MicroBio Exam 2
Cas
(CRISPR-associated) proteins, are endonucleases as defense, and incorporate new spacer regions
(-)ssRNA animal virus influenza virus
-(-) strand, RNA synthetase make complementary RNA strand, which then is the mRNA -RNA synthase also makes (-) strand again from (+) strand as part of replication -Enveloped, pleomorphic (non-uniform), budding virus with segmented genome (separate pieces)
Genomes
-A cell's entire complement of genetic information -Bacterial genomes ca. 1-5 Mbp -Includes genes, regulatory sequences, and no-coding DNA
(+)ssRNA animal virus poliovirus
-Belongs to rhinoviruses, also including coronavirus (+) strand is mRNA -Gets immediately translated when in cytoplasm -A 5'-end guide protein, and 3'-end is poly(A)-tail like on cellular mRNAs -Production of polyproteins that are self-cleaving into individual proteins (like genes of a bacterial operon)
anoxygenic phototrophic
-Carotenoids, e.g., B-carotene -Typically absorb blue light and reflect yellow, red, brown or green
Genome comparison can reveal which of the following?
-Chromosomal islands. -Genes of the core and pan genome. -Regions of putative horizontal gene transfer.
ssDNA bacteriophage ⏀X174
-Contains a circular single-stranded DNA genome inside an icosahedral virion -Contains the (+) strand, so has to make (-) strand/replicative form -From (-) strand mRNA is generated, and more (+) strand(chromosome) to pack into new virions
Basic genetic engineering techniques
-DNA amplification (PCR) -Gel Electrophoresis -Nucleic acid hybridization (probes) -Molecular cloning -Targeted mutagenesis
external influence(mutagenic agents)
-Exposure to radiation -Exposure to chemicals that chemically modify DNA -Infection with an integrating virus
dsDNA animal herpesvirus
-Large group causing different disease in humans, including cold sores, chicken pox, shingles, and infectious mononucleosis -Able to remain latent for extended periods of time (years; reside in nerve cells) -Get re-activated under stress or weakened immune system, can cause nerve damage
dsDNA bacteriophage Mu
-Large virus with an icosahedral head, helical tail, and six tail fibers, genome is linear -Host range is controlled by tail fibers: one type allows infection only of E. coli; other allows infection of other enteric bacteria -Performs lytic and lysogenic pathway -Always integrates: replicated by transposition, at many possible sites of the boat chromosome, via enzyme transposase (brings it to the host)
light reaction needs
-Light-harvesting/antenna pigments in the membrane --Chlorophyll (cyanos) --Bacteriochlorophyll (others) -Light-active molecules have a conjugated electron system -Production of different pigments allows different the kinds phototrophs to absorb different wavelengths of light, while coexisting in the same habitat
Locations for Ammonium- electron donor
-Manure (animal urine) -Produced by bacteria in water and soil as end product of plant and animal waste decomposition -Usually low nontoxic concentrations in soil, air and water and serves an indirect N source for plants -Leaves the system by drainage via groundwater, or, microbial oxidation (nitrification)
What can a genome tell us?
-Metabolic capabilities of an organism -Molecular adaptations to specific environments, e.g., encoding enzymes with features stable at high temperatures -Identify virulence factors -Detecting horizontal gene transfer -Monitoring and diagnosing disease outbreaks
Information on mutations
-Mutations are random changes in ---DNA sequence occurring over time -Most mutations are neutral or deleterious, some are beneficial
Microbial functions in the nitrogen cycle
-Nitrogen has a total of nine oxidation states -Bacteria can perform oxidation or reduction reactions starting at several levels -The ecosystem conditions govern what is possible
Fertilization
-No external electron acceptor -No respiration/membranes involved -No energy conservation with an ion gradient (H+, no proton motive force) -Internal electron cycling, regeneration of NAD+ to achieve redox balance -Substrate-level phosphorylation -Partially oxidized end produced
oxygenic phototrophs
-Phycocyanin absorbs around 550 nm, looks red -Phycocyanin absorbs around 620 nm, looks blue
Why RNA to DNA transition?
-Protect against host ribonucleases (RNAses) -Develop own DNA replication machinery that is foregin to host cell -DNA is more stable than RNA (spontaneous hydrolysis of RNA) -Mutation rate is higher in RNA, because RNA sequence repair mechanisms are less prevalent
what do dark reactions require?
