Restriction Enzymes
Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonucleases.
In order to be able to sequence DNA, it is first necessary to cut it into smaller fragments. Many DNA-digesting enzymes (like those in your pancreatic fluid) can do this, but most of them are no use for sequence work because they cut each molecule randomly. This produces a heterogeneous collection of fragments of varying sizes. What is needed is a way to cleave the DNA molecule at a few precisely-located sites so that a small set of homogeneous fragments are produced. The tools for this are the restriction endonucleases. The rarer the site it recognizes, the smaller the number of pieces produced by a given restriction endonuclease.
A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. For example, the bacterium Hemophilus aegypticus produces an enzyme named HaeIIIthat cuts DNA wherever it encounters the sequence
5’GGCC3′
3’CCGG5′
The cut is made between the adjacent G and C. This particular sequence occurs at 11 places in the circular DNA molecule of the virus φX174. Thus treatment of this DNA with the enzyme produces 11 fragments, each with a precise length and nucleotide sequence. These fragments can be separated from one another and the sequence of each determined.
| Link to page describing DNA sequencing. |
HaeIII and AluI cut straight across the double helix producing “blunt” ends. However, many restriction enzymes cut in an offset fashion. The ends of the cut have an overhanging piece of single-stranded DNA. These are called “sticky ends” because they are able to form base pairs with any DNA molecule that contains the complementary sticky end. Any other source of DNA treated with the same enzyme will produce such molecules.
Mixed together, these molecules can join with each other by the base pairing between their sticky ends. The union can be made permanent by another enzyme, a DNA ligase, that forms covalent bonds along the backbone of each strand. The result is a molecule of recombinant DNA (rDNA).
The ability to produce recombinant DNA molecules has not only revolutionized the study of genetics, but has laid the foundation for much of the biotechnology industry. The availability of human insulin (for diabetics), human factor VIII (for males with hemophilia A), and other proteins used in human therapy all were made possible by recombinant DNA.
| Link to discussion of recombinant DNA. |
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12 March 2011
DNA Sequencing
DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA.
The most popular method for doing this is called the dideoxy method or Sanger method (named after its inventor, Frederick Sanger, who was awarded the 1980 Nobel prize in chemistry [his second] for this achievment).
DNA is synthesized from four deoxynucleotide triphosphates. The top formula shows one of them: deoxythymidine triphosphate (dTTP). Each new nucleotide is added to the 3′ -OH group of the last nucleotide added.
| Link to discussion of DNA synthesis. |
The dideoxy method gets its name from the critical role played by synthetic nucleotides that lack the -OH at the 3′ carbon atom (red arrow). A dideoxynucleotide (dideoxythymidine triphosphate — ddTTP — is the one shown here) can be added to the growing DNA strand but when it is, chain elongation stops because there is no 3′ -OH for the next nucleotide to be attached to. For this reason, the dideoxy method is also called the chain termination method.
| The bottom formula shows the structure of azidothymidine (AZT), a drug used to treat AIDS. AZT (which is also called zidovudine) is taken up by cells where it is converted into the triphosphate. The reverse transcriptase of the human immunodeficiency virus (HIV) prefers AZT triphosphate to the normal nucleotide (dTTP). Because AZT has no 3′ -OH group, DNA synthesis by reverse transcriptase halts when AZT triphosphate is incorporated in the growing DNA strand. Fortunately, the DNA polymerases of the host cell prefer dTTP, so side effects from the drug are not so severe as might have been predicted. |
The Procedure
The DNA to be sequenced is prepared as a single strand.
This template DNA is supplied with
- a mixture of all four normal(deoxy) nucleotides in ample quantities
- dATP
- dGTP
- dCTP
- dTTP
- a mixture of all four dideoxynucleotides, each present in limiting quantities and each labeled with a “tag” that fluoresces a different color:
- ddATP
- ddGTP
- ddCTP
- ddTTP
- DNA polymerase I
Because all four normal nucleotides are present, chain elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide (shown as colored letters) instead of the normal deoxynucleotide (shown as vertical lines). If the ratio of normal nucleotide to the dideoxy versions is high enough, some DNA strands will succeed in adding several hundred nucleotides before insertion of the dideoxy version halts the process.
