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Genetics Part-II

What is the evidence for semi-conservative replication?
Classical experiments of Meselson and Stahl. Label DNA with *heavy isotope* N15 and allow replication in light N14: distinguish heavy, light and hybrid DNA by centrifugation.

Results: after 1 generation, each genome contains a hybrid N15-N14 DNA; after 2 generations, there are 2 hybrid and 2 light (N14-N14) genomes.

• Each strand of DNA serves as a template for the synthesis of its complement.

• The strands separate and each is used as a template for the synthesis of a daughter strand.

• The two new double helices each contain half the parental DNA.

• This process produces a replication fork

Is replication uni-directional or bi-directional?

• Bi-directional

• Two replication forks proceeding from the origin.

DNA replication, transcription and translation.
What is the major replication enzyme?

DNA polymerase III, a DNA-directed DNA polymerase

• Synthesis is 5'-->3'

• Substrates are deoxynucleoside triphosphates (to make deoxyribonucleic acid)

• Proof reading , errors removed by 3'--5' exonuclease

• Processivity is very high (the ability of the enzyme to replicate a large tract of DNA before *falling* off)

• Replication requires DNA unwinding by enzymes termed helicases: these enzymes unwind the DNA helix before the replication fork and wind it up again afterwards.

There are large numbers of different enzymes and proteins involved at the replication fork in the replisome.

DNA damage by UV radiation or chemicals is repaired by other DNA polymerases. UV-damage results in adjacent T residues in one strand becoming covaletly linked to each other, producing a thymine dimer. This causes the double helix to become distorted -- kinky. Xeroderma pigmentosa is a genetic disorder in which patients cannot carry out UV-radiation repair. They are very prone to skin cancer from an early age.

What is Transcription?
• Copying a gene as RNA

• DNA-directed RNA synthesis from a gene

What is a gene?

• There is no good definition of a gene!

• A sequence of DNA that is transcribed from specific start to specific stop base sequences.

• Beadle and Tatum, working with the eukaryote mold Neurospora crassa, concluded that one gene codes for one protein.

• But what about genes that code for RNA's like rRNA and tRNA?

• A gene is a sequence of DNA that is transcribed into a single RNA as defined by specific start and stop sequences of bases.

• Note the circularity of the argument!

• But the single RNA may be polycistronic!

What does that mean?

• A cistron is synonymous with a gene.

• A polycistronic RNA results from the transcription of an operon.
DNA replication, transcription and translation.
What's an operon?

• A genetic unit containing several genes with related functions: the bacterial operon for lactose (milk sugar) metabolism contains 3 genes coding for 3 different proteins.

• An operon is transcribed as a single unit, a polycistronic messenger RNA (mRNA) that codes for more than one gene product.

Name 4 types of RNA. What are their functions?

• mRNA, messenger RNA that is translated into protein

• rRNA, ribosomal RNA that, together with ribosomal proteins, forms a structural scaffold for the translation of mRNA, the ribosome

• tRNA, transfer RNA, a specific carrier of amino acids

• snRNA, small nuclear RNA involved in processing of mRNA in the nucleus

What is the major transcription enzyme?

RNA polymerase, a DNA-directed RNA polymerase

• RNA synthesis is 5'-->3'

• substrates are ribonucleoside triphosphates ( to make ribonucleic acid)

• begins at the promoter, 5' end of the gene

• processivity is very high, proceeds to 3' end of gene without stopping or falling off the gene

• proof reading by precise Watson-Crick base pairing, A=U and G=C

Regulation of transcription of a gene is at the 5'-end of the gene at region(s) termed operators

• Transcription of some genes is constitutive = housekeeping genes

• Transcription of other genes is in response to a stimulus = inducible genes

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