MOLECULAR GENETICS AND CONTROL SYSTEMS

Molecular Genetics

Proteins, in their role as enzymes directly determine how the cell operates by determining which

chemical reactions occur. Consequently, in a sense the enzyme makeup of a cell defines the

cell’s metabolic processes. Thus, the inheritance which a dividing cell leaves to daughter cells

must include the information for directing the production of the same protein constituents as

found in the parent cell. The study of transmission of such information is called genetics.

The Process of Gene Expression

The DNA molecule is a very long double helix consisting of two chains. Both chains are

polymers constructed from four nucleotides, each characterized by a particular nitrogeneous

base: adenine (A), guanine (G), cytosine (C), thymine (T). moreover, the two chains in the DNA

double helix have complementary sequences: A in one strand is always paired with T in the

other, and G and C are likewise paired.

A gene is a DNA segment that codes for a functional product. Expression of a gene to

form the amino acid sequence of the corresponding protein (the gene product) proceeds by a

two-step process in which a corresponding messenger ribonucleic acid (mRNA) serves as an

intermediate. In the first step if gene expression, which is called transcription, a complex

oligomeric enzyme called RNA polymerase catalyzes synthesis of an mRNA molecule to direct

the synthesis of a peptide chain with a corresponding amino acid sequence. This process, which

is known as translation, involves many different components including ribosomes, the sites and

organizers of peptide synthesis, and several different transfer RNA (tRNA) molecules, which
bring chemically, activated amino acids into the peptide synthesis process in the sequence

prescribed by the mRNA.

Transcription begins by binding of the enzyme RNA polymerase to a signal sequence

called a promoter on one of the complementary double strands of DNA. The strand possessing

this enzyme binding sequence becomes the DNA template strand for synthesis of an mRNA

molecule. After some unwinding of the DNA double helix in the vicinity of the RNA polymerase

moves along the DNA template strand in the 3’ to 5’ direction. Using DNA-RNA base pairing

rules, the nucleotide sequence in the DNA template strand is employed to construct a

complementary nucleotide sequence in a (single-stranded) mRNA molecule. The mRNA being

synthesized runs antiparallel to the DNA template strand; that is the mRNA is synthesized from

the 5’ to the 3’ end.

Turning to the specificity of RNA, At the base of the lower loop of the tRNA molecule is

a sequence of three nucleotides called an anticodon. This anticodon base sequence is

complementary to a three-nucleotide segment of mRNA known as a codon. Biochemical

research has revealed that each codon is a “word” in the genetic message; each codon specifies

one amino acid. Since there are only four letters in the chemical alphabet of RNA (the four bases

A, C, G, U) and there must be at least 20 words (one for each amino acid), a language or code is

required for transmission of genetic information.

A different view of translation which focuses on the transmission of information from the

mRNA nucleotide sequence to the corresponding polypeptide amino acid sequence is shown

below
 A second summary of

the expression process,

reviewing and emphasizing

the control elements, is worthwhile since proper orchestration of these controls is essential for

success in recombinant DNA technology. An overall schematic of expression which tracks the

transmission of genetic information and the functions of control sequences is shown below:

At the outer flanks of the gene are the promoter and terminator sequences which control

initiation and termination of transcription. The DNA must also contain the translation control

signals so that these can be transcribed into the mRNA. Finally, during translation, the

portion of mRNA complementary to the structural gene, which contains the code for a particular

amino acid sequence, is employed to guide polypeptide synthesis.

 Often more than one ribosome is attached to a single mRNA chain at a time.

Polyribosomes are clusters containing several individual ribosomes. This arrangement, which

can be highly organized, permits simultaneous translation of information carried by different

portions of mRNA and thus lends speed and efficiency to protein synthesis.

The molecules of tRNA and ribosomal ribonucleic acid (rRNA), like mRNA, are

synthesized using a portion of the DNA chain as a template. Thus, the information carried by

DNA includes the structure of the translation devices for protein synthesis as well as the coded

information for protein primary structure.

