Microbial Genetics (DNA Replication & Protein Synthesis), Recombinant DNA, Genetic Engineering
I. THE BASIS OF HEREDITY
All information necessary for life is stored in an organism’s chromosomes, which are made up of DNA (exception: some viruses only have RNA). A chromosome is circular (prokaryotes) or linear (eukaryotes). Remember the organic molecules stuff we covered at the beginning of the semester? Nucleic acids (DNA & RNA) are made up of building blocks called nucleotides (more on structure below). In DNA, nucleotides are arranged in a twisted double chain called a helix. The particular nucleotide sequence spells out the “genetic code” that provides information for the synthesis of new DNA (DNA replication necessary for cell division) and for the synthesis of proteins.
A typical prokaryotic cell contains a single circular chromosome. Bacteria may also have a small, circular piece of extrachromosomal DNA called a plasmid. Human body cells, examples of eukaryotic cells, have 46 linear chromosomes.
A gene, the basic unit of heredity, is a liner sequence of DNA nucleotides that form a functional unit of the chromosome or plasmid. All information for the structure and function of an organism is coded in its genes. The information in specific gene is not always the same; different versions of the same gene are called alleles. Using humans as an example, the hair color gene is always found at the same location on a chromosome, but the different versions or alleles that can exist for hair color are blond, brunette, red, etc. Because prokaryotes only have one chromosome, so they generally only have one allele for a particular gene. Many eukaryotes have 2 sets of chromosomes and thus 2 alleles of each gene, which may be the same or different. For example in humans, we have 46 chromosomes or 23 pair. In each pair, you get one chromosome from your mom and one for your dad. You may have a blonde hair allele from your mom and a dark hair allele from your dad (so you get dark hair since the dark hair allele is dominant).
II. DNA STRUCTURE – THE WATSON-CRICK MODEL [DNA = deoxyribonucleic acid]
In the Watson-Crick model, the DNA molecule is a double-stranded helix, shaped like a twisted “ladder.” Remember that nucleic acids (DNA & RNA) are made up of building blocks called nucleotides. Each nucleotide is made up of a sugar, a phosphate, and a nitrogenous base. When we put these nucleotides together to build a DNA ladder, the sides of the ladder are composed of alternating phosphate groups & sugar molecules. The rungs of the ladder are made up of paired nitrogenous bases joined in the middle by hydrogen bonds. The nitrogenous bases are adenine, thymine, guanine, & cytosine; adenine always pairs with thymine (A-T or T-A) & guanine always pairs with cytosine (G-C or C-G) [This is called complementary base pairing]. These 4 bases spell out the genetic message or code!
DNA enters into 2 kinds of reactions:
1.) Replication – replicates the DNA before cell division, so that each new daughter cell will receive a copy.
2.) Protein Synthesis (Gene Expression); 2 steps: transcription & translation
[Remember bacteria have a circular chromosome.]
Replication begins by an enzyme breaking the hydrogen bonds between the nitrogenous bases in the DNA molecule; the double stranded DNA molecule “unzips” down the middle, with the paired bases separating. As the 2 strands separate, they act as templates, each one directing the synthesis of a new complementary strand along its length.
If a nucleotide with thymine is present on the old strand, only a nucleotide with adenine can fit into place in the new strand; if a nucleotide with guanine is present on the old strand, only a nucleotide with cytosine can fit into place in the new strand, & so on. This is called complementary base pairing. DNA replication is called semiconservative replication since half of the original DNA molecule is conserved in each new DNA molecule. Like other biochemical reactions, DNA replication requires a number of different enzymes, each catalyzing a particular step in the process.
