EGG CELL -> -> -> -> - -> ANATOMY of the BODY
DNA Base Sequences -> -> -> ANATOMY of the BODY
(Genetic Information) (Geometric arrangements of differentiated cells)
How do genes cause anatomical structures to form?
NOT by having different kinds of cells recieve different genes
(Which was what scientists guessed at first)
Skin cells have the same genes as nerve cells etc. All the cells get all the chromosomes & genes
This fact is called "Genomic equivalence"
One of the experimental methods used to prove this was nuclear transplantation.
Briggs & King (1952) sucked nuclei out cells of early frog embryos, and injected these nuclei into one-cell stage egg cells, whose nuclei had been removed
Some of these egg cells developed into tadpoles (with all the normal cell types: nerves, skin, liver)
This could not have happened unless all the early embryonic cells had still contained all the genes, the same as in the original egg.
For unknown reasons, all these tadpoles died before becoming frogs
In the 1970s, Gurdon repeated these experiments using Xenopus frogs (instead of Rana) and got "clones" of adult frogs. (by nuclear transplanation)
{Notice that the word "cloning" is used for a lot of different biological techniques that don't really have much relationship to each other!
"Cloning of genes", "Monoclonal antibodies", "Cloning of animals" by nuclear transplanation.
Actually, if you grow plants from cuttings, that's cloning too}
In 1997, Wilmut in Scotland used nuclear transplantation of sheep tissue culture cells into a sheep egg-cell (oocyte),
putting the oocyte back into the uterus of another sheep, and produced Dolly the sheep.
The newspapers got very excited by this! "This means that the body is caused by genes!!"
Embryologists had proven this 100 years before.
Dan Rather & a few newspaper editors were the only people in the whole world who had not already known that all cells contain all the genes.
Spemann in Germany had done experiments that were logically equivalent to nuclear transplantation in the 1890s.
There were also a few cases in which cells of one differentiated cell type can change into cells of a completely different cell type.
(Which obviously couldn't occur unless that had the genes for being this other cell type)
Pigment cells into lens cells; Pigment cells into nerve cells of the eye;
In general however, especially in animals, differentiation is almost permanent:
once cells differentiate, some mechanism keeps them locked in to that one type.
Plant cells often switch from one cell type to another. That is much more unusual in animal cells.
Whole plants were grown from single carrot tissue culture cells in the 1960s.
Wilmut's key gimmick was to get nuclei from tissue culture cells, in G1 of their cell cycles.
Since then cows, mice, cats etc. have been cloned (in the sense of being produced by nuclear transpnatation into oocytes).
Such production of horses and dogs have been claimed; and aarvarks may not be far behind.
Notice the very high death rates (99% or more) of embryos developing with transplanted nuclei (Dolly one of 146 attempts)
(remember that when you read claims that Elvis has been cloned)
Also, most of the cloned animals that are born have defects & die young (as did Dolly herself, last year)
Some special cases in which some cells don't have the same genes (DNA base sequences) as all the other cells
1) Mammal red blood cells expel their whole nucleus (when mature, are anucleate)
But bird, reptile, amphibia, fish red blood cells have nuclei
2) Amphibian oocytes make hundreds of extra copies of the DNA coding for ribosomes.
3) Some insect larvae make extra copies of certain genes.
4) (and medically most important) Lymphocytes (the kind of cell that makes antibodies)
cut and splice ("rearrange") the DNA that codes for the binding sites of antibody proteins.
( Don't confuse this with the cutting and splicing of RNA, by which introns are removed) (Also don't confuse it with mismatch repair of DNA)
During your embryonic development, random splicing of DNA alternate "V", "D" and "J" regions
randomly generated billions of genetically different clones of B-lymphocytes,
each clone making antibodies with its own special shaped binding sites!
Antibodies "against" smallpox
Antibodies against pollen
Antibodies against type A blood group antigen
Antibodies against DNA
etc. etc. billions of different kinds of antibodies
Even if you have type A blood, your embryo starts out with B-lymphocytes that make antibodies whose binding sites specifically bind to the A blood group antigen.