At the end of the incubation period, the fragments are separated by length from longest to shortest. The resolution is so good that a difference of one nucleotide is enough to separate that strand from the next shorter and next longer strand. Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam and an automatic scanner provides a printout of the sequence.
| If you wish to see a representative example of a DNA sequence (455 nucleotides of the lysU gene of E. coli) which was generated by an automated sequencing device, LINK HERE. (The file size is 172K.) (The image is courtesy of Pharmacia Biotech Inc., Piscataway, NJ.) |
| External Link |
| Animation of the procedure |
| Please let me know by e-mail if you find a broken link in my pages.) |
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2 February 2011
Genome Sizes
The genome of an organism is the complete set of genes specifying how its phenotype will develop (under a certain set of environmental conditions). In this sense, then, diploid organisms (like ourselves) contain two genomes, one inherited from our mother, the other from our father.
The table below presents a selection of representative genome sizes from the rapidly-growing list of organisms whose genomes have been sequenced.
| Base pairs | Genes | Notes | |
|---|---|---|---|
| φX174 | 5,386 | 11 | virus of E. coli |
| Human mitochondrion | 16,569 | 37 | |
| Epstein-Barr virus (EBV) | 172,282 | 80 | causes mononucleosis |
| Nanoarchaeum equitans | 490,885 | 552 | This parasitic member of the Archaea has the smallest genome of a true organism yet found. |
| nucleomorph of Guillardia theta | 551,264 | 511 | all that remains of the nuclear genome of a red alga (a eukaryote) engulfed long ago by another eukaryote |
| Mycoplasma genitalium | 580,073 | 485 | two of the smallest true organisms |
| Mycoplasma pneumoniae | 816,394 | 680 | |
| Chlamydia trachomatis | 1,042,519 | 936 | this bacterium causes the most common sexually-transmitted disease (STD) in the U.S. |
| Rickettsia prowazekii | 1,111,523 | 834 | bacterium that causes epidemic typhus |
| Treponema pallidum | 1,138,011 | 1,039 | bacterium that causes syphilis |
| Mimivirus | 1,181,404 | 1,262 | A virus (of an amoeba) with a genome larger than the six cellular organisms above |
| Pelagibacter ubique | 1,308,759 | 1,354 | smallest genome yet found in a free-living organism (marine α-proteobacterium) |
| Borrelia burgdorferi | 1.44 x 106 | 1,738 | bacterium that causes Lyme disease [Note] |
| Campylobacter jejuni | 1,641,481 | 1,708 | frequent cause of food poisoning |
| Helicobacter pylori | 1,667,867 | 1,589 | chief cause of stomach ulcers (not stress and diet) |
| Thermoplasma acidophilum | 1,564,905 | 1,509 | These unicellular microbes look like typical bacteria but their genes are so different from those of either bacteria or eukaryotes that they are classified in a third kingdom: Archaea. |
| Methanococcus jannaschii | 1,664,970 | 1,783 | |
| Aeropyrum pernix | 1,669,695 | 1,885 | |
| Methanobacterium thermoautotrophicum |
1,751,377 | 2,008 | |
| Haemophilus influenzae | 1,830,138 | 1,738 | bacterium that causes middle ear infections |
| Streptococcus pneumoniae | 2,160,837 | 2,236 | the pneumococcus |
| Neisseria meningitidis | 2,184,406 | 2,185 | Group A; causes occasional epidemics of meningitis in less developed countries. |
| Neisseria meningitidis | 2,272,351 | 2,221 | Group B; the most frequent cause of meningitis in the U.S. |
| Encephalitozoon cuniculi | 2,507,519 | 1,997 | (plus 69 RNA genes); a parasitic eukaryote. |
| Propionibacterium acnes | 2,560,265 | 2,333 | causes acne |
| Listeria monocytogenes | 2,944,528 | 2,926 | 2,853 of these encode proteins; the rest RNAs |
| Deinococcus radiodurans | 3,284,156 | 3,187 | on 2 chromosomes and 2 plasmids; bacterium noted for its resistance to radiation damage |
| Synechocystis | 3,573,470 | 4,003 | a marine cyanobacterium (“blue-green alga”) |
| Vibrio cholerae | 4,033,460 | 3,890 | in 2 chromosomes; causes cholera |
| Mycobacterium tuberculosis | 4,411,532 | 3,959 | causes tuberculosis |
| Mycobacterium leprae | 3,268,203 | 1,604 | causes leprosy |
| Bacillus subtilis | 4,214,814 | 4,779 | another bacterium |
| E. coli K-12 | 4,639,221 | 4,377 | 4,290 of these genes encode proteins; the rest RNAs |
| E. coli O157:H7 | 5.44 x 106 | 5,416 | strain that is pathogenic for humans; has 1,346 genes not found in E. coli K-12 |
