Gordon College - Microbiology Exam 2

Genetics, genomics, antibiotic resistance, viruses, and Student presentations

Genome

Entire complement of genetic information (genes that encode proteins, RNAs, regulatory sequences + non-coding DNA)

Genomics

•Mapping, sequencing, analyzing, and comparing genomes

•Requires molecular biology + computing power

•Analysis is difficult and time consuming, but incredibly informative

mutations

change in nucleotide sequence (genotype), may alter phenotype

ways other than a mutation by which genomes can change

horizontal gene transfer, gene duplicaitons, transposable elements, population changes

silent mutation

doesn’t affect phenotype of cell

always in 3rd base codon bc of genetic code degeneracy

missense mutation

informational “sense” in polypeptide has changed

if at critical location in polypeptide chain, protein could be inactive

nonsense mutation

premature termination of translation leading to incomplete polypeptide

change is from sense (coding) codon to a nonsense (stop) codon

frameshift

insertion/deletion of nucleotide base in numbers that aren’t multiples of 3

transposable elements

Genes that can move throughout genome. shape genomes through their mobilization

gene duplication

Become functionally diploid.

One remains functional and the other can accumulate mutations.

horizontal gene transfer

plasmids between bacteria.

movement of genetic information between organisms

insertion sequence (IS)

a portion of DNA that codes for transposes and has terminal repeats

transposon

like an insertion sequence, but has addition genes between terminal repeats

IS and transposons are transposable elements T/F

T

inverted terminal repeats

parts recognized by enzymes when cutting, also sites of recombination, 3’ and 5’ ends of same strand can also connect with each other

How transposable elements can change genomes

replicative transposition and non-replicative transposition

replicative transposition

transposes cuts at terminal repeats

Cut ends of transposable element attacks target sequence at 5’ ends

DNA polymerase fills in the gaps

DNA ligase seals the cuts

Recombination results in original TE staying the same but copy is inserted in between two copies of the target sequence

non-replicative transposition

Transposase brings inverted repeats together making hairpin structure

Carrier DNA is ejected and the elements attack target DNA at 5’ ends

DNA polymerase fills in gaps at 3’ ends (3→5)

DNA ligase seals cuts

Result is transposable element removed from original seuqnece and inserted between two copies of target sequence

types of base-change mutations

insertion, deletion, substitution

insertion

nucleotides added causing frameshift

deletion

nucleotides removed causing frameshift

Type of base-change mutation: substitution

nucleotides switched, can be silent, cause missense or nonsense

Base pair substitutions: effects of silent

mutated codon codes for same amino acid, no change in protein

Base pair substitutions: effects of missense

mutated codon codes for different amino acids, changes protein structure

effects of nonsense

mutated codon codes for stop codon, curing polypeptide short (stop codons: UAA, UGA, UAG)

effects of transposable elements of the genome

can change reading frame causing different kinds of mutations and regulation of gene expression

horizontal gene transfer concept

method of integrating DNA sequence from one bacterial genome into another bacterial genome

conjugation, transformation, transduction

vertical gene transfer concept

genome replication and cell division

conjugation

cell-cell contact (pili) allows DNA transfer following rolling circle replication

transformation

free DNA taken up by competent cell

transduction

how a bacteriophage transfers

mechanism of conjugation: steps

• Pili connects to other cell

• Pulls cell close

• Circular plasmid gets nicked in one strand

• One strand goes through pili into other cell

• Complementary strand is synthesized in recipient cell, transferred strand is replaced in donor cell by complementary strand synthesis

• Cells separate 

mechanism of transformation: steps

• Free DNA binds to DNA-binding protein on recipient cell

• Proteins inside facilitate single strand movement through cytoplasm into bacterial genome

• New strand is added to the genome via recombination

  • Forms heteroduplex —-------->>>>

• Genome replaces other strand to fix circle

mechanism of transduction

• Via bacteriophage

• Transfers DNA from donor to recipient

• When bacteriophage is repackaged, some host genome gets packaged

• When bacteriophage goes to infect new cell, injects genome (with hosts genome) into new recipient cell

importance of conjugation and horizontal gene transfer in biotech

We can add genes to certain bacteria to make then produce certain proteins for us

