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An Introduction to Biotechnology

Once Homo sapiens starts to understand something, it seems to be in his/her nature to try to manipulate it to better understand how it works, and perhaps to bend it to our use.
Why should genes be exempt from our tinkering?

Over evolutionary time, genes change via random (or not so random) mutation and via recombination during meiosis. These processes might seem random to us (though they are often directed by selective forces we cannot easily fathom, since they are many and complex!). Since the 1970's however, humans have been devising techniques by which they can manipulate genes and modify them in (somewhat) predictable and directed ways.

  • Recombinant DNA technology =
  • Genetic engineering =
  • Gene cloning

    Goals of the DNA Technologist include

    Recombinant DNA

    The idea is simple if the practice is not:

    In the early 1970's, Herbert Boyer, Stanley Cohen, Paul Berg and their graduate students slave...er...colleagues ushered in the era of Recombinant DNA Technology when they first began inserting foreign pieces of DNA in to host cells and cloned those host cells to produce multiple copies of the inserted DNA.

    Today, nearly 30 years later, there are many sophisticated techniques available for doing essentially the same thing: inserting DNA from one species into another species, and allowing that recipient species to replicate, producing multiple copies of the new recombinant DNA.

    Mosaics and Chimeras

    Mosaicism is a a condition in which cells within a single individual organism differ in their genetic makeup. In its broadest application, this genetic variation can include

    An animal consisting of more than one genetically-distinct population of cells can be either a

    Many individual mammals--including humans--are mosaics. An early somatic mutation can cause two different cell lines to proliferate during embryogenesis, and the earlier this mutation occurs, the greater the proportion of cells with the new, mutant genotype.
    For example, a normal female mammal embryo (46 XX) might have one cell undergo a nondisjunction of the X chromosome resulting in some cells with a genotype of 47 XXX. The earlier this happens during ontogeny, the greater the proportion of cells with 47 XXX.

    In a case such as duplication of the X chromsome, little or no damage will be done to the individual because of Barr Body formation. But in some cases, a potentially harmful genotype in a cell line can arise. If this happens early in embryo development, a larger proportion of cells will carry this deleterious genotype, and the individual may manifest signs of a genetic disorder. On the other hand, a sufficiently large population of normal cells can rescue an embryo from a lethal condition.
    For example, women with Turner syndrome can have one of three different genotypes, and one of these is causes Mosaic Turner Syndrome. Because not all cells of the body lack the X chromosome, the symptoms of this condition are less pronounced.

    In biotechnology, the artificial production of chimeras is a major research tool.

    Concepts and Definitions in Recombinant DNA Technology

    How is it done?

    Separating Restriction Fragments and Visualizing DNA

  • Once DNA is cleaved by restriction enzymes, the resulting restriction fragments can be isolated from one another via electrophoresis, a process by which molecules of different sizes and chemical/electrical properties migrate differentially through an electrically charged gel or other substrate.

    Amplifying DNA: The Polymerase Chain Reaction (PCR)

    Multiple copies of desired DNA can be made by inserting them into bacteria and then allowing replication.

    But these days, a far more cost-effective and quick alternative to cloning via bacterial hosts is the Polymerase Chain Reaction, affectionately known as PCR.

  • PCR allows rapid, efficient amplification of DNA sequences of interest.
  • Let us look at a nice movie... But I actually like this video better.

    Transgenics: Changing Genomes at the Individual Organism Level

    In transformation a host cell's DNA molecule has taken up and incorporated a piece of DNA from an outside source.

    Creating transgenic organisms involves transformation of host DNA, not with DNA from the same species, but with DNA from a different species.

    Some bacterial species will readily take up foreign DNA, and are said to be competent.

    However, other useful bacterial species can be "forced" to take up DNA fragments by methods such as exposure to a salt solution (e.g., calcium chloride), or heat shocking.

    Transforming eukaryotic cells isn't as simple. A few methods include:

    electroporation

    biolistics

    microinjection

    cloning vectors

    Eukaryotic Vectors

    For certain purposes, plasmid DNA and bacterial vectors are not sufficient. For example, E. coli lacks some of the enzyme systems that allow post-transcriptional/post-translational modification of proteins.