-Requires reducing power, so the cell also needs to oxidize an external substrate (electron donor) to create NADH -Donor is not successively oxidized like in chemotrophy, but right at the membrane -Oxygenic photosynthesis: oxidation of H2O produces O2(cyanobacteria) -Anoxygenic photosynthesis: all other phototrophic bacteria (use H2S, H2,...)
Ethanolic Femrenetaiton
-Saccharomyces cerevisiae ("common baker's yeast) --Microbial eukaryotes --Single celled fungi; also called yeasts --Used in bread baking (sourdough) and alcoholic beverage industry --End products: CO2 and ethanol
Locations for nitrate (NO3-) = e- acceptor
-Terrestrial -Aquatic -Oxic and anoxic layers -Used up in upper sediment and soil layers just below the oxic zone
Calculating distance matrix form alignment
-The first step in making a tree is to align sequences -Second step is a distance matrix is calculated from the number of sequence differences -Third step is the tree is constructed by adding nodes to join lineages that have the fewest differences
human virome
-Their function as transduction agents may have major impact on bacterial evolution -May confer new metabolic or other beneficial properties (retained when beneficial in habitat)
Goals of systems biology
-Understand the functions of a specific organism on all possible molecular levels, and, ideally at all levels in its natural environment -Understand the structure and function of microbial communities on all possible molecular levels and, ideally, at all levels in their natural environments
Why did the nucleus develop?
-formation may be associated with evolution of RNA processing -Spliceosomes remove introns from eukaryotic mRNAs
Genotypic Analysis
-genome -Challenge: getting clean and complete genome information
phenotypic analysis
-morphological, metabolic, physiological, chemical characteristics -Challenge: requires the ability to culture, AND getting a pure culture
Genetic engineering include laboratory techniques such as
-nucleic acid hybridization. -molecular cloning. -restriction enzyme digestion.
Microarrays allow for
-the analysis of global gene expression. -the hybridization of cDNA (converted from mRNA) with DNA affixed to a solid-state support. -a comparison of expression of different genes under different growth conditions.
phylogenetic analysis
-using a gene marker showing evolutionary distance -challenge for unknown organisms PCR primers/probes may not bind
Heterofermentative
-yields lactate, ethanol, CO2 and 1 ATP/glucose --Lactobacillus brevis --Sauerkraut and pickles
Homofermentative
-yields lactic acid only and 2 --ATP/glucose --Lactobacillus delbrueckii --Milk and dairy products
What do light reactions produce?
ATP
Photoautotrophy requires
ATP production and CO2 reduction
What the organisms oxidize...
Chemoorganotrophs: are at top of pH scale Chemolithotrophs: are in middle of pH scale Phototrophs: bottom of the pH scale
Bacteria are mainly infected by
Class I viruses
Eukarya are mainly infected by
Class I, II, and IV
CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats that protect bacteria from bacteriophage infection
__________ is a process that involves cell-to-cell contact to move the plasmid from a donor cell to a recipient cell.
Conjugation
Photosynthesis
Conversion of light energy from the sun into chemical energy.
Bacteria are mainly infected by _____ viruses, while animal cells are infected by _________ viruses.
DNA / RNA
Restriction enzymes recognize specific nucleotide sequences and cut them as part of a defense mechanism in bacteria. They are cutting
DNA double-strands
specialized transduction
DNA form a specific region of the host genome(genes), integrated among the virus genome
generalized transduction
DNA from any region of the host genome is packaged typically filling entire virion, random, often not whole genes
In order to apply PCR for DNA-based diagnostics, we make use of the principle that (a) _________ operates only when the last nucleotide of the __________ on the 3'-end can bind, so polymorphisms in a DNA sequence can be detected via this mechanism.
DNA polymerase / primer
Pandoravirus is the largest known virus, and has a _______ genome that is ________ than the smallest bacterial genome.
DNA/larger
Place organism in hierarchical system:
Domain Phylum Class Order Family Genus Species
Oxygenic photosynthesis
Employs 2 reaction centers, quinone-pool and Fe-S complex
Natural sources for mutations can be a result of
Error during DNA replication -most often, a single or few nucleotide is/are different
Which of the following statements is true?
