The Central Dogma of molecular biology describes the directional flow of information;
DNA -> RNA -> Protein
- DNA replication
- DNA
- RNA synthesis (Transcription)
- RNA
- Protein synthesis (Translation)
- Protein
What is Genetic Material?
DNA (and sometimes RNA) as genetic material
Genetic material must have certain characteristics….
What are the Key Characteristics of genetic material? 4
- Must contain COMPLEX information
- Must REPLICATE faithfully
- Must encode the PHENOTYPE
- Must have the capacity to VARY
How do we know that DNA is the genetic material?
– the Hershey-Chase Experiment (1952)
- T2 bacteriophage (DNA/protein) – infect bacteria
- how was phage genetic material transmitted to bacteria (protein or DNA?)
- Grew E.coli infected with T2 phage in either 32P or 35S, -> phage with radiolabelled DNA (32P) and proteins (35S).
- Used these phage to infect unlabelled
E.coli. - Then isolated the cells… only the 32P labelled phage produced progeny phage that were radiolabelled, showing DNA not protein was passed on
RNA can also be genetic material
– Fraenkel-Conrat & Singer (1956)
- Used tobacco mosaic virus (TMV) uses RNA.
- Mixed RNA and protein from different TMV strains
- The viral progeny were identical to the strain of the RNA donor, not the protein donor.
- Proved that the RNA, not the protein, is the genetic material in TMV
Information flow in biological systems – exceptions to the central dogma = 2
- Major information pathways
- DNA
- DNA REPLICATION - DNA POLYMERASE
- Information is transferred from one DNA molecule to another
- TRANSCRIPTION - RNA POLYMERASE
- information is transferred from DNA to an RNA molecule
-TRANSLATION - RIBOSOME
- information is transferred from RNA to a protein through a code that specifies the amino acid sequence. - SPECIAL INFORMATION PATHWAYS
DNA - RNA - PROTEIN
- RNA DEPENDENT RNA polymerase; …or to another RNA molecule
- REVERSE TRANSCRIPTION
in some viruses, information is transferred from RNA to DNA.
DNA STRUCTURE: Primary, Secondary, Tertiary
- Primary: nucleotide structure
- Secondary: DNA’s stable three-dimensional configuration
- Tertiary: packing of double-stranded DNA in chromosomes
DNA and RNA are composed of polynucleotide strands - what are they in a nucleotide?
Nucleotide:
- Deoxyribose
(DNA) or Ribose (RNA) sugar
- Phosphate group
- Nitrogenous base (purine: A and G, pyramidine: C and U/T)
DNA polynucleotide strands vs RNA polynucleotide strand
DNA polynucleotide strands
1. T-A have 2 HYDROGEN BONDS
- PHOSPHODIESTER linkage connects the 5’-phosphate group and the 3’-OH group of adjoining nucleotides.
- C-G pairs have 3 HYDROGEN BONDS
- The strands run in opposite direction; they are antiparallel
- DNA has deoxyribose sugar (no oxygen here).
RNA Nucleotide strand
- In RNA, uracil (U) replaces THYMINE (T)
- RNA has ribose sugar (an OH group here)
The 3D shape of the double stranded DNA molecules are?
What is the most common one?
- The 3- dimensional shape of the double stranded DNA molecules can vary
- The B-DNA structure is the predominant structure in the cell
- B-DNA helix = right-handed helix with approximately 10 BASES PER TURN.
Special Secondary Structures Can Form in DNA and RNA - What are they?
- Hairpin; loop + stem
- a hairpin consisting of a region of paired bases (with form the stem) and a region of unpaired bases between the complementary sequences (which form a loop at the end of the stem) - Stem = a stem with no loop
- Complex secondary structure; secondary structure of the RNA component of RNase P of E.coli.
- RNA molecules often have complex secondary structures.
DNA molecules are very long.
How are they packed into a tiny cell? 3.
Overrotated, Underrotated, Topoisomerases
Supercoiling results from strain produced when rotations are added to a relaxed DNA molecule or removed from it.
- Overrotated: positive supercoiling;
underrotated: negative supercoiling
- Overrotated: positive supercoiling;
- *Topoisomerases ADD/REMOVE ROTATIONS by BREAKING the NUCLEOTIDE STRANDS
- Most DNA FOUND in cells is NEGATIVELY SUPERCOILED
- SEPARATION of the TWO STRANDS of DNA easier during REPLICATION and TRANSCRIPTION
- SUPERCOILED DNA can be PACKED INTO a SMALLER SPACE THAN RELAXED DNA
Positive Supercoil vs Negative supercoil DNA process
- Relaxed circular DNA
***Supercoild DNA is overwound or underwound, causing it to twist on itself.
a.) Add two turns (OVERROTATE)
a.) Positive supercoil -
a.) Positive supercoiling occurs when DNA is overrotated; the helix twists on itself
b.) Remove tow turns (UNDERROTATE)
b.)Negative supercoil
b.)negative supercoiling occurs when DNA is underrotated; the helix twists on itself in the opposite direction
Within a cell, DNA is associated with specialised proteins that organise it and give it structure
PROKARYOTIC CELL
- DNA, Wrapped around histone protein, chromosome,
nucleotide: (irregularly-shaped region within the cell of a prokaryote that contains all or most of the genetic material.)
- plasmid:( is a small, circular, double-stranded DNA molecule that is distinct from a cell’s chromosomal DNA.)
EUKARYOTIC CELL
- Chloroplast DNA
- Nuclear DNA
- Mitochondrial DNA
Chromatin is a highly complex structure with several levels of organisation - WHAT ARE THEY?
7.
