Exam 4

Viral Defense Mechanisms of Bacteria and Archaea

**Prokaryotes and viruses compete for survival -Prokaryotes using: 1) Restriction endonucleases to target and destroy foreign DNA. 2) Using the CRISPR-Cas system to destroy foreign DNA. 3) Altered viral receptor sites. -Viruses respond by: 1) Substituting modified bases into the sites recognized by restriction endonucleases. 2) Evolving mechanisms to evade the CRISPR system by targeting host protective measures or inhibiting activity of Cas proteins. 3) Substituting a phage gene into existing toxin-antitoxin modules such that the cell has to maintain the prophage to survive.

Subviral Agents: Viroids and Prions

*Viroids and prions are infectious agents that resemble viruses but lack either nucleic acid or protein so are not viruses. -Viroids: Infectious RNA molecules with no protein; small, circular ssRNA molecules that range in size from 246-399 nucleotides and share homology. Cause a number of plant diseases, but are not known to infect animals or microorganisms. Consist of naked ssRNA with extensive secondary structure that allows it to be sufficiently stable to exist outside of a host cell. Enters plant cells via wounds and move between cells using plasmadesmata. Contain no protein-encoding genes so viroid is dependent upon the host for replication. Plants use an RNA polymerase with RNA replicase activity to replicate the viroid in a large molecule that self-cleaves into individual viroids. Viroids are thought to interfere with plant small regulatory RNAs (may have evolved from these molecules!). -Prions: Distinct extracellular form consisting solely of protein, no nucleic acid. Infectious and causes diseases in animals and humans collectively called transmissible spongiform encephalopathies (TSEs). Includes scrapie, BSE (mad cow disease), chronic wasting disease, kuru, and Creutzfeldt-Jakob disease (CJD). Prion that causes BSE in cows can infect humans; led to variant CJD. Prion proteins and the prion infectious cycle...

Recording and control of genetically modified E. coli

-All TAG stop codons were replaced with TAA stop codons. -The AUG anticodon is now chared with a synthetic amino acid (sAA).

Biocontainment of Genetically Modified Organmisms

-Although applications of genetic engineering have been beneficial, there are concerns about genetically modified genes into wild populations. Have introduced a synthetic amino acid into some GMOs such that if the modified amino acid is not available, the organism cannot grow. Since the synthetic amino acid is not available in the environment, the organism cannot grow there.

The Human Virome

-Animal viruses typically take up the entire virus particle while many prokaryotes just take up the genome. -There are also more potential outcomes of viral infection in animal cells (lytic, persistent infection, latent infection, transformation) than in prokaryotic cells (lytic, lysogenic). -The human virome is the entire population of viruses present on and in the human body. -For healthy humans, the virome is dominated by viruses with DNA genomes, including a number of plant viruses that are likely transmitted in food and bacteriophage (particularly in the gut).

Viruses of Archaea

-Both DNA and RNA archaeal viruses have been discovered but most are dsDNA. -There are some unusual structures found in archaeal viruses, including spindle-shaped virions that cluster in rosettes and rigid helical rod-shapes. -One spindle-shaped virus is lemon-shaped when it is released from its host cell but then produces long thin "tails" at each end. As the tails grow, the virion becomes thinner with a reduced volume.

Molecular Methods for Mutagenesis

-Conventional mutagens introduce mutations in random locations. -Site Directed Mutagenesis: Uses synthetic DNA in combination with cloning techniques to introduce mutations at specific locations. -If a target gene is denatured into ssDNA and hybridized with a mutated synthesized probe, the dsDNA will contain the mutation. -After transformation of the vector into host cells, the semiconservative replication of the vector into daughter cells means that one copy will get the mutation while the other will receive the wild-type gene.

Biotechnology

-Genetic engineering that transforms microorganisms into factories to produce valuable products. Many human proteins can be produced in microbial cells. -Insulin was the first human protein produced in bacteria but somatotropin, a growth hormone, is another. The human somatotropin gene was cloned as cDNA from mRNA and espressed in a bacterial expression vector. -Recombinant bovine somatotropin is used in the dairy industry to stimulate milk production.

The Bacterial and Archaeal Virosphere

-In seawater, there are about 1 million prokaryotic cells per milliliter and 10 million viruses. -Estimated that 5-50% of the bacteria are killed by viruses each day and most of the others are eaten by protozoa. -If host cells are rare, lysogeny would be favored over lytic infections until host population size increases. This strategy would work for dsDNA viruses that can become lysogenic but we don't know how ssDNA and RNA viruses survive low host cell availability.

Positive-Strand RNA Viruses

-In these viruses, the sequence of their genome and mRNA is the same. -Bacteriophage MS2 is an icosahedral virus that infects E. coli by attaching to the pilus. Its overlapping genome encodes 4 proteins: coat protein, lysis protein, maturation protein, and 1 subunit of RNA replicase (the other subunits come from the host cell). Its genome is translated directly upon entry to the cell by the host ribosomes. Once replicase is made, it makes copies of -RNA using the +strands as templates. The - strands can make more + strands which make more viral proteins. MS2 regulates protein synthesis by controlling access of host ribosomes to translational start sites on its RNA. Of the 4 possible viral AUG start sites, the most accessible are those for the coat protein and replicase, so translation begins at these sites early in infection but as coat protein molecules accumulate, they bind to the RNA around the start site, blocking synthesis of replicase. The AUG site for the maturation protein has limited access due to extensive folding of the RNA so only a few copies are made. -Poliovirus: Cause disease in humans and other animals, including polio, common cold, respiratory syndromes, and hepatitis A. One of the smallest icosahedral structures with a linear genome. A protein called VPg is attached covalently to the 5' end and a poly(A) tail is on the 3' end of the viral RNA. The genome is the mRNA and the VPg protein helps the RNA to bind to host ribosomes. Translation of the mRNA yields a polyprotein, a single large protein that self-cleaves into ~20 smaller proteins (post-translational cleavage). Replication of viral RNA begins shortly after infection via the polioviral RNA replicase. Both + and - strands have the VPg protein on the 5' end which also functions as a primer for RNA synthesis. Host lysis and release of new virions occurs within 5 hours of infection.

