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BSci 121M.

CYTOGENETICS
UNIVERSITY OF SAN AGUSTIN
COLLEGE OF PHARMACY AND MEDICAL TECHNOLOGY

Cyto = Cell
Genes= Elements of heredity (carrying inherited traits) that are transmitted from
parents to offspring during reproduction .
Genetics= The study of biologically inherited traits./ The study of genes.
Genomics= The study of all the genes in an organism to understand their
molecular organization, function, interaction and evolutionary history.
Chapter 1.The Genetic Code of Genes & Genomes
History:
1860s- Existence of genes and the rules governing their transmission from
generation to generation were discovered by Gregor Mendel. His work with
garden peas represents the beginning of what would become the science of
Genetics.
1869- Friedrich Miescher discovered a new type of weak acid abundant in the
nuclei of WBCs that turned out to be the chemical substance of which genes are
made. These acid would later be known (until today) as Deoxyribonucleic acid.
1870s- The nuclei of the male and female reproductive cells were observed to fuse
in the process of fertilization. This later led to the discovery of thread-like objects
with characteristic splitting behavior inside the nucleus that become visible in the
light microscope when stained with dyes. These would later be known
as
Chromosomes.
1900s- Chromosomes are indeed carriers of genes.
1920s- DNA and other various types of proteins are present in chromosomes.
DNA- Deoxyribonuleic acid. Considered as the Molecule of Heredity.
- According to studies by James Watson and Francis Crick at Cambridge
University (1953), DNA consists of two long chains of subunits twisted
around one another to form a double-stranded helix.
- The subunits of each strand are called nucleotides, each of which
contains any one of four chemical constituents called bases (Adenine;
Thymine; Guanine and Cytosine).
- The base pairing between A and T and between G and C is said to be
complementary base pairing; the complement of A is T, and the
complement of G is C. The complementary pairing in the duplex molecule
means that each base along one strand of the DNA is matched with a base
in the opposite position on the other strand.
- Each DNA strand has a polarity or directionality, like a chain of circus
elephants linked trunk to tail. In this analogy, each elephant corresponds
to one nucleotide along the DNA strand. The trunk end of the strand is
called the 5 end of the strand, and the tail end is called the 3 end. In
double stranded DNA, the paired strands are oriented in opposite
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directions: The 5end of one strand is aligned with the 3 end of the other.
The oppositely oriented strands are said to be Antiparallel.
Replication- The copying process in which a single DNA molecule becomes two
identical molecules. In
this process, each parental DNA strand directs the synthesis of a new
partner strand.
For most genes, the genetic information contained in the nucleotide
sequence specifies a particular type of protein. Proteins control the chemical
and physical processes of cells known as metabolism. Many proteins are
enzymes , a term introduced in 1878 to refer to the biological catalysts that
accelerate biochemical reactions.
Archibald Garrod- A British physician who studied genetic diseases caused by
inherited defects in
metabolism. He concluded that an inherited defect in metabolism results
from an inherited defect in an enzyme.
- Example: PKU (Phenylketonuria) results from the absence of (or a defect
in) the enzyme Phenylalanine hydroxylase (PAH). When this step in the
pathway is blocked, phenylalanine accumulates. The excess phenylalanine
is broken down into harmful metabolites that cause defects in myelin
formation that damage a childs developing nervous system and lead to
severe mental retardation.
The Central Dogma of Molecular Genetics
The details of how genes code for proteins were not understood until the 1960s
when the Dogma was formulated. Since then, it has become the fundamental
principle of molecular genetics because it summarizes how the genetic information
in DNA becomes expressed in the amino acid sequence in a polypeptide chain.

DNA

transcription

RNA

translation

PROTEIN

The main concept in the central dogma is that DNA does not code for protein
directly but rather acts through an intermediary molecule of Ribonucleic acid. The
structure of RNA is similar to, but not identical with, that of DNA.

