Gene mapping in eukaryotes

Mapping the genes in a correct order helps to know the location of the genes on the chromosomes. Various techniques achieved success in mapping the genes. Mapping techniques achieved success with eukaryotic organisms such as Drosophila and plants before humans. Gene mapping not only helps us in knowing the exact location of the gene but also helps in conducting various other experiments based on the gene location. Thomas Morgan worked with Drosophila strains and found out recombination mechanisms. An experiment sometimes gives an idea of other hidden strategies. For example, gene mapping studies revealed the mechanism of recombination. The studies revealed that the progeny obtained by crossing some strains of the eukaryotic organisms also showed phenotype differing from the parental phenotype.

Morgan’s experiment:

Thomas Morgan and his colleagues worked with Drosophila strains. They cultured Drosophila with a particular X-linked phenotype. The experimenters selected certain strains of fruit flies. The female flies had two X chromosomes with linked genes. The males had one X and one Y chromosome. The female flies had a phenotype of white eyes and miniature wings. The male flies were wild-type flies. The cross between these two types of flies gave rise to an F1 generation having wild-type females, and white-eyed, miniature winged male flies. Interbreeding of the F1 progeny gave different kinds of flies. There were total 2241 flies in the F2 generation. Out of these, total 900 flies had a non-parental phenotypic combination of white eyes and normal wings. Other types of non-parental strains included red-eyed, miniature winged flies. The non-parental ones are known as recombinants. The recombinants arise due to the crossing over between the homologous chromosomes. The theory involves two key concepts. The first one is the site of physical exchange. It is known as the chiasma. The second one involves the genetic recombination between the linked genes. It is known as crossing over. It also involves a reciprocal exchange of chromosome segments.

Image 2: Morgan's experiment

Stern’s Experiment:

Stern worked with X-linked gene loci in Drosophila. The experimenters conducted a cross between the wild-type bar eyed females and carnation type round-eyed males. The female flies had two X chromosomes. One of them additionally had a detached piece of X chromosome. The other X chromosome had an additional attachment of a piece of the Y chromosome. The chromosomes in the males flies had no extra pieces attached. The interbreeding of the F1 progeny gave rise to different types of flies. Four main types of progeny observed included carnation bar, red round, carnation round, and red bar eyed males and females respectively. The results of the experiment revealed the genetic recombination and exchange of identifiable segments. 

Barbara McClintock’s corn experiment:

The corn species selected for the experiment consisted of heterozygotes for the two genes on the 9^th chromosome. One of the genes gave a phenotype of colored versus colorless. The other type of genes resulted in the phenotypes such as standard type starch with amylose and amylopectin versus waxy plants having the only amylopectin. The chromosomes had genes cWx giving normal phenotype. The homologs of the chromosomes having genes cWx had the genotype of Cwx. These homologs had a large double stained knob and a piece of 8^th chromosome attached near the wx gene. It was a translocated segment. These features are known as the cytological markers. Hence, the corn experiments revealed the process of genetic recombination associated with the physical exchange between the parts of the homologous chromosomes.

Linkage studies using testcross:

A cross involving a normal individual with an individual who is homozygous recessive for all the genes is known as a testcross.

·        Two point test cross

Consider the autosomal recessive individuals. Suppose there involves a cross between the double heterozygotes with a genotype of a⁺b⁺/ a⁺b⁺ and double homozygous recessives with a genotype of ab/ab. The F1 generation revealed progeny with a wild-type a⁺b⁺/ab genotype. Upon conducting a testcross with double homozygous recessives, the progeny had 50% parental non-recombinants and 50% recombinant progeny. The formula for the recombination frequency involves (Number of recombinants/ Number of testcross progeny) x 100. The recombination frequency cannot exceed 50%.