Molecular Cloning

-Isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated. -Vector:Small, simple genetic element such as a plasmid or virus. -Molecular cloning results in recombinant DNA (DNA from 2 or more sources) and involves 3 steps: 1) Isolation and fragmentation of source DNA. 2) Insertion of the DNA fragments into a cloning vector. 3) Introduction of the cloned DNA into a host organism. -Cloning vectors are usually designed to allow insertion of foreign DNA at a restriction site. -Restriction Enzymes (or Endonucleases): Enzymes that recognize specific base sequences (restriction sites) within DNA and cut the DNA. -The sites that the restriction enzymes recognize are inverted repeats called palindromes (they are the same if read from 5' to 3' on either strand). Most are homodimeric and one subunit cuts each strand, resulting in a double-stranded break, often leaving short, single-stranded overhangs on each fragment called sticky ends. These sequences have been protected by methylation and cannot be cut.

Expression Foreign Genes in Bacteria

-Once genes are cloned, they can be transcribed and translated to produce proteins. -To make a eukaryotic protein in a prokaryotic cell, the following issues must be addressed: 1) The genes must be placed under control of a bacterial promoter. 2) Any introns must be removed as prokaryotes do not do a lot of post-transcriptional modifications. 3) Codon usage must conform to the producing organism. 4) Many eukaryotic proteins require post-translational modification that is not available in prokaryotic cells. -Expression Vector: Vector that allows and controls the expression of the cloned gene(s) in a new host; often require enhanced recognition of promoters. -If a cloned gene contains introns, the correct protein will not be made in a prokaryotic host. -Instead, if mRNA is isolated, this should give the correct protein as the introns should be removed. The mRNA is converted to DNA using RT-PCR and can now be cloned into a vector and inserted into a prokaryotic host. It will still need a promoter and other upstream regulatory sequences (EX: Shine-Dalgarno). -For small proteins (EX: insulin), the entire gene is artificially synthesized to allow the gene, promoters, and other regulatory sequences to be present as well as "fix" codon bias issues.

Hosts for Viruses for Each Baltimore Class

-Only 2 of the 7 classes of viruses infect Archaea and only 4 infect Bacteria. -All 7 classes are capable of infecting animal cells. *dsDNA are the major class infecting prokaryotes. *ssRNA in the plus sense are the major class infecting eukaryotic cells. -The reasons for host preference are unclear.

Manipulating DNA: PCR and Nucleic Acid Hybridization

-Polymerase Chain Reaction (PCR): DNA replication in vitro; copies segments of DNA in a process called amplification to yield large amounts of specific DNA fragments. Employs a DNA polymerase and artificially synthesized oligonucleotide DNA primers to amplify a portion of DNA. There are 3 main steps that are repeated for each cycle: 1) dsDNA template DNA is denatured 2) DNA primers anneal to the now ssDNA template 3) DNA polymerase extends the primers using the ssDNA as the template. -This powerful tool has revolutionized biology, allowing millions to billions of copies of specific DNA fragments to be generated within hours in a machine called a thermocycler. Because high temperatures are necessary to denature the dsDNA, a thermostable DNA polymerase is used (Taq is the most common). Variations on PCR led to reverse transcription PCR (RT-PCR), which allows for copying information in mRNA into DNA via reserve transcriptase; very useful for gene expression studies or to get an intron-free copy of a eukaryotic gene for expression in a prokaryote. *Gel Electrophoresis and Nucleic Acid Hybridization* -Gel Electrophoresis: Mechanism to separate fragments of nucleic acids using migration in an electrical field; rate of migration is determined by the charge on the molecule and its size and shape. Gels are made of a porous material, typically agarose. The DNA has a negative charge and moves toward the positive electrode, with small molecules moving more rapidly than large molecules. Utilize a sample containing nucleic acid fragments of known size called a ladder. Pieces cut by restriction endonucleases can now be separated by length via electrophoresis. -Nucleic Acid Hybridization: Uses complementary base pairing of single strands of DNA or RNA from 2 different sources to give a hybrid double strand. -Nucleic Acid Probes: Segments of single-stranded DNA or RNA that are used in hybridization and for which the sequence is known. -The exactness of the required match can be adjusted by changing the environmental conditions. -Often use radioactivity or fluorescence for easy detection of the hybridization. -There are 3 main types of hybridization used: 1) Southern Blot: Uses DNA as the target and RNA or DNA as the probe. 2) Northern Blot: Uses RNA as the target and RNA or DNA as the probe; used for gene expression. 3) Western Blot: Uses protein as the target and an antibody as the probe. -If you know the sequence of the gene you are looking for, you can "probe" the gels for its presence.

Viruses that Use Reverse Transcriptase

-Retroviruses contain RNA genomes and hepadnaviruses have DNA genomes that are replicated via a DNA intermediate. "Retro" means backwards and the viruses were named because they transfer information from RNA to DNA using the enzyme reverse transcriptase. -Have been shown to cause cancer. -Enveloped viruses with several proteins in the viral envelope and typically 7 internal proteins; 4 structural and 3 enzymes (reverse transcriptase, integrase, and a protease). -Also contain specific tRNA molecules for replication and 2 identical ss-RNA molecules in the + orientation.

Double-Stranded RNA Viruses

-Rotavirus is an example of Reoviruses and causes diarrhea particularly in infants. -Also include other animal and plant viruses. - Have to bring their own RNA replicase to replicate the dsRNA genome, which consists of 10-12 segments of linear dsRNA. Virions bind to cell receptors, are endocytosed and transported to lysosomes where instead of being killed, the outer protein coats are removed, activating the viral RNA replicase and releasing the nucleocapsid into cytoplasm. *All replication occurs within the nucleocapsid:why? Once viral proteins are formed in the cytoplasm, they aggregate to form new nucleocapsids, trapping RNA replicase inside. The correct complement of dsRNA genome segments is achieved by taking up molecules of ssRNA that are replicated to dsRNA in the nucleocapsid using the RNA replicase. Virions are released by budding or cell lysis. Employ a unique nucleic acid replication mechanism that is neither semiconservative nor rolling circle. •Reovirus Life Cycle: Note that all replication and transcription occurs inside the nucleocapsids.

