BIOL 402 Chapter 19 & 20

Which is least likely to alter the rate at which a DNA fragment moves through a gel during electrophoresis?

Alternating the nucleotides sequence of the DNA fragment

Bacterial Recombination

DNA fragments from one bacterium enter another bacterium and recombine to produce progeny with shared characteristics. 3 different ways: transformation, conduction, transduction.

Which procedure would produce RFLPs?

Incubating DNA with restriction enzymes

To cause a human pandemic, the H5N1 avian flu virus would have to

become capable of human-to-human transmission.

How does a bacterial cell protect its own DNA from restriction enzymes?

by adding methyl groups to adenines and cytosines

In the video, we used the Southern blot method in the analysis of the RFLP that distinguish individual #1 from individual #2. How were we able to detect the different DNA fragments on the nylon filter "blot" of the gel?

by using a labeled ssDNA probe

DNA sequencing

the process of determining the precise order of nucleotides within a DNA molecule.

Gene cloning

the production of multiple copies of a single gene Steps: 1. Restriction enzyme cuts the sugar-phosphate backbones. 2. DNA fragments added from another molecule cut by the same enzyme. Base pairing occurs. 3. DNA ligase seals the strands. - Gene cloning is use to make recombinant DNA - In order to clone a gene, we use transformation or PCR.

DNA fragments from a gel are transferred to a nitrocellulose paper during the procedure called Southern blotting. What is the purpose of transferring the DNA from a gel to a nitrocellulose paper?

to attach the DNA fragments to a permanent substrate

Agarose gel electrophoresis

- To separate and visualize the fragments, researchers carry out a technique called gel electrophoresis, which uses a gel made of a polymer that has microscopic holes of different sizes, through which shorter fragments can travel faster. The gel works as a molecular sieve to separate out a mixture of nucleic acid fragments by length. - DNA has a negative charge on the phosphate group. DNA molecules are separated by length in a gel made of a polysaccharide called agarose. - (-) charged DNA fragments will move through a gel in an electric field, anions are attached to (+) anode, - smaller DNA fragments move faster through the gel so will separated from larger fragments.

bacterial transformation

- ability of bacteria to alter their genetic makeup by uptaking foreign DNA from another bacterial cell and incorporating it into their own. - Bacterial transformation is a key step in molecular cloning, the goal of which is to produce multiple copies of a recombinant DNA molecule. - Many bacteria have cell-surface proteins that can capture DNA from closely related species. - In transformation, the DNA (usually in the form of a plasmid) is introduced into a competent strain of bacteria, so that the bacteria may then replicate the sequence of interest in amounts suitable for further analysis and/or manipulation.

Southern Blot Hybridization

- allows the detection specific DNA fragments in a sample of genomic DNA - a method to detect RFLPs in genomic restriction digests, hybridize a DNA probe to identify homologous DNA fragments. - DNA fragments are separated based on their size and charge during electrophoresis. - this method is used to detect disease.

List or describe the components of enveloped and non-enveloped viruses

- Enveloped viruses: influenza viruses have an outer envelope, with glycoprotein spikes, around a capsid that contains 8 double-helical RNA-protein complexes, each associated with viral polymerase. - Non-enveloped viruses: tobacco mosaic virus has a helical capsid, made up of capsomeres, surrounding a single helical RNA molecule, with the overall shape of a rigid rod.

Identify the specific type of host cell and receptor targeted by HIV infection.

- HIV infect T4 lymphocytes - HIV surface glycoproteins bind to CD4 receptor - envelope is fused with the T-cell plasma membrane - uncoating release HIV genome into the cell - exit by budding & then assembly within the envelope. - REVERSE TRANSCRIPTION (RNA to DNA): When the viral oncogene infects another cell, an enzyme called reverse transcriptase copies the single-stranded genetic material into double-stranded DNA, which is then integrated into the cellular genome.

Tools for gene cloning

- Restriction enzymes - Cloning vectors: plasmids, viruses - Southern blot - PCR - Ligation - Transformation

Explain the lytic vs. lysogenic bacteriophage infection cycles.

