MICRO. 555 (555 Microbial Molecular Genetics)

Dr.Afaf Ibrahim ShehataBotany and Microbiology Department

King Saud University

MOLECULAR GENETICS

molecular basis of inheritance Genes ---> Enzymes ---> Metabolism (phenotype)

Central Dogma of Molecular Biology* DNA -transcription--> RNA -translation--> Protein

Student CD Activity - 13.2 Events Protein Synthesis: INFORMATION FLOW

What is a GENE = ? DNA is the genetic material... [ but what about, retroviruses, as

HIV & TMV, contain RNA ] - a discrete piece of deoxyribonucleic acid

- linear polymer of repeating nucleotide monomers nucleotides* --> A adenine, C cytosine

T thymidine, G guanine --> polynucleotide*

Understanding Genetics

INFORMATION PROCESSING & the CENTRAL DOGMA

- the letters of the genetic alphabet... are the nucleotides A, T, G, & C of DNA

- the unit of information is CODON = genetic 'word'

a triplet sequence of nucleotides 'CAT' in a polynucleotide

3 nucleotides = 1 codon (word) = 1 amino acid

- the definition of (codon) word = amino acid

- Size of Human Genome: ≈ 3,000,000,000 base pairs or 1.5b in single strand of DNA genes

≈ 500,000,000 possible codons (words or amino acids)

- average page your textbook = approx 850 words thus, human genome is equal to 590,000 pages

or 470 copies of bio text book reading at 3 bases/sec it would take you about

47.6 years @ 8h/d - 7d/w WOW... extreme nanotechnology

µ Mice & humans (indeed, most or all mammals including dogs, cats, rabbits, monkeys, & apes)

have roughly the same number of nucleotides in their genomes -- about 3 billion

bp.

Experimental Proof of DNA as Genetic Material... 1. Transformation Experiments of Fred Griffith... (1920's)

Streptococcus pneumoniae - pathogenic S strain & benign R transforming 'principle'* (converting R to S cells) is the genetic

element

2. Oswald Avery, Colin MacLeod, & Maclyn McCarty... (1940's) suggest the transforming substance* is DNA molecules, but...

3. Alfred Hershey & Martha Chase's* 1952 bacteriophage experiments

*... VIRAL REPLICATION* [ pic 1 phage infection & pic-2* & lytic/lysogenic ]

a genetically controlled biological activity (viral reproduction) they did novel experiment... 1st real use radioisotopes in biology*

CONCLUSION - DNA is genetic material because (32P) nucleic acid not (35S) protein guides* viral replication

Sumanas, Inc. animation - Life cycle of HIV virus

Replication of DNA... (Arthur Kornberg - 1959 Nobel - died 10/26/07)

copying of DNA into DNA is structurally obvious??? [figure*]

Patterns of Replication* = conservative, semi-conservative, & dispersive

Matt Meselson & Frank Stahl 1958 - experimental design* can we separate 15N-DNA from 14N-DNA - (OLD DNA from

NEW DNA)? sedimentation of DNA's (sucrose gradients -->

CsCl gradients* & picture*) we can predict results... figure* & overview & all possible

results Sumanas, Inc. animation - Meselson-Stahl

DNA Replications

DNA polymerase: enzyme that copies DNA... prokaryotic Pol I-IV eukaryotic a & d

Pol III (pic) req: 4-deoxy-NTP's & ssDNA template piece reads template and adds a complimentary nucleotide*

reads 3' to 5' and synthesizes in 5' to 3' direction...

[quicktime movie] proofreads* & bidirectional synthesis*... & EM pic*

Replication forks - leading & lagging strands - Campbell figure*

Arthur Kornberg - 1st to synthesize DNA in test tube, died 26 Oct 2007

Model of Replication is bacterial with DNA polymerase III...

several enzymes* form a Replication Complex (Replisome) & include:

helicase - untwists DNA topoisomerase [DNA gyrase] - removes supercoils,

single strand binding proteins - stabilize replication fork, Primase - makes RNA primer

POL III - synthesizes new DNA strands DNA polymerase I - removes RNA primer 1 base at a time,

adds DNA bases DNA ligase repairs Okazaki fragments (seals lagging strand 3'

open holes)