    Also, if you want to study the function of the eukaryotic genome in vivo, the only way to do so is to work with eukaryotic cells.
    And finally, if we want to manipulate eukaryote genes for medical and economic reasons, we have to use eukaryotic vectors.

    There are several eukaryotic vectors in use.

    The Yeast Artificial Chromosome (YAC)

    In nature, Baker's yeast (Sacchromyces cerevisieae) contain a small plasmid. However, the inclusion of the plasmid is unstable, and the wee thing tends to get lost as the yeast divide.

    To solve this problem, geneticists have succeeded in manufacturing an artificial version of this plasmid by inserting into a the plasmid

  • a yeast centromere
  • the yeast origin of DNA replication (known as ARS--autonomously replicating sequence).
  • sometimes, telomeric sequences (easy cutting spots for making linear chromosomes out of the circular plasmid)

    This modified plasmid is called a Yeast Artificial Chromosome (YAC). It can carry comparatively large pieces of DNA (from a donor eukaryote) to be inserted into eukaryotic cells. (Note that whereas a cosmid can carry only about 50 kb, a YAC can carry as much as 800 kb).

    Vectors Specific to Animals

  • DNA tumor virus SV40 (simian vacuolating virus) - This can transform normal eukaryote cells into cancer cells.

  • SV40 can carry foreign DNA.
  • Can be replicated in eukaryotic cells for cloning of carried DNA

    Many other viruses with an affinity for animal cells can be used, as we already mentioned.

    Vectors Specific to Plants

    A soil bacterium known as Agrobacterium tumefaciens causes gall tumors in dicot plants. The causative agent of the tumors is a plasmid named Ti, which transforms the normal plant cell into a cancerous cell when it is integrated into the plant's DNA. (This hints that the Ti plasmid could be a good vector!).

    Geneticists can insert foreign DNA into this plasmid, and use its affinity for dicot plant DNA to facilitate insertion of new genes into dicot plants.

  • When foreign DNA is inserted into a eukaryotic genome, transfection is said to have occurred.
  • This is similar to bacterial transformation, but renamed to distinguish it from eukaryotic "transformation" which generally refers to a cell becoming cancerous.
  • A eukaryotic organism that has taken up foreign DNA with a vector is said to be transgenic.

    Transgenics transcends taxonomic relationships. Very distantly related organisms can be artifically implanted with each other's DNA.

    A few examples

  • Transgenic mice are routinely made by injecting vectors containing cloned DNA into oocytes or even one- or two-celled embryos which are then re-implanted.
  • Note that only in about 15% of these operations is the foreign DNA actually incorporated into the host's genome. It doesn't always work.

  • The first transgenic eukaryote ever made (1988) was a mouse that carried a gene that predisposed it to cancer. It is used as a model to study cancer.

  • Since it's sometimes difficult to determine whether an inserted gene is "turned on" geneticists sometimes will insert a reporter gene alongside the gene of interest. Such a reporter gene is one which is easily detectable in phenotype. When it is functioning, its product's presence is a good indication that the adjacent gene of interest is also working.

    One such reporter gene that gives rather spectacular results is the luciferase gene, which causes the transgenic organism expressing it to bioluminesce. When you see pictures of glowing mouse embryos or glowing tobacco plants, you're seeing the results of a genetic marker that's used to detect the possible activity of the gene in its vicinity that's really the one of interest.

    The race is on. There are transgenic organisms of many kinds, most created in order to study a particular human function or disorder.

    Things Aren't Always Perfect

    Vectors do not always insert exactly where desired! In fact, there are three general scenarios that may occur if donor DNA is actually incorporated into a recipient cell.

    And in many instances, the DNA of interest isn't accepted at all.

    Genomics: Characterizing Entire Genomes

    Genomics is the characterization of entire genomes: determining sequence, and--eventually--discovering the function of all components of the genome.

