CHEM 365 Chapter 3: Nucleotides, Nucleic Acids, and Genetic Information
Define modification methylase.
A bacterial enzyme that methylates a specific sequence of DNA as part of a restriction-modification system.
Define restriction endonuclease
A bacterial enzyme that recognizes a specific DNA sequence and cleaves the DNA as part of a restriction-modification system.
Define Nucleoside
A compound consisting of a nitrogenous base and a five carbon sugar (ribose or deoxyribose) in N-glycosidic linkage.
Define dNTP
A deoxyribonucleoside triphosphate
Define gene knockout
A genetic engineering process that deletes or inactivates a specific gene in an animal.
Define restriction-modification system
A matched pair of bacterial enzymes that recognize a specific DNA sequence: a modification methylase that methylates bases in that sequence, and a restriction endonuclease that cleaves the DNA if it has not been methylated in that sequence. It is a defensive system that eliminates foreign (e.g., viral) DNA
Define Deoxyribonucleotides
A nucleotide in which the pentose is 2' -deoxyribose. Also known as a deoxynucleotide.
Define Ribonucleotides
A nucleotide in which the pentose is ribose.
Define ADP
Adenosine diphosphate; molecule that ATP is formed from.
Define ATP
Adenosine triphosphate; a nucleotide containing adenine, ribose, and a triphosphate group; energy carrier or energy transfer agent
Define allele
An alternate form of a gene; diploid organisms contain two alleles for each gene, which may or may not be identical.
Define severe combined immunodeficiency disease (SCID)
An inherited disease that greatly impairs the immune system. One such defect is a deficiency of the enzyme adenosine deaminase.
Define template
DNA strand can act as a template for the synthesis of its complementary strand and hence that hereditary information is encoded in the sequence of bases on either strand.
Define Purine
Derivatives of the compound purine, a planar aromatic, heterocyclic compound. Adenine and guanine, two of the nitrogenous bases of nucleotides, are purines.
Define Pyrimidine
Derivatives of the compound pyrimidine, a planar aromatic, heterocyclic compound. Cytosine, uracil, and thymine, three of the nitrogenous bases of nucleotides, are pyrimidines.
Define luciferase,
firefly enzyme, which generates a flash of light. Triggered when pyrophosphate is released during pyrosequencing
Define overproducers
genetically engineered organisms/proteins that may reach 30% of the host's total cellular protein
What are in vitro and in vivo?
in the test tube and in living systems
List all the components and explain their purpose in the reaction mixture used for the dideoxy DNA sequencing method.
includes the single-stranded DNA to be sequenced (the template), a primer, the four deoxynucleotide triphosphates (represented as dATP, etc.), and small amounts of the four fluorescently labeled dideoxynucleoside triphosphates (ddATP, etc.).
Define Baculoviruses
infect insect cells, are used for cloning in cultures of insect cells.
Define ferritin
iron-storage protein
Define yeast artificial chromosomes (YACs).
linear DNA molecules that contain all the chromosomal structures required for normal replication and segregation during yeast cell division
Describe what happens during Translation.
mRNA goes to ribosome made of rRNA. Each set of three nucleotides in the mRNA pairs with three complementary nucleotides in a tRNA which had its corresponding amino acid attached to it. Amino acids are added to the growing protein chain according to the order in which the tRNA molecules bind to the mRNA. When a new amino acid is added, the preceding tRNA is ejected, and the ribosome proceeds along the mRNA.
Define transcriptome,
mRNA molecules
Define wild-type
naturally occurring
Define Gene therapy
the transfer of new genetic material to the cells of an individual in order to produce a therapeutic effect.
Define transgene
the transplanted foreign gene in a transgenic organism
What is the job of DNA Polymerase?
to make DNA from nucleotides, the building blocks of DNA by Copying a Template Strand
What are some of the challenges of expressing a eukaryotic gene in a prokaryotic host cell?