Transformation

• Steps to clone gene into many bacteria

  • Cut DNA with restriction enzyme

  • Add vector cut with same restriction enzyme

  • Add DNA ligase to form recombinant molecules

  • Introduce recombinant vector into host

Hallmarks of horizontal gene transfer

• Gene found in distantly related species

• Flanked by inverted repeats (hints has transposition)

• Gene clusters

Causes of population-level changes in genomes

Selection, Genetic drift, and Bottleneck

Cause of population-level changes in genomes: selection

mutation helps organism survive, reproduce

Cause of population-level changes in genomes: Genetic drift

some have more offspring than others (random)

Cause of population-level changes in genomes: Bottleneck

populations size is reduced greatly, removes some phenotypes

Information that can be revealed from the genome

Evolutionary history

Essential genes (use comparative genomics)

• How an organism functions

  • Virulent, metabolism, niche role, etc.

core genome

portion shared by all strains

accessory genome

optional genes in some strains

pan genome

core + all optional genes (not all in strains)

genomic or chromosomal island

gene clusters for specialized function (pan)

pathogenicity island

genomic island that codes for virulence factors

usefulness of transcriptomic information

insights into evolutionary history

uses mRNA transcripts (rather than genome) to analyze genome

molecular phylogeny

evolutionary history of organisms inferred from homologous genes

sequence similarity

% of nucleotide positions shared by any to sequences (shared/total)

homology

genes inherited from common ancestor (binary trait, either inherited or not)

suitability of 16S rRNA sequences for constructing phylogenetic trees

• Encodes a small subunit of ribosomal RNA molecule

• 16S rRNA sequence found in all bacteria and archaea, so can compare any species

• Has 9 hypervariable regions (V1-V9)

importance of gaps in sequence alignments

• Not all DNA changes are point mutations

• Establishes positional homology

phylogenetic tree

depicts evolutionary history of an organism

branch tips

species that exist today

node

where ancestor diverged into 2 new lineages

how to interpret phylogenetic trees

the closer the branches (connection wise not proximity), the more related

factors that may confound construction of phylogenetic trees

• Choosing one of two equally supported trees

• Convergent evolution

• Horizontal gene transfer

convergent evolution

two species having a shared sequence not due to common ancestry

categories of antibiotic resistance

• Development of resistant biochemical pathway

  • Ex. dihydrofolate synthase mutated to bind less efficiently to sulfonamide (antibiotic)

• Antibiotic inactivation

  • Ex. beta-lactamasde splits beta-lactam ring of penicillin

• Reduced membrane permeability

  • Ex. reduce porB, a porin that allows entry of penicillin

• Efflux

  • Ex. tetA, a tetracycline (antibiotic) pump to pump out of cell

Events leading to genome-altering mutations

DNA replication errors

size of virus genome and reasons for its size

size: 2-350 genes; 2000-1.2 million base pairs

reason: crucial niche selection and specified transmission routes in the susceptible host

diverse structures of viruses

helical (rod-shaped), icosahedral (polyhedral), or complex

factors contributing: capsid, envelope, and genome type

components of viruses

nucleic acid genome (DNA or RNA) enclosed within a protein coat called a capsid

similarities and differences between these components and their cellular counterparts

similarities: genetic material, evolution, interaction with other organisms

differences: cellular structure, reproduction, metabolism, size, host dependence, genetic material, infection mechanism

5 main stages of viral infection (using T4 as example

T4 uses lytic life cycle: lysis of the host cell and release of the new viral progenies

attachment

entry

uncoating

replication

maturation

release

Attachment

controls host specificity

surface factors interact w/ host receptor

surface factor = protein/lipid w/ other function

outcomes of viral infection in bacterial and animal cells

direct cell damage and death

differences between viral and bacterial growth curves

occurs when visions are released from the lysed host cell at the same time

sense vs antisense strand terminology

two strands of DNA that make up a double-stranded DNA molecule

Baltimore scheme of classifying viruses

classifying viruses based on the type of genome and its replication strategy

examples of viruses from groups I, IV, V, VII of the Baltimore classification scheme