It is important to recognize that protein synthesis mechanisms of eucaryotes and

procaryotes are different. Synthesis of mRNA and translation of mRNA to proteins on the

ribosomes occur at almost the same point within the nuclear region of the bacterial cell. In

eucaryotes transcription occurs in the nucleus. The mRNA molecules, after modification diffuse

through pores in the nuclear membrane into the cytoplasm where, at the ribosomes, translation is

accomplished. In eucaryotes and to a lesser degree in procaryotes, the translation product is

frequently modified to give the final, functional form of the molecule.

Split Genes and mRNA modification in Eucaryotes

Eucaryotic messenger RNA is modified at the 5’ end to add a structure called a cap which

helps stabilize the mRNA against attack by phosphatases and nucleases. Attached at the 3’

end of most mRNAs in eucaryotes are poly A tails which contain 150 to 200 residues of

deoxyadenylate. This property can be used to advantage to isolate eukaryotic mRNAs using a

column packed with particles to which poly T oligonucleotides are covalently attached.
 It is now conclusively established that many eukaryotic genes are mosaics of nucleotide

sequences which code for protein structure and of sequences which do not. Sequences of the

former class are sometimes called exons. The interjected noncoding sequences are designated

introns or intervening sequences (IVS). Intervening sequences are found between exons;

separating protein synthesis coding sequences with what appears superficially to be useless

information. Clearly, eukaryotic cells must have mechanisms to ensure that exons and introns are

identified and distinguished during gene expression so that only the exon sequences are

ultimately employed to direct translation.

The basic features of eukaryotic gene structure and expression are illustrated below

During transcription, the DNA template strand guides synthesis of an mRNA which is

complementary to the complete template. That is, both the exon and intron sequences are

transcribed into this mRNA.

 A number of splicing steps follow, apparently unique to eucaryotes, in which the regions

in the primary transcript derived from the introns are clipped out. The remaining regions, all

complementary to exon sequences, are spliced together in the same order as found in the primary

transcript. The mature mRNA which results may then be translated as described above to give

the protein gene product.

Introns can be very large, and the total amount of intron DNA in eucaryotes is probably

more than one-half of the cell’s total DNA.

Post Translational Modifications of Proteins

The polypeptide produced by mRNA translation is often not the final, biologically active form

of the molecule. The N-terminal methionine is sometimes removed. Two cysteine residues may

be oxidized to give a disulfide bond which can be significant factor in protein tertiary structure.

Other types of polypeptide chemical modifications include hydroxylation, phosphorylation,

and acetylation.

Some polypeptides have at their N terminus a short (15-30 residues) sequence of

hydrophobic residues. These signal sequences are important components in protein transport

across cell membranes. Secreted proteins generally contain such signal sequences. The prefix

“pre-” to the name of a protein to indicate the form which includes the signal sequence (for

example, prelysozyme).

Proteins may contain other amino acid sequences which are cleaved away to yield the

active form of the molecule. This device and the protein secretion process just outlined allow the
cell to compartmentalize protein synthesis and activation of different specific proteins at

different locations within or outside the cell.

Induction and Repression: Control of Protein Synthesis

Control devices, Induction and Repression cause change in the rates of protein synthesis

(and consequently in the amount of enzyme present) and act at the level of the gene. The greatest

similarity between these two kinds of controls is their sensitivity to the concentration of

relatively small molecules.

Bacteria are free living, isolated cells which consequently have almost no influence on

their external environment. Therefore, they must be very adaptable. Many bacteria indeed

possess such versatility. They can synthesize enzyme systems for effective utilization of many

different types of nutrients. Different enzymes are generally required for each nutrient.

Consequently, the bacterium must carry genetic information for them all. The sum total of all

information carried in the chromosomes is called the cell’s genotype.

However, such a bacterium will not require the same mix of enzymes for all these

nutrients, and synthesis of any extra protein represents a waste of valuable energy and

intermediary metabolites. Thus, for maximum efficiency in a particular environment, only a

portion of the total genetic information is expressed (actually synthesized) as protein. The term

phenotype denotes the observable features of an organism. The phenotype arises from a

combination of two factors, the genotype and the organism’s environment. Constitutive

enzymes are synthesized independent of the cell’s surroundings.

 The biosynthesis rates of inducible enzymes, however, are sensitive to environmental

influence. A common example is β-galactosidase synthesis. This enzyme catalyzes the

hydrolysis of the disaccharide lactose to its component monosaccharides, glucose and galactose.

