Mapping the genome of bacteria

Genome mapping


Introduction: locating of a specific gene to
particular region of a chromosome and
determining the location of and relative distances
between genes on the chromosome.



There are two types of maps: genetic linkage map
and physical map.

Genome mapping


The genetic linkage map shows the arrangement
of genes and genetic markers along the
chromosomes as calculated by the frequency with
which they are inherited together.

Genome mapping


The physical map is representation of the
chromosomes, providing the physical distance
between landmarks on the chromosome, ideally
measured in nucleotide bases.

Genome mapping


Physical maps can be divided into three general
types: chromosomal or cytogenetic maps,
radiation hybrid (RH) maps, and sequence maps.



The ultimate physical map is the complete
sequence itself.

Law of independent assortment


Mendel: States that genes are transmitted from
parents to offspring independently of one
another.



If a person has blood group A (e.g. genotype AO) and brown eyes (e.g.
genotype Bb, where B is the allele for brown and b is the allele for
blue eyes), the AO alleles are transmitted to the offspring
independently of the Bb alleles.



However, not all genes are inherited independently of one another.

Linked genes


Genes that are located on the same chromosome
and are described as linked genes.



If each chromosome were to be transmitted from parent to offspring
as a whole and unaltered structure, it would be expected that all the
genes located on the same chromosome would be transmitted
together as a block and not independently of one another as proposed
in Mendel's law.

Recombination


However, linked genes are not always transmitted
en bloc because of the phenomenon of
recombination.



One of the fundamental events that occur in
meiosis is crossing over in which homologous
chromosomes exchange segments causing a
reshuffling of genes.

GENETIC DISTANCE


If genes are far apart on the same chromosome, it
is likely that recombination occurs. Conversely, if
they are very close together, they are more likely
to be transmitted as a block .

Frequency of recombination


The frequency of recombination of two genes is
proportional to the distance between them.



The frequency with which recombination occurs
in the offspring is expressed as a percentage.



Genes which are very close together (closely
linked) will have a very small recombination
frequency (e.g. 1%).



A recombination frequency of 1% means that only
one out of 100 offspring was the combination of
two genes different from that in their parents.



In contrast, genes that are very far apart on the
same chromosome or those that are on different
chromosomes are equally likely to be transmitted
together or separately and so would have a
recombinant frequency of 50%.

centi-Morgan


The centi-Morgan which is defined as the distance
between two genes in which recombination occurs
with a frequency of 1%.



The unit of gene distance is also called
a map unit.

Genetic Markers


Genes can be mapped by linkage studies with
polymorphic markers, which are nucleotide
sequences identifiable at specific sites along the
genome.



Numerous markers have been identified
throughout the genome using restriction
endonucleases and so it is possible to construct
maps of disease genes in relation to closely linked
markers.

Restriction endonuclease


Restriction endonucleases are naturally occurring
enzymes produced by bacteria as a defence
against invasion by viruses. The bacterial
endonucleases cut the viral DNA thus restricting
its further proliferation.

Restriction endonuclease


Using a large number of restriction endonucleases,
it is likely that one finds one or more RFLPs close
to the gene of interest.



Such RFLPs are then used as markers for linkage
studies with known genes. Linkage studies have
been one of the most important tools for gene
mapping.

Marker!!


Although the gene causing a particular trait may
not be known it is possible to identify markers
which are very closely linked to it.

Cotransduction


If two genes are close together along the
chromosome, a bacteriophage may package a
single piece of the chromosome that carries both
genes and transfer that piece to another
bacterium.

Cotransduction


In genetic mapping studies, cotransduction is used
to determine the order and distance between
genes that lie fairly close to each other.

Cloning vector


A cloning vector is a small piece of DNA, taken
from a virus, a plasmid, or…, that can be stably
maintained in an organism, and into which a
foreign DNA fragment can be inserted for cloning
purposes.

Cloning vector


The vector therefore contains features that allow
for the convenient insertion or removal of DNA
fragment in or out of the vector, for example by
treating the vector and the foreign DNA with a
restriction enzyme.

Types of cloning vectors


There are many types of cloning vectors, but the
most commonly-used ones are genetically
engineered plasmids.



Cloning is generally first performed using
Escherichia coli, and cloning vectors in E. coli
include plasmids, bacteriophages (such as phage
λ), cosmids, and bacterial artificial chromosomes
(BACs).