group I (dsDNA): HSV, adenovirus, poxvirus

group IV (+ssRNA): Poliovirus, corona, rotavirus

group V(-ssRNA): Influenza virus, rabies virus

group VII (dsDNA-RT): hepatitis B virus

Baltimore classification scheme: how each of these viruses replicate

group 1: Double-stranded DNA (dsDNA) viruses

group 2: single-stranded DNA (ssDNA) viruses

group 3: double-stranded RNA (dsRNA) viruses

group 4: positive-sense single-strand (+ssRNA) viruses

group 5: negative-sense single-stranded RNA (-ssRNA) viruses

Baltimore classification scheme: proteins encoded by these viruses/unique features of each virus

group 1: DNA polymerase, thymidine kinase, capsid proteins, immediate early proteins, latency-associated proteins

group 2: replication initiator proteins, capsid proteins, nonstructural proteins

group 3: RNA-dependent RNA polymerase, structural proteins, outer shell proteins

group 4: RNA-dependent RNA polymerase, Protease, Capsid proteins, envelope glycoproteins, accessory proteins

group 5: RNA-dependent RNA polymerase, Nucleocapsid proteins, envelope glycoproteins, matrix proteins, accessory proteins

group 6: Reverse Transcriptase, integrate, protease, capsid proteins, envelope glycoproteins, regulatory proteins

group 7: reverse transcriptase, capsid proteins, surface glycoproteins, x proteins

SARS-CoV2 structure

positive sense RNA virus.

structural proteins of SARS-CoV2

envelope, membrane, spike, and nucleocapsid

Non-structural proteins of SARS-CoV2

NSP1 to NSP11 and NSP12 to NSP16

accessory proteins of SARS-CoV2 and their functions

ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, and ORF10, essential for controlling the hosts response to COVID infection

Replication cycle of SARS-CoV2

opening double helix and separation of DNA strands, priming of the template strand, and assembly of the new DNA segment

Replication cycle of SARS-CoV2: Where and how each of the 5 main stages of the viral life cycle is accomplished by SARS-CoV2

virus entry

translation of viral replication machinery

replication

translation of viral structure proteins

virion assembly

release of virus

Penetration

bacteria: entry of nucleic acid + specific viral proteins

animal: entry of full virion

synthesis

using host cell machinery, but directed by virus; early proteins, middle proteins, late proteins

assembly

package genome into preassembled capsids

release

package genome into preassembled capsids (late proteins breach host cytoplasmic membrane and peptidoglycan layer

sense (coding strand)

a strand of DNA that carries the code for making a protein

anitsense strand

non-coding DNA strand of gene. serves as template for producing mRNA, directs the synthesis of a protein

Which of the following events is an example of a mutation?

A.       Phosphorylation of an amino acid that is part of an enzyme increases the efficiency of that enzyme

B.       A mistake is made by DNA polymerase during DNA replication resulting in the change of an ‘A’ to a ‘C’ in the genome of a cell

C.      An mRNA molecule is degraded by nucleases

D.   A protein changes its conformation upon binding to another protein

B

which of the following mutations is likely to have the smallest impact on a cell?Explain your answer

A.       Insertion

B.       Deletion

C.      Substitution

D.      Frameshift

C. because it changes just one base in the DNA, which may not alter the protein if the new codon codes for the same amino acid.

In which of the following examples will there be two copies of the gene present at the end of the process. Select all that apply (1) Explain your answer (1)

A. Conjugation

B. Conservative transposition

C. Generalized transduction

D. Transformation

A. Conjugation and C. Generalized transduction result in two copies of the gene because genetic material is transferred while the donor retains its original copy. In B. Conservative transposition and D. Transformation, only one copy of the gene is present at the end.

Transcriptomics gives us information about the sequence of which molecule?

A.       RNA

B.       DNA

C.      Proteins

D.      Carbohydrates

A. RNA because Transcriptomics studies the RNA molecules made from DNA, giving information about gene expression.

Which of the following features is not identified as part of a typical bioinformatics workflow used to generate genomic information (1)

 A.       Start codons

B.       Stop codons

C.      Ribosome binding sites

D.      Post-translational modifications

D. Post-translational modifications. This feature occurs after protein synthesis and is not part of the genomic information workflow, unlike start codons, stop codons, and ribosome binding sites.

At which stage of the viral life cycle would you expect to detect the largest amount of virions in the infected tissue of a patient? (1) Explain your answer (1)

A.       Attachment

B.       Penetration

C.      Synthesis

D.      Release

D. Release. This is when newly formed virions are expelled from infected cells, resulting in the highest concentration of viruses in the tissue.