Genetika Molekuler (2)

Sutarno

Genetika Ragi• Tujuan :• To convey how we use genetic analysis to study

biological problems in yeast. • to analyze single genes, then move on to how we

analyze/order pathways and determine the relationships between genes.

CLASSICAL AND REVERSE GENETIC ANALYSIS

• Classical: biological process -> identify mutants -> clone the gene -> specific biochemical function

• Reverse: Gene in hand -> create mutants -> what is the phenotype? -> biological process

Ragi (Saccharomyces cerevisiae

• Technically, YEAST is a generic (almost meaningless) term, like “tree”, that refers to a single celled fungi in the phylum Ascomycetes. It is not a species name.

• But for these lectures yeast = Saccharomyces cerevisiae= baker’s yeast

• There are other yeasts: Schizosaccharomyces pombe and pathogenic yeasts, but we will not be talking about them in these lectures.

• The big question: why would anybody want to study yeast, when really, what most of us are interested in is solving problems in human health and understanding human biology?

• The answers:• It’s the best organism to study basic aspects of cell

biology. (MANY advantages)• regardless of what organism or what protein or what

gene you are studying, you have to be familiar with what is known about homologs in other species. If you aren’t familiar with those studies, if you can’t understand the experiments done in other organisms, then you are limiting yourself, and you are at a disadvantage.

• techniques used in yeast will be used over and over in other organisms, or simply adapted to use in other organisms. The more you understand those techniques, and why they are useful, the better off you are to adapt those techniques in other organisms. (e.g k.o technologies, 2 hybrid system, genomic techniques, etc)

• The historical perspective: genetics starts with the big 3 M’s Mendel, Morgan, and McClintock, who worked on peas, flies, and corn. Little progress for decades, then the era of prokaryotic genetics starts when it is realized that bacteria and their phage have genes too. Now the Golden Era starts, and HUGE progress occurs, finding what genes are, how they are passed on, what mutations are, the Central Dogma (DNA makes RNA makes protein),the uncovering of the genetic code, the identification of regulatory genes. All essentially accomplished in bacteria. But the question remained: do those findings apply to humans and other eukaryotes? Plus, some processes can’t be studied in bacteria, since they don’t have a nucleus, or organelles, don’t go through meiosis, etc. A eukaryotic organism was needed that had the advantages of bacteria, in particular, the ability to do sophisticated genetic analysis.

• Yeast Advantages:• 1. small size• 2. rapid growth (bacteria double in 20 min., human cell

culture ~24 hrs, yeast 90 min)• Examples:• can be grown on Petri dishes, similar to bacteria (plus

they smell better than bacteria)• ~1/2 a colony on the head of a toothpick holds more

yeast then people in the whole NYC metropolitan area• A 30 ml saturated culture holds more yeast than the

entire world population• Yeast radius = 1.5 mm; human cell 10 mm; bacterium

= ??• 3. sophisticated genetics (good system to start, but

then gets even better later on…transformation, high recombination rate, new vectors)

• 4. small genome• 5. still amenable to biochemistry

• Yeast has all this, but is still a typical eukaryote: organelles, cytoskeleton, secretory pathway, receptors and second messenger systems, chromosomal elements (CEN, TEL, ARS), mitosis and meiosis, cell cycle and all the proteins involved in transcription, translation, replication, repair, recombination, splicing are virtually identical. In many cases they can be exchanged biochemically and genetically.

• Yeast should be thought of not as a large bacterium, but as a very small human.

• Yeast can do anything humans can. Except play basketball.

• Genome Project• Small: 1.2 x 107 bp (compare to other organisms in

chart) (= 12,000 kb)• Key points:• First eukaryote sequenced. Completely sequenced!

(second overall? H. influenza was first)…E. coli was not even sequenced by that time!

• Completed in April, 1996.• ~5800-6100 ORFs (greater than 100 amino acids)• Q: why don’t we know the exact number of genes yet

despite having the sequence since 1996?• arbitrary ORF cutoff of 100 amino acids• structural RNAs (tRNAs, rRNAs, snRNAs, snoRNAs, etc)• sequencing errors (if 99.9% accuracy, then 67% of ORFs

have and error, if 99.99%,then 15%)

• few introns: 5819 with none• 263 with one intron• 10 with two introns• Average gene =1922 bp (1450 bp average ORF (483

amino acids) + 472 bp intergenic region)• Largest = 4910 aa, • Smallest =PMP1 40 amino acids plasma

membrane protein• RPB12 = 70 aa essential (small does not mean

unimportant!)• 1 cM =2kb (in humans 1 cM = 700 kb • Humans and yeast have about the same genetic size

3,000-4,000 cM, but humans have 900x as much DNA! Drosophila has only 400 total cM, but 40x as much DNA.