    The quest to sequence and identify the function of a particular species is undertaken via multi-lab Genome Projects, such as the Human Genome Project.

  • The eukaryotic genome contains (1) unique genes (2) gene families (3) "non-functional" elements (which probably aren't).
  • Stage one: identify gene loci on chromosomes
  • Stage two: map and sequence each gene
  • The payoff: Once the genome is characterized, mapped and sequenced, genes of interest can be isolated for study of function. This is of special interest when it comes to genes in which mutations cause disease.
  • Investigators in Genomics seek not only to elucidate gene location, but ultimately to understand the function and expression of genes at all stages of development, growth, aging, etc., and under a variety of environmental conditions.
  • In short, it's the science of trying to determine how and why genes create us, and what factors can act on the innate plasticity of the genome.

  • Structural Genomics - The study of the physical nature of the genome, such as the physical location of the genes on the chromosomes.

  • Functional Genomics - The study of overall patterns of gene expression, including environmental influence.

    The Vocabulary of Structural Genomics

    Eukaryotic genomes usually include
    • genes that are present in only one copy per genome
    • genes and elements that are present in multiple copies per genome
    • "spacer" DNA - (apparently) not transcribed or translated

      Here's an overview.

    Some types of Functional Repetitive Sequences...

    • Dispersed Gene Families - a gene family is one which codes for a particular type of protein or product. (Examples: albumin, histones, immunoglobins, hemoglobins, etc.) The genes are found throughout the genome in different locations (they are dispersed!). Some families include a few members which apparently no longer code for a functional product, but are clearly derived from the genes of the family. Such a nonfunctional mutant "leftover" is called a pseudogene.
      (Recall: "Proteome" is the term used to describe all the genes within a genome which code for proteins.)
    • Tandem Gene Family Arrays - These are genes which code for a product needed in great quantities in the cell, and are arranged in adjacent repeats along a chromosome. (Example is the Nucleolar Organizer regions)
    • Non-coding Functional Sequences - These are sequences that have no product, yet serve a definite function. For example, the telomere sequences allow replication without reduction in telomere size via their affinity for telomerase.

    There are also sequences with no known function

    • Highly Repetitive Centromerit DNA - tandem repeats in the (untranscribed) heterochromatin flanking the centromeres; function unknown.
    • VNTRs - Variable Number Tandem Repeats are 1-5 kb long, and consist of variable numbers of adjacent repeats. No one knows what they do, but they are highly variable among individuals. These are the fragments of DNA that are used as DNA fingerprints, and are used extensively in criminal forensics.
    • Transposed Sequences - Multiple copies of small DNA segments called transposable genetic elements exist throughout the genome, and some can excise and move to other parts of the genome. Their function is not known, and some suspect that they could be remnants of "parasitic" or "selfish" DNA that simply goes along for the ride without regard for the host.
    • Spacer DNA - Apparently not transcribed, but this is not certain. Function unknown.

    Genomic Data Bases: DNA Libraries

    Bioinformatics is the use of computers to store and analyze genetic data, such as DNA Libraries.

    A restriction fragment is to a DNA library as a single book is to a regular library: it's only one piece of an entire collection of such information.

    Every organism has a genome, and theoretically, we could have a DNA library for every species. A DNA library is a collection of cloned restriction fragments from a single organism's genome. The goal is to have a library containing clones of ALL the organism's genes.

    Genomic Libraries

  • A genomic library is a DNA library containing an organism's complete genome, in the form of small DNA fragments (oligonucleotides) representing known genes.

    cDNA Libraries

  • A cDNA library (for "complementary" DNA) is a DNA library made up of DNA clones reconstructed from the organism's mRNA molecules.
  • mRNA is removed from a living cell, and reverse transcriptase is used to manufacture a DNA sequence complementary to it.
  • This DNA fragment is inserted into a vector, and then into an E. coli.
  • The reconstructed fragments together make up the cDNA library

    The benefit of a cDNA clone is that it can be translated into functional protein if it's inserted into a bacterium. This way, the gene's function in a eukaryotic cell can actually be determined.

