Genetics Exam 3

Advantages of Using Bacteria and Viruses for Genetic Studies

• Reproduction is rapid

• Many progeny can be produced

• The haploid genome allows all mutations to be expressed directly 

• Asexual reproduction simplifies the isolation of genetically pure strains 

• Growth in the laboratory is easy and requires little space

• Genomes are small 

• Techniques are available for isolating and manipulating their genes 

• They have medical importance

• They can be genetically engineered to produce substances of commercial value 

Prokaryotes

All prokaryotes are unicellular and lack a membrane-bound nucleus and membrane-bound organelles 

Eubacteria

“True Bacteria”, single-celled prokaryotes found in many environments, such as E. coli 

Archaea

Single-celled prokaryotes that are generally distinct from bacteria and often live in extreme environments, such as Halobacterium

Characteristics of Bacteria

• Diverse shapes and sizes 

• Some are photosynthetic

• Replication occurs prior to binary fission 

Minimal Medium

Medium that contains the nutrients required by only prototrophic bacteria – typically a carbon source, essential elements such as nitrogen and phosphorus, certain vitamins, and other required ions and nutrients

Complete Medium

A medium that contains all the substances required by bacteria for growth and reproduction. Auxotrophic mutants can grow on a complete medium

Plasmids

Small, usually circular DNA molecule that is distinct from the bacterial chromosome; capable of replicating independently of the bacterial chromosome, replicate independently of its bacterial chromosome

Episomes

Plasmid capable of replicating freely and able to integrate into a bacterial chromosome 

F (Fertility) Factor of E. coli 

Episome of E. coli that controls conjugation and gene exchange between E. coli cells. The F factor contains an origin of replication and genes that enable the bacterium to undergo conjugation

The Bacterial Genome

Mostly single, double stranded, circular DNA molecule/chromosome 

Conjugation

Mechanism by which genetic material can be transferred from one bacterium to another. In conjugation, two bacteria lie close together and a connection forms between them. A plasmid, or sometimes a part of the bacterial chromosome, passes through this connection from one cell to another (direct transfer of DNA from one bacterium to another, not a reciprocal exchange)

Transformation

Mechanism by which DNA found in the environment is taken up by a cell. After transformation, recombination may take place between the introduced genes and those of the bacterial chromosome (bacterium takes up free DNA)

Transduction

Type of gene exchange that takes place when a virus carries genes from one bacterium to another. After it is inside the cell, the newly introduced DNA may undergo recombination with the bacterial chromosome. (bacterial viruses take DNA from one bacterium to another) 

Pili

Extension of the cell membrane of some bacteria that allows conjugation to take place. When a pilus on one cell makes contact with a receptor on another cell, the pilus contracts and pulls the two cells together 

Competent

The ability to take up DNA from the environment (to be transformed) 

Transformants

Cell that has received genetic material through transformation 

Cotransformed

Process in which two or more genes are taken up together during cell transformation

Horizontal Gene Transfer

Transfer of genes from one organism to another by a mechanism other than reproduction

The F factor contains a number of genes that?

Regulate transfer into a bacterial cell, replication, and insertion into the bacterial chromosome 

F+ Cells

Donor cells containing F factor

F- Cells

recipient cells lacking F factor

Sex pilus

Connects F+ and F- cells during bacterial conjugation

Hfr (high frequency recombination) cells

donor cells with F factor integrated into the donor bacterial chromosome

F’ (F prime) cells

contain F plasmid carrying some bacterial genes

Bacterial genes may be transferred from an Hfr cell to

an F- cell in conjugation

The F factor is transferred during conjugation between?

an F+ and F- cell

An Hfr cell may be converted into an F’ cell when?

