Concepts of Biology

Introduction to Cell & Molecular Biology (BIOL121) - Dr. S.G. Saupe (ssaupe@csbsju.edu); Biology Department, College of St. Benedict/St. John's University, Collegeville, MN 56321

Molecular Genetics: Mutations

I. Definition ‑ heritable change in DNA sequence

II. Kinds of mutations

Point ‑ substitutions of a single base. These may be "silent" (not have an effect on the protein product) or not. Example: if the normal DNA sequence is ATTACG, then a point mutation might be represented by ATCACG.
Insertions/deletions ‑ extra bases are added/deleted. These mutations usually have a significant impact on the protein product; often deleterious. The reason is because these mutations cause a shift in the reading frame of the codons (i.e., frameshift).
Chromosomal ‑ see below

III. Hemoglobin, Sickle Cell Anemia and Thalassemia ‑ Some examples
Hemoglobin, the oxygen carrying protein in blood cells, is a quarternary protein comprised of four chains, 2 alpha (α) and 2 beta (β) chains. The molecule also has a heme unit that contains an iron. The gene sequence of the anti‑sense strand for the β-chain of hemoglobin was provided in class (click here). Some take home lessons from the gene:

The anti‑sense is often reported because it is easy to convert to a protein (its sequence is identical to mRNA except change T's to U's)
Transcription converts the message in DNA into RNA. RNA polymerase is the enzyme responsible.
RNA polymerase binds at the promoter region. A specific sequence of nucleotides (TATA box) are important for this recognition
Transcription begins about 20 nucleotides downstream from the TATA box
Approximately 100 nucleotides 'upstream' from the beginning of transcription are sequences of DNA that serve to enhance or silence transcription by binding general transcription factors
There is a transcription termination signal at the end of the gene.
The gene has leader and trailer sequences ‑ for punctuation and ease in handling
Translation converts the message in mRNA into a sequence of amino acids in a polypeptide (protein). This occurs at the ribosome.
AUG is the first codon to be translated. Thus, methionine is the first amino acid in a protein. However, it may be removed after translation (as in this example).
There are about 146 amino acids in the β-chain of hemoglobin
There are 3 exon regions (coding into amino acids) and 2 intron regions in the gene. In fact, at least half of the nucleotides in the gene don't code for an amino acid. Thus, the mRNA gets processed after it is produced during transcription. The original mRNA is called pre‑mRNA (pre for premature, or not ready to be translated) and the processed mRNA is called mature mRNA.
Sickle cell anemia ‑ this disease is ultimately caused by a single point mutation where an A is substituted for a T. This causes a change in the protein from glutamic acid to valine. Note glutamic acid is charged, valine is more neutral and hence, the protein now has slightly different chemical properties, hence, it precipitates when oxygen tensions decrease.
Thalassemia ‑ is a disease also caused by defective hemoglobin. In one form, there is an insertion of an A after codon 71. Note that this insertion mutation causes a frame-shift and results in a severely shortened version of hemoglobin ‑ not too healthy.

IV. Chromosomal mutations ‑ larger scale changes in nucleotides at the level of the chromosome. Several kinds. (not on exam)

A. Transposons ‑ first identified by B. McClintock at Cold Spring Harbor, NY (1951). She won a Nobel prize for her work. She studied kernal color in maize (corn). She discovered two families of transposons (movable elements, or jumping genes). One she called AcDs (activator/dissociator). These transposons are regions of chromosomes that could move from one place to another. Holy Mendel, Batman. This was a heretical idea at the time and virtually ignored. Ac can move by itself, but Ds only moves in the presence of Ac. A schematic representation of what happens (diagrams on overhead are a little better):

‑‑‑‑‑‑‑‑‑Ds‑‑‑‑‑‑‑‑‑Gene‑‑‑‑‑‑‑‑     (makes purple pigment, no Ac to cause Ds to move)
‑‑‑Ac‑‑‑‑‑‑‑‑‑‑‑‑Ge Ds ne‑‑‑‑‑‑‑‑     (no purple pigment, presence of Ac causes Ds to jump; if it by chance lands in the pigment gene as in this example, then no pigment is produced)
‑‑‑Ac‑‑‑‑‑‑‑‑‑‑‑‑‑Gene ‑‑‑‑‑‑‑‑‑‑    (spots; Ac can also cut Ds out of gene, reactivating the purple pigment in certain cells)

Nina Federoff et al ‑ studied the molecular biology of this system. They found that Ac was 4563 nucleotides long with inverted repeats on each end. The inverted repeats are mirror image sequences of nucleotides. There were a variety of Ds elements, varying sizes, but all similar in nucleotide sequence to Ac. In one Ds she found, it was identical to Ac except for a 194 piece segment missing. This suggested that something in that piece is necessary for movement. Deduced it was a protein called transposase ‑ cuts out elements with inverted repeat sequences and reattaches at raondom. Thus, Ds is essentially a crippled version of Ac. Ac produces the transposase that frees Ds to reinsert in other places.

