GENETICS

Genetics is the study of heredity. Heredity is a biological process where a parent passes certain genes onto their children or offspring. Every child inherits genes from both of their biological parents and these genes in turn express specific traits. Some of these traits may be physical for example hair and eye color and skin color etc. On the other handsome genes may also carry the risk of certain diseases and disorders that may pass on from parents to their offspring.

 

Gregor Johann Mendel was a scientist and Augustinian friar and abbot of St. Thomas' Abbey in Brno who gained posthumous fame as the founder of the modern science of genetics. Though farmers had known for centuries that crossbreeding of animals and plants could favor certain desirable traits, Mendel's pea plant experiments conducted between 1856 and 1863 established many of the rules of heredity, now referred to as the laws of Mendelian inheritance.

 

Mendel worked with seven characteristics of pea plants: plant height, pod shape and color, seed shape and color, and flower position and color. With seed color, he showed that when a yellow pea and a green pea were bred together their offspring plant was always yellow. However, in the next generation of plants, the green peas reappeared at a ratio of 1:3. To explain this phenomenon, Mendel coined the terms “recessive” and “dominant” in reference to certain traits. (In the preceding example, green peas are recessive and yellow peas are dominant.) He published his work in 1866, demonstrating the actions of invisible “factors”—now called genes—in providing for visible traits in predictable ways in the journal “Annual Proceedings of the National History Society” of Brunn. However, his paper was not accepted till 1990.

 

TERMINOLOGY

Gene: The basic unit of heredity; a segment of DNA which contains the information for a specific characteristic or function.

Allele: An alternative form of a gene that occurs at the same locus on homologous chromosomes, e.g., A, B, and O genes are alleles.

 

Genotype: the genetic make-up (the assemblage of alleles) of an individual.

 

Phenotype - the physical or chemical expression of an organism’s genes.

 

Homozygous – Individual possessing a pair of identical alleles for a particular locus (gene).

 

Heterozygous - Individual possessing a pair of unlike alleles for a particular locus (gene).

Gamete - a haploid (n) sex cell in plants and animals (egg or sperm)

Haploid - the condition of having only one set of chromosomes per cell (n)

Diploid - the condition of having two sets of chromosomes per cell (2n)

Dominant allele - an allele that is always expressed when present, regardless of whether the organism is homozygous or heterozygous for that gene.

Recessive allele - an allele that is only expressed when the organism is homozygous for that allele and not expressed when heterozygous (when paired with a dominant allele).

Parent generation (P) - the generation that supplies gametes to the filial generation.

Filial generation (F) - the generation that receives gametes from the parental generation (Filial means brother in Latin).

Linkage: The tendency for genes or segments of DNA which are located close together on the same chromosome to be inherited together.

Progeny Testing: It is a test of the value for selective breeding of an individual's genotype by looking at the progeny produced by different matings.

Cross pollination (fertilization) - Pollination by genetically different plants

 

Self pollination (fertilization) - The natural or artificial pollination of a female flower with pollen from the same genotype.

 

Wild type (WT): It refers to the phenotype of the typical form of a species as it occurs in nature.

 

Mutant: An organism, gene, or chromosome that is different from the wild type by one or more.

 

Mendel’s Law:

There are about three laws proposed by Mendel.  They are

 

Mendel's first law (also called the law of segregation) states that during the formation of reproductive cells (gametes), pairs of hereditary factors (genes) for a specific trait separate so that offspring receive one factor from each parent.

 

Mendel's second law (also called the law of independent assortment) states that chance determines which factor for a particular trait is inherited.

 

Mendel's third law (also called the law of dominance) states that one of the factors for a pair of inherited traits will be dominant and the other recessive, unless both factors are recessive.

Some alleles do not have complete dominance and instead have incomplete dominance by expressing an intermediate phenotype, or codominance by expressing both alleles at once.

Figure 2-1. A pea flower with the keel cut and opened to expose the reproductive parts.