Features
1. Sugar
2. Strandedness

DNA
Deoxyribose
Double stranded
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RNA
Ribose
Single stranded

3. Base

Thymine

Uracil

Three Types of RNA:


1.) mRNA- carries the genetic information from DNA and is used as a template
for polypeptide synthesis.
2.) rRNA- the major constituents of the cellular particle Ribosomes on which
polypeptide synthesis takes place.
3.) tRNA- the carriers of particular amino acids for polypeptide formation. As
each tRNA participates in translation, its amino acid becomes the terminal
subunit of the growing polypeptide chain.
Transcription- is the production of an RNA strand that is complementary in base
sequence to a DNA strand (template).
Translationmolecule.

the synthesis of a polypeptide under the direction of an mRNA

Examples of Standard Genetic Code (which comes in groups of 3 bases or CODON):


CUA- Leucine
GUU- Valine
CGG- Arginine

Chapter 2. Transmission Genetics: Heritage from Mendel


Transmission genetics- the study of the patterns of inheritance from generation
to generation. In eukaryotic organisms, transmission genetics is often called
Mendelian genetics.
Gregor Mendel
- Realized that each parent contributed to its progeny a number of separate
and distinct elements of heredity (factors as he called them; in modern times
genes)
- He also realized that each of these parental factors remain unchanged as it
was passed from one generation to the next.
- He selected peas for his experiments for two reasons:
1.) He had access to varieties that differed in observable alternative
characteristics (round vs wrinkled seeds, yellow vs green seeds)

2.) His preliminary studies had indicated that peas normally reproduce by
self- fertilization, in which pollen produced in a flower is used to fertilize the eggs in
the same flower. Left alone, pea flowers always self- fertilize .
- He did a crossbreeding between two different varieties by opening the keel
petal (which encloses the reproductive structures), removing the immature anthers
(the pollen- producing structures) before they shed pollen, and dust the stigma(part
of the female structure) with mature pollen taken from a flower on a different plant.
- He established true- breeding varieties in which the plants produced only
progeny like themselves when allowed to self- fertilize. (Ex. One true- breeding
variety always yielded round seeds, whereas another true- breeding variety always
yielded wrinkled seeds.)
-For his experiments, Mendel chose seven pairs of varieties, each of which
was true- breeding for a different trait. The contrasting traits affected: seed shape
(round vs wrinkled), seed color (yellow vs green), flower color (purple vs white), pod
shape (smooth vs constricted), pod color (green vs yellow), flower and pod position
(axial vs terminal) and stem length (standard vs dwarf). When two varieties that
differ in one or more traits are crossed, the progeny constitute a hybrid between
the parental varieties. Crosses in which the parental varieties differ in one, two or
three traits of interest are called: Monohybrid, Dihybrid and Trihybrid
respectively.
Geneticists call the true- breeding parents the P1 generation and the hybrid
filial seeds or plants the F1 generation.
One pair of traits studied was round vs wrinkled seeds. When pollen from a
variety of plants with wrinkled seeds was used to cross- pollinate plants from a
variety with round seeds, all of the resulting hybrid seeds were round. When plants
from the variety with round seeds were used as the pollen parents and those from
the variety with wrinkled seeds as the female parents (reciprocal cross), all of the F1
seeds turned out to be round.
Generalizations:
1.) The Traits expressed in the hybrids were called the Dominant traits;
while the traits not expressed in the hybrids were called Recessive
traits.
2.) Two plants with the same outward appearance (for example with round
seeds) might nevertheless differ in their hereditary makeup.
3.) The normal gene encodes an enzyme, starch-branching enzyme I (SBEI),
required to synthesize a branched chain form of starch known as
Amylopectin. As pea seeds dry, they lose water and shrink. Round seeds
contain amylopectin and shrink uniformly.Wrinkled seeds lack amylopectin
and shrink irregularly. In other words, wrinkled peas have an inborn error
in starch metabolism. The molecular basis of the wrinkled mutation is that
the SBEI gene has become interrupted by the insertion of aDNA sequence
called a transposable element. These are DNA sequences that are
capable of moving (transposition) from one location to another within a

