Enm

Cytosine

Figure 7. The base pairing between A:T and G:C in DNA.

3' direction. DNA helicases are essential motor proteins that function to unwind double-stranded (duplex) DNA by an active, rolling mechanism to yield the transient ssDNA intermediates for replication, recombination and repair (11). During the synthesis of DNA each deoxynucleotide is joined to the previous one through a phosphodiester bond. DNA polymerization occurs at a maximum speed of 100 and 1000 nucleotides per second for bacterial and animal cells, respectively. The open ends of the DNA at 3'-hydroxyl and 5'-phosphoryl position could be joined by DNA ligase (12). In certain mammalian cells, there are polynucleotide kinases that possess both 5-phosphotransferase and a 3-phosphatase activity that can restore DNA strand breaks, with 5-hydroxyl termini or 3-phosphate termini, or both, to a form that supports the subsequent action of DNA repair polymerases and DNA ligases, that is, 5'-phosphate and 3-hydroxyl termini (13).

A gene is a segment of DNA or, as in certain viruses, RNA made of a stretch of bases that, respectively, code for an RNA and a polypeptide molecule. Synthesis of a polypeptide from the transcript of a gene has been visualized (Fig. 8). It requires translational machinery to synthesize proteins from N-terminal to C-terminal by joining amino acids through their carboxy-terminals. In some viruses and eukaryotic organisms some genes and hence their RNAs are made of translatable and intervening sequences (known as exons and introns, respectively). The genetic code is read as a codon of three bases at a time. There are 64 codons, of which 61 code for amino acids and 3 for termination. There is some degeneracy in certain codons; for example, the amino acid glycine is coded for by the triplets GGA, GGG, GGU, and GGC. Different organisms have a bias in codon usage; for example, they may prefer to use one set of these over the other for the synthesis of polypeptides. Polypeptides in their nascent form are self-assembled into functional structures through the physi-cochemical properties of the constituent amino acids. Other polypeptides require a special class of proteins called nucleoplasmins, heat-shock proteins, and chaperon-

Figure 8. Transcription of bacterial DNA into an mRNA and its translation by polysomes into polypeptide. Source: Photograph courtesy of B. A. Hamakalo.

ins for assembly. Chaperonins are multifunctional proteins that catalyze the correct folding of other proteins by preventing side reactions such as aggregation (14). They are not themselves components of the final functional proteins. Different members of the chaperonin family assist in folding in a concerted manner. Some, but not all, molecular chaperonins are heat-shock or -stress proteins. Chaperonins act in the assembly of other proteins where they are not components of the final structure (14). Protein structure, folding, and enzymatic stability are dictated by co-valent, disulfide linkages, and by noncovalent forces and interactions (15). Mutational replacement of amino acids within certain regions of a protein could affect its conformation and, hence, function, for example, thermostability and reaction rates (15).

Mutations and Selections

Most heritable variation of phenotypes is explained by mutations in genotypes. Improved mutant organisms have been used in industrial microbiology and food and fermentation technology. The initial attempts at strain improvements for food technology were through selection for spontaneously occurring variants from the original strain (16). Subsequently, fundamental studies on the occurrence and induction of mutations, enrichment and isolation of mutants, site-specific mutagenesis, and use of genetic recombination have made this process more manageable (15,16). In vivo mutations in microorganisms can occur spontaneously or with the mediation of mutagens at frequencies in the 10_<7_9) or 10_(4_6) range, respectively. Mutations alter DNA base sequences, by adding, deleting, or substituting base(s) and often affect the structure, and hence the function, of proteins. Mutagenic agents fall into three classes: physical (UV and X-ray irradiation), chemical (nitrous acid or nitrosoguanidine), and biological (transposons and mutagenic bacteriophages) (16).

