complex species tend to have more genes. A second reason is that species vary with regard to the amount of repetitive DNA sequences in their genomes.
121
What is a reason for gene duplications within eukaryotes?
Gene duplications are important because they provide raw material for the addition of more genes into a species’ genome. Such duplications produce homologous genes, two or more genes that are derived from the same ancestral gene. Over the course of many generations, each version of the gene accumulates different mutations, resulting in genes with similar but not identical DNA sequences.
122
What are homologous genes?
Genes derived from the same ancestral gene that have accumulated random mutations that make their sequences slightly different.
123
How do gene duplications occur? See saved image.
One mechanism that produces gene duplications is a misaligned crossover (Figure 21.13b). In this example, two homologous chromosomes have paired with each other during meiosis, but the homologs are misaligned. A crossover produces one chromosome with a gene duplication, one with a gene deletion, and two normal chromosomes. Each of these chromosomes is segregated into different haploid cells. If a haploid cell carrying the chromosome with the gene duplication participates in fertilization with another gamete, an offspring with a gene duplication is produced. In this way, gene duplications can form and be transmitted to future generations.
During evolution, gene duplications can occur several times. Two or more homologous genes within a single species are also called paralogous genes, or paralogs. Multiple gene duplications followed by the accumulation of mutations in each paralog result in a gene family—a group of paralogs that carry out related functions. A well-studied example is the globin gene family found in animals. The globin genes encode polypeptides that are subunits of proteins that function in oxygen binding. Hemoglobin, which is made in red blood cells, carries oxygen throughout the body. In humans, the globin gene family is composed of 14 paralogs that were originally derived from a single ancestral globin gene (Figure 21.14). According to an evolutionary analysis, the ancestral globin gene duplicated between 500 and 600 mya. Since that time, additional duplication events and chromosomal rearrangements have occurred to produce the current number of 14 genes on three different human chromosomes. Four of these are pseudogenes—genes that have been produced by gene duplication but have accumulated mutations that make them nonfunctional, so they are not transcribed into RNA.
The accumulation of different mutations in the various family members has produced globins that are specialized in their function. For example, myoglobin binds and stores oxygen in muscle cells, whereas the hemoglobins bind and transport oxygen via red blood cells. Also, different globin genes are expressed during different stages of development. The zeta (ζ)-globin and epsilon (ε)-globin genes are expressed very early in embryonic life. During the second trimester of gestation, the alpha (α)-globin and gamma (γ)-globin genes are turned on. Following birth, the γ-globin genes are turned off, and the β-globin gene is turned on. These differences in the expression of the globin genes reflect the differences in the oxygen transport needs of humans during the embryonic, fetal, and postpartum stages of life
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How do the proteins encoded by different members of the globin gene family vary in their affinity for oxygen?
The proteins produced by family members at early stages of development (embryonic and fetal stages) have a higher affinity for oxygen than the proteins produced in an adult. This allows the embryo and fetus to obtain oxygen from the mother’s bloodstream.
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What is a 13-year international effort coordinated by the U.S. Department of Energy and the National Institutes of Health that characterized and sequenced the entire human genome?
The Human Genome Project
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What were the goals of the Human Genome Project?
To obtain the DNA sequence of the entire human genome. The first draft of a nearly completed DNA sequence was published in February 2001, and a second draft was published in 2003. The entire human genome is approximately 3.2 billion base pairs in length.
To identify all human genes. This involved mapping the locations of genes throughout the entire genome.
To develop technology for the generation and management of human genome information. Some of the efforts of the Human Genome Project have involved improvements in molecular genetic technology, such as gene cloning, DNA sequencing, and so forth. The Human Genome Project has also developed computer tools that allow scientists to easily access up-to-date information from the project and analytical tools to interpret genomic information.
To analyze the genomes of model organisms. These include E. coli, S. cerevisiae, D. melanogaster, C. elegans, A. thaliana, and M. musculus.
To develop programs focused on understanding and addressing the ethical, legal, and social implications of the results obtained from the Human Genome Project. The Human Genome Project raised many ethical issues regarding genetic information and genetic engineering. Who should have access to genetic information? Should employers, insurance companies, law enforcement agencies, and schools have access to our genetic makeup?
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Why are researchers interested in sequencing eukaryotic genomes?
Multiple select question.
They can use this information to develop breeds of livestock and crops with improved traits for greater yields.
It makes it easier for researchers to identify and characterize the genes of model organisms.
They can use this information to better identify and treat human diseases that are caused by mutation.
The information can be used to better understand evolutionary relationships among organisms.
Most diseases are caused by eukaryotic organisms.
They can use this information to develop breeds of livestock and crops with improved traits for greater yields.
It makes it easier for researchers to identify and characterize the genes of model organisms.
They can use this information to better identify and treat human diseases that are caused by mutation.
The information can be used to better understand evolutionary relationships among organisms.
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What are DNA sequences that are present in many copies in a genome?
Repetitive sequences. They are found in bacterial, archaeal, and eukaryotic genomes.
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What are the two broad categories of repetitive sequences?
