Biotechnology

I.
Biotechnology includes a large variety of techniques applied to the manipulation and utilization of genetic information—particularly human genetic information.
A.
A variety of testing and screening techniques have arisen which use the tools of biotechnology.
1.
Sophisticated mapping techniques have been developed which allow us to locate genes on chromosomes without performing controlled mating studies (which are essentially impossible to do with humans).
2.
Certain kinds of genetic screening were impossible before the growth in biotechnology skills.  For example, until recent years it was impossible to screen for the presence of the Huntington's allele.
3.
DNA comparison tests which depend upon biotechnology can allow far more certain identification than tools like blood typing and fingerprinting.
B.
Two very important enzymatic tools have been largely responsible for the development of our biotechnological skills.
1.
RNA directed DNA polymerase (reverse transcriptase) is an enzyme which will use an RNA template to construct a DNA.  This is done via the production of an RNA/DNA hybrid, which is then used to produce another RNA/DNA hybrid as well as a double stranded DNA which carries the sequence of the original RNA.  This enzyme was discovered because of its role in the infection cycles of retroviruses.  A retrovirus in an RNA-containing virus which, in order to complete its reproductive cycle, must cause its host cell to manufacture a DNA version of its genome.  Interestingly, the reverse transcriptase gene is sometimes part of the host's genome.  No one is quite sure why the host would carry such a gene, since modern genetic practices in cells do not apparently include no episode of RNA directed DNA synthesis.
2.
Restriction endonucleases are a large (and growing) group of enzymes manufactured by bacteria.  They apparently function in the bacteria as part of their defense mechanisms.
a.
A nuclease is an enzyme which digests DNA or RNA.
i.
An exonuclease begins at one end (either the 3' or 5') and cleaves off one nucleotide at a time.
ii.
An endonuclease cleaves the molecule in the middle somewhere, cutting a long DNA or RNA into shorter pieces.
b.
The restriction endonucleases are DNA digesting enzymes.  The reason their discovery has been so very important is that each of them has a very specific recognition site, and will cleave the DNA only when they find that site.
i.
The sites vary in length from 3 or 4 base pairs log up to 11 or 12 base pairs long.  The most common length is apparently 6 base pairs long.  Many restriction sites are palindromic—the 5' to 3' sequences of the two strands have exactly the same sequences.
ii.
Some restriction endonucleases cleave both strands of the DNA at the same place, producing a blunt cut.
iii.
Some restriction endonucleases cleave the two strands in different place, producing a staggered cut, and leaving behind "sticky ends" (short single stranded ends).

II.
Restriction Fragment Length Polymorphisms (RFLPs) are studied by comparing the restriction digests of similar DNA molecules.
A.
Since the cleavage of restriction enzymes is always sequence specific, if the same DNA sequence is exposed to the same endonuclease over and over again, it will be cut in exactly the same places every time, producing the same number and sizes of fragments.
B.
Very similar DNA's may give almost identical cleavage patterns, and the difference(s) in cleavage pattern can safely be assumed to reflect the differences between the two molecules.
C.
This phenomenon was one of the most significant tools which allowed the localization of the Huntington's disease gene. Huntington's disease is a late onset genetic disorder caused by a dominant allele.  To this day, the normal function of the Huntington's gene is unknown.  It has thus been impossible to screen the at-risk offspring of a Huntington's parent to see if they carry the allele or not (no symptoms generally manifest until the 30's or 40's). Since we know no normal function for this gene, and there are no known genetic markers which seem to be closely linked to it, classical tools of genetics couldn't locate the gene or provide a screening technique.
1.
The key study which located this gene and provided our first screening method began with the discovery of a family in Venezuela whose members have carried Huntington's for seven generations.  The gene was introduced from a single source, and had been proliferated via a large amount of close breeding within a small community.  That the gene came from a single source is significant because it means that all of the people in the family who suffer from Huntington's should carry the same version of the gene, and probably the same version of the nearby chromosome material.
2.
The investigators prepared a gigantic family tree showing as much as possible the relationships between the individuals in the family.
3.