chromosome or between chromosomes. Many spontaneous mutations


result from the insertion of transposable elements into a gene.
Terminologies deduced from Mendels experiment:
1. Gene- hereditary determinant of a trait
2. Alleles- the different forms of a particular gene. (Ex. The alleles of the
gene for seed shape are W for round seeds and w for wrinkled seeds. W
and w are alleles because they are alternative forms of the gene for seed
shape. Alternative alleles are typically represented by the same letter or
combination of letters, distinguished either by upper case vs lower case or
by means of superscripts or subscripts or some other typographic
identifier.)
3. Genotype- the genetic constitution of an organism or cell- its molecular
makeup. With respect to seed shape in peas, WW, Ww and ww are
examples of the possible genotypes for the W and w alleles. Because
gametes contain only one allele of each gene, W and w are examples of
genotypes of gametes.
4. A genotype in which the members of a pair of alleles are different, as in
the Ww hybrids, is said to be heterozygous. A genotype in which the
two alleles are alike is said to be homozygous. A homozygous organism
may be homozygous dominant (WW) or homozygous recessive (ww). The
terms homozygous and heterozygous can not apply to gametes because
gametes contain only one allele of each gene.
5. The observable properties of an organism including its visible traits
constitute its phenotype. Round seeds and wrinkled seeds are
phenotypes. So are yellow seeds and green seeds. The phenotype of an
organism does not necessarily imply anything about its genotype. For
example, a seed with the phenotype round could have either the
genotype WW or the genotype Ww.
6. A dominant trait is that expressed in the phenotype when the genotype is
either heterozygous or homozygous. A recessive trait is that expressed in
the phenotype when a genotype is homozygous for the alternative allele.
The presence of a dominant trait masks a recessive trait.
7. Wildtype form- most common form of a trait occurring in a natural
population. (Represented as W., ex the round peas.)
8. Mutant form- any form that differs from the wildtype.(Ex. The wrinkled
peas).
A way to identify the W and w forms of gene is the procedure gel
electrophoresis. It is used for separating DNA molecules of different sizes.
Samples containing relatively small fragments of duplex DNA are placed into slots
near one edge of a slab of a jelly-like material (agarose) which is then submerged in
a buffer solution and subjected to an electric field. DNA fragments in the samples
move in response to the electric field in accordance with their lengths. Shorter
fragments move faster and farther than long fragments. The W fragment moves
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farther than the w fragment because the w fragment is larger owing to the insertion
of the transposable element. The separation of the fragments is indicated by the
dark rectangles called bands.
9. Morphological trait- one that is manifest, plainly shown, and readily
perceived by the senses. Frequently dominant or recessive.
10.Molecular trait- one that can be perceived only by means of special
methods(such as gel electrophoresis)that enable differences between
molecules to be visualized. Often (but not always) codominant.
11.Codominant genes- Alternative forms of a gene (W and w) which can
both be detected when they are present in a cell or organism using
special methods (e.g. gel electrophoresis showing single rapidly migrating
band of the true- breeding strain with round seeds; and the single slowly
migrating band of the true-breeding strain with wrinkled seeds; and the
progeny of the cross which has round seeds BUT exhibit BOTH bands ).

Mendelss hypothesis of genetic transmission:


Genes are physical entities that come in pairs, separate in gametes and
join randomly in fertilization.
In the first generation of hybrids, the recessive visible trait disappeared,
only to reappear in the next generation, after the hybrid progeny were allowed to
undergo a self fertilization. The progeny seeds produced by self- fertilization of the
F1 generation constitute the F2 generation. Mendel found that the dominant and
recessive traits appear in the F2 progeny in the proportions 3 round: 1 wrinkled
(ratio of dominant: recessive is 3:1).
The recessive trait that seemingly disappeared in the F1 generation
reappeared again in the F2 generation. Not only did the recessive trait reappear, it
was in no way different from the trait present in the recessive P1 plants. Thus,
Mendel concluded that the hereditary determinants for the traits in the
parental lines were transmitted as two different elements that retain their
purity in the hybrids. In other words, the hereditary determinants do not mix
or contaminate each other.
Mendelss hypothesis of genetic transmission:
1.) Each reproductive cell (or gamete) contains one representative of each kind
of hereditary determinant in the plant.
2.) When an F1 plant is self- fertilized, the W and the w determinants separate
from one another and are included in the gametes in equal numbers. This
separation of the hereditary elements is the heart of Mendelian genetics. The
principle is called Segregation. The hereditary determinants are
completely unaltered after separation by their having been paired in the
previous generation.
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3.) The gametes produced by segregation come together in pairs at random