Image 2: Two-point test cross

·        Three-point test cross:

Consider a cross between the triple heterozygotes with a genotype of a⁺b⁺c⁺/abc and triple homozygous recessives with a genotype of abc/abc. These crosses reveal the genetic recombination. Consider another example of flowering plants having three linked genes controlling the fruit phenotype. The recessive p allele gives a purple phenotype versus the wild-type yellow phenotype. The recessive r allele gives a round shape versus the wild-type elongated one. The recessive j allele gives juicy phenotype versus the wild-type dry fruit. The order of genes gets determined through a three-point test cross. Two parentals and six recombinants arise due to crossing over. The frequency of the double crossovers was found less than the frequency of the single crossovers.

Gene-centromere distance studies in Neurospora crassa:

The products of meiosis get a specialized arrangement depicting the four chromatids of each of the homologous pair of chromosomes. It usually reflects during the metaphase I. Neurospora consists of ordered tetrads. Meiotic and the mitotic divisions in the tetrads help in studying the process of recombination. It becomes easy to map the distance between the gene and the centromere using the ordered tetrads. The first division segregation tetrad consists of a parental type occupying half the ordered tetrad and another parental type in the other half of the tetrad. It occurs when there is no crossover. A single crossover between the gene and the centromere gives different types of tetrad segregation patterns (the second division segregation). The percentage of the second division tetrads divided by 2. It is known as the gene-centromere map distance. Tetrad analysis also helps in mapping two linked genes.

Mitotic recombination:

Crossing over is also known as genetic recombination between the linked genes or the reciprocal exchange of chromosome segments. It occurs during the mitosis as well as meiosis. The mitotic crossing over is also known as mitotic recombination. It leads to the production of the progeny cells having a combination of genes differing from the diploid parental cell entering the mitotic cycle. A classic example of the mitotic recombination includes fungus Aspergillus nidulans. It has a parasexual cycle of genetic systems. The genetic recombination in Aspergillus occurs through the processes other than regular alteration of meiosis and fertilization. The heterokaryon forms due to the mycelial fusion and the fusion of the two haploid nuclei. It gives rise to a diploid nucleus. The parasexual cycle also consists of mitotic crossing over within the diploid nucleus or haploidization of the diploid nuclei without meiosis. It becomes easy to calculate the gene order and the map distances.

Human gene mapping:

Physical mapping techniques help in mapping human genes. This technique mainly involves large genomes. It is not possible to set up a testcross for human genes since the human genome is vast. We obtain the recombination data from the pedigree analysis in humans. Gene mapping involves the use of gene markers and DNA markers.

                       
References:
[1] Genetics: Analysis of Genes and Genomes, Daniel L. Hartl, Elizabeth W. Jones
[2] Biology, Raven
[3] Biology, Pages 172-180, Neil A. Campbell, Jane B Reece
            

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Generation of detailed maps using sequence tagged sites

A map designed for a sequencing project to accurately locate the genes and assign the gene function adds direct value to mapping studies. The smallest functional units or genes are present on the chromosome. Hence it is important to study their position and locate them. The recombination frequencies or the number of recombinants found out of the total number of progenies guide in assessing the relative distances between the loci. The mathematical relationship between the map distance and recombination frequencies help to determine map function. Linkage of genes mainly tests whether the genes are present on the same chromosome. Therefore a map is constructed based on the relative positions of the genes. For large genomes, constructing a physical map would add value to the sequencing projects. However, if the purpose is to clone the individual genes, a different approach may be used. The physical mapping techniques do not rely on the presence of alleles to map the genome. The genetic mapping requires the alleles for a given marker. Unavailability of a map leads to an error-prone assembly of the genome sequence. The genetic maps have a poor resolution and inaccuracy. These properties are refined using a physical map.

Image 1: STS Mapping (Cloned DNA fragments showing markers)

Molecular biology techniques are used to construct a genome map. The plethora of physical mapping involves restriction mapping, FISH, and STS mapping. Restriction sites in small DNA molecules are possible in restriction mapping with a limitation in eukaryotic chromosomes. FISH is a good choice for mapping genomes. However, it takes a lot of time for mapping large genomes. More specific approach for mapping large genomes is required. Sequence tagged sites (STS) guide very accurately in physical mapping. This type of mapping is known as STS mapping. A sequence tagged site though seems complicated by its name, is not so complicated. In fact, it is a very specific approach. It is any site in the chromosome that is identified by a known unique DNA sequence. Mapping large genomes require a high resolution and rapid technique with less demand in technicality.

