I) Be sure to remember the distinction between
transcription as opposed to translation
making RNA copies of DNA Making proteins, by "decoding" RNA

Also: don't forget! What is "replication"

II) Several different kinds of RNA are made:
1-2% of weight of average cell nearly all r-RNA
Ribosomal RNA a giant ribozyme the ribosome molecular weight > million
bound to ~ 50 specific proteins
r-RNA genes at nucleolus structure
(polyribosomes in cytoplasm)

III) another kind of RNA: snRNA and snoRNA small nuclear RNA and small nucleolar RNA
These serve as ribozymes catalysing splicing of messenger RNAs, before the m-RNAs leave nucleus

IV) Transfer RNA "tRNA"
These are the adaptors that carry amino acids and bind to the m-RNA at their anticodon sites. very short: only about 75 bases long; kinked into clover-leaf: 3 double strand bends About one in 10 bases gets chemically changed
sulfur added, deamination, reduce=bonds etc. p339
3' end ribose covalently bonded to COOH of AA
anticodon of t-RNA base pairs with codon (if codon were GGG, then anticodon is CCC glycine)

V) Messenger RNA = "transcripts" code for amino acid sequences of all proteins.

Very simple in procaryotes Absurdly complex in eucaryotes (introns/caps/ export from nucleus)

VI) Messenger RNA processing in eucaryotes; Before m-RNA is exported from the nucleus

Splice out intron "Lariats"

Multiple versions of many eucaryote proteins
(for example: fibronectin
One gene, many different lengths
Splice out different combinations of introns

Add methyl-guanosine "cap" to start end (5')
Add polyadenylate tail to 3' end
(controls life-span of m-RNA until As digested)

VII) Special initiator t-RNA
always methionine in eucaryotes/ methylmethionine in procaryotes

VIII) Folding of proteins, catalysed by
Heat shock proteins chaperonins

IX) Life span of proteins, before digestion
n-terminal signal to be destroyed Arg, Lys, His, etc.
Ubiquitin 76 amino acid long carboxy end to lysine

Proteosome: large protein complex for digesting

X) The danger of proteins that have folded so as to leave hydrophobic amino acids on their surface!
aggregation Alzheimer's Huntington's disease
also: prion diseases: domino effect alpha helix->beta sheet
Mad Cow disease scrapie in sheep
Creutzfeldt-Jacob disease in humans

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Seventh chapter topics: Today's amino acid is arginine page 132

I) Mechanisms have evolved for turning genes on and off (at the level of transcription) by binding of special proteins (transcription factors) selectively to where every the DNA has certain base sequences.

Procaryote examples:
Operons, for control of sets of enzymes that are used for the digestion or synthesis of certain sugars, amino acids etc.
Lac repressor etc. controls galactose digesting enzymes
(if no lactose, then wasteful to make enzymes to digest it)

Lambda repressor (self-inhibition by lambda virus)

Eucaryote example:
Differentiation of cells in developing embryos
(The concept of the differentiated cell type....liver cells etc.)

Nuclear transplantation, in frogs; fusionof cells to oocytes (Dolly)

II) There are many examples of transcription factor proteins.
Binding sites have lots of lysine, arginine, histidine
Also, these binding sites often protrude or have loops.
"Helix-turn-helix" including Homeodomain proteins

Note, many transcription factors are dimers
and DNA binding sites have palindromic sequences
Twice the binding energy, thereby squares the affinity constant
(if one half bound with 1.4 kCal, then 2 halves 2.8 kCal etc.)

"Helix-loop-helix", Zinc finger proteins,
Leucine zipper proteins, and even beta sheet binding sites.

III) What kinds of methods are used to identify and isolate such DNA binding proteins?

a) DNA affinity chromatography:
either with DNA of many sequences
or with just DNA having a specific base sequence
Proteins bind until washed out with concentrated salt solutions

b) Gel mobility assay: electrophoresis of DNA fragments
Those fragments that bind to proteins are slowed down

In procaryotes: control by binding just "upstream" of gene
in location where DNA polymerase binds and starts m-RNA

In eucaryotes: control partly just upstream ("promoter regions"
but also by more distant sites, further & even downstream!!
"Enhancer" regions. (DNA folding & complex formation)
(also the rather new concept of the insulator regions)

V) Spatial patterns of (some) genes' transcription has been analysed in detail in Drosophila: (homeodomain discovered this way)
5 families of genes (mostly coding for transcription factors)
that control where in the early embryo each is transcribed.
"bicoid", kruppel, hunchback, even-skipped, antennapedia

Historical note: How were these genes discovered? By studying recessive mutations that cause fly embryos; to "abort" spontaneously at various specific early stages.
Eric Wieschhaus & Cristine Nusslein-Volhard

The great riddle of co-linearity! can you suggest explanations?