Structure of DNA polymerase III* copies both strands simultaneously, as DNA is

Threaded Through a Replisome* a "replication machine", which may be

stationary by anchoring in nuclear matrix Continuous & Discontinuous replication

occur simultaneously in both strands

EVENTS: 1. DNA pol III binds at the origin of replication site in the template strand 2. DNA is unwound by replisome complex using helicase & topoisomerase

3. all polymerases require a preexisting DNA strand (PRIMER) to start replication,

thus Primase adds a single short primer to the LEADING strand and adds many primers to the LAGGING strand

4. DNA pol III is a dimer adding new nucleotides to both strands primers direction of reading is 3' ---> 5' on template

direction of synthesis of new strand is 5" ---> 3' rate of synthesis is substantial 400 nucleotide/sec

5. DNA pol I removes primer at 5' end replacing with DNA bases, leaves 3' hole

6. DNA ligase seals 3' holes of Okazaki fragments on lagging strand the sequence and DNA Repair*

Rates of DNA synthesis: myDNAi movie of replication* native polymerase: 400 bases/sec with 1

error per 109 bases artificial: phophoramidite method

(Marvin Caruthers, U.Colorado); ssDNA synthesis on polystyrene bead @ 1 base/300 sec with

error rate of 1/100b

GENE Expression the Central Dogma of Molecular Biology depicts flow of genetic

information Transcription - copying of DNA sequence into RNA

Translation - copying of RNA sequence into protein

DNA sequence -------> RNA sequence -----> amino acid sequence

TAC AUG MET triplet sequence in DNA --> codon in mRNA ----> amino acid

in protein

Information : triplet sequence in DNA is the genetic word [codon]

Compare Events: Procaryotes* vs. Eucaryotes* = Separation of labor

Differences DNA vs. RNA (bases & sugars) and its single stranded

Flow of Gene Information (FIG*) - One Gene - One enzyme (Beadle & Tatum

Transcription - RNA polymerase RNA*polymerase - in bacteria Sigma factor* binds promoter

& initiates* copying* [pnpase] Student CD

Activity 15.1 - DNA Regulatory Regions transcription factors* are needed to recognize specific DNA

sequence [motif*], binds to promoter DNA region [ activators & transcription

factors*] * makes a complimentary copy* of one of the two DNA

strands [sense strand] Quicktime movie of transcription* myDNAi Roger

Kornberg's movie of transcription (2006 Nobel)*

Kinds of RNA [table*] tRNA - small, 80n, anticodon sequence, single strand with

2ndary structure* function = picks up aa & transports it to ribosome

rRNA - 3 individual pieces of RNA - make up the organelle =

RIBOSOME primary transcript is processed into the 3 pieces of rRNA pieces (picture*) & recall structure of

ribosome

hnRNA - heterogeneous nuclear RNA : large Primary Transcript RNA function - is the precursor of mRNA in eukaryotes

hnRNA - heterogeneous nuclear RNA : large Primary Transcript RNA function - is the precursor of mRNA in eukaryotes

mRNA - intermediate sizes - 100n to 400n ( split genes*) primary transcript & mRNA

function - codes for amino acid sequence were not same size?

processing (cutting) of introns & exons*

Splicesome splicing of eucaryotic genes* [glossary] (Sumanas, Inc. advanced animation)

structure of mRNA* - caps & tails

role of 5' CAP and Poly-A Tails* [glossary] luciferase

summary of eukaryotic RNA processing*

Other classes of RNA:

small nuclear RNA (snRNP's) - plays a structural and catalytic role in spliceosome*

there are 5 snRNP's making a spliceosome [U1, U2, U4, U5, & U6];

they and participate in several RNA-RNA and RNA-protein interactions

SRP (signal recognition particle) - scRNA is a component of the protein-RNA complex

that recognizes the signal sequence of polypeptides targeted to the ER - figure*

small nucleolar RNA (snoRNA) - aids in processing of pre-rRNA transcripts for

ribosome subunit formation in the nucleolus

micro RNA's (micro-RNA) - also called antisense RNA & interfereing RNA; c7-fig 19.9*

short (20-24 nucleotide) RNAs that bind to mRNA inhibiting it. figure*

present in MODEL eukaryotic organisms as: roundworms, fruit flies, mice, humans, & plants

(arabidopsis); seems to help regulate gene expression by controlling

the timing of developmental events via mRNA action also inhibits translation of target mRNAs. ex: siRNA

--> [BARR Body*]

TRANSLATION - Making a Protein

process of making a protein in a specific amino acid sequence from a unique mRNA sequence... [ E.M.

picture* ] polypeptides are built on the ribosome (pic) on a polysome [ animation*]

Sequence of 4 Steps in Translation...