The limitations of FISH being difficult to conduct and accumulate the data in a single experiment, STS mapping may meet the requirement of providing map positions more than three markers in one go. A short DNA sequence with 100-500 base pairs is easy to recognize because it occurs only once in the chromosome or a genome. Such a site or a sequence is useful for physical mapping. STS mapping requires such a collection of overlapping fragments from the genome. The entire chromosome is used to obtain the mapping reagent or a collection of overlapping fragments. It helps to identify the marker position.

Properties of Sequence Tagged Sites:

As discussed earlier, sequence tagged sites are small DNA sequences that are unique. Two main properties determine sequence tagged sites. The DNA sequence must be known. Hence, a PCR assay can be set up. A PCR assay of known DNA sequence determines the STS on different fragments. The DNA fragment must have a unique location on a chromosome. If the STS are positioned more than once in the genome, it becomes difficult to map. Mainly repetitive DNA has high chances of having more than once positioned sequences. Thus STS mapping does not include repetitive DNA sequences. The reason for using PCR instead of hybridization lies in its automatic mechanisms and efficiency. It is difficult to include those fragments whose sequence is not known to us. Therefore the foremost criteria for a genome mapping are to know a sequence. The probability that the two closely linked markers determined by the fragments found on the same chromosome.

Distantly linked markers may be present in different fragments. A collection of fragments involves many fragments with closely linked or distantly linked markers. The frequency at which breaks occur between the two markers decides the map distance.

Image 2: Fragment collection (STS mapping)

Sources of sequence tagged sites (STS):

Three main sources of sequence tagged sites include expressed sequence tags (EST), SSLPs, and random genomic sequences.

·        Expressed sequence tags (ESTs): The cDNA clones are analyzed to obtain short sequences known as expressed sequence tags or ESTs. The sequence derived from the cDNA library is unique. A sequence transcribed in some tissue or at some stage of the developmental process is used to derive a unique sequence. Thus, an EST mapped through a specific mapping procedure identifies a unique gene locus. EST markers are produced using PCR. It involves oligonucleotide primers based on cDNA sequence. They correspond to protein-coding genes. So, the unique ESTs are capable of becoming STS.

·        SSLPs: Simple sequence length polymorphisms are arrays of repeat sequences displaying length variations. SSLPs contain alleles with a different number of repeat units that are multi-allelic. Two main types of SSLPs include minisatellites and microsatellites. Polymorphic SSLPs are usually preferred.

·        Random genomic sequences: They are randomly cloned sequences. Randomly spanning the available online databases help to obtain random genomic sequences. However, they are known sequences.

Fragment collection for STS mapping:

The collection of DNA fragments are known as a mapping reagent. The fragments are present in the entire chromosome. Each point has an average of five fragments. The markers may be near or far on the fragments. Mapping reagents assemble in two ways such as clone library and radiation hybrids. Rodent cell lines support the radiation mapping techniques. Different fragments of the second genome consist of the rodent cell line. Irradiation techniques are used to construct these cell lines. Hence they are mapping reagents in studying large genomes. A clone library is another mapping reagent. It is a collection of clones representing an entire genome. These clone collections supply individual clones of interest. Let us know the two mapping reagents in detail.

Radiation hybrids:

The human chromosomes paved way in the development of radiation hybrids. During the early 1970’s, experimenters exposed human cells to 3000-8000 rad doses of X-rays, leading to chromosome breakage. However, this treatment was lethal for human cells. The irradiated cells propagated on fusion with non-irradiated hamster cells. Polyethylene glycol or Sendai viral exposure led to the fusion of both the cells. However, all hamster cells are not capable of accepting human chromosomes. The hamster cells, therefore, need to undergo a selection process. Some hamster cells are unable to make thymidine kinase or hypoxanthine phosphoribosyl transferase. So the cells are grown in a medium containing hypoxanthine, aminopterin and thymidine medium (HAT). The fused cells are cultured in the HAT medium.