(1) Bacteria can produce eukaryotic proteins only if the recombinant DNA that carries the protein-coding sequence also includes bacterial transcriptional and translational control sequences. (2) eukaryotic genes are large and contain introns that are transcribed and excised before translation ; bacteria lack the machinery to excise the introns. (3) eukaryotic proteins are posttranvslationally modified by the addition of carbohydrates or by other reactions
Describe the activities of the enzymes required to construct a recombinant DNA molecule.
(1) The cloning vector and the foreign DNA are cut by the same restriction endonuclease. (2) The sticky ends of the vector and the foreign DNA fragments anneal and are covalently joined by DNA ligase. (3) The result is a chimeric DNA containing a portion of the foreign DNA inserted into the vector.
Define haploid
Having one set of unique chromosomes.
Define expression
How often a gene is transcribed and translated
Explain the structure of RNA
Identical to a DNA strand except for the presence of 2'-OH groups and the substitution of uracil for thymine—
Define monomers
Individual units of a polymer
Define DNA ligase.
Joins Two DNA Segments; An enzyme that catalyzes bond formation coupled with the hydrolysis of ATP.
Define phosphodiester bond
Linkage between individual nucleotides because the phosphate is esterified to two ribose units
Define RNA world
Many of the processes essential for life began through the chemical versatility of small polynucleotides
Define transgenic
Multicellular organisms expressing a gene from another organism
Define diploid
Organism contains two equivalent sets of chromosomes, one from each parent.
Define genome
Organisms unique DNA content
Define Ribose
Pentose in Ribonucleotides
Describe the bases of nucleotides
Planar, aromatic, heterocyclic molecules that are derivatives of purine or pyrimidine
Define mutations
Proteins with altered structures and functions.
Define bacteriophages
Viruses that are specific for bacteria
Define Gel electrophoresis
a charged molecule moves in an electric field with a velocity proportional to its overall charge density, size, and shape. Primarily dependent on size.
What is a DNA library?
a collection of cloned DNA segments that can be screened to find a particular gene.
Define X-linked adrenoleukodystrophy
a defect in a membrane transport protein leads to brain damage
Define polyacrylamide
a more rigid cross-linked synthetic polymer
Define expression vector
a plasmid that contains properly positioned transcriptional and translational control sequences required for the production of a foreign DNA gene product (RNA or protein) in a host cell.
Define Leber's congenital amaurosis
a rare form of blindness
Define recombinant DNA technology
also called molecular cloning or genetic engineering, makes it possible to isolate, amplify, and modify specific DNA sequences.
Define Beta-carotene
an orange pigment that is the precursor of vitamin A
Define dideoxy method
another name for the chain-terminator method
Define Plasmids
are circular DNA molecules of 1 to 200 kb found in bacteria or yeast cells. Can act as molecular parasites, but can also benefit the host by provision functions like antibiotic resistance
What does DNA polymerase do?
assembles the four deoxynucleoside triphosphates (dNTPs), dATP, dCTP, dGTP, and dTTP, into a complementary polynucleotide chain that it elongates in the 5' S 3' direction - add deoxynucleotides only to the 3' end of a polynucleotide
How does The polymerase chain reaction amplify a DNA segment?
by repeatedly synthesizing complementary strands.
Define agarose
carbohydrate polymers that form a loose mesh
Define sticky ends
cleaved by a restriction endonuclease that cuts the two strands of DNA at positions that are staggered, producing DNA fragments with complementary single-strand extension
What is the purpose of cloning?
cloning provides materials (nucleic acids and proteins) for other studies and also provides a means for studying gene expression under controlled conditions.
Define X-gal
colorless compound that was cleaved by the encoded enzyme B-galactosidase
Define gamma c cytokine receptor
essential for proper immune system function
Define substrates
reactants in enzymatic reactions
Define clone
refers to the collection of cells that contain the vector carrying the DNA of interest or to the DNA itself
Define Cloning
refers to the production of multiple identical organisms derived from a single ancestor
Define bacterial artificial chromosomes (BACs)
replicate in E. coli, are derived from circular plasmids that normally replicate long regions of DNA and are maintained at the level of approximately one copy per cell
Define transformation
(1) The permanent alteration of a bacterial cell's genetic message through the introduction of foreign DNA. (2) The genetic changes that convert a normal cell to a cancerous cell.