IV. GENE EXPRESSION – PROTEIN SYNTHESIS
A. FROM DNA TO PROTEIN: THE ROLE OF RNA
By the 1940’s biologists realized that all biochemical activities of the cell depend on specific enzymes; even the synthesis of enzymes depends on enzymes! Remember that the DNA molecule is a code that contains instructions for biological function & structure. Proteins (enzymes) carry out these instructions. The linear sequence of amino acids in a protein determines its 3-D structure & it is this 3-D structure that determines the protein’s function. The big question was: How does the sequence of bases in DNA specify the sequence of amino acids in proteins? The search for the answer to this question led to the discovery of RNA (ribonucleic acid), which is similar in structure to DNA (deoxyribonucleic acid).
Three types of RNA:
1. messenger RNA (mRNA) – single stranded; contains codons (3 base codes); mRNA is constructed to copy or transcribe DNA sequences.
2. ribosomal RNA (ribosomes!) (rRNA) – ribosomes “read” the code on the mRNA molecule & send for the tRNA molecule carrying the appropriate amino acid.
3. transfer RNA (tRNA) – clover leaf shaped; at least one kind for each of the 20 a. a. found in proteins; each tRNA molecule has 2 binding sites – one end, the anticodon (also a 3 base code), binds to the codon on the mRNA molecule; the other end of the tRNA molecule binds to a specific amino acid; each tRNA & its anticodon are specific for an a. a.!!
Differences between RNA & DNA:
1. RNA nucleotides contain a different sugar than DNA nucleotides. (ribose vs. deoxyribose).
2. RNA is single stranded – DNA is double stranded.
3. In RNA, uracil replaces thymine. There is no thyamine in RNA!!! But, there is adenine.
B. TWO MAJOR EVENTS IN PROTEIN SYNTHESIS:
1. Transcription [mRNA copies or transcribes DNA sequences]
This process is similar to what occurs in DNA replication. A segment of DNA uncoils unzips. Free RNA nucleotides, are then added one at a time to one end of the growing RNA chain. Cytosine in DNA dictates guanine in mRNA, guanine in DNA dictates cytosine in mRNA, adenine in DNA dictates uracil in mRNA, thymine in DNA dictates adenine in RNA. This complementary base pairing is just like what occurs in DNA replication. An enzyme catalyzes this process. After transcription the mRNA goes out in search of a ribosome. This mRNA molecule will now dictate the sequence of a. a. in a protein in the next step called translation.
2. Translation – actual synthesis of polypeptides or proteins; translate information from one language (nucleic acid base code) into another language (amino acids); remember, the sequence of amino acids (the protein’s primary structure) determines what the protein’s 3-D globular structure is going to be & structure determines function.
a. Initiation – Begins when the ribosome attaches to the mRNA molecule, reading its first or START codon. The first tRNA comes into place to pair with the initiator codon of mRNA (it occupies the peptide site in the ribosome). The START codon is AUG, which specifies the amino acid methionine. All newly synthesized polypeptides have to start with methionine.
b. Elongation – The second codon of the mRNA molecule is then read and a tRNA with an anticodon complementary to the second mRNA codon attaches to the mRNA molecule; with its a. a. this second tRNA molecule occupies the aminoacyl site of the ribosome. When both the P & A sites are occupied, an enzyme forges a peptide bond between the 2 a. a. & the first tRNA is released. The first tRNA cannot be released until this peptide bond is formed, as it will take its a. a. with it!! The second tRNA is then transferred from the A site to the P site & a third tRNA is brought into the A site. The ribosome continues to move down the mRNA molecule in this fashion, “reading” the codons on the mRNA molecule & adding amino acids to the growing polypeptide chain.
c. Termination – Toward the end of the coding sequence on the mRNA molecule is a codon that serves as a termination signal. There are no tRNA anticodons to complementary base pair with this codon. Translation stops and the polypeptide chain is freed from the ribosome. Enzymes in the cell then degrade the mRNA strand.
[In eukaryotic cells, the polypeptide is taken up by the rough e.r. & is modified into a 3-D protein; the proteins are then packaged into transport vesicles (a piece of the e. r. pinches off around the protein); these vesicles transport the proteins to the golgi complex for further modification; the finished protein is pinched off in a piece of golgi membrane (another vesicle) and is transported to the part of the cell where it is needed. In the prokaryotic cell, none of these organelles exist, modification/processing of the polypeptide into a protein occurs in the cytoplasm.]