Hybrid hamster cells grow on this medium. It indicates that these cells have accepted human chromosomes. The hybrid cells consist of human DNA inserted into hamster chromosomes. These fragments are 5-10 Mb in size. The collection of hybrids is known as radiation hybrid panels. It is a mapping reagent. The rodent cells may also be used to obtain radiation hybrid panels. The rodent cell lines consist of human DNA fragments in the rodent nucleus. Sometimes the hybrid rodent cells are fused with hamster cells. Such hybrids may contain both human and mouse chromosomes or a mixture of both. Specific probes help to identify hybrid cells containing human DNA. The probes are specific sequences identifying human DNA. Examples include SINEs or short interspersed nuclear element called as Alu. The Alu elements have a copy number of over a million. Two types of radiation hybrid panels are known so far. They include single chromosome panels and whole genome panels. Let us know the difference between the two.

Single chromosome panel	Whole genome panel
Only a few hybrids are required	A few hundred hybrids are required.
PCR screening involved convenience in handling	Involved less convenience in handling
Involved irradiation of mouse cell containing more mice DNA and less human DNA.	Irradiation of human DNA
Human DNA hybrid is less in content	Higher human DNA hybrid content
The human genome project avoided the approach due to less human DNA hybrid content.	The human genome project utilized the approach due to high human DNA hybrid content.

 Table: Difference between single chromosome panel and the whole genome panel

Clone library:

A collection of clones represents the human genome. They are used to obtain individual clones of interest. A clone library is obtained by breaking the genome into fragments and thereby cloning them into the vector. Hence, a clone library consisting of large genome fragments is a mapping reagent. A clone library is a chromosome specific library. It is possible to separate chromosomes using a clone library. They have sufficient information for STS mapping. The STS analysis determines the clones consisting of overlapping DNA fragments enabling clone contigs to build-up.

References:
[1] Molecular Biology, David P. Clark, Nanette J. Pazdernik
[2] Human Molecular Genetics 3, Volume 3, T. Strachan, Andrew P. Read

© Copyright, 2018 All Rights Reserved.

Human Genome Project

The human genome is the total genetic material in the cells of a human being. It contains billions of nucleotide base pairs. The genetic material is present in the chromosomes.  Human Genome Project involves sequencing of all the DNA base pairs and mapping of several genes. The project started in 1991 in the USA. It was the largest project in human genetics. It was completed by April 14, 2003. The National Institutes of Health-funded primarily for this project. The project covered 99% euchromatic genome with 99.99% accuracy. Human genome sequencing benefitted a lot. It helped in understanding the diseases, genotyping of specific microbes, identification of mutations, cancer genetics, drug designing, and biotechnology. Specific databases were designed to store the sequences of the DNA. The National Center for Biotechnology Information (NCBI) consists of all the database information in GenBank. It is a hub for gene sequence information, protein sequences, and related information.

Image: Human Genome Project

Objectives of the Human Genome Project:

1.     Human Genome Sequencing:

It is used to figure out the order of nucleotides or bases such as adenine, guanine, cytosine or thymine.

2.     Human Gene Mapping:

It is used to identify a locus of a gene and the distance between the genes. Human gene mapping places a collection of molecular markers on their respective genome positions.

3.     Mapping of human inherited Diseases:

It helps to identify genes and biological processes. It is used to understand the molecular basis of inheritance.

4.     Development of new DNA technologies:

Human genome project developed new DNA technologies. They consisted of recombinant techniques for studying diseases and drug designing.

5.     Development of bioinformatics:

It helped in assembling DNA sequences, finding genetic landscape features, genome mining, the study of genetic variation and disease.

6.     Comparative genomics:

A computer-based analysis is used to compare the entire genome sequence. Comparative genomics is used to study similarity and difference regions.

