Genetics of Microorganisms Exam 1

Friedrich Miescher

• identified DNA + RNA (nucleic acids) in 1869 in waste surgical bandages

• named it “nuclein” - material from nucleus of cell

• later research showed DNA (and RNA) release H⁺ in water, making them acids

• led to the rename “nucleic acids”

nucleotides

• form repeating unit of nucleic acids, linked by ester (covalent) bonds, repeating structural unit of RNA/DNA

• composed of phosphate group, pentose sugar (ribose/deoxyribose) and a nitrogenous base

• base + sugar + phosphate; AMP, ADP, ATP

phosphodiester linkage

phosphate connects 5′ C of nucleotide to 3′ C of adjacent nucleotide

nucleoside

• base + sugar

• adenine + ribose = Adenosine

• adenine + deoxyribose = Deoxyadenosine

DNA

• 3D structure from folding and bending of double helix

• interaction with proteins produces chromosomes

• 5′ to 3’, all sugar molecules oriented in same direction

• phosphates and sugar form backbone of nucleic acid strand and bases project from backbone

DNA structure

• 10 base pairs (bp) in each strand and 3.4 nm per complete turn of helix

• 2 strands are antiparallel, one is 5′ to 3′ and other 3′ to 5′

• right-handed, as it spirals away, it turns clockwise

• A-T bonded by 2 H-bonds and G-C by 3 H-bonds

• Base stacking, bases oriented so flattened regions face each other

• 2 asymmetrical (major/minor) groves on outside that allow for protein interaction with specific sequences

Ball-and-stick vs space-filling model of DNA

structure of deoxyribonucleic acid (DNA) vs ribonucleic acid (RNA)

James Watson and Francis Crick

• In 1953, they elucidated double helical structure of DNA

• initially tried to build ball-and-stick models that incorporated all known experimental observations

• original model had sugar-phosphate backbone on outside and bases projecting toward each other with bases forming H bonds with identical bases in the opposite strand

• later realized that H bonding of A to T was structurally similar to that of C to G

Linus Pauling

• in 1950s, proposed that regions of protein can fold into a secondary structure called an α-helix

• built ball-and- stick models to elucidate this structure

• incorrectly proposed triple helix DNA structure

Rosalind Franklin

• used X-ray diffraction to study wet fibers of DNA

• diffraction pattern is interpreted (using mathematical theory) to provide info about the structure of a molecule

• made advances in X-ray diffraction techniques with DNA

• diffraction pattern showed DNA was helical, had more than 1 strand and 10 base pairs per complete turn

Erwin Chargaff

• pioneered biochemical techniques for isolation, purification and measurement of nucleic acids from cells

• analyzed base composition of DNA isolated from many different species

• when base compositions from different tissues within same species were measured, there were similar results

Chargaff’s rule

% adenine = % thymine

% cytosine = % guanine

B-form DNA (DNA secondary structure)

• predominant form found in living cells

• bases relatively perpendicular to central axis

A-form DNA (DNA secondary structure)

RNA and DNA-RNA hybrids

Z-form DNA (DNA secondary structure)

• Left-handed helix, 12 bp per turn

• formation favored by

  • alternating purine/pyrimidine sequences (GCGCGCGCGC), at high salt concentrations

  • cytosine methylation, at low salt concentrations

  • Negative supercoiling

• may have role in transcription and chromosome structure

• recognized by cellular proteins, may alter chromosome compaction

• bases substantially tilted relative to central axis

• sugar-phosphate backbone follows zigzag pattern

RNA

• usually single-stranded, can form short double-stranded regions

• double-stranded secondary structure forms due to complementary base-pairing (A/U and C/G)

• double helices are right-handed and have 11-12 base pairs per turn

4 RNA Loop Structures

1. Bulge loop

2. Internal loop

3. Multibranched loop

4. Stem loop

Non-canonical base pairs

• Planar hydrogen bonded pairs of nucleobases

• H-bonding patterns that differ from patterns observed in Watson-Crick base pairs (classic double helical DNA)

Recombinant DNA technology

• use of in vitro molecular techniques to isolate and manipulate fragments of DNA