- At the simplest level, chromatin is a DOUBLE-STRANDED HELICAL STRUCTURE OF DNA
- DNA is complexed with HISTONES to form NUCLEOSOMES
- Each nucleosome consists of 8 HISTONE proteins around which the DNA wraps 1.65 times
- The nucleosomes fold TO PRODUCE A 30-NM FIBER…
- ..that FORMS LOOPS averaging 300 nm in LENGTH
- The 300-nm LOOPS are COMPRESSED and FOLDED to PRODUCE a 250-nm-wide fibre.
- TIGHT COILING of the 250-nm fibre produces the CHROMATID of a CHROMOSOME.
Eukaryotic DNA packaging is dynamic & changes throughout the cell cycle
What is it usually? When does it change and what does it change to?
- Mostly, CHROMATIN IS RELATIVELY UNCONDENSED.
the DNA can be ACCESSED relatively EASILY by cellular machinery. - CONDENSATION TAKES PLACE WHEN THE CELL IS ABOUT TO DIVIDE. Chromosomes progress from a HIGHLY PACKAGED STATE to a state of EXTREME CONDENSATION, which is necessary for chromosome MOVEMENT IN MITOSIS AND MEIOSIS.
- DNA packing also changes locally DURING TRANSCRIPTION AND TRANSLATION, when the TWO NUCLEOTIDE STRANDS must UNWIND so that PARTICULAR BASE SEQUENCES ARE EXPOSED.
What are the 2 basic types of Chromatin?
The two basic types of chromatin are:
euchromatin (most of the chromosome, active)
- legs of the chromsomes
and heterochromatin (densely packed)
- middle, centromere and telomeres
LOOK AT DIAGRAM - LECTURE 1 SLIDE 19
What is the Characteristic of EUCHROMATIN? (3)
Found on Legs of the chromosome.
- Most of the chromosome
- CONDENSATION and
DECONDENSATION with the cell cycle - TRANSCRIPTIONALLY ACTIVE
What are the Characteristics of Heterochromatin? (4)
Found in Middle, centromere and tips and tops of the chromosome legs, TELOMERES
- Remains in a HIGHLY CONDENSED state throughout the cell cycle, even during interphase
- Constitutive (PERMANENT) heterochromatin at the telomeres and centromeres
- FACULTATIVE heterochromatin at certain DEVELOPMENTAL STAGED
- Repeated sequences, few genes
Sensitivity to DNase I shows that chromatin structure changes with gene activity…
EXPLAIN HOW
- DNase I = enzyme that digests DNA
- Its ability to digest DNA depends on chromatin structure (histone-bound DNA is less sensitive)
- GLOBIN genes ENCODE HEMOGLOBIN & are expressed in the ERYTHROBLASTS of chicks at different stages of development
- Experiments on globin genes in chick embryos show that DNase SENSITIVITY is CORRELATED with GENE ACTIVITY
DNase I = enzyme that digests DNA
- ability to digest DNA depends on chromatin structure
- DNA sensitivity is CORRELATED WITH GENE ACTIVITY
EXPERIMENT:
QUESTION: is chromatin structure altered in transcription?
METHOD? RESULTS? CONCLUSION?
METHOD: DNA’S Sensitivity to DNase I was tested on different tissues and at different times in development.
RESULTS:
1. Before Haemoglobin synthesis, none of the globin genes are sensitive to DNase I.
- After Globin synthesis has begun, all genes are sensitive to DNase I, But the embryonic globin gene U is the most sensitive.
- In the 14-day-old embryo, when only adult haemoglobin is expressed, adult genes are most sensitive and the embryonic gene is insensitive.
- Globin genes in the brain-which does not produce globin - remain insensitive throughout development.
CONCLUSION:
Sensitivity of DNA to digest by DNAse I is correlated with gene expression, suggesting that chromatin structure changes in the course of transcription.
DNase I sensitivity is correlated with the transcription of globin genes in chick embryos.
What are Epigenetic changes?
STABLE CHANGES IN GENE ACTIVITY
NOT CAUSED BY CHANGED IN DNA SEQUENCE ITSELF BUT IT I ENVIRONMENTAL
Epigenetic changes are stable changes in gene activity which are not caused by changes in the DNA sequence itself
….WHY? (2)
- DNA METHYLATION is a common type of epigenetic modification in eukaryotes – the CYTOSINE BASED in DNA are METHYLATED
- CHEMICAL MODIFICATION of the HISTONE PROTEINS leads to changes in CHROMATIN STRUCTURE
All the Genetic Information Must Be Accurately Copied Every Time a Cell Divides.
WHY?
- ensures that each DAUGHTER CELL gets a copy of the genome, and therefore, SUCCESSFUL INHERITANCE of GENETIC TRAITS
- essential for INHERITANCE of TRAITS that DEFINE the PHENOTYPE of CELLS and the ORGANISM
-
Lecture 129
-
Lecture 2: DNA REPLICATION IN EUKARYOTES AND PROKARYOTES40
-
Gene Mutations I and II60
-
Mechanisms of Gene Mutation31
-
Lecture 325
-
Lecture 4: RNA molecules and processing30
-
Mechanisms of Gene Mutation 226
-
Transgenic 136
-
Transgenics 214
-
Transgenics 330
-
Molecular Techniques31
-
Molecular techniques 222
-
Molecular Techniques 321
-
Transposons I44
-
L1 Cancer Genetics Regulated and unregulated cell proliferation49
-
Dissection of Gene Function part I: Forward Genetics49
-
Dissection of Gene Function part II: Reverse Genetics and Mutant Analysis49
-
Immunogenetics 1 and 251
-
Cancer Genetics Regulated and unregulated cell proliferation L256
-
Mechanisms of DNA Repair L1 AND L259
-
TRANSPOSABLE ELEMENTS 2 IN EUKARYOTES43