Pathogens and Antibodies as Engineered Anticancer Therapeutics

-Targeting tumor cells have been a problem with cancer treatments. -Now trying to use some weakly pathogenic strains of Listeria monocytogenes to grow in tumor cells, delivering toxic drugs or radioisotopes to the tumor cells while being expelled from healthy cells. -Antibodies can bind to foreign substances inside cancer cells and trigger the body's immune response to kill the cells but the antibodies can't freely enter the cells. -Can use a toxin found in Bacillus anthracis to "carry" an anticancer antibody into these cells.

Genome Editing and CRISPRs

-The CRISPR-Cas system can recognize and cleave a specific DNA sequences within cells and foreign DNA could be inserted into the cut site which allows for genome editing. By designation the Cas9 protein and binds to the desired target DNA sequence, the CRISPR-Cas system can be used to cut specific DNA sequences in the genome of virtually any cell. Being used to target HIV genome and viral RNAs.

Gene Mining

-The process of identifying and isolating potentially useful genes from the environment without the need to culture the producing organism. -DNA or RNA isolated directly from the environment and cloned into suitable vectors to construct a metagenomic library. The library is screened to identify clones with the activity of interest. -If the property being screened for requires a large cluster of genes (so a large segment of DNA), the vectors used are bacterial artificial chromosomes (BACs) because they can hold more genetic information than a plasmid. -This approach has led to the discovery of a number of lipases, chitinases, esterases, and other enzymes with industrial applications.

Cloning Vectors

-There are various cloning vectors, including viruses, cosmids, plasmids, and artificial chromosomes. -Multiple Cloning Site: Often have locations within the vector containing cut sites for a variety of restriction enzymes. -Good example is pUC19 that provides a visual indicator of recombination through insertional inactivation of the lacZ gene.

Cassette Mutagenesis and Gene Disruption

-To make larger mutations, synthetic DNA fragments called DNA cassettes can be used in a process called cassette mutagenesis. These cassettes can be inserted into the middle of a gene, disrupting its correct synthesis (called gene disruption) or a knock-out mutation. -Often add a gene for antibiotic resistance with the mutation(s) in the gene(s) of interest to allow easy selection of those cells that received the cassette. -If a gene is knocked-out, this allows us to determine if that gene is essential for survival.

DNA Tumor Viruses

-Viruses of the polyomavirus family and some herpesviruses, all dsDNA viruses, can induce cancers. -Polyomavirus SV40 is a naked icosahedral virus with a circular genome that is too small to encode its own DNA polymerase so uses that of its host; has overlapping genes due to small genome size. Upon infection, there are 2 possible outcomes: 1) In a permissive host, virus infection results in new virus particles and lysis of the host. 2) In a nonpermissive host, the viral DNA becomes integrated into the host's DNA, genetically altering the cells. -These genetically altered cells can become malignant in a process called transformation (different than the process of taking up free DNA from the environment). Viral proteins bind to and inactivate host cell proteins that control cell division, thus allowing uncontrolled cell development (cancer). -Herpesviruses can remain latent in the body for long periods and become active under stress or when the immune system is compromised. Replicate the genome in the nucleus via rolling circle replication. Viral envelope is acquired via budding from the nuclear membrane (not the cytoplasmic membrane). •Herpesvirus Replication: The linear genome circularizes the host.

Virus Life Cycle

-Viruses replicate only inside living cells. -Bacterial viruses are the easiest to grow and have served as model systems. -Cells that support the complete replication cycle of a virus are permissive for that virus. -Viral replication cycle has 5 steps: 1) Attachment of virion to the host cell. 2) Penetration of viral nucleic acid into the host cell. 3) Synthesis of viral nucleic acid and proteins. 4) Assembly of capsids and packaging of viral genomes into new virions. 5) Release of virions from the cell. -Viral growth has a one-step growth curve (no virus presence until release from host cells).

Negative-Strand RNA Animal Viruses

-Viruses with (-) ssRNA genomes need to have a (+) strand made to serve as mRNA. Two major examples: 1) Rabies: Bullet-shaped with an extensive and complex envelope; virions contain several enzymes that are essential for infection including an RNA replicase. Their genome must first be transcribed by the replicase before being translated; occurs in the cytoplasm and generates two classes of RNAs: 1 for mRNAs and the other for the genome template. Assembly of rhabdoviruses is complex, requiring 2 different coat proteins for the nucleocapsid and envelope. The nucleocapsid assembles first around the viral genome and the envelope is acquired when the virion buds from the host cell, picking up viral glycoproteins that were inserted into the host cell's membrane. 2) Influenza Virus: Only found in animals and has caused a number of pandemics, including one in 1918 that killed millions of people. Enveloped virus with a segmented genome (multiple pieces of (-) ssRNA make up the genome). (Influenza A is the most infectious for humans, has 8 genome segments). The envelope is acquired as the virus buds from the host cell and contains a number of viral-specific glycoproteins including: 1) Hemagglutinin 2) Neuraminidase Influenza viruses must have 2 other enzymes, an RNA replicase to convert the (-) strand genome into a (+) strand, and an RNA endonuclease which cuts the cap from the host mRNAs and puts them on viral mRNAs to allow translation by the host. Uncoating activates the RNA replicase and transcription of the 10 proteins on the 8 segments begin. Segmented genomes have practical consequences and cause antigenic shift when a cell is infected with 2 different viral strains that are packaged incorrectly, with segments from each of the 2 viruses in progeny.

Viral Evolution

-When did viruses first appear on Earth and what is their relationship to cells? Because all known viruses require a host for replication, the natural conclusion is that they evolved after cells were present. However, there are other hypothesis, including that viruses are relics of the "RNA world", a time when RNA is thought to have been the sole carrier of genetic information, or that viruses were once cells that lost enough genes that they became dependent upon a host for replication. We will likely never know the answers! -Also want to know why viruses exist: is it a mechanism to quickly move genes in nature? RNA viruses hypothesized to have evolved DNA as a modification mechanism to protect their genomes from cellular ribonucleases; would have used an enzyme like reverse transcriptase for this process. DNA is also more stable than RNA.

