Genetika I

Silabus

Struktur dan fungsi apparatus genetika

Dogma sentral dan ekspresi gen
Regulasi ekspresi gen
Menganalisa kelainan gen
Macam-macam dan mekanisme mutasi gen
Contoh mutagen
cara untuk memeriksa kelainan gen
Rekayasa genetika

DNA as the genetic material

H. J. Muller postulated that genetic
material, must have three fundamental
properties :
-it must encode information for the
production of compounds that determine
phenotypic features.
-it must be able to replicate.
-it must undergo change that can be
perpetuated.

DNA
Functions
1. Storage of genetic information
2. Self-duplication & inheritance.
3. Expression of the genetic message.
DNAs major function is to code
for proteins.
Information is encoded in the order
of the nitrogenous bases.

DNA
DNA is a nucleic acid, made of repeating subunits
called nucleotides
Nucleotides have three parts: a simple sugar, a
phosphate group, and a nitrogenous base.
Phosphate
group
Nitrogenous
base
Sugar
Phosphate
group

Nitrogenous base
(A, G, C, or T)

Nucleotide

Thymine (T)

Sugar
(deoxyribose)

DNA nucleotide
Polynucleotide

Sugar-phosphate backbone

 DNA has four kinds of bases, A, T, C, and G

Thymine (T)

Cytosine (C)
Pyrimidines

Adenine (A)

Guanine (G)
Purines

The structure of
nucleotides
Base pairing rule:
A always pairs with T
G always pairs with C

A-T
G-C

RNA

Different sugar (Ribose)

U instead of T

Single strand, usually

Nitrogenous base
(A, G, C, or U)
Phosphate
group

Uracil (U)

Sugar
(ribose)

DNA is a double-stranded helix

James Watson and Francis Crick worked out
the three-dimensional structure of DNA,
based on work by Rosalind Franklin

DNA and RNA

Chromosomes

DNA and Chromosomes

Chromosome

E. Coli Bacterium
Bases on the
Chromosomes

Copyright Pearson Prentice Hall

Eukaryotic Chromosome Structure

Chromosome

Nucleosome
DNA
double
helix
Coils

Supercoils

Histones

Copyright Pearson Prentice Hall

Chromosomes
Prokaryotic
Circular
Very small
1 chromosome per
cell
Some enzymes and
proteins are
associated with the
DNA.
Not housed in a
nucleus.

Eukaryotic
Linear
Fairly long
Several
chromosomes per cell.
Histone
proteins---spools.
Same in all
eukaryotes
Housed in a nucleus.

Homologous Pairs
two
copies
of
every chromosome
one from mother
one from father
Homologous pairs
have the same
genes
but
not
always the same
form of the gene

The number of chromosomes

per cell
varies in different species

Genes
Functional
units of DNA
Code for
proteins

Human Genome
Refers to all the
DNA in human
cells
Contains 3 Billion
base pairs
Sequencing
(HGP) completed
April 25th 2003

Genes and Proteins

The sequence of nucleotides in DNA
contain information.
This information is put to work through the
production of proteins.
Proteins
fold
into
complex,
three
dimensional shapes to become key cell
structures and regulators of cell functions.
Thus, by encoding the instructions for
making proteins, DNA controls cells.

The central dogma of

genetics
DNA replication: makes DNA copies that are transmitted
from cell to cell and from parent to
offspring.

Gene

Chromosomal DNA: stores information in

units called genes.

Transcription: produces an RNA copy of a gene.

Messenger RNA: a temporary copy of a gene

that contains information to
make a polypeptide.
Translation: produces a polypeptide using the
information in mRNA.
Polypeptide: becomes part of a functional protein
that contributes to an organism's traits.

DNA Replication

Before a cell divides, it duplicates its DNA

in a process called replication.
Replication ensures that each resulting
cell will have a complete set of DNA.
Each strand of the DNA double helix has
all the information needed to reconstruct
the other half by the mechanism of base
pairing.
Copyright Pearson Prentice Hall

DNA Replication Model

Conservative
proposed both strands of one copy would be
entirely old DNA, while the other copy would
have both strands of new DNA.
Dispersive
dsDNA might fragment, replicate dsDNA, and then
reassemble, creating a mosaic of old and new
dsDNA regions in each new chromosome.
Semiconservative
DNA strands separate, and a complementary
strand is synthesized for each, so that sibling
chromatids have one old and one new strand.
This model was the winner in the Meselson and
Stahl experiment.