 

 

 

 

The seven character differences studied by Mendel:

Applications of Mendel’s Law

Mendel’s laws can be easily applied

a.       In breading experiments of plants and animals

b.      In improving the varieties of plants and animals

c.       In studying the various physical, physiological and pathological traits in human beings

d.      In the improvement of eugenics

 

Results of All Mendel’s Crosses in Which Parents Differed in One Character

Parental phenotype

F1

F2

F2 ratio

1. Round×wrinkled seeds

All round

5474 round; 1850 wrinkled

2.96:1

2. Yellow×green seeds

All yellow

6022 yellow; 2001 green

3.01:1

3. Purple×white petals

All purple

705 purple; 224 white

3.15:1

4. Inflated×pinched pods

All inflated

882 inflated; 299 pinched

2.95:1

5. Green×yellow pods

All green

428 green; 152 yellow

2.82:1

6. Axial×terminal flowers

All axial

651 axial; 207 terminal

3.14:1

7. Long×short stems

All long

787 long; 277 short

2.84:1

Law of dominance experiment (Monohybrid Ratio)

The phenotypic ratio of different types of individuals occurring in the F2 generation of the monohybrid cross is called the monohybrid ratio. In the Mendelian monohybrid experiments, this ratio was always 3:1 ( i.e., 75% is dominant and 25% is recessive). For example, for one of his monohybrid crosses, Mendel selected true breeding homozygous parents showing contrasting characters for the height of the plant. He performed the experiment in three stages as described.

 

According to Mendel, each sexually reproducing diploid organism possesses two 'factors' (genes) for each character; one factor is received (inherited) from male parent and the other factor is inherited from the female parent. These two factors for a particular character are called alleles or allelomorphs. When an offspring receives identical alleles from both parents, it is called homozygous, pure or true breeding for the character. On the other hand, when the offspring receives dissimilar alleles from two parents, it is called heterozygous, impure or a hybrid for that character.

 

The pure tall is crossed with the pure dwarf parent. According to Mendel, when the diploid individual (having both the alleles/factors) produces gametes, each gamete receives only one of the two factors/alleles of a character. No gamete receives both the alleles of a character. Thus, pure tall parent produces only one type of gametes, i.e. all the gametes possess only (T) factor for tallness. Similarly, all gametes produced by pure dwarf are of one type only and possess (t) factor. The fusion of (T) and (t) gametes (fertilization) results in the F1 offspring with (Tt) genotype. It is heterozygous or a hybrid. Its phenotype (external appearance) is tall because the factor for tallness (T) is dominant and expresses itself. The factor for dwarfness (t) is present in F1 hybrid but, being recessive, does not express itself (remains hidden).

Mendel allowed hybrids to self-fertilize or inbreed to raise F2 generation. The F1 hybrid has dissimilar alleles (Tt). Therefore, it will produce two types of gametes in equal number i.e. 50% gametes will have (T) factor and remaining 50% will have (t) factor. Since the pea flower is bisexual, it produces both male and female gametes. Thus, the F1 hybrid will produce two types of male gametes (T) and (t) in equal numbers. Similarly, there will be two types of female gametes (T) and (t) in equal numbers. During self fertilization, the fusion between these male and female gametes occurs at random. For example, each type of male gamete has an equal chance to fuse with either (T) or (t) female gametes and vice-versa. This chance fusion, between two types of male and two types of female gametes will produce a maximum of four combinations (genotypes) in the F2 progeny. This is shown in the checker board or Punnet’s Square. These four combinations fall into three categories of the genotypes as follows : 1 (TT), 2 (Tt) and 1 (tt) i.e.

1 Pure tall : 2 Hybrid tall : 1 Pure dwarf

      (TT)           2(Tt)                (tt)

 

This is called 1:2:1 genotypic ratio of a monohybrid cross. However, phenotypically, the progeny shows 3 Tall and 1 Dwarf individuals (75% Dominant and 25% recessive characters) or 3:1 ratio. This is called monohybrid ratio or phenotypic ratio of a monohybrid cross.

 

The result indicates that even though the recessive character was not seen in hybrid, it was present there and reappeared in pure form in 25% individuals of the progeny. This result also enabled Mendel to conclude that the two factors (alleles) come together in the hybrid but do not mix or fuse with each other. They simply remain together without diluting or contaminating each other. In other words, factors maintain their purity. As the gamete always receives only one factor (alleles) for a trait, it is always pure for the character. This is called purity of gametes.

 

Reason for tallness as dominants:

The growth of body is controlled by pituitary hormone or somatotrophic or growth hormone.  Dominant gene for tallness is responsible to develop normal pituitary and to produce sufficient growth hormone that results normal size or tallness.  Even heterozygous condition is enough to produce the sufficient hormone.  In homozygous recessive condition neither normal pituitary is developed nor sufficient growth hormone secreted that is why they become dwarf.