Define ligated
spliced together
The Watson-Crick model of DNA has the following major features:
(1) Two polynucleotide chains wind around a common axis to form a double helix (2) The two strands of DNA are antiparallel, but each forms a right-handed helix. (3) The bases occupy the core of the helix and sugar-phosphate chains run along the periphery, thereby minimizing the repulsions between charged phosphate groups. The surface of the double helix contains two grooves of unequal width: the major and minor grooves. (4) Each base is hydrogen bonded to a base in the opposite strand to form a planar base pair.
Summarize the steps required to amplify a given segment of DNA in vivo and in vitro.
(1) a DNA fragment is generated either by a restriction enzyme, by PCR, or by chemical synthesis. (2) The fragment is incorporated into another DNA molecule known as a vector (3) The vector is introduced into cells and replicates (4) Cells containing the desired DNA are identified, or selected.
Describe the chain-terminator method
(1) obtain single polynucleotide strands (2) polynucleotide fragments that terminate at positions corresponding to each of the four nucleotides are generated (3) the fragments are separated and detected
Explain how site-directed mutagenesis can be used to produce an altered protein in bacterial cells.
(1) oligonucleotide with identical portion of gene of interest used to direct replication (2) oligonucleotide hybridizes to the corresponding wild-type (3) Extension of the oligonucleotide, called a primer, by DNA polymerase yields the desired altered gene (4) The altered gene can then be inserted into an appropriate vector.
What information was derived about DNA from the X-ray diffraction photograph taken of it?
(a) that DNA is a helical molecule and (b) that its planar aromatic bases form a stack that is parallel to the fiber axis.
What does DNA consist of? What does RNA consist of?
- DNA contains adenine, guanine, cytosine, and thymine deoxyribonucleotides, - RNA contains adenine, guanine, cytosine, and uracil ribonucleotides.
Explain what the Watson-Crick structure did
- Provided a model of DNA - Suggested the molecular mechanism of heredity
Identify the purines and pyrimidines commonly found in nucleic acids.
- Purines: adenine (A) and guanine (G) - Pyrimidines: cytosine (C), uracil (U), and thymine (T)
What is the difference between manipulating a gene for gene therapy and for producing a transgenic organism?
- gene therapy: - transgenic organism:
Define molecular cloning or genetic engineering
- made it possible to purify specific DNA sequences and to prepare them in quantities sufficient for study - can be used to manipulate genes for protein expression or for the production of transgenic organisms.
Define DNA polymerase I
- one of the enzymes that participates in replication of bacterial DNA - Using a single DNA strand as a template, the enzyme elongates the primer by stepwise addition of complementary nucleotides. Incoming nucleotides pair with bases on the template strand and are joined to the growing polynucleotide strand in the 5' S 3' direction
Compare the properties of cloning vectors such as pUC18, bacteriophage lambda, and BACs.
- pUC18: E. coli plasmid that has multiple restriction sites and 3 genes: amp^R (resistance to the antibiotic ampicillin); lacZ, which encodes the enzyme Beta- galactosidase; and lacI, which encodes a factor that controls the transcription of lacZ - Bacteriophage lambda: During phage infection, DNA contained in the "head" of the phage particle enters the bacterial cell, where it is replicated 100 times and packaged to form progeny phage. - BACs (bacterial artificial chromosomes): replicate in E. coli, are derived from circular plasmids that normally replicate long regions of DNA and are maintained at the level of approximately one copy per cell
Define primer
- short polynucleotide - An oligonucleotide that serves as a starting point for additional polymerization reactions catalyzed by DNA polymerase to form a polynucleotide. A primer base pairs with a segment of a template polynucleotide strand so as to form a short double-stranded segment that can then be extended through template-directed polymerization.
What is the overall strategy for sequencing any polymer of nonidentical unit
1. Cleave the polymer into fragments that are small enough to be fully sequenced. 2. Determine the sequence of residues in each fragment. 3. Determine the order of the fragments in the original polymer by aligning fragments that contain overlapping sequences.
