MadSci Network: Development
Query:

Re: I want to know about the cell differentiation takes place in the body.

Date: Fri Sep 9 18:12:59 2005
Posted By: Amit Agarwal, Grad student, Neuroscience/Neurogenetics,
Area of science: Development
ID: 1124971987.Dv
Message:

Dear Amol,
From your question i wished I could have talked to you face to face. 
As there are many things, I feel I could have explained you better in 
person. As I don’t know the level of your knowledge, if i try to explain 
you from scratch it would take me lot more space which would have 
boared you anyway. I have tried my best to be as basic as possible 
and from next time i would suggest you to break you questions into 
many small fragements instead of one large chunk. One thing i must 
mention that your question is indeed interesting. I have not dewelled 
into the details of lot many things as they are in themselves a large 
topic, and details of which you can very easily find in many standard 
text books in your university library.

So, coming directly to the point, I would like to clarify two term for you.
 
i) During differentiation, certain genes are turned on, or become 
activated, while other genes are switched off, or inactivated. This 
process is intricately regulated by the expression of other genes and 
the process of alternate splicing. As a result, a differentiated cell will 
develop specific structures and perform certain functions. 
Differentiation can involve changes in numerous aspects of cell 
physiology; size, shape, polarity, metabolic activity, responsiveness to 
signals, and gene expression profiles can all change during 
differentiation. Usually in a cell there is no change at the gross 
genomic level, with few exceptions which includes B cells (where 
rearragment takes place).

ii) Recombination generally refers to the molecular process by which 
alleles at two genes in a linkage group can become separated. In 
this process alleles are replaced by different alleles from the same 
genes thereby preserving the structure of genes. One mechanism 
leading to recombination is chromosomal crossing over (during 
meiosis). In analog fashion exchange of alleles is possible between 
homologous sites within one DNA molecule. If the structure of genes 
is changed in that process it is called unbalanced recombination. 
Enzymes called recombinases catalyze this reaction.

So during differentiation mainly rearrangment of genes takes place 
instead of recombination, which is highly specific molecular process. 
Note word recombination should not be confused or used 
interchangibly with rearrangment

Before going futher i would here like to give you an insight about how 
B cells can synthesize antibody against any antigen they combat with. 
Here, I would like you to take note that even  in B-cells, 
rearrangement of gene takes place and not the recombination, even 
though the enzymes involved in this process are called as 
recombinases.

There are an estimated 109 (10 raised to the power of 9) different 
antibody molecules, and thus 109 (10 raised to the power of 
9)distinct B cell clones in a single individual. Most intriguing quest 
is that, how is this astonishing diversity generated?
This problem has been resolved over the past 20 years by the 
demonstration that Ig (immunoglobulin) genes rearrange during B 
cell development. The heavy chain genes don’t have complete exon 
encoding the V (Variable) region domain, instead this is split into 
arrays of gene segments. Light chain genes are similarly organised 
on different chromosomes but they don’t have D (Diversity) gene 
segments. There are 51 functional VH (Variable heavy chain) genes 
and 41 Vk (Variable light chain) genes. D (diversity) and J (junctional) 
genes code for amino acids at the carboxyl end of V regions including 
CDR3 (Complementarity determining regions).
It was first noted that the organization of Ig genes in mature B cells 
was different from that in embryonic tissues. The pieces of genomic 
DNA containing antibody genes in embryonic tissue, or adult non-B 
cells, are larger that those from mature B cells. This implies that the 
DNA from mature B cells has been rearranged resulting in the 
excision of some DNA. It is now known that Ig genes rearrange 
segments with the looping out of intervening DNA. This is done in a 
precise order. First the heavy chain rearranges: i) D -> J and then ii) V 
-> DJ.
If a functional heavy chain (always IgM initially) results (many joins 
are out of frame) the light chains rearrange also in order, first kappa 
then if kappa is unproductive (or cannot pair with the heavy chain) 
lambda i.e. V -> J.
(Note: there are two light chain isotypes (kappa and lambda); one B 
cell will only ever express one light chain isotype).
Expression of the first µ heavy chain (heavy chain of IgM is termed as 
µ) prevents rearrangement on the other chromosome in a process 
called allelic exclusion designed to prevent expression of two 
antibodies by the same B cell. The same is true for the light chain.
The enzymes involved in rearrangement are recombinases 
(lymphocyte specific), exonucleases and ligases. The recombinases 
recognise conserved heptamer and nonamer sequences found 
adjacent to the V, D and J exons. Two recombination activating genes 
(RAG1 and RAG2) have been shown to affect rearrangement of both 
genes for Ig and the T cell receptor.