 

Exceptions from Mendel’s Law: (Deviations from Law of Dominance)

There are some exceptions to the “rules” of Mendel. They are truly exceptions in that they do not alter any of his conclusions or application of his ideas.

 

Incomplete Dominance: In some cases, there is no clear dominant or recessive allele. For example, some heterozygous flowers display an intermediate coloration. This occurs in such cases because both genes keep functioning. Since these genes code for a colored protein, a heterozygote ends up making half of each color. Note that it is fortunate that Mendel’s pea plants weren’t subject to this phenomenon, or he might not have come to his amazing conclusions.

It is the situation in which both alleles of the heterozygote influences the phenotype.  The phenotype is usually intermediate between the two homozygous forms.  In the snapdragon (Antirrhinum majus) or the Four O clock plant (Mirabilis jalapa), a cross between red and white flowered plants produces an all pink F1 progeny.  This intermediate inheritance or partial inheritance is called incomplete dominance.  The F1 when selfed gives F2 progenies which are red, pink and white in the ration of 1:2:1.  Pink is therefore heterozygous phenotype.  The phenotypic and genotypic ratios are the same in the case of incomplete dominance. However, incomplete dominance is not an evidence for blending inheritance.  One fourth each of the F2 progeny have the parental red and white phenotypes, which would not be possible if blending truly occurred.  The alleles of the genes are therefore discrete or particulate.

 

This phenomenon is also seen in Andulsian fowl, when a homozygous black feathered fowl is crossed to a homozygous white feathered hen, the F1 generations are hybrids showing blue color of feathers.  On selfing, these F1 individuals, F2 generations show 1 black : 2 blue : 1 white feathered individuals.

 

Codominance:

The situation in which a heterozygote shows the phenotypic effects of both alleles equally.  When the dominant character is not able to suppress, even incompletely the recessive character and both the characters appear side by side in F1 hybrids, the phenomenon is called as codominancy.

It can be seen in leaves pattern in Trifolium species (Clover plants), coat color in cattle and blood groups in humans.

The best examples come from cattle.  In cattle, if cattle with black coat is crossed with white coat, the F1 heterozygotes are found to possess roan coat.  In roan coat, black and white patches appear separately but no hair has intermediate color of black and white.

 

Co-Dominance of Multiple Alleles Humans have three alleles that contribute to blood type: A, B or neither, which is termed “O.” An individual only has two of the possible alleles (one on each homologous chromosome). However, none of the alleles are dominant. Rather, they are all fully expressed, which is referred to as co-dominance. An individual inheriting two A alleles will have a blood type of AA, two B alleles will have BB, an A and a O will have AO, a B and an O will have BO, an O and an O will have type OO, and finally an A and a B will have type AB. This so far is like a simple Punnett square analysis. However, the blood type protein is one of those “self-recognition” proteins on the surface of blood cells, and receiving a transfusion of the “wrong” blood type can induce a serious immune reaction requiring treatment. Functionally, since O is “null”, AA and AO are the same, BB and BO are the same, and AB is unique. Someone with type A (AA or AO) can receive AA, AO or OO. Someone with type B can receive BB, BO or OO. Someone with type AB can receive any blood type, while someone with type O can only receive OO.

 

Another “exception” to Mendel is Polygenic Inheritance. This refers to phenotypes that result from the combined action of more than one gene. For example, the size of a bird’s beak results from the interplay of a number of genes, each of which has a dominant and recessive allele.

The same is true for human height. As a result of the contribution of multiple dominant and recessive alleles of different genes, we observe a gradation of height, rather than the distinct tall or short forms of Mendel’s pea plants.

 

 

GENE MAPPING IN HAPLOIDS AND DIPLOIDS

Gene mapping describes the methods used to identify the locus of a gene and the distances between genes.  There are two distinctive types of "Maps" used in the field of genome mapping: genetic maps and physical maps. While both maps are a collection of genetic markers and gene loci, genetic maps' distances are based on the genetic linkage information, while physical maps use actual physical distances usually measured in number of base pairs.  Genetic markers themselves usually consist of DNA that does not contain a gene. But because markers can help a researcher locate a disease-causing gene, they are extremely valuable for tracking inheritance of traits through generations of a family.