The reaction is essential if the cell is to employ lactose as a nutrient since only the hydrolysis

products can be used in subsequent reactions. The E. coli bacterial cell regulates synthesis of β-

galactosidase in response to the need for this enzyme. Thus, the substrate induces the formation

of the enzyme.

An analogous situation arises with the repressible enzyme. For example, although E. coli

can make all the enzymes necessary to synthesize all 20 amino acids from simpler precursors,

these particular enzymes are not found in significant quantity when the amino acids are supplied

to the cell as nutrients. In this instance the end product of a biosynthetic pathway represses

synthesis of the enzymes for that pathway.

Although this discussion has emphasized regulation of bacterial enzyme synthesis,

similar mechanism applies for control of synthesis of other proteins in bacteria and in higher

cells. The set of proteins synthesized by cells and the associated biological and catalytic

activities can often do change in response to changes in the cell’s environment. This adaptation

ability poses problems in formulation of kinetics for cell reactions and in bioreactor design which

are unparalleled in reaction engineering for synthetic catalysis.

DNA Replication and Mutation

Since DNA contains the information required for proper development and function of the

living cell, it is essential that there be an almost foolproof mechanism for copying DNA. When a
cell reproduces, each of the offspring must receive a complete set of genetic data in the form of

DNA.

Actually, DNA replication occurs in a slightly more complex fashion than indicated above.

It is not built up in one continuous sequential pass; instead an enzyme called DNA

polymerase conducts synthesis of daughter strands in the 5’ – 3’ direction. This proceeds

relatively smoothly on the parental strand running 3’ – 5’. On the other parental strand, DNA

polymerase adds fragments of the daughter strand in the 5’ – 3’ direction which are then

covalently coupled by the enzyme DNA ligase. This device achieves overall daughter strand

synthesis in the 3’ – 5’ direction.

There are some differences in DNA storage and duplication between prokaryotes and

eukaryotes. In the ill-defined nucleus region of prokaryotes, there is a single chromosome, or

carrier of genetic information, which consist of circular double strand of DNA. A chromosome
in eukaryotes consists of a DNA molecule associated with proteins and possibly some RNA.

Eukaryotic cells typically contain several chromosomes.

A mutation is a change in DNA which is passed to succeeding generations. In molecular

terms, mutation involves an alteration in nucleotide sequence of DNA. To some extent mutation

is a spontaneous process which is constantly occurring. The rate of spontaneous mutation is

rather low. A typical value is 1 error for every 106 gene duplications.

The importance of a gene-copying error depends on its nature. There are several

postulated mechanisms for spontaneous mutation. First the nucleotide bases of DNA have

several different structural forms, known as tautomers. Another possible cause of spontaneous

mutation is interference with the enzymes necessary for DNA synthesis and repair. Finally, some

intermediates of normal cellular metabolism, e.g, peroxides, nitrous acid, and formaldehyde, are

mutagens, i.e., chemicals which interfere with DNA function.

Another common cause of mutation is radiation. In particular, ultraviolet light is strongly

absorbed by DNA, to such extent that exposure to ultraviolet light rapidly kills most cells. The

surviving cells exhibit a high rate of mutation. All cells possess enzymatic machinery to repair

DNA occasionally damaged by ultraviolet light. These repair enzymes, in a rather elaborate

process, replace the damaged segment, which contain covalently linked pyrimidine residues.

The phenomenon of mutation occupies several important niches in biochemical

engineering practice. Mutation in microorganisms can improve them for our use. Thus, mutagens

and exposure to ultraviolet light have been employed in attempts to obtain mutated, more

productive protists. On the other hand, mutations can create processing difficulties.
Alteration of Cellular DNA

In addition to mutation, a variety of processes change the genetic material within a cell. These

have both positive and negative implications from the stand point of industrial biotechnology.

From the former perspective, ant method for altering a cell’s DNA content provides an

opportunity for development of more productive strains. On the other hand, an unplanned

modification of a desired species’ DNA can cause expensive failures of industrial bioprocess.