There are 4 types of mechanism involved in generating antibody 
diversity.

i) Pairing of different combinations of Ig heavy and light chains.
ii) Recombination of V, D and J segments (VJ for light chains) the 
arrangement of Ig gene in segments.

 	
Segment	                                         Kappa       Lambda   Heavy Chain

Variable (V)	                                       40	               31	                51
Diversity (D)	                                       0                 	0	                 25
Joining (J)	                                          5	                 4	                  6
                           
Max No. Combinations	                 200	            124	             16218

Number of possible HL combinations are about 5.2 Million.

Together these potentially generate some 5x106 different antibodies.

Note: for the heavy chain the functional coding frame is fixed for V 
segments and for J segments i.e. each hasonly a single useful 
frame. D segments however can be read in more than 1 frame. As 
the human locus has 25 functional D segments, the total possible 
number of D products would be 75 if all D segments could be read in 
all frames. In fact only 53 possible reading frames exist (that is there 
are in-frame stop codons within the other 22 D-frames). That is why 
the number of possible heavy chain V regions is 16218 (51 x 53 x 6) 
and not 7650. It also means that instead of 1/3 rearrangements 
being in-frame the true number is 1/4.25 (23.5%).

iii) Variability in the joins of the recombined DNA segments.
This arises from: a) Imprecise joining by the recombinational 
machinary
b) the addition of extra random nucleotides by Terminal 
deoxynucleotide transferase (N region addition, not seen in light 
chains)

iv)  Somatic hypermutation, a poorly understood mechanism for 
introducing mutations into V regions of activated B cells (antigen 
driven).

Antibody molecules show the greatest sequence diversity at the V -> 
C junction. This forms the CDR3 and is encoded largely by D and J 
gene segments and thus benefits from both recombinational and N 
region diversity.

Now we come to our second question, that is, if all the cells have 
same genetic material? 

I would say YES!! Take any cell either heart, liver, skin or kidney from 
an individual and if you do a genomic DNA sequencing from them 
and you will find the same sequence (watch out!! B-celles there you 
have DNA rearrangment). So i would say 'human genome sequence 
is the treasure’ and for sure it has made possible for life science to 
take a giant leap.

But, in your question there is one more mystery hidden and that is 
'Transposons’ or i always like to call them "jumping genes". The first 
transposons were discovered in the 1940s by Barbara McClintock 
who worked with maize (Zea mays, called "corn" in the U.S.). It took 
about 40 years for other scientists to fully appreciate the significance 
of Barbara McClintock's discoveries. She was finally awarded a 
Nobel Prize in 1983.
Transposons are segments of DNA that can move around to different 
positions in the genome of a single cell. In the process, they may
i) cause mutations
ii) increase (or decrease) the amount of DNA in the genome. 
There are three distinct types:
a)Class II Transposons consisting only of DNA that moves directly 
from place to place.
b) Class III Transposons; also known as Miniature Inverted-repeats 
Transposable Elements or MITEs.
c)  Retrotransposons (Class I) that
          o first transcribe the DNA into RNA and then
          o use reverse transcriptase to make a DNA copy of the RNA to 
insert  in a new location.

Transposons are mutagens or i would call them 'Natural Genetic 
Engineers’. They can cause mutations in several ways:
a ) If a transposon inserts itself into a functional gene, it will probably 
damage it. Insertion into exons, introns, and even into DNA flanking 
the genes (which may contain promoters and enhancers) can 
destroy or alter the gene's activity.
b) Faulty repair of the gap left at the old site (in cut and paste 
transposition) can lead to mutation there.
c) The presence of a string of identical repeated sequences presents 
a problem for precise pairing during meiosis.

Two major transposing elements in human beings are:

i) LINEs (Long interspersed elements)

a) The human genome contains some 850,000 LINEs (representing 
some 21% of the genome).
b) Most of these belong to a family called LINE-1 (L1).
c) These L1 elements are DNA sequences that range in length from 
a few hundred to as many as 9,000 base pairs.
d)Only about 50 L1 elements are functional "genes"; that is, can be 
transcribed and translated.
e)The functional L1 elements are about 6,500 bp in length and 
encode three proteins, including
          o an endonuclease that cuts DNA and a
          o reverse transcriptase that makes a DNA copy of an RNA 
transcript. 