[glossary]

1. add an amino acid to tRNA -- > aa-tRNA - ACTIVATION*

2. assemble players [ribosome*, mRNA, aa-tRNA] - INITIATION*

3. adding new aa's via peptidyl transferase - ELONGATION*

4. stopping the process - TERMINATION

Review the processes - initiation, elongation, & termination myDNAi real-time movie of

translation* & Quicktime movie of translation Review figures & parts: Summary fig*

[ components, locations, AA-site, & advanced animation ]

[ Nobel Committee static animations of Central Dogma

GENETIC CODE...

...is the sequence of nucleotides in DNA, but routinely shown as a mRNA code*

...specifies sequence of amino acids to be linked into the protein

coding ratio* - # of n's... how many nucleotides specify 1 aa 1n = 4 singlets, 2n= 16 doublets, 3n = 64 triplets

Student CD Activity - 11.2 - Triplet Coding

S. Ochoa (1959 Nobel) - polynucleotide phosphorylase can make

SYNTHETIC mRNA Np-Np-Np-Np <----> Np-Np-Np + Np

Marshall Nirenberg (1968 Nobel) - synthetic mRNA's 5'-UUU-3' = phe U + C --> UUU, UUC, UCC,

CCC UCU, CUC, CCU, CUU

the Genetic CODE* - 64 triplet codons [61 = aa & 3

stop codons] universal (but some anomalies), 1 initiator

codon (AUG), redundant but non-ambiguous, and exhibits

"wobble*".

GENETIC CHANGE - a change in DNA nucleotide sequence

- 2 significant ways mutation & recombination

1. MUTATION - a permanent change in an organism's DNA*that results in

a different codon = different amino acid sequence Point mutation - a single to few nucleotides change...

- deletions, insertions, frame-shift mutations* [CAT] Student CD Activity - 11.2 -

Triplet Coding - single nucleotide base substitutions* :

non-sense = change to no amino acid (a STOP codon) UCA --> UAA ser to non

mis-sense = different amino acid UCA --> UUA ser to leu

Sickle Cell Anemia* - a mis-sense mutation... (SCA-pleiotropy)

another point mutation blood disease - thalassemia

- Effects = no effect, detrimental (lethal), +/- functionality, beneficial

2. Recombination (Recombinant DNA) newly combined DNA's that [glossary]*

can change genotype via insertion of NEW (foreign) DNA molecules into recipient cell

1. fertilization* - sperm inserted into recipient egg cell --> zygote [n + n = 2n] 2. exchange of homologous chromatids via crossing over* = new gene

combo's 3. transformation* - absorption of 'foreign' DNA by recipient cells changes

cell 4. BACTERIAL CONJUGATION* - involves DNA plasmidsg* (F+ & R =

resistance) conjugation may be a primitive sex-like reproduction in

bacteria [Hfr*] 5. VIRAL TRANSDUCTION - via a viral vector ( lysogeny* &

TRANSDUCTION* ) general transduction - pieces of bacterial DNA are

packaged w viral DNA during viral replication restricted transduction - a temperate phage goes lytic

carrying adjacent bacterial DNA into virus particle

6. DESIGNER GENES - man-made recombinant DNA molecules

Designer Genes - Genetic Engineering - Biotechnology RECOMBINANT DNA TECHNOLOGY...

a collection of experimental techniques, which allow for

isolation, copying, & insertion of new DNA sequences into

host-recipient cells by A NUMBER OF laboratory protocols & methodologies

Restriction Endonucleases-[glossary]*... diplotomic cuts* at unique DNA sequences,