Types of cloning vectors


Some DNA however cannot be stably maintained
in E. coli, for example very large DNA fragment,
and other organisms such as yeast may be used.
Cloning vector in yeast include yeast artificial
chromosomes (YACs).

Cosmid


Cosmids are plasmids that incorporate a segment
of bacteriophage λ DNA that has the cohesive end
site (cos) which contains elements required for
packaging DNA into λ particles.



It is normally used to clone large DNA fragments
between 28 to 45 Kb.

Fosmid


Fosmids are similar to cosmids but are based on
the bacterial F-plasmid.



Fosmids can hold DNA inserts of up to 40 kb in
size; often the source of the insert is random
genomic DNA.

F’ Factors
► If

excision of F from the chromosome is not
precise, a small section of host chromosome
may be carried with the plasmid, creating an F’
(F-prime) plasmid. An F’ plasmid is named for
the gene(s) it carries, e.g., F’ (lac).

F’ Factors
►

F’ cells can conjugate with F- cells, and thus
introduce the bacterial gene(s) it carries. The
recipient already has a set of bacterial genes, and
so will be merodiploid (partially diploid) for those that
are introduced. This is F-duction (sometimes called
sexduction).

Bacterial artificial chromosome (BAC)


A bacterial artificial chromosome (BAC) is a DNA
construct, based on a functional fertility plasmid
(or F-plasmid), used for transforming and cloning
in bacteria, usually E. coli.



The bacterial artificial chromosome's usual insert
size is 150-350 kbp.



A similar cloning vector called a PAC has also been
produced from the bacterial P1-plasmid.



BACs are often used to sequence the genome of
organisms in genome projects, for example the
Human Genome Project.



A short piece of the organism's DNA is amplified as
an insert in BACs, and then sequenced. Finally,
the sequenced parts are rearranged in silico,
resulting in the genomic sequence of the
organism.

Common gene components of BAC
oriS, repE - F
for plasmid replication and regulation of copy number.
parA and parB
for partitioning F plasmid DNA to daughter cells during
division and ensures stable maintenance of the BAC.
A selectable marker
for antibiotic resistance; some BACs also have lacZ at the
cloning site for blue/white selection.
T7 & Sp6
phage promoters for transcription of inserted genes.



Electroporation



Transformation



Transfection



Microinjection



Transfection is the process of deliberately introducing
nucleic acids into cells. The term is used notably for nonviral methods in eukaryotic cells.



Microinjection refers to the process of using a glass
micropipette to insert substances at a microscopic or
borderline macroscopic level into a single living cell. It is
a simple mechanical process in which a needle roughly 0.5
to 5 micrometers in diameter penetrates the cell
membrane and/or the nuclear envelope.

Yeast artificial chromosome


A yeast artificial chromosome (YAC) is a vector
used to clone DNA fragments larger than 100 kb
and up to 3000 kb.



YACs are useful for the physical mapping of
complex genomes and for the cloning of large
genes.

Yeast artificial chromosome


A YAC is built using an initial circular plasmid,
which is typically broken into two linear
molecules using restriction enzymes; DNA ligase is
then used to ligate a sequence or gene of interest
between the two linear molecules, forming a
single large linear piece of DNA.

Yeast artificial chromosome advantage


Yeast expression vectors, such as YACs, YIps (yeast
integrating plasmids), and YEps (yeast episomal
plasmids), have an advantage over bacterial
artificial chromosomes (BACs) in that they can be
used to express eukaryotic proteins that require
posttranslational modification.



BUT YACs are significantly less stable than BACs.

Using Conjugation to Map Bacterial Genes



Conjugation experiments to map genes begin with appropriate
Hfr strains selected from the progeny of F+ X F- crosses.

Interrupted-mating experiments with a variety of Hfr strains, showing that
the E. coli linkage map is circular

Genetic Mapping in Bacteria by Transformation


Transformation is used to map genes in situations where
mapping by conjugation or transduction is not possible.


Donor DNA is extracted and purified, broken into fragments, and added to
a recipient strain of bacteria. Donor and recipient will have detectable
differences in phenotype, and therefore genotype.



If the DNA fragment undergoes homologous recombination with the
recipient’s chromosome, a new phenotype may be produced.
Transformants are detected by testing for phenotypic changes.