2 mechanisms of transposition

1) Conservative: Transposon is excised from 1 location and reinserted at another location so copy number remains the same. 2) Replicative: A new copy of the transposon is made and inserted at the 2nd location so the copy number increases with each transposition.

2 major types of transposable elements in Bacteria

1) Insertion Sequences (IS): Simplest type; short DNA segments ~1 kbp long and contain inverted repeated of 10-50 bp; also carry a gene encoding transposase (enzyme for transposition). 2) Transposons: Larger than IS but have inverted repeats at both ends and a gene for transposase; any gene within the inverted repeats is thus moved (often antibiotic resistance genes). *The inverted repeats and the transposase are essential for transposition; transposase recognizes, cuts, and ligates the DNA. *When transposable elements are inserted into target DNA, a short sequence in the target DNA at the site of insertion is duplicated as a result of single-strand DNA breaks made by transposase.

Lysis or Lysogeny?: 2 key repressor proteins that can accumulate in infected cells controls this decision.

1) Lambda repressor or cI protein when present in high levels represses that transcription of other lambda-encoded genes and allows integration of the lambda genome into the host's chromosome causing lysogeny. -If cI is at a low level, other viral genes are transcribed, including Cro, which sends the virus down the lytic pathway as it represses a protein needed to activate the synthesis of cI. -Also uses a cascade of other regulatory systems.

3 mechanisms of genetic exchange within prokaryotes

1) Transformation: Free DNA is taken up by another cell. 2) Transduction: DNA transfer mediated by a virus. 3) Conjugation: DNA transfer involves cell-to-cell contact and a conjugative plasmid in the donor cell.

3 possible fates for incoming DNA

1)Can be degraded by restriction enzymes. 2) It may replicate by itself (if it has an origin of replication). 3) It may recombine with the host chromosome.

Uniquely Replicating DNA Animal Viruses

2 groups of dsDNA viruses show unusual replication strategies: 1) pox viruses: In pox viruses, all replication events, including DNA replication occur in the cytoplasm instead of the nucleus. Important historically and medically (smallpox, cowpox) and are among the largest of all known viruses. Uncoating of pox genomes requires the activity of a viral protein that is synthesized after infection (the gene is transcribed by a viral RNA polymerase within the virion). Other viral genes are also transcribed, including genes for a DNA polymerase that synthesizes copies of the viral genome. The viral genome is incorporated into virions that accumulate in the cytoplasm and are released via host cell lysis. Vaccinia virus, a pox virus is used as a carrier of proteins from pathogenic viruses, as it elicits a strong immune response and stimulates antibody production. 2) adenoviruses: In adenoviruses, replication of the genome proceeds in a leading fashion on both DNA template strands. They are small, naked icosahedral viruses with a linear dsDNA genome. They are of minor health importance but have a unique mechanism for replicating their genomes. At the 5' ends of the genomic DNA are adenoviral terminal proteins that are essential for replication. The complementary strands also have inverted terminal repeats that are important. Following infection, the adenoviral nucleocapsid is released into the host cell nucleus and transcription of early genes initiates using the host cell's RNA polymerase. Once the viral DNA polymerase has been made, replication of the linear genome begins at either end of the genome, facilitated by the terminal protein with a covalently bound cytosine that serves as the primer for DNA polymerase. Products of initial replication are a complete dsDNA viral genome and a ss (-) DNA molecule. The ssDNA molecule forms a stem-loop structure using the inverted terminal repeats and a complementary ss (+) DNA molecule is synthesized from the 5' end without a lagging strand as occurs normally in semiconservative DNA replication.

Lysogen

A cell harboring a temperate virus.

Transgenic Organisms in Agriculture and Aquaculture -Transgene

A gene from another organism. -Because genetically engineered plants or animals contain a gene or genes from other organisms, they are referred to as transgenic organisms or genetically modified organisms (GMOs). -Recombinant viruses are ineffective but not virulent and can carry genes from a pathogenic virus, inducing host immunity to the pathogenic virus. -Different combinations of this approach can yield polyvalent vaccines which immunize against multiple disease at the same time.

Plaque

A zone of lysis seen as a clear area when a virion initiates an infection on a layer of host cells growing on a flat surface. *The plaque assay (counting areas of clearing on a host "lawn" of cells) is a common way to measure virus infectivity. Lawn can be bacterial cells or animal cells in tissue culture.

Animal Virus Infection

Animal viruses differ from bacterial viruses in the entire animal virus typically enters the host cell and many viruses replicate in the nucleus of their eukaryotic host cell. After attaching to their host cell, animal viruses often fuse with the host cell's cytoplasmic membrane or are endocytosed. The virus must then lost the protein coat to allow the genome to function. -If the viral genome is DNA, it passes through the nuclear membrane into the nucleus for replication. -If it has an RNA genome, it often remains inside the nucleocapsid for replication. *Unlike prokaryotic cells, animal cells lack a cell wall, allowing viruses to be more easily released. -After the viral genome is packaged into the capsid, many animal viruses remove part of the animal cell's lipid bilayer as they pass through the membrane via budding, thus acquiring their envelope. -Have more possible outcomes of infection than just lytic or lysogenic. These possible outcomes are: 1) Virulent infection results in destruction of the host cell (most common). 2) However, enveloped viruses are often released via a budding process, which is slow and may not lyse the host cell, allowing it to continue making more virus particles (persistent infection). 3) Latent infection of the host involves a delay between infection and host cell lysis (EX: fever blisters caused by herpes reappear sporadically when the virus emerges from latency). 4) Can convert normal host cells into tumor cells (transformation).

Penetration

Attachment of a virus to its hosts results in changes to both the virus and the cell surface to facilitate penetration (entry). There are a variety of ways for this to occur; in some, the entire virus enters the cell (via endocytosis) while in others, only the proteins and/or nucleic acids enter the host cell. -Uncoating: The process in which the virion loses its outer coat and the viral genome is exposed; can occur inside the cell or outside the cell. *Bacteriophage T4 has a complex penetration mechanism!

Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR System)

Bacteria and Archaea not only produce restriction endonucleases to destroy foreign DNA, they also have an RNA-based defense program to destroy invading nucleic acids from viral infection or horizontally transferred genes. This rudimentary immune system helps to preserve the stability of genome and is called the CRISPR System. CRISPR region on the bacterial chromosome is a memory blank of incoming nucleic acid sequences used as surveillance against foreign nucleic acids. This region consists of many different segments of foreign DNA called spacers alternating with identical repeating sequences. Once incorporated into CRISPR region, the spacers provide resistance to incoming nucleic acids that have the same or nearly the same sequence. The Cas proteins of the CRISPR system (named CRISPR-associated) are found upstream of the CRISPR DNA sequences and perform 2 roles: 1) Participate in obtaining and storing segments of foreign DNA as spacers. 2) Use the stored sequence information to recognize and destroy intruding DNA. -The CRISPR region is transcribed as a single long RNA molecule that is cleaved in the middle of each repeated sequences by Cas nuclease activity, making small RNAs called crRNAs. If these crRNAs base-pairs with invading nucleic acid, it is destroyed by Cas proteins nuclease activity. *CRISPR is widely distributed in Bacteria and Archaea and is highly effective but mutations in the sequence of the invading nucleic acid can limit its efficacy.

Non-homologous recombination or Lateral/Horizontal Gene Transfer VERSUS Homologous Recombination

Bacterial genomes are commonly considered to be composed of a core genome, consisting of genes essential to survival that are shared by all related strains and species, and an accessory pan genome, consisting of non-essential genes that might or might not be present within a given strain. LGT (or HGT) transfers genes that make up the accessory genome, whereas homologous recombination affects both the accessory genome and the core genome. -For homologous recombination to generate new genotypes, the 2 homologous sequences must be related but genetically distinct. -In prokaryotes, only a part of a chromosome is transferred so if recombination does not occur, the DNA fragment will be lost as it cannot replicate independently. -To detect physical exchange of DNA segments, the cells resulting from recombination must be phenotypically different from both parents. -Genetic crosses usually depend upon recipient strains that are lacking some selectable character that recombinants gain.

Single-stranded DNA Bacteriophages

Before an ssDNA genome can be transcribed, a complementary strand of DNA must be synthesized forming a ds replicative form. One strand serves as the mRNA and the other as a genome copy. Two examples 1) ΦX174: The icosahedral bacteriophage ΦX174 has a circular ssDNA genome with only a few genes that are overlapping (parts of the genome must be read more than once in different reading frames). Upon infection, the genome is converted into a double-stranded form by host enzymes and several copies are made. This replicative form is used to make copies of the phage genome using rolling circle replication. In this case, there is no complementary second strand that must be added as in lambda phage. The rolling circle facilitates the continuous production of + strands from the replicative form. The - strand serves as the template to make mRNA. 2) M13: Filamentous phage with helical symmetry that attaches to the pilus of its host cell. Can be released without lysing the host cell via budding; acquires coat proteins as it moves across the cytoplasmic membrane. Has a very small ssDNA genome with a dsDNA replicative form. Part of the genome does not encode protein so can be replaced with foreign DNA, making it an effective cloning vector.

Biofuels

Biologically produced fuels that are in demand with growing concerns about fossil fuel supplies and climate change. Current biofuels include ethanol, biodiesel, hydrogen, and methane. Although microorganisms make many of these, their production often is associated with the accumulation of toxic by-products thus, they have been genetically modified to optimize production of biofuels. Trying to get away from the fermentation of corn sugars by yeast so have now discovered a bacterium that breaks down the cellulose in swtichgrass to sugars which are fermented to ethanol. *Have also engineered E. coli to convert glucose into propane or other hydrodarbons.

Preserve genomic integrity through

CRISPR Interference

Mining Genomes and Engineering Pathways

Complex environments such as soils contain many uncultured organisms with potentially useful genes,.

Gene Fusions

Constructs that contain segments from 2 different genes; usually involves a regulatory region (EX: promoter) and a coding sequence (often a reporter gene); used to study gene regulation.

3 mechanisms of genetic exchange within prokaryotes 3) Bacterial Conjugation

DNA transfer involves cell-to-cell contact and a conjugative plasmid in the donor cell. Considered mating and is a mechanism of genetic transfer involving cell-to-cell contact. Uses a donor (contains conjugative plasmid) and recipient cell (does not have a conjugative plasmid) and also requires a sex pilus produced by the donor cell. -Use rolling circle replication for DNA of the plasmid. -Most use an F plasmid (F = Fertility) of ~100 kbp that contains genes for DNA replication and several transposable elements to permit integration into the host chromosome. -tra genes used for transfer functions between hosts. -Very efficient and rapid process (~5 minutes for the F plasmid); under favorable conditions, nearly 100% of recipient cells gain a plasmid. -If plasmid genes are expressed in the recipient cell, it becomes a donor and can transfer the plasmid to other recipients. **Conjugation between an Hfr cell and an F- cell leads to transfer of genes from the donor cell chromosome (rolling circle replication continues into the host's chromosome), allowing mobilization of the cell's genome.

3 mechanisms of genetic exchange within prokaryotes 2) Transduction

DNA transfer mediated by a virus/Virus transfers DNA from one cell to another. There are 2 mechanisms for transduction: 1) Generalized Transduction: DNA from any portion of the host genome is packaged into the virion in place of the virus genome. Occurs when host DNA is accidentally packaged into a virion; this virion is called transducing particle. Some bacterial cells are infected with transducing particles rather than virus particles and the DNA can recombine with the new cell's chromosome; has very low efficiency. While a normal virion contains viral genes, a transducing particle contains host genes. 2) Specialized Transduction: DNA from a specific region of the host chromosome is integrated directly into the viral genome (replacing viral genes). Extremely efficient but is highly selective and transfers only a small region of the bacterial chromosome. DNA of a temperate virus excises from the chromosome incorrectly and takes adjacent host genes with it. *These processes usually render the virus non-infectious as viral genes or the entire genome have been replaced.

Biobricks

Each of the individual parts (EX: promoters, enhancers, operators, riboswitches, regulatory proteins, etc). -Are now manufacturing vallillin in E. coli rather than from the seed pods of orchids in the genua Vanilla --> called Synbio vanillin and far less expensive than the "real" vanillin -Also producing artemisinin, an antimalarial drug, S. cerevisiae as E. coli couldn't be altered to produce the product.