• Recombinant DNA technology and gene cloning have been fundamental to our understanding of gene structure and function

recombinant DNA molecules

• chimeric molecules, first constructed in 1970s by researchers at Stanford

• can be introduced into living cells where they are replicated to make many identical copies, led to era of gene cloning

gene cloning

• technique of isolating and making many copies of a gene; refers to use of vectors

• devised during the early 1970s

• includes DNA sequencing, DNA probes and expression of cloned genes

Chromosomal DNA

• serves as source of DNA segment of interest

• must obtain cellular tissue from organism, break open cells, and extract and purify DNA using a biochemical techniques to obtain

Vector DNA

• carrier for DNA segment that is to be cloned

• can replicate independently of host chromosomal DNA

vector

• host cell harbors the vector

• when a vector is replicated inside host cell, the DNA that it carries is also replicated

• the sequence of ORC determines if a vector can replicate in a particular host cell

• originally derived from two natural sources

  • Plasmids: have selectable markers; genes conferring antibiotic resistance to host cell

  • Viruses: infect living cells and propagate by taking control of host cell’s machinery

restriction endonucleases/enzymes

• enzymes that cut DNA, allowing insertion of chromosomal DNA into vector

• Discovered 1960-70s by Werner Arber, Hamilton Smith and Daniel Nathans

• made naturally by many species of bacteria

• protect bacterial cells from invasion by foreign DNA, particularly bacteriophages

• bind to specific DNA sequences and cleave DNA at 2 defined locations, one in each strand

• some digest DNA into fragments with “sticky ends” or generate blunt ends; NaeI cuts in middle of recognition sequence

Recognition sequences

• palindromic, identical when read in opposite direction in complementary strand

• several hundred different restriction enzymes are available commercially

DNA ligase

covalently links sugar-phosphate backbone of DNA molecules with sticky or blunt ends

polymerase chain reaction (PCR)

• developed by Kary Mullis in 1985

• can copy DNA without vectors and host cells

• must know enough about gene of interest to have sequence of 2 short primers

• specific DNA segment can be amplified

• also used to amplify chromosomal DNA nonspecifically which uses mix of primers with many different random sequences

• these will anneal randomly throughout genome and amplify most chromosomal DNA

• used to amplify very small samples, ex) crime scene DNA

4 starting materials for PCR

1. Template DNA: contains region to be amplified

2. Oligonucleotide primers: complementary to sequences at ends of DNA fragment to be amplified, synthetic, 15-20 nucleotides long

3. Deoxynucleoside triphosphates (dNTPs): provides precursors for DNA synthesis

4. Taq polymerase (or other polymerase): DNA pol isolated from bacterium Thermus aquaticus; thermostable enzyme, used bc PCR involves heating steps that inactivates most other DNA pols

PCR steps

• carried out in thermocycle, all reagents in one tube

• sequential process of denaturing-annealing-synthesis repeated for many cycles, typically 20-30 cycles of replication, taking a few hours

• after 20 cycles, target DNA sequence increase 2²⁰-fold (1 million-fold)

• after 30 cycles, target DNA sequence increase 2³⁰-fold (1 billion-fold), assuming 100% efficiency (not real)

Reverse transcriptase PCR (RT-PCR)

• used to detect and quantitate amount of RNA in live cells

• RNA is isolated from sample then mixed with reverse transcriptase

• primer will anneal to 3’ end of RNA, generating single-stranded cDNA that can be used as template DNA in PCR

• extraordinarily sensitive, can detect expression of small amounts of RNA in a single cell

Quantitative PCR (qPCR)

• used to quantitate amount of specific gene or mRNA in a sample in real time

• can be used to quantify the amount of DNA or RNA present in a specific gene or mRNA

• carried out in thermocycler that can measure changes in fluorescence emitted by detector molecules in the PCR reaction mix

• will increase in proportion to amount of PCR product produced

Cycle Threshold (Ct) in qPCR

• reached when accumulation of fluorescence is significantly greater than background fluorescence

• depends on initial concentration of template DNA

5 steps of qPCR

1. Initially, little product is made so no increase in fluorescence detected

2. When reagents are not limiting, product doubles with every cycle (exponential phase); Cycle Threshold (Ct) is reached.