Techniques used in the Human Genome Project:

Genome annotation technique used in the human genome project identified boundaries between genes and other features in a DNA. The bioinformatics domain stores the genome annotated sequences. RNA-Seq is a new technology introduced to sequence a messenger RNA in the cells. RNA-Seq was more accurate than annotation.

Highlights of the findings:

·        The Human genome project identified approximately 22,300 protein-coding genes.

·        Human genome sequencing identified 3.2 billion base pairs.

·        A human body consists of 26,000 to 35,000 genes.

·     Genes constitute only 5% of the human genome. Over 95% of the human genome is known as junk DNA. The non-coding DNA is known as junk DNA.

·        The junk DNA constitutes repeating DNA segments.

·        Only 7% of protein families were vertebrate specific.

·        Genes function as complex networks.

The Mapping Phase of Human Genome Project:

The gene mapping involved restriction fragment length polymorphisms (RFLPs). These RFLPs are highly polymorphic DNA markers. It comprised of 393 RFLPs. It also consisted of ten polymorphic markers. The marker density was 10 Mb. The RFLP map consisted of single strand length polymorphisms (SSLPs). Clone contigs were primarily used to develop physical mapping. Methods used were STS screening and clone fingerprinting.

A clone contig map consisted of 33,000 Yeast Artificial Chromosomes (YACs). However, there was a limitation of YAC. It contained few pieces of non-contiguous DNA. STS markers were mapped using radiation hybrid mapping. STS maps included 7000 polymorphic SSLPs.

Human Genome Sequencing:

Since YACs consisted of non-contiguous DNA, the entire focus switched over to BACs. The scientists cloned and mapped the Bacterial artificial chromosomes (BACs). A library of three lakh BAC clones was generated and mapped into the genome. The ready map of BAC was a primary foundation for the sequencing project. The shotgun sequencing method was used to replace the clone contig method.

There are many advantages to the human genome project. It helps to study the genes and mutations associated with them. It is easier to diagnose, predict and prevent the disease using HGP principles. Medicines can be developed based on the individual’s response to treatment. There is a wide scope for personalized medicine and drug designing. Using the databases generated during HGP, it is easy to carry out research activities. The gene databases help the molecular and cytogeneticists to study specific gene mutations, chromosomal abnormalities, and sequencing strategies. DNA fingerprinting techniques are useful in forensic medicine and crime investigation. With the help of gene mapping, it becomes easier to study inherited diseases and conduct efficient genetic counseling and prenatal diagnosis. However, there are very few disadvantages to the human genome project. Knowing one’s genome and possible risks that could result in a disease in the future may create an environment for genetic discrimination. However certain laws have been implemented such as GINA act to prevent genetic discrimination.

Cancer genomics:

With approximately millions of cancer cases worldwide, the survival rates of cancer patients are decreasing every year. The response of patients to the current treatment methodologies is not satisfactory. Moreover, the drugs and radiations given to the patients have been fraught with severe toxicity and side effects thereby limiting their applications in the cancer therapeutics. The human genome project had an objective of genome sequencing and mapping. Thus the research can be directed toward the development of new anti-cancer products. These products might target the signaling pathways, apoptosis, metastasis or migration of cancer cells. An increasingly rapid DNA analysis with the help of the human genome project may establish new therapeutic targets and facilitate effectiveness.

Enable Technologies:

An evolutionary improvement in the existing genome sequencing technologies through Human genome project may have a revolutionary impact on the genetic research. The human genome project would be helpful in designing treatment strategies for various diseases. The past, present and the future are genome based. Overcoming those few disadvantages of HGP might equip the human population to adopt the changes in the future. Though the human genome project is a well-established one, we know that studying the entire genome would never be complete. As the environment changes, the conditions may change and so the genes. Thus human genome would always be a topic for study. Chances of new mutations would be high. Thus, studying a human genome is a coordinated effort of the researchers to carry out the mapping and sequencing. High-throughput revolutionary technologies developed with the synergy of advanced computerization, automated machines, and robotics work excellent with microarrays, modern screening, and imaging techniques.