- The lytic cycle involves the reproduction of viruses using a host cell to manufacture more viruses; the viruses then burst out of the cell. With lytic phages, bacterial cells are broken open (lysed) and destroyed after immediate replication of the virion. As soon as the cell is destroyed, the phage progeny can find new hosts to infect. - The lysogenic cycle involves the incorporation of the viral genome into the host cell genome, infecting it from within. The lysogenic cycle does not result in immediate lysing of the host cell. Those phages able to undergo lysogeny are known as temperate phages. Their viral genome will integrate with host DNA and replicate along with it fairly harmlessly, or may even become established as a plasmid. The virus remains dormant until host conditions deteriorate, perhaps due to depletion of nutrients; then, the endogenous phages (known as prophages) become active. At this point they initiate the reproductive cycle, resulting in lysis of the host cell. An example of a bacteriophage known to follow the lysogenic cycle and the lytic cycle is the phage lambda of E. coli.

Translation of viral RNA of Coronavirus

- 5' end (ORF1a, ORF1b) immediately translated by ribosomes - the polyproteins (pp1a, pp1b) contain the replicase, proteases, & nsp - these long polypeptides are cut apart by one of the proteases - the replicase is an RNA-dependent RNA polymerase (RDRP) - RDRP transcribe the (+) RNA of the second half of the genome, produces subgenomic RNA templates encoding the structural proteins - The RDRP also produces (+) RNA genomes for assembly - Coronavirus replication occurs within the double membrane compartments - nsp 3,4 induce curvature of ER, produces DMVs - the host cell RNAases can't degrade the dsRNA - the new (+) RNA genomes assemble with nucleocapsid (N) proteins - the spike proteinm other surface proteins are synthesized by rough ER - large flattened Golgi vesicles contain viral proteins - the vesicle membranes fuse to form a double-membrane structure - the vesicle fuses with the plasma membrane of the cell, releasing the enveloped virus (exocytosis of exosomes).

Conjugation

- DNA is transferred cell to cell through a "mating bridge" or conjugation tube. - Sex pili can form conjugation tubes with other cells 1. F plasmid DNA is replicated 2. ssDNA is transferred to F- cell 3. ssDNA is replicated to produce ds F plasmid - The F plasmid can integrate into the bacterial genome of the F+ cell. Hfr = high frequency recombination.

Genetic engineering

- modify gene: alter protein, gene therapy - modify gene expression (alter fruit ripening) - add gene from different organism (transgenic crops)

Identify two mechanisms by which new Influenza A viruses can emerge to create epidemics.

- newly appeared as a disease in human populations - mutation of existing viruses - travel and exposure from isolated populations - spread from other animals: animals as natural reservoir for viruses. - Epidemics occur when genetic changes allow a new viral strain to be easily transmitted between humans. - Influenza viruses can change quickly because they have a genome made up of nine segments of RNA rather than a single RNA molecule. When an animal is infected with multiple strains of influenza virus, the RNA molecules making up the viral genomes can mix and match during viral assembly, resulting in new genetic combinations.

Viruses

- obligate intracellular parasites of living organisms: replicated only within a host cell, the viral genome is replicated, new capsomeres are produced, new virus particles are assembled. - aggregates of biological molecules. - lack numerous of characteristics of life: no metabolism (do not conserve energy), lack cellular structure (no cytoplasm, no ribosomes), cannot produce on their own.

Recombinant DNA technology

- technology that combines genes from different sources into a single DNA molecule - Eg: bovine growth hormone, insulin, enzyme, interferon. The human GH1 gene was cloned from mRNA: - cDNA was obtained from the mRNA genes expressed in pituitary gland. - to clone only the mRNA produced, not genomic DNA - How? First, isolate the mRNA from the cells in the pituitary gland; then use reverse transcriptase to produce cDNAs. Next, attach R.E recognition sites, cut with RE to produce sticky ends. Ligation to insert cDNA in plasmids. Finally, transformation of E.coli with recombinant plasmid DNA.

