Chapter 19 Microbial Models: The Genetics of Viruses and Bacteria

• Chapter 19• Microbial Models: The

Genetics of Viruses and Bacteria

Viral structure

• Virus: “poison” (Latin); infectious particles consisting of a nucleic acid in a protein coat

• Capsid; (viral envelopes); DNA or RNA

• Bacteriophages (phages)

Viral reproduction: Lytic Cycle• Host range: infection of a

limited range of host cells (receptor molecules on the surface of cells)

• The lytic cycle: 1- attachment 2- injection 3- hydrolyzation 4- assembly 5- release

• Results in death of host cell• Virulent virus (phage

reproduction only by the lytic cycle)

Viral reproduction: Lysogenic Cycle

• Genome replicated w/o destroying the host cell

• Genetic material of virus becomes incorporated into the host cell DNA (prophage DNA)

• Temperate virus (phages capable of using the lytic and lysogenic cycles)

• May give rise to lytic cycle

RNA viruses

• Retroviruses: transcribe DNA from an RNA template (RNA--->DNA)

• Reverse transcriptase (catalyzing enzyme)

• HIV--->AIDS

Viroids and Prions

• Viroids: tiny, naked circular RNA that infect plants; do not code for proteins, but use cellular enzymes to reproduce; stunt plant growth

• Prions: “infectious proteins”; “mad cow disease”; trigger chain reaction conversions; a transmissible protein

Bacterial genetics

• Nucleoid: region in bacterium densely packed with DNA (no membrane)

• Plasmids: small circles of DNA

• Reproduction: binary fission (asexual)

Bacterial DNA-transfer processes

• Transformation: genotype alteration by the uptake of naked, foreign DNA from the environment (Griffith expt.)

• Transduction: phages that carry bacterial genes from 1 host cell to another

•generalized - random transfer of host cell chromosome

•specialized - incorporation of prophage DNA into host chromosome

Conjugation

direct transfer of genetic material; cytoplasmic bridges; pili; sexual

Bacterial Plasmids• Small, circular, self-replicating DNA separate from the bacterial

chromosome• F (fertility) Plasmid: codes for the production of sex pili (F+ or F-)• R (resistance) Plasmid: codes for antibiotic drug resistance

• Transposons: transposable genetic element; piece of DNA that can move from one location to another in a cell’s genome (chromosome to plasmid, plasmid to plasmid, etc.); “jumping genes”

Operons, I• Repressible (trp operon): • tryptophan (a.a.) synthesis • promoter: RNA polymerase binding

site; begins transcription • operator: controls access of RNA

polymerase to genes (tryptophan not present)

• repressor: protein that binds to operator and prevents attachment of RNA polymerase - coded from a regulatory gene (tryptophan present - acts as a corepressor)

• transcription is repressed – when tryptophan binds to a

regulatory protein

Unit of genetic function consisting of coordinately related clusters of genes with related functions (transcription unit)

Operons, II

• Inducible (lac operon): - lactose metabolism

• lactose not present: repressor active, operon off; no transcription for lactose enzymes

• lactose present: repressor inactive, operon on; inducer molecule inactivates protein repressor (allolactose)

• Transcription is stimulated when inducer binds to a regulatory protein

Unit of genetic function consisting of coordinately related clusters of genes with related functions (transcription unit)

• Chapter 19 • The Organization and

Control of Eukaryotic Genomes

Chromatin• Def: complex of DNA and proteins• DNA Packing

•histone protein (+ charged amino acids - phosphates of DNA are - charged)

• Nucleosome •”beads on a string”; basic unit of DNA

packing• Heterochromatin

•highly condensed interphase DNA (can not be transcribed)

• Euchromatin•less compacted interphase DNA

(can be transcribed)

Molecular Biology of Cancer

• Oncogene •cancer-causing genes

• Proto-oncogene •normal cellular genes

• How? 1-movement of DNA; chromosome fragments that have rejoined incorrectly 2-amplification; increases the number of copies of proto-oncogenes

3-proto-oncogene point mutation; protein product more active or more resistant to degradation

• Tumor-suppressor genes •changes in genes that prevent uncontrolled cell growth (cancer growth stimulated by the absence of suppression)

• Chapter 20 and 21

BioTechnology & Genomics

O.J. Simpson capital murder case,1/95-9/95

• Odds of blood in Ford Bronco not being R. Goldman’s:• 6.5 billion to 1

• Odds of blood on socks in bedroom not being N. Brown-Simpson’s:• 8.5 billion to 1

• Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson:

• 21.5 billion to 1• Number of people on planet earth:

• 6.1 billion• Odds of being struck by lightning in the U.S.:

• 2.8 million to 1• Odds of winning the Illinois Big Game lottery:

• 76 million to 1 • Odds of getting killed driving to the gas station to buy a lottery ticket

• 4.5 million to 1• Odds of seeing 3 albino deer at the same time:

• 85 million to 1• Odds of having quintuplets:

• 85 million to 1• Odds of being struck by a meteorite:

• 10 trillion to 1

Recombinant DNA

• Definition: DNA in which genes from 2 different sources are linked

• Genetic engineering: direct manipulation of genes for practical purposes

• Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

Bacterial plasmids in gene cloning

DNA Cloning

• Restriction enzymes (endonucleases):in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific

• Restriction site:recognition sequence for a particular restriction enzyme

• Restriction fragments:segments of DNA cut by restriction enzymes in a reproducable way

• Sticky end:short extensions of restriction fragments

• DNA ligase: enzyme that can join the sticky ends of DNA fragments

• Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)

Steps for eukaryotic gene cloning

• Isolation of cloning vector (bacterial plasmid) & gene-source DNA (gene of interest)

• Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase

• Introduction of cloning vector into cells (transformation by bacterial cells)

• Cloning of cells (and foreign genes)• Identification of cell clones carrying

the gene of interest

DNA Analysis & Genomics

• PCR (polymerase chain reaction)

• Gel electrophoresis• Restriction fragment

analysis (RFLPs)• Southern blotting• DNA sequencing• Human genome project

Polymerase chain reaction (PCR)

• Amplification of any piece of DNA without cells (in vitro)

• Materials: heat, DNA polymerase, nucleotides, single-stranded DNA primers

• Applications: fossils, forensics, prenatal diagnosis, etc.

DNA Analysis

• Gel electrophoresis: separates nucleic acids or proteins on the basis of size or electrical charge creating DNA bands of the same length

Restriction fragment analysis

• Restriction fragment length polymorphisms (RFLPs)• Southern blotting: process that reveals sequences and the

RFLPs in a DNA sequence• DNA Fingerprinting

Southern Blotting• Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence

• Southern blotting is a laboratory technique used to detect a specific DNA sequence in a blood or tissue sample. A restriction enzyme is used to cut a sample of DNA into fragments that are separated using gel electrophoresis. The DNA fragments are transferred out of the gel to the surface of a membrane. The membrane is exposed to a DNA probe labeled with a radioactive or chemical tag. If the probe binds to the membrane, then the probe sequence is present in the sample.

Southern Blotting

DNA Sequencing

• Determination of nucleotide sequences (Sanger method, sequencing machine)

• Genomics: the study of genomes based on DNA sequences

• Human Genome Project

Practical DNA Technology Uses

• Diagnosis of disease• Human gene therapy• Pharmaceutical products

(vaccines)• Forensics• Animal husbandry (transgenic

organisms)• Genetic engineering in plants• Ethical concerns?

GENOMICS

AP Biology Chap 21

• Genomes – set of genes and their interactions

• Bioinformatics – computational methods of gene analysis

- NCBI National Center Biotechnology Information – database of DNA sequences and proteins (proteomes)

NCBI HomePage

• The most ambitious mapping project to date has been the sequencing of the human genome

• Officially begun as the Human Genome Project in 1990, the sequencing was largely completed by 2003

• The project had three stages:– Genetic (or linkage) mapping

– Physical mapping

– DNA sequencing

Fig. 21-2-4

Cytogenetic map

Genes locatedby FISH

Chromosomebands

Linkage mapping1

2

3

Geneticmarkers

Physical mapping

Overlappingfragments

DNA sequencing

Fluorescence In Situ Hybridization

• A linkage map (genetic map) maps the location of several thousand genetic markers on each chromosome

• A genetic marker is a gene or other identifiable DNA sequence

• Recombination frequencies are used to determine the order and relative distances between genetic markers

Fig. 21-3-3

Cut the DNAinto overlappingfragments short enoughfor sequencing

1

2

3

4

Clone the fragmentsin plasmid or phagevectors.

Sequence eachfragment.

Order thesequences intoone overallsequencewith computer software.