A special class of genetic elements that are capable of insertion into any DNA are known as insertion sequences (ISs) and transposons (TNs). Insertion sequences constitute an important component of most genomes. More than 500 individual ISs have been described (17). The organization of a typical IS includes discrete DNA sequences of 0.8 to 1.4 kbp with a short DNA sequence at each terminus or 30 to 40 bp inverted repeats (IRs). Transposons are a class of genetic elements that contain at least one gene such as gene(s) for metabolism, antibiotic resistance, or synthesis of toxins (class I TNs), and in addition the trans-posase (class II TNs) (18). Insertion of a TN into the genome of a host requires the presence in the recipient DNA of ISs. Two functions are associated with a TN DNA once inserted into DNA molecules in a host cell. First, TN through its own insertion causes disruption of the sequence of a gene leading to a deletion-type mutation (18,19). Second, TN can affect the regulation of the expression of genes in the neighborhood of their integration site; for example, by inactivation of one gene and increased transcription of another adjacent gene (20). Another example is the deactivation of a regulatory phenotype by TN insertions in the lac A gene, encoding an endogenous /?-galactosidase of B. subtilis, leading to inactivation of its negative regulator, ZacR (21). The mechanism of such phe-notypic change is due to effects involving one or several elements internal to the TN. On the other hand, the loss and restoration of mutator TN activity in maize has shown evidence against dominant-negative regulator associated with loss of activity (22).

As expected the exact removal of a TN DNA should restore the gene's function. This form of mutagenesis is called transposon mutagenesis and has wide application in genetic engineering (23). The end result of in vivo mutagenesis is alterations in DNA sequence(s), which must be fixed, replicated, and segregated. The rare mutant clone is then screened, enriched, purified, and characterized. One problem with in vivo mutagenesis is the difficulty in targeting specific genes. Certain industrially useful mutants are hard to grow or maintain because of the multiple or deleterious mutations they acquire during induced, but random mutagenesis. These problems can be eliminated by using in vitro mutagenesis. Here, a gene sequence is isolated, cloned into a suitable vector, and usually treated with a chemical mutagen, to specifically alter a base. More commonly, an oligonucleotide with a modified sequence is synthesized and then inserted into a gene (15,23). Through these approaches it is now possible to generate gene- or site-specific mutations. Recombination between two mutants with different characteristics can generate new organisms for industrial exploitation (6).

Elements and Regulation of Gene Expression

Gene expression requires template DNA, which is divided into regulatory and structural regions, and RNA transcription to produce the intermediate RNA (mRNA, rRNA, and tRNA). Although the primary function of mRNA is to serve as a blueprint for translation of a genetic message into a polypeptide (23), certain RNA molecules, called ribozymes, can function as self-splicing catalysts (24). The discovery of ribozymes has changed our dogmatic views of catalysis being solely the domain of protein enzymes.

To regulate their growth and metabolism, organisms must determine when and how much cellular constituents are needed. This task is achieved by controlling gene expression (3,23). The following elements regulate and control prokaryotic gene expression (Fig. 9). The operator is a DNA sequence where a repressor protein binds and pre-

Operator

Repressor / Structural genes

Figure 9. Gene expression in prokaryotic organisms.

vents transcriptional occurrence. Repressor-operator recognition involves a complex code of DNA base-pair sequences. Induction refers to expression of a gene subsequent to the removal of the repressor, usually by the addition of specific compound(s) called inducers. The promoter is located immediately in front of the gene and is the RNA polymerase recognition and transcriptional initiation site. Natural promoter efficiency varies, some are stronger than others. During the transcription of prokaryotic genes, RNA polymerase identifies in the 5' to 3' direction of the sense strand of the promoter before the initiation co-don, a recognition sequence of bases, TTGACA or -35bps sequence, and a TATAAT (the Pribnow box) sequence at -10bps for its subsequent binding. Mutationally induced base changes in these two sequences can have mild or severe effects on transcription. Through its specificity for DNA transcriptional initiation sites, binding of RNA polymerase generates a localized melting and separation of the two strands of a small segment of the DNA leading to the initiation of RNA synthesis usually starting with GTP or ATP. DNA topoisomerases contribute to this process by relaxing DNA tension (25). There is continuous polymerization of ribonucleotides into an RNA transcript until the terminus of the gene is reached. At the terminus, either a sequence of six uridine residues and a hairpinlike structure or a sequence lacking such bases but requiring a termination factor, rho, to signal the stoppage of transcription. Some structural genes contain a leader and a trailer sequence, respectively, immediately after the promoter and before the terminator regions. At the end of transcription, RNA polymerase is freed to repeat the cycle.

