Chpt 15 Genes and How They Work

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What is the difference between minimal and complete media?

Minimal media contains only the basic essentials needed to grow a wild type organism whereas complete media contains minimal media plus all other components needed for growth.

What does X-rays do to DNA?

Genes exposed to X-ray/UV light will suffer damage and small deletions.

What evidence did Beadle and Tatum provide to come up with the "one gene-one enzyme" hypothesis?

In 1941, Beadle and Tatum used wild type Neurospora (bread mold) as a model system to determine whether genes encode proteins. Wildtype Neurospora can synthesize all neceassary nutrients required to survive. However, they need a source of carbon, vitamins and inorganic salts, which can be obtained from Minimal Media.

Wildtype neurospora was then exposed to X-rays, which causes small deletions in DNA. Some of these deletions could possibly occur in genes that encode enzymes needed to synthesize necessary nutrients. After that, they grew the Neurospora mutants on complete media, selected one of the spores to test it on minimal media to confirm that it had a mutation (no growth on MM).

Since the mutant could grow on CM, but not on MM, CM appeared to supplement the mutant with a nutrient not present in MM. They identified and separated each component present in CM, and added each component separately to a tube of MM. To identify what was missing in each mutant, they were grown on several MM tubes each supplemented with a component of CM.

One of the mutants only grew on a MM tube suplemented with arginine, therefore is an arg mutant. They found that there were three classes of arg mutants, depending on where on the chomosome the deletion occured. Each was mutant for a different enzyme in the arginine biosynthesis pathway (which was already well known). Beadle and Tatum proposed that each enzyme of the Arginine pathway was encoded by a separate gene. Therefore, proposed the one gene = one enzyme hypothesis

Why did the "one gene- one enzyme" hypothesis change to "one gene- one polypeptide"?

1. All proteins are not enzymes.

2. Some proteins are made of more than one polypeptide, each encoded by a gene.

What is the Central Dogma? Are there exceptions?

Proposed by Francis Crick, it states that information flows in only one direction: DNA is transcribed into RNA which is translated into protein. This mechanism is used for all organisms, bacteria to human. The only exceptions to this rule are RNA viruses, which use viral RNA as a template for DNA synthesis.

What do you know about the Genetic code?

What experiment/ evidence from Crick and Brenner proved that the code is triplet?

Crick and Brenner manipulated a viral gene by adding or deleting 1,2 or 3 bases that were close to each other. The gene was then transcribed and translated into polypeptide. They wanted to determine whether the deletion of these bases would alter only a single amino acid or all amino acids after the deletion.

They found that when 1 or 2 nucleotides were deleted ,all amino acids after the deletion were altered. When 3 bases were deleted, only one aa was altered and the rest of the protein was normal.

Briefly describe 2 methods used by Nirenberg and Khorana to decipher the genetic code?

1 - Synthetic RNA in a cell-free system (an extract from cells that supports translation): All U's attached to Phe-Phe-Phe-etc..

2 - Triplet binding assay: Tri-nucleotide RNA sequences (codons) were bound to beads in a column. Radioactive labeled amino acids were poured down the column. The amino acid that bound the 3-nucleotide sequence stuck to the column. (Ex - Met bound to AUG)

The finding that the genetic code is practically universal has implications in genetic engineering. Explain

The genetic code is nearly universal because the genetic code is the same for ALL living organisms from the smallest, most primitive bacteria to the most complex organisms such as humans, whales, trees, giraffes, etc. This is true because there is VERY strong evidence that all living organisms evolved from one common ancestor. Because of this, we were able to advance in genetic engineering by taking genes from one organism and inserting it into another and then a functional protein is made from that. Example- a baby piglet has a jellyfish gene inserted and thus the baby piglet's nose and cloven hooves tend to be florescent. This has also helped tremendously in medicine- such as recombinant insulin, industry and agriculture.

Define and describe a promoter in a prokaryotic gene.

The promoter is a stretch of DNA immediately upstream of the gene and contains the specific DNA sequences at -10bp and -35bp that are recognized and bound by the sigma factor subunit of RNA polymerase. The promoter is upstream of the start site (+1), where transcription begins.

Compare and contrast DNA and RNA polymerase.

RNA does not require a primer. RNA is transcribed off the 3'-5' template DNA strand and is synthesized in a 5'-3' direction RNA polymerase adds Ribonucleotides to the 3' end

In prokaryotes, transcription and translation are coupled. Explain

There is no membrane bound nucleus, so the ribosomes can immediately to the available 5' end of the mRNA, beginning translation.

What are the components of a ribosome? Where are the subunits assembled?

The large and small subunit, composed of ribosomal RNA and protein. They are assembled in the nucleolus.

Describe a tRNA. What is its function? Name the enzyme that attaches amino acids onto tRNAs?

How do proteins that are to be secreted directed to the RER?

How does gene expression differ in prokaryotes and eukaryotes?

Gene expression differs in prokaryotes and eukaryotes as follows: -Eukaryotic gene expression is more complex than prokaryotic. -Prokaryotes have operons (groups of genes have just one promoter) -Eukaryotes contain introns that are spliced immediately after primary transcription and before translation begins. This splicing can lead to modifications in the gene transcription. -Prokaryotes,on the other hand, have coupled gene expression (transcription is coupled with translation) due to the fact that they have no nucleus so ribosomes attach immediately following transcription. This process does not include splicing or gene modification. -Eukaryotes have a 5'(prime) cap which helps in locating the start position while prokaryotes have a 5' UTR (helps attach mRNA to small subunit)with no cap and the poly-A tail

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