Transformation in Bacillus subtilis

Chapter 14 slide 42

Transformation experiments are used to determine:
 Whether

genes are linked (physically close on the bacterial
chromosome).

 The
 The

order of genes on the genetic map.

map distance between genes. Recombination
frequencies are used to infer map distances.

Demonstration of determining gene order by cotransformation

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

Transduction Mapping of Bacterial Chromosomes


Closely linked genes are cotransduced at high frequency,
allowing a detailed genetic map to be generated. For
example:
(1) Of the leu+ selected transductants, 50% have aziR and
2% have thr+.
(2) Of the thr+ selected transductants, 3% have leu+, and
0% have aziR.
(3) This gives the map order: thr--leu------azi.

Specialized Transduction



Specialized transduction is useful for moving specific genes
between bacteria, but not for general genetic mapping.
Some phages transduce only certain regions of the chromosome,
corresponding with their integration site(s). An example is λ in E.
coli:
i. Excision is usually precise.
ii. Rarely excision results in genetic exchange, with a fragment
ofλDNA remaining in the E. coli chromosome, and some bacterial
DNA (e.g., gal+) added to theλchromosome.
iii. The resulting transducing phage is designated λd gal+ (d for
defective, since not all phage genes are present).
iv. λd gal+ can replicate and lyse the host cell, since allλgenes are
present either on the phage or bacterial chromosome.

Specialized Transduction


Because transducing phage are only rarely produced, a
low- frequency transducing (LFT) lysate results. Infection
of gal bacterial cells results in two types of transductants:
i.

ii.

Unstable transductants result when wild-type
λintegrates first at its normal attλ site. λd gal+ then
integrates into the wild-typeλ, producing a double
lysogen with both types of λ integrated.
Stable transductants are produced when a cell is
infected only by a λd gal+ phage, and the gal+ allele
is recombined into the host chromosome by double
cross-over with gal.

Specialized transduction by bacteriophage

Mapping Genes of Bacteriophages
1. Phage genes are mapped by 2-, 3- or 4-gene crosses,
involving bacteria infected with phages of different
genotypes.
a. Progeny phage are counted using a plaque assay in which each
phage produces a cleared area in a bacterial lawn.
b. Distinguishable phage phenotypes include mutants with
different plaque morphology. An example is strains of T2
differing in plaque morphology and/or host range.
c. The h and r genes are mapped by infecting E. coli strain B
simultaneously with two phages, h+ r and h r+.

The principles of performing a genetic cross with bacteriophages

Fine-Structure Analysis of a Bacteriophage
Gene


Intragenic mapping determines mutation sites within
genes.

Fine-Structure Analysis of a Bacteriophage
Gene


Benzer’s fine-structure mapping of phage T4
used similar experiments involving the rII gene.
a. Different rII mutations of T4 were used,
each with the characteristic large clear
plaques and limited host range.
b. T4 with the wild-type r+ gene infects E. coil
strains B and K12(λ). For rII T4, strain B is
permissive but K12(λ) is nonpermissive.

Recombination Analysis of rII Mutants
1.

2.
3.

Benzer’s fine-structure mapping involved 60
independently isolated rII mutants, which were
crossed in all possible combinations, using E. coli B as
the permissive host.
A linear map was constructed from the recombination
data from all crosses of the 60 rII mutants.
Later experiments have observed recombination
between adjacent base pairs, indicating that the base
pair is both the unit of mutation and the unit of
recombination. This replaced the older idea that the
gene was indivisible.

Benzer’s general procedure for determining the number of r+ recombinants
from a cross involving two rII mutants of T4

Reversion
To Wild tye

Preliminary fine-structure genetic map of the rII region of phage T4 derived
by Benzer from crosses of an initial set of 60 rII mutants

Chapter 14 slide 57
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

台大農藝
系遺傳
學 601
20000

Deletion Mapping
1. Benzer eventually mapped over 3,000 rII mutants. A
deletion mapping technique was used to simplify
these studies. a. Some of the mutants did not revert, nor did they recombine
to produce r+ phage in crosses with a variety of rII mutants.
These were deletion mutants.
b. The systematic approach crossed each unknown rII mutant
with a set of seven standard deletion mutants defining the
seven main segments of the rII region.
c. Once a region for the mutation was known, the new mutant
was crossed with members of the relevant secondary set of
reference deletions. Analysis of recombination or
nonrecombination enabled more precise localization of the
mutation site

Benzer’s experiment: Segmental subdivision of the rII region of phage T4
by means of deletion

Fine-structure map of the rII region derived from Benzer’s experiments

Defining Genes by Complementation (Cis-Trans)
Tests
1. The complementation test determines how many genes
are involved in a set of mutations that produce a given
phenotype.
2. The T4 rII region has two genes, rIIA and rIIB. A
mutation in either gene produces the rII phenotype for
both plaque morphology and host range.

