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frederick griffithThe Pneumonia Guy

In 1928, a medical official at the Ministry ofHealth in London, England studied thepossibility of creating a vaccine against a typeof pneumonia called Streptococcus

pneumonia. His name was Frederick Griffith.The disease Frederick was working with was aserious cause of death in this period of time.

Griffiths experiments contained two strains

of a type of bacteria commonly calledpneumococci, which mainly caused his killerdisease. One of the two strains was deadly tohumans and the other was harmless.

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The only physical difference Griffith could find was that the deadly strain had asmooth-coated surface made of sugar surrounding it, and the harmless strain hada rough-coated surface with nothing protecting it. The reason the sugar-coatedstrain was so lethal was because the human white blood cells had trouble

penetrating the smooth, sugary coat.When testing his experiments, Griffith used lab mice to prove his theories. First,

Griffith injected the smooth-coated strain into the mice. As expected, they allcaught pneumonia and died. Then, Griffith tried to kill some of the deadly strainwith heat and injected it into the mice. Amazingly, the mice didnt getpneumonia. Next, he injected the rough-surfaced bacteria into the

mice.

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Griffith's experiment discovering a "transforming principle" in heat-killed virulent smoothpneumococcus that enables the transformation of rough non-virulent rough pneumococcus.

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Bacteria have no sexual reproduction in the

sense that eukaryotes do. The

haveno alternation of diploid and haploid

generations

no gametesno meiosis

But the essence of sex is genetic

recombination, and bacteria do have three

mechanisms to accomplish that:

transformation

conjugationtransduction

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Transformation

Many bacteria can acquire new genes by

taking up DNA molecules (e.g., a plasmid) fromtheir surroundings. The ability to deliberately

transform the bacterium E. coli has made

possible the cloning of many genes

including human genes and the

development of the biotechnology industry.

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Streptococcus pneumoniae (pneumococci)

growing as colonies on the surface of a culture

medium. Left: The presence of a capsulearound the bacterial cells gives the colonies a

glistening, smooth (S) appearance. Right:

Pneumococci lacking capsules have produced

these rough (R) colonies. (Courtesy of Robert

Austrian,J. Exp. Med. 98:21, 1953.)

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The first demonstration of bacterial transformation was done with Streptococcus

pneumoniae and led to the discovery that DNA is the substance of the genes. The path

leading to this epoch-making discovery began in 1928 with the work of an English

bacteriologist, Fred Griffith.

The cells ofS. pneumoniae (also known as the pneumococcus) are usually surroundedby a gummy capsule made of a polysaccharide. When grown on the surface of a solid

culture medium, the capsule causes the colonies to have a glistening, smooth

appearance. These cells are called "S" cells. However, after prolonged cultivation on

artificial medium, some cells lose the ability to form the capsule, and the surface of

their colonies is wrinkled and rough ("R"). With the loss of their capsule, the bacteria

also lose their virulence. Injection of a single S pneumococcus into a mouse will kill themouse in 24 hours or so. But an injection of over 100 million (100 x 106) R cells is

entirely harmless.

The reason? The capsule prevents the pneumococci from being engulfed

(by phagocytosis) and destroyed by the scavenging cells

neutrophils and macrophages of the body. The R forms are completely at the mercy

of phagocytes.

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(S. pneumoniae apanhados emflagrante

delicto)

Streptococcus pneumoniae

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Streptococcus_pneumoniae.jpg

Scanning ElectronMicrograph of Streptococcus

pneumoniae. Pneumococcus, Streptococci

http://upload.wikimedia.org/wikipedia/comm

ons/2/20/Streptococcus_pneumoniae.jpg

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Chlamydiae

Chlamydiae are also obligate intracellular parasites (they cannot

make their own ATP).Chlamydia trachomatisIts genome contains

1,042,519 bp of DNA encoding 938 genes.

In 1998, this organism infected 604,420 people in the U. S. The

infection is usually spread by sexual intercourse making it the

most common sexually-transmitted disease (STD). It is easily cured

if diagnosed, but many infections remain untreated and, in

females, are a major cause ofpelvic inflammatory disease. This

causes scarring of the uterus and fallopian tubes and often results

in infertility.Mothers can pass the infection on to their newborn

babies causing serious eye disease and pneumonia. To avoid this,

pregnant women are usually tested for chlamydia and treated

with antibiotics if they are infected.

