Messenger RNA Populations in Differentiating Muscle Cells
Messenger RNAs, as the immediate readout of gene expression, provide a direct indication of how a cell uses its genetic information as its developmental status changes. Working backward from the mRNA to the gene leads to the underlying question of transcriptional control. After my thesis, I went to Paris to the Pasteur Institute as a postdoc in the lab of François Gros, to work on messenger RNA. At that time he and François Jacob, after the characterization of mRNA in bacteria (Jacob & Monod, 1961), were turning their attention to eukaryotes. Jacob was primarily interested in early embryonic development and was beginning to work with teratocarcinomas. Trying to unravel what might be happening for single genes at the level of the embryo was a herculean task with the tools available, and in vitro cell systems appeared more tractable. With F. Gros, we focussed on the progression of a tissue-specific precursor cell to formation of the tissue, so-called terminal differentiation. Muscle tissue provided an attractive model since the precursor cells, myoblasts, could be physically separated from differentiated muscle fibers which were large syncythia formed by myoblast cell fusion. Primary cultures of mononucleated cells were enriched in myoblasts and devoid of differentiated muscle cells. Furthermore, David Yaffe had succeeded in cloning myoblasts and establishing mouse and rat muscle cell lines (Richler & Yaffe, 1970).
Myoblasts spontaneously differentiate when the cultures become confluent. It is thus possible to follow their progression from proliferating precursor cells to the onset of differentiation, followed by the formation of muscle fibers. In our initial studies, we characterized changes in mRNA populations by plotting Rot curves based on hybridization kinetics (Bishop, 1969) and changes in the location of mRNAs in polysomes or ribonucleoprotein particles separated on sucrose gradients. The identification of 3′polyadenylation as a feature of mRNAs had made it possible to separate this class of RNA on polyA/U affinity columns. Radioactive labeling of mRNAs in cultured cells and pulse-chase experiments provided information on mRNA dynamics (e.g., Buckingham, Caput, Cohen, Whalen, & Gros, 1974), showing, for example, that some mRNAs move from the RNP compartment into polysomes at the onset of differentiation when messenger stability also increases. Even this level of general information was conceptually useful in the uncharted domain of cell differentiation. More precise information on the mRNAs came from in vitro translation experiments with analysis of the protein products by two-dimensional gel electrophoresis. The major muscle proteins present in fibers were well known and we could now separate different isoforms of contractile proteins such as actins or myosin light chains and follow the switch to those specific for differentiating muscle cells (e.g., Daubas, Caput, Buckingham, & Gros, 1981).
I.a. The DNA, RNA and ProteinsDNA or other wise called deoxyribonucleic acid is the building block of the life. It contains the information the cell requires to synthesize protein and to replicate itself, to be short it is the storage repository for the information that is required for any cell to function. Watson-Crick has discovered the current-structure of DNA in 1953.The famous double-helix structure of DNA has its own significance. There are basically four nucleotide bases, which make up the DNA. Adenine (A), Guanine (G), Thymine (T) and Cytosine(C). A DNA sequence looks some thing like this "ATTGCTGAAGGTGCGG". DNA is measured according to the number of base pairs it consists of, usually in kBp or mBp(Kilo/Mega base pairs). Each base has its complementary base, which means in the double helical structure of DNA, A will have T as its complimentary and similarly G will have C. nbsp; DNA molecules are incredibly long. If all the DNA bases of the human genome were typed as A, C, T and G, the 3 billion letters would fill 4,000 books of 500 pages each! The DNA is broken down into bits and is tightly wound into coils, which are called chromosomes; human beings have 23 pairs of chromosomes. These chromosomes are further broken down into smaller pieces of code called Genes. The 23 pairs of chromosomes consist of about 70,000 genes and every gene has its own function. As I have mentioned earlier, DNA is made up of four nucleotide bases, finding out the arrangement of the bases is called DNA sequencing, there are various methods for sequencing a DNA, it is usually carried out by a machine or by running the DNA sample over a gel otherwise called gel electrophoresis. A typical sequence would look like this "ATTTGCTGACCTG".
Fig 1.1.1. Sample genetic code with complementary strands.
Determining the gene's functionality and position of the gene in the chromosome is called gene mapping. Recent developments show that scientists are mapping every gene in the human body. They named their project Human Genome Project (HGP), which involves careful study of all the 70,000 genes in human body. Whew! That's some thing unimaginable. When there is a change in the genetic code it is called mutation.
The significance of a DNA is very high. The gene's sequence is like language that instructs cell to manufacture a particular protein. An intermediate language, encoded in the sequence of Ribonucleic Acid (RNA), translates a gene's message into a protein's amino acid sequence. It is the protein that determines the trait. This is called central dogma of life.
Fig 1.a.2 Central dogma of life.
Notes: Genes are DNA sequences instruct cells to produce particular proteins, which in turn determine traits. Chromosomes are strings of genes. Mutations are changes in gene's DNA sequence.
RNA is somewhat similar to DNA; they both are nucleic acids of nitrogen-containing bases joined by sugar-phosphate backbone. How ever structural and functional differences distinguish RNA from DNA. Structurally, RNA is a single-stranded where as DNA is double stranded. DNA has Thymine, where as RNA has Uracil. RNA nucleotides include sugar ribose, rather than the Deoxyribose that is part of DNA. Functionally, DNA maintains the protein-encoding information, whereas RNA uses the information to enable the cell to synthesize the particular protein.