Reproductive cloning

- technology that produces genetically identical individuals. - nuclear transfer, enucleated eggs, diploid cell from donor. Eg: dolly sheep first cloned mammal. Method: nuclear transfer

Transformation methods of animals

- transgenic animals: transformation of animal cells - DNA is injected into in vitro fertilized eggs by microinjection: zygotes -> embryos, embryos implanted into surrogate mother. Eg: bovine growth hormone boost milk production 25%

RFPLs (restriction fragment length polymorphisms)

- used to detect single nucleotide differences among individuals - are the result of the loss/gain of restriction sites - indicate DNA sequence variation - RFLP methods can be used to detect an allele.

Describe the steps of a viral infection cycle

1. Attachment, entry: the virus enters the cell and is uncoated, releasing viral DNA and capsid proteins. - endocytosis (virus w/o membrane) - fusion (virus w/ membrane) & uncoating to release viral genome. 2. Host enzymes replicate the viral genome (DNA or RNA) 3. Meanwhile, host enzymes transcribe the viral genome into viral mRNA, which host ribosomes use to make more capsid proteins, surface proteins, and enzyme. 4. Viral genomes and capsid proteins self-assemble into new virus particles, which exit the cell by: - lysis: host cell burst opens, localized area, lesions. - budding: viruses emerge from the host cell, surrounded by host cell membrane, infection can be chronic, possibly oncogenic (DNA viruses), cell cycle is pushed into S-phase for replication.

steps for splicing foreign DNA into a plasmid and inserting the plasmid into a bacterium

1. Extract plasmid DNA from bacteria cells 2. Cut the plasmid DNA using RE 3. Hydrogen-bond the plasmid DNA to nonplasmid DNA fragments 4. Use ligase to seal plasmid DNA to nonplasmid DNA 5. Transform bacteria with recombinant DNA molecule

Explain/diagram the infection cycle of an enveloped animal virus, eg. influenza.

1. Glycoproteins on the viral envelope bind to receptors on the host cell. For some viruses, the envelope fuses with the plasma membrane, others enter by endocytosis. 2. The capsid and viral genome enter the cell. Digestion of the capsid by cellular enzymes releases the viral genome. 3. The viral genome functions as a template for synthesis of complementary RNA strand by a viral RNA polymerase. 4. New copies of viral genome RNA are made using the complementary RNA strands as templates. 5. Complementary RNA strands also function as mRNA, which is translated into both capsid proteins in the cytosol and glycoproteins for the viral envelope in the ER and Golgi body. 6. Vesicles transport envelop glycoproteins to the plasma membrane. 7. A capsid assembles around each viral genome molecule. 8. Each new virus buds from the cell, its enveloped studded with viral glycoproteins embedded in membrane derived from the host cell.

How to clone a gene

1. Isolate a DNA segment with your gene of interest from genomic DNA Isolation methods: a. restriction digests, agarose gel, southern blot hybridization & then another agarose gel or b. PCR c. start from mRNA transcript 2. cut the isolated DNA segment with a restriction enzyme to produce a restriction fragment with sticky ends. Complementary sticky ends can anneal. 3a. choose a plasmid (small circular DNA from bacteria) vector b. cut the plasmid vector with the same restriction enzyme 4. Ligation: - mix cut plasmid DNA + cut restriction fragment - cool to anneal (complementary base-pairing between sticky ends) - to form covalent bonds (phosphodiester linkage) between plasmid, fragment add DNA ligase 5. Transformation of E.coli with ligation DNA mix - selection for ampicillin resistance. 6. choose white colonies, transfer and keep on LB amp.

Transformation methods of plants

1. Tiplasmid, TMV virus (special plant vectors) by Agrobacterium tumefaciens, bacteria that carries a Ti plasmid, infects plant. Eg: golden rice. 2. The gene gun, "biolistics": particle bombardment, shoot DNA-coated gold or tungsten particles at cells, the DNA enters the nucleus. Eg: Bt gene (insect resistant crops), roundup ready = glyphosate - Transgenic plants with glyphosate-resistance gene from glyphosate-degrading bacteria.

Gene therapy method

1. isolate stem cells from patient 2. infect cells with recombinant virus (w/normal allele) 3. inject child with transformed cells.