Plaque Forming Units

Each plaque is assumed to result from infection by a single virus particle of infectious unit called plaque forming units and based on the number of plaques, an estimation of the virus titer can be made.

Reporter Genes

Encode a protein that is easy to detect and assay; examples include lacZ, luciferase, and grren fluorescent protein (GFP); used to report the presence/absence of a particular genetic element or to show gene expression.

Heterologous Expression

Expression of a gene from one organisms in a different host organism.

Cells containing an F plasmid that is not integrated into the chromosome

F+

3 mechanisms of genetic exchange within prokaryotes 1) Transformation

Free DNA is taken up by another cell/Incorporation of free DNA into a recipient cell. *Some bacteria and archaea are naturally transformable; called competent cells. -Process was first discovered by Frederick Griffith in the late 1920s in Streptococcus pneumoniae. This was prior to understanding the structure of DNA and how it was used as genetic material. -Process of being competent is highly regulated and requires special proteins; most bacteria are not naturally transformed. -Electroporation: Technique used to get DNA into organisms that are not readily transformed; uses electrical pulses to make the cell envelope permeable. -Transforming DNA binds at the recipient cell surface by DNA-binding proteins. Either 1 strand or both strands are taken up depending up on the organism. Once inside, the DNA is bound by a competence-specific protein to protect it from degradation. Once delivered to the chromosome, RecA takes over and integrates the DNA into the host cell's chromosome via recombination.

Virus

Genetic element that cannot replicate independently of a living (host) cell but that possess an extracellular form. They are obligate intracellular parasites. **Extracellular form is called a virus particle or a virion; this form facilitates transmission from one host cell to another. -There are at least 10 times as many viruses as living cells on this planet and they infect all types of cells.

Homologous Recombination

Genetic exchange between homologous DNA sequences (not quite the same but very similar) from 2 different sources. It is started by nicking one strand of the donor DNA with an endonuclease; the strands are then separated by helicase and maintained by single-stranded binding protein. *RecA protein (discussed in the SOS repair system) is a key player in homologous recombination. -For homologous recombination to generate new genotypes, the 2 homologous sequences must be related but genetically distinct.

Size and Structure of Viral Genomes

Genome size varies dramatically, from 1.75 kb to 2.5 megabase (million pairs). *RNA genomes are typically smaller than DNA viral genomes. -David Baltimore developed a classification scheme for viruses based on the relationship of the viral genome to its mRNA; has 7 classes. *By convention, viral mRNA is considered to be in the + configuration so need to know what has to happen to get to + mRNA as all viruses need to go through transcription. -ssDNA viruses can be either positive-strand or negative-strand viruses. Transcription of a + strand would yield a - strand so must make a complementary DNA strand first --> replicative form. -Host cell RNA polymerases do not make RNA from an RNA template (they use a DNA template) so RNA viruses must have their own RNA-dependent RNA polymerase called RNA replicase. -The genome of + strand RNA viruses is also mRNA while for - strand RNA viruses, RNA replicase must synthesize a + strand from the - strand template. -Retroviruses are animal viruses with a + ssRNA genome that replicate through a dsDNA intermediate; the process of copying the information found in RNA into DNA is called reverse transcription and is catalyzed by reverse transcriptase.

Hepadnaviruses

Have tiny genomes that are partially ds and include overlapping genes. Use reverse transcriptase to form the incomplete dsDNA genome from viral mRNA. The DNA genome is replicated through an RNA intermediate. Upon infection, the partial dsDNA genome is completed to a full dsDNA genome and transcription uses the host RNA polymerase.

Cells with an F plasmid integrated into the chromosome

Hfr (high frequency recombination) cells. *Because there are several distinct insertion sequences on the chromosome, different Hfr strains are possible. Each strain always donates genes in the same order; different strains transfer genes in different orders.

Prion Proteins and the Prion Infectious Cycle

Host cells contain a gene (Prnp = prion protein) which encodes the native form of the prion protein in healthy individuals (PrP^C). Pathogenic form of the protein is PrP^Sc (Sc for scrapie, the first prion disease discovered). PrP^Sc is identical in amino acid sequence to PrP^C but has a different conformation (the native is largely alpha helices while the pathogenic forms contain more beta pleated sheets). Native prion proteins (PrP^C) are converted to the pathogenic form (PrP^Sc) by inducing misfolding when a PrP^Sc form enters a host cell. As pathogenic prion proteins accumulate, they form insoluble aggregates in nerve cells which leads to disease symptoms that are neurological and due to the destruction of brain or related nervous tissue. PrP^C functions as a cytoplasmic membrane glycoprotein but is not required for survival; membrane attachment of PrP^Sc is necessary for disease symptoms. Exposure to prions can be infectious (transmitted between animals), sporadic (random misfolding in a healthy individual), or inherited (mutation increases transformation into pathogenic form). Other vertebrates possess genes homologous to the Prnp gene but do not have pathogenic misfolded versions. Have found prion-like proteins in fungi that are not pathogenic; instead allow the fungus to adapt to altered environmental conditions. Still results in the accumulation of insoluble protein aggregates but without pathogenicity. Humans also have beneficial prions such as MAVS, a protein that is part of the innate immune response. Its aggregation triggers the production of immune modulators called interferons which increase the prevalence of phagocytic cells.

Provirus

Is made when the dsDNA genome (from retrovirus) enters the host nucleus with the integrase enzyme, which incorporates the viral dsDNA into the host genome. The provirus is transcribed by host RNA polymerase to form copies of the genome and mRNA to make proteins/enzymes. Eventually, the nucleocapsid is assembled and the genome and enzymes packaged inside and the virus buds from the host cell and is free to infect neighboring cells.

Hosts for Cloning Vectors

Most useful hosts are easy to grow and transform with engineered DNA, genetically stable in culture, and have necessary enzymes to allow replication of the vector. Include: -Escherichia coli: Gram negative is not ideal for gene expression and secretion due to the other membrane. -Bacillus subtilis: Because of E coli, this is often used. -Saccharomyces cerevisiae: used to clone DNA from eukaryotic sources as they have the necessary RNA and post-translation processing systems.