(subject to chance variation) to yield the progeny of the next generation.
Testcross- A cross between an organism of dominant phenotype and an organism
of recessive phenotype (homozygous recessive) which yielded a progeny with Ww
and ww ratio of 1:1.

The Human ABO blood groups illustrate both Dominance and codominance.
Incomplete dominance- the phenotype of the heterozygous genotype is
intermediate between those of the homozygous genotypes. It is more frequent for
morphologic traits than for molecular traits. (Ex. The color pink of snapdragon
flower that is formed is intermediate between red and white from the parent
flowers.)
Codominance- the heterozygous genotype exhibits the traits associated with
both homozygous genotypes. It is more frequent for molecular traits than for
morphologic traits.
ABO blood groups- determined by polysaccharides (polymers of sugars) present on
the surface of red blood cells. Both the A and B polysaccharides are formed from a
precursor substance that is modifeied by the enzyme product of either the IA or the
IB allele. The gene products are transferase enzymes that attach either of two types
of sugar units to the precursor.
People of genotype IAIA- produce RBCs having only the A polysaccharide and
are said to have blood typeA.
People of genotype IBIB- produce RBCs having only the B polysaccharide and
are said to have blood type B.
Heterozygous IAIB people have RBCs with both A and B polysaccharides and
are said to have blood type AB. This genotype illustrates codominance because
heterozygous genotype has the characteristic of both homozygous genotypes- in
this case, the presence of both the A and the B carbohydrate on the RBCs. Although
the polypeptides encoded by the IA and the IB differ in only 4 out of 355 amino
acids, these differences are at strategic positions in the molecules and change their
substrate specificity. Both the IA and the IB are dominant to the recessive allele IO.
The IO allele has a single base deletion in codon 86 that shifts the translational
reading frame of the mRNA resulting in an incomplete, inactive enzyme. The
precursor substrate remains unchanged and neither the A nor the B type of
polysaccharide is produced.
People of genotype IOIO therefore lack both the A and the B polysaccharide
and thus said to have blood type O.
In IAIO heterozygotes, presence of the IA allele results in production of the A
polysaccharide, and correspondingly in IBIO heterozygotes, presence of the IB allele
results in production of the B polysaccharide. Thus persons with IAIO have blood
type A and persons with IBIO have blood type B.
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Chapter 3. The Chromosomal Basis of Heredity

Mitosis
Meiosis

The Sex chromosomes an exception to the rule that all chromosomes of diploid
organisms are present in pairs of morphologically similar homologs. As early as
1891, microscopic analysis showed that one of the chromosomes in males of some
insects (grasshoppers) does not have a homolog. This unpaired chromosome was
called the X chromosome and it was present in all somatic cells of the males but in
only half the sperm cells. The biological significance of these observations became
clear when females of the same species were shown to have two X chromosomes.
In other species in which the females have two X chromosomes, the male has
one X chromosome along with a morphologically different chromosome, later known
to be the Y chromosome. The difference in the chromosomal constitution of males
and females is a chromosomal mechanism for determining sex at the time of
fertilization. Whereas every egg cell contains an X chromosome, half the sperm cells
contain an X chromosome and the rest contain a Y chromosome. Fertilization of an
X- bearing sperm results in an XX zygote which normally develops into a female;
and fertilization by a Y- bearing sperm results in an XY zygote, which normally
develops into a male. The result is a criss- cross pattern of inheritance of the X
chromosome in which a male receives his X chromosome from his mother and
transmits it only to his daughters.