Define recombinant, or chimera
A DNA molecule constructed by combining DNA from different sources.
Define vector
A DNA molecule such as a plasmid, virus, or artificial chromosome that can accommodate a segment of foreign DNA for cloning.
Define introns
A portion of a gene that is transcribed but excised prior to translation. Also called an intervening sequence.
Define pyrosequencing
A procedure for determining the sequence of nucleotides in DNA by copying a DNA strand in such a way that a flash of light is generated when the correct nucleotide is incorporated.
Define stem-loop
A secondary structural element in a single-stranded nucleic acid, in which two complementary segments form a base-paired stem whose strands are connected by a loop of unpaired bases.
Define DNA fingerprinting
A technique for distinguishing individuals on the basis of DNA polymorphisms, such as the number of short tandem repeats (STR).
Define Site-directed mutagenesis
A technique in which a cloned gene is mutated in a specific manner; mimics natural process of evolution
Define chromosomes
A threadlike structure of nucleic acids and protein found in the nucleus of most living cells, carrying genetic information in the form of genes.
Define polymorphism
A variation in DNA or amino acid sequences between individuals.
Define palindrome
A word or phrase that reads the same forward or backward. In a palindromic DNA segment, the sequence of nucleotides is the same in each strand, and the segment is said to have twofold symmetry
Define exonuclease
Cleaves a nucleic acid by removing one of its terminal residues
Define endonuclease
Cleaves a nucleic acid within the polynucleotide strand
Define STR (short tandem repeats)
Clusters of nearly identical sequences of up to 10 bp that are repeated thousands of times; the sequences are present at 106 copies per haploid genome.
How can the single strands in the chain-terminator method be obtained?
Complementary DNA strands can be separated by heating, which breaks the hydrogen bonds between bases
Does DNA or RNA direct the synthesis of proteins?
DNA
Define DNA and RNA
DNA - Deoxyribonucleic acid. A polymer of deoxynucleotides whose sequence of bases encodes genetic information in all living cells. RNA - Ribonucleic acid. A polymer of ribonucleotides. The major forms of RNA include messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).
Define transcription
DNA -> RNA The process by which RNA is synthesized under the direction of a DNA template, thereby transferring genetic information from the DNA to the RNA. Transcription is catalyzed by RNA polymerase as facilitated by numerous other proteins.
Define dimer, trimer, tetramer, & oligomer
Dimer: An assembly consisting of two monomeric units (promoters). Trimer: An assembly consisting of three monomeric units Tetramer: An assembly consisting of four monomeric units. Oligomer: A short polymer consisting of a few linked monomer units.
Define Bacteriophage lambda
During phage infection, DNA contained in the "head" of the phage particle enters the bacterial cell, where it is replicated 100 times and packaged to form progeny phage.
Define pUC18
E. coli plasmid that has multiple restriction sites and 3 genes: amp^R (resistance to the antibiotic ampicillin); lacZ, which encodes the enzyme Beta- galactosidase; and lacI, which encodes a factor that controls the transcription of lacZ
Define nucleotide residue
Each nucleotide that has been incorporated into the polynucleotide
Define tautomers
Easily converted Isomers that differ only in the positions of their hydrogen atoms and double bonds.
What does a Nucleotide consist of?
Eight varieties of: A nitrogenous base, a ribose or deoxyribose sugar, and one or more phosphate groups.
Define gene expression
Focuses on the set of mRNA molecules, or transcriptome, that is transcribed from DNA under any particular set of circumstance
Give an example of how diploid and haploid relate
For example, humans are diploid organisms that carry 46 chromosomes per cell; their haploid number is therefore 23.
Why do the smallest fragments of DNA move the farthest during electrophoresis?
For molecules with a relatively homogeneous composition (such as nucleic acids), shape and charge density are constant, so the velocity depends primarily on size. Longer molecules can get more easily caught up in the mesh agarose.
What is released with each nucleotide addition in a polymerase-catalyzed reaction?
Pyrophosphate
Define translation
RNA -> amino acids -> protein The process by which a polypeptide is synthesized under the direction of the sequence information contained in a messenger RNA as specified by the genetic code. Translation is catalyzed by ribosomes and requires the additional participation of messenger RNA, transfer RNA, and a variety of protein factors.