References:
[1] Genomes, T.A. Brown
[2] Human Genome Project- Wikipedia
[3] Genetics Home Reference

© Copyright, 2018 All Rights Reserved.

Bacterial conjugation process

The review article focuses on the plasmid-mediated conjugation process in E. coli bacteria. A unidirectional transfer of the genetic material through a contact between the two bacterial cells is known as conjugation. A physical bridge between the two cells mediates the DNA transfer. In prokaryotes, such as bacteria, the transfer of the genetic material mostly involves a one-way process. Thus, the process of conjugation helps to transfer the genetic material from one cell to another, enabling the process of copying the genetic material. Among the bacteria, most widely used ones for genetic analysis involve Escherichia coli bacteria. 
Lederberg and Tatum first conducted a conjugation experiment on E. coli cells. William Hayes demonstrated the theory of the unidirectional transfer of the genetic material in E. coli. Bernard Davis independently conducted a U-tube experiment. With the help of the above studies, various researchers came up with different findings in bacterial genetics. Conjugation studies also help to map the genes. In the late 1950’s, Francis Jacob and Elie Wollman studied the transfer of genetic material from Hfr strains to F^- strains. Most of the conjugation occurs through plasmids in the bacterial cells.

A plasmid is an extrachromosomal genetic material present in the bacterial cell. These plasmids exhibit a property of transferring the genetic material. Hence they are used as vectors in the process of cloning and recombinant DNA technology. The contact between the two cells involves a physical bridge between the two cells. Thus, a segment of the chromosome from one cell transfers to another cell thereby undergoing genetic recombination. Hence, the cells receiving the DNA are known as trans-conjugants. The essential genetic element for a bacterial conjugation is known as a conjugon. Unlike prokaryotes, the process of conjugation in protozoa involves a two-way process.

Image 1: Conjugation in bacteria

Lederberg and Tatum experiment:

Two E. coli strains to differ in their nutritious environments were studied. Note that even bacteria require nutrients for carrying out various cellular activities.  The two bacterial strains were labeled as strain A and strain B respectively. The amino acid synthesizing bacteria do not require a supplemented medium. Such type of bacteria labeled as “+” strains, synthesize the required nutrients. The strain A had a genotype known as met bio thr⁺ leu⁺thi⁺. The strain A bacteria grew on a medium supplemented with methionine and biotin. Without these two functional molecules, the strain A would not have grown. The strain B had met⁺bio⁺thr leu thi genotype. It required threonine, leucine, and thiamine to grow. Both the strains were mixed and plated on a minimal medium. The mixed culture gave rise to the prototrophic colonies. No colonies were visible on the minimal medium after plating the strains individually. It is due to the auxotrophic cells.

A mutant organism capable of growing only on a minimal medium with the growth factor supplementation not required by the wild-type strains is known as an auxotroph. A strain of microorganisms not requiring any additional nutrient to grow is known as a prototroph. The prototrophic colonies occurred at a frequency of 1 in 10 million cells. These colonies were recombinants arising due to the exchange of the genetic material between the two cells.

Davis U-tube experiment:

Bernard Davis showed physical contact between the two bacterial cells using a U-tube apparatus. He placed both the bacterial strains in a liquid medium poured into either side of the tube separated by a filter. The medium moved between the compartments. It was later on, plated on a minimal medium. None of the colonies grew. Hence, through this experiment, Davis demonstrated the cell to cell contact of the bacteria mediated gene transfer.

William Hayes experiment:

The genetic exchange in the E. coli occurred in one direction. One cell acted as a donor and the other like a recipient. Sex factor or the F factor-mediated the transfer of the genetic material. There are two types of bacterial cells such as the donor and the recipient cells. The donor cells are the one giving the genetic material. The recipient cells accept the genetic material from the donor cells. F-factor is a plasmid capable of replicating independently. Hence, the donor bacteria are known as F+ strains. The recipient bacteria are known as F- strains. Two same types of bacteria do not undergo conjugation. The F+ and F- strains only undergo conjugation.