  • An inserted cDNA gene lacks upstream and downstream regulators which turn protein synthesis on and off in the actual eukaryote in which it exists, and it's already had its introns removed, so the protein made by E. coli with the cDNA insert is usually a good indicator of the actual, finished mRNA found in the eukaryote. This mRNA can be reverse-transcribed into cDNA.

    Note that a cDNA library is good for studying which proteins are actually made by which genes, but only a genomic library can be used to study the function of regulatory sequences and the nature of how introns/exons affect the final protein product of a gene.

    Expression Libraries

    Expression libraries are made up of expression vectors. These are cloning vectors that contain the required regularly elements for gene expression, such as the promoter region.
    • In an E. coli expression vector, an E. coli promoter is placed next to a unique restriction site where DNA of interest can be inserted.
    • If successful, insertion of the foreign gene into the correct reading frame will result in the gene's being transcribed and translated by the host E. coli cell.

    DNA libraries can be sorted various ways, such as with a flow cytometer, or via pulsed field gel electrophoresis (PFGE), which sort nucleic acids or proteins by size.

    DNA Sequencing

    DNA sequencing, first introduced in 1975, provides some of the most basic information needed in Genomics and other areas of genetic study. This is the determination of the actual base sequence in a DNA segment of interest.

    The most commonly used method for sequencing DNA is the Sanger or Dideoxy Method .

    Here's an additional diagram of the Dideoxy Method that you may find useful.

    The Main Idea: Once you have completed your dideoxy binding and made your Sanger sequencing gel (via electrophoresis), you will have a huge number of DNA fragments, each one nucleotide longer than the last. The nucleotide sequence can essentially be read directly from the gel.

    This is usually done by a sequencing machine that also performs the polymerization reactions described above.

    A Catalog of Biotechnology

    The tools and techniques we use to analyze genomes are varied in both function and purpose. Consider this next section an overview of some important terms and procedures used in labs all over the world in an effort to dissect and understand how our genes make us what we are.

    Probes

    A genetic probe is a radioactively labeled nucleic acid fragment of known sequence that allows precise location of a complementary DNA sequence. DNA of interest is denatured, and the probe is allowed to anneal with complementary regions.

    How do you make a labeled probe? Here's one way:

      1. Select a protein product of interest, and determine its aa sequence.
      2. From aa sequence, mRNA sequence can be extrapolated and isolated from the cell.
      3. Reverse transcriptase is used to manufacture DNA from the isolated mRNA
      4. Radioactive nucleotides (usually labeled with 32P) are provided as raw material for this synthesis
      5. The resulting DNA-RNA hybrid, when denatured, will yield a radioactive DNA probe that will bind to the DNA that coded for the mRNA of interest.
      6. The probe can then be cloned.

      (Note that the mRNA lacks introns, but as long as there are complementary regions of the gene, the probe should work, even if the introns form "outloopings" in the probe-DNA hybrid.)

    Blot Tests

    One important method for isolating and probing DNA clones is the Southern Blot.

  • Recall that an autoradiograph is an image taken on radiography film in which the exposure is made by radioactivity present in the sample itself.

  • Northern Blot: Similar technique used to assay RNA.

  • Western Blot: Similar technique used to assay proteins (via antibody binding)

    Functional Complementation

    Genes of interest can be located for cloning through their ability to restore wild type phenotype to a mutant organism. Here's how it works...
    • Select a protein of interest, and a wild type organism (Let's say "Species X" that produces its normal product.
    • Create a DNA library from that wild type organism, using an appropriate vector
    • Take library samples and introduce them to mutant colonies of Species X which cannot produce the protein of interest.
    • Plate out your (hopefully) transformed Species X, and select only the ones that exhibit the wild type phenotype. Those are the ones that have been successfully transformed.
    • Use the transformed colonies to recover and clone the wild type gene, which you know is present because its product is being manufactured.
    • Remember: the vectors are supplying only a fragment of the Species X genome, which is why this works!