Eukaryotic genomes are composed mostly of noncoding DNA, whereas bacterial genomes have mostly coding DNA.
A silent mutation is the most common single nucleotide change.
False
If a change in nucleotide sequence information does not change the cell's phenotype compared to wild type it is not considered a mutation.
False
Mutation rates of DNA-containing cells are higher than DNA- or RNA-containing viruses.
False
The "tree of life" that shows the three domains Bacteria, Archaea, and Eukarya is the phylogenetic reconstruction of the evolutionary relationship between all organisms, and thanks to modern genetic technology it is now considered 100% correct.
False
The reason for the variability of the influenza virus - resulting in the necessity of developing a new vaccine basically every replicative season (~12 months) - lies in the virus alternating between an enveloped and unenveloped virion.
False
With the onset of the human microbiome project we learned that our body does not contain many microorganisms, and that especially our intestinal tract has a very low density of bacteria.
False
What is a metagenome?
Genome sequences from all the organisms in a particular environment.
Characterizing large pools of molecules
Genomics Transcriptomics Proteomics Metabolomics Secretomics Interactomics
Goal of genetic engineering
Heterologous expression- expression of a gene in a different host
naturally competent bacteria
Streptococcus Bacillus Vibrio
Which of the following is NOT a palindrome (recognition site for a restriction enzyme)?
TAGGAT
Transduction
Transfer of DNA from one cell to another by a bacteriophage
A suicide vector encodes a gene that kills the host when expressed or selected for its function. Only vectors that have been successfully inserted with the gene of interest (suicide gene is disrupted) can grow under selective conditions.
True
Antigenic shifts in the influenza genome generates hybrid viruses with strongly modified surface proteins, triggering new outbreaks of influenza after long periods of apparent immunity.
True
Bacteria of distantly related lineages can have some highly identical genes because they have means to share part of their genetic information via horizontal gene transfer.
True
In transduction, DNA is accidentally moved from one bacterium to another by a bacteriophage.
True
One important advantage of eukaryotic cells as hosts for cloning vectors is that they already possess the complex RNA and posttranslational processing systems required for the production of eukaryotic proteins.
True
Restriction endonucleases are naturally occurring enzymes in bacteria to cleave foreign/viral DNA.
True
The core genome is the collection of genes that is shared by all sub-lineages of a group of interest, while the pan genome includes all variants in that sub-lineage, and also includes the genetic information of plasmids.
True
The lactate end product from lactic acid fermentation is only partially oxidized and can be used by other bacteria in syntrophy, e.g., Propionibacterium, which further oxidizes lactate into acetate, propionate and CO2.
True
The multiple cloning site of a vector is within the lacZ gene. During the ligation of the gene of interest into this site the lacZ gene gets disrupted. Addition of X-Gal allows for screening. What is a correct description of a color result?
White colonies means that the lacZ gene has been disrupted.
Phages are viruses that infect
bacteria
Assembly
bringing the fragments together like a puzzle (overlapping regions, coverage), forming scaffolds, and ultimately the entire genetic molecule
Which molecule is used for the hybridization to DNA that is bound on a microchip?
cDNA
CO2 is a potent greenhouse gas, and it
can be accessed as carbon source by many bacteria, including phototrophic bacteria, as well as phototrophic eukaryotes (algae and plants).
Which of the following is NOT a feature of nitrogen fixation?
can be performed by phototrophic bacteria only
Facultative aerobes
can use with oxygen or nitrate
Bacteria usually do not...
carry much "unnecessary" DNA ("genetic streamlining")
Taxonomy
characterization, classifying.ranking, naming organisms
pan genome
core + individual variants in a given sub-lineage, can also include (multiple) plasmids
Consider a mutation in which the change is from UAC to UAU. Both codons specify the amino acid tyrosine. Which type of mutation is this?
silent mutation
ssRNA
single stranded RNA complementary to mRNA
Some RNA viruses carry a ___________ RNA molecule that serves directly as mRNA - one reason why the infectious cycle of these viruses is initiated very __________.
single-stranded / rapidly
Agarose gel electrophoresis separates DNA fragments from each other based on
size
EcoRI is a restriction enzyme that recognizes a six base-pair sequence and creates ends.