Genetic maps are useful in many ways: they allow us to understand the overall complexity and genetic organization of a particular species, improve our understanding of the evolutionary relationships among different species, can be used to diagnose, and perhaps, someday to treat inherited human diseases,  can help in predicting the likelihood that a couple will produce children with certain inherited diseases and provide helpful information for improving agriculturally important strains through selective breeding programs. Mapping the genes of different types of organisms (diploid, haploid, eukaryotic, prokaryotic) gives geneticists insight into genetic processes.

Figure 5.23. Not all restriction sites are polymorphic.

Restriction mapping

Genetic mapping using RFLPs as DNA markers can locate the positions of polymorphic restriction sites within a genome, but very few of the restriction sites in a genome are polymorphic, so many sites are not mapped by this technique. 

The simplest way to construct a restriction map is to compare the fragment sizes produced when a DNA molecule is digested with two different restriction enzymes that recognize different target sequences. An example using the restriction enzymes EcoRI and BamHI is shown in figure. First, the DNA molecule is digested with just one of the enzymes and the sizes of the resulting fragments are measured by agarose gel electrophoresis. Next, the molecule is digested with the second enzyme and the resulting fragments again sized in an agarose gel. The results so far enable the number of restriction sites for each enzyme to be worked out, but do not allow their relative positions to be determined. Additional information is therefore obtained by cutting the DNA molecule with both enzymes together. In the example shown in figure, this double restriction enables three of the sites to be mapped. However, a problem arises with the larger EcoRI fragment because this contains two BamHI sites and there are two alternative possibilities for the map location of the outer one of these. The problem is solved by going back to the original DNA molecule and treating it again with BamHI on its own, but this time preventing the digestion from going to completion by, for example, incubating the reaction for only a short time or using a suboptimal incubation temperature. This is called a partial restriction and leads to a more complex set of products, the complete restriction products now being supplemented with partially restricted fragments that still contain one or more uncut BamHI sites. In the example shown in figure, the size of one of the partial restriction fragments is diagnostic and the correct map can be identified.

 

 

A partial restriction usually gives the information needed to complete a map, but if there are many restriction sites then this type of analysis becomes unwieldy, simply because there are so many different fragments to consider. An alternative strategy is simpler because it enables the majority of the fragments to be ignored. This is achieved by attaching a radioactive or other type of marker to each end of the starting DNA molecule before carrying out the partial digestion. The result is that many of the partial restriction products become ‘invisible’ because they do not contain an end-fragment and so do not show up when the agarose gel is screened for labeled products. The sizes of the partial restriction products that are visible enable unmapped sites to be positioned relative to the ends of the starting molecule.

 

Figure 5.24. Restriction mapping.

The objective is to map the EcoRI (E) and BamHI (B) sites in a linear DNA molecule of 4.9 kb. The results of single and double restrictions are shown at the top. The sizes of the fragments given after double restriction enable two alternative maps to be constructed, as explained in the central panel, the unresolved issue being the position of one of the three BamHI sites. The two maps are tested by a partial BamHI restriction (bottom), which shows that Map II is the correct one.

Figure 5.25. The sequence 5′-CG-3′ is rare in human DNA because of methylation of the C, followed by deamination to give T.

The scale of restriction mapping is limited by the sizes of the restriction fragments

Restriction maps are easy to generate if there are relatively few cut sites for the enzymes being used. However, as the number of cut sites increases, so also do the numbers of single, double and partial restriction products whose sizes must be determined and compared in order for the map to be constructed. Computer analysis can be brought into play but problems still eventually arise. A stage will be reached when a digest contains so many fragments that individual bands merge on the agarose gel, increasing the chances of one or more fragments being measured incorrectly or missed out entirely. If several fragments have similar sizes then even if they can all be identified, it may not be possible to assemble them into an unambiguous map.

 

Restriction mapping is therefore more applicable to small rather than large molecules, with the upper limit for the technique depending on the frequency of the restriction sites in the molecule being mapped. In practice, if a DNA molecule is less than 50 kb in length it is usually possible to construct an unambiguous restriction map for a selection of enzymes with six-nucleotide recognition sequences. Fifty kb is of course way below the minimum size for bacterial or eukaryotic chromosomes, although it does cover a few viral and organelle genomes, and whole-genome restriction maps have indeed been important in directing sequencing projects with these small molecules. Restriction maps are equally useful after bacterial or eukaryotic genomic DNA has been cloned, if the cloned fragments are less than 50 kb, because a detailed restriction map can then be built up as a preliminary to sequencing the cloned region.