3. Changes to linear phase when reagents are somewhat limiting

4. Reaction eventually plateaus when one or more reagents are used up

5. Concentration of unknown amount of starting DNA (or RNA) can be determined by comparing Ct with known standards by adding a known amount of DNA or amplifying another gene in sample

Chromosomes

structures that contain genetic material, complexes of DNA and proteins

genome in proks vs euks

• all genetic material an organism possesses

• proks: single circular chromosome, chloroplast genome

• euks: one complete set of nuclear chromosomes, mitochondrial genome

4 things DNA sequences are necessary for

• Synthesis of RNA and cellular proteins

• Replication of chromosomes

• Proper segregation of chromosomes

• Compaction of chromosomes so they can fit within cells

Prokaryotic Chromosomes

• most are circular w/ a few million nucleotides/bp in length

• bacterial chromosome contains thousands of genes, majority of which is protein-coding genes

• most contain single type of chromosome, but it may be in multiple copies

• several thousand different genes and repetitive sequences interspersed throughout

• at least 1 ORC to initiate DNA replication

intergenic regions

non-transcribed DNA between adjacent genes

nucleoids

• where prok chromosomes are found

• not bounded by membrane

• DNA in direct contact with cytoplasm

loop domains (microdomains)

• allows chromosomal DNA to be compacted 1000-fold to fit within bacterial cell

• 10,000 bp

macrodomains

• # of loops varies according to size of bacterial chromosome and species

• E. coli has 400-500 microdomains

• organizes adjacent microdomains in E.coli, 800-1000 kbp

nucleoid-associated proteins (NAPs)

• DNA-binding proteins used by bacteria to form microdomains and macrodomains

• facilitate chromosome compaction and organization

• bend DNA or act as bridges for DNA to bind to other DNA regions

• facilitate chromosome segregation

• involved in gene regulation

Archaeal Chromosomes

• structure varies, depends on DNA-binding proteins expressed

• some produce bacterial-like nucleoid-associated proteins or eukaryotic-like histone proteins

• DNA wrapped around histone proteins to form nucleosomes and organized into loop domains

• # of histone proteins varies among diff species, in some, structure is similar to eukaryotic chromatin

DNA supercoiling

• way for prokaryotic chromosomes to become more compact

• formation of additional coils due to twisting forces where the 2 strands within DNA already coil around each other

• caused by both underwinding and overwinding of DNA double helix

negative DNA supercoiling

• formed when DNA given a turn that unwinds helix (left)

• can also cause fewer turns, topoisomers

• chromosomal DNA in bacteria is negatively supercoiled

• In E. coli, 1 - supercoil per 40 turns of double helix

• Helps compaction of chromosome

• In localized regions, creates tension that may be released by DNA strand separation which it also promotes

positive DNA supercoiling

• formed when DNA given a turn that overwinds the helix (right)

• can also cause more turns

• topoisomers

DNA gyrase (DNA topoisomerase II)

• introduces - supercoils using energy from ATP

• relax + supercoils when they occur

• can untangle intertwined DNA molecules

• crucial for bacterial survival

DNA topoisomerase I

relaxes - supercoils

2 DNA Gyrase Inhibitors

• blocking gyrase can treat bacterial diseases

• Quinolones: ex) Ciprofloxacin: used in treatment of anthrax, Coumarins

• do not inhibit euk topoisomerases

Eukaryotic Chromosomes

• contain ≥1 sets of chromosomes composed of several different linear chromosomes, many species are diploid

• contains a single, linear molecule of DNA with tens of to hundreds of millions of bps and a few hundred to several thousand genes interspersed throughout

• in simpler euks, genes are short (several hundred bp)

• in complex euks, genes are long with many introns w/ lengths from <100 to >10,000 bp