sticky
Transcriptomics
study of RNA patterns -Total RNA extracted during a certain growth condition -Reverse transcription mRNA→cDNA (complementary strand) --DNA is more stable than RNA, easier to work with -Sequencing of all transcribed mRNA sequences -Map to genome to see which genes they are
Mitochondria are
the cell's respiration machinery (with O2)
mitochondria are:
the cell's respiration machinery (with O2)
lactic acid fermentation
the chemical breakdown of carbohydrates that produces lactic acid as the main end product
Which gene is used for the reconstruction of the phylogenetic relatedness of all living cells?
the gene for the RNA in the small ribosomal subunit, 16S/18S rRNA
Denaturation of DNA refers to the process when
the hydrogen bonds between double-stranded DNA weaken, so the DNA separates into the single strands.
Via genetic engineering, the vaccinia virus was altered to produce a foreign protein to be used as a vaccine against
the smallpox virus
Metagenomics
the study of genetic material recovered directly from environmental samples -looking at all DNA from an environment --Resolve metabolic capabilities --unraveling different identities
Restriction endonucleases cut specific sequence regions in DNA molecules. Which molecule(s) do you have to cut with the same restriction enzyme for the purpose of molecular cloning?
the vector and the insert
Endosymbiotic or parasitic microorganisms usually have very small genomes because
they streamline their genetic content, keeping only genes necessary to produce what is not provided by the host.
The process of importing free DNA into bacterial cells is known as __________
transformation
Which of the following methods may introduce foreign DNA into a bacterial recipient?
transformation, transduction, and conjugation
The bacteriophage Mu always integrates its entire DNA chromosome into a specific site (5-nucleotide sequence) of the bacterial chromosome, thereby creating direct repeats on either side of the inserted viral DNA. This process is called
transposition
Bioinformatics
use of computer and specially designed programs to store and analyze sequences and structures of nucleic acids and proteins
genetic engineering
using in vitro techniques to alter genes in the lab
Viral Evolution
viruses contain genetic material but are not cellular in nature and cannot reproduce. It is theorized that viruses are a precursor to life and "bridge the gap" between non life and the UCA
Two step oxidation across two types of bacteria, occurring in aggregates:
1. Ammonium-oxidation to nitrate Nitrosomonas 2. Nitrite-oxidation to nitrate Nitrospira
Identifying an ORF
1. Computer finds possible start codons 2. Computer finds possible stop codons 3. Computer counts codons between start and stop 4. Computer finds possible RBS 5. Computer calculates codon bias in ORF 6. Computer decides if ORF is likely to be genuine 7. List of probable ORFs
Steps towards a whole genome
1. Genome extraction, fragmentation and sequencing 2.assembly 3.annotation
combining the gene of interest and vector
1.Cut DNA with restriction enzyme 2.Add vector cut with same restriction enzyme 3.Add DNA ligase to form recombinant molecules 4.Introduce recombinant vector into a host
Anoxygenic Photosynthesis
1.Light generates high potential electrons from bacteria/chlorophyll in reaction center 2. Elections flow through the membrane, analog to respiratory membrane, and generate proton motive force
Genome extraction, fragmentation and sequencing
several methods, differ in price, fragment length, and accuracy (error rate)
Mycobiome:
60+ fungi present on skin, in oral cavity, and other moist surfaces
The Baltimore Scheme
7 classes taxonomy based on nucleic acids
missense mutation
A base-pair substitution that results in a codon that codes for a different amino acid. -leads tp replacement of amino acids in a polypeptide (eg. UAC to AAC).
Mutation
A change in a gene or chromosome.
silent mutation
A mutation that changes a single nucleotide, but does not change the amino acid created. -do not affect sequence of polypeptide or phenotype, are typically at third base of codon
nonsense mutation
A mutation that changes an amino acid codon to one of the three stop codons, resulting in a shorter and usually nonfunctional protein.
polymerase chain reaction (PCR)
A technique for amplifying DNA in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides.
Conjugation
A temporary union of two organisms for the purpose of DNA transfer.
What is a suicide vector in molecular cloning?
A vector containing a gene that, when being expressed or selected for, kills the host. Only vectors that have been successfully inserted with the gene of interest can grow under selective conditions.
Which statement is true?
All organisms differ in their genome size, and, their amount of genes.
Human Genome Project
An international collaborative effort to map and sequence the DNA of the entire human genome.