Virus and Phages: Lysogeny and Transduction

We know that viruses are agents of human disease. Also, several commercial biological

processes employing bacteria (including cheese-making and antibiotic production) can be

severely disrupted by viral infection of the bacteria. The subgroup of viruses which infect

bacteria are called bacteriophages or just phages.

Viruses are constructed of protein and nucleic acid and also may include lipoproteins.

The function of the protein is to house and protect the nucleic acid viral component and

sometimes to attach the virus to a living cell. The nucleic acid component which may be either

DNA or RNA, is ultimately responsible for the infection and its aftermath.

Outside a living cell of the proper type, the virus is an inert particle which cannot

reproduce by itself. Multiplication of the virus occurs after it has infected a host cell. Thus,

viruses are parasites.

Considering the virus called phage λ, which infects the E. coli bacterium. After attaching

to the cell wall, the phage injects its DNA into the cell’s interior. At this point two alternative
courses are possible One option is a state of lysogeny, where the phage DNA, now called the

prophage, is integrated into the bacterial chromosome. If this occurs, the cell lives and

reproduces normally, at the same time copying the prophage and creating more lysogenic cells.

Phages, like λ, which can enter lysogenic relationship with their hosts are called temperate

phages.

The other possible outcome of temperate-phage infection, called the lytic cycle,

invariably kills the host cell. During the lytic cycle the phage is said to be in a vegetative state:

the phage DNA literally takes over control of the cell. It first directs the ribosomes to synthesize

enzymes which destroy the host cell’s DNA and which will multiply the phage DNA. Then the

protein components necessary to create an intact phage particle are synthesized. These proteins

spontaneously join with phage DNA to form complete, highly organized bacteriophage particles.

After this self-assembly of many new phages, the enzyme lysozyme is synthesized.

It is clear why phage infestation of commercial cultures can be a serious problem. The

phage particles are very small, they can multiply rapidly in the right environment, and they can

hide in the relatively dormant state of lysogeny.

Occasionally, during reproduction of the phage within its host, some phage particles are

formed which contain a small portion of the host cell’s chromosome. When one of these

transducing phages injects its DNA into another bacterium, the DNA derived from the first host

may cross over with a fragment of the new host’s chromosomes. In this process, called

transduction, the genetic characteristic of the new host may be altered.

Bacteria Transformation and Conjugation

Transformation is a process of genetic transfer by free DNA. Here a double-stranded DNA

fragment enters recipient cells which, because of their physiological state, are competent to take

up external DNA. If the transformed fragment is similar to a fragment to the recipient’s DNA,

alteration of the recipient chromosome by crossing-over occurs rapidly.

Besides chromosomes fragments, plasmids may be introduced into fiving bacteria by

transformation. A plasmid is a DNA molecule that is separate from the bacterial

chromosome and duplicates independently. While usually nonessential for normal cell

function, plasmids can confer useful properties upon the cell they inhabit. Closely related to

plasmids are episomes, DNA molecules which may exist either integrated into the cell

chromosomes or separate from it. One of the better known episomes is the F, or fertility, factor

which characterizes the partners in conjugating E. coli cells. During conjugation of E. coli, cells

containing the F factor (F+ cells) transmit it to F- cells, and occasionally some of the chromosome

of the F+ partner is transferred to the F- cell. R (resistance) factors may be transmitted in a similar

fashion.
Cell Fusion

Multiple genetic modifications, including crossing of genetic material from different species, can

be achieved by fusing different cells together. The first step as shown above is preparation of

protoplasts, cells that have been stripped of their outer cell wall and which are contained only in

the cell membrane. Various cell wall hydrolase enzymes are applied to remove the cell walls,
and hypotonic medium is employed at this stage to minimize breakage of the osmotically

sensitive protoplasts. Incubation of the protoplasts results in cell wall regeneration and reversion

to normal cell morphology. This is a critical property since, after fusing protoplasts from

different strains, we wish to recover an organism which can be cultured further by normal

methods, potentially on a large scale. Protoplast fusion is induced and accomplished in solutions

of polyethylene glycol, and selection methods similar to those used in traditional microbial

genetics are used to isolate protoplast fusion products.

