Textbook assignment: Chapter 8, Pages 244 - 253.
Major concepts:
Gene amplification: Our textbook does not explore this topic in depth. The only reference to gene amplificaiton I have found is on page 755, where drug induced amplification of the DHFR gene (see drug-resistant tumors, below) has been used to achieve deliberate amplification of a linked gene to increase production of the product of that gene. References have been given to material from the previous textbook for this course (on reserve in Norlin). However, you do not need to know this material beyond the level of coverage in these notes.
Amplification only occurs in a few special circumstances: In most discussions of control over level of gene expression, we start with the assumption that the genome contains an invariant number of copies of the gene in question. While this is usually the case, there are important exceptions, as discussed on pages 556-558 of Klug and Cummings, Concepts of Genetics, 5th Edition (Norlin Reserve). (You may also want to refer back to previous mentions of these phenomena on pages 245, and 341 of Klug and Cummings). Four major examples are discussed briefly.
Amplification of ribosomal RNA coding genes: Amphibian embryos develop from rather large eggs that support a large amount of protein synthesis over a relatively short period of time. This requires accumulation of a huge number of ribosomes in the egg, which in turn requires a very high rate of transcription of ribosomal RNA. The mechanism that is used to achieve such synthesis is extrachromosomal amplification of the DNA sequences that the ribosomal RNA is transcribed from.
Amplification of genes coding for chorion proteins in Drosophila eggs: Amplification of genes coding for egg shell proteins occurs during oogenesis in Drosophila, with the region of amplification extending well beyond the immmediate limits of the genes that are directly involved.
Chromosomal puffs: In some species of insects, the polytene chromosomes exhibit "puffs", in which particularly active genes are amplified beyond the extent of amplification of the entire genome in the polytene chromosomes (Figure 19.21, Klug and Cummings).
Drug-resistant tumors: Gene amplification does not appear to be a normal developmental phenomenon in mammals. However, aneuploid (chromosomally altered) tumor cells may respond to chemotherapeutic drugs by greatly amplifying the genes whose products are inhibited by the drugs, sometimes overwhelming the ability of the drugs to control the malignant growth. One example is amplification of the genes coding for dihydrofolate reductase (DHFR) in response to treatment with methotrexate. Our textbook briefly discusses use of this phenomenon to achieve amplification of a second gene that has been linked to the DHFR gene through use of recombinant DNA technology (page 755, second column). In some cases, the amplificaiton products are seen as extrachromosomal structures that have come to be called double minute chromosomes.
Alternative splicing: Nearly all eukaryotic genes contain introns that are "spliced out" during processing of the initial transcript to generate the final mRNA. One fairly common mechanism for altering the properties of the final protein product is to modify the pattern of splicing, such that certain exons are included in some versions of the protein and excluded from others (figure 8.29). Often this phenomenon is controlled in a tissue specific manner. In an extreme case, 64 different alternatively spliced forms of the muscle protein troponin T have been identified. Similarly, there are 10 different forms of alpha tropomyosin. In some cases, different peptide sequences with different biological functions may be included or excluded in different tissues. An example of this is shown in Figure 19.23 of Klug and Cummings, where the coding sequence for tachykinen K may or may not be retained in a larger mRNA.
Sex determination in Drosophila: The cascade of regulatory mechanisms that determines whether a Drosophila develops as a male or a female includes several steps of directed alternative splicing of mRNAs for regulatory proteins that ultimately determine which pathway is followed (See figures 19.24 and 19.25 of Klug and Cummings). This pathway will be examined in detail in MCDB 4650.
Message stability: Another interesting control over gene expression is achieved by changes in message stability. A message with a long half-life will be translated far more times before it is degraded than a message with a short half life. The textbook describes the stabilizing effect of prolactin (a hormone that stimulates milk production) on the mRNA for casein, a milk protein. Increased message stability is a mechanism that is frequently used in differentiated mammalian cells that produce very large amounts of particular proteins.
