Advanced
Genetics,
(Modified 10:45 am, August 25, 2009
L. Curtis (Curt) Hannah
Building 710 (Plant Physiology building -- east of Fifield Hall across
the
parking lot)
phone: 352-392-6957, personal cell 352-219-5943
Reference for Mendels original paper. http://www.esp.org/foundations/genetics/classical/gm-65.pdf
The purpose of this section is two-fold:
(a) Familiarize students with an historical understanding of the concept of a gene, starting before Mendel, focusing on Mendel, then Benzer, the lac operon and our present understanding of the gene in molecular terms. We will actually start with the last point. In doing this, the central dogma of genetics (which hopefully you know very well) will be reviewed.
(b) Reacquaint students with genetics. This will involve analysis of data, following alleles of genes through meiosis and fertilization in diploid organisms, simple concepts of recombination, etc. In many ways, this point is more important than the first. The ability to think genetically is critically important to today's biological sciences. All too often, scientists know molecular biology or physiology but very little genetics. This is often seen in grant proposals and submitted manuscripts.
The purpose of the second section is also two-fold:
(a) Familiarize students with transposable elements. We will cover the classic work that first detected these elements and then subsequent studies that showed the ubiquity of them and their importance in reshaping genomes.
(b) Cover ways transposable elements (te) can be used for gene isolation. This will involve what is called "forward" genetics (isolate a te-induced mutant to isolate the gene) as well as what is called "reverse" genetics (have a cloned gene and isolate a te-induced mutant).
Grading for this section involves two components:
Homework and unannounced quizzes: Homeworks can be found on a sister website or will be provided via hard copy. Each set counts 10% of the grade. Unannounced quizzes may also be used. These would be 10 minutes at the end of the lecture. Each counts 10% of this grade. The examination at the end of this section accounts for the remainder. This section counts for ~15% of your total grade; so each homework is worth ~1.5%.
Examination will be in the evening as described on the schedule provided by Dr. Chase.
Homework:
The purpose of the homework is to make sure the students are familiar with and competent in the use of the material presented in class as well as background material learned in former genetics courses. In some cases, a reference Genetics text book will be helpful. Dr. Chase has provided a list of general genetics books. Homework will clearly test your ability to do diploid genetics.
Collaboration among students on the
homework is
permissible. If you use a source for some of your
answers,
give appropriate citation. Plagiarism is illegal and is
clearly
against the rules of the
Keys to the homework will be posted the lecture day following receipt of your homework. I will keep your homework. If, by chance, the same question appears on the homework and on the exam, and if, by chance, the answer on the homework is given higher credit than the answer on the exam, the grade on the homework will be lowered to the grade on the exam for that question. This will ensure that you understand the answer you give on the homework.
For reference purposes, the homework assignments
and the
first exam from last year are posted on the homework website along with
homework for this year. Use the older ones
for
practice. Some of these questions could end up on your
examination this
year!
Lecture 1
What is a gene?
Our thoughts concerning the gene have changed throughout the years and, to a large extent, they depend on the technology used. Next time we will begin at the beginning and go up to modern times with the focus on genetic analysis. Today though we will describe the gene in modern molecular terms.
How many genes
does an
organism have?
Humans and other
closely
related animals = ~35,000 (likely now much lower)
Arabidopsis
~23,000
Flowering
plants
~ 23,000
C. elegans
~18,000
Drosophila melonogaster
~13,000
yeast
6 - 7,000
How much DNA does an organism have? Corn and man have about 3 X 109 base pairs or about 1 meter of DNA per haploid genome. If an average gene is 2 X 103 bp, then there is 14 X 107 bp taken up by coding information. Or about 5% of the genome is actually composed of genes. An interesting fact is that while the amount of DNA can vary up to 100 fold, the number of genes pretty much remains the same among higher eukaryotes. This points to the fact that during evolution, lots of non-genic DNA has been added to genomes.