The F factor excises from the bacterial chromosome and carries bacterial genes with it 

Restriction Modification Systems

A type of bacterial defense that uses restriction enzymes to cleave and foreign DNA. The bacteria protects its own DNA from cleavage by modifying the sequences recognized by the restriction enzymes 

CRISPR-Cas Systems

A system used by bacteria and archaea as a defense mechanism against foreign DNA. has been adapted for use as a molecular tool for precisely cutting DNA, providing a powerful way of editing the genome 

CRISPR Array

A series of repeated DNA sequences called clustered regularly interspaced short palindromic repeats located in the bacterial or archaeal chromosomes. Fragments of foreign DNA are inserted into the array; these are later transcribed into CRISPR RNA

Protospacer-Adjacent Motif (PAM)

Adjacent DNA sequence required for binding by a CRISPR-Cas effector complex; the complex associates with the PAM and unwinds the DNA nearby

CRISPR-Cas immunity takes place in three steps:

adaptation, expression and interference

Virus

Non-cellular replicating structure consisting of nucleic acid surrounded by a protein coat; can replicate only within a host cell 

Virulent Phages

Bacteriophage that reproduces strictly through the lytic cycle and kills its host cell 

Temperate Phages

Bacteriophages and Prophages

Bacteriophage

 (bacterial infection virus) that can undergo the lysogenic cycle, in which the phage DNA integrates into the bacterial chromosome and remains in an inactive state

Prophage

Phage genome that is integrated into a bacterial chromosome 

Bacteriophage Characteristics

• always kills the host cell

• have two alternative life cycles, lytic and lysogenic

Plaque

Clear patch of lysed cells on a continuous layer of bacteria on the agar surface of a petri dish plate; each plaque represents a single original phage that multiplied and lysed many cells

Generalized Transduction

Transduction in which any gene may be transferred between bacteria by viruses 

Specialized Transduction

Transduction in which genes near special sites on the bacterial chromosome are transferred from one bacterium to another; requires lysogenic bacteriophages 

Transducing Phages

Phage that contains a piece of the chromosome of its bacterial host inside the phage coat

Transductants

Bacterial cell that has received genes from another bacterium through transduction 

Cotransduced

Process in which two or more genes are transferred together from one bacterial cell to another by a virus. Only genes located close together on a bacterial chromosome will be cotransduced 

Retroviruses

Virus that injects its RNA genome into a host cell, where reverse transcription produces a complementary, double-stranded DNA molecule from the RNA template; the DNA copy then integrates into the host chromosome to form a provirus

HIV-1 evolved from?

A similar gene found in chimpanzees

HIV attacks?

helper T cells

Emerging Viruses

Arose recently and are more likely to cause infection than previous strains, leads to loss of human life and public alarm, seen with SARS-CoV-2

Influenza caused by RNA viruses

Three main types: Influenza A, B and C

• Most cases are influenza A 

• Divided into subtypes based upon expression of hemagglutinin (HA) and neuraminidase (NA)

Coronaviruses

• Precise evolutionary origin is unknown 

• Single-stranded RNA genome of 29,903 nucleotides and five main genes 

Reverse Transcriptase

Enzyme that can synthesize complementary DNA from either an RNA or DNA template

Provirus

A viral genome incorporated into a host chromosome 

Integrase

Enzyme that inserts prophage, or proviral, DNA into a host chromosome 

Oncogenes

Dominant-acting gene that stimulates cell division, leading to the formation of tumors and contributing to cancer; arises from a mutated copy of a normal cellular gene (proto-oncogene)

Antigenic Drift

The appearance of new strains of a rapidly evolving virus because of mutations

Antigenic Shift

Major change in a viral genome through the reassortment of genetic material from two different strains of the virus

Key characteristics of Genetic Material

Must contain complex information, replicate faithfully, must encode the phenotype, and have the capacity to vary 

Chargaff’s rules

Adenine is always equal to thymine (A = T) 

Guanine is always equal to cytosine (G = C) 

Griffith’s Experiments Demonstrated Transformation in Bacteria

• If Type IIIS (virulent) bacteria are injected into a mouse it leads to the death of the mouse and the type IIIS (virulent) bacteria is recovered 