• require ORC, centromeres, and telomeres for chromosomal replication and segregation

intron

noncoding intervening sequences

Origins of replication

• chromosomal sites necessary to initiate DNA replication

• eukaryotic chromosomes contain many origins

• interspersed every 100,000 base pairs

centromeres

• regions that play a role in segregation of chromosomes

• forms a recognition site for kinetochore proteins

kinetochore

required for centromere linkage to spindle apparatus during mitosis and meiosis

Telomeres

• specialized regions at the ends of chromosomes

• important in replication and for stability

• contain specialized sequences located at both ends of linear chromosome

repetitive sequences

commonly found near centromeric and telomeric regions, but they may also be interspersed throughout chromosome

eukaryotic Genomes size

• total amount of DNA much greater than bacterial cells

• vary substantially in size, variation not related to complexity of species

• difference in size due to accumulation of repetitive DNA sequences which do not code proteins

• the plant Tmesipteris oblanceolata has 160 billion bps

Sequence complexity

• number of times a particular base sequence appears in genome

• unique/non-repetitive, moderately repetitive, highly repetitive

Unique/non-repetitive sequences

• found ≥1 times in genome

• includes protein-encoding genes and intergenic regions

• in humans, makes up 41% of genome

Moderately repetitive sequences

• found a few hundred to several thousand times

• Genes for rRNA and histones

• Sequences that regulate gene expression and translation

• Transposable elements

Highly repetitive sequences

• found tens of thousands to millions of times

• each copy is relatively short (a few nucleotides to several hundred in length)

• some sequences interspersed throughout genome, ex) Alu family in humans, 300 bp, 10% of genome, found every 5000–6000 bp

• other sequences clustered together in tandem arrays, ex) AATAT and AATATAT sequences in Drosophila found in centromeric regions

Transposition

• integration of small segments of DNA into a new location in genome

• can occur at many different locations in genome

• since many outcomes are likely to be harmful, it is highly-regulated

• occurs only in few individuals under certain conditions

transposable elements (TEs) or transposons

• “jumping genes” or small, mobile DNA segments

• first identified by Barbara McClintock in 1950s in corn

• move by different transposition pathways; simple transposition or retrotransposition

• can enter genome of organism and proliferate quickly

• ex) Drosophila melanogaster: TE called P element introduced in 1950s and expanded throughout D. melanogaster populations worldwide. The only strains without P element are in labs collected prior to 1950

• have effects on chromosome structure and gene expression

• Agents such as radiation, chemical mutagens and hormones stimulate the movement

Simple transposition

• used widely by transposons in bacterial and euks

• TE removed from original site and transferred to new target site, cut-and-paste mechanism

• occurs after replication fork passes through TE, so there are 2 copies of TE

• one TE can transpose ahead of fork where it is copied again

• one chromosome will still have one TE, but the other will now have two copies

Retrotransposition (retrotransposons)

transposable elements that move via an RNA intermediate and is transcribed into RNA , found only in euks

direct repeats (DRs)

identical base sequences oriented in same direction and repeated, flank TEs

insertion element

simplest TE; flanked by inverted repeats, moves by simple transposition

Inverted repeats

• DNA sequences that are identical but run in opposite directions

• 9-40 bp

• may contain gene for transposase

simple transposon

carries >1 genes not required for transposition, moves by simple transposition

LTR Retrotransposons

• evolutionarily related to known retroviruses

• retain ability to move around genome but mostly do not produce mature viral particles

• contain long terminal repeats (LTRs) at both ends

• few hundred bps in length

• code virally related proteins, reverse transcriptase and integrase, required for retrotransposition process

• move by retrotransposition via RNA intermediate

Non-LTR retrotransposons

• do not resemble retroviruses in having LTR sequences

• may contain gene that encodes protein that functions as both reverse transcriptase and an endonuclease

• some are evolutionarily derived from normal eukaryotic genes

• Alu family of repetitive sequences found in humans is derived from single ancestral gene, 7SL RNA gene which has been copied by retrotransposition many times, w/ current # of copies being 1 million

• move by target-site primed reverse transcription

transposase

• catalyzes transposition event, removal of a TE and its reinsertion at another location

• recognizes inverted repeats at ends of TE and brings them close together

Reverse transcriptase

uses RNA as template to synthesize double-stranded DNA molecule

Integrase

Recognizes LTRs at ends of DNA, makes cuts at target site in host chromosome and catalyzes insertion of TE into site

target-site primed reverse transcription

• allows non-LTR retrotransposons to move

• retrotransposon transcribed into RNA with 3′ polyA tail

• target DNA recognized by endonuclease

• PolyA tail binds to nicked site

• reverse transcriptase uses target DNA of primer and makes DNA copy of RNA

LINEs (Long interspersed elements)