The Central Dogma of Genetics (as coined by Francis Crick) :
DNA<--------transcription -------> RNA ---translation---------> protein.
(The first step is reversible while the second step is not. A
challenging question to ask yourself is
what causes
this difference in the two reactions)
The second property of a gene is replication. This involves enzymes
that
faithfully copy the DNA, using the complementary nature of the strands
to
accomplish this.
<>Gene Structure (in eukaryote) :
Size of a eukaryotic gene: usually around 2000bp although lots of variation. I know of one that is approximately 20 Kbp with 121 exons!!!
Promoter: Site of beginning of transcription, binding of RNA polymerase and many other interesting proteins: One can think of this as the engine of the train. Where is goes, the train follows. It is critically important in determining tissue specific transcription and responding to cellular stimuli. Much of the current research deals with factors that turn genes on and off in various tissues.
If you look at your own body, ask why is your finger a finger and your nose is a nose? They both have identical genetic information and both have been exposed to approximately the same environment. Shouldn't they be the same? The reason they are not is because of differential gene expression. Some genes are expressed in some tissues while others are expressed in other tissues. (In the case of plants, gene duplications have been used extensively through evolution to provide gene function needed in various tissues. One duplicate is expressed in one tissue while a second in a second tissue, a third in a third tissue, etc.)
Promoters and gene regulation are extremely intense areas of
research.. Dr. William Gurley, a
member of the
Terminator: is involved in terminating transcription. It also involves specific sequences.
Transcription occurs in the 5' to 3' direction using the strand running 3' to 5' as a template.
Following transcription, the RNA is modified through a process called pre-mRNA processing:
In the case of eukaryotes this involves capping or alteration in the 5' base, the addition of polyA tail (some eukaryotic messages do not have a poly A tail), and removal of introns (not all eukaryotic genes have introns) to leave exons spliced together in the mature transcript. Introns were big surprise and led to at least one Nobel prize.
The origin of intron (intron early versus intron late or, in other words, whether introns came before or after formation of a particular gene) and their possible roles are extremely interesting questions.
The next step in information flow is diagrammed
below. It
is termed translation.
Translation is the process by which information is translated from
RNA to
protein.
This involves the processed
mRNA, ribosomes (composed of two parts -
each made up of RNA and
protein), transferRNA
(tRNA) and amino acids.
Each tRNA molecule is "activated" in that it is attached to a particular amino acid by an "activating" enzyme. This is specific to each tRNA and amino acid. Activated tRNAs then flow to the ribosome which is attached to the mRNA. Translation (protein synthesis) starts with the amino acid methionine. Every protein begins with methionine, although in many cases the methionine is cleaved off following synthesis. Subsequent amino acids are added and a peptide bond is formed between the carboxyl terminus of methionine with the amino group of the second amino acid. Thus the amino group of the first methionine remains free while the carboxyl group of the last amino acid is not involved in a covalent bond. Because of these groups, the first "end" of the protein synthesized is termed the amino terminus while the last "end" synthesized is termed the carboxyl terminus. So, remember that 5' and amino refer to the "front end" of these macromolecules while 3' and carboxyl refer to the "rear end" .
The process is called "translation" for good reason. Just like translating one language to another, the information here initially is in nucleic acid (RNA) and it is changed or translated into protein. Given that there are only four bases in RNA (A, U, C and G) and 20 amino acids, it was clear that more than one base was needed to specify an amino acid. Furthermore, on first principles, we know that "the code" must involve more than two bases, since two bases could only specify 16 amino acids. (It is extremely important that you understand why this is so. If problems, see me).
Given this logic, the minimum number of bases per code was three (This would specify 64 combinations and hence is adequate to encode the 20 amino acids.) Experimental work (leading to some Nobel prizes) showed that the number of bases per amino acid, termed the codon, is, in fact, three. One codon (AUG) specifies the start of translation while three others (UGA, UAA, UAG) stop translation.