Viral Phylogeny

Most viral genes retrieved from nature are of unknown function. Insightful phylogenetic trees for viral genes have not been possible; there are only a few groups where phylogenies can be reliably traced. Using genomes has not been helpful either, as there is so much diversity that useful trees are lacking.

Virus Symmetry

Nucleocapsids are constructed in highly symmetrical ways -2 major kinds of symmetry in viruses which correspond to the 2 primary shapes. 1) Rod-shaped viruses have helical symmetry. 2.) Spherical viruses have icosahedral symmetry. -Icosahedron: Symmetric structure containing 20 triangular faces and 12 vertices that is roughly spherical; most efficient arrangement of subunits because it uses the smallest number of capsomeres (common viruses often range from 60 to 360 capsomeres). *Some viruses are complex and are composed of several parts, each with a different shape and symmetry (EX: icosahedral head and helical tail). -Enveloped viruses: Have a lipoprotein membrane surrounding the nucleocapsid with proteins (usually glycoproteins) embedded in it that can be used for attachment. Membrane is usually derived from the membranes of the animal host cell but contain viral proteins embedded in the membrane. Envelope is what makes initial contact with the host cell so has some control over attachment and penetration as well as release of the virion after replication. -Viruses are considered to be metabolically inert but some virions contain enzymes that play important roles in infection (EX: lysozymes to make a hole in the host bacterial cell wall for infection and later to lyse the host cell). -RNA viruses bring their own RNA-dependent RNA polymerase (RNA Helicase) that replicates the RNA genome and produces viral-specific mRNA. Without this enzyme, the host cell has no mechanism to make RNA from an RNA template. -RNA viruses that replicate via a DNA intermediate also need their own enzyme (reverse transcriptase) to copy their RNA genome into DNA.

Temperate viruses

Other dsDNA viruses that can infect and establish a long-term stable relationship

Recombination

Physical exchange of DNA between genetic elements.

What is a virus?

Possess a nucleic acid genome encoding the information necessary for replication but rely on their host cell for energy and materials for this replication. -Capsid: Protein shell surrounding the nucleic acid; usually composed of protein molecules arranged in a precise and repetitive pattern around the nucleic acid. -Nucleocapsid: Complex of nucleic acid and protein packaged in the virion. *Naked viruses have no envelope around the nucleocapsid while enveloped viruses have a phospholipid bilayer around the nucleocapsid. -Two possible outcomes from infection: 1) Virulent or lytic infection that lyses the host cell. 2) Lysogenic infection where the viral genome inserts into the host cell's genome. -Unlike cells, viral genomes can be either DNA or RNA and either single-stranded or double-stranded and either circular or linear. -ssRNA genomes can be either plus sense (exact base sequence as mRNA so will be immediately translated) or minus sense (complementary to mRNA so will need to be copied before translation). *RNA viruses do not follow the central dogma of molecular biology (DNA --> RNA -->Protein) but genetic information still flows from nucleic acid to protein. -Viral genomes are usually very small (2 up to 350 genes) and encode primarily proteins whose functions the virus cannot get from its host cell. -Viruses can also be classified based on what hosts it infects: Bacteria, Archaea, animal, plant, and other viruses. -Bacteriophage (phage): Virus that infects bacteria. -Viruses classified into families that share similar morphology, genome structure, and/or replication strategy.

Restriction and Modification

Prokaryotes possess several weapons against viruses. 1) Toxin-antitoxin modules 2) CRISPR 3) Restriction endonucleases: cleave foreign DNA at specific sites. Only effective against double-stranded DNA viruses and some of these viruses have found ways to protect themselves from digestion by substituting modified bases that are resistant to cleavage at the recognition sites. -Host DNA protection is conferred by modification of host DNA typically by methylation at the sites recognized by the restriction enzymes.

Attachment and Entry of Bacteriophage T4

Proteins on the external surface of the virion interact with specific host cell receptors (usually proteins, carbohydrates, glycoproteins, lipids, or lipoproteins). If there is no receptor, there is no attachment so the host range of a virus is determined by the presence of suitable receptors. Receptors have normal functions for the cell. *Flagella and pili can also serve as receptors.

Horizontal gene transfer

Results of closely related organisms often exhibiting different phenotypes that are based on differences in their genomes.

Retroviruses

Retroviruses are animal viruses that contain an RNA genome. This genome is replicated inside the host cell using the DNA intermediate reverse transcriptase. They are enveloped viruses that carry enzymes within the virion including reverse transcriptase, integrase, and a protease. --Retroviruses are animal viruses with a + ssRNA genome that replicate through a dsDNA intermediate; the process of copying the information found in RNA into DNA is called reverse transcription and is catalyzed by reverse transcriptase. -Reverse transcriptase: Enzyme that allows RNA to be copied to DNA. -The genome consists of 2 identical ssRNAs in the + sense and reverse transcriptase converts this to a ssDNA which then becomes dsDNA.

Activity of Reverse Transcriptase

Reverse transcriptase is a type of DNA polymerase and thus must have a primer. This primer is a specific tRNA encoded by the host cell (the exact tRNA varies by virus) and packaged into the virion from the previous host cell. Process of reverse transcription results in long terminal repeats (LTRs) that participate in the integration process into the host genome. The integrated genome is called a provirus and cannot be removed from the host genome. ~8% of the human genome is sequence from retroviruses. Translation of retroviral mRNA and protein processing, the gag/pol region is removed to get translation of the env polyprotein (2 proteins).

Lysogeny

State in which most viral genes are not transcribed and the viral genome is replicated in synchrony with the host chromosome and passed to daughter cells.

Transposable Elements

Stretches of DNA that can move from one site to another; always inserted into another DNA molecules and do not possess their own origin of replication, thus can only replicate when the host DNA molecule is replicated. Moved by process called transposition and its frequency varies by transposable element. *Transposition is site-specific recombination as transposase recognizes specific DNA sequences (inverted repeats); different from homologous recombination where homologous regions of DNA are recognized via base pairing.