Moderately and highly repetitive
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What is the category of repetitive sequence of a DNA sequence that is repeated a few hundred to several thousand times in a genome?
Moderately repetitive sequence
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What are some examples of moderately repetitive sequences?
In some cases, these sequences are multiple copies of the same gene. For example, the genes that encode ribosomal RNA (rRNA) are found in many copies. The cell needs a large amount of rRNA for its cellular ribosomes. This is accomplished by having and expressing multiple copies of the genes that encode rRNA.
In addition, other types of functionally important sequences can be moderately repetitive. For example, multiple copies of origins of replication are found in eukaryotic chromosomes. Other moderately repetitive sequences may play a role in the regulation of gene transcription and translation.
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What is the category of repetitive sequence of a DNA sequence that is repeated tens of thousands or even millions of times throughout a genome?
Highly repetitive sequence
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How long is typically a highly repetitive sequence?
Each copy of a highly repetitive sequence is relatively short, ranging from a few to several hundred nucleotides in length.
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What is a tandem array for highly repetitive sequences?
Very short nucleotide sequence is repeated many times in a row. In Drosophila, for example, 19% of the chromosomal DNA is highly repetitive DNA found in tandem arrays.
Ex. AATAT AATATAT AATAT AATAT AATATAT
Theses tandem arrays can be up to 1 million bp in length!
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What is the family of sequences within humans and primates that has been widely studied and is a highly repetitive sequence?
Alu family of sequences roughly 300 bp long.
This sequence derives its name from the observation that it contains a site for cleavage by a restriction enzyme known as AluI.
It represents about 10% of the total human DNA and occurs (on average) approximately every 5,000–6,000 bases.
This sequence occurs over 1 million times!
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What is the percentage of protein-encoding genes and noncoding genes within the human genome?
Exons, the protein-encoding genes make up only 2% of the genome! 98% is composed of noncoding sequences.
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What does the 98% of noncoding sequences consist of?
Intron DNA = 24% of human genome.
Unique noncoding DNA = 15%
Repetitive DNA = 59%
Only a small percentage of the human genome is involved with encoding the proteins that are largely responsible for human traits.
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What is the overall goal of the Encyclopedia of DNA Elements (ENCODE)?
The overall goal of the ENCODE Project is to identify functional sequences in the human genome. In 2012, the researchers announced that they were able to assign function to approximately 80% of the human genome. Much of the previously described “junk DNA” appears to play a role in a complex network of gene regulation.
Project has involved more than 440 researchers in 32 laboratories.
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What is a segment of DNA that can move from one site to another within a genome?
Transposable element (TE)
The Alu sequence discussed earlier is an example of a TE. The process in which a TE moves to a new site in a genome is called transposition. TEs range from a few hundred to several thousand base pairs in length. They have sometimes been referred to as “jumping genes,” because they are inherently mobile.
American cytogeneticist Barbara McClintock first identified TEs in the late 1940s from her studies with corn plants. She identified a segment of DNA that could move into and out of a gene that affected the color of corn kernels, producing a speckled appearance. Since that time, biologists have discovered many different types of TEs in nearly all species they have examined.
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What are the two TEs that fall into two groups which have been discovered in bacteria, archaea, and eukaryotes?
DNA transposons and retrotransposons based on different mechanisms of movement.
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What is a type of transposable element that moves as a DNA molecule?
DNA transposon.
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What is a characteristic of DNA transposons?
Both ends are inverted repeats (IRs)—DNA sequences that are identical (or very similar) but run in opposite directions.
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What is the length of IRs in transposoms?
9 to 40 bp in length.
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What is the enzyme that facilitates transposition?
Transposase.
Transposase first recognizes the inverted repeats (IR) in the transposon.
It then cleaves both ends of the DNA transposon and removes it from its original site.
Next, the transposase/transposon complex moves to a new location, where transposase cleaves the target DNA and inserts the transposon into the site.
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How does transposons increase prevalence in a cell?
Transposition may occur when a cell is in the process of DNA replication. If a TE is removed from a site that has already replicated and is inserted into a chromosomal site that has not yet replicated, the transposon will increase in number after DNA replication is complete. This is one way for transposons to become more prevalent in a genome.
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What form of transposition is very common but is only found in eukaryotic species?
The transposable elements are called retrotransposons.
A type of transposable element that moves via an RNA intermediate.
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What is an example of a retrotransposon in humans?
The Alu sequence
The integration of retrotransposons can occur at many locations within the genome. Furthermore, because a single retrotransposon can be copied into many RNA transcripts, retrotransposons may accumulate rapidly within a genome. This explains how the Alu sequence in the human genome was able to proliferate and constitute 10% of the human genome.
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What do retrotransposons have that facilitate their recognition?
Terminal repeats
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Based on their mechanism of movement, which type of TE do you think would proliferate more rapidly in a genome, DNA transposons or retrotransposons?
Retrotransposons. A single retrotransposon can be transcribed into multiple copies of RNA, which can be converted to DNA by reverse transcriptase, and inserted into multiple sites in the genome.