Note also that more than one codon encodes a particular amino
acid. It turns out that this is not random. Organisms
differ in the
frequency in which they use a particular codon. This is called "codon
bias".
Another fundamentally important concept in genetics is mutation.
To me, a mutation is any change in the genome. This can involve the insertion, deletion, substitution, or inversion of one or more base pairs in the DNA.
What I would call macro-changes such as polyploidy, aneuploidy, chromosomal interchanges or translocations, etc., are also lumped into "mutation" by some investigators. While correct, I tend to think of these types of changes as something other than true mutation.
Mutations can be classified in a number of way:
recessive: Clearly these are most frequent and almost always involve loss of (nearly) total gene function. (Note that dominance/recessive relationships can only be determined in tissues containing diploid or higher levels of genes. Later in this section of the course, you will get into a more detailed discussion of this. Dr. Chase will also touch on this in discussions of nuclear/cytoplasmic interactions and male sterility.)
dominant: These are rare, and sometimes involve a gain of some function. In some cases, for example in the case of a family of duplicate genes all expressed in one tissue, these are the only types of changes detectable by the eye.
polar: affect genes downstream in a polygenic mRNA. (These will be discussed in lectures dealing with the Lac operon.)
frame shift: shift the reading frame or phase of codons used, i.e. instead of reading bases 13, 14 and 15 together, a deletion of one base has occurred such that bases 12, 13, and 14 are read together. Note that a insertion mutation can also be a frameshift mutation.
nonsense: leads to chain termination during translation. Mutation is such that the codon is now changed to UGA, UAA, or UAG. These are also called chain termination mutations. Note that a frameshift mutation can lead to a nonsense codon downstream.
missense: Changes the amino acid incorporated into the protein
transitions : purine for purine, pyrimidine for pyrimidine
transversions : purine for pyrimidine or vice verse.
silent: does not change the sequence of the protein and hence has no effect on protein structure.
neutral : in evolutionary terms, it has no consequence. Some, but not all, silent mutations would be neutral
Intragenic suppressors (intra meaning within): mutation occurring within the gene that restores, at least partially, gene function. Usually this involves a change in an amino acid that somehow compensates for the change induced by the first mutation that destroyed gene function.
Intergenic suppressors : mutation occurring in a separate or different gene that suppresses the original mutation. A classic case involves a change in a tRNA molecule usually involved in chain termination that now inserts an amino acid. Non-sense mutants were sorted out quickly in the early days of genetics by the fact that they were suppressible by these types of intergenic suppressors. These will discussed in the context of the Lac operon and the proof for a single but poly-genic transcript arising from the operon.
Conditional mutants : expresses the mutant phenotype only under certain conditions. A classic example are microbial mutants that exhibit the mutant phenotype only when grown at elevated temperatures. Molecularly, these usually correspond to changes in the protein structure involving folding, etc. The protein can fold correctly at lower temperatures but not at higher temperatures.
Auxotrophic: These require something for growth that the wildtype version does not require. Usually used with microbes.
null versus leaky (very important): null mutants abolish total gene function whereas leaky mutants are partially active. A deletion of the gene, for example, would be a null mutant. Some single base change mutants can be null mutants if the change is in a protein motif critically important for gene function. On the other hand, some frameshift mutants and deletion mutants can be leaky if they occur towards the 3' end of the gene and the carboxyl terminus of the resulting protein is important but not critically so. (A question: what kind of single base changes do you believe would likely give rise to a null mutation?).
reverting versus non-reverting : single base change mutants have the ability to revert back to wildtype whereas something like a deletion does not have such ability. This distinction is important and is exploited in the work of Benzer in a later part of this section.
epistasis
suppression of the mutant phenotype determined by one gene
by the
presence of a mutant gene at another locus. We will return to
epistasis
in a later lecture.
Note that the classifications above are not mutually exclusive.