Hemophilia A- a classic example of a human trait with an X-linked pattern of


inheritance.
- A severe disorder of blood clotting determined by a recessive allele.
- Affected persons lack a blood-clotting protein called factor VIII that is
needed for normal clotting, and they suffer excessive, often life
threatening bleeding after injury.
X-linked inheritance in human pedigrees shows several characteristics that
distinguish it from other modes of genetic transmission:
1. For any rare trait due to an X-linked recessive allele, the affected individuals
are exclusively, or almost exclusively, male. There is an excess of affected
males because females carrying the rare X-linked recessive allele are almost
exclusively heterozygous and so do not express the mutant phenotype.
2. Affected males who reproduce have normal sons. This follows the fact that a
male transmits his X chromosome only to his daughters.

3. A woman whose father was affected has normal sons and affected sons in the
ratio of 1:1. This is true because any daughter of an affected male must be
heterozygous for the recessive allele.

Chapter 5. Human Chromosomes and Chromosome Behavior

In most species, organisms with an extra chromosome or a missing chromosome


usually have developmental or other types of abnormalities. Some organisms are
found to have a variation in chromosome structure (missing segment, duplicated,
reversed in orientation or attached to a different chromosome.) Generally speaking,
animals are much less tolerant of chromosomal changes than are plants.
- Human beings have 46 chromosomes in 23 pairs.
Chromosome painting - technique in labeling chromosomes. Different colors are
painted on each chromosome by hybridization (formation of duplex molecules) with
DNA strands labeled with different fluorescent dyes.
a. Metaphase spreading- chromosomes are arranged just as they appear in the
cytological preparation
b. Karyotyping- more conventional representation; autosomes (22 pairs in
humans) in the metaphase spread are rearranged systematically in pairs,
from longest to shortest and numbered 1 (longest) through 22. The sex
chromosomes (X and Y or X and X) are usually set off at the bottom right.
Each human chromosome is linear and has a single centromere.
Classification of chromosomes according to the relative position of centromeres:
1. Metacentric chromosomes ( yield V-shaped daughter chromosomes) - middle
location
2. Submetacentric chromosome (yield J- shaped daughter chromosomes)Centromere is off center
3. Acrocentric chromosome (yield I- shaped daughter chromosomes)Centromere is very close to one end
Acentric chromosome- A chromosome that lacks a centromere. Genetically
unstable because they can not be maneuvered properly during cell division and are
lost.
Dicentric chromosome- A chromosome with two centromeres. Genetically
unstable because it is not transmitted in a predictable fashion.

The Principle of Dosage Compensation


For all the organisms with XX-XY sex determination, there is a problem of the
dosage of genes on the X chromosome because females have two copies of this
chromosome whereas males have only one. A mechanism of Dosage compensation
has evolved in which the unequal dosage in the sexes is corrected either by
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increasing the activity of genes in the X chromosome in males or by reducing the


activity of genes in the X chromosome in females. In the early cleavage divisions of
the embryo, one and only one X chromosome in each cell (chosen at random)
remains genetically active and any other X chromosomes that may be present in
the cell undergo a process of X inactivation (and any inactivated X chromosome
remains inactive in all the descendants of that cell). The process of X- chromosome
inactivation takes place in all embryos with two or more X chromosomes including
normal XX females. The inactivation process is one of chromosome condensation
initiated at a site called XIC (X- inactivation Center) near the centromere on the long
arm between Xq11.2 and Xq21.1.
Consequences of X-chromosome Inactivation:
1. Results in Dosage compensation. It equalizes the number of active copies of
X-linked genes in females and males. Single active X- principle (proposed
by Mary Lyon).
2. A normal female becomes a mosaic for the expression of X-linked genes.
(Genetic mosaic= an individual that contains cells of two or more different
genotypes.) A normal female is a mosaic for gene expression because the X
chromosome that is genetically active can differ from one cell to the next.
Approximately 15 % of all recognized pregnancies in human beings terminate in
spontaneous abortions, and in about half of all spontaneous abortions, the fetus has
a major chromosome abnormality.
Definition of terms:
1. Trisomic- otherwise diploid organism that has an extra copy of an individual
chromosome.
2. Monosomic- otherwise diploid organism but having a missing copy of an
individual chromosome. Generally, is more frequent and more harmful than
chromosome gains .
3. Diploid- Two sets of chromosomes are present (46 chromosomes)
4. Triploids- Three sets of chromosomes are present (69 chromosomes)
5. Tetraploids- Four sets of chromosomes are present (92 chromosomes)