Replication of Bacteriophage T4

T4 encodes its own DNA polymerase rather than using the host cell's polymerase. Each copy of the viral genome contains the same genes but they are arranged in a different order, a result of circular permutation. Circularly permutated genome have some DNA sequences duplicated on both ends of the DNA molecule. T4 genome is replicated as a unit and then several units are combined to form a longer DNA molecule called a concatemer. The concatemer is cut into lengths sufficient to fill the head of the phage. Shortly after infection, T4 DNA is transcribed and translated to initiate new virion synthesis. This process is complete in less than 30 minutes. Within a minute following the introduction of T4 DNA, host cell synthesis of DNA and RNA stops and synthesis of viral genes begins. Within 4 minutes of infection, phage DNA replication has started. The genome has 3 sets of proteins: early proteins, middle proteins, and late proteins. Timing refers to their appearance in the host cell and use in the viral replication process. T4 does not encode its own RNA polymerase; instead T4 proteins modify the host RNA polymerase to only recognize viral promoters. Modification proteins are part of the early genes and are transcribed by the host RNA polymerase. Phage has an anti-sigma factor that binds to host RNA polymerase sigma factor and prevents it from recognizing promoters on host genes so only transcribes phage genes. When the genome is complete, it is forcibly pumped into a preassembled capsid using a packaging motor. Once completed and the motor has been discarded and the capsid sealed, the tail, tail fibers, and other components are added by self assembly. Two very late enzymes are used to allow virus escape from the cytoplasmic membrane and peptidoglycan layer. Osmotic pressure causes cell lysis, and the newly formed virions are released and can infect neighboring host cells.

Horizontal Gene Transfer in Archaea

The development of gene transfer systems in Archaea lags behind Bacteria. There is no model species for Archaea (often extremophiles and do not respond to common antibiotics) but examples of transformation, transduction, and conjugation have been found. Although transformation works reasonably well in members of the Archaea, transduction is extremely rare and 2 types of conjugation have been detected.

Integrated F plasmids

The orientation of integration of the F plasmid also determines which genes are transferred. Sometimes when integrated F plasmids are excised from the host's chromosomal genes with them; these plasmid are called F' plasmids and they transfer the chromosomal genes at high frequency to recipient cells.

High frequency of recombination strains

The recipient cell does not become Hfr or F+ as only part of the plasmid chromosome is transferred.

Genetic Engineering

The use of in vitro techniques to alter genetic material in the laboratory; can be inserted into the original source organism or another organism. *Requires that specific DNA be isolated, purified, and further manipulated.

Double-Stranded DNA Bacteriophages

Two examples: 1) T7: A relatively small DNA virus that infects E. coli; has an icosahedral head and short tail. Early genes are transcribed by host RNA polymerase. One gene transcribed makes its own RNA polymerase that only recognizes T7 promoters and enzymes that inhibit additional host RNA polymerase activity. Genome replication begins at an origin of replication and proceeds directionally with its own DNA polymerase (consisting of 1 phage protein and 1 host protein). Has terminal repeats at both ends of the molecule that are used to form concatemers that are cut by a phage specific endonuclease (genomes are identical). 2) Mu: Temperate phage with the ability to replicate via transposition facilitated by transposase. Mu named because it generates mutations when it integrates into the host cell chromosome. Has an icosahedral head, helical tail, and several tail fibers; host range is determined by the kind of tail fibers made. Replicates in a completely different manner than all other bacteriophage as its genome is replicated as part of a larger DNA molecule; integration into the host chromosome is necessary for both lytic and lysogenic development and requires transposase.

Synthetic Biology

Use of genetic engineering, to create novel biological systems from available parts, often from several different organisms.

Culturing, Detecting, and Counting Viruses

Usually easier to quantify viral effects on their host cells than counting them with an electron microscope thus, a virus infectious unit is the smallest unit that causes a detectable effect when added to a susceptible host cells. -Titer: The number of infectious units per volume of fluid.

Prophage

Viral DNA that replicates along with the host cell while viral genes for virulence are repressed. *A repressor protein is used to maintain the lysogenic state. If this repressor is inactivated or its synthesis is stopped, the prophage can be induced to revert to the lytic stage (where is lyses its host after viral replication and assembly). If incorporated into the host genome, it is excised prior to transcription and translation of viral genes. *Various stress conditions (EX: damage to host DNA) can induce a prophage to enter the lytic pathway.

Structure of the Virion

Virions are virus particles; Extracellular form that facilitates transmission from one host cell to another. There structure... -Virions come in many shapes and sizes, most are smaller than prokaryotic cells (range from 0.02 to 0.3 micrometers or 20-300 nm). Commonly measured in nanometers and only properly characterized with electron microscopy. Viral genomes are usually smaller than those of cells; some only contain <5 genes and some are segmented into multiple nucleic acid molecules. -Capsid is composed of protein molecules called capsomeres. *Structure is quite diverse, varying in size, shape, and chemical composition.

Utility of Transposon Mutagenesis

When a transposon inserts itself within a gene, the DNA is altered and a mutation occurs in that gene. This can occur naturally but is a convenient way to create a mutation. Typically done with transposon carrying an antibiotic resistance gene that inserts into the bacterial genome.

Bacteriophage Lambda

dsDNA virus with a head and a tail that infects E. coli. The genome has 12bp single-stranded regions at the 5' end of each strand that are complementary so when the virus enters a host cell, they base-pair to form the cos site and a circular genome. -If the virus enters the lytic pathway, the genome is copied using rolling cycle replication where 1 strand is nicked and rolled out to use as a template for synthesis of the complementary strand. The resulting concatemer is cut into genome-sized lengths at the cos site and packaged into phage heads. -Sometimes lambda also packages a few chromosomal genes from its host and can transfer these genes to a second host; this process of genetic exchange is transduction. -Instead of being lytic, lambda can also integrate its genome into that of it's host's chromosome. Requires integrase, a phage-encoded enzyme that recognizes genome attachment sites and facilitates integration of the lambda genome. -Environmental triggers can initiate the lytic cycle again; requires excision protein to excise the lambda genome from the host chromosome.

Bacteriophage T4

is a virulent virus that always kills its host.

Both the F plasmid and the chromosome have insertion sequences which provide

the needed homology for integration.

DNA transfer

typically occurs in only one direction - from donor to recipient.