VI) Another puzzle: Fetal hemoglobin, and all that...
Although the genes for alpha hemoglobin and beta hemoglobin
are on different chromosomes, in humans
each are near genes for fetal and embryonic forms of hemoglobin
Fetal hemoglobin protein binds oxygen slightly stronger.
The time sequences of synthesis of these different hemoglobin genes are in the same spatial sequences on the chromosomes.
NOTE! Some rare people continue to make fetal hemoglobin all through their life, but have no serious symptoms!!
Sickle cell anemia is caused by a single amino acid substitution
that makes heterozygous people resistant to malaria;
but causes aggregations of hemoglobin in homozygous people!

If there were some way to switch on the fetal hemoglobin gene
and switch off the adult form, that would cure sickle cell anemia
(somebody please figure out how to do that & I will split the Nobel money!)

VII) RNA Interference RNA i (a puzzle recently solved)
If you inject cells with double stranded RNA with the same base sequence as some particular gene, then the result is that cell will permanently stop making the protein coded by that gene
(alternatively: inject cells with anti-sense RNA)

For experimenters, this is a useful way to "turn off" one gene at a time, to test what changes occur in phenotype.

The cause seems to be a mechanism for defending against viruses
Special RNA-digesting enzymes cut up any double stranded RNA
and retain single-stranded fragments of this RNA
which then base pair with m-RNAs that have complementary seq.
so that the enzyme continues to find & destroy any RNA with those sequences! forever!? Spreads from cell to cell in plants.

NOTE: Anti-sense RNA treatments developed for some cancers

There is also a "nonsense-mediated m-RNA surveillance"
(selective enzymatic digestion of RNAs with stop codons)
"Exactly how this is accomplished is not understood in detail, but it is easy to understand why ribosomes must play a part..."
which sounds to me like they don't have much more than a clue!

VIII) Some species have very small introns: much less DNA
good example is Fugu, the puffer fish. 400 million base pairs
(Other species: Newts, Amphiuma, have much more DNA than humans)
{Volumes of each cell are linearly proportional to DNA amount}
(but no one can figure out the mechanism)

IX) DNA methylation (cytosine, specifically) is used as part of the mechanism of gene control in vertebrates
CG pairs: when the cytosine is methylated, then an enzyme
GC automatically methylates the C on the opposite strand
(this mechanism makes the methylation state self-perpetuating)
(but deamination of methyl-cytosine makes thymine
which defeats part of the repair mechanisms; therefore causing especially large mutation rates at methylated GC regions)
[The "CG-rich islands" are mostly in genes that are never methylated because they are housekeeping genes, expressed in all differentiated cell types, and never turned off; therefore they were never subject to this tendency to mutate from CG to AT]

X) "imprinting" = a weird & unexpected phenomenon in which genes coming from sperm are treated differently from those coming from the egg cell. Discovered by accident in experiments on nuclear transplants: What will happen if both sets of chromosomes came from sperm? Or both from eggs?
Both kinds of embryos developed abnormally!
The mechanism is based on DNA methylation of certain genes.

XI) X chromosome inactivation in mammals (dosage compensation) is done by coating one or the other X with many copies of a certain RNA (never translated) "Xist"

XII) Mechanisms have evolved for trapping certain RNAs into certain parts of egg cells (or other cells)
an example is the RNA transcript of the bicoid gene in flies.

XIII) Recombination of different exons (& processing) can produce different versions of many proteins.

But note that this is NOT how antibodies get their specificity!
(but imagine a DNA equivalent to exon splicing, & you would be close)

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Eighth chapter topics: Today's amino acid is Valine page 133

I) Tissue culture (both plants and animals) Tissue culture medium primary cultures secondary cultures permanent cell lines HeLa etc. "in vitro"

Immortalization of cell lines telomerase also mutations in cancer causing genes

Cell separation methods;
* protein digesting enzymes
* chelators of calcium ions and magnesium ions

Keeping cells alive: Freezing cells in liquid nitrogen etc.
Stem cell lines (from early embryos of mammals)(political controversy)

II) Cell fusion (damage plasma membranes, so that cytoplasms run together; one cell with two nuclei (can be two species!)
3 methods of fusion: *viruses, *chemicals, *electric sparks

Heterocaryons (fused cells, with nuclei from different species)
for example, mouse-human heterocaryon tissue culture cells.

III) Monoclonal antibodies hybridomas

IV) Homogenization of cells: methods; grinding; forcing through tiny holes; osmotic shock; ultrasonic vibration

V) Centrifugation and ultracentrifugation

VI) "cell free systems" = mixtures of isolated enzymes that are able to carry out complex sets of reactions, such as Krebs' cycle enzymes, or ribosomes & t-RNAs etc. capable of protein synthesis

VII) Chromatography different forms of chromatography
complete separation of molecules (could be anything!)
based on small differences in properties such as relative solubilities in pairs of solvents, sizes of molecules, charges, etc.
gel chromatography, gas chromatography, you name it...!