They then began what could have been a terrifically long and tedious screening of their chromosomes for RFLPs which were associated with the inheritance of Huntington's.
4.
The got very lucky and found what they were looking for relatively quickly. (23 pairs of chromosomes provide a huge bunch of DNA to screen for this.)
5.
They were able to localize the Huntington's gene to a 500,000 base-pair-long portion of chromosome 4.
6.
Now that the gene is localized, it is theoretically possible to screen for it in any family which carries Huntington's.
a.
In order to do this, there must be members of the family available who are known to have inherited the trait from the same source as the person(s) who are to be screened, as well as healthy members of the same family.
b.
A RFLP study is done of the significant region of chromosome 5 in these family members.  The hope is to determine just which RFLP pattern is characteristic of that region of chromosome 4 in the specific chromosomes which are carrying Huntington's.
c.
The chromosomes of the person(s) who are being screened are then compared to the RFLP patterns of the chromosomes from other family members.  If they are found to have a chromosome 4 with the Huntington's associated RFLP pattern, they have a high probability of having inherited the gene as well.
d.
NOTE:  This assumption is based upon the same principles of linkage and crossing over which we used for linkage mapping.  500,000 base pairs is a relatively short segment of DNA, and not a great deal of recombination is expected to go on within it.  However, it is possible for crossing over to have occurred, so the possession of the suspect RFLP pattern is not a 100% guarantee of possession of the Huntington's allele.

III.
Biotechnology has enabled us to implant human genes in bacteria and induce the bacteria to express those genes.
A.
For instance, the human insulin gene has been transferred into E. coli. This is an important step forward in treating insulin dependent diabetics. In the past, humans who don't possess a properly functioning insulin gene were treated by periodic injections of pig or calf insulin, isolated from the pancreases of slaughtered animals. While this was a reasonably good substitution, pig or cow insulin is not exactly identical to human insulin.  It would have been preferable to use insulin isolated from humans of chimps (their insulin is identical to ours). Unfortunately, since the donor must be slaughtered to get its pancreas, these are not really useful alternatives.  However, inserting the gene into E. coli allows us to manufacture human insulin in vitro (literally, "in glass").
B.
There are several problematic steps necessary to achieve this end.
1.
First, the gene of interest must be located and isolated.
2.
The gene must then be attached to a vector which will deliver it into the bacterial cell.  Gene and vector must be in a form which will avoid the cell's defense mechanisms.
3.
We must be able to control the activity of a eukaryotic gene existing inside a prokaryotic cell.
4.
We must then be able to replicated organism and gene to provide a relatively high volume production system.
C.
The first problem was initially rather difficult.  To "fish out" one gene from the approximately 30,000 human genes is a formidable challenge.  It requires using a probe to locate the gene.
1.
This was first achieved with genes which operate under rather special circumstances which made it relatively easy to acquire the mRNA transcribed from a gene, which could then be used as your probe.  For example, the globin genes of hemoglobin.
a.
Hemoglobin is produced by erythrocytes.  Mammalian erythrocytes are produced in the bone marrow.  One of the last events in their differentiation is the loss of their nuclei.  The life of the erythrocyte then becomes devoted exclusively to the production of hemoglobin and the carrying of oxygen through the blood stream.
b.
Before the loss of the nucleus, the DNA of the immature erythrocyte manufactures vast quantities of globin mRNA—and almost nothing else.  When the nucleus is finally lost, the cytoplasm of the cell is packed full of ribosomes and globin mRNA.  There is very little mRNA in the cytoplasm which is not globin message.
c.
Biotechnologists are able to isolate this essentially pure globin mRNA and utilize it as a probe to extract the globin genes from DNA.  This is done by taking advantage of the complementarity between an mRNA and the template strand of the gene from which it was transcribed. Human DNA is extracted and cut into relatively small pieces. The DNA is then gently melted (heated until the strands separate) and mixed with the mRNA. The temperature is slowly lowered, allowing the molecules to anneal.  Some of the DNA template will pair with mRNA instead of DNA, producing RNA/DNA hybrids. It is possible to separate such hybrids from pure DNA.  The hybrids are then treated with an enzyme which will digest away the single strands ends of the DNA, leaving a double stranded molecule containing globin mRNA and just the template region of the DNA.  Replication will then yield pure DNA—the globin gene.
d.