The control of gene expression occur through transcriptional and translational control. One mechanism of control of transcription of genes is through induction of specific mRNA synthesis when an inducing substance is present (to turn on gene expression) and its repression (or turning off) once such inducers are removed. For example, when lactose is present in the growth medium of E. coli, the enzyme yS-galactosidase is synthesized and, conversely, when it is absent, the repression of the enzyme synthesis occurs through a repressor protein. This modality of regulation is called negative control. In addition, several positive regulators of RNA polymerase are involved in control. For example, intracellular regulator cyclic AMP (cAMP) through its binding to cAMP binding protein (CAP) creates a complex, cAMP-CAP, which binds to specific promoters to activate certain operons. Finally, through a process called attenuation, the biosynthesis of the amino acid tryptophan is controlled. In this case, the transcription of the biosyn-thetic genes begins in the usual manner. However, instead of a complete transcript of the genes, an incomplete transcript of relatively short mRNA (162 bases) responsible for the tryptophan leader protein is made. The leader protein is tryptophan rich. When intracellular tryptophan supply is high, the mRNA makes a structure to block further continuation of transcription, by stalling (attenuating) the translation. With low tryptophan levels, a different structure in the stem-loop region of mRNA develops, which allows the continuation with transcription to the end of the operon. Therefore, in the case of tryptophan operon, the removal of repressor and attenuation generates a 70-fold (de repression) and another 8- to 10-fold (attenuation) or an overall of 600- to 700-fold increase in the expression (23).

In certain eukaryotic genes and hence their mRNAs (Fig. 10) there are some sequences of about 1000 bp inserted (introns) between the coding sequences (exons). Transcription begins at the promoter (5') of a gene and proceeds through the exons and introns (if present) to the 3' terminus, producing a primary mRNA transcript or heterogeneous nuclear RNA. In eukaryotic cells, several processing steps must occur to change the primary mRNA transcript into functional mRNA and allow its translation into proteins. Any introns present are subsequently removed or spliced out. The actual ending of the transcription in some cases could be several hundred base pairs beyond the polyadenylation site of the 3' end of a mRNA. The third step required to change a primary mRNA transcript to a functional mRNA is the addition of a 5' cap.

Eukaryotic promoters are recognized by specific DNA-binding proteins of less than 100 amino acid residues in size that bind to DNA and activate or repress gene transcription. Most bacterial RNA polymerases are large multimeric proteins; for example, E. coli enzyme contains two a, one fS, and one /?' subunits. This holoenzyme has additional subunits, the d 70, o, and a subunits, which are integral to its functioning. In sporulating bacteria (eg, B. subtilis), RNA polymerase subunits are responsible for the selection of sporulation promoters. The RNA polymerases of eukaryotic organisms differ from the preceding both in molecular size and number of enzyme species. As many as two to four different types of RNA polymerases can be found in eukaryotic cells, and in several cases their ft and fi' subunits have conserved sequences. In higher eukary-otes, RNA polymerase I initiates transcription for rRNA genes from nucleolus, and RNA polymerases II and III transcribe mRNA, tRNA, and 5S rRNA from nuclear matrix (26). The transcriptional machinery of eukaryotic—as compared with the prokaryotic—cells is much more com-

Exon 1 Intron 1 Exon 2 Intron 2 DNA /II II -

Transcription hnRNA

I + Spliced fragment

Protein

Figure 10. Transcription of an eukaryotic gene containing introns and exons.

plex (27) and can involve allosteric control by DNA in the case of selective gene transcription (28). Functions of RNA can be regulated by antisense RNA, which is an RNA complementary to the sequence in mRNA that through complementary, stable base pairing, prevents its translation (29). Antisense RNA also regulates or even prevents gene expression in prokaryotic, plant, and animal cells.