• If Type IIR (nonvirulent) bacteria are injected into a mouse, then the mouse will live and no bacteria will be recovered 

• If heat-killed type IIIS bacteria are injected into a mouse, then the mouse will live and no bacteria will be recovered 

• If a mixture of type IIR bacteria and heat-killed type IIIS bacteria is injected into a mouse, then the mouse will die and Type IIIS (virulent) bacteria will be recovered 

• A substance in the heat-killed virulent bacteria genetically transformed the type IIR bacteria into live, virulent type IIIS bacteria 

• The smooth bacteria has a gene that makes a protective sugar-based (glycocalyx layer) coat gene to the bacteria and immune to the response of the mouse, the rough bacteria did not have this coat, letting the immune system of the mouse kill it 

Chemical Nature of Transforming Substances

1. Use heat to kill virulent bacteria, homogenize and filter

2. Treat samples with enzymes that destroy proteins, RNA, or DNA 

3. Add the treated samples to cultures of type IIR bacteria 

4. Cultures treated with protease or RNase contain transformed type IIS bacteria but the culture treated with DNase does not 

5. Because only DNase destroyed the transforming substance, the transforming substance is DNA 

T2 is a bacteriophage that infects E. coli, therefore?

• Phage genome is DNA 

• All other parts of the bacteriophage are protein 

1. Phage attaches to E. coli and injects its chromosome 

2. Bacterial chromosome breaks down, and the phage chromosome replicates 

3. Expression of phage genes produce phage structural components 

4. Progeny phage particles assemble 

5. Bacterial wall lyses, releasing progeny phages 

How did Hershey and Chase demonstrate that DNA carries the genetic information in bacteriophages?

1. Infect E. coli grown in medium containing 35S OR infect E. coli grown in medium containing 32P 

2. 35S is taken up in phage protein, which contains S but not P/32P is taken up in phage DNA which contains P but not S 

3. Phages with 35S infect unlabeled E. coli/Phages with 32P infect unlabeled E. coli 

4. Shear off protein coats in blender and separates protein from cells by centrifuging 

5. After centrifugation, 35S is recovered in the fluid containing the virus coats/after centrifugation infected bacteria form a pellet containing 32P in the bottom of the tube

6. No radioactivity is detected, indicating that protein has not been transmitted to the progeny phages/the progeny phages are radioactive, indicating DNA has been transmitted to the progeny phages 

What is the genetic material in bacteriophages?

DNA, not protein

How was DNA determined the source of genetic information?

• Watson and Crick’s discovery of the three-dimensional structure of DNA 

• X-ray diffraction image of DNA 

How does X-Ray Diffraction Provides Information About the Structures of Molecules

1. Crystals of a substance are bombarded with X-rays, which are diffracted (bounced off) 

2. The spacing of the atoms within the crystal determines the diffraction pattern, which appears as spots on a photographic film 

3. The diffraction pattern provides information about the structure of the molecule 

Nucleotides

Repeating unit of DNA or RNA made up of a sugar, a phosphate and a nitrogenous base 

Transforming Principle

Substance responsible for transformation. DNA is the transforming principle

Isotopes

Different forms of an element that have the same number of protons and electrons but differ in the number of neutrons in the nucleus

X-ray Diffraction

Method for analyzing the three-dimensional shape and structure of a chemical substance in which crystals of the substances are bombarded with X-rays, which hit the crystals, bounce off, and produce a pattern of spots on a detector 

Primary structure of DNA

• Deoxyribonucleotides

• Nucleotides 

• Sugar 

• A phosphate 

• A nitrogenous base (A, U, C, G) 

• A purine or pyrimidine base 

How does DNA get its negative charge?