PCR (polymerase chain reaction)

A laboratory technique for amplifying DNA in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides. In the PCR procedure, a three-step cycle causes a chain reaction that produces an exponentially growing population of identical DNA molecules. During each cycle, the reaction mixture is heated to high temperatures to denature (separate) the strands of the double-stranded DNA and then cooled to allow annealing (hydrogen-bonding) of short, single-stranded DNA primers complementary to sequences on opposite strands at each end of the target sequence; finally, a special DNA polymerase extends the primers in the 5'-3' direction. This cycle is then repeated 30-40 times. The key to automating PCR was the discovery of an unusual heat-stable DNA polymerase enzyme called Taq polymerase. PCR is speedy and very specific. Only a minuscule amount of DNA need be present in the starting material, and this DNA can be partially degraded, as long as there are a few copies of the complete target sequence. The key to the high specificity is the pair of primers used for each PCR amplification. The primer sequences are chosen so that they hybridize only to sequences at opposite ends of the target segment, one on the 39 end of each strand. Despite its speed and specificity, PCR amplification cannot substitute for gene cloning in cells to make large amounts of a gene. This is because the polymerases that are used have no proofreading function, and occasional errors during PCR replication limit the number of good copies and the length of DNA fragments that can be copied. Instead, PCR is used to provide the specific DNA fragment for cloning.

reverse transcriptase

An enzyme encoded by certain viruses (retroviruses) that uses RNA as a template for DNA synthesis.

Bacteriophage

Bacteriophage - a virus that infects bacteria: - DNA viruses - Protein coat with capsid - many much more complex - limited host range, determined by host surface molecules that the phage must bind. - tail fibers bind complementary to receptors. - binding induces conformation change in tail fibers, contacting base plate with cell wall, enzyme digests a hole in cell wall, DNA injected into cell.

Transformation of plants/animals

Challenges for plant/animal transformation: - genes in a nucleus (eukaryotic cells) - eukaryotic gene expression (regulatory sequences, etc.) - multicellular organisms: transform single cell (in tissue culture) cell -> embryo -> adult. STEPS: 1. clone gene: recombinant DNA in E.coli 2. transformation eukaryotic cells: methods - gene gun, Ti plasmid, viral infection, microinjection 3. Cell in tissue culture -> adult organism or transform embryo Result: transformation of whole organism (heritable) to produce genetically modified organism (GMOs). - transgenic: if gene from a different organism Plants are easier to transfer because they have totipotent cells: single cell -> undifferentiated cells -> whole plant!

How is the COVID-19 pandemic different compared to the SARS or MERS coronaviruses?

SARS CoV-1 - high death rate (7-9%) but did not spread easily - no transmission until 24-36 hours after symptoms - no asymptomatic cases, contract tracing worked MERS CoV - very high death rate (34%) - in hospital transmission, or camel to human transmission - slow spread, managed SARS CoV-2 - mostly mild cases, many symptomatic cases - death rate 0-50% depending on age - easier transmission - by large and small droplets, surfaces.

Explain the components of HIV and their role in the infection cycle, including RNA genome, capsid, envelope, glycoproteins, reverse transcriptase, integrase

Entry: glycoprotein knobs bind to specific molecule - envelop fuse with host plasma membrane - RNA genome is reversely transcribed into DNA using a viral enzyme called reverse transcriptase. This drives the reverse transcription which transcribe RNA to complementary DNA (cDNA). That single-stranded DNA is again reverse transcribed into a double-stranded DNA. At that time, another enzyme that has come in with the virus in the beginning called integrase essentially grabs hold of that double stranded DNA and carries it through a nuclear pore into the nucleus of the cell. Within the nucleus of the cell, it finds the host chromosome, the integrase enzyme makes a nick in the host DNA and allows for HIV to insert itself into the host chromosome. The viral particles then exit cell by budding, the capsids tear off a surrounding piece of the host plasma membrane that then become the envelope of the virus.

In the gene cloning video, you learned how a DNA fragment can be spliced into a plasmid vector. In the example used in the video, which plasmid gene was disrupted by ligation of the fragment?

GFP

What is the function of reverse transcriptase in retroviruses?

It uses viral RNA as a template for DNA synthesis.