These ‘rare cutters’ fall into two categories:

The potential of restriction mapping is therefore increased by using rare cutters. It is still not possible to construct restriction maps of the genomes of animals and plants, but it is feasible to use the technique with large cloned fragments, and the smaller DNA molecules of prokaryotes and lower eukaryotes such as yeast and fungi.

 

If a rare cutter is used then it may be necessary to employ a special type of agarose gel electrophoresis to study the resulting restriction fragments. This is because the relationship between the length of a DNA molecule and its migration rate in an electrophoresis gel is not linear, the resolution decreasing as the molecules get longer. This means that it is not possible to separate molecules more than about 50 kb in length because all of these longer molecules run as a single slowly migrating band in a standard agarose gel. To separate them it is necessary to replace the linear electric field used in conventional gel electrophoresis with a more complex field. An example is provided by orthogonal field alternation gel electrophoresis (OFAGE), in which the electric field alternates between two pairs of electrodes, each positioned at an angle of 45° to the length of the gel. The DNA molecules still move down through the gel, but each change in the field forces the molecules to realign. Shorter molecules realign more quickly than longer ones and so migrate more rapidly through the gel. The overall result is that molecules much longer than those separated by conventional gel electrophoresis can be resolved. Related techniques include CHEF (contour clamped homogeneous electric fields) and FIGE (field inversion gel electrophoresis).

 

Figure 5.26. Conventional and non-conventional agarose gel electrophoresis.

 (A) In standard agarose gel electrophoresis the electrodes are placed at either end of the gel and the DNA molecules migrate directly towards the positive electrode. Molecules longer than about 50 kb cannot be separated from one another in this way. (B) In OFAGE, the electrodes are placed at the corners of the gel, with the field pulsing between the A pair and the B pair. OFAGE enables molecules up to 2 Mb to be separated.

Direct examination of DNA molecules for restriction sites

(A) To carry out gel stretching, molten agarose containing chromosomal DNA molecules is pipetted onto a microscope slide coated with a restriction enzyme. As the gel solidifies, the DNA molecules become stretched. It is not understood why this happens but it is thought that fluid movement on the glass surface during gelation might be responsible. Addition of magnesium chloride activates the restriction enzyme, which cuts the DNA molecules. As the molecules gradually coil up, the gaps representing the cut sites become visible. (B) In molecular combing, a cover slip is dipped into a solution of DNA. The DNA molecules attach to the cover slip by their ends, and the slip is withdrawn from the solution at a rate of 0.3 mm s-1, which produces a ‘comb’ of parallel molecules.

 

It is also possible to use methods other than electrophoresis to map restriction sites in DNA molecules. With the technique called optical mapping, restriction sites are directly located by looking at the cut DNA molecules with a microscope. The DNA must first be attached to a glass slide in such a way that the individual molecules become stretched out, rather than clumped together in a mass. There are two ways of doing this: gel stretching and molecular combing. To prepare gel-stretched DNA fibers, chromosomal DNA is suspended in molten agarose and placed on a microscope slide. As the gel cools and solidifies, the DNA molecules become extended. To utilize gel stretching in optical mapping, the microscope slide onto which the molten agarose is placed is first coated with a restriction enzyme. The enzyme is inactive at this stage because there are no magnesium ions, which the enzyme needs in order to function. Once the gel has solidified it is washed with a solution containing magnesium chloride, which activates the restriction enzyme. A fluorescent dye is added, such as DAPI (4,6-diamino-2-phenylindole dihydrochloride), which stains the DNA so that the fibers can be seen when the slide is examined with a high-power fluorescence microscope. The restriction sites in the extended molecules gradually become gaps as the degree of fiber extension is reduced by the natural springiness of the DNA, enabling the relative positions of the cuts to be recorded.

 

In molecular combing, the DNA fibers are prepared by dipping a silicone-coated cover slip into a solution of DNA, leaving it for 5 minutes (during which time the DNA molecules attach to the cover slip by their ends), and then removing the slip at a constant speed of 0.3 mm s-1). The force required to pull the DNA molecules through the meniscus causes them to line up. Once in the air, the surface of the cover slip dries, retaining the DNA molecules as an array of parallel fibers. Optical mapping was first applied to large DNA fragments cloned in YAC and BAC vectors. More recently, the feasibility of using this technique with genomic DNA has been proven with studies of a 1-Mb chromosome of the malaria parasite Plasmodium falciparum, and the two chromosomes and single megaplasmid of the bacterium Deinococcus radiodurans.