What the organisms reduce... what they respire!
Anaerobic respiration Aerobic respiration
Host-virus defense mechanisms
Bacteria produce restriction endonucleases that cleave foreign DNA signature sequences
where are pigments located?
Bacteria/Chlorophyll pigments and accessory pigments are located within special membranes
mixed solvent fermentation
From cheap sources/waster Acetone, butanol, ethanol (solvent) production Microbial production of biofuels
chromosomal islands
Region of bacterial chromosome of foreign origin that contains clustered genes for some extra property such as virulence or symbiosis -Can be identified by genome comparison of close relatives --Clusters of genes for specialized functions not essential for survival --But can give selective advantage e.g., for biodegradation, nitrogen fixation, magnetosomes
gene transfer
Movement of genetic information between organisms
gel electrophoresis
Procedure used to separate and analyze DNA fragments by placing a mixture of DNA fragments at one end of a porous gel and applying an electrical voltage to the gel
nitrogen fixation
Process of converting nitrogen gas into ammonia
propionic acid fermentation
Propionibacterium and related prokaryotes produce propionic acid as a major fermentation product
There is scientific evidence that the first cell-like "virocells" and the first viruses on Earth contained _____ genomes.
RNA
What is NOT an ingredient of a PCR reaction?
RNA polymerase
Mutation rates
RNA viruses: 10^-3 DNA viruses: 10^-7 Bacteria: 10^-8 Eukarya: 10^-9
light reactions
The first of two major stages in photosynthesis (preceding the Calvin cycle). These reactions, which occur on the thylakoid membranes of the chloroplast or on membranes of certain prokaryotes, convert solar energy to the chemical energy of ATP and NADPH, releasing oxygen in the process.
What assumption is made for using a specific gene for the reconstruction of an evolutionary model of species divergence?
The gene has to be present in all species, and the mutation rate of the gene is constant.
Which of the following is NOT true of plasmids?
They are found in many eukaryotes.
The light reaction involves photosystems with light-active molecules, such as chlorophyll, that are localized ___________ of the photorophic cell.
in specially folded membranes
Point mutations
a single nucleotide is exchanged for another
Polyvalent vaccine
a single vaccine that immunizes against two different infections, e.g., an attenuated virus is also carrying an additional gene from another pathogen (attenuated + vector)
Phototrophic bacteria have a large variety of light-harvesting and accessory pigments, so they can _______ certain wavelengths of the visible light spectrum for energy generation.
absorb
nomenclature
agreed words and word stems to be used for the ranks
During genome assembly, you first have to __________, and then ________ .
align overlapping DNA fragments / ideally reconstruct the entire chromosome
Part of the development of modern-day Eukarya was that a cell of the archaeal lineage underwent endosymbiosis with a(n) _______, and turned it into a ________.
alphaproteobacterium / mitochondrium
In anaerobic soils
ammonium is respired (reduced) back to NH3 or to N2 gas -Ammonium is preferred nitrogen source for bacteria -Potential loss of N
In well-aerated soils
ammonium is turned over into nitrate by microorganisms -Nitrate is the preferred nitrogen source for plants -biofertilizer
Carboxysomes
inclusions with protein shell containing and improving efficiency of RubisCO in many autotrophs
Mutations happen randomly in the chromosomes of all organisms, and those that ________ the fitness of the organism in a given environment will be retained in the lineage.
increase
Genomics
discipline of sequencing,analyzing and comparing genomes
dsRNA
double stranded RNA
Most plasmids are
double-stranded DNA, though a few are not.
Cyclic photophosphorylation:
electrons move within closed loop; no net input or consumption
Larger genomes...
encode more transcriptional regulation and signal transduction
Smaller genomes...
encode more translational processes
RNA synthase
enzyme that catalyzes transcription process.
mutation rate
establishment of differences in nucleotide sequence in a population(diversification)
Annotation
find open reading frames (OFs)= "genes"; DNA sequence that codes for a polypeptide). Check nucleotide and amino acid sequence against databases, similarities suggest function of the ORF
how did chloroplasts arise?
from stable incorporation (endosymbiosis) of a cyanobacterium-like cell into the cytoplasm of a eukaryotic cell, leading to eukaryotic photosynthesis (algar, plants)
how did mitochondria arise?
from stable incorporation (endosymbiosis) of an aerobic respiring Alpha Proteobacterium-like cell into the cytoplasm of early eukaryotic cells
Vector vaccine
genes from a pathogenic virus is engineered to the genome of a harmless carrier virus, induces immunity to pathogen
core genome
genes shared by all sub-lineages
Lactate, ethanol and CO2 are end products:
heterofermentative/heterolactic fermentation
The study of gene origin can be extremely difficult in Bacteria, because they do not only pass genes to their progeny cells. Bacteria also perform a extensive amount of active
horizontal gene transfer.