Fig. 3.1 Three models for the replication of DNA

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

The Meselson-Stahl experiment

DNA Replication
In most prokaryotes, DNA replication
begins at a single point and continues in
two directions.
In
eukaryotic
chromosomes,
DNA
replication occurs at hundreds of places.
Replication proceeds in both directions
until each chromosome is completely
copied.
The sites where separation and replication
occur are called replication forks.
Copyright Pearson Prentice Hall

DNA Replication

Copyright Pearson Prentice Hall

Initiation of DNA Replication

1. Replication starts at origin of replication
2. Events in initiating DNA synthesis:
a. Gyrase (a type of topoisomerase) relaxes
supercoils in the region.
b. Initiator proteins attach.
c. DNA helicase binds initiator proteins on the
DNA, and denatures the region using ATP as
an energy source.
d. DNA primase binds helicase to form a
primosome, which synthesizes a short RNA
primer.

Initiation of Replication

Events occurring around a single

replication fork

Events occurring around a single

replication fork

DNA Replication
Helicase denaturing DNA causes tighter winding in
other parts of the circular chromosome. Gyrase
relieves this tension.
Single-strand DNA-binding proteins (SSBs) bind the
ssDNA formed by helicase, preventing re-annealing.
Primase synthesizes a primer on each template
strand.
DNA polymerase III adds nucleotides to the 3 end of
the
primer,
synthesizing
a
new
strand
complementary to the template.
Leading strand is synthesized continuously, while
lagging strand is synthesized discontinuously, in the
form of Okazaki fragments. DNA replication is
therefore semi-discontinuous.
Okazaki fragments are gradually joined together to
make a full-length dsDNA chromosome using DNA
polymerase I and DNA ligase

DNA ligase sealing the gap

DNA Replication in
Eukaryotes

DNA replication is very similar in both

prokaryotes and eukaryotes, except that
eukaryotes
have
more
than
one
chromosome.
Each eukaryotic chromosome must be
copied, so both DNA and histones must be
doubled in each cell cycle.

Eukaryotic Replication
Enzymes
DNA polymerases
are known in mammalian

Five
cells:
(alpha) is nuclear, uses RNA primers, is
involved in nuclear DNA replication and has not
been shown to proofread.
(beta) is nuclear, serves in DNA repair and
does not show proofreading activity.
(delta) is nuclear, uses RNA primers, is
involved in nuclear DNA replication and is
capable of proofreading.
(gamma) is mitochondrial, replicating the
mitochondrial DNA using RNA primers and
proofreading.
(epsilon) is nuclear, has proofreading activity
and may be used for DNA repair.

Replicon

The DNA replicated from a single origin

is called a replicon, or replication unit
Replicon size is smaller in eukaryotes
than prokaryotes, and each chromosome
contains many replicons

FTemporal ordering of DNA replication initiation events in replication units

of eukaryotic chromosomes

601

Chapter 3 slide 40

Peter
J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
20000

Replicating the Ends of

Chromosomes
When the ends of chromosomes are replicated
and the primers are removed from the 5 ends,
there is no adjacent DNA strand to serve as a
primer, and so a single-stranded region is left
at the 5 end of the new strand.
If the gap is not addressed, chromosomes
would become shorter with each round of
replication.
This problem compensated by the addition of
the telomere repeats.