From phosphate

Adenine

A, deoxyadenosine 5’-monophosphate, dAMP, deoxyadenosine, dA

Guanine

G, deoxyguanosine 5’-monophosphate, dGMP, deoxyguanosine, dG

Thymine

T, deoxythymidine 5’-monophosphate, dTMP, deoxythymidine, dT

Cytosine

C, deoxycytidine 5’-monophosphate, dCMP, deoxycytidine, dC

DNA structure

The DNA backbone is deoxyribose sugars linked by phosphate 

Central Dogma

Concept that genetic information passes from DNA to protein in a one-way information pathway

Major Information Pathways 

• In transcription, information is transferred from DNA to an RNA molecule

• In DNA replication, information is transferred from one DNA molecule to another 

• In translation, information is transferred from RNA to a protein through a code that specifies the amino acid sequence 

Special Information Pathways 

• In reverse transcription, in some viruses, information is transferred from RNA to DNA or to another RNA molecule

What holds DNA together?

Hydrogen bonds

Because DNA is very thin, its main problem is?

Raveling it into a small space in an ordered fashion where genes can be located and expressed as needed 

How many bases of DNA per turn?

10 bases

Phosphodiester Linkages

A strong covalent bond that joins the 5’-phosphate group of one nucleotide and the 3’-hydroxyl group of the next nucleotide in a polynucleotide strand 

Polynucleotide Strand

Series of nucleotides linked together by phosphodiester bonds 

B-DNA Structure

Right-handed helical structure of DNA that exists when water is abundant; the secondary structure described by Watson and Crick and probably the most common DNA structure in cells

A-DNA Structure

Right-handed helical structure of DNA that exists when little water is present 

Z-DNA Structure

Secondary structure of DNA characterized by 12 bases per turn, a left-handed helix, and a sugar-phosphate backbone that zigzags back and forth 

DNA must be tightly packed to?

Fit in small spaces

DNA in E. coli is how long?

About 1000 times as long as the cell itself

Supercoiling

Tertiary structure that forms when strain is placed on a DNA helix by overrotation or underrotation 

Supercoiled DNA is?

Overwound, or underwound, causing it to twist on itself

What does topoisomerase do with supercoiling?

It is the enzyme responsible for adding and removing turns in the coiling 

Relaxed State

Energy state of a DNA molecule when there is no structural strain on the molecule 

Positive Supercoiling

Tertiary structure that forms when strain is placed on a DNA helix by overrotating

Negative Supercoiling

Tertiary structure that forms when strain is placed on a DNA helix by underrotating 

Bacterial DNA is highly folded into?

A series of twisted loops 

• Most bacterial genomes have a single circular DNA molecule

• DNA appears as a clump called the nucleoid 

DNA Packing

• Eukaryotic chromosomes

• Chromatin structure: euchromatin, heterochromatin, histone proteins

Chromatin has a highly complex structure with several levels of organization:

• Nucleosome 

• Linker DNA 

• High-order chromatin structure: 30 nm fiber, 300 nm loops, 250 nm-wide fiber 

1. At the simplest level, chromatin is a double-stranded helical structure of DNA 

2. DNA is complexed with histones to form nucleosomes 

3. Each nucleosome consists of eight histone proteins around which the DNA wraps 1.65 times

4. The nucleosomes fold up to produce a 30 nm fiber that forms loops averaging 300 nm in length

5. The 300 nm loops are compressed and folded to produce a 250 nm-wide fiber

6. Tight coiling of the 250-nm fiber produces the chromatid of a chromosome 

The Nucleosome is the fundamental repeating unit of chromatin:

1. A small amount of nuclease cleaves the “string” between the beads releasing individual beads attached to about 200 bp of DNA 

2. More nuclease destroys all the unprotected DNA between the beads leaving a core of proteins attached to 145-147 bp of DNA 

Epigenetic Changes

Chromatin structure – changes in chromatin structure

• Epigenetic changes: methylation; capable of being reversed and often due to environmental factors 

• Variation in DNA methylation at the agouti locus produces different coat colors in mice

Topoisomerase

Enzyme that adds or removes rotations in a DNA helix by temporarily breaking nucleotide strands; controls the degree of DNA supercoiling