Principles Of Genetics

The hereditary material of most cellular organisms is DNA, although in many bacteriophages and plant or animal viruses it could be either DNA or RNA. DNA is largely the basis for the preservation of the instructions for the organization, structure, and functioning of living cells. Historically, it was realized that the passage of hereditary traits to one's offspring depended on those that were found a b c iirtl*» * i < t l i » i c ■ ( i a b c iirtl*» * i < t l i » i c ■ ( i

Figure 1. DNA obtained from human genome (a) has been hybridized by either (b) DNA or (c) RNA by Southern blotting method. Source: Photograph courtesy of J. M. Mac Pherson.

in the parents. In the early 1860s the Austrian monk Gregor Mendel not only experimentally confirmed this in pea plants, but also established the principal rules governing the appearance of specific traits, that is, the parental factors (subsequently these became known as genes) and their assortment into individual progeny. He also made the distinction between the external appearance of such traits (later named phenotypes) and those that are reflections of the composition (later named genotypes). Mendel also advanced the idea of the expression of traits when they were in single copies from those that required two copies (later named dominant and recessive genes). The advent of microscopy and the discovery of dyes that could differentially stain cellular parts permitted the examination of the anatomical features of cells including the nucleus. The hereditary material, housed in distinct and organized structures, were named chromosomes. The number of chromosomes per cell are constant in any species. In most cells, the chromosomes are found in pairs of homologous structures, varying in number from a few two to three dozen. The concept of ploidy was developed to reflect the presence of haploid (N) and diploid (2N) cells, such as those found in bacteria and gametes or sex cells and in nonso-matic or fertilized cells, respectively. The N was defined to be the number of nonhomologous chromosomes found in a cell.

Analysis of Hereditary Materials and Exchanges

In the 1940 to 1950s the nature of DNA was elucidated by the use of quantitative genetic experiments, application of radioisotopes to analytical biochemistry, and the discovery of the electron microscope. These tools permitted the visualization of DNA and demonstrated its many activities: replication, repair, exchange, and recombination.

The structures of prokaryotic (eg, bacterial) and eukaryotic (eg, yeasts, fungi, plant, and animal) cells, which are both of significance in food science, and their genetic apparati have been investigated in great detail (3,4). The best-studied prokaryotic cell, the workhorse of many genetic engineering technologies and a causative of certain foodborne illnesses, is that of the bacterium Escherichia coli. This bacterial cell is cylindrical in shape, 1 X 0.5 //m in size (Fig. 2), and can grow and double in number every 20 min. The E. coli chromosome is in a covalently closed circular (ccc) form about 1 mm long and is made of 4,600,000 base pairs (4600 kilobase pairs, kb). The chromosome of this bacterium is mapped and completely sequenced.

We are beginning to realize that the structure of the genome in bacteria is more dynamic and diverse than once thought and accepted. Analysis of more than 150 bacterial species and isolates for genome size indicates a range, 580 kbp for Mycoplasma genitalium and 9200 kbp for Myxo-coccus xanthus, the smallest and the largest known genomes, respectively (3). These two organisms carry a gene number of 470 and more than 10,000, respectively, representing the lifestyles of an obligate parasite within living hosts and a metabolic generalist, undergoing sporulation, mycelial growth, and differentiation. The geometry of chromosomal DNA is found in a double-stranded circular, lin

Figure 2. The simple binary fission of Escherichia coli cells along with the production of DNA-less minicells. Source: Ref. 30.

ear, and folded configuration called the nucleoid or folded chromosome. Some bacteria have more than one set of chromosomes; for example, Deinococcus radiodurans has four chromosomes per cell in its stationary phase, suggesting that the classification of bacteria as haploid is an oversimplification (3). The current view of bacterial chromosome replication is that the DNA replication machinery is held at a relatively fixed position (at inner cell membrane) through which chromosomal DNA is threaded to produce two daughter chromosomes (5).