The problem of replicating completely a linear

chromosome in eukaryotes

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

Synthesis of telomeric DNA by telomerase

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

Transcription
RNA
polymerase

RNA nucleotide

Direction of
transcription
Template
strand of DNA
Newly made RNA

RNA polymerase

In transcription,
DNA helix unzips
RNA
nucleotides
line up along one
strand
of
DNA,
following the basepairing rules
single-stranded
messenger
RNA
peels away and
DNA strands rejoin

DNA of gene
Promoter
DNA
Initiation

Terminator
DNA

Elongation

Termination
Growing
RNA

Completed RNA
RNA
polymerase

The Stages of Transcription

Transcription occurs in three stages

Initiation
Elongation
Termination

These steps involve protein-DNA interactions

Proteins such as RNA polymerase interact
with DNA sequences

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

DNA of a gene

Transcription

Promoter

Terminator

5 end of growing
RNA transcript
Open complex

RNA polymerase

Initiation: The promoter functions as a recognition

site for transcription factors (not shown). The transcription
factor(s) enables RNA polymerase to bind to the promoter.
Following binding, the DNA is denatured into a bubble
known as the open complex.

Elongation/synthesis of the RNA transcript:

RNA polymerase slides along the DNA
in an open complex to synthesize RNA.

Termination: A terminator is reached that causes RNA

polymerase and the RNA transcript to dissociate from
the DNA.

Completed RNA
transcript

RNA
polymerase

Bases preceding the

start site are
numbered in a
negative direction

Most of the promoter region is

labeled with negative numbers,
play a key role in transcription

There is no base
numbered 0

Coding strand

Transcriptional
start site

Promoter region

35 sequence

16 18 bp

10 sequence

+1

5
T TGA CA
AACTGT

TA TAA T
ATAT TA

A
T

3
Template strand

5
Sometimes termed the
Pribnow box, after its
discoverer
Bases to the right are
numbered in a
positive direction

5
A

RNA

Transcription

Initiation

RNA polymerase is the enzyme that catalyzes

the synthesis of RNA.
In E. coli, the RNA polymerase holoenzyme is
composed of Core enzyme and Sigma factor
The RNA polymerase holoenzyme binds loosely
to the DNA scans along the DNA, until it
encounters a promoter region, When it does, the
sigma factor recognizes both the 35 and 10
regions
The binding of the RNA polymerase to the
promoter forms the closed complex. The open
complex is formed when the TATAAT box in the
-10 region is unwound

A short RNA strand is made within the open

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

RNA polymerase
Promotor region
factor

35

10

After sliding along the DNA,

factor recognizes a promoter, and
RNA polymerase holoenzyme
forms a closed complex.
35

RNA polymerase
holoenzyme

10

Closed complex
An open complex is formed, and
a short RNA is made.
35
10

Open complex
factor is released, and the
core enzyme is able to proceed
down the DNA.
RNA polymerase
35
core enzyme
10

factor
RNA transcript

Elongation

The RNA transcript is synthesized during the

elongation stage
The DNA strand used as a template for RNA
synthesis is termed the template strand
The opposite DNA strand is called the coding
strand,it has the same base sequence as the
RNA transcript Except that T in DNA
corresponds to U in RNA
The open complex formed by the action of RNA
polymerase is about 17 bases long, Behind the
open complex, the DNA rewinds back into a
double helix

On average, the rate of RNA synthesis is about

Termination

Occurs when the short RNA-DNA hybrid of

the open complex is forced to separate
This releases the newly made RNA as well
as the RNA polymerase
E. coli has two different mechanisms for
termination
rho-dependent termination, requires a
protein known as r (rho)
rho-independent
termination,does not
require r

Transcription in Eukaryotes
The basic features of gene transcription are
very similar in bacteria and eukaryotes
Gene transcription in eukaryotes is more
complex
Larger, more complex cells (organelles)
Added cellular complexity means more
genes that encode proteins are required
Multicellularity adds another level of
regulation

Eukaryotic RNA Polymerases

DNA is transcribed by three different RNA

polymerases
RNA
pol I, transcribes all rRNA genes
(except for the 5S rRNA)
RNA pol II
Transcribes
all
structural
genes,
synthesizes all mRNAs
Transcribes some snRNA genes
RNA pol III
Transcribes all tRNA genes
And the 5S rRNA gene

 From Patrick Cramer, David A. Bushnell, Roger D. Kornberg. "Structural Basis of

Transcription: RNA Polymerase II at 2.8 ngstrom Resolution." Science, Vol.
292:5523, 1863-1876, June 8, 2001.

From Seth Darst, Bacterial RNA polymerase.

Current Opinion in Structural Biology.
Reprinted with permission of the author.