NOTE: An alternative method would be to use the mRNA as a template for reverse transcriptase to create the complementary DNA. This method sidesteps the problem of introns, which are present in eukaryotic genes, but not in prokaryotic genes.  If we are to put our new gene into a bacterium to function, it must not contain introns.
2.
Later, techniques were developed which allowed biotechnologists to isolate any gene they wanted, as long as they knew the amino acid sequence of the gene product.
a.
Because we know the genetic code, if we know the amino acid sequence of a protein we can make a pretty good guess at most of the base sequence of its gene.
b.
Biotechnologists are capable of constructing artificial nucleic acids one nucleotide at a time, thus producing a molecule with any desired sequence.
c.
The probe for a gene is constructed by best-guessing the sequence necessary to code for the protein. This is then used to locate the gene in the whole DNA, or to fish out its mRNA from the cytoplasmic contents of the cell.
D.
Once you have the gene, you must then attach it to a vector which will make it possible for you to get it intact into a bacterial cell.
1.
Two basic types of vectors have been used.
a.
Some viruses can serve as vectors for inserting genes into cells.
i.
Viral transduction was a relatively early discovery in molecular genetics. What investigators discovered was that in a small percentage of cases, viruses would be improperly constructed within a host cell, and would end up carrying chunks of the host cell's DNA instead of (or in addition to) the normal viral DNA.  These bacterial genes would then be injected into the next host the virus attacked.  The virus would generally not contain enough viral DNA to complete its own life cycle, and so would not damage the cell.  However, the DNA carried in from the previous host cell was capable of recombining with and altering the DNA of the new host, thus introducing new genetic characteristics without sexual activity.
ii.
It has become relatively easy for biotechnologists to construct viruses in the laboratory, and to select just what DNA they insert into the coat protein.  The engineered virus thus serves as a "bullet" to deliver new DNA to a cell.
iii.
If the virus used is a temperate virus, and you make sure that the included DNA contains its insertion recognition sequences and the appropriate genes to initiate lysogeny, then you can target your DNA for insertion into a specific location in the new cell's DNA.
iv.
This leads to a new problem—how do you combine together two particular pieces of DNA in exactly the way you want?  This question is dealt with below.
b.
Increasingly, genetically engineered bacterial plasmids are used as vectors for delivering genes into bacterial cells.
i.
There are a large number of natural plasmids which have been extensively studied and sequenced.  From these, a library of artificial, engineered plasmids has been developed. Each of these plasmids has in its sequence a known arrangement of restriction endonuclease sites, generally on site per plasmid. They also carry one or more antibiotic resistance genes which can be used as markers to determine whether a cell has received the plasmid or not.  They also carry sequences such as the controlling sequences of the lactose operon to provide a means of controlling the activity of any gene inserted into the plasmid.
ii.
To insert a gene into a plasmid, the gene and its surrounding DNA are analyzed for sequence and location of restriction sites.
a.
Sometimes, the biotechnologist will simply cleave the gene out of its normal position with a selected restriction endonuclease, cleave the plasmid with the same endonuclease, and allow the sticky ends to base pair. The addition of a little ligase will seal up the breaks, and you will have inserted your gene into the plasmid. This has a few problems.  One of the biggest problems is that all of your DNA fragments have the same sticky ends.  A lot of the plasmids will simply heal up; the genes may circularize and base pair their own sticky ends; you may get chains of plasmids, chains of genes, etc. All of these undesired outcomes must be sorted out.  Also, since most restriction endonucleases leave only about 4 bases unpaired at the ends, the connections are pretty fragile.
b.
A more efficient tool is terminal transferase.  This is an enzyme which adds nucleotides one at a time to the 3' end of a DNA strand.  The scientist selects the plasmid and cleaves it with a chosen restriction endonuclease. The cut plasmids are then treated with terminal transferase with, for instance, only Thymine-containing nucleotides available. This will create single stranded poly-T tails on the 3' ends of the two strands of the plasmid DNA. The desired gene is then treated the same way, except that it has poly-A added. Thus, the only pairing that can occur is between an end of the plasmid and an end of the gene.
iii.