 

Bacterial Gene Transfer

1. Gene transfer in bacteria can be accomplished through conjugation, transformation, and transduction by phages.

 

2. Conjugation, transformation, and generalized transduction have in common one important property. Each process introduces a DNA fragment into the recipient cell; then a double-crossover event must take place if the fragment is to be incorporated into the recipient genome and subsequently inherited. Unincorporated fragments cannot replicate and are diluted out and lost from the population of daughter cells.

 

3. Sexduction by F′ factors and specialized transduction by phages are similar processes in that, in each case, a specific and limited set of bacterial genes is introduced into the recipient cell. After transfer, the F′ factor replicates in the bacterial cytoplasm as a separate entity, whereas specialized transducing-phage DNA is recombined into the bacterial chromosome by a phage-encoded recombination system. In both cases, a partial diploid (merozygote) results.

 

4. Gene transfer can be used to map the bacterial chromosome. Hfr crosses are first used to localize a mutation to a region of the chromosome. Then, recombinant frequency, generalized transduction, or transformation provides a more exact localization.

 

Discovery of conjugation

Conjugation was proved in 1946 by the elegantly simple experimental work of Joshua Lederberg and Edward Tatum, who studied two strains of Escherichia coli with different nutritional requirements. Strain A would grow on a minimal medium only if the medium were supplemented with methionine and biotin; strain B would grow on a minimal medium only if it were supplemented with threonine, leucine, and thiamine. Thus, we can designate strain A as met− bio− thr+ leu+ thi+ and strain B as met+ bio+ thr− leu− thi−. The Figure displays in simplified form the concept of their experiment. Here, strains A and B are mixed together, and some of the progeny are now wild type, having regained the ability to grow without added nutrients.

Figure 9-25. Recombination processes in bacteria.

Recombination processes in bacteria. Bacterial recombination requires that a bacterial cell receive an allele obtained from another cell. (a) In conjugation, a cytoplasmic element such as the fertility factor (F) integrates into the chromosome of a bacterial cell. During cell-to-cell contact, the integrated factor can transfer part or all of that chromosome to another cell whose chromosome carries alleles of genes on the transferred chromosome. The transferred segment recombines with a homologous segment in the recipient cell’s chromosome; in the example shown here, allele B thereby replaces allele b. (b) In transformation, a DNA segment bearing a particular allele is taken up from the environment by a cell whose chromosome carries a matching allele; the alleles (in our example, B and b) are then exchanged by homologous recombination. (c) In transduction, after a phage has infected a bacterial cell, one of the newly forming phage particles picks up a bacterial DNA segment instead of viral DNA. When this phage particle infects another cell, it injects its bacterial DNA, which recombines with a homologous segment in the second cell, thereby exchanging any corresponding alleles (in our example, A and a).

 

 

Figure 7-2. Demonstration by Lederberg and Tatum of genetic recombination between bacterial cells.

Demonstration by Lederberg and Tatum of genetic recombination between bacterial cells. Cells of type A or type B cannot grow on an unsupplemented (minimal) medium (MM), because A and B each carry mutations that cause the inability to synthesize constituents needed for cell growth. When A and B are mixed for a few hours and then plated, however, a few colonies appear on the agar plate. These colonies derive from single cells in which an exchange of genetic material has occurred; they are therefore capable of synthesizing all the required constituents of metabolism.

 

Lederberg and Tatum plated bacteria into dishes containing only unsupplemented minimal medium. Some of the dishes were plated only with strain A bacteria, some only with strain B bacteria, and some with a mixture of strain A and strain B bacteria that had been incubated together for several hours in a liquid medium containing all the supplements. No colonies arose on plates containing either strain A or strain B alone, showing that back mutations cannot restore prototrophy, the ability to grow on unsupplemented minimal medium. However, the plates that received the mixture of the two strains produced growing colonies at a frequency of 1 in every 10,000,000 cells plated (in scientific notation, 1 × 10−7). This observation suggested that some form of recombination of genes had taken place between the genomes of the two strains to produce prototrophs.

 

Figure 9-3. Some properties of the fertility (F) factor of E.