(a) Structure of a bacterial

RNA polymerase

Structure of
RNA polymerase

Structure of a eukaryotic
RNA polymerase II (yeast)

3
Transcribed DNA
(upstream)

Exit

Lid

Clamp
Rudder

Entering DNA
(downstream)
3

Wall

Mg2+

5
Bridge

Catalytic
site
NTPs enter
through a pore

(b) Schematic structure of RNA polymerase

Jaw

Transcription

Eukaryotic Structural Genes

Eukaryotic promoter sequences are more variable

and often more complex than those of bacteria
For structural genes, at least three features are
found in most promoters
Regulatory elements
TATA box
Transcriptional start site
Core promoter

TATA box

Coding-strand sequences:

100

DNA

Common location for

regulatory elements such
as GC and CAAT boxes

50

TATAAA

25

Transcriptional
start site

Usually an
adenine

Py2CAPy5

+1

Transcription

Transcription in Eukaryote

Proteins required for basal transcription to occur

at the promoter
RNA polymerase II
Five different proteins called general
transcription factors (GTFs)
A protein complex called mediator

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TFIID binds to the TATA box. TFIID is
a complex of proteins that includes the
TATA-binding protein (TBP) and several
TBP-associated factors (TAFs).
TFIID
TATA box
TFIIB binds to TFIID.
TFIID

TFIIB

TFIIB acts as a bridge to bind

RNA polymerase II and TFIIF.
TFIID

TF
IIB

TFIIF

RNA polymerase II
TFIIE and TFIIH bind to RNA
polymerase II to form a preinitiation
or closed complex.
TF
IIB

Preinitiation complex
TFIIF
TFI
IH

TF
IIE

TFIID

A closed complex

TFIIH acts as a helicase to form an

open complex. TFIIH also phosphorylates
the CTD domain of RNA polymerase II.
CTD phosphorylation breaks the contact
between TFIIB and RNA polymerase II.
TFIIB, TFIIE, and TFIIH are released.

Released after the

open complex is
formed

TFIID
TFIIF
Open complex

TFIIB
TFIIE

RNA pol II can now

proceed to the
elongation stage

TFIIH

PO4
PO4

CTD domain of
RNA polymerase II

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Transcriptional termination
Pre-mRNAs are modified by cleavage near
their 3 end with subsequent attachment of a
string of adenines
Transcription
terminates
500
to
2000
nucleotides downstream from the poly A
signal

Possible mechanisms for Pol II termination

RNA polymerase II transcribes a gene
past the polyA signal sequence.

5
3
PolyA signal sequence
The RNA is cleaved just past the
polyA signal sequence. RNA
polymerase continues transcribing
the DNA.

3
3

Torpedo model: An exonuclease

binds to the 5 end of the RNA
that is still being transcribed and
degrades it in a 5 to 3 direction.

Allosteric model: After passing the polyA signal sequence,

RNA polymerase II is destabilized due to the release of
elongation factors or the binding of termination factors (not
shown). Termination occurs.
5

3
Exonuclease catches
Exonuclease
up to RNA polymerase II
and causes termination.

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Eukaryotic RNA Processing

Exon Intron

Noncoding
segments,
introns,
are
spliced out
A cap and a
tail are added
to the ends

Exon

Intron

Exon

DNA
Cap
RNA
transcript
with cap
and tail

Transcription
Addition of cap and tail

Introns removed

Tail

Exons spliced together

mRNA
Coding sequence
NUCLEUS

CYTOPLASM

Splicing

Three different splicing mechanisms have been

identified
Group I intron splicing
Group II intron splicing
Spliceosome
All three cases involve
Removal of the intron RNA
Linkage of the exon RNA by a phosphodiester
bond

Benefits of Splicing
Allows for genetic recombination
Link exons from different genes together to
create a new mRNA

Also allows for 1 primary transcript to

encode for multiple proteins by
rearrangement of the exons

Capping

Most
mature
mRNAs
have
a
7methylguanosine covalently attached at their
5 end
The 7-methylguanosine cap structure is
recognized by cap-binding proteins
Cap-binding proteins play roles in the
Movement of some RNAs into the cytoplasm
Early stages of translation
Splicing of introns

Tailing

Most mature mRNAs have a string of adenine

nucleotides at their 3 ends. This is termed the
polyA tail
The polyA tail is not encoded in the gene
sequence It is added enzymatically after the
gene is completely transcribed

Polyadenylation signal sequence

5

Consensus
sequence in higher
eukaryotes

AAUAAA
Endonuclease cleavage occurs
about 20 nucleotides downstream
from the AAUAAA sequence.