Several million people in the desert regions of Asia, Africa, and the

Near East have been blinded by trachoma. This eye infection is

caused by a strain of C. trachomatis (and is responsible for its

name).Chlamydia psittaci usually infects birds, but can infect their

human contacts causing psittacosis (a.k.a. ornithosis).

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(a) Scanning electron microscope (SEM) ofStaphylococci(b) Staphylococci haemolysis on

blood agar(BA) (c) Electron microscope picture

ofS. aureus

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(a) Mannitol salt agar (b) Nutrient agar (c) Blood agar

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(a) SEM ofStaphylococcus aureus (b) Electron

microscope (EM) ofVibrio cholera (c) EM of

Streptococcus pneumoniae (d) Ourgroup's

sample location (e) Group 12'ssample location(f) Group 20'ssample location

Staphylococcus aureusVibrio cholera Streptococcus pneumoniae

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transformation

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Figure 362. Model for the structure of the

nucleosome,

in which DNA is wrapped around the surface ofa

flat protein cylinder consisting of two each of

histones

H2A, H2B, H3, and H4 that form the histone

octamer.

The 146 base pairs of DNA, consisting of 1.75superhelical

turns, are in contact with the histone octamer.

This

protects the DNA from digestion by a nuclease.

The position

of histone H1, when it is present, is indicatedby

the dashed outline at the bottom of the figure.

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Figure 363. Shown is the extent of DNA

packaging in metaphase chromosomes (top)

to noted duplex DNA (bottom).Chromosomal DNA is packaged and organized

at several levels as shown (see Table 362).

Each phase of condensation

or compaction and organization (bottomto

top) decreasesoverall DNA accessibilityto an

extentthatthe

DNA sequences in metaphase chromosomes

are almost totally transcriptionally inert. In

toto, these five levels of

DNA compaction result in nearly a 104-fold

linear decrease in end-to-end DNA length.

Complete condensation and

decondensation of the linear DNA in

chromosomes occur in the space of hours

during the normal replicative cell

cycle (see Figure 3620).

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Figure 365. The two sister chromatids of

human chromosome 12 (. 27,850). The

locationof the A+T-rich centromeric region connecting

sister chromatids is indicated, as are two of the

four telomeres residing at the very ends of the

chromatids that are attached one to the other

at

the centromere. (Modified and reproduced,with

permission, from DuPraw EJ: DNA and

Chromosomes.

Holt, Rinehart, and Winston, 1970.)

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Figure 366. A human karyotype (of a man

with a normal 46,XY constitution), in

which the metaphase chromosomes have been

stained by the Giemsa method and

aligned according to the Paris Convention.

(Courtesy of H Lawce and F Conte.)

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Figure 367. The relationship between

chromosomal DNA and mRNA. The human

haploid DNA complement of 3 . 109 base pairs

(bp) is distributed between 23 chromosomes.Genes are clustered on these chromosomes.

An average gene is 2 . 104 bp in

length, including the regulatory region

(hatched area), which is usually located at the

5

end of the gene. The regulatory region is

shown here as being adjacent to the

transcription

initiation site (arrow).Most eukaryotic genes

have alternating exons and introns. In

this example, there are nine exons (dark blue

areas) and eight introns (light blue areas).

The introns are removed from the primary

transcript by the processing reaction, and the

exons are ligated together in sequence to form

the mature mRNA. (nt, nucleotides.)

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DNAREPLICATION begins at a defined origin, is bidirectional, and is semiconservative

(one new chain, one old chain in daughter DNA), and chain growth

occurs in the 5 to 3 direction.

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Hierarchical Organization of a Multicel-

lular Organism: The Human Being

Multicellular organisms have several levelsof organization: Organ systems, organs, tis-

tissues, cells, organelles, molecules, and

atoms. The digestive system and one of its

component organs (the liver) are shown.

The liver is a multifunctional organ that has

several digestive functions. For example, itproduces bile, which facilitates fat diges-

digestion, and it processes and distributes the

food molecules absorbed in the small intes-

intestine to other parts of the body. DNA, one

molecule found in cells, contains the genetic

information that controls cell function.

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DNA

(a) A diagrammatic view of

DNA. The sugar-phosphate

backbones of the double helix

are represented by colored

ribbons. The bases attached

to the sugar deoxyribose are on

the inside of the helix, (b) An

enlarged view of two base

pairs. Note that the two DNA

strands run in opposite

directions defined by the 5' and

3' groups of deoxyribose. Thebases on opposite strands form

pairs because of hydrogen

bonds. Cytosine

always pairs with guanine;

thymine always pairs with

adenine.

