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• Allele
• One copy of a gene.
• Antibody
• A protein (also called immunoglobulin) produced by B lymphocytes that binds to a foreign molecule.
• Antigen
• A molecule against which an antibody is directed.
• antisense nucleic acid
• A nucleic acid (either RNA or DNA) that is complementary to an mRNA of interest and is used to block gene
expression.
• bacterial artificial chromosome (BAC)
• A type of vector used for cloning large fragments of DNA in bacteria.
• cDNA
• A DNA molecule that is complementary to an mRNA molecule, synthesized in vitro by reverse transcriptase.
• central dogma
• The concept that genetic information flows from DNA to RNA to proteins.
• Chromosome
• A carrier of genes, consisting of long DNA molecules and associated proteins.
• Codon
• The basic unit of the genetic code; one of the 64 nucleotide triplets that code for an amino acid or stop sequence.
• Cosmid
• A vector that contains bacteriophage λ sequences, antibiotic resistance sequences, and an origin of replication. It
can accommodate large DNA inserts of up to 45 kb.
• CRISPR/Cas system
• A system for introducing targeted mutations into mammalian genes. It consists of CRISPR RNAs that recognize
specific target sequences and Cas proteins that cleave the targeted DNA.
• Dideoxynucleotide
• A nucleotide that lacks the normal 3′ hydroxyl group of deoxyribose and is used as a chain-terminating nucleotide
in DNA sequencing.
• Diploid
• An organism or cell that carries two copies of each chromosome.
• DNA ligase
• An enzyme that seals breaks in DNA strands.
• DNA polymerase
• An enzyme catalyzing the synthesis of DNA.
• Dominant
• The allele that determines the phenotype of an organism when more than one allele is present.
• dominant inhibitory mutant
• A mutant (also called dominant negative mutant) that interferes with the function of the normal allele of the gene.
• Electroporation
• The introduction of DNA into cells by exposure to a brief electric pulse.
• embryonic stem (ES) cell
• A stem cell cultured from an early embryo.
• expression vector
• A vector used to direct expression of a cloned DNA fragment in a host cell.
• gel electrophoresis
• A method in which molecules are separated based on their migration in an electric field.
• Gene
• A functional unit of inheritance, corresponding to a segment of DNA that encodes a polpeptide or RNA molecule.
• gene transfer
• The introduction of foreign DNA into a cell.
• genetic code
• The correspondence between nucleotide triplets and amino acids in proteins.
Cell Biology Ch. 4 Overview
• Genotype
• The genetic composition of an organism.
• Haploid
• An organism or cell that has one copy of each chromosome.
• homologous recombination
• Recombination between segments of DNA with homologous nucleotide sequences.
• Immunoblotting
• A method that uses antibodies to detect proteins separated by SDS-polyacrylamide gel electrophoresis. Also
called Western blotting, it is a variation of Southern blotting.
• in situ hybridization
• The use of radioactive or fluorescent probes to detect RNA or DNA sequences in chromosomes or intact cells.
• in vitro mutagenesis
• The introduction of mutations into cloned DNA in vitro.
• in vitro translation
• Protein synthesis in a cell-free extract.
• Knockout
• Inactivation of a chromosomal gene by homologous recombination with a cloned mutant allele.
• Liposome
• A lipid vesicle used to introduce DNA into mammalian cells.
• Meiosis
• The division of diploid cells to haploid progeny, consisting of two sequential rounds of nuclear and cellular division.
• molecular cloning
• The insertion of a DNA fragment of interest into a DNA molecule (vector) that is capable of independent
replication in a host cell.
• monoclonal antibody
• An antibody produced by a clonal line of B lymphocytes.
• Mutation
• A genetic alteration.
• next-generation sequencing
• New methods that allow rapid sequencing of billions of bases of DNA.
• Northern blotting
• A method in which mRNAs are separated by gel electrophoresis and detected by hybridization with specific
probes.
• nucleic acid hybridization
• The formation of double-stranded DNA and/or RNA molecules by complementary base pairing.
• origin of replication
• A specific DNA sequence that serves as a binding site for proteins that initiate replication.
• P1 artificial chromosome (PAC)
• A vector used for cloning large fragments of DNA in E. coli.
• Phenotype
• The physical appearance of an organism.
• Plasmid
• A small, circular DNA molecule capable of independent replication in a host cell.
• polymerase chain reaction (PCR)
• A method for amplifying a region of DNA by repeated cycles of DNA synthesis in vitro.
• real-time PCR
• A method in which the polymerase chain reaction (PCR) is used to quantitate the amount of target DNA or RNA
present in a sample.
• Recessive
• An allele that is masked by a dominant allele.
• recombinant DNA library
• A collection of genomic or cDNA clones.
Summary
Heredity, Genes, and DNA
• Genes and chromosomes: Chromosomes are the carriers of genes.