Then some UNKNOWN MECHANISM weeds out those B-lymphocytes whose antibody binding sites fit any "self" antigen (molecule that is a normal part of your body)
You keep all the other billions of B-lymphocyte clones.
"northern blotting"
Isolate RNA from embryo.
Then all the RNAs are transferred to a thin sheet of nylon
Then radioactive DNA with certain base sequences is put into solution and the nylon sheet is put in this solution for a while.
PCR = Polymerase Chain Reaction
Small amounts of DNA are caused to duplicate themselves selectively in parts bounded by certain base sequences.
Heat DNA, so the 2 strands separate.
Cool DNA in presence of DNA "oligonucleotide" primers
Add DNA polymerase enzyme, plus deoxyATP, CTP, GTP & TTP
Then heat again and repeat the process.
A special form of the DNA polymerase from hot springs bacteria (found in Yellowstone National Park !) is not destroyed by the heating,
It's so easy, even molecular biologists can do it!
Other related methods
In situ hybridization:
fixing tissues, and often sectioning them
Microarrays:
Glass chips;
Chips then soaked in solutions of nucleic acids
If both bind: then glows yellow;
Antisense RNA
RNA interference "RNAi"
Vaccines work by stimulating growth of those antibody clones that happen to make antibodies against that certain germ. Allergies result from B-lymphocytes whose antibody binding sites happen to fit some molecule that would otherwise be harmless. Autoimmune diseases result from the failure to weed out some of the lymphocytes whose antibodies fit self molecules.Lupus, Multiple Sclerosis, many forms of arthritis result from your immune system attacking you:what amounts to an allergy to yourself. These are terribly cruel diseases, that ruin the lives of millions of people, and every year kill hundreds of thousands.
If you are thinking about going into medical research as a career, I suggest that you try to discover the unknown mechanism that normally eliminates the anti-self clones. This is ripe for discovery, and is one of the biggest medical contributions anyone could hope to make. Most textbooks claim that the immune system works by detecting the foreigness of molecules, but that is idiotically wrong. Foreign molecules do not have any particular properties in common with all other foreign molecules, so foreigness cannot be detected. The immune system also doesn't work by detecting "self"! If you want to know more about this subject, see my website for "Unsolved Problems", which is taught every fall. It is not really part of embryology. This random generation of genes for antibody binding sites results from certain "recombinase" enzymes.
These genes are expressed (m-RNA transcribed) ONLY in embryonic lymphocytes. (in addition to B- lymphocytes, there are T lymphocytes)A medically important effect is that sometimes the DNA gets cut at the antibody genes, but then gets reconnected (by mistake!) just upstream of some gene coding for a protein that controls cell growth.
Most cases of the kind of cancer called lymphoma have such a cause. When these cells try to make antibodies, this activates transcription of some gene that either stimulates cell growth, or inhibits cell death. SOME METHODS USED TO STUDY GENE EXPRESSION
(= to map or measure which genes are transcribed to make m-RNA)
Please note, these are the areas that I know least about, so I will just outline the main points well enough that we can later discuss evidence based on these kinds of methods.
(this name is a joke; because a Prof. Southern invented the equivalent method for studying DNA. Doing it with proteins is called a "western.")
Put RNA in narrow holes in a gel.
Pull RNA molecules with a electric field (voltage gradient)
(the textbook is misguided to say that it's the current: The key thing is the voltage!)
THIS GENERAL METHOD IS CALLED ELECTOPHORESIS
The shorter RNAs travel faster.
(this is the process of "blotting") (nitrocellulose was also used)
The purpose is to lock the RNA into whatever relative positions they had moved to in the gel.
If you left them in the gel, they would diffuse around.But to nylon, they bind strongly, and stay put.
By random diffusion, these find and bind selectively to any RNA that has the complementary base sequences
These will pair with any DNA base sequences complementary to them
CGATTGGCC would pair with AAAAGCTAACCGGTTTT
DNA strands will be copied starting at the places where primers are bound
Each cycle doubles the number of the selected DNA sequences
so you don't have to keep adding more enzyme; heating and cooling over and over is sufficient.
