BY322- CH9

pick up at Erwin Chargaff

characteristics of genetic material

characteristics of genetic material

replicate, storage of information, expression of information, allow variation by mutation

the central dogma

DNA→ RNA→ Protein

Central dogma of molecular genetics is made up of two processes:

1. Transcription: synthesis of RNA from information in DNA. Involves: Messenger RNA (m RNA), Ribosomal RNA (r RNA), Transfer RNA (t RNA)

2. Translation: Uses information in m RNA to synthesize proteins.

Miescher (1868)

isolated cell nuclei; credited with discovering DNA. Derived acid substance containing DNA: nuclein. Found to be present in chromosomes. Thought to lack chemical diversity needed for genetic information.

tetranucleotide hypothesis

In 1910, Phoebus Levene proposed the tetranucleotide hypothesis: DNA contains equal amounts of four nucleotides. Chargaff (1940) later showed this was incorrect (instead, G equals C and A equals T). Definitive evidence favoring DNA was first obtained in 1944 during the study of bacteria and bacteriophages; publication on chemical nature of transforming principle in bacteria.

Frederick Griffith (1928)

Transformation Studies: Experiments with Diplococcus pneumonia (formerly Streptococcus pneumoniae) and mice. Virulent strain: S (smooth; with a polysaccharide capsule); nonvirulent strain: R (rough; no capsule). [In our textbook: “Serotype IIR” (rough, no capsule) is avirulent; “Serotype IIIS” (smooth, with capsule) is virulent.] Experimental design: Part 1: inject virulent strain (S) into mouse: death; inject nonvirulent strain (R): mouse survives. Part 2: Boil S and inject the heat-killed cells into mouse: mouse survives; Boil S strain and inject heat-killed cells + live R strain: death. Via some unknown mechanism, Griffith had transferred hereditary material from dead cells to live cells. The cellular debris from the S strain was able to convert the R strain into the virulent form. This process is now called transformation

transformation

modification of a genome via the external application of DNA originating from a different genotype

Avery, MacLeod, & McCarty (1944)

Extended Griffith’s results. Destroy all the major categories of chemicals in the extracts of the dead S cells. For each experimental condition, only one category of chemicals would be destroyed. For example: RNase (RNA); Protease (proteins); also polysaccharides and lipids; DNase (DNA). When DNA was destroyed, the mouse survived. Taken together, these experiments strongly implicated DNA as the molecule that carries hereditary information. They reported obtaining the transforming principle, and demonstrated it was DNA, not protein. We now know that fragments of the DNA carrying the virulence trait become inserted into the bacterial chromosome (replacing the nonvirulent form of gene)

Hershey-Chase Experiments (1952)

Alfred Hershey and Martha Chase experimented with Escherichia coli and bacteriophages (viruses that infect bacteria; T2 virus). In one experiment, bacteria were infected with virus particles labeled with a radioisotope of sulfur (35S). The sulfur only labeled viral proteins. The viruses were dislodged from the bacteria by whirling the mixture in a kitchen blender. Most of the radioactive sulfur was detected in the viruses, not inside the bacterial cells; thus, the viruses had not injected protein into the bacteria. In another experiment, bacteria were infected with virus particles labeled with a radioisotope of phosphorus (32P). The phosphorus only labeled viral DNA. When the viruses were dislodged from the bacteria, radioactive phosphorus was detected inside the bacterial cells. The viruses had injected DNA into the cells, providing evidence that DNA is the genetic material of the virus. Protein parts of viruses, labeled with 35S (sulfer 35), stayed outside the bacteria; DNA of viruses, labeled with 32P (phosphorus 32), entered bacteria. Conclusion: DNA, not protein, is the material that stores hereditary information.

indirect evidence 1: DNA distribution

Proteins are found everywhere in the cell, and abundant in cytoplasm; DNA is not. DNA is only found where primary genetic function is known to occur. Also, mitochondria and chloroplasts perform genetic functions; DNA is present in these organelles

indirect evidence 2: mutagenesis

UV light: most mutagenic at wavelength 260 nanometer. Action spectrum of UV can be compared to absorption spectrum of molecules. DNA and RNA absorb UV at 260 nanometers, whereas Protein absorbs UV at 280 nanometers. 280 nanometers: wavelength at which no significant mutagenic effects are observed. Molecule serving as genetic material expected to absorb at mutagenic wavelength.

DNA structure

The structure of DNA holds the key to understanding its function. Prior to the current understanding of DNA structure, three key properties for DNA were assumed:

1. DNA must allow for successful replication of the genetic material at every cell division.

2. DNA must encode all the information needed for the assembly of proteins expressed by an organism.

3. DNA must be able to change (mutations must be possible).

nucleotide

A nucleic acid monomer consisting of a five-carbon sugar (a “pentose” sugar: ribose or deoxyribose), a phosphate group, and one of four nitrogenous bases:

Two pyrimidines: thymine and cytosine. These have single ring structures. Two purines: adenine and guanine. These have double ring structures.

DNA bases

A, C, T, G,

RNA bases

A, C, U, G. Only DNA contains thymine, while only RNA contains uracil

RNA during gene expression

These originate as complementary copies of DNA: ribosomal RNAs (rRNAs) are structural components of ribosomes for protein synthesis during translation; messenger RNA (mRNA) is the template for protein synthesis; carries genetic information from gene to ribosome; transfer RNAs (tRNAs) carry amino acids to ribosome for protein synthesis. Other RNAs perform various roles: telomerase RNA and RNA primers are involved in DNA replication at chromosome ends (telomeres); small nuclear RNA (snRNA) process mRNAs; antisense RNA, microRNA, small interfering RNA (siRNA), and long noncoding RNAs (lncRNAs) involved in gene regulation.

nucleoside

contains a nitrogenous base and a pentose sugar. Nucleosides and nucleotides are named for specific nitrogenous base (A, G, C, T, or U).

nucleoside monophosphates (NMP)

: A nucleotide with one phosphate group

nucleoside diphosphates (NDP)

: nucleotide with two phosphate groups

nucleoside triphosphates (NTP)

nucleotide with three phosphate groups

ATP and GTP

adenosine triphosphate and guanine triphosphate) are important in cell bioenergetics. Triphosphates serve as precursors during nucleic acid synthesis; large amounts of energy involved in adding/removing terminal phosphate group. Once nucleotides are incorporated into DNA, they lose two phosphates

phosphodiester bonds

Linkage between two mononucleotides involves a phosphate group linked to two sugars. Phosphoric acid joins two alcohols by ester linkage on both sides. Nucleotides are linked by phosphodiester bonds between phosphate group at C-5’ position and OH group on C-3’ position.

oligo- and polynucleotides

Short chains consisting of around 20 nucleotides are called oligonucleotides; longer chains are called polynucleotides; store vast amounts of genetic information and give rise to extraordinary variation.