    Knockout Technology

    One way to study the function of a normal gene in a wild type organism is to disrupt that gene's function with intentional mutations to "knock out" the function of the gene in an organism that inherits the mutation. \

    The Amazing Knockout Mouse

    Mice have been used extensively in transgenic studies, and are often the organism of choice for producing clones of DNA in which a specific gene has been targeted and inactivated ("knocked out"). In most cases, a gene transfected into a mouse cell is randomly incorporated into the genome. We don't know exactly where it goes.
  • Once in a while, the donor gene completely replaces the mouse locus where it inserts (this happens if the foreign gene lines up with its homolog before crossing over, and is taken up instead of the actual homologous gene.
  • This property is at the center of Knockout Technology.
  • To make a Knockout Mouse, the geneticist transfects a wild type mouse oocyte with a gene that is a defective version of the one s/he wishes to study.

  • If a successfully transfected oocyte is fertilized with sperm from a normal, wild type mouse and the embryo grows into a new mouse, that mouse will be heterozygous for the mutant gene.

  • Breeding two such heterozygotes together should give you 25% homozygous recessives.

  • By studying these homozygotes (which express the mutant, knockout condition), the scientist can determine whether the gene in question is essential, or what its normal functions are by noting the deficiencies in the knockout mice.

    In Vitro Mutagenesis

      As always, the function of a wild type gene can be elucidated best by studying what happens when there is a mutation of that gene. If the sequence of a wild type gene is known (and its function is not), then in vitro mutagenesis can be used to destroy the wild type gene's function in a controlled fashion, so that the effect on mutant organisms (usually bacteria) can be studied.
    • a short, complementary sequence known as an oligonucleotide can be made
    • a site-specific mutation can be induced in the oligonucleotide
    • the oligonucleotide can contain a mutation of any desired type, including base pair substitution, insertion, deletion, etc.
    • when taken up by a phage vector (usually phage M13), it can then be inserted into bacteria for cloning and further study.

    Reverse Genetics

      In the Good Old Days, an investigator would discover an organism with a mutant phenotype, determine that it had a particular mutant allele, determine the DNA sequence for that allele and then infer the amino acid sequence of the faulty protein.

      Today...

      • a protein or a gene of unknown function is isolated.
      • an ORF ("open reading frame") is a segment of DNA flanked by a start and stop codon. Though it has been sequenced and found, its function and product are not known. It is a putative gene.
      • To determine the function of the ORF, a site-specific mutation can be induced (as described above)
      • This can then be radioactively labeled, inserted into a vector and cloned.
      • The radioactive clones can be used as probes to find the relevant gene.
      • Insertion of the mutant sequence into the genome of a bacterium can be used to determine the function of the disrupted gene, since the bacterium will be unable to manufacture the product of the disrupted gene.
      • This works because a eukaryote gene inserted into a bacterium will always be expressed (whether wild type or mutant), since the bacterium lacks the eukaryotic gene's regulatory sequences that will turn it on or off.
      • This technology is important in gene knockout, which we'll discuss shortly.
      • Also, genetically engineered bacteria (and fungi) like this can be used to produce a eukaryotic gene product in great quantities--a commercial boon.

    RFLP - Restriction Fragment Length Polymorphisms

    • We've already seen that restriction endonucleases cut the genome into varying sizes
    • One might expect that since the genes within a species are homologous, the restriction fragments will show a constant size for any given fragment type.
    • However, this is not the case. Probes do not always bind the same way in all individuals.
    • This is probably due to neutral mutations of some type, often resulting in restriction fragments of different lengths in different individuals--even though they are the same class of restriction fragment.
    • The apparent "presence" or "absence" of a restriction site are often considered to be markers akin to different alleles of that site.
    • The presence of various forms of a particular restriction fragment within a species is known as RFLP - Restriction Fragment Length Polymorphism.

      What's so important about RFLPs?

      • heterozygosity of restriction fragments within a single individual can be used as a genetic marker.
      • RFLPs in related individuals can be used as a marker (as in mapping!) for diagnosing disorders which might be related to that RFLP's location.
      • Evolutionary history of related species can be studied by examination of the RFLPs of the species. A measure of the total RFLP difference can be used as an index of evolutionary divergence from a common ancestor.

