| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
Outline:
A. What is Molecular Biology?
1. Understanding of Biological
Processes at Molecular Level, ie
via PhysicoChemical Laws
Cells to Organisms ... little yet in Ecology or Pop Bio
2. Mol Bio grew out of Genetics
and Biochemistry
Biological Processes
involve molecules that can form complex biological structures (organelles,
membranes, tissues, organs), that these interact via molecules and the molecules
are present in the organism ultimately due to expression of Information residing in
the Genetic Material
Molecular Biology gets back to: a Molecular understanding of Inheritance
or Heredity
Example:
vision as a biological process
molecules comprising the organ (eye) and how they interact
where the molecules come from:
proteins, synthesized as a result of Gene Expression
proteins as Enzymes to catalyse synthesis of other molecules:
lipids, etc
Regulation in time: when the molecules are made, how they come
together
Biochemistry of how an eye works ... what do the molecules do?
Signal transduction: how a light photon signal is transduced into
signal for brain to "see"
Genetics:
to understand a Biological Process at the Molecular level, one first does Genetics:
isolate and characterize Mutants ...
Gene by Gene Genetics: Map the Phenotype of each Mutant
in Genetic Crosses
Genome Genetics: Characterize phenotypes of all
mutants in single experiment
Biochemistry: understanding of molecules
involved in what's happening in Genetics
Gene by gene Biochemistry: what molecule(s) is changed
in each Mutational process?
how does this change affect molecular events leading to the phenotype
observed?
How to do this?
Purify molecules: enzymology
Study enzyme and other molecular properties
Reconstitute biological process in vitro (in the test tube),
compare with in vivo (in
the cell) data
Genome Biochemistry: what are all the molecules
involved in the process?
What are the total changes in gene expression for each
and every Mutation obtained?
... High ThroughPut (HTP) analyses
or for a change in environment that turns the process on or off?
(light vs no light for vision...)
What changes in immediate expression of genes into mRNA occurs?
... gene expression arrays
What changes in expression of proteins occurs?
... in modifications of these proteins?
... in interactions between these proteins? ... proteomics
Answers to these types of questions are based in a Molecular Understanding of Inheritance called the Chromosomal Theory of Inheritance.
Chromosomal Theory of Inheritance
This theory encompasses the following principles:
1. Inheritance is encoded in units called
"Genes" which are found in DNA molecules
2. The complete set of DNA molecules for an organisms is called
the "Genome" of the organism.
3. Except for organelle DNA, e.g. mitochondrial DNA, these DNA
molecules are found in the Chromosomes in the Nuclei of each Cell
of the organism:
... Chromosomal Theory of Inheritance
4. Expression of these Genes, yielding Proteins or Stable RNA
molecules, is the process which determines the phenotypic properties
of each cell, tissue, organ, and organism.
5. The molecular basis of Gene Expression is focused within the
... Central Dogma of Molecular Biology:
Central Dogma of Molecular Biology (Brown, Fig 1.2):
DNA makes RNA
makes Protein
(DNA also makes DNA: replication)
(DNA makes RNA: transcription)
(RNA sometimes makes DNA: reverse transcription)
(RNA makes Protein: translation)
Proteins then catalyze synthesis
of other molecules
Regulation thereof
The above view of the Central
Dogma is simplistic for eukaryotes; see Brown, Fig. 1.2
B. Genetic Material is Nucleic
Acid, usually found in Chromosomes: experimental evidence
1. Chromosomal Theory
of Inheritance
Early correlation of Genetic Maps with physical organelles
called Chromosomes
Chiasmata observed during Meiosis (recombination)
Gross mutational aberrations (translocations, etc) <-->
Physical change in Chromosome
Thus the theory: Chromosomes contain the Genetic Material
2. Could be most likely either Protein or DNA, the main constituents
of Chromosomes
Most people favored Protein:
20 amino acids vs only 4 bases in DNA: higher potential Information
Content
Tetranucleotide theory: DNA as repeat of tetramer (4-mer): GATCGATCGATC
BUT:
a. DNA content in all Diploid Cells was the same
b. DNA content in Haploid Cells was half that in Diploid Cells
c. Nucleic Acid implicated as the Target of UV light in UV mutagenesis
Key Experiments: ... [Brown, p. 148, Research Briefing 7.1]
3. Avery, MacCleod, McCarty: 1940s ... ... [Brown, p. 148,
Research Briefing 7.1]
a. Based on earlier Griffith experiments
Virulent pneumococci ... smooth colonies on petri dishes -->
mouse: mouse dies
Avirulent pneumococci ... rough colonies on petri dishes -->
mouse: mouse lives
Heat-killed virulent pneumococci --> mouse: mouse lives
Heat killed virulent + Avirulent --> mouse: MOUSE DIES !!!
