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: circles of DNA

small

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 cells genome (chromosome to plasmid, plasmid to plasmid, etc.); jumping genes

Operons, I

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

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

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? 1movement 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. Goldmans: 6.5 billion to 1 Odds of blood on socks in bedroom not being N. Brown-Simpsons: 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
Fluorescence In Situ Hybridization

Chromosome bands

Genes located by FISH

Linkage mapping Genetic markers Physical mapping Overlapping fragments

DNA sequencing

 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

1 Cut the DNA into overlapping fragments short enough for sequencing
2 Clone the fragments in plasmid or phage vectors. 3 Sequence each fragment.

4 Order the sequences into one overall sequence with computer

 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. 21-7

Exons (regions of genes coding for protein or giving rise to rRNA or tRNA) (1.5%)

Repetitive DNA that includes transposable elements and related sequences (44%)

Introns and regulatory sequences (24%)

L1 sequences (17%)

Unique noncoding DNA (15%) Repetitive DNA unrelated to transposable elements (15%) Large-segment duplications (56%)

Alu elements (10%) Simple sequence DNA (3%)

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

Transposon DNA of genome

New copy of transposon

Transposon is copied

Insertion

Mobile transposon (a) Transposon movement (copy-and-paste mechanism)

Retrotransposon

New copy of retrotransposon

RNA Insertion Reverse transcriptase

(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 McClintocks breeding experiments with Indian corn

Fig. 21-8

Genes
Many eukaryotic genes are present in one copy per haploid set of chromosomes More than occur in multigene families such as for RNA products and hemoglobin

Fig. 21-10

DNA RNA transcripts Heme -Globin

Nontranscribed spacer

Hemoglobin

Transcription unit
-Globin -Globin gene family Chromosome 16 18S 5.8S 28S -Globin gene family Chromosome 11
G

DNA

rRNA 5.8S

2 1
2 1

28S
18S (a) Part of the ribosomal RNA gene family Embryo Fetus and adult Embryo Fetus Adult

(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 we Are 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

Gene Transposable element Nonsister chromatids Crossover

Incorrect pairing of two homologs during meiosis

and

 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!

Comparing evolutionary developmental processes evo-devo

Fig. 21-17

Adult fruit fly

Fruit fly embryo (10 hours)

Fly chromosome

Mouse chromosomes

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 legbearing segments of crustaceans and insects

for example, flies with feet in place of antennae.