Tests
3. In Benzer’s work, nonpermissive strain K12(λ) was
infected with pairs of rII mutants. Neither can grow
alone in this strain.
a. If progeny are produced, the two mutants have complemented
each other by providing different gene functions, either by
genetic recombination (producing a few plaques) or
complementation (lysing the entire lawn) (Figure 14.23).
i. Infect bacterium with two phage genomes. Genotype of
one is rIIA+ rIIB, and of the other is rIIA rIIB+.
ii. One phage provides the rIIA product, the other the rIIB
product, and so the phage lytic cycle occurs.
b. If no progeny are produced, both mutations are in the same
functional unit. Both mutants produce the same defective
product (e.g., the rIIA product), and so the phage lytic cycle
cannot occur.

Tests
c.Benzer’s work showed two functional units for the rII
phenotype, the complementation groups rIIA and rIIB.
Both gene products must be produced for the lytic
cycle to occur.
d. Alleles may be arranged two different ways in cistrans complementation experiments:
e. When the mutant alleles are on two different
chromosomes, as in the complementation
experiment above, they are in the trans
configuration.

Complementation tests for determining the units of function in the rII
region of phage T4

4. Benzer called the genetic unit of function defined by a
cis-trans complementation test a cistron. Defined as
the smallest segment of DNA encoding an RNA, cistrons
are now usually referred to as genes.

References


http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/ApplMapping.sht
ml



Strachan T. (2011). Human molecular genetics / Tom Strachan and Andrew
Read, 4th ed.



Haldi M, Perrot V, Saumier M, Desai T, Cohen D, Cherif D, Ward D, Lander ES.
Large human YACs constructed in a rad52 strain show a reduced rate of
chimerism. Genomics. 1994 Dec;24(3):478-84.



Bronson SK, Pei J, Taillon-Miller P, Chorney MJ, Geraghty DE, Chaplin
DD.Isolation and characterization of yeast artificial chromosome clones
linking the HLA-B and HLA-C loci.Proc Natl Acad Sci U S A. 1991 Mar
1;88(5):1676-80.

References


O'Connor M, Peifer M, Bender W (1989). "Construction of large DNA segments in
Escherichia coli". Science 244 (4910): 1307–1312. doi:10.1126/science.2660262.
PMID 2660262.



Shizuya H, Birren B, Kim U-J, Mancino V, Slepak T, Tachiiri Y, Simon M (1992).
"Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in
Escherichia coli using an F-factor-based vector". Proc Natl Acad Sci USA 89 (18):
8794–8797. doi:10.1073/pnas.89.18.8794. PMC 50007. PMID 1528894.



Shizuya, H; Kouros-Mehr H (2001). "The development and applications of the
bacterial artificial chromosome cloning system". Keio J Med. 50 (1): 26–30.
PMID 11296661.



Stone NE, Fan J-B, Willour V, Pennacchio LA, Warrington JA, Hu A, Chapelle
A, Lehesjoki A-E, Cox DR, Myers RM (1996). "Construction of a 750-kb bacterial
clone contig and restriction map in the region of human chromosome 21 containing
the progressive myoclonus epilepsy gene". Genome Research 6 (3): 218–225.
doi:10.1101/gr.6.3.218. PMID 8963899.

References


Physical Mapping of Bacterial Genomes, MICHAEL FONSTEIN AND ROBERT
HASELKORN*, Department of Molecular Genetics and Cell Biology, The
University of Chicago, Chicago, Illinois 60637, JOURNAL OF BACTERIOLOGY,
June 1995, p. 3361–3369 Vol. 177, No. 12, 0021-9193/95/$04.0010, Copyright
q 1995, American Society for Microbiology

Mapping the genome of bacteria

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