Human chromosome abnormalities


A. Deletion/ Deficiency- chromosomes arise in which a segment or segments
is/ are missing.
- Generally harmful to the organism. The larger the deletion, the greater the
harm.
- Very large deletions are usually lethal, even when heterozygous with a
normal chromosome.
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Small deletions are often viable when they are heterozygous with a
structurally normal homolog, because the normal homolog supplies gene
products that are necessary for survival.
- However, even small deletions are usually lethal when both members of a
pair of homologous chromosomes carry the deletion.
Example: ASD (Autism Spectrum Disorders)- characterized by communication
deficits, social impairment and repetitive behaviors occurring prior to the age of 3,
usually requiring extensive family support and medical intervention. Males: Females
ratio 5:1; Identical twin chance of acquiring the defect: 70 90% if the other twin is
affected.
Deletions can be formed in 2 major ways:
1. Chromosome breakage and reunion- Chromosome breaks result from doublestranded breaks in the DNA backbone. May occur spontaneously at a slow
rate or can be induced by xrays and chemicals.
2. Ectopic recombination- pairing and homologous recombination between
repeated DNA sequences (direct repeats) present at different sites along the
DNA results in deletion of the material between the repeats.
B. Duplication- Abnormal chromosomes having a region that is present twice.
- Tandem duplication- the duplicated segment is present in the same
orientation
immediately adjacent to the normal region in the
chromosome. This produces even more copies of the duplicated region by
means of a process called unequal crossing over.
Human color blindness a result of unequal crossing over
- Human color vision is mediated by 3 light- sensitive protein pigments
present in the cone cells of the retina. Each of the pigments is related to
Rhodopsin, the pigment found in he rod cells that mediates vision in dim
light. The light sensitivities of the cone pigments are toward blue, red and
green (primary colors).Perception of all other colors are brought about by
mixtures of these primaries. The gene for the blue- sensitive pigment is in
chromosome 7, whereas the genes for the red and green pigments are in
the X chromosome (arose from the duplication of a single ancestral
pigment gene, are still 96% identical in amino acid sequence and are
similar enough that they can pair and undergo unequal crossing over).

Red- green color blindness- one of the most common inherited


conditions in humans.
- 5% of males
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Preponderance of affected males immediately suggests X-linked


inheritance (have normal sons and 50% carrier daughters).
Protanopia- inability to perceive red; Protanomaly- impaired ability to
perceive red
Deuteranopia- inability to perceive green; Deuteranomaly- impaired
ability to perceive green

C. Inversion- A chromosome in which the linear order of a group of genes is the


reverse of the normal order. Can be formed by 2 methods:
- 1. Two- break event in a chromosome in which the middle segment is
reversed in orientation before the breaks are healed.
2. Ectopic recombination between DNA sequences that are inverted
repeats, resulting in a
chromosome with an inversion in the order of the gens between the
repeats.
In an organism that is heterozygous for an inversion, one chromosome is
structurally normal (wildtype) and the other carries an inversion. These
chromosomes pass through mitosis without difficulty because each chromosome
duplicates and its chromatids are separated into the daughter cells without regard
to the other chromosome. However, there could be problems in meiosis. In Prophase
I in which there is gene for- gene pairing taking place everywhere along the
length of the chromosome, one or the other of the chromosomes must twist into a
loop in the region in which the gene order is inverted, forming an inversion loop.
When there is crossing over within the inversion loop, the chromatids involved in
the crossing over become physically joined and the result is the formation of
chromosomes containing large duplications and deletions.
Chapter 7. The Genetics of Bacteria and Their Viruses
Bacteria and their viruses (bacteriophage) have unique and diverse reproductive
systems with multiple and novel mechanisms of genetic exchange.
A high percentage of bacteria isolated from clinical infections are resistant to one or
more antibiotics. The widespread antibiotic- resistance genes almost never originate
from new mutations in the bacterial genome, but they are actually acquired, usually
several at a time, in various forms of Mobile DNA
(sequences in bacteria which are mobile and can be transferred between DNA
molecules and from one cell to another, between individuals and among species).
Plasmids- nonessential DNA molecules that exist inside bacterial cells. They
replicate independently of the bacterial genome and segregate to the progeny when
a bacterial cell divides, so they can be maintained indefinitely in a bacterial lineage.
Many are circular DNA molecules but others are linear. They range in size from a few
kilobases to a few hundred kilobases.