Often DNA of extra chromosomal origin, called plas-mids, which are autonomously replicating pieces of DNA about 1/100 of the chromosome size, are found in the cytoplasm. These plasmids frequently contain genes for antibiotic resistance, conjugation, and production of proteins generally deemed nonessential to normal cell functioning. Some bacterial plasmids are nonconjugative, yet others are conjugative and permit their and/or other plasmids' transfer within the species or a broad range of hosts from different genera. Certain conjugative plasmids can transfer DNA between kingdoms; for example, the Ti plasmids of the bacterium Agrobacterium tumefaciens can be transferred to dicotyledonous and monocotyledenous plants or to the yeast Saccharomyces cerevisiae. In all cases, transfer of plasmids requires cell-to-cell contact and presence of transfer and mobilizing genes.

Cells of the yeasts S. cerevisiae and Kluyveromyces marxianus (Fig. 3), which are used in fermentation, bread making, and food-grade enzyme production, are quasi-spherical, 3 to 5 fim in diameter, and can double hourly. Yeasts may be haploid or diploid, depending on their life-cycle stage. All eukaryotic cell chromosomes contain some basic proteins or histones, which wrap around the DNA to form nucleosomes. S. cerevisiae contains 17 linear chro

Figure 3. The yeast Kluyveromyces marxianus showing budding of a daughter cell and the location of previous bud scars.

mosomes ranging from 150 to 2500 kb. The collaboration of more than 600 laboratories in the United States, Canada, Europe, and Japan to sequence 12 billion bases and arrange 6000 genes of this yeast has been one of the largest decentralized experiments in molecular biology to date. A whole issue of the magazine Nature was devoted in 1997 to the yeast genome. DNA duplication in eukaryotic cells occurs during the S-phase of the cell cycle. Here the nuclear DNA will contain more than 100 putative replication factories, each with 300 or so replication points (forks) (6). Individual chromosomes can be separated by pulsed-field gel electrophoresis technique. Eukaryotic cells may also contain membrane-bound intracellular organelles such as mitochondria (Mtc), endoplasmic reticulum (Er), or chlo-roplasts (Chi). In Chi and Mtc (Fig. 4) a ccc DNA is compartmentalized and expressed. mtDNA are believed to have been derived from some prokaryotic cell genome. During cell division, all DNA, whether organellic, plasmid borne, or chromosomal, are divided equally and partitioned between the two daughter cells.

Bacterial cells are able to accept DNA from another parent or donor and undergo in vivo recombination. This was studied initially in the 1950s through the 1970s. These studies used whole cells and relied on natural exchange such as mating, transformation, and recombination occurring in vivo between donor DNA and recipient cells. During the 1960 to 1980s, after many of the requirements of DNA metabolism were discovered, it became possible to perform in vitro the synthesis, breakage, and joining the DNA from homologous or heterologous origins. These discoveries gave birth to the science and production of in vitro recombinant DNA (rDNA) molecules (7), which when transferred into living cells could express new traits.

Figure 4. Covalently closed circular DNA molecules from the mitochondrion of a fungus and E. coli plasmid pBR322.

The terms transformation, transduction, transfection, and conjugation are used to indicate "natural" gene transfer into a cell (4). Transformation and transfection involve transfer of donor DNA into a recipient cell, whereas conjugation and transduction require the presence of a donor cell and a bacteriophage, respectively. Both natural and CaCl2+ heat-induced transformation of many microorganisms by rDNA are now possible. Natural transformation of certain bacteria (eg, Bacillus subtilis) by linear duplex chromosomal DNA, and induced transformation of others (eg, E. coli) using ccc-plasmid DNA, has become common practice in genetic engineering (4). DNA can also be introduced into a cell through one of many laboratory-devised techniques of gene transfer, such as electroporation, particle gun or biolistics, microinjection, microlaser technique, and liposome fusion (see later). Transformation of many animal and plant cells is also possible, although the underlying mechanisms are quite different from those in bacteria. Transformation of cells can occur by intergeneric fusion of two individual cells from, in the order of their discovery, plant, microbial, and animal origins (8). Microbial and plant-cell fusion requires the production of protoplasts or cells without external surface layer(s). Protoplasts are osmotically unstable; in hypotonic environments they lyse, but in the presence of stabilizers (sugars, salts) they remain intact. When two protoplasts are brought into contact in the presence of a fusogenic substance (eg, Polyethylene glycol 6000) their membranes fuse, causing cytoplasmic and nuclear mixing events to occur. A transient fusant contains chromosomes from both parents; subsequently, karyogamy and recombination of nuclear materials and chromosomes can take place. With animal cells the fusogen could be an animal virus (8).