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Membranes in Living Cells Membranes are an

essential feature of living cells. Most

biochemical

processes occur in or near these dynamic and

complex supramolecular structures.

Cells are the structural units of all living

organisms. One remarkable feature of cells is

their diversity. For example, the human body

contains about 200 types of cells. This

great variation reflects the variety of functions

that cells can perform. However, no

matter what their shape, size, or species, cells

are also amazingly similar. They are allsurrounded by a membrane that separates

them from their environment. They are all

composed of the same types of molecules.

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Homologous sequences. Orthologs and

Paralogs are two types of homologous

sequences. Orthology describes genes in

different species that derive from a common

ancestor. Orthologous genes may or may not

have the same function. Paralogy describes

homologous genes within a single species that

diverged by gene duplication.

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Diverse living organisms share common

chemical features.

Birds, beasts, plants, and soil microorganismsshare with humans

the same basic structural units (cells) and the

same kinds of

macromolecules (DNA, RNA, proteins) made

up of the same kinds of

monomeric subunits (nucleotides, aminoacids). They utilize the same

pathways for synthesis of cellular components,

share the same genetic

code, and derive from the same evolutionary

ancestors. Shown here

is a detail from The Garden of Eden, by Janvan Kessel the Younger

(16261679).

or nucleoid (bacteria)

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or nucleoid (bacteria)

Contains genetic materialDNA and

associated proteins. Nucleus is

membrane-bounded.

Plasma membrane

Tough, flexible lipid bilayer.Selectively permeable to

polar substances. Includes

membrane proteins that

function in transport,

in signal reception,

and as enzymes.Cytoplasm

Aqueous cell contents and

suspended particles

and organelles.

Supernatant: cytosol

Concentrated solutionof enzymes, RNA,

monomeric subunits,

metabolites,

inorganic ions.

Pellet: particles and organelles

Ribosomes, storage granules,

mitochondria, chloroplasts, lysosomes,

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The universal features of living cells. All

cells have a

nucleus or nucleoid, a plasma

membrane, and cytoplasm. The cytosol

is defined as that portion of the

cytoplasm that remains in the

supernatant

after centrifugation of a cell extract at

150,000 gfor1 hour.

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Bacterial PU1

In this electron micrograph, a sex pilus con-connects two conjugating E. coli cells. Note the

numerous smaller pili covering the surface

of one of the cells.

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ExocytosisProteins produced in the ER and

processed by the Golgi apparatus are

packaged into vesi-

vesicles that migrate to the plasma

membrane and merge with it.

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Receptor-Mediated Endocytosis

(a) Extracellular substances may enter the cell during

endocytosis, a process in which receptor molecules in the

plasma membrane bind to the

specific molecules or molecular complexes called ligands.Specialized regions of plasma membrane called coated pits

progressively invaginate to

form closed vesicles. After the coat proteins are removed,

the vesicle fuses with an early endosome, the precursor of

lysosomes. The coat proteins

are then recycled to the plasma membrane. Duringendosomal maturation, the proton concentration rises and

the ligands are released from their

receptors which are subsequently also recycled back to the

plasma membrane. As endosomal maturation continues,

lysosomal hydrolases are

delivered from the Golgi apparatus. Lysosomal formation iscomplete when all the hydrolases have been transferred to

the late endosome and the

Golgi membrane has been recycled back to the Golgi

apparatus, (b) Electron micrographs illustrating the initial

events in endocytosis.

FIGURE Z Z4

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FIGURE Z.Z4

TheMitochondrion

(a)Membranes and crista. (b) Mitochondria

from adrenal cortex.

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Replication of a Mitochondrion by Binary Fission

1.Mitochondria and chloroplasts are similar in size to many

modern prokaryotes.

2. These two organelles reproduce by binary fission, as do bac-

bacteria and the archaea (Figure 2A).

3. The genetic information (DNA) and the protein-synthesizing

capability of mitochondria and chloroplasts are similar to

those of prokaryotes. For example, both mitochondrial and

chloroplast DNA are circular and "naked" (i.e., not com-

plexed with histone proteins as nuclear DNA is). (There is

insufficient genetic information on these chromosomes to

account for all organelle components. However, the nuclear

genes that are responsible for synthesis of mitochondrial

components resemble prokaryotic genes.)