• That’s why mapping was so important before the genome was sequenced…if you mapped a gene within a few cM of a previously cloned gene in yeast, your gene might be on somebody else’s plasmid (the easy “clone by phone” strategy)

• For the 5800 ORFs,• ~1/3 have clear-cut sequence homologs in other

organisms• ~1/3 have some motifs or clues (Zn finger,

ATPase, transmembrane domain, etc)• ~1/3 have no clue whatsoever• Comment: three goals of genetics in my mind:

» Find genes » Map their location» Identify their function

• Sequencing therefore does a great job for #1 and #2 (on C-II, 92 genes were found by genetics, 410 by sequence), but incomplete for #3. Now that is where we should be concentrating. This is the most important question, and the most fun one to answer. The other two were essentially mindless grunt work that we no longer have to do. Sequencing is not the end of genetics, but now lets us concentrate on the important questions. It just changes the emphasis to that of studying the function and regulation of individual genes and how they link with each other into biological pathways.

• BASIC YEAST BIOLOGY / THE YEAST LIFE CYCLE• There are three versions of yeast (in the lab):haploids

of the a mating type• haploids of the a mating type• a/a diploids (non-maters)• Each type is mitotically stable

• The major differences:• Haploids:

– produce mating pheromones– respond to mating pheromones– arrest in G1 when starved

• (a cell makes secretes a factor, and produces a cell surface receptor that binds a factor)

• Diploids:– don’t produce mating pheromones– don’t respond to pheromones (therefore non-

maters)– undergo meiosis and sporulation in response to

starvation• When diploids go through meiosis, the produce 4

haploid progeny (spores) (two a, two a)• The result is recombination of markers, both by (1)

crossing over, and (2) random assortment of chromosomes.

• THIS LIFE CYCLE IS THE HEART OF APOYG.• Awesome Power Of Yeast Genetics• Why?• Haploids allow easy isolation of recessive mutations• MAJOR advantage, since the phenotype DIRECTLY

reflects the genotype

• Genome Project• Small: 1.2 x 107 bp (compare to other organisms in

chart) (= 12,000 kb)• Key points:• First eukaryote sequenced. Completely sequenced!

(second overall? H. influenza was first)…E. coli was not even sequenced by that time!

• Completed in April, 1996.• ~5800-6100 ORFs (greater than 100 amino acids)• Q: why don’t we know the exact number of genes yet

despite having the sequence since 1996?• arbitrary ORF cutoff of 100 amino acids• structural RNAs (tRNAs, rRNAs, snRNAs, snoRNAs, etc)• sequencing errors (if 99.9% accuracy, then 67% of ORFs

have and error, if 99.99%,then 15%)• few introns: 5819 with none• 263 with one intron• 10 with two introns• Average gene =1922 bp (1450 bp average ORF (483

amino acids) + 472 bp intergenic region)• Largest = 4910 aa, • Smallest =PMP1 40 amino acids plasma

membrane protein• RPB12 = 70 aa essential (small does not mean

unimportant!)• 1 cM =2kb (in humans 1 cM = 700 kb • Humans and yeast have about the same genetic size

3,000-4,000 cM, but humans have 900x as much DNA! Drosophila has only 400 total cM, but 40x as much DNA.

• That’s why mapping was so important before the genome was sequenced…if you mapped a gene within a few cM of a previously cloned gene in yeast, your gene might be on somebody else’s plasmid (the easy “clone by phone” strategy)

• For the 5800 ORFs,• ~1/3 have clear-cut sequence homologs in other

organisms• ~1/3 have some motifs or clues (Zn finger,

ATPase, transmembrane domain, etc)• ~1/3 have no clue whatsoever• _____________________________________________

_________•

Comment: three goals of genetics in my mind: Find genes

• Map their location• Identify their function• Sequencing therefore does a great job for #1 and #2 (on

C-II, 92 genes were found by genetics, 410 by sequence), but incomplete for #3. Now that is where we should be concentrating. This is the most important question, and the most fun one to answer. The other two were essentially mindless grunt work that we no longer have to do. Sequencing is not the end of genetics, but now lets us concentrate on the important questions. It just changes the emphasis to that of studying the function and regulation of individual genes and how they link with each other into biological pathways.

• The genome projects also allow new technologies (practical aspects):

• Microarrays• Complete set of knockout strains (Research

Genetics)• Perfect libraries• Mass spec identification of proteins• Web addresses: • YPD search

http://www.proteome.com/databases/YPD/YPDsearch-long.html

• SGD home http://genome-www.stanford.edu/Saccharomyces/

• (search for your favorite proteins or genes or gene categories)

• ______________________________________________________

• BASIC YEAST BIOLOGY / THE YEAST LIFE CYCLE• There are three versions of yeast (in the lab):haploids

of the a mating type• haploids of the a mating type• a/a diploids (non-maters)• Each type is mitotically stable• The major differences:• haploids produce mating pheromones• respond to mating pheromones• arrest in G1 when starved• (a cell makes secretes a factor, and produces a cell

surface receptor that binds a factor)• (a cell makes secretes a factor, and produces a cell

surface receptor that binds a factor)• diploids don’t produce mating

pheromones• don’t respond to pheromones

(therefore non-maters)• undergo meiosis and sporulation in

response to starvation•

When diploids go through meiosis, the produce 4 haploid progeny (spores) (two a, two a)

• The result is recombination of markers, both by (1) crossing over, and (2) random assortment of chromosomes.

• THIS LIFE CYCLE IS THE HEART OF APOYG.• Awesome Power Of Yeast Genetics• Why?• Haploids allow easy isolation of recessive mutations• MAJOR advantage, since the phenotype DIRECTLY

reflects the genotype