The genetic code. The mRNA codons for the 20 universal amino acids.
See the table in your text of mRNA codons for the 20 amino acids. The 3-base codons are written to the left and the abbreviations of the amino acids they correspond to are written to the right.
The amino acid abbreviations in the table are: Ala – alanine; Arg – arginine;
Asn – apararagine; Asp – aspartamine; Cys – cysteine; Glu – glutamic acid; Gln – glutamine;
Gly – glycine; His – histidine; Ile – isoleucine; Leu – leucine; Lys – lysine; Met – methionine; Phe – phenylalanine; Pro – proline; Ser – serine; Thr – threonine; Trp – tryptophan;
Tyr – tyrosine; Val – valine.
The code has been proven to be the same for all organisms from humans to bacteria – it’s known as the universal genetic code.
Notice that most of the amino acids have more than one code (ex. Arg has 6 codes!). However, each code is specific for an amino acid (ex. UUU only codes for the amino acid Phe).
Three of the 64 codons do not specify amino acids. Instead they indicate STOP or termination of the translation process (they say “This is the end of the polypeptide.”)
The START codon is AUG, which specifies the amino acid methionine. All newly synthesized polypeptides have to start with methionine. Since AUG is the only codon for methionine, when it occurs in the middle of a message, it is ignored as a START codon and is simply read as a methionine-specifying codon.
A. A mutation is any chemical change in a cell’s genotype (genes) that may or may not lead to changes in a cell’s phenotype (specific characteristics displayed by the organism). Many different kinds of changes can occur (a single base pair can be changed, a segment of DNA can be removed, a segment can be moved to a different position, the order of a segment can be reversed, etc.). Mutations account for evolutionary changes in microorganisms and for alterations that produce different strains within species. Mutations often make an organism unable to synthesize one or more proteins. The absence of a protein often leads to changes in the organism’’ structure or in its ability to metabolize a particular substance.
B. Spontaneous mutations – occur by chance, usually during DNA replication. Only about one cell in a hundred million (108) has a mutation in any particular gene. Since full-grown cultures contain about 109 cells per milliliter, each milliliter contains about 10 cells with mutations in any particular gene. Because the bacterial chromosome contains about 3,500 genes, each ml of culture contains about 35,000 mutations that weren’t present when the culture started growing. Wow, when you think about it that’s a lot of mutations in just one ml!
C. Induced mutations are caused by chemical, physical, or biological agents called mutagens.
1. Chemical Mutagens – ex. Nitrates and nitrites are added to foods such as hot dogs, sausage, and lunch meats for antibacterial action. Unfortunately these same compounds have been proved to cause similar mutations and cancer in lab animals
2. Physical Mutagens – Include UV light, X-rays, gamma radiation, & decay of radioactive elements; heat is slightly mutagenic.
D. Consequences of Mutations – Most mutations do not change the cell’s phenotype. If the mutation changes the codon to another that encodes the same amino acid, the protein remains the same. For example if the DNA code is changed from AGA to AGG, the mRNA codon would change from UCU to UCC. Check your table! The amino acid would not change. The amino acid would stay serine. In this case the genotype is altered, but the phenotype stays the same. Having more than one codon for each amino acid allows for some mutations to occur, without affecting an organism’s phenotype. A mutation that changes a codon to one that encodes a different a. a. may alter the protein only slightly if the new a. a. is similar to the original one. However, if a mutation changes an a. a. to a very different one, there may be a drastic change in the structure of the protein, causing major complications for the cell. For example, if the structure of an enzyme called DNA polymerase was greatly altered, the cell would not be able to replicate its DNA and thus would not be able to multiply.
