Antibody genes are rearranged at the genomic level to allow a high diversity of antibody types to be produced. This can lead a few hundred germ line genes to be shuffled into ten to a hundred million different types of antibodies. This involves "combinatorial joining" of different segments to generate a wide diversity of antibody types. Somatic mutation is another mechanism which contributes to antibody diversity.
Another type of DNA alteration is methylation of the C of 5’ CpG 3’sequences. This can be detected using restriction digestion with MspI (which cuts CCGG) or with HpaII (which cuts CCGG only if the interior C is not methylated). There is a correlation between methylation of these Cs and inactive genes, but it is difficult to determine what is cause vs. effect.
Molecular methods can be used to monitor variations in gene expression. These include dot blots to measure differences in quantity of genes, Southern blots to measure differences in gene structure, Northern blots and RT(reverse transcriptase)-PCR to measure differences in RNA type and quantity.
Transcriptional control in eukaryotes most often appears to involve positive regulation of transcription. Some genes are "on" (expressed) in most cell types; these are sometimes referred to as "housekeeping" genes and may be used as controls in gene expression studies. Examples: actin,, histones. Other genes are turned on under specific conditions (developmental stages, tissue types).
Transcriptional activator proteins bind to DNA and facilitate transcription of specific genes in eukaryotes. Two classes of these proteins are "helix-turn-helix" proteins and "zinc finger" proteins, both of which can recognize specific DNA sequences. Helix-turn-helix proteins contain alpha-helices, which can bind into the major groove of DNA.. The zinc finger proteins bind the ion zinc and also bind to DNA.
These proteins may bind small molecules such as the steroid cortisol. The complex then would move into the nucleus and bind a specific site on the DNA. In this way a signal molecule like cortisol could target a large number of different genes.
The targets of these proteins are often DNA sequences called enhancers. These are short sequences, usually < 20 bp. They are usually upstream from the gene, but may be in introns or even downstream. The same enhancer sequence may be near more than one gene. Multiple enhancers may be associated with a given gene. This sytem provides a means of regulating different genes at different locations in response to s simple signal. This thus accomplishes the same goal as is achieved in prokaryotes through production of a polycistronic message.
The transcription complex in eukaryotes is more complex than that of prokaryotes. It includes RNA polymerase enzyme. It also includes a number of proteins called "general transcription factors". Finally, it includes one or more proteins specific to the gene, the "transcriptional activator proteins". Combining a variety of transcriptional activator proteins recognizing various enhancers, with a diversity of enhancers which might be associated with specific genes, allows a great variety of expression patterns. This is referred to as "combinatorial control".
Other levels of control are present in eukaryotes. RNA processing patterns may vary for the same mRNA. Differences in splicing of the alpha-amylase gene between liver and salivary gland can result in different rates of translation in the two tissues. Some messenger RNAs have greater stability than others, leading to specific timing of expression or greater levels of protein expression. Masked mRNAs in oocytes may only be expressed after fertilization. Casein mRNA in mammary glands has a high stability, leading to enhanced casein expression. Silk fibrion mRNA has enhanced stability (3 days vs. 3 hours average eukaryotic mRNA life span) in silk worms.
Understanding the genetic analysis of development is one of the great challenges of modern genetics. The basic approach we will discuss is to isolate or induce mutants in the developmental process. This is similar in principle to what was done with the lac operon. Development is more complex (more genes, more tissue types) but additional tools like sequencing and RNA and protein expression assays are now available. Outline of coverage for this section: organisms studied, interesting mutants, general principles.