Could isolate Live Virulent penumococci from the dead mice ...
CONCLUSION:
Heat-killed bacteria provide a TRANSFORMATION ACTIVITY
that "transforms"
avirulent bacteria into Virulent bacteria ...
b. Avery et al purified the Transformation Activity; showed it
was DNA, not Protein
Basic Experiment:
1. Cell Extract from Virulent Pneumococci ... add to Avirulent
pneumococci ... incubate ... get Virulent bacteria
2. Purify crucial Molecules from the Cell Extract: DNA
a. Purified Material was sensitive to DNase
b. Purified Material was INsensitive to proteases
Not believed by many people ...
DNase could have Protease activity, of a type that degrades the
Transformation Activity
Transformation Activity could be resistant to the Proteases used
4. Hershey and Chase, 1952:... [Brown, p. 148, Research
Briefing 7.1]
Bacterial virus ... Bacteriophage ... T2
Radioactively labeled with P32 (in DNA) and S35
(in Protein)
Infection of E. coli as host:
RESULT: Only P32 enters E. coli, no S35
enters ...
THUS: Genetic Material must be Phage DNA,
since progeny phage emerge from lysed E. coli
5. Exceptions to the Rule: RNA can be Genetic Material
Tobacco Mosaic Virus (TMV):
Plant virus ... long rod: coat protein a single molecule repeated
over 1000 times
Nucleic acid: single molecule of RNA
Critical Experiment: Purify RNA and Protein
Rub each on tobacco leaves:
RNA is infectious, protein is not
6. Retroviruses: RNA as Genetic Material, proceed through DNA
intermediate
7. Recombinant DNA Technology
Purify DNA, transform cells ... Plasmids --> Bacteria,
Cultured cells from any organism
C. Nucleic Acid Structure
- Review Of Primary and Secondary
Overall Nucleic Acid Structure:
Primary: all covalent bond structure
Secondary: all helical structure ... H bonds
Tertiary: anything else, eg linear, circular, covalently
closed, supercoils
1. Primary - Covalent Bond Structure
Primary Structure of DNA: DeoxyriboNucleic Acid - [Brown, Fig
1.1]
Built from basic subunit ... DeoxyRiboNucleotide ... Nucleotide
for short
Nucleotide has 3 parts: Phosphate - DeoxyRibose - Nitrogenous
Base
Phosphate: PO4
DeoxyRibose: sugar ... monosaccharide ... 5 carbons ...
2' lacks Oxygen
Base: nitrogenous ...
4 such Bases: [Brown, Fig 1.1]
2 purines - Adenine & Guanine
2 pyrimidines - Thymine & Cytosine
Joining of Nucleotides: always 5',3'-PhosphoDiEster linkage
[Brown, Fig 1.1]
Condensation reaction [Brown, Fig 7.1]
Result of Single Linkage: no Branching
Thus: long linear macromolecule as basic structure of Single Stranded
DNA (ssDNA)
Primary Structure of RNA: RiboNucleic Acid
similar to that of DNA...
Similarities:
1. Nucleotides as basic subunits:
Phosphate, Ribose, 4 nitrogenous Bases
2. always a 5',3'-PhosphoDiEster linkage --> no branching;
long linear macromolecule
Differences: [Brown, Fig 7.2]
1. DeoxyRibose --> Ribose
2. Thymine = 5-Methyl-Uracil --> Uracil
3. RNA is mainly Single Stranded; DNA is mainly Double Stranded
(dsDNA)
2. Secondary - Helical Structure: DNA Double Helix
Double Stranded Plectonemic
Coil - two strands wound around each other
Watson-Crick Double Helix - [Brown, Fig 1.1, 7.3, 7.4;
Table 7.1]
Secondary Structure of RNA: Single Stranded
Yet: much Helical Structure ... Intrastrand Helix-loop Structure
DNA Helix: B form of Nucleic Acid
Right-handed helix [Brown, Fig 7.4]
Pitch: 34 Å = 3.4 nm
Distance between Residues (nucleotides): 3.4 Å = 0.34 nm
Residues / turn: 10.5
Other Double-Helical Nucleic Acid Structures:
A form: commonly seen
in RNA intrastrand helical structure, e.g. tRNA [Brown,
Fig 7.4]
Z form: left-handed double-stranded DNA helix [Brown, Fig
7.4]
Joining of the Two DNA Strands: H-bonds between bases [Brown,
Fig 7.3]
Sugar-Phosphate on outside ...