• Genes and enzymes: Genes specify the amino acid sequence of proteins.
• Identification of DNA as the genetic material: DNA was identified as the genetic material by bacterial transformation
experiments. Bacterial Transformation
• A pathogenic S strain of Pneumococcus forms smooth colonies on Petri plates, because the bacteria are
surrounded by slippery polysaccharide capsules. A nonpathogenic R strain lacks the capsule and forms rough colonies.
• An R strain can be transformed into an S strain. In this procedure, DNA is extracted from the S strain. The purified
S DNA is added to a culture of the R strain bacteria.
• The bacteria take up fragments of DNA from the environment. The DNA passes through the cell wall and plasma
membrane. One strand of the foreign DNA degrades.
• Recombination between the foreign DNA strand and the bacterial chromosome is accomplished by a pair of
crossing over events. The result is a double-stranded DNA in which the two strands are not complementary because part of one strand came from a different source. The leftover DNA is degraded.
• Both strands of the chromosome replicate, so the two new DNA molecules are genetically different. One daughter
cell is genetically the same as the original cell, and the other contains a DNA code that results in the formation of a capsule around the bacterium, resulting in pathogenicity.
Avery, MacLeod, and McCarty
• In 1944, Avery, MacLeod, and McCarty performed experiments to determine the chemical nature of the
transforming principle, which in today's terms is genetic material. They prepared an active transforming principle from a heat-killed S strain of Pneumococcus bacteria. A live S strain is pathogenic and kills mice.
• A bioassay was performed in which this active transforming principle was added to a nonpathogenic R strain of
bacteria, and then the bacteria were used to inoculate mice. In such an assay, the bacteria were transformed— they had become pathogenic.
• In an important experiment, they first treated the transforming principle with a protease, a protein-destroying
enzyme. Although it hydrolyzed the proteins in the bacterial extract, this treatment did not inactivate the transforming principle, which in a bioassay was still capable of transforming a nonpathogenic R strain of Pneumococcus into a pathogenic one.
• Then they treated the transforming principle with a carbohydrase. This hydrolyzed the carbohydrates in the
extract, but did not inactivate the transforming principle.
• Treatment with RNase (ribonuclease) hydrolyzed RNAs in the extract, but did not inactivate the transforming
principle.
• However, treatment with DNase (deoxyribonuclease) both hydrolyzed DNA and destroyed the transforming
principle. In the bioassay, the bacteria were not transformed—they did not become pathogenic. From these experiments, it certainly seems reasonable to conclude that the transforming principle is DNA.
• The structure of DNA: DNA is a double helix in which hydrogen bonds form between purines and pyrimidines on
opposite strands. Because of specific base pairing—A with T and G with C—the two strands of a DNA molecule are complementary in sequence.
• Replication of DNA: DNA replicates by semiconservative replication in which the two strands separate and each serves
as a template for synthesis of a new progeny strand.
Expression of Genetic Information
• Colinearity of genes and proteins: The order of nucleotides in DNA specifies the order of amino acids in proteins.
• The role of messenger RNA: Messenger RNA functions as an intermediate to convey information from DNA to the
ribosomes, where it serves as a template for protein synthesis.
• The genetic code: Transfer RNAs serve as adaptors between amino acids and mRNA during translation. Each amino
acid is specified by a codon consisting of three nucleotides.
The Central Dogma
• The central dogma of molecular biology, as first stated by Francis Crick, asserts that information flows from DNA
to RNA, and then from RNA to protein. Information never flows from protein back up the line.
DNA Mutations
• The genetic information of DNA is transcribed into mRNA, which is translated into a polypeptide chain.
• If the wild-type DNA suffers a change in one base pair, the change will be transcribed in the mRNA, and the
resulting codon may code for a different amino acid at this point in the forming polypeptide.
• Alternatively, some DNA mutations result in the appearance of a stop codon in the resulting mRNA. The stop
codon causes premature termination of polypeptide synthesis.
• RNA viruses and reverse transcription: DNA can be synthesized from RNA templates, as first discovered in
retroviruses.
• HIV, the human immunodeficiency virus, is a retrovirus. An HIV particle attaches to the host cell by way of
receptor proteins on the membrane.
• The cell removes the viral coat as it takes up the virus by endocytosis.
• The reverse transcriptase brought in by the virus copies the viral RNA to make a complementary DNA strand. The
viral RNA is degraded, and the DNA is the template for a second DNA strand.
• The double-stranded DNA enters the host cell’s nucleus and integrates into a chromosome as a provirus.
• Eventually, transcription produces viral RNA. The viral RNA travels to the host cell’s cytoplasm, where some is
reserved for new viral genomes and some is translated, producing new viral proteins.