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The presence of plasmids can be detected physically by electron microscopy or by


gel electrophoresis of DNA samples. Some can be detected because of phenotypic
characteristics that they confer on the host cell (like antibiotic resistance).
(Text book: Fig. 7.1)
Plasmids rely on the DNA- replication enzymes of the host cell for their reproduction,
but the initiation of replication is controlled by plasmid genes.
High- copy-number plasmids- found in as many as 50 copies per host cell.
Replication is initiated multiple times during replication of the host genome.
Low-copy-number plasmids- present in 1 to 2 copies per cell. Replication is initiated
only once per round of replication of the host genome.
Pilus (plural: pili)- a tube-like structure formed between the cells through which the
plasmid DNA passes as they are being transferred between cells.
Conjugation- the joining of bacterial cells in the transfer process. Plasmids that can
be transferred in this manner are called conjugative plasmids. Not all plasmids
are conjugative.
Most small plasmids
are nonconjugative. They can be
maintained in a bacterial lineage as the cells divide, but they do not contain the
approximately 20 genes necessary for pilus assembly or those for DNA transfer.
Hence they are unable to be transferred on their own.
(Text book: Fig. 7.2)
The pilus between the E. coli cells above is an F pilus whose synthesis results from
the presence of a conjugative plasmid called the F factor (F stands for fertility).
Cells that contain the F plasmid (low-copy number plasmid) are donors and are
designated the F+ cells (F plus); those lacking F are recipients and are designated
the F- cells (F minus.)
Conjugation begins with physical contact between a donor cell and a recipient cell.
Once the pilus contacts the F- cell, the pilus retracts and the cell membranes of the
donor and recipient are brought into close proximity. Then the donor DNA moves
through a pore in the membrane from the donor to the recipient. The transfer is
always accompanied by replication of the plasmid. Contact between an F+ and an
F- cell initiates rolling circle replication which results in the transfer of a singlestranded linear branch of the rolling circle to the recipient cell. During transfer, DNA
is synthesized in both donor and recipient. When transfer is complete, the linear F
strand becomes circular again in the recipient cell. After the transfer, both cells
contain F and can function as donors. The F- cell has been converted into an F+ cell.
(Text book: Fig. 7.3)
Transposable elements are DNA sequences that can jump from one position to
another or from one DNA molecule to another. Bacteria contain a wide variety of
transposable elements. The smallest and simplest are insertion sequences or IS
elements, which are typically 1-3 kb in length and usually encode only the
transposase protein required for transposition and one or more additional proteins
that regulate the rate of transposition.

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Other transposable elements in bacteria contain one or more genes unrelated to