VIII) Electrophoresis (starch gel, etc.) [Oliver Smithies]
SDS polyacrylamide gel electrophoresis size of proteins
sodium dodecyl sulfate beta mercaptoethanol (doesn't work for glycoproteins)

IX) Isoelectric focusing and two-dimensional PAGE
Blotting onto nitrocellulose paper antibody localization
("Western" blotting) (also peptide fragments)

X) Mass spectrometry (time of flight)

XI) DNA sequencing became very "easy" in the late 1970s
(so it is now much easier to figure out amino acid sequences based on the genes' DNA base sequences & the genetic code
WHAT IS THE BASIC METHOD?
Agarose gel electrophoresis (separate DNA 123 bases long from 124 bases long!)
& selective termination of DNA replication by di-deoxyribose nucleotides (see page 505)
for example,
if those partial copies that are 100 bases long have an adenine as their last base,
and those that are 101 bases long have a guanine as their last base,
and those that are 102 bases long have a cytosine as their last base
and those that are 103 bases long have a guanine as their last base
etc.
that tells you that this part of the original DNA's base sequence
must have been ..AGCG...

to test if you understand this concept, then suppose that the
sequence was ..AGCGCCAAGCTGCTTGCGCGGCC

Then figure out which base would have been at the very end of those fragments that were 104 bases long? those that were 105 bases long? 106 bases long? etc.

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Chapter 9 "visualizing cells" mostly about optical methods. Amino acid Aspartic Acid

Sizes of things

...one meter = 39 inches (3 feet, plus)

...one millimeter 1 thousandth of a meter
the thickness of a microscope slide (pencil lead, etc.)

...one micrometer 1 thousandth of a millimeter


= one millionth of a meter
about the diameter of a mitochondrion & a bacterium
an average human cell ~ 12- 15 micrometers in diameter
(old-fashioned biologists tend to say "microns")

...one nanometer 1 thousandth of a micrometer
(the same thing as a "millimicron")
the wave-length of visible light is 400-700 nanometers
the smallest distance light microscopes can resolve is 200 nm
a cell membrane is about 10 nm thick
an average amino acid is about 1 nanometer in diameter.

a tenth of a nanometer is called an Angstrom unit 10^-10 meters
individual atoms are a couple or 3+ angstroms in diameter

II) contrast (by staining, or by "phase contrast") ; specificity
Histological sectioning (embedding in paraffin, or freezing.) & slicing
phase contrast microscopy Nomarski DIC = differential interference contrast
video microscopy = light microscopy using TV Camera to observe image,
and using various electronic tricks to enhance contrast, etc.
Other exotic new forms of microscopy: scanning acoustic microscopy (sound waves!)
fluorescent staining; staining with fluorescently labeled antibodies
Green Fluorescent Protein
  Confocal fluorescence Microscope

III) Fluorescent analogue cytochemistry

Works as in the following example:

1) isolate microtubule protein from brains from slaughterhouse
2) covalently attach rhodamine to the individual tubulin proteins
3) microinjectthe labeled tubulin into tissue culture cells
4) observe with a fluorescent microscopeas the labeled pig tubulin
adds itself onto the ends of microtubules inside the living
tissue culture cells:

V) electron microscopy ; often called just "EM" or Transmission EM
use magnetic "lenses" (or sometimes electric fields)
since electrons have much smaller effective "wave lengths" than light
the diffraction limit is only a nm or less, although the low NAs of lenses
result in the real resolution being very far from the theoretical limit
Can only be used to observe killed cells in a vacuum.

specimen preparation:

embedding in wax (for light microscopy) slice with steel knife
embedding in plastic (for electron microscopy) diamond knives!
For TEM: metal grids instead of slides (high vacuum inside e.m. scope!)
stain with heavy metals, such as lead and osmium (for contrast)

SEM= scanning electron microscopy
is fundamentally rather different;
much lower resolution; don't focus image;
but does need vacuum and metal deposition

VI) "atomic force microscopy" !!! mechanical touching!
and can resolve distances much smaller than light, or even T.E.M.

6 1/2) Laser forceps: can exert tiny sideways forces by light rays! =(Laser tweezers)

VII) X-ray diffraction if there were some kind of lens that you could focus X-rays with, then maybe you could build an X-ray microscope, with very high magnification!
Mathematical calculations are used to deduce the molecular structure:
Angstrom resolutions
(and also electron diffraction)
NMR nuclear magnetic resonance also gives information about tertiary structures.

VIII) Electron Microscope Tomography

IX) Ion Sensitive Indicators Calcium ion concentrations, etc.
(also luminescent proteins: Aequorin)

X) radioactive tracers pulse chase experiments