E. Repair of DNA Damage – Bacteria & other organisms have enzymes that repair some mutations.
VI. GENETIC TRANSFER
Gene transfer refers to the movement of genetic information between organisms. In most eukaryotes, it is an essential part of the organism’s life cycle and usually occurs by sexual reproduction. Male and female parents produce sperm and egg which fuse to form a zygote, the first cell of a new individual. Of course, sexual reproduction does not occur in bacteria, but even they have mechanisms of genetic transfer. Gene transfer is significant because it greatly increases the genetic diversity of organisms. We’ve already discussed how mutation account for some genetic diversity, but gene transfer between organisms accounts for even more. In recombinant DNA technology, genes from one species of organism are introduced into the genetic material of another species of organism. For example, human genes can be inserted into the bacterial chromosome.
A. BACTERIAL PLASMIDS & CONJUGATION
Most bacteria carry additional DNA molecules known as plasmids:
1. Plasmids are circular DNA molecules, much smaller than the bacterial chromosome.
2. Plasmids can move in and out of the bacterial chromosome.
3. Two important plasmids are fertility (F) plasmids and drug resistant (R) plasmids.
1. The F Plasmid – This plasmids contains about 25 genes, many of which control the production of F pili. F pili are long, rod-shaped protein structures that extend from the surface of cells containing the F plasmid. Cells that lack the F plasmid are known as female (recipient) or F(-) cells. Cells that possess the F plasmid are known as male (donor) or F(+) cells. F(+) cells attach themselves to F(-) cells by their pili and transfer a copy of an F plasmid to the F(-) cells through a pilus. The once F(-) cells are now F(+) and will now produce pili, because they now have the F plasmid that contains the plasmid genes that code for these pili. This transfer of DNA from one cell to another by cell-to-cell contact is known as conjugation and is a form of sexual recombination because new genetic material is introduced into the cell. This is as close to sex as bacteria get!
2. The R Plasmid – In 1959 a group of Japanese scientists discovered that resistance to certain antibiotics and other antibacterial drugs can be transferred from one bacterial cell to another. It was subsequently found that genes conveying drug resistance are often carried on plasmids. Over the last few decades, R factors have proliferated to the point that some infections are difficult to cure with antibiotics.
Note: Plasmids are very important to scientists involved in recombinant DNA research. Genes of interest can be inserted into plasmids. The plasmids are introduced to bacteria and the bacteria take them up by endocytosis. As the bacteria reproduce themselves by mitosis, they replicate the plasmid during interphase and pass it to their daughter cells. The plasmids can then be isolated from all of these bacterial cells and the gene of interest can be excised. In this way a large quantity of a gene of interest can be produced. We’ll talk about this more later.
B. TRANSFORMATION – A genetic change in which DNA leaves one cell, exists for a time in the aqueous extracellular environment, & then is taken into another cell where it may become incorporated into the genome. Ex. Extracts from killed, encapsulated, virulent (disease causing) bacteria, when added to living, harmless, unencapsulated bacteria, can convert the latter to the virulent type. By endocytosis, the living, nonvirulent bacteria pick up the DNA from the dead, virulent bacteria and incorporate the DNA into their own DNA. The nonvirulent bacteria now have the genes that code for proteins that transform them into virulent bacteria.
A LITTLE ABOUT VIRUSES
Viruses consist essentially of a molecule of nucleic acid (RNA or DNA) enclosed in a protein coat called a capsid. No cytoplasm, ribosomes, or other organelles are present. Viruses move from cell to cell, utilizing the host cell’s chromosomes, enzyme systems, and organelles to replicate the viral nucleic acid and synthesize new capsid proteins. They are obligate parasites in that they can’t multiply outside the host cell. Viruses that infect bacteria are called bacteriophages.
A virus will attach to a host cell and inject its nucleic acid into the host cell. The viral nucleic acid takes over the host cell’s genetic material and causes it to help the virus replicate the viral nucleic acid and to produce its capsids. The new viruses are assembled in the host cell (nucleic acids are inserted into the capsids) and the host cell is lysed to release the new viruses to go and attack other cells. This cycle is known as the lytic cycle. Viruses that infect bacterial cells are called bacteriophages.
