Mapping the genome of bacteria

Genome Mapping

Dr.NasrMeisam.Roozbahani

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.

CONSTRUCTING A GENE MAP

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.

► 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).

F’ Factors

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 BACoriS, repE - F

for plasmid replication and regulation of copy number.

parA and parBfor partitioning F plasmid DNA to daughter cells during division and ensures stable maintenance of the BAC.

A selectable markerfor antibiotic resistance; some BACs also have lacZ at the cloning site for blue/white selection.

T7 & Sp6phage 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 non-viral 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.

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

Using Conjugation to Map Bacterial Genes

Interrupted-mating experiment

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

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.

Genetic Mapping in Bacteria by Transformation

Chapter 14 slide

42

Transformation in Bacillus subtilis

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

The order of genes on the genetic map.

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

Transformation experiments are used to determine:

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

Demonstration of determining gene order by cotransformation

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.

Transduction Mapping of Bacterial Chromosomes

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. 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.

ii. 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

Specialized transduction by bacteriophage

Specialized transduction by bacteriophage

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+.

Mapping Genes of Bacteriophages

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 Mutants1. 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.

2. A linear map was constructed from the recombination data from all crosses of the 60 rII mutants.

3. 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

ReversionTo Wild tye

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Chapter 14 slide

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

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

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.


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.


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 cis-trans 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

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.shtml

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

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Mapping the genome of bacteria

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