    Practical Application of Biotechnology

    Sequencing and identifying functional components of the genome is a goal unto itself, as such knowledge will open the door to various uses. But even now, techniques in biotechnology are being applied in the commercial world in the hope of making things better. (The jury's still out on whether this is always the case.)

    Genetic Engineering of Agricultural Species

    Foreign genes are under study for insertion into commercial plant species to confer...
  • selective herbicide resistance
  • herbivore resistance
  • increased yield
  • improved nutrient balance
  • plant-grown vaccines and pharmaceuticals
  • insert your Brave New Idea here

    Application of Recombinant DNA Technology: Pandora's Box?

    Problems?
  • possible allergic reactions in humans to foreign proteins
  • jumping of plasmids from commercial crops to "weed" species resulting in
    • herbicide-resistant weeds
    • increased use of herbicides --> increased human exposure
    • toxicity to commercially important pollinators and their larvae
    • eco-mayhem not yet predicted?

    Transgenic animals are becoming more common every day.

  • transfection accomplished at zygote stage affects all future generations
  • transfection accomplished in target cells affects only the individual, and not future generations

    This dichotomy is at the root of the future of human gene therapy. If we alter human disease genes, do we plan to do it at the zygote stage--or the somatic stage? Major bioethical implications!

    Prenatal Genetic Testing

    Recombinant DNA probes can be used in tissue cultures obtained via amniocentesis

    or chorionic villus sampling to detect genetic abnormalities in human (or other species) fetuses.

  • Usually, detection of disorders is done via expression of normal protein products present or not present in the cultured cells.

  • Recombinant DNA could be used for greater accuracy, since cloned probes of the normal gene could be used to detect abnormalities at the level of the DNA.

  • Example: Sickle cell fetuses, because of the GAG-to-GTG mutation resulting in the disorder, also disrupt a restriction site normally present in normal fetuses. Detection of the lack of this restriction site could be useful in prenatal diagnosis of Sickle Cell.
  • Once the human genome project reveals normal sequences for various genes, altered sequences will be detectable via oligonucleotide probes specific to the gene of interest.

    Gene Therapy

    Using the sequencing, cloning and vector-insertion techniques we've discussed, scientists hope to be able to deliver working versions of genes to individuals who were born with deleterious, disease-causing mutant versions of particular genes.

    As you might guess from the low success rates of vector transfection, this is a dicey procedure, and the techniques are still crude, at best.

    Still, technologies are improving at an exponential rate, and it is true that some devastating genetic diseases might some day be cured by the insertion of wild type genes to replace the faulty, non-functional ones.

  • Germ Line Gene Therapy - this is the transfection of germ cells with "desired" genes, and it does have evolutionary consequences. Not only is a person carrying a deleterious allele cured, but some of his/her gametes might also be "cured"--meaning the end to the heritability of the particular disorder by that individual's offspring.

    Problem: transfection fragments often insert haphazardly (ectopically, i.e., not in its usual locus) which means not only that normal gene function could be disrupted if the insertion takes place in the middle of a normal gene, but also that even if the normal gene inserts in a non-disruptive spot, it has not replaced the disease gene. It can still be passed on to progeny, even if the parental organism is functional.

  • Somatic Gene Therapy - This targets only the affected body cells, and has no direct evolutionary consequences. Only some somatic cells are transfected--enough to effect a cure or confer effective function. (Note that unless one transfects a one-celled embryo, gene therapy cannot correct genetic errors in every cell of the body. Hence, this type of therapy is useful only for disorders that are highly tissue-specific and treatable if some functional cells are present. Diabetes is one such disorder, as functional Islet of Langerhans cells can be created transgenically so that the individual can produce insulin in some cells, even if many of his/her other cells are not functional.

    The difference between these two types of therapy is illustrated HERE.

    A panacea or a dangerous road? Only time will tell.