C. Temperance or Lysogeny – This mechanism gives viruses the capacity to set up long-term relationships with their host cell. Instead of going through the lytic cycle, the virus nucleic acid remains integrated in the host cell’s chromosome. The virus may remain in this latent stage for long periods of time before initiating a lytic cycle. The problem with this type of cycle is that the integrated viral nucleic acid gets replicated along with the host cell’s chromosome during cell division & is passed to daughter cells (& then they pass it to their daughter cells, & so on). Something (ex. temperature change, stress) may later trigger these latent viruses to go into the lytic cycle all at once, destroying all of the infected host cells.
D. Transduction – Viruses can serve as vectors or carriers of genetic information from one bacterium to another. During the reproduction of the virus in the bacterium, fragments of bacterial DNA instead of viral nucleic acid may become accidentally incorporated into a viral capsid. Such “viruses” may be able to infect a new host cell, but they are not able to complete a lytic cycle. The genes they carry from a previous bacterial host may become incorporated into the chromosome of the new bacterial host, possibly giving the bacterium new characteristics (ex. drug resistance).
VII. GENETIC ENGINEERING
Genetic engineering refers to the purposeful manipulation of genetic material to alter the characteristics of an organism in a desired way. One of the most useful of all techniques of genetic engineering is the production of recombinant DNA – DNA that contains information from two different species of organisms.
For example a particular human gene can be removed from a human chromosome. Recombinant DNA is then constructed by inserting that gene into a bacterial plasmid, which serves as a carrier. The recombinant DNA is then introduced host bacterium, which takes up the plasmid. The host bacterial cell then divides and its daughter cells divide, producing millions of cells that all contain a copy of the human gene of interest. This process serves at least 2 purposes:
1.) Large quantities of the human gene of interest are produced.
2.) The bacteria can read the human gene of interest, producing the protein coded for on the gene by protein synthesis. The genetic code is universal! We can obtain large quantities of a particular protein using bacteria.
B. MEDICAL APPLICATIONS – (See Table 8.3 p. 211 for a more extensive list) – Products such as insulin to treat diabetes, human growth hormone to treat dwarfism, blood-clotting proteins to treat hemophilia, antibiotics, and vaccines have all been produced by bacteria using recombinant DNA technology.
a. Vaccines – Immunization means deliberately introducing an antigen into the body that can provoke an immune response & the production of memory cells. The first injection elicits a primary immune response, which provokes the production of antibodies & memory cells to provide long-lasting protection against disease. Many vaccines are made from killed pathogens (called inactivated vaccines); pathogens can be killed/inactivated by heat or chemicals (such as formaldehyde). Too much heat denatures proteins (changes their shape). When the body comes into contact with the real thing it won’t have the right antibodies and memory cells & the person may get the disease. If the pathogens aren’t heated enough, some live pathogens may be injected in the vaccine! Attenuated (weakened) pathogens are also used in vaccines; these have been cultured in abnormal conditions so that they are no longer pathogenic. Sometimes these organisms cause adverse side effects & can sometimes revert to their pathogenic forms, causing the disease.
Using “genetically engineered viruses:” Recently, genetic engineering & recombinant DNA technology has allowed us to use bacteria to produce the protein antigens found in the protein capsids of certain viruses (remember, viruses don’t have phospholipid cell membranes – they have proteins coats or capsids). Scientists determine the genetic code for these proteins & insert the gene into the chromosome of bacterial cells. The bacteria produce the proteins coded for on the inserted genes when they go through their regular process of protein synthesis. These proteins can then be injected as a vaccine (your body doesn’t care if the proteins are in the real viral capsid or if they were made by a bacterium; they are the same proteins & your body’s immune system will respond to these antigens in the same way). “Genetically engineered viruses” (ex. hepatitis B, influenza, rabies) do not pose the same risks as inactivated and attenuated viruses!