In recombinant DNA methods, the term vector can refer to

a plasmid used to transfer DNA into a living cell

Steps in transformation of E.coli procedures:

R plasmids carry antibiotic resistance genes. 1. Prepare transformation competent cells: - transfer E.coli single colonies to ice cold CaCl2 - keep on ice 2. add 10ml plasmid DNA to CaCl2/wells, leave on ice for 15-30 mins. 3. heat shock in 42˙C water bath for 90 sec. 4. put back on ice, then add 100ml LB broth to each tube, incubate at 37˙C for 15 minutes. 5. spread 100 ml of each sample onto selective media.

Explain why RNA viruses are more likely to evolve rapidly, while DNA viruses may cause cancer or a recurring infection.

RNA viruses appear to have higher rates of mutation because replication of their genomes does not involve proofreading and because the single stranded genetic material develops mutations more frequently than DNA viruses.

RT-PCR (reverse transcriptase-polymerase chain reaction)

RT-PCR begins by turning sample sets of mRNAs into double-stranded DNAs with the corresponding sequences. First, the enzyme reverse transcriptase is used to synthesize a complementary DNA copy (a reverse transcript) of each mRNA in the sample. The mRNA is then degraded by addition of a specific enzyme, and a second DNA strand, complementary to the first, is synthesized by DNA polymerase. The resulting double-stranded DNA is called complementary DNA.

STRs (short tandem repeats)

Short tandem repeats (STRs) are short nucleotide sequences that are repeated in tandem, - composed of different numbers of repeating units in individuals and - used in DNA profiling. - the most commonly used VNTRs are called STR.

Explain the role of the spike protein, ACE-2, and the host cell protease in the CoV-2 infection cycle.

Spike protein (S1) must bind to the ACE2 receptor on host cell. Binding with ACE-2 must be followed by a cutting of parts of spikes by host protease called TMPRSS2. This protease bends over, cleaves the spike protein, exposing the S1 fusion domain. The S1 fusion domain initiates fusion/endocytosis as it grabs the membrane and bring them together. ssRNA, once free, is translated to produce RNA replicase complex and 2 different viral proteases.

Genome editing with CRISPR-Cas9 System

The Crisper technique is derived from a newly understood bacterial immune system.

Therapeutic cloning

The cloning of human cells by nuclear transplantation for therapeutic purposes, no implantation of embryo. Embryonic: stem cells removed from a blastocyst Stem cells: undifferentiated cells, proliferate in culture, divide by mitosis, pluripotent.

Identify the components of the CoV-2 virus, and specify their role in infection, including ssRNA (+), nucleocapsids, envelope, Spike (S) protein, M protein, E protein

The coronavirus virion consists of structural proteins, namely spike (S), envelope (E), membrane (M), nucleocapsid (N). The positive-sense, single-stranded RNA genome (+ssRNA) is encapsidated by N, whereas M and E ensure its incorporation in the viral particle during the assembly process. S trimers protrude from the host-derived viral envelope and provide specificity for cellular entry receptors.

restriction fragments.

The most commonly used restriction enzymes recognize sequences containing fourto eight nucleotide pairs. Because any sequence that is this short usually occurs (by chance) many times in a long DNA molecule, a restriction enzyme will make many cuts in such a DNA molecule, yielding a set of restriction fragments.

Which of the following statements describes the lysogenic cycle of lambda (λ) phage?

The phage genome replicates along with the host genome.

DNA ligation

The process of joining two pieces of DNA to a single piece through the use of ligase (DNA enzyme).

sticky ends

The resulting double-stranded restriction fragments have at least one single-stranded end, called a sticky end. 3' overhanging ssDNA, can reanneal to base pair with complementary ssDNA which resulting in recombinant DNA. These short extensions can form hydrogen-bonded base pairs with complementary sticky ends on any other DNA molecules cut with the same restriction enzyme The associations formed in this way are only temporary but can be made permanent by DNA ligase, an enzyme that catalyzes the formation of covalent bonds that close up the sugar-phosphate backbones of DNA strand

Distinguish between the receptor binding and fusion domains of the CoV-2 Spike protein.