• A complete haploid set of human chromosomes consists of 3.2 billion base pairs

By summer 2007, genomes had been sequenced for 500 bacteria, 45 archaea, and 65 eukaryotes including vertebrates, invertebrates, and plants

What do we know?

• Humans have 20,488 genes• With alternate gene splicing, we can make

75,000 polypeptides• Genomes of most bacteria and archaea range

from 1 to 6 million base pairs (Mb); genomes of eukaryotes are usually larger

• Free-living bacteria and archaea have 1,500 to 7,500 genes

• Unicellular fungi have from about 5,000 genes and multicellular eukaryotes from 40,000 genes

• Number of genes is not correlated to genome size

• Humans and other mammals have the lowest gene density, or number of genes, in a given length of DNA

Table 21-1

About the human genome…

• Only 1.5% codes for proteins, rRNA and tRNA• The rest is used for • regulatory sequences and introns 24% • pseudogenes (nonfunctioning genes) 15% • repetitive DNA 59%

Fig. 21-7Exons (regions of genes coding for proteinor giving rise to rRNA or tRNA) (1.5%)

RepetitiveDNA thatincludestransposableelementsand relatedsequences(44%)

Introns andregulatorysequences(24%)

UniquenoncodingDNA (15%)

RepetitiveDNAunrelated totransposableelements (15%)

L1sequences(17%)

Alu elements(10%)

Simple sequenceDNA (3%)

Large-segmentduplications (5–6%)

Repetitive DNA• 44% transposable elements (jumping genes) • - Transposons - cut and paste (ex Alu in

primates)• - Most of these are retrotransposons –

cut, copy to RNA, RT to DNA, and paste (ex Line1 or L1)

• 15% – large segment and simple sequence DNA• - small ones STR - Short Tandem Repeats

often used in centromeres and telomeres

Fig. 21-9

TransposonNew copy of transposon

Insertion

Transposonis copied

Mobile transposon

DNA ofgenome

(a) Transposon movement (“copy-and-paste” mechanism)

RetrotransposonNew copy of

retrotransposon

Insertion

Reversetranscriptase

RNA

(b) Retrotransposon movement

Animation Quiz 5 - Transposons: Shifting Segments of the Genome

“Jumping Genes”

• The first evidence for wandering DNA segments came from geneticist Barbara McClintock’s breeding experiments with Indian corn

Fig. 21-8

Genes

• Many eukaryotic genes are present in one copy per haploid set of chromosomes

• More than ½ occur in multigene families – such as for RNA products and hemoglobin

Fig. 21-10

DNARNA transcripts

Nontranscribedspacer Transcription unit

18S

28S

5.8S 28S

5.8S

rRNA

18S

DNA

(a) Part of the ribosomal RNA gene family

Heme

Hemoglobin

-Globin

-Globin

-Globin gene family -Globin gene family

Chromosome 16 Chromosome 11

2

12 1

G A

AdultFetusEmbryoFetus

and adultEmbryo

(b) The human -globin and -globin gene families

Genomic Evolution

• Duplication of chromosome sets (polyploidy)• Chromosome alteration – duplications,

inversions• Exon shuffling• Transposons

• Humans have 23 pairs of chromosomes, while chimpanzees have 24 pairs

• Following the divergence of humans and chimpanzees from a common ancestor, two ancestral chromosomes fused in the human line

Why weAre

Smarter!

• The rate of duplications and inversions seems to have accelerated about 100 million years ago

• This coincides with when large dinosaurs went extinct and mammals diversified

How transposons affect genomes

• Multiple copies may facilitate crossing-over• Insertion may block protein sequence• Insertion may affect promoters• Insertion may carry new genes to an area• May create new sites for alternative splicing in

RNA

Fig. 21-12

Transposableelement

Gene

Nonsisterchromatids

Crossover

Incorrect pairingof two homologsduring meiosis

and

Comparing evolutionary developmental processes

“evo-devo”• Homeobox – 180 nucleotides that regulate

gene expression during development• Found in many organisms, both inverts and

verts• Called “hox genes” in mammals• You should read “Our Inner Fish”!

Fig. 21-17

Adultfruit fly

Fruit fly embryo(10 hours)

Flychromosome

Mousechromosomes

Mouse embryo(12 days)

Adult mouse

• Sometimes small changes in regulatory sequences of certain genes lead to major changes in body form.

• For example, variation in Hox gene expression controls variation in leg-bearing segments of crustaceans and insects

for example, flies with feet in place of antennae.