Figure 7-11. Summary of the various events that take place in the conjugational cycle of E.

Summary of the various events that take place in the conjugational cycle of E. coli.

Figure 9-6. Transfer of single-stranded fragment of donor chromosome, and recombination with recipient chromosome.

Transfer of single-stranded fragment of donor chromosome, and recombination with recipient chromosome. note: double crossovers can occur in any location; those shown are examples.

Transfer of F during Conjugation

Figure 7-5. (a) During conjugation, the pilus pulls two bacteria together.

Recombinant genotypes for marker genes are relatively rare in bacterial crosses, Hayes noted, but the F factor apparently was transmitted effectively during physical contact, or conjugation. A kind of “infectious transfer” of the F factor seemed to be taking place. We now know much more about the process of conjugation and about F, which is an example of a plasmid that can replicate in the cytoplasm independently of the host chromosome. The F plasmid directs the synthesis of pili, projections that initiate contact with a recipient and draw it closer, allowing the F DNA to pass through a pore into the recipient cell. One strand of the double-stranded F DNA is transferred and then DNA replication restores the complementary strand in both the donor and the recipient. This replication results in a copy of F remaining in the donor and another appearing in the recipient

Figure 9-5. The transfer of E.

The transfer of E. coli chromosomal markers mediated by F. (a) Occasionally, the independent F factor combines with the E. coli chromosome. (b) When the integrated F transfers to another E. coli cell during conjugation, it carries along any E. coli DNA that is attached, thus transferring host chromosomal markers to a new cell. (c) In a population of F+ cells, a few cells will have F integrated into the chromosome; these few cells can transfer chromosomal markers. Therefore, when a population of F+ cells is mixed with a population of F− cells, a few F− cells will acquire markers from the donor. (d) Occasionally, the integrated F can leave the chromosome and return to the cytoplasm. In rare cases, F can carry host genes with it, incorporating them into the circular F, which is now termed an F′. The F′ can transfer these genes at high efficiency to other cells, because they are part of the F′ genome.

Interrupted-mating conjugation

Figure 9-7. Interrupted-mating conjugation experiments with E.

Interrupted-mating conjugation experiments with E. coli. F− cells that are strr are crossed with Hfr cells that are strs. The F− cells have a number of mutations (indicated by the genetic markers azi, ton, lac, and gal) that prevent then from carrying out specific metabolic steps. However, the Hfr cells are capable of carrying out all these steps. At different times after the cells have been mixed, samples are withdrawn, disrupted in a blender to break conjugation between cells, and plated on media containing streptomycin. The antibiotic kills the Hfr cells but allows the F− cells to grow and to be tested for their ability to carry out the four metabolic steps. (a) A plot of the frequency of recombinants for each metabolic marker as a function of time after mating. Transfer of the donor allele for each metabolic step depends on how long conjugation is allowed to continue. (b) A schematic view of the transfer of markers over time.

 

Transformation

Some bacteria have another method of transferring DNA and producing recombinants that does not require conjugation. The conversion of one genotype into another by the introduction of exogenous DNA (that is, bits of DNA from an external source) is termed transformation. Transformation was discovered in Streptococcus pneumoniae in 1928 by Frederick Griffith; in 1944, Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty demonstrated that the “transforming principle” was DNA. Bacterial transformation can be demonstrated by using the genes for drug resistance. The exogenous transforming DNA is incorporated into the bacterial chromosome by a double-crossover process analogous to that depicted for Hfr × F− crosses.

 

Figure 7-16. Bacterium undergoing transformation (a) picks up free DNA released from a dead bacterial cell.

Bacterium undergoing transformation (a) picks up free DNA released from a dead bacterial cell. As DNA-binding complexes on the bacterial surface take up the DNA (inset), enzymes break down one strand into nucleotides; meanwhile the other strand may integrate into the bacterium’s chromosome (b).

Transformation can be used to assess linkage. When the DNA from the donor is extracted for use in a transformation experiment, some breakage into smaller pieces is inevitable. If two donor genes are located close together on the chromosome, then there is a greater chance that they will be carried on the same piece of transforming DNA and hence will cause a double transformation. Conversely, if genes are widely separated on the chromosome, then most likely they will be carried on separate transforming segments and the frequency of double transformants will equal the product of the single-gene transformation frequencies. Thus, it should be possible to test for close linkage by testing for a departure from the product rule. Hence, if a+ b+ donor DNA is used to transform a− b− recipient cells, then, if a and b are closely linked, the proportion of a+ b+ double transformants, or cotransformants, should exceed the product of the proportions of single a+ and b+ transformants. Relative map distances of closely linked genes can be deduced from cotransformation percentages in an approach similar to that used in transduction.