Cell fusion may also be used to obtain genetically crossed animal or plant cells. An

example of this type of manipulation, which has numerous current and potential practical

implications, is the formation of hybridoma cells by fusion of antibody-producing white cell with

a myeloma (skin cancer) cell of a mouse or other animal. Each hybridoma cell synthesizes a

single molecular species of antibody. Cells cultured from a single such hybridoma produce a

monoclonal antibody, in pure form. Monoclonal antibodies are bases of many diagnostic

methods in current practice or under development. In immobilized form, monoclonal antibodies

provide highly specific affinity adsorbents for laboratory and large-scale purification of antigen.

Also, monoclonal antibodies may be widely used in the future for tumor imaging and for drug

delivery to specific cell types in the body.

Commercial Application of Microbial Genetics and Mutant Population

Often, improved productivity of a mutual microorganism has a straightforward

interpretation in terms of basic cellular control systems. Understanding of the mechanisms

controlling biosynthesis of the desire’s product has another important application: deciding how

to formulate and control the growth medium so that productivity is maximized.

Cellular Control Systems:

Implication for Medium Formulation

There are two different approaches to altering cellular productivity via choice of medium

composition: (1) add inducers and (2) decrease amount of repressor present. Both

approaches are rather obvious on the surface, but in practice some sophistication is

required to achieve optimal results.

Beyond the enzyme’s substrate itself, non-metabolizable substrate analogs can be

extremely effective inducers for enzyme production. For example, β-galactosidase specific in E.

coli can be increased more than 1000-fold by galactosides.

Catabolite repression is known to decrease product biosynthesis in many important

processes. Availability of a rapidly consumed substrate such as glucose at high concentrations

fosters rapid growth but limits production of the antibiotics, penicillin, mitomycin, bacitracin,

and streptomycin. Other examples of repressors whose concentration can be directly

controlled by medium formulation include inorganic phosphate and ammonia.

Maintenance of low repressor concentrations is more difficult when the repressor is

synthesized by the microorganism. One useful approach in this case is to alter the cell’s

membrane so that the repressing substance quickly diffuses from the cell’s interior to the

medium.

Utilization of Auxotrophic Mutants

 Auxotrophic mutants lack enzyme activity for one or more steps in a biosynthetic

pathway. As a result, one or several end products of the pathway are not synthesized. In order for

such a mutant to survive, it must be fed the unsynthesized metabolites. For example, a

tryptophan auxotroph must be fed tryptophan in order to grow. A strain which synthesizes its

own tryptophan is said to be prototrophic for tryptophan.

Since the missing metabolites are supplied in the growth medium and are not synthesized

by the auxotroph, the engineer rather than the microorganism has control of the metabolites’

concentrations. Clearly this can be a desirable situation from a practical viewpoint when the

unsynthesized end product is a repressor.

As shown above, auxotrophic mutants lacking repressor synthesis can be made to overproduce

an intermediate metabolite. By keeping the concentration of repressor E low in the medium,

feedback inhibition and repression of pathway enzyme are minimized. The intermediate C will

then achieve much higher concentrations than in the native organism.

 The role of auxotrophy in commercial –lysine manufacture using C. glutamicum is

illustrated above. The productive mutant lacks homoserine dehydrogenase, so that the inhibitory

effect of threonine on lysine synthesis (via aspartokinase) is eliminated. Since the auxotropic

mutant does not synthesize threonine or methionine, both these amino acids must be supplied in

the growth medium.

Organisms with similar biosynthetic pathways do not necessarily utilize identical control

systems. Differences may confer commercial advantage.

Mutants with Altered Regulatory Systems

Several other types of genetic manipulation and selection have provided commercially superior

strains by altering controls at the enzyme and or at the gene level. Mutants of this type can be

used to increase yields of metabolites and enzymes.

 To obtain overproduction of a metabolite which acts as an inhibitor and or repressor of its

biosynthesis, we seek mutant organisms whose relevant allosteric enzymes and operons are

insensitive to the metabolite’s presence. Such mutants are often isolated using antimetabolites,

which are toxic analogs of the metabolite in question. Normal cells will not usually grow in a

medium containing antimetabolite since the antimetabolite represses or inhibits biosynthesis of

the necessary metabolite without serving as a substitute for the unsynthesized metabolite in

subsequent pathways. On the other hand, strains with deficient feedback controls will not alter

their biosynthesis patterns or rates in response to the antimetabolite and will therefore survive in

its presence. The table below lists some of the microbial products whose yields can be enhanced

by this technique.
 Although resistance to an antimetabolite may involve a variety of control system

alterations, one possibility is elimination or reduction of repression of enzymes in the

metabolite’s biosynthesis pathway.