B. Chromosomal (pericentric) inversions. Chromosome breaks and piece reinserts itself upside down.

C. Robertsonian translocation. Chromosomes fuse end to end, especially in small acrocentric chromosomes.

V. Sources/Causes of mutations

A. Errors during replication

B. Mutagens such as chemicals (replace correct nucleotides, increase rate of error by DNA polymerase) or physical agents (such as xray, UV ‑ thymine dimers)

VI. Importance of Mutations

A. From the individual perspective, a mutation can be good, bad or indifferent. Frequently, a mutation is selected against.

B. From the perspective of a population, mutations are good because they are the raw material necessary for evolutionary change.

VII. Molecular Biology of Evolution ‑ Chimps and Humans (not on exam)

A. DNA similarity/differences can show: (1) phylogeny ‑ the more similar the DNA, the more closely related; and (2) provide indication of when species evolved (time of divergence; a sort of molecular clock).

B. The Third Chimpanzee (Jared Diamond)
In this book Diamond describes some recent studies concerning the evolution of humans. Cites a study by Sibley and Ahlquist (1984) that compared DNA of several primates.

DNA hybridization studies ‑ DNA melts (strands separate as increase temperature. Can mix DNA from different species. The more similar the DNA the higher the temperature it will take to melt (spearate the strands, because they form a lot of hydrogen bonds with their complement nucleotides). As a rule of thumb, for every 1 degree lowering of melting point there is a 1% difference in the DNA. Conclusions from Sibley/Ahlquist studies:

93% of DNA of humans and other primates is similar
DNA of chimps and pygmy chimps is 99.3% similar (pygmy chimps differ in size, more slender, longer legs proportionately, more copulatory positions, and both sexes intiate copulation).
Humans show 98.4% similarity with chimps.
Our closet relative is a chimp, not a gorilla
The difference between humans and chimps (upright, speech, big brains, less hair) due to 1.6% of the DNA, and much of that is "junk."
Humans and chimps diverged from a common ancestor relatively recently. Evidence: (1) Fossil data show that monkeys and apes diverged around 30 mya. Since they differ by 7.5% of DNA, thus there is a 1% change in DNA approximately every 4 million years (30/7.5); (2) Fossil data show that orangutans/gorillas diverged around 16 mya. Since there DNA differs by 3.6%, there is a 1% change in DNA approx every 4.4 my (16/3.6). Thus, note using two independent estimates the molecular clock in primates is constant ‑ every 4 million years or so there is a 1% change in DNA. These data tell us that humans and chimps diverged about 6 mya (4 my x 1.6).

VIII. Mechanisms of molecular evolution: An example
This example features the evolution of two human sex hormones, β-LH (luteininzing hormone) which stimulates ovulation and β-HCG (human chorionic gonadotropin) which is produced by embryo and is the basis of pregnancy test. Both hormones are proteins, each with 2 chains, an alpha and beta. The alpha chain of both are identical and coded by chromosome #6. The beta chain of both are similar. They are coded by a series of genes on chromosome 19. There are 8 genes side by side, 7 code for HCG and one for LH. It is hypothesized that an ancestral LH gene duplicated (occurs during meiosis when there is an unequal cross over with one chromatid getting extra, the other missing stuff). One of the duplicated LH genes was conserved and is our current LH gene. The other diverged, accumulating mutations, and became an HCG gene. This HCG gene was duplicated additional times resulting in our current arrangement on chromo 19. Two HCG genes have been sequenced and they differ by two nucleotides. One change is silent; the other results in a difference of one amino acid between the two protein products (beta chain). The beta LH protein is 121 amino acids long. The beta HCG is 145. The first 112 amino acids of both are the same and they have the same length piece of DNA coding for both. For more details, check out the assignment posted on line.