The diversity of LINEs between individual human genomes make 
them useful markers for DNA "fingerprinting".

ii) SINEs (Short interspersed elements)
SINEs are short DNA sequences (100–400 base pairs) that 
represent reverse-transcribed RNA molecules originally transcribed 
by RNA polymerase III; that is, molecules of tRNA, 5S rRNA, and 
some other small nuclear RNAs.The most abundant SINEs are the 
Alu elements. There are over one million copies in the human 
genome (representing about 11% of the total DNA).Alu elements 
consist of a sequence of 300 base pairs containing a site that is 
recognized by the restriction enzyme AluI. They appear to be reverse 
transcripts of 7S RNA, part of the signal recognition particle. Most 
SINEs do not encode any functional molecules and depend on the 
machinery of active L1 elements to be transposed; that is, copied and 
pasted in new locations.
SINEs (mostly Alu sequences) and LINEs cause only a small 
percentage of human mutations. (There may even be a mechanism 
by which they avoid inserting themselves into functional genes.) 
However, they have been found to be the cause of the mutations 
responsible for some cases of human genetic diseases, including:

a) Hemophilia A (Factor VIII gene) and Hemophilia B [Factor IX gene]
b) X-linked severe combined immunodeficiency (SCID) [gene for part 
of the IL-2 receptor]
c) Porphyria
d) predisposition to colon polyps and cancer [APC gene]
e) Duchenne muscular dystrophy [dystrophin gene] 

Then a question arises, what good are transposons?

We don't know. They have been called "junk" DNA and "selfish" DANN 
(here i would recommend you to read books like  'Selfish Genes’ and 
'Genome’ by Matt Ridley).

i) "selfish" because their only function seems to make more copies of 
themselves and ii)  "junk" because there is no obvious benefit 
to 
their host.

Retrotransposons cannot be so selfish that they reduce the survival 
of their host. Perhaps, they even confer some benefit. Some 
possibilities:

Retrotransposons often carry some additional sequences at their 3' 
end as they insert into a new location. Perhaps these occasionally 
create new combinations of exons, promoters, and enhancers that 
benefit the host.

      

Example:
     
 Thousands of our Alu elements occur in the introns of 
structural 
genes. Some of these contain sequences that when transcribed into 
the primary transcript are recognized by the spliceosome. These can 
then be spliced into the mature mRNA creating a new exon, which 
will be transcribed into a new protein product. 
 Alternative splicing can provide not only the new mRNA (and 
thus 
protein) but also the old. In this way, nature can try out new proteins 
without the risk of abandoning the tried-and-true old one. 
 Telomerase, the enzyme essential for maintaining 
chromosome 
length, is closely related to the reverse transcriptase of LINEs and 
may have evolved from it. 
 RAG-1 and RAG-2. The proteins encoded by these genes are 
needed to assemble the repertoire of antibodies and T-cells 
receptors (TCRs) used by the adaptive immune system. The 
mechanism resembles that of the cut and paste method of Class II 
transposons , and the RAG genes may have evolved from them. If so, 
the event occurred some 450 million years ago when the jawed 
vertebrates evolved from jawless ancestors. Only jawed vertebrates 
have an adaptive immune system and the RAG-1 and RAG-2 genes 
that make it possible.
 In Drosophila, the insertion of transposons into genes has 
been 
linked to the development of resistance to DDT and 
organophosphate insecticides.

From the above example we can infer that every gene cannot act as 
trasposons, jumping from one location to the other, there are in infact 
specialized class of DNA sequence. So, if you say all the oncogenes 
are present in chromosome 11 then they will be on cromosome 11 in 
every human being. But, there will be the possiblity of some 
modifications in these genes within and among different individuals 
due to random mutations, recombination (during cell division), 
rearrangment (during differentiation), and some time integration of 
stray transposon (which is very rare). We say most of our DNA is junk, 
but i would say that here word 'junk’ is misnomer, the correct way  to 
say is that most of these unknow DNA sequences are 'holes’ in our 
current knowledge, as i personally feel that mother nature would 
never select something junk and propagate it using an energy 
intesive process such as DNA replication.

I hope that i didn’t end up confusing you, since u have asked 
specifically not to site references I had to elaborate lot of things. If you 
are more confused and still have mindbogling questions then you 
can get back to me and i would love to take you questions.

Best wishes,
Amit








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