Other methods can be used to identify and isolate constitutive mutants. In these species,

normally induced or repressed enzymes are produced whether or not inducer or repressor is

present. Among the enzymes whose yields can be improved with constitutive mutants are β-

galactosidase, catalase, phosphates, proteases and amidase.

Recombinant DNA Technology

The tools of recombinant DNA permit precise construction of new genetic instructions

which can be inserted into and utilized by living cells.

A brief schematic of a method for cloning a segment of DNA is shown above. Cloning means

obtaining a colony of genetically identical cells which contain the DNA segment of interest.

Cloning of a DNA fragment provides enough of that DNA sequence for detailed analysis

and for use as a reagent in subsequent biochemical and genetic manipulation.

Enzymes for Manipulating DNA

Several enzymes which can be used to cut, alter, and join DNA molecules in the test tube have

been discovered and subsequently their commercial supply. Prominent among these crucial

enzymes are the restriction endonucleases which recognize and cut specific nucleotide

sequences within the DNA molecule.

The utility of restriction enzymes in recombinant DNA technology derives from their

specificity in cleaving DNA at particular sites. Also, because recognition sites of 6 or 4

nucleotides in length are not found frequently in a DNA molecule, the DNA fragments obtained

by restriction enzyme hydrolysis, typically several hundred nucleotides in length, are sufficiently

long to contain useful genetic information and sufficiently short to allow convenient physical and

biochemical in vitro manipulation.

Vectors for Escherichia coli

Vectors are DNA molecules which provide propagation of a DNA fragment in a growing cell

population. A useful vector for cloning should have the following properties:

1. Ability to replicate in the host cell

 2. Ability to accommodate foreign DNA of various sizes without damage to replication

functions

3. Easy insertion in host cell after in vitro DNA manipulations.

4. Contains a selection marker to facilitate rapid, positive selection of cells which contain

the vector.

5. Contains only one target site for one or more different restriction endonucleases.

Two different classes of vectors – plasmids and bacteriophages – have been developed for

cloning DNA in E. coli.

Plasmids are introduced into E. coli by transformation. Since a transformed E. coli cell

may receive only a single plasmid and characterize, and utilize DNA sequences of interest, it is

absolutely essential that the plasmid be able to replicate in the growing bacterial cell. This

requires inclusion in the plasmid of an origin of relication – a nucleotide sequence, which directs

and regulates replication so that each cell contains a reasonably consistent number of plasmid

copies.

Selection markers are also important. By including, for example, a gene for antibiotic

resistance in the plasmid, we can rapidly and positively select for cells containing plasmids. This

is done by allowing the cells on medium containing a level of the antibiotic which kills plasmid-

free cells but which allows growth of cells containing plasmids and hence the antibiotic

resistance gene. Another common strategy for selection is to use a mutant host lacking an

enzyme required for growth in a particular medium and to include on the recombinant plasmid

the normal gene for that enzyme. The enzyme expressed from the plasmid gene complements the

genetic lesion in the host.

Selection markers in plasmids can serve two quite distinct function in recombinant DNA

technology. Fist, laboratory investigations are greatly accelerated by positive selection

procedures which rapidly identify vector-containing colonies. Second, in subsequent growth of

cells containing the plasmid, imposing selection pressure – by adding antibiotic to growth

medium, for example – minimizes competition from any plasmid-free cells that may be born

during population growth.

Characterization of Cloned DNAs

Suppose now that recombinant plasmids have been transformed into a population of E. coli cells,

and those cells containing plasmids have been identified by growth on agar plates made with

selective medium.

First, we can distinguish between clones which have plasmids with inserts and those

which contain only reclosed plasmids by applying insertional inactivation during the cloning

procedure.