Only lactate is end product:
hormofemrnetation/homolatic fermentation
Photosynthesis originated:
in bacteria -First: anoxygenic photosynthesis (not producing O2) -Later: oxygenic photosynthesis (O2 is a waste product)
molecular cloning
isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated
DNA instead of thymine:
letal!
Systematics
links phylogeny (relationship of genes) with taxonomy (ranking this diversity of organisms with name categories)
attenuated vaccine
modified pathogen via genetic engineering, virulence factors have been deleted, but retained features to elicit immune responses (recombinant, infective)
Bottleneck
molecular biologists today need to speak computer language(s) and/or be able to work on the computer console (python, R, mothur,...)
Estimations state that the Earth's virome counts ________ virus particles than bacterial cells, and most of those viruses are _______.
more / bacteriophages
where does photosynthesis occur in bacteria?
pigments are integrated into complex invaginations of cytoplasmic membrane: --Vesicular chromatophores or lamellae in purple bacteria --Chlorosomes in green sulfur bacteria --Thylakoids in cyanobacteria
Proofreading activity is intrinsic to the __________ enzyme to _________ the accumulation of mutations during a replicative cycle.
polymerase / minimize
Herpesviruses are a large collection of different viruses that infect humans. Once infected, they typically remain latent in the body for an entire lifetime, and may break out occasionally (enter lytic stage). Where do these viruses reside?
nerve cells
New traits can evolve quickly in bacteria due to their high mutation rate and short generation time. But only mutations that are _______ or _______ for the fitness of the organism at a given selective pressure result in a retaining of the mutation(s) in the lineage.
neutral / favorable
Mutations cause
new genes and alleles to arise -heritable changes
Phototrophs
obtain energy from light -use energy from light to reduce CO2 to organic compounds
Deletions
one or more nucleotides are deleted
insertions
one or more nucleotides are inserted
During genome annotation, you first have to identify __________, and then ________ by comparison to databases.
open reading frames / assign functions to them
The genes individual to each strain within a bacterial species is called the
pan genome
The polyphasic approach to ultimately describe a true new species of Bacteria comprises a _________, _________, and _________ characterization.
phenotypic / genotypic / phylogenetic
where does photosynthesis occur in Eukarya
photosynthesis occurs in chloroplasts (intracellular organelles containing thylakoids: stacked, sheet like membrane systems)
A polymerase chain reaction (PCR) amplifies a certain genetic segment bordered on each end with a(n) ________
primer
autotrophy
process by which CO2 is reduced and assimilated into cells
Transformation
process in which one strain of bacteria is changed by a gene or genes from another strain of bacteria -Genetic transfer process by which free DNA is incorporated into a recipient cell and brings about genetic change
Sequencing of the mitochondrial genome indicates that mitochondria are most closely related to
proteobacteria
Purple sulfur bacteria
quinone pool as intermediate e-acceptor and H+-translocation
A molecule of the group of __________ is used as a "molecular clock" to determine the approximate time since two lineages of organisms diverged.
rRNAs
Carl Woese chose to examine phylogeny using rRNA sequences because
rRNAs are part of ribosomes - molecules present in all and function similar in all living organisms.
Calvin Cycle
reactions of photosynthesis in which energy from ATP and NADPH is used to build high-energy compounds such as sugars
The CRISPR system
recognizes foreign DNA sequences that have previously entered the cell and directs the Cas proteins to destroy them.
In the experiment of bacterial evolution we selected for the gain of function (ability to produce tryptophan) in those bacteria that have
recombined externally provided DNA into their chromosome.
dark reactions
reduces atmospheric CO2 to cell material for growth
The DNA of herpesviruses replicates by the __________ method.
rolling-circle