Define antiparallel
Run in opposite directions.
Define messenger RNA, or mRNA
Simplification: mRNA directs the creation of polypeptide A ribonucleic acid whose sequence is complementary to that of a protein-coding gene in DNA. In the ribosome, mRNA directs the polymerization of amino acids to form a polypeptide with the corresponding sequence.
Define ribosomal RNA, or rRNA
Simplification: site of polypeptide creation The RNA molecules that constitute the bulk of the ribosome, the site of polypeptide synthesis. rRNA provides structural scaffolding for the ribosome and catalyzes peptide bond formation.
Define transfer RNA, or tRNA
Simplification: tRNA delivers amino acids to mRNA through complementarity. The small L-shaped RNAs that deliver specific amino acids, which have been esterified to the tRNA's 3' ends, to ribosomes according to the sequence of a bound mRNA. The proper tRNA is selected through the complementary base pairing of its three-nucleotide anticodon with the mRNA's codon, and the growing polypeptide is transferred to its aminoacyl group.
Explain how restriction enzymes generate either sticky ends or blunt ends.
Sticky ends: Cut in a staggered way -> Very often they cut the two DNA strands four base pairs from each other, creating a four-base 5' overhang in one molecule and a complementary 5' overhang in the other. Blunt Ends: restriction endonucleases cleave the two strands of DNA at the symmetry axis to yield restriction fragments with fully base-paired blunt ends
Define 2' -deoxyribose
Sugar in Deoxyribonucleotides
Define Blunt ends
The fully base-paired ends of a DNA fragment that has been cleaved by a restriction endonuclease that cuts the DNA strands at opposing sites.
Define minor grooves
The groove on a DNA double helix whose bounding glycosidic bonds from each base pair form an angle of < 180 . In B-DNA, this groove is narrower than the major groove.
Define major Grooves
The groove on a DNA double helix whose bounding glycosidic bonds from each base pair form an angle of > 180 . In B-DNA, this groove is wider than the minor groove.
What is 2,3-dideoxynucleoside triphosphate (ddNTP) role in the chain-terminator method?
The key component of the reaction mixture: When the dideoxy analog is incorporated into the growing polynucleotide in place of the corresponding normal nucleotide, chain growth is terminated because addition of the next nucleotide requires a free 3' -OH.
Define Chargaff 's rules
The observation, first made by Erwin Chargaff, that duplex DNA has equal numbers of adenine and thymine residues and equal numbers of guanine and cytosine residues.
Define central dogma of molecular biology
The paradigm that DNA directs its own replication as well as its transcription to RNA, which is then translated into a polypeptide. The flow of information is from DNA to RNA to protein.
What does the The polymerase-catalyzed reaction require?
The polymerase-catalyzed reaction requires a free 3' -OH group on the growing strand.
Define replication
The process of making an identical copy of a DNA molecule. During DNA replication, the parental polynucleotide strands separate so that each can direct the synthesis of a complementary daughter strand, resulting in two complete DNA double helices.
Define codon
The sequence of three nucleotides in DNA or RNA that specifies a single amino acid.
Define base pair (bp)
The specific hydrogen-bonded association between nucleic acid bases. The Watson-Crick base pairs are A * T and G * C.
Define transcriptomics
The study of gene expression
Define genomics
The study of the genome's size, organization, and gene content
Define proteomics
The study of the proteins (the proteome) produced as a result of transcription and translation.
Define 3' end
The terminal residue whose C3' is not linked to another nucleotide
Define 5' end
The terminal residue whose C5' is not linked to another nucleotide
How can the problems of expressing a eukaryotic gene in a prokaryotic host cell by mitigated?
These problems can be overcome by using expression vectors that propagate in eukaryotic hosts, such as yeast or cultured insect or animal cells.
Define kilobase pairs (kb)
Thousands of base pairs
Define anneal
To maintain conditions that allow loose base pairing between complementary single polynucleotide strands so that properly paired double-stranded segments form.