Consequently, clones containing recombinant plasmids may be identified by first

growing tetracycline plates (to eliminate the background of cells with no plasmids). Then, the

replica plating method is applied in which a felt pad or nitrocellulose paper is pressed on the

original, master plate and then pressed onto a second plate, inoculating the replica plate with an

exact copy of the colony distribution on the master. In this example, the medium used for the

replica plates would contain ampicillin. Colonies present on the master plate which do not grow

in the replica plate are those which contain plasmids with inserts in the ampicillin resistance

gene.
 Screening for specific nucleotide sequences in the clone may be accomplished by several

different procedures which are all based on hybridization of denatured DNA from the clone with

a nucleic acid probe. The nucleotide sequence of the probe whether mRNA or DNA, is

complementary to a portion of the sequence of the DNA segment of interest, mRNA probes are

obtained by isolating mRNA pools from cells enriched in the message of interest, while DNA

probes are obtained by an earlier cloning step or perhaps by direct chemical synthesis.

Plasmid DNA may be isolated by ultracentrifugation in a cesium chloride density

gradient, then treated with a restriction enzyme. The resulting DNA fragments provide a kind of

fingerprint of the plasmid DNA. These fragments may be sized electrophoresis is 0.5 to 1.5

percent agarose gels, where the fluorescent dye ethidium bromide, specific for double-stranded

nucleic acids, is frequently used to visualize the bands corresponding to DNA fragments of

different sizes. By analysis of the fragment sizes obtained with other restriction enzymes and by

multiple restriction enzymes together, a restriction map showing the relative positions of various

restriction sites can be generated. Restriction mapping is extremely useful to verify DNA

insertions at particular target points in the plasmid and also provides a basis for further

manipulation of the recombinant plasmid.

The radiolabelled probes mentioned earlier may be used to identify particular restriction

fragments of interest. In the Southern blotting method, the DNA in the gel is denatured with

alkali, and the gel is covered with cellulose nitrate filter paper. Buffer solution drawn from moist

filter paper below this sandwich to dry filter paper above it carries DNA from the gel to the

cellulose nitrate paper where it binds. The bound, denatured DNA is then exposed to probe and
then to x-ray film as before to identify gel bands containing nucleotide sequences

complementary to that of the probe.

Two different methods for rapid and efficient sequencing of DNA, the Maxam-Gilbert

and Sanger techniques, were invented in the 1970s and have had tremendous impact in the

biological sciences and biotechnology.

In the Maxam-Gilbert method, the double-stranded DNA is first labeled radioactively at

one end of each strand. The DNA is then denatured, and a preparation of one of the two type of

strands is divided into four aliquots, each of which is treated with different chemicals. Each of

the four chemical treatments destroys selectively one or two bases and cleaves the DNA at those

points. It is critical to the method that this destruction be incomplete –ideally, we would like the

chemical treatment to cleave each strand only once. The fragments resulting from these four

parallel treatments are then run on a long polyacrylamide gel. Gel band positions are visualized

by exposure to x-ray film. Sequences of the order of 200 nucleotides may be routinely

determined on a single experiment using this method. The Sanger DNA sequencing is usually

used when sequencing extensive domains of DNA.

Genetic Engineering Using Other Host Organisms

E. coli is the workhorse of cloning and genetic engineering because it is the organism, we know

best at the molecular level. However, E. coli is not a familiar industrial microorganism and does

not ordinarily secrete proteins into the surrounding medium. Also, as a prokaryote, E. coli cannot

conduct RNA splicing or post-translation protein modification characteristic of eukaryotic cells.

For these and other reasons, there is a great interest in developing cloning and expression
systems for organisms other than E. coli. In all cases it is important to keep in mind the following

potential barriers to expression of a foreign gene:

1. Host cell nuclease attack on foreign DNA or RNA

2. Vector replication malfunction

3. Poor activity of promoter or transcription terminator

4. Incomplete mRNA splicing

5. Inefficient translation

6. Protease attack.

Also, we need an efficient procedure for introducing the vector into host cells with high yields.