Once you have engineered your custom plasmids you can induce bacteria to pick them up by treating the bacterial culture to transforming conditions (as studied by Griffith).  You select only the bacteria which have picked up the plasmid by treating the culture with the antibiotic(s) whose resistance gene(s) are carried on your plasmid. All of the bacteria which do not carry the plasmid will be susceptible and will die.  Only the plasmid (and human gene) carrying ones will remain.
E.
The key to being able to control the activity of an inserted gene is in the selection of the plasmid that carries it. The most common approach is to use a plasmid which contains the lacI gene as well as the promoter and operator for the lactose operon, but which does not carry the structural genes which are normally controlled by these sequences.  The plasmid would have a restriction site immediately following the lacO site, so that your human gene will be inserted in the position which would normally be occupied by the lacZ, lacY and lacA genes.  Thus you will be able to manipulate the activity of the human gene with the same signals you could use to turn the lac operon on and off (eg, high lactose, low glucose environment).  The lac promoter and lac operator will thus "think" they are turning on the lac digestion genes, but will instead be controlling, for instance, the insulin gene. This is, in fact, how insulin was handled.
F.
To replicate your plasmid is easy, once it is inside a bacterium.  Bacteria reproduce very quickly.  (E. coli, if it is happy, will double every 20 minutes.)  They duplicate their plasmids right along with the rest of the cell. There are also some plasmids which duplicate within a cell, so that the cell will end up containing a dozen or more copies of the same plasmid.  Thus, selection of the plasmid to utilize is significant in this context as well.

IV.
The most obvious goal of biotechnology is to perform "gene surgery" on human beings.  If you suffer from hereditary diabetes, it would certainly be preferable to have the genetic defect corrected in your own cells, rather than having to inject yourself with insulin for the rest of your life.
A.
This is a far more difficult task than genetically engineering bacteria. Humans are eukaryotic, and eukaryotic cells are much more complex structurally and genetically than prokaryotic cells. In addition, there is a great deal we don't yet know about gene control in eukaryotic cells. It is much more difficult to insert DNA into the chromosomes than into the circular DNA of a bacterium, and it is much more difficult to manipulate the operation of the DNA once it is inserted.
B.
There are at least two levels at which such gene surgery is considered.
1.
Somatic cell surgery would be attempts to correct genetic problems in the bodies of human beings.
a.
One level would be to attempt to adjust the genetic constitution of a fetus as it develops, either in the womb or in an artificial gestation system (something which has not been tried on humans).
b.
A second level would be to attempt to perform gene surgery on post-natal humans.  This has actually been attempted with some success on a very children who suffer from SCIDS (severe combined immune deficiency syndrome).
2.
Germ line genetic surgery would be an attempt to revise the genetic information carried in gametes.
a.
One form of such intervention would be to attempt to alter the gametes and gametogenic cells of adults.
b.
A second and much more accessible (and thus practical) approach would be to remove gametes from potential parents, genetically alter them, perform in vitro fertilization, and reimplant the resulting embryos in the mother.
C.
Many of the technical problems with gene surgery are the same as III above, although there are a number of additional ones. A variety of possible vectors has been considered, though the most commonly suggested are viruses, since they are already designed to get DNA into a cell, and many carry the information necessary to get that DNA into chromosomes. However, exactly how to control the cellular targets of these vectors is not always clear.  In order to insure that your virus vector would affect a large percentage of cells in a body, you'd have to inject a huge number of virus particles.  The usual pattern of a virus is to infect relatively few cells, then have the viruses produced by those cells attack additional cells, etc. Since the engineered virus wouldn't be capable of such activities, you would be likely to get only a small percentage of your target cells affected.  We are probably quite a bit short of routing implementation of these activities.
D.
Of all of the potential developments in biotechnology, this last—genetically tampering with human beings—is the most loaded in terms of ethical questions. It is to be hoped that we will be wise enough to consider these issues before we attempt a wide-scale application of genetic engineering on humans.




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