• usually 1,000-10,000 bp long

• occur in 20,000-1,000,000 copies per genome

• 17% of human genome

SINEs (Short interspersed elements)

• > 500 bp in length

• ex) Alu (Arthrobacter luteus restriction endonuclease) sequence present in 1,000,000 copies in human genome (10% of genome)

2 theories of biological significance of transposons

1. selfish DNA theory: TEs exist because they can! They can proliferate within host as long as they don’t harm the host by disrupting survival

2. TEs exist because they offer some advantage. Bacterial TEs carry antibiotic-resistance genes

exon shuffling

• TEs cause genetic variability through recombination

• TEs cause insertion of exons into coding sequences of protein-coding genes

• lead to evolution of genes with more diverse functions

nucleus

2-4 μm diameter, DNA is tightly compacted to fit within

chromatin

• compaction of linear DNA in euk chromosomes involves DNA-protein complex

• proteins bound to DNA subject to change during life of cell, affecting degree of chromatin compaction

nucleosome

• repeating structural unit within euk chromatin

• composed of double-stranded segment of DNA wrapped around an octamer of histones

• histone octamer composed of 2 copies each of 4 different histone proteins

• 146 bp of DNA make 1.65 negative superhelical turns around octamer

Histones

• basic, contain + charged AAs; lysine and arginine

• these AAs bind to - charged phosphates along DNA backbone

• have globular domain and flexible, charged amino terminus or ‘tail’

core histones

H2A, H2B, H3 and H4; there are 2 of each, making up the octamer

linker histone

• H1, binds to DNA in linker region

• less tightly bound to DNA than core histones

• helps to organize adjacent nucleosomes

how do salt concentrations affect H1?

• moderate salt concentrations: H1 is removed, classic beads-on-a-string morphology

• low salt concentrations: H1 remains bound, beads associate together into a compact morphology

zigzag model

• proposed model for interactions of nucleosomes

• linker DNA is relatively straight, and nucleosomes form a zigzag arrangement

• zigzag arrangement only occurs over short distances, such as 2-4 nucleosomes

• a former model (30nm fiber model) depicted long-range interactions of nucleosomes to form a fiber; this model is no longer accepted

loop extrusion model

• Chromatin can be further compacted by folding segments of nucleosomes into loops/loop domains

• 2 proteins play a role in loop formation: SMC proteins (structural maintenance of chromosomes) and CCCTC binding factor (CTCF)

SMC proteins (structural maintenance of chromosomes)

• forms a dimer that can wrap itself around 2 DNA segments and promote formation of a loop

• use energy from ATP to catalyze loop formation

CCCTC binding factor (CTCF)

after loop has formed due to SMC proteins, 2 different CTCFs bind to DNA and then bind to each other to stabilize loop

topologically associating domains (TADs)

• regions that chromatin is organized into

• 100 kb - 1 Mb in length

• segments of DNA within are more likely to interact with each other than they are with segments in other neighboring TADs

topologically associating domains (TADs) boundaries

• determined by SMC proteins and CTCFs

• promote interactions within

• act as insulators, preventing interactions between different TADs

Heterochromatin

• Tightly compacted regions of chromosomes

• Transcriptionally inactive (in general)

• Loop domains compacted even further

• constitutive and facultative

Constitutive heterochromatin

• regions that are always heterochromatic

• permanently inactive with regard to transcription

• usually contain highly repetitive sequences

Facultative heterochromatin

Regions that can interconvert between euchromatin and heterochromatin

Euchromatin

• Less condensed regions of chromosomes

• Transcriptionally active

• Loop domains are less compacted

chromosome territory

• each chromosome in cell nucleus is in a discrete territory

• shown in studies by Thomas and Christoph Cremer and others through fluorescent staining in which each chromosome is shown in a different color

level 1 of chromosome organization

• at scale of an entire nucleus

• chromosomes occupy distinct territories

• interchromosomal and chromosomal interactions with other nuclear structures (ex: nuclear lamina) play a role in chromosomal arrangements