Bases joined A to T, C to G via H bonds in middle of DNA double-helix
Bases nearly perpendicular to axis of double-helix
3 H-bonds between C and G, but only 2 between A and T
Chargaff rules:
(A) = (T)
(C) = (G)
%(A+T): varies from
about 25% to about 75% throughout Bio Kingdom
%(A+G) = %(C+T) = 50% always
Pairing is "Anti-Parallel"
Nomenclature: 5' end to Left-hand side
D. Genes as Coding Units
within Genomes
... [Brown, Fig 1.5,
1.7, Box 1.1 ... many other figures in textbook]
Genetic information is encoded
in units along this DNA molecule, each of which encodes a protein
... units are Genes
Genes then are the fundamental unit of function.
Genetic mutants are (usually) changes in the nucleotide
sequence in a Gene
A Gene then is a region of a DNA molecule (or chromosome) that encodes a Protein or Structural RNA molecule (tRNA, rRNA, snRNA, ...)
Definitions:
Cistron: basic (smallest) unit of function,
based on genetic Cis-Trans Complementation test
... same as Gene
Recon: basic (smallest) unit of Recombination in genetic
crosses ... same as Nucleotide
Muton: basic (smallest) unit of Mutation ... same as Nucleotide
1. Prokaryotic Genes -
Operons ... [Brown,
Fig 1.7]
Genes encoding proteins involved catalytically in the same process are often found immediately adjacent to each other, e.g. trp biosynthetic and lac catabolic genes.
Control of expression of these genes is such that they are all turned on or turned off together; such groups of genes that show coordinate expression, together with their control elements, are called Operons ... an Operon is often expressed using a single mRNA molecule for all of its genes, e.g. the trp Operon ... [Brown, Fig 6.14]
In bacteria and prokaryotes,
most of the DNA is used to comprise Genes.
DNA not used for a gene is called Intergenic DNA.
2. Eukaryotic Genes - Introns
and Exons ... [Brown,
Fig 1.5, 1.7; Box 1.1]
Eukaryotic genes are often very different
from Prokaryotic genes:
Prokaryote: unicellular
organism with no true nucleus, e.g. bacteria
Eukaryote: unicellular, e.g. protazoa, or multicellular,
e.g. humans, organisms with cells that have a true nucleus (an
organelle with a nuclear membranethat contains the chromosomes).
DNA encoding a Gene is found in "pieces" on the total DNA molecule:
Regions encoding the Gene are called Exons
or EXpressed regions.
Regions between the Exons are called Introns or INTervening
sequences.
Pseudo genes, eg in human DNA, are DNA regions that arose in evolution via tandem duplication recombination events of existing genes that subsequently mutated sufficiently extensively that they lost their original function (the original function, eg beta-globin function, is still provided by the original gene that was duplicated).
Exon sizes: a few bp to a few hundred bp
Intron sizes: a few 10s of bp to a few thousand bp
Exons are those regions of the DNA that are
encoded in the final mRNA. Note that usually not all of
the mRNA is translated into the resulting Protein.
The region of the DNA (or mRNA) actually encoding amino acids
found in the Protein is called the Coding Sequence (cds).
Thus, the initial RNA made from the DNA contains Introns. This RNA is enzymatically "processed" to yield the final mRNA, the RNA used during translation for protein biosynthesis. In some of these processing events, the Introns are "spliced out" of the initial RNA
A Gene then includes all DNA sequences
found in the initial RNA transcript.
Note that this definition of a Gene does not include all DNA control
elements for expression of the gene. These Control Elements
are Protein binding sites on the DNA where proteins involved
in control of expression of the gene bind, usually to "turn
on" expression in eukaryotes (Activator Proteins)
These binding sites can be as far as 50,000
bp (50 kb) either upstream or downstream from the Exons and Introns
of the gene.
These binding sites are often found clustered in regions called
Enhancers.
Upstream: in
the direction 5' from the Exon-Intron region ... [Brown, Box 1.1]
Downstream: in the direction 3' from the Exon-Intron region
Eukaryotic genes are usually expressed as monocistronic
units: one mRNA for each gene
This is in contrast to the operon structure of expression
of clusters of genes found in prokaryotes
Some eukaryotic genes can yield more than one protein, due to alternative splicing sites for processing of the initial RNA transcript
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug Smith