transposition that can be mobilized along with the transposable element; this type
of element is called a transposon. Much of the widespread antibiotic resistance
among bacteria is due to the spread of transposons that include one or more
antibiotic- resistance genes. When a transposon mobilizes and inserts into a
conjugative plasmid, it can be widely disseminated among different bacterial hosts
by means of conjugation.
Some transposons have composite structures with antibiotic resistance sandwiched
between insertion sequences, as is the case with the Tn5 element (illustration
below), which terminates in two IS 50 elements in inverted orientation. Transposons
are designated by the abbreviation Tn followed by an italicized number (ex. Tn5).
For example, Tn5( neo- r ble-r str-r) contains genes for resistance to three different
antibiotics: Neomycin, Bleomycin and Streptomycin.
(Text book: Fig. 7.4)
Nonconjugative and conjugative plasmids typically coexist in the same cell along
with host genomic DNA, and when a transposable element is mobilized, all of the
DNA molecules present are potential targets for insertion. Many nonconjugative and
conjugative plasmids present in a bacterial cell come to carry one or more copies of
the same transposable element. Because these copies are homologous DNA
sequences, they can serve as substrates for recombination. When 2 plasmids
undergo recombination in a region of homology, the result is as shown below. The
recombination forms a composite plasmid called a cointegrate. By this
mechanism, nonconjugative plasmids can temporarily ride along with conjugative
plasmids and be transferred from cell to cell.
(Text book: Fig. 7.5)
In the evolution of multiple antibiotic resistance, bacteria have also made liberal use
of a set of enzymes known as Site- specific recombinases which were present in
bacterial populations and functioned in the evolution of other traits long before the
antibiotic era. Each type of site specific recombinase binds with a specific
nucleotide sequence in duplex DNA. When the site is present in each of two duplex
DNA molecules, the recombinase brings the sites together and catalyzes a
reciprocal exchange between the duplexes. Site specific recombinases are used in
the assembly of multiple antibiotic-resistance units called integrons. An integron
is a DNA element that encodes a site- specific recombinase as well as a recognition
region that allows other sequences with similar recognition regions to be
incorporated into the integron by recombination. The elements that integrons
acquire are known as cassettes. In the context of integrons, a cassette is a circular
antibiotic- resistance- coding region flanked by a recognition region for an integron.
Because the site specific recombinase integrates cassettes, the integron
recombinase is usually called an integrase.
(Text book: Fig. 7.7)
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In nature, a conjugative plasmid can accumulate different transposons containing


multiple independent antibiotic- resistance genes, or transposons containing
integrons that have acquired multiple antibiotic- resistance cassettes with the result
that the plasmid confers resistance to a large number of completely unrelated
antibiotics. These multiple resistance plasmids are called R plasmids. The
evolution of R plasmids is promoted by the use of and overuse of antibiotics which
selects for resistant cells because in the presence of antibiotics, resistant cells have
a growth advantage over the sensitive cells. The presence of multiple antibiotics in
the environment selects for multiple drug resistance. Serious complications result
when plasmids resistant to multiple drugs are transferred to bacterial pathogens or
agents of disease.
Transduction- bacterial DNA is transferred from one bacterial cell to another by a
phage particle containing the DNA. Such a particle is called a transducing phage.
Two types of transducing phages are known:
1.) Generalized transducing phage- produces some particles that contain only
DNA obtained from the host bacterium, rather than phage DNA; the bacterial
DNA fragments can be derived from any part of the bacterial chromosome.
2.) Specialized transducing phage- produces particles that contain both phage
and bacterial genes linked in a single DNA molecule, but the bacterial genes
are obtained from a particular region of the bacterial chromosome.
Bacteriophage life cycles:
Lytic cycle- the reproductive cycle of a phage. Phage DNA enters a cell and
replicates repeatedly, bacterial ribosomes are used to produce phage protein
components, the newly synthesized phage DNA molecules are packaged into
protein shells to form progeny phage, and the bacterium is split open (lysis),
releasing the progeny phages from the cell.
Lysogenic cycle- The alternative to the lytic cycle. No progeny particles are
produced, the infected bacterium survives, and a phage DNA molecule is
transmitted to each bacterial progeny cell when the cell divides. All phage species
can undergo a lytic cycle. Those phages that are also capable of the lysogenic cycle
are called temperate phage , and those capable of only the lytic cycle are called
virulent phage. In the lysogenic cycle, a replica of the infecting phage DNA becomes
inserted, or integrated into the bacterial chromosome. The inserted DNA is called a
prophage, and the surviving bacterial cell is called a lysogen.
(Text book: Fig. 7.22)
A mutation is any heritable change in the genetic material.
(Text book: Table 12.1)

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