Question

Part III: Dr. Hernandez sits down with Ann and her partner. The doctor is sorry to report that Ann has breast cancer; however, because it was caught early and responds to estrogen and progesterone, the prognosis is good. At this point it looks like the cancer is stage 1. However, as Ann is young, the doctor wants to perform a couple tests. The first test is a genetic sequencing test for BRCA1 and BRCA2, as well as testing for other genes known to increase the risk of cancer. The other test is an Oncotype DX test on the tumor cells. This is a gene expression test, where the results correlate with how aggressive the cancer cells are. Ann is overwhelmed, but wants to do everything she can to get rid of this disease.

1. a. As a result of your discussions, you know that mutations in BRCA1 and BRCA2 increase the risk of breast cancer. Because of this, a positive test for these mutations would mean different treatment for Ann, including removal of both her breasts (bilateral mastectomy) as well as her ovaries to prevent ovarian cancer. Ann decides to have the test done, but she is a little hazy on the biology of DNA. a. Describe the basic structure and function of a gene. INSERT ANSWER

b. At the molecular level, what is a mutation? Are all mutations harmful? What type of mutation does not lead to a change in protein structure? INSERT ANSWER

c. The polymerase chain reaction (PCR) is a method that can be used to detect mutations. It uses machinery similar to your cells to replicate a specific DNA sequence, which in this case would be only the mutated gene. Describe the basic process of DNA replication in a cell. INSERT ANSWER

Answer 1

a) Structure and function of Gene. Gene is the functional unit of hereditary . Each gene is a segment of DNA that give rise to protein product or RNA . Gene exists in alternative forms called alleles . Chromosomes carry genes .

PROKARYOTIC Gene structure Genes based on their activity 1.House keeping genes 2.Specific genes STRUCTURAL FEATURES: Simple gene structure. Small genomes(0.5 to 10 million bp) Prokaryotic genes are collinear with their proteins a. CODING REGION b. PROMOTER ELEMENTS c. TERMINAL REGION OR TERMINATOR

a. Coding region- Starts with an initiator codon and ends with termination codon No introns (uninterrupted). Collinear to its mRNA. Transcribed sequenceterminator Prokaryotic AUG Stop codon 5 UTR Coding sequence 3 UTR RNA-coding sequence DNA Promoter Terminator Transcription Protein-coding sequence Leader Tratler 5 MRNA ranslation Polypeptide

b Promoter elements- The upstream elements from the start of the coding region include promoter sequences. 50 to 100 ntds upstream of the start codon- transcriptional initiation site or START site. (any nucleotide present on the left is denoted by (-)symbol and the region is called upstream element. E.g. -10,-20,-35 etc. Start site symbolized by +1 Any sequence to the right of the start is downstream elements and numbered as +10,+35 etc.)

At -10 there is a sequence TATAAT or PRIBNOW BOX. At -35 another consensus sequence TTGACA These two are the most important promoter elements. Recognized by transcription factors. aryote om Ooter ypica RNA Sta -10 Region 35 Region TTGACA TATAAT ATG Protein Start At -65 to -60 activator elements. Activation of the polymerase. At -200 to -1000 enhancer sequence. Enhances transcription by 100 to 200 folds. -10 -200 -65-60 -35 IA Enhancers/ Activator TTGACA TATAT

Terminal region of the gene Sequences for the termination of transcription It takes place by Rho dependent mode or Rho independer mode

Eukaryotic gene structure *Exons Introns ※Promoter sequences ※Terminator sequences *Upstream sequences *Downstream sequences *Enhancers and silencers(upstream or downstream) »Signals (Upstream sequence signal for addition of cap Downstream sequences signal for addition of poly A tail.)

Eukaryotic gene 5' 3 intron exon exon intron exon intron untranslated region (UTR) coding sequence (CDS) RNA-coding sequence DNA Promoter Terminator Transcription Cap Poly(A) tai Exon Intron Exon Intron Exon 5 Pre-mRNA Trailer Leader RNA processing introns removed Protein-coding sequence MRNA Translation Polypeptide

OEXONS-coding sequence, transcribed and translated. Coding for amino acids in the polypeptide chain. Vary in number ,sequence and length. A gene starts and ends with exons.(5' to 3'). Some exon includes untranslated(UTR)region OINTRONS- coding sequences are separated by non- coding sequences called introns Any nucleotide sequence that are removed when the primary transcript is processed to give the mature RNA are called introns All introns share the base sequence GT in the 5'end and AG in the 3'end. Introns were 1st discovered in 1977 independently by Phillip Sharp and Richard Roberts.

OPROMOTERS- A promoter is a regulatory region of DNA located upstream controlling gene expression 1.Core promoter - transcription start site(-34) Binding site for RNA polymerase. General transcription factor binding sites. 2. Proximal promoter-contain primary regulatory elemer Apprx. -250,specific transcription factor binding sites. OTATA box or hogness box (-30 to -80)and CAAT(upstream TATA) are two distinct sequences. These together are responsible for binding of RNA polymerase Il which is responsible for transcription mRNA CCAA -200 bp -100 bp -30 bp

UPSTREAM(5'END). 5'UTR serve sevral functions including mRNA transport and initiation of translation. Signal for addition of cap(7 methyl guanisine) to the 5'end of the mRNA The cap facilitates the initiation of translation. Stabilization of mRNA. ODOWNSTREAM(3'END)-3 UTR serves to add mRNA stability and attachment site for poly-A-tail The translation termination codon TAA AATAA sequence signal for addition of poly A tail.

OTERMINATOR- recognized by RNA polymerase as a signal to stop transcription DENHANCER-enhances the transcription of a gene. Upto few thousand bp upstream SILENCERS-reduce or shut off the expression of a near by gene.

b) A gene mutation is a permanent alteration in the DNA sequence that makes up a gene, such that the sequence differs from what is found in most people. Three types of mutation are :-

- base substitution

- deletion

- insertion

A point mutation does not lead to a change in protein structure . A mutation affecting only one or very few nucleotides in a gene sequence.

C) The basic process of DNA replication in a cell :-

1. Replication Fork is the breaking of the H bonds in the base of the two antiparallel strands. The unwounding of the two strands is the starting point. The A-T pairs slipt. That is because there are only two bonds between Adenine and Thymine (there are three hydrogen bonds between Cytosine and Guanine). Helicase is the enzyme that splits the two strands. The initiation point where the splitting starts is called "origin of replication".The structure that is created is known as "Replication Fork".

2 - One of the most important steps of DNA Replication is the binding of RNA Primase. RNA Primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3'-5' strand due to the hydrogen bonds between the bases. RNA nucleotides are the primers (starters) for the binding of DNA nucleotides.

3 - The elongation process is different.

5'-3' Template: The 3'-5' proceeding daughter strand -that uses a 5'-3' template- is called leading strand because DNA Polymerase can "read" the template and continuously adds nucleotides (complementary to the nucleotides of the template, for example Adenine opposite to Thymine etc).

4 - In the lagging strand the DNA Pol I -exonuclease- reads the fragments and removes the RNA Primers. The gaps are closed with the action of DNA Polymerase (adds complementary nucleotides to the gaps) and DNA Ligase (adds phosphate in the remaining gaps of the phosphate sugar backbone).

Each new double helix is consisted of one old and one new chain. This is what we call semiconservative replication