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FIGURE 3.1 O

The Hydrophobic Effect.When nonpolar molecules and water are

mixed, a solvation sphere composed of many

lay-

layers of highly ordered hydrogen-bonded

water molecules forms around the

hydrophobic mol-molecules. Although nonpolar molecules,

when in close proximity, are attracted to each

other

by van der Waals forces, the driving force in

the formation of the solvation spheres is the

strong tendency of water molecules to form

hydrogen bonds among themselves. Nonpolar

molecules are excluded because they cannot

form hydrogen bonds.

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8. Stress response. The capacity of living

organisms to survive a variety of

abiotic stresses is mediated by certain

proteins. Examples include cytochrome

P450, a diverse group of enzymes found in

animals and plants that usually con-

convert a variety of toxic organic contaminants

into less toxic derivatives, and met-

allothionein, a cysteine-rich intracellular

protein found in virtually all mammalian

cells that binds to and sequesters toxic metalssuch as cadmium, mercury, and sil-

silver. Excessively high temperatures and other

stresses result in the synthesis of a

class of proteins called the heat shock proteins

(hsps) that promote the correct

refolding of damaged proteins. If such proteinsare severely damaged, hsps pro-

promote their degradation. (Certain hsps

function in the normal process of protein

folding.) Cells are protected from radiation by

DNA repair enzymes.

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Phage T2 is a virus that infects the bacterium E.

coli. When phage particles are added to

bacteria, they adsorb to the outside surface,some material enters the bacterium, and then

~20 minutes later each bacterium bursts open

(lyses) to release a large number of progeny

phage

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Lunar excursion module (LEM) ofApollo 16: courtesy NASA

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In a first experiment, they labelled the

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p , y

DNA of phages

withradioactive Phosphorus-32 (the

element Phosphorus is present in DNAbut not present in any of the 20 amino

acids from which proteins are made).

They allowed the phages to infect E.

Coli, then removed the protein shellsfrom the infected cells with a blender

and a centrifuge. They found that the

radioactive tracer was visible only in the

bacterial cells and not in the proteinshells.

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In a second experiment, they labelled the

phages with radioactive Sulfur-35 (Sulfur is

present in the amino

acids Cysteine andMethionine, but not in

DNA). After separation, the radioactive tracer

then was found in the protein shells, but not in

the infected bacteria, confirming that the

genetic material which infects the bacteria is

DNA.

Hershey shared the 1969 Nobel Prize in

Physiology orMedicine for his discoveries

concerning the genetic structure of viruses.

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The Hershey-Chase experiments were a series

ofexperiments conducted in1952 by AlfredHershey andMartha Chase, confirming

that DNA was thegenetic material, which had

first been demonstrated by Oswald

Avery in 1944.

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Alfred Hershey andMartha Chase

Alfred Hershey andMartha Chase

Independent Functions of Viral Protein

and Nucleic Acid in Growth ofBacteriophage. September 20, 1952.

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Life Cycle:

The virus attaches to the E coli cell (a) This requires a precise molecular interaction

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The virus attaches to the E. coli cell (a). This requires a precise molecular interaction

between the fibers and the cell wall of the host.

The DNA molecule is injected into the cell (b).

Within 1 minute, the viral DNA begins to be transcribed and translated into some of

the viral proteins, and synthesis of host proteins is stopped.At 5 minutes, viral enzymes needed for synthesis of new viral DNA molecules are

produced (c).

At 8 minutes, some 40 different structural proteins for the viral head and tail are

synthesized.

At 13 minutes, assembly of new viral particles begins (d).

At 25 minutes, the viral lysozyme destroys the bacterial cell wall and the viruses burstout ready to infect new hosts (e).

If the bacterial cells are growing in liquid culture, it turns clear.

If the bacterial cells are growing in a "lawn" on the surface of an agar plate, then

holes, called plaques, appear in the lawn

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Bacteriophage.jpg

The contractile tail sheath of phage T4

ofEscherichia coli, one of the best understood

phages. This computer-generated image is

based on the data of J. Lepault and K. Leonard

(46) and was generated by StevenMcQuinn.

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Bacteriophage phi 29 ...

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VIRIONS are virus particles: they are the INERT

CARRIERS of the genome, and

are ASSEMBLED inside cells, from virus-

specified components: they do not GROW,

and do not form by DIVISION

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