Whether prokaryotic or eukaryotic, the mRNAs are translated into proteins using ribosomes, charged tRNA molecules, and auxiliary proteins, beginning with codons ATG or GTG (23). The ribosome is a large multifunctional complex composed of both RNA and proteins. Accumulating evidence suggests that rRNAs play a central role in the critical ribosomal functions of tRNA selection, and binding, translocation, and peptidyl transferase (30). Usually several ribosomes (or a polysome) are present on a mRNA. In prokaryotic cells transcription and translation occur concurrently within the cytoplasm; but in eukaryotic cells, translation occurs outside the nucleus. Translational control includes the ribosome-binding sequence (Shine-Delgarno sequences) and attenuation, which is prevention of translation of mRNA because of its folding back on itself. After its transcription, the mRNA is degraded. Messenger stability, among other things, depends on the presence of AU-rich sequences at 3'-end, resulting in shorter half-lives than those lacking such sequences. If a nonsense codon is reached either at the end or through mutations within a gene, the translation is halted and a partial polypeptide results. Finally, protein stability and half-life depends on proteases that degrade not only normal but also for certain heterologous and abnormal (mutant) proteins. In eukaryotic cells protein stability depends on the small protein ubiquitin, which when conjugated to proteins leads to their rapid degradation.

Genetic Exchange and Recombination in Vivo

Prior to the discovery of genetic engineering in vitro, it was known that genetic information can be exchanged in vivo among various organisms (3,4). The simplest forms of genetic exchange—transformation, transduction, and conjugation—are those found among bacteria. Only conjugation requires cell-to-cell contact. Conjugation was initially discovered in the common enteric bacterium E. coli, but it occurs in many genera of bacteria and between members of two kingdoms, for example, A. tumefaciens and many plants species (31), as well as E. coli and S. cerevisiae (32). In certain bacteria such as streptococci, mating depends on the presence of special mating pheromones, which are small polypeptides promoting mating aggregation. In fungi, mating occurs when two haploid cells of different mating types fuse to allow genetic exchange to occur. In yeasts this requires special mating control locus (MAT) and switching loci (HML or HMR), which are silent cassettes that introduce the a and a mating-cell types through direct transposition of required genetic cassettes. In the absence of sexual cycle, certain fungi use parasexual mating events. In transformation, whether natural or induced, naked DNA or DNA contained in membranous vesicles called transformosomes are transferred into a recipient cell (4). In generalized and specialized transduction, DNA

packaged into a bacteriophage (Fig. 11) is injected into a bacterial cell. The latter two modes of genetic exchange are used in recombinant DNA technology for packaging of rDNA (eg, bacteriophage X) or transformation of commercially available competent E. coli cells. Once inside a cell, homologous DNA can undergo recombination using recom-binational enzymes to generate hybrid DNA molecules. All mechanisms of genetic exchange are currently used in food research.

GENETIC ENGINEERING TECHNOLOGIES Instrumentation and Tools

Central to and fundamental in the process of genetic engineering is the isolation, manipulation, insertion into, and expression of DNA in a host cell (33). The instrumentation and tools needed for the technical tasks are shown in Table 1.

Organisms

Microorganisms, plants, and animals are used organisms in food production and processing technologies. While the production of microbial cells through fermentation technology has been a long-established art, that of plant cells in cultures is rather new. In the 1970s several groups studied the ability of plant cells and tissues to grow in liquid nutritive media. Cultures from diverse origins such as (1) organs (roots, flowers, anthers), (2) meristems (shoot, leaf), (3) callus (undifferentiated cell mass), (4) cells (homogenized tissues), and (5) protoplasts were found to grow on defined salts media containing a carbon source, vitamins, plant hormones, and various other nitrogenous substances. Plant cell biotechnology and economic value-added or value-derived products have matured more rapidly with advances in plant cell culture systems, construction of transgenic plants, and other applications of biotechnology (see later).

Figure 11. The bacteriophage lambda of E. coli ejecting its DNA.

1206 GENETIC ENGINEERING: PRINCIPLES AND APPLICATIONS Table 1. Basic Steps in a Simple Genetic Cloning

Step Tools or systems needed

1206 GENETIC ENGINEERING: PRINCIPLES AND APPLICATIONS Table 1. Basic Steps in a Simple Genetic Cloning

Step Tools or systems needed

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