• These proteins are assembled to form new capsids and envelopes. The assembled retroviruses bud from the
plasma membrane. The new viruses can parasitize more host cells.
Recombinant DNA
• Restriction endonucleases: Restriction endonucleases cleave specific DNA sequences, yielding defined fragments of
DNA molecules.
• Restriction enzymes cut specific recognition sequences in DNA. The enzyme Eco RI recognizes the sequence 5′-
GAATTC-3′ and produces a staggered cut with 5′ single-stranded overhangs.
• The enzyme Pst I recognizes the sequence 5′-CTGCAG-3′ and produces a staggered cut with 3′ single-stranded
overhangs.
• The enzyme Sma I recognizes the sequence 5′-CCCGGG-3′ and produces blunt ends on the DNA.
• Generation of recombinant DNA molecules: Recombinant DNA molecules consist of a DNA fragment of interest ligated
to a vector that is able to replicate independently in an appropriate host cell.
• The basic strategy in molecular cloning is to insert a DNA fragment of interest (such as a segment of human DNA)
into a DNA molecule (called a vector) that is capable of independent replication in a host cell. In this example, the vector DNA is a loop of DNA called a plasmid.
• The human DNA is cut with a restriction endonuclease, which recognizes a specific base sequence, called a
recognition site. If there are multiple recognition sites on the DNA, it will be cut into pieces of various lengths.
• The same endonuclease is used to cut the vector. If the cut human DNA and the vector are mixed in the presence
of DNA ligase, some of them will join to form recombinant DNA molecules.
• The recombinant molecule is introduced into E. coli, where it is replicated. In this way, large quantities of the
inserted human DNA can be obtained.
• In the next phase, the temperature is raised to 72 degrees Celsius. Taq polymerase functions optimally at this
temperature and begins polymerization, adding nucleotides to the 3' end of each primer attached to a DNA strand. After one complete cycle, there are two double-stranded copies of the target DNA.
• The PCR reaction mixture contains many copies of the primers and an abundant supply of nucleotides to perform
many addition cycles. After a second cycle, there are four copies of the target DNA.
• After cycle 3 is finished, there are eight copies of the double-stranded target DNA sequence. Note that only two of
the double-stranded copies consist of just the target fragment. The others also include flanking DNA regions.
• As the number of cycles increases, the products consist of a greater proportion of fragments with just the target
DNA.
• With each additional cycle, the number of copies of our target sequence doubles. At the end of cycle 25 there are
more than 33 million copies of this double-stranded target region.
• Nucleic acid hybridization: Nucleic acid hybridization allows the detection of specific DNA or RNA sequences by base
pairing between complementary strands.
Nucleic acid hybridization
• The key to detecting a specific nucleic acid sequence in a mixture of different DNA molecules is in the base
pairing between complementary strands. The base pairs are held together by hydrogen bonds. A cytosine and guanine pair form three hydrogen bonds. A thymine and adenine pair form two hydrogen bonds.
• When DNA is heated, the molecules gain energy and begin to jiggle. As the temperature reaches 95 degrees
Celsius, the molecules move vigorously enough to break hydrogen bonds. The complementary DNA strands separate in a process called denaturation.
• A labeled DNA probe is then added to the denatured DNA. The probe has a sequence that is complementary to a
DNA molecule of interest. When the temperature is lowered to about 65 degrees Celsius, complementary strands can renature by pairing with each other. The label on the probe allows the detection of the DNA of interest.
Southern Blotting
• These four cells represent different clinical isolates of the same species of bacteria. Each contains plasmid DNA,
but the plasmids in one isolate may not be the same as the plasmids in another isolate. We can purify the plasmid DNA from the isolates and use two procedures—gel electrophoresis and Southern blotting—to compare these plasmids.
• As a first step, we can use gel electrophoresis to examine the plasmids in their native form. Blue loading buffer is
added to an aliquot of the plasmid sample, and the mixture is loaded into wells of a jello-like slab made of a polysaccharide material called agarose. The agarose contains microscopic pores through which the DNA can travel.
• An electric current draws the negatively charged DNA through the gel toward the positive electrode. The positions
of the DNA on the agarose gel indicate the sizes of the plasmids. Smaller pieces of DNA migrate faster than larger pieces through the agarose pores, because larger pieces meet with more resistance. In this example, the plasmids in lanes A through D are similar in size.
• The plasmid in lane C migrates slower than the others and is therefore a little larger. Although the plasmids in A,
B, and D appear to be the same, we must perform other tests before we can come to this conclusion. Our next step is to add enzymes called restriction enzymes to the plasmids.
• An example of a restriction enzyme is Eco RI. A restriction enzyme cuts the DNA at a particular recognition
sequence. Eco RI recognizes and cuts the sequence GAATTC. If the plasmids in the different tubes are identical in sequence, they should have the same number of Eco RI recognition sequences.