When PCR was first invented, more enzyme was added in each cycle.
& incubating these with labeled single-stranded nucleic acids;
that selectively stick at any locations where there are m-RNA with complementary sequences
Robot arms are used to spot thousands of different nucleic acid samples, each at a certain location.
Some bound to fuorescein (glows green in UV)
Others bound to rhodamine (glows red in UV)
if binding mostly only by first set, glows green.
if binding mostly only by second set, glows red.
This is a method for selectively blocking synthesis of a certain protein, by binding another RNA to the messenger RNA for this protein. It was invented long before RNA-i, but I suspect that many or all cases where genes were blocked by anti-sense RNA, the real reason for the blockage was RNA i. Notice that the textbook is oblivious to this possibility! Molecular biology tend to attract dimmer minds, who don't notice such implications. The idea is somehow to synthesize or isolate RNA whose base sequence is complementary to some particular messenger RNA that you are interested in. In other words, if the messenger had the sequence ACTGAGGCCTT etc, then the complementary sequence would be TGACTCCGGAA. These two strands would pair selectively, forming double-stranded RNA, and tending to prevent the translation of the messenger. The net result is as if you had knocked out that particular gene. Thousands of experiments of this kind were done, including even treatment of certain kinds of cancer with anti-sense RNA to block expression of certain concogenes.
To everyone's surprise, putting a little bit of double stranded RNA into a cell will induce the formation of enzymes that will selectively digest this and any other RNAs with the same sequences. This was a big surprise and a bigger mystery! The mechanism is now believed to be enzymes that defend cells from RNA viruses. These viruses selectively digest any double stranded RNA, and retain fragments of the RNAs they have digested, by which they selectively bind to any single stranded RNA that has base sequences complementary to what they previously digested. No such possibility had occurred to those who invented and use the antisense RNA method. But when antisense strands bind to messenger RNA, that creates a double stranded RNA, and will thereby set off the RNA-i mechanism. It is a good question how one could determine which of these mechanisms was the real cause of blocking gene action in a given case. Most of the anti-sense RNA literature should be reexamined. Specific criteria could include how long the blockage lasted (after the antisense RNA was put in the cells), whether the blockage would still occur in organisms that lacked the normal genes for the enzyme that digests the double stranded RNA, and also perhaps effects of antibiotics that specifically inhibit RNA synthesis. These questions are beyond the scope of this course, but worth thinking about. Gene transformation: DNA from some other source can sometimes bcome inserted in among genes of some organism. This is equivalent to moving a base sequence from the genes of one organism to the genes of another organism, even putting animal genes into plants, etc. Many methods have been invented to get the DNA into the other organism:
* Simply putting the DNA in the culture medium; * Injecting solutions of the DNA into cells;
* Shooting tiny heavy metal beads (coated with DNA) into cells, originally using gunpowder;
* Inducing phagocytosis of goopy stuff with DNA in it, into tissue culture cells;
* Electrical sparks, that temporarily puncture the plasma membranes;
there are other methods, and biologists will try anything! Transgenic mice
and transgenic animals of other species not to mention transgenic plants, which the public and news media calls "genetically modified". [Of course, mutations are another form of genetic modification. But the current excitement is about non-random changes, specifically transfer of base sequence from one organism to another.] There are some nifty tricks for finding the one mouse in a thousand in which the transgene got incorporated into the host chromosomes, or for getting it to insert where you want it. You can learn those in other courses. Chimeric mice (or other chimeric animals, or plants) are partly made out of cells from one set of parents, mixed with cells from another set of parents. For example, a sheep/goat chimera has been made. It would not be impossible to make a human/animal chimera; there would be no reason to do this, & I only mention it to emphasize what is possible. Embryologists have been making chimeric frogs and salamanders (and other animals) since the 1920s). The first chimeric mice were made in the mid-1960s. Chimeras are equivalent to people with transplanted kidneys, hearts, or other organs (and analogous to Frankenstein's monster) except that the foreign tissues are mixed during embryonic development. Note that the immune system does not attack tissues that have been in you since embryonic development; nor do these tissues attack your body.
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