Methods exist for molecular coloning and gene expression in several bacteria. Perhaps

the most extensively studied is Bacillus subtilis, a gram positive, nonpathogenic, nonparasitic,

industrial microorganism used before the advent of genetic engineering to manufacture several

enzymes and polypeptide antibiotics. B. subtillis secretes some of its protein into the surrounding

medium. Product secretion is a desirable feature for some genetic engineering applications since

secreted proteins in the medium will not be contaminated by large quantities of closely related

intracellular proteins. Also, if the product protein is secreted, it may be possible to obtain higher

product densities than is possible with an intracellular protein.

Several plasmids and bacteriophages are available for cloning in B. subtilis. Transformation,

transduction, and protoplast fusion methods can be used to introduce foreign DNA.

The last example introduces a new theme – metabolic engineering. With genetic engineering we

can not only produce proteins useful in their own right, but we can also add to a living cell more
of some particular enzymes, regulatory proteins, permeases, or any other protein. Further, we

may be able to give to the cell completely new enzymatic, regulatory, or transport activity which

we would never expect to find in nature or through use of random mutagenesis. Accordingly, we

now have the capability to redesign and rebuild in a carefully controlled and directed fashion

some parts of the metabolic network of the cell.

Growth and Reproduction of a Single Cell

Growth of an organism may be defined as an orderly increase in all its chemical

constituents. When most single-celled organisms grow, they eventually divide. Consequently,

growth of a population usually implies an increase in the number of cells as well as mass of

cellular material: either parameter may be used to investigate cell growth quantitatively.

The life cycle of a single cell may be represented schematically by either a circular or a

linear map as shown below:

In either case, cell division is the key benchmark. This point is conventionally the right hand end

of a linear diagram of the cell cycle, with the left hand denoting the smallest or youngest progeny

cell.

The Cell Cycle of E. coli

The simple prokaryote E. coli dividesby binary fission and can, in rich medium, attain doubling

times of 20 minutes or less. E. coli is a rod shaped bacterium whose length provides a reasonable

measure of xell volume and cell mass. Microscopic observation of elongation of individual E.

coli cells indicate that length increase occurs continuously over the cell cycle and appears to be

well approximated by an increasing exponential function of time. Total protein increases through

the cell cycle in a similar fashion.

Under many growth conditions giving different doubling times, the time for replication of

the E. coli chromosomes is approximately constant at a value around 40 minutes. During DNA

synthesis the rate of advance of replication fork is also constant. Hence, the rate of DNA

synthesis is directly proportional to the number of active replication forks. DNA replication

always begins at a particular initiation point, the origin of replication, on the chromosome. Two

forks move in opposite direction from the origin of replication around the circular E. coli

chromosome and converge at the terminus, at which point replication is complete.

Cell division control is coupled with DNA synthesis : the bacterial cell divides

approximately twenty minutes after replication fork reaches the terminus of the chromosome.

DNA synthesis takes place at a constant rate for the first firty minutes of the cell cycle, and the

cell synthesizes no DNA during the final twenty minutes of growth before division. However
growth of an E. coli cell with a doubling time of fifty minutes implies a more complicated

pattern of DNA synthesis in which a new set of replication forks begin synthesizing DNA 10

minutes before the cell divides. Consequently, DNA is synthesized during the final 10 minutes of

cell growth at double the rate observed at any time in the slower growth case.

The Eukaryotic Cell Cycle

There is no such thing as a typical eucaryote, however this imaginary cell does serve as a useful

device when discussing general features common to many eucaryotes.

In the eukaryotic cell cycle, there is more differentiation between various parts of the

cycle than in prokaryotes. The standard pattern of the eukaryotic cycle is given below:
It is subdivided into four phases, G1, S, G2, and M whose relative durations are indicated in the

figure. In the G1 phase, protein and RNA are actively synthesized, but DNA is not. Following the

G1 phase, the chromosomes are replicated in the S phase. The significance of the G2 phase is not

fully understood at present. After its completion, cell division starts (M phase). Division in

eukaryotic proceeds by a well orchestrated proceess called mitosis. During mitosis, the two

sets of chromosomes are separated and partitoned into each of the progeny cells.

In contrast to the prokaryotic linear pattern, enzyme synthesis is almost periodic in

eukaryotes. A particular enzymes increases sharply in amount at a definite point of the cell cycle.

Bailey and Ollis, 1986 (Biochemical Engineering Fundamentals)