The spike (S) protein of SARS-CoV-2, which plays a key role in the receptor recognition and cell membrane fusion process, is composed of two subunits, S1 and S2. The S1 subunit contains a receptor-binding domain that recognizes and binds to the host receptor angiotensin-converting enzyme 2, while the S2 subunit mediates viral cell membrane fusion by forming a six-helical bundle via the two-heptad repeat domain.

Identify two separate ways that the CoV-2 virus uses the rough ER of the host cell during infection.

Upon translation, the S, E, and M structural proteins are inserted into the rough ER. From there, the proteins travel along the secretory pathway to the endoplasmic reticulum-Golgi apparatus intermediate compartment, the site of CoV particle assembly. Afterwards, the virus is released from the cell via exocytosis.

Retroviruses

Viruses that have single-stranded RNA that acts as a template for DNA synthesis.

CRISPR

a collection of DNA sequences that tells Cas9 exactly where to cut

Sanger method of DNA sequencing (dideoxy sequencing)

a method of DNA sequencing that uses dideoxyribonucleotides to terminate the growth of DNA strands

Animal virues

a virus infects animals. - can be DNA or RNA viruses - can be double-stranded or single-stranded - can be naked or enveloped. 1. DNA, naked - adenoviruses - ds DNA - largest naked virus, icosahedral shape - enters by endocytosis - replicates in nucleus by host DNA polymerase - exits by lysis 2. DNA, enveloped: - herpes, chickenpox (shingles) - entry by fusion - provirus: latency -DNA can remain in host nucleus, genome, may recur - exit by budding 2. RNA, naked (capsid only): - hepatitis A, rabies, rhinovirus - enter by endocytosis - replicated in the cell (+) or (-) RNA - exit by lysis 3. RNA, enveloped: - influenza (flu), measles, Ebola, coronaviruses - enter by fusion - exit by budding - RNA genomes replicated by viral replicases, no proofreading so mutations likely. - most RNA viruses evolved rapidly, new viral forms emerge 4. Retroviruses - HIV - tumor-causing virus - can be latent - evolve rapidly

One of the last steps in gene cloning is transformation of E. coli. For the cloning example on the video, which of these describes the results on the LBAMP plate that was spread with the ligation DNA?

both green and white colonies

Bacteria containing recombinant plasmids are often identified by which process?

exposing the bacteria to an antibiotic that kills cells lacking the resistant plasmid

Restriction fragments of DNA are typically separated from one another by which process?

gel electrophoresis

RNA viruses require their own supply of certain enzymes because

host cells lack enzymes that can replicate the viral genome.

The host range and specificity of a virus is determined by whether

it has surface proteins that recognize host cell receptors

Emerging viruses arise by

mutation of existing viruses, the spread of existing viruses to new host species, and the spread of existing viruses more widely within their host species.

DNA fingerprint (profile)

pattern of DNA fragments obtained by analyzing a person's unique sequences of noncoding DNA. To prepare DNA profile: 1. isolate DNA from food, saliva, skin samples, etc. To detect DNA segment by PCR 2. amplify DNA segment by PCR 3. electrophoresis to detect length difference.

RFLP analysis can be used to distinguish between alleles based on differences in which of the following?

restriction enzyme recognition sites between the alleles

SCIDS (severe combined immunodeficiency syndrome)

result of a mutation in an X-linked gene, affected T lymphocytes, compromising the immune system.

A gene that contains introns can be made shorter (but remain functional) for genetic engineering purposes by using

reverse transcriptase to reconstruct the gene from its mRNA

Transduction

transfer of DNA from one cell to another by a bacteriophage

Explain how we can select for transformants.

transformants are E.coli clones, harbouring a pGT4 plasmid, or a pGT4 derived plasmid.

Restriction enzymes

types of nucleases that cut DNA molecules at a limited number of specific locations. - exonuclease - cut nucleotides off ends of DNA or RNA - endonuclease - cut nucleotides at an interior location; Restriction enzymes are endonuclease which cut DNA at specific sequences known as recognition sequences aka restriction sites. - Bacteria uses these to defend against phages. - Many restriction enzyme sequences are palidromic.