F+ and F- matings:

The process of conjugation involves mating between F+ and F- strains. The F factor of the donor bacteria has a nick at one of the strands extending through the sex pilli or a physical bridge. The nicked strand gets transferred to the recipient where the remaining strand gets copied. Hence, the transfer and the synthesis of the DNA gets completed. Once the transfer of the genetic material from F+ to F- strains gets completed, the F- strain with the genetic material now becomes a donor or an F+ strain. It becomes a donor with a very high frequency.

Three types of plasmids based on their mobility include conjugative, mobilizable, and non-mobilizable plasmids. A protein gets involved in the conjugative machinery. It is known as relaxase. It is an important protein capable of recognizing the origin of transfer (OriT). The OriT is a short DNA sequence required in the cis position. Relaxase catalyzes the initial and final stages of conjugation. It resembles rolling circle replication proteins. The mobilizable plasmids thus carry OriT, relaxase gene, and nicking auxiliary proteins. Though conjugative and mobilizable plasmids appear similar in their properties, still they exhibit a difference in the machinery required for gene transfer.

Hfr strain:

The high-frequency recombination strains (Hfr) originate by rare crossovers. The Hfr strain arises due to the integration of the F factor into the bacterial chromosome. Such type of F factors is known as episomes. Hence, it replicates as a part of the bacterial chromosome. The Hfr cells conjugate with the F- strains. The nicked strands in the integrated factor F get transferred to the recipient F- strain, thereby transferring the bacterial genes. The transferred strand gets copied along with the genes. Recombination occurs in the recipient. Though the genes get copied, an F- strain never acquires Hfr phenotype because a complete copy of the F factor of the Hfr strain does not retain. Only a part of the F factor gets transferred.

Occasionally, the Hfr cell may not be efficient in excision of the F factor. The host chromosome adjacent to the F factor sometimes gets integrated into it due to an aberrant excision. Not only one but many segments get aberrantly inserted into it. During this excision, the F factor plus bacterial genes loop out of the chromosome. It leads to the formation of F’ factor. This type of conjugation is known as F-duction or sexduction.  

Image 2: Hfr strain

Bacterial gene mapping using conjugation:

The interrupted mating experiment helped in mapping the bacterial genes. It involved a cross between the F – and Hfr strains.

1.     Hfr strain had genes such as Hfr H thr⁺ leu⁺ azi^R ton^R lac⁺ gal⁺str^s

2.     The recipient had genes such as F^- thr leu azi^S ton^S lac gal str^R

“S” indicates sensitive and the “R” indicates resistant. The generation of the recombinants results from a double crossover. At various time intervals, the conjugating pairs broke apart and the transconjugants plated on a selective agar medium. It helped in studying the gene transfer. A single F factor gets integrated into Hfr strain. The interrupted mating experiment revealed the circular structure of the E. coli linkage map.

What is an inter-kingdom conjugation?

Nitrogen-fixing bacteria undergo an inter-kingdom conjugation. Agrobacterium tumefaciens and Agrobacterium rhizobium undergo inter-kingdom conjugation. A few pieces of evidence also report the inter-kingdom conjugation between the bacteria and the yeast. Hence, it is not necessary for the bacteria to undergo conjugation between their species. Inter-kingdom gene transfer is an example of horizontal gene transfer between two species, or different organisms.

Applications in genetic engineering:

The transfer of the genetic material through the process of conjugation involves convenience. It is possible to transfer genes from one bacterium to another, from bacteria to the yeast, plants or other cells. With conjugation, it is possible to use or synthesize a metabolite. Conjugative bacteria show the ability to pick up new plasmids from the environment. Recombinant DNA technology uses plasmids as cloning vectors. 

References:
[1] Int Std Ed-General Biology, Peter Russel
[2] Genetics of Bacteria, Sheela Srivastava
[3] Introduction to Genetics: A Molecular Approach, Terry Brown

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