• The type of plasmid in this particular sample can be cut in four places with Eco RI, producing four fragments of
different sizes, represented here as different colors. When the fragments are loaded on a gel and electrophoresed, the fragments separate from each other. The slower bands on the gel contain larger fragments, and the faster bands contain smaller fragments.
• The patterns of fragments in lanes A and B appear identical. On the other hand, the patterns of fragments in lanes
C and D appear unique. Are the DNA sequences in fragments in lanes A and B the same (or nearly so), and is there any similarity between them and any of the fragments in lanes C and D? To answer this question, we perform what is called a Southern Blot.
• An apparatus is set up to transfer the DNA in the gel onto a solid support, such as nitrocellulose paper. The gel is
placed on top of a sponge soaking in an alkaline solution. Nitrocellulose paper is placed on top of the gel, and a
stack of paper towels is added on top of the nitrocellulose. The solution is drawn up the stack. The DNA denatures in the alkaline solution as it is carried up the stack.
• The DNA stops traveling when it reaches the nitrocellulose. If we could see the DNA on the nitrocellulose paper, it
would show the same pattern of DNA bands as existed on the agarose gel.
• We place the nitrocellulose paper into a plastic bag and add a solution containing a radioactive, single-stranded
DNA, called a probe. In this case, the probe consists of all of the fragments from plasmid A. During an incubation period, the probe anneals to complementary sequences, forming base pairs with the DNA in some of the bands on the paper.
• The excess probe is washed off. The only probe that remains on the nitrocellulose paper is the probe that has
annealed to complementary DNA on the paper.
• After the probe is washed off, the paper is overlaid with x-ray film. The film and nitrocellulose are kept in the dark
for a period of time, and then the researcher develops the film. The radioactivity in the probe creates black bands on the x-ray film, which is called an autoradiograph.
• The autoradiograph provides more evidence that plasmids A and B are the same or very similar. However,
plasmid D is unrelated to plasmid A, from which the radioactive probe was derived. Plasmid C has some similarity to plasmid A, but the similar sequences are found on different-sized DNA fragments. The autoradiograph gives us more information than the agarose gel on its own.
Colony Hybridization
• The colonies on this plate consist of clones of bacterial cells. The plasmids found in each colony contain
fragments of DNA from a different organism, such as a human. As such, the colonies make up part of a human DNA library. Using a technique analogous to Southern blotting, called colony hybridization, a researcher can identify a colony containing a DNA insert of interest.
• In colony hybridization, nitrocellulose paper is overlaid onto the Petri plate and then lifted off. Portions of all the
colonies stick to the nitrocellulose paper, producing a replica of the Petri plate colonies.
• The colonies on the Petri plate are incubated so that they regrow. Meanwhile, the nitrocellulose paper is treated
with chemicals so that the bacteria burst, or "lyse," and the double-stranded DNA from the bacteria denatures to form single-stranded DNA.
• The DNA, which is now permanently affixed to the nitrocellulose, can be incubated with radioactive DNA called a
probe. The probe is chosen so that it is complementary to a DNA sequence of interest somewhere in the DNA library. In this example, the inserted DNA in one colony is complementary to the probe, while the inserted DNA in another is not.
• Excess probe is washed off the nitrocellulose. Only the plasmid DNA containing the appropriate insert retains the
radioactive probe. The presence of radioactivity can be detected by x-ray film. The radioactivity exposes overlying areas of the film. These areas turn black when the film, called an autoradiograph, is developed.
• The single positive signal shown in this autoradiograph represents a single colony on the original Petri plate. The
orientations of the nitrocellose paper, the film, and the Petri plate have been marked so that the film and Petri plate can now be lined up to find the colony of interest. The bacteria from the colony can be grown for the large- scale production and analysis of this particular plasmid.
• Antibodies as probes for proteins: Antibodies are used to detect specific proteins in cells or cell extracts.
Monoclonal Antibodies
• The first step in making a hybridoma is to generate antibody-producing B cells. This is done by immunizing a
mouse against the antigen of interest. Typically, multiple immunizations are performed over a period of weeks until an appropriate antibody titer is achieved. Intraperitoneal (IP) injections are the most common method for delivering the antigens into mice.
• Next it must be determined if the mouse is producing antibodies of interest. Test bleeds are performed and
examined for the presence of antibodies. Note that while some B cells will produce the antibodies that bind to epitopes on the antigen of interest (red and blue), others will produce "nonspecific" antibodies that do not recognize the injected antigen.
• If the host is producing the desired antibody, the spleen is removed and dissociated in culture medium to release
the resident B cells. The culture medium also includes cells from a special mouse myeloma cell line. These tumor cells can divide indefinitely, but do not produce antibodies.
• When polyethylene glycol (PEG) is added to the mixture, some of the two types of cells fuse to form hybridomas.