| Agrobacterium tumefaciens | 4,674,062 | 5,419 | Useful vector for making transgenic plants; shares many genes with Sinorhizobium meliloti |
| Salmonella enterica var Typhi | 4,809,037 | 4,395 | + 2 plasmids with 372 active genes; causes typhoid fever |
| Salmonella enterica var Typhimurium | 4,857,432 | 4,450 | + 1 plasmid with 102 active genes |
| Yersinia pestis | 4,826,100 | 4,052 | on 1 chromosome + 3 plasmids; causes plague |
| Schizosaccharomyces pombe | 12,462,637 | 4,929 | Fission yeast. A eukaryote with fewer genes than the four bacteria below. |
| Ralstonia solanacearum | 5,810,922 | 5,129 | soil bacterium pathogenic for many plants; 1681 of its genes on a huge plasmid |
| Pseudomonas aeruginosa | 6.3 x 106 | 5,570 | Increasingly common cause of opportunistic infections in humans. |
| Streptomyces coelicolor | 6,667,507 | 7,842 | An actinomycete whose relatives provide us with many antibiotics |
| Sinorhizobium meliloti | 6,691,694 | 6,204 | The rhizobial symbiont of alfalfa. Genome consists of one chromosome and 2 large plasmids. |
| Saccharomyces cerevisiae | 12,495,682 | 5,770 | Budding yeast. A eukaryote. |
| Cyanidioschyzon merolae | 16,520,305 | 5,331 | A unicellular red alga. |
| Plasmodium falciparum | 22,853,764 | 5,268 | Plus 53 RNA genes. Causes the most dangerous form of malaria. |
| Thalassiosira pseudonana | 34.5 x 106 | 11,242 | A diatom. Plus 144 chloroplast and 40 mitochondrial genes encoding proteins |
| Neurospora crassa | 38,639,769 | 10,082 | Plus 498 RNA genes. |
| Naegleria gruberi | 41 x 106 | 15,727 | This free-living unicellular organism lives as both an amoeboid and a flagellated form. 4,133 of its genes are also found in other eukaryotes suggesting that they were present in the common ancestor of all eukaryotes. The great variety of functions encoded by these genes also suggests that the common ancestor of all eukaryotes was itself as complex as many of the present-day unicellular members. |
| Caenorhabditis elegans | 100,258,171 | 21,733 | The first metazoan to be sequenced. |
| Arabidopsis thaliana | 115,409,949 | ~28,000 | a flowering plant (angiosperm) See note. |
| Drosophila melanogaster | 122,653,977 | ~17,000 | the “fruit fly” |
| Anopheles gambiae | 278,244,063 | 13,683 | Mosquito vector of malaria. |
| Tetraodon nigroviridis (a pufferfish) | 3.42 x 108 | 27,918 | Although Tetraodon seems to have more protein-encoding genes than we do, it has much less “junk” DNA so its total genome is about a tenth the size of ours. |
| Rice | 3.9 x 108 | 28,236 | |
| Sea urchin | 8.14 x 108 | ~23,300 | |
| Zebrafish | 1.2 x 109 | 15,761 | |
| Dogs | 2.4 x 109 | 19,300 | |
| Humans | 3.3 x 109 | ~21,000 | [Link to more details.] |
| Mouse | 3.4 x 109 | ~23,000 | |
| Amphibians | 109–1011 | ? | |
| Psilotum nudum | 2.5 x 1011 | ? | Note |
Note: The gene total for Borrelia burgdorferi is based on 853 genes on its single chromosome (of 910,724 base pairs) plus 430 genes on 11 of the 17 plasmids it contains.
Arabidopsis thaliana is a plant (in the mustard family) that has the smallest genome known in the plant kingdom and for this reason has become a favorite of plant molecular biologists.
Even though Psilotum nudum (sometimes called the “whisk fern”) is a far simpler plant than Arabidopsis (it has no true leaves, flowers, or fruit), it has 3000 times as much DNA. No one knows why, but 80% or more of it is repetitive DNA containing no genetic information. This is also the case for some amphibians, which contain 30 times as much DNA as we do but certainly are not 30 times as complex.
The total amount of DNA in the haploid genome is called its C value. The lack of a consistent relationship between the C value and the complexity of an organism (e.g., amphibians vs. mammals) is called the C value paradox.
How many genes does it take to make an organism?
The scientists at The Institute for Genomic Research (now known as the J. Craig Venter Institute) who determined the Mycoplasma genitalium sequence have followed this work by systematically destroying its genes (by mutating them with insertions) to see which ones are essential to life and which are dispensable. Of the 485 protein-encoding genes, they have concluded that only 381 of them are essential to life.
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3 February 2011
dodi ' s blog biology 1 