Transduction

Some phages are able to “mobilize” bacterial genes and carry them from one bacterial cell to another through the process of transduction.

 

Discovery of transduction

In 1951, Joshua Lederberg and Norton Zinder were testing for recombination in the bacterium Salmonella typhimurium by using the techniques that had been successful with E. coli. The researchers used two different strains: one was phe− trp− tyr−, and the other was met− his−. When either strain was plated on a minimal medium, no wild-type cells were observed. However, after the two strains were mixed, wild-type cells appeared at a frequency of about 1 in 105. Thus far, the situation seems similar to that for recombination in E. coli.

There are two kinds of transduction: generalized and specialized. Generalized transducing phages can carry any part of the chromosome, whereas specialized transducing phages carry only restricted parts of the bacterial chromosome.

Generalized transduction

Figure 7-26. The mechanism of generalized transduction.

Phages P1 and P22 both show generalized transduction. These temperate phages have different fates in the cell: P22 inserts into the host chromosome as a prophage, whereas P1 remains free like a large plasmid. However, both act as generalized transducers because, in the course of lysis, bacterial DNA is accidentally “stuffed” into the phage head. Inside the recipient cell, the transducing fragments integrate by double crossover utilizing the host recombination system. Generalized transduction can transfer any host gene. It occurs when phage packaging accidentally incorporates bacterial DNA instead of phage DNA.

 

Linkage date from transduction

Generalized transduction allows us to derive linkage information about bacterial genes when markers are close enough that the phage can pick them up and transduce them in a single piece of DNA. For example, suppose that we wanted to find the linkage between met and arg in E. coli. We might set up a cross of a met+ arg+ strain with a met− arg− strain. We could grow phage P1 on the donor met+ arg+ strain, allow P1 to infect the met− arg− strain, and select for met+ colonies. Then, we could note the percentage of met+ colonies that became arg+. Strains transduced to both met+ and arg+ are called cotransductants.  Linkage values are usually expressed as cotransduction frequencies. The greater the cotransduction frequency, the closer two genetic markers are.

 

Specialized transduction

Specialized transduction is due to faulty separation of the prophage from the bacterial chromosome, so the new phage includes both phage and bacterial genes. The transducing phage can transfer only specific host genes.  Lambda is a good example of a specialized transducing phage. As a prophage, λ always inserts between the gal region and the bio region of the host chromosome. In transduction experiments, λ can transduce only the gal and bio genes. 

Figure 9-23. Specialized transduction mechanism in phage λ.

Specialized transduction mechanism in phage λ. (a) The production of a lysogenic bacterium takes place by crossing-over in a specialized region. (b) The lysogenic bacterial culture can produce normal λ or, rarely, an abnormal particle, λdgal, which is the transducing particle. (c) Transduction by the mixed lysate can produce gal+ transductants by the coincor-poration of λdgal and a λ helper phage or, more rarely, by crossovers flanking the gal gene. The blue double squares are bacterial integration sites, the red double squares are λ integration sites, and the pairs of blue and red squares are hybrid integration sites, derived partly from E. coli and partly from λ.

 

The recombination between regions of λ and the bacterial chromosome is catalyzed by a specific enzyme system. This system normally ensures that λ integrates at the same point in the chromosome and, when the lytic cycle is induced (for instance, by ultraviolet light), it ensures that the λ prophage excises at precisely the correct point to produce a normal circular λ chromosome. Very rarely, excision is abnormal and can result in phage particles that now carry a nearby gene and leave behind some phage genes. In λ, the nearby genes are gal on one side and bio on the other. The resulting particles are defective due to the genes left behind and are referred to as λdgal (λ-defective gal), or λdbio. These defective particles carrying nearby genes can be packaged into phage heads and can infect other bacteria. In the presence of a second, normal phage particle in a double infection, the λdgal can integrate into the chromosome at the λ-attachment site. In this manner, the gal genes in this case are transduced into the second host. Because this transduction mechanism is limited to genes very near the original integrated prophage, it is called specialized transduction.

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