Analysis of the DNA as Genetic Code and Its Functions

The flow of genetic information is in general from DNA through transcription to RNA (messenger, transfer, and ri-bosomal RNA) and through translation of mRNA to proteins (Pig. 5). This was known as the central dogma of mo lecular biology until the discovery of the enzyme reverse transcriptase, which could synthesize a complementary DNA (cDNA) from an mRNA molecule. DNA and RNA molecules contain four bases: two purines, adenine (A) and guanine (G); and two pyrimidines, cytosine (C) and thymine (T) in DNA and uracil (U) in RNA. These bases are connected to the sugar deoxyribose (DNA) or ribose (RNA) to form a deoxynucleoside or nucleoside and are phosphor-ylated to form deoxyribonucleotides (DNA) or ribonucleotides (RNA), respectively (Fig. 6). The double helical or Watson-Crick form of base pairing for A:T and G:C in DNA was the first to be discovered (Fig. 7). Other types of Watson-Crick base pairing called k and n with a hydrogen bonding pattern (9) have increased the genetic alphabet from four to six letters. Many of the natural bases can be modified by addition of organic groups, for example, meth-ylcytosine. Nucleotides are linked through phosphodiester linkage, and each polymer of DNA has a 5' to 3' polarity. The ratio of (G + C)/(A + T) is known by the designation (G + C) content or % (G + C) and reflects taxonomic re-latedness and molecular characteristics. The (G + C) content of two different DNA molecules determines their separation during buoyant density-gradient centrifugation. Additionally, double-stranded (ds) DNA of higher %(G + C) will have a higher melting temperature (Tm) than that with a higher %(A + T). Through melting, dsDNA can be dissociated into two single-stranded (ss)DNAs, and through cooling down, they will regain the complementary ds structure. This process forms the basis for several DNA:DNA or DNA:RNA hybridization techniques. In vitro, every DNA molecule has a topological feature. The open-ended DNA molecule is either in ss or ds form and can be in rod shape. DNA molecules can bend; for example, a small polymer of242 base pairs (bp) or larger can go from a linear (open-ended) to a circular (ccc) form by the joining of its open ends with ligase. More complex forms of dsDNA are also found. DNA molecules can assume many forms, including winding or unwinding, depending on their physical and enzymatic environments, for example, presence of enzymes called topoisomerases (for twisting) and endonucleases (for nicking and relaxing the twisted DNA). In addition, the sequence of bases within a ssDNA could create structures of their own. When a sequence of -AAAAAAAGCTTTTTTT- from a DNA duplex is allowed to separate into two ss polymers, each can generate a hairpinlike structure through base pairing of all A and T residues. Such sequences are thought to be recognition segments within DNA for its interaction with regulatory proteins. RNA structures also have unique organizations such as folding on itself and forming of a hairpinlike structure (eg, tRNA).

All DNA synthesis is enzymatic and utilizes the four deoxyribonucleotides. The pathways for the biosynthesis of deoxyribonucleotide triphosphates and degradation of DNA are well known. The enzyme responsible for DNA biosynthesis, DNA polymerase, appears in the forms I, II, and III in prokaryotic and a, ft, and y in eukaryotic cells and, along with several ancillary proteins, comprise the DNA synthesis machinery (6,10-12). DNA polymerase is used either for repair, in a 3' to 5' direction (which could also be due to exonuclease activity) or replication in a 5' to

Figure 5. Flow of genetic information as perceived in molecular biology: (1) DNA replicates through DNA polymerase system; (2) transcription of DNA into RNA occurs via RNA polymerase system; (3) RNA is copied into DNA via reverse transcriptase action; (4) RNA as an enzyme can act on itself; (5) RNA being translated into proteins by the translational system.

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