AAUAAA
PolyA-polymerase adds
adenine nucleotides
to the 3 end.

AAUAAA
Appears to be important in the
stability of mRNA and the
translation of the polypeptide

AAAAAAAAAAAA.... 3
PolyA tail
Length varies between species
From a few dozen adenines to
several hundred

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Eukaryotic vs
Procaryotic

Procaryotic
No CAP; have specific ribosome binding site upstream of
AUG
Polycistronic multiple proteins from same mRNA

Eucaryotic
Monocistronic one polypeptide per mRNA

Expression of
Genes

Some genes are

transcribed
in
large quantities
because we need
large amount of
this protein
Some genes are
transcribed
in
small quantities
because we need
only a small
amount of this
protein

Translation
Translation is the process of turning mRNA
into protein
Translate from one language (mRNA
nucleotides) to a second language
(amino acids)
Genetic code nucleotide sequence that
is translated to amino acids of the protein
Take place at ribosome in cytoplasm

Degener
ate DNA
Code

Nucleotides read 3 at a time meaning that

there are 64 combinations for a codon (set
of 3 nucleotides)
Only 20 amino acids
More than 1 codon per AA degenerate
code with the exception of Met and Trp

Transfer RNA
Molecules

Translation requires an
adaptor molecule that
recognizes the codon
on mRNA and at a
distant site carries the
appropriate amino acid
Intra-strand
base
pairing allows for this
characteristic shape
Anticodon is opposite
from where the amino
acid is attached

Attachment of AA to tRNA
Aminoacyl-tRNA synthase
is
the
enzyme responsible for linking the amino
acid to the tRNA
A specific enzyme for each amino acid
and not for the tRNA

Ribosomal Subunits

1 large subunit catalyzes the formation of the

peptide bond
1 small subunit matches the tRNA to the mRNA
Moves along the mRNA adding amino acids to
growing protein chain

Binding sites
mRNA binding site
Peptidyl-tRNA binding site (P-site), holds
tRNA attached to growing end of the peptide
Aminoacyl-tRNA binding site (A-site), Holds
the incoming AA
Exit site (E-site)

Ribosomal Assembly
Initiation Phase
Initiation factors (IFs) catalyze
the steps not well defined
Step 1 small ribosomal
subunit with the IF finds the
start codon AUG
Moves 5 to 3 on mRNA
Initiator tRNA brings in the 1st AA
which is always Met and then
can bind the mRNA

Step 2 IF leaves and then

large subunit can bind
protein synthesis continues
Met is at the start of every
protein until post-translational
modification takes place

Elongation Phase
Step 1 aminoacyl-tRNA comes
into empty A-site next to the
occupied P-site; pairs with the
codon
Step 2 C end of peptide chain
uncouples from tRNA in P-site and
links to AA in A-site
Peptidyl
transferase
responsible for bond formation
Each AA added carries the
energy for the addition of the
next AA
Step 3 peptidyl-tRNA moves to
the P-site; requires hydrolysis of
GTP
tRNA released back to the
cytoplasmic pool

Protein Release
Protein released when a
STOP
codon
is
encountered (UAG, UAA,
UGA)
Cytoplasmic
release
factors bind to the stop
codon that gets to the Asite; alters the peptidyl
transferase and adds H2O
instead of an AA
Protein released and the
ribosome breaks into the 2
subunits to move on to
another mRNA

Regulation of gene expression

in bacteria
Natural selection has favored bacteria that
produce only the products needed by that
cell
A cell can regulate the production of
enzymes by feedback inhibition or by gene
regulation
Gene expression in bacteria is controlled by
the operon model