Short half-life mRNAs and proteins: There are also some messages that have inherently short half lives. This is particularly important for mRNAs that code for regulatory protiens. For any signaling system that is capable of responding quickly to change, it is important both for the signal molecules to be generated rapidly and for them to go away rapidly after they have transmitted the needed message. In many cases, the mRNAs and the proteins they code for are both unstable and degrade rapidly after the regulatory signal has been sent. This has the effect of clearing the system so it is ready for a new signal. Short half life mRNAs tend to have AU rich sequences in their 3'-untranslated regions. Short half life proteins tend to contain sequences rich in proline, glutamic acid, serine, and serine. These are sometimes called "PEST" sequences, based on the one letter codes for these four amino acids (described in Tamarin, Principles of Genetics, 5th Edition, pages 378-380, available at Norlin reserve desk).
Degradation of pre-mRNA: Our textbook points out that 75% of all eukaryotic transcripts are degraded in the nucleus without ever being processed. This is yet another level at which selective degradation or stabilization of specific RNA sequences can influence the ultimate level of gene expression.
Translational regulation: There are a number of situations in which fully processed cytoplasmic mRNAs are held in an inactive state for later translation. One example is the storage of maternally-derived mRNAs in egg cytoplasm for later use during early embryonic development after fertilization. There are also mechanisms for selectively increasing the translation of specific mRNAs. The example cited in our textbook is the one used by the AIDS virus to selectively increase translation of its mRNA in infected cells.
Allosteric feedback inhibition: We have already seen several examples of allosteric changes in protiens caused by the binding of small molecules. In each case, the allosteric changes have had profound effects on the biological activities of the proteins. Allolactose causes the lactose repressor to be unable to bind to the operator of the lactose operon. Tryptophan activates the tryrtophan apo-repressor, converting it into an active repressor that can bind to the operator and stop transcription of the tryptophan operon. Binding of a steroid hormone to its receptor converts the receptor into a functional transcription factor that can bind to the appropriate HRE and turn on (or off) the transcription of a particular hormone-responsive gene. In feedback inhibition, the end product of a biosynthetic pathway (often a specific nutrient) can bind to an early enzyme in the pathway, causing it to lose its activity. This allows the end product to control the rate of its own synthesis. When the end product accumulates, synthesis is reduced, and when it is in short supply, the inhibition is released and more synthesis occurs.
Rapid response versus efficiency: Allosteric feedback inhibition allows rapid "fine-tuning" of levels of synthesis without the delays that are encountered when regulatory systems act through transcriptional or translational control mechanisms. However, for responses to lasting changes in conditions, transcriptional controls are more efficient in that they save the energy that would otherwise be used for synthesis of mRNA and its translation into enzyme proteins.
Branched pathways: The patterns of feedback inhibition can become quite complex in branched biosynthetic pathways. The textbook cites an example where lysine inhibits an entire pathway that branches into lysine, threonine, isoleucine and methionine. An excess of lysine normally causes cultured plant cells to fail to grow for lack of methionine. Mutant strains that lack this inhibition and grow without methionine in the presence of high lysine will sometimes accumulate higher levels of lysine when grown in the absence of supplements. This has been used to generate strains of plants (regenerated from the cultures) that produce more lysine (which tends to be at lower levels than ideal for human nutrition in many grain products).
Bigger picture: Although the basic central dogma of molecular biology (Figure 3.1) seems relatively simple, there are actually many opportunities for regulatory controls in the overall flow of information from genomic DNA to final functional gene product. The brief summary presented in these notes has not even come close to summarizing all of the possibilities. In particular, we have barely touched on the many types of post-translational modifications that determine how gene products ultimately function. In addition to feedback inhibition and the various allosteric modificaitons that we have explored, there are many additional ways in which the biological activities of proteins can be altered. One example is by phosphorylation, which is a very common theme in cell signaling regulatory cascades. As you progress through the MCDB major, you will encounter many more of these regulatory mechanisms and gain an increased appreciation of just how complex eukaryotic gene regulatory mechanisms actually are (and perhaps be thankful that you did not have to learn all of them in this course).