Operons: The Basic Concept

A cluster of functionally related genes can be under
coordinated control by a single on-off switch
The regulatory switch is a segment of DNA called an
operator usually positioned within the promoter
An operon is the entire stretch of DNA that includes
the operator, the promoter, and the genes that they
control
The operon can be switched off by a protein
repressor
The repressor prevents gene transcription by binding
to the operator and blocking RNA polymerase
The repressor is the product of a separate regulatory
gene

General structure of an OPERON

Repressible and Inducible

Operons: Two Types of Negative
Gene Regulation
A repressible operon is one that is usually
on; binding of a repressor to the operator
shuts off transcription
The trp operon is a repressible operon
An inducible operon is one that is usually
off; a molecule called an inducer
inactivates the repressor and turns on
transcription

trp
operon
trp operon is

By default the
on and the
genes for tryptophan synthesis are
transcribed
When tryptophan is present, it binds to the
trp repressor protein, which turns the
operon off
The repressor is active only in the
presence of its corepressor tryptophan;
thus the trp operon is turned off
(repressed) if tryptophan levels are high

trp operon
Promoter

Promoter
Genes of operon

DNA

trpE

trpR

Regulatory
gene

trpD

trpC

trpB

trpA

Operator
3

RNA
polymerase

mRNA

Start codon

Stop codon

mRNA 5

5
E
Protein

Inactive
repressor

Polypeptide subunits that make up

enzymes for tryptophan synthesis

(a) Tryptophan absent, repressor inactive, operon on

DNA
No RNA
made
mRNA

Protein

Active
repressor
Tryptophan
(corepressor)

(b) Tryptophan present, repressor active, operon off

The lac
operon
The lac operon
is an inducible operon and

contains genes that code for enzymes

used in the hydrolysis and metabolism of
lactose
By itself, the lac repressor is active and
switches the lac operon off
A molecule called an inducer inactivates
the repressor to turn the lac operon on

Regulatory
gene
DNA

Promoter
Operator

lacI

lacZ
No
RNA
made

3
mRNA

RNA
polymerase

Active
repressor

Protein

(a) Lactose absent, repressor active, operon off

lac operon
DNA

lacI

lacZ

lacY

lacA

RNA polymerase
3

mRNA
5

mRNA 5

-Galactosidase

Protein
Allolactose
(inducer)

Inactive
repressor

(b) Lactose present, repressor inactive, operon on

Permease

Transacetylase

Eukaryotic gene regulation occurs

at several levels

Control at DNA level by DNA

methylation
Small percentages of newly synthesized
DNAs (~3% in mammals) are chemically
modified by methylation.
Methylation
occurs
most
often
in
symmetrical CG sequences.
Transcriptionally active genes possess
significantly lower levels of methylated
DNA than inactive genes.
Methylation results in a human disease
called fragile X syndrome; FMR-1 gene is
silenced by methylation.

Control at DNA level by gene

amplification

Repeated rounds of DNA replication yield

multiple
copies of a particular chromosomal
region.

Control at transcription initiation

By using different sequences (promoter, enhancer
or silencer sequences) and factors, the rate of
transcription of a gene is controlled

gene
X

promoter

Control at mRNA stability

The stem loop at 3 end is an iron response
element.
The stem loop is stabilised by a 90 kDa protein in
the absence of iron and protects the mRNA from
degradation.
In90the
of iron,
transferrin receptor protein
kDa presence
iron sensing protein
(aconitase)
No iron :
synthesis is reduced.
mRNA is
Transferrin receptor
mRNA

AUG

UA
A

+ iron

Transferrin receptor mRNA

Degraded by 3 nuclease

Fe

translated
into
protein

+ iron
stimulates

Control at mRNA stability

Some hormones which enhance the
production of proteins also increase the
half life of the proteins mRNA.
Estrogen : ovalbumin
>24hr
Prolactin : casein
92hr

t1/2 from 2- 5hr to

t1/2 from 5 hr to

Control at initiation of
translation
3 UTR

5 UTR
AUG

UAA

 Peter J. Russel, 2009, iGenetics A

Molecular Approach 3rd Edition
Jack J. Pasternak, 2005, An
Introduction to Human Molecular
Genetics Mechanisms of Inherited
Diseases 2nd Edition, Wiley, New
Jersey.