Eco-R1-figure* mostly palindromes... [Never odd or even] ▼

5' GAATTC 3' 5' G . . . . . + AATTC 3' 3' CTTAAG 5' 3' CTTAA . . . . G 5'

▲ campbell 7/e movie* DNA's cut this way have sticky (complimentary) ends & can be

reannealed or spliced* w other DNA molecules to produce new genes

combos and sealed via DNA ligase. myDNAi movie of restriction

enzyme action

Procedures of Biotechnology? [Genome Biology Research]

A. Technology involved in Cloning a Gene... [animation* & the tools of genetic analysis]

making copies of gene DNA

1. via a plasmid* [ A.E. fig & human shotgun plasmid cloning & My DNAi movie]

2. Librariesg... [ library figure* & Sumanas animation - DNA fingerprint library ]

3. Probesg... [ cDNAg & reverse transcriptaseg

cDNA figure* & cDNA library* ]

4. Polymerase Chain Reactiong & figure 20.7* & animation* + Sumanas, Inc. animation*

PCR reaction protocol & Xeroxing DNA & Taq polymerase

B. Detection of a Gene... Locating a gene (or its activity) - Restriction Maps.

1. Restriction mapsg... via gel electrophoresis* &

DNA-electropherogram*

2. DNA fingerprintg... CSI Miami - how to make one* a murder case* & a rape case* + DNA prints in

Health & Society & DNA Forensic Science 3. DNA Probe Hybridizationg... to detect specific DNA

with a probe fig 20.5*

4. Comparing Restriction Fragments... to a probe Southerng Blotting fig* Sumanas, Inc. animation - DNA electrophoresis & blotting* one can detect specific gene sequence

in samples by binding to labeled probes

5. DNA micro-arrays - monitor gene expression in thousands of genes & changes

by passing cDNA of the cell's mRNA over slide with ssDNA of all cell's genes;

DNA microchips are fabricated by high speed robotics akin to Intel chip making

cDNA (mRNA's) are fluorescently tagged so easy to see in slide's wells.

[microchips arrays made simultaneously by phopshoramidite method of Caruthers]

Sumanas animation - DNA chip technology* & myDNAi DNA microarrays

5. Gene Sequencing - Human Genome Project

strategy - shotgun approach* developed by Celera Genomics

random fragments are sequenced and then ordered

relative to each other via overlap & supercomputing

Student CD Activity - 16.1 - Sequencing Strategies

methodology dideoxy procedure* (development by Fred Sanger)

Surprising Size Estimates of Human Genome & figure* NHGRI researchers* have confirmed the

existence of 19,599 protein-coding genes in the human genome and identified another

2,188 DNA segments that are predicted to be protein-coding genes =

21,787 genes

mtDNA & Y-chromosome DNA aid in search for our human ancestry

Practical Applications of DNA Technology - Some examples of What's been Do

1. Medical... disease often involves changes in gene expression a. disease/infection diagnosis:

PCR & labeled DNA probes from pathogens can help identify microbe types...

isolate HIV RNA --RT--> cDNA --PCR--> probe can id... AIDS infection

b. RFLP - Restriction Fragment Length Analysis - markers often inherited with disease

what is RFLP* genetic testing & polymorphism ---> RFLP markers to disease

DdeI cuts Sickle gene* (also MST II cuts Sickle Cell)

fragment analysis (DNA fingerprinting) also used for paternity testing

c. Gene Therapy... idea is to replace defective genes via microinjection of DNA*

requires VECTORS - fig 20.16* (patient: ADA Deficiency & Ashanti DeSilva update)

SCID (severe combined immunodeficiency - a single gene enzyme defect):

clinical trials in 2000 resulted in 2 of 9 cured, but they developed leukemia:

a retroviral vector inserted a repair gene in bone marrow cells

near genes involved in blood cell division, thus leukemia. trials stopped.

2. Pharmaceutical Products... manufactured drugs

Recombinant bacteria* = Humulin & protropin (an ethical dilemma)* Student CD Activity - 17.1

- Producing Human Growth Hormone

Control of Gene Expression How do we know a gene has been active

(turned on) within cells???? we look for gene's

product, i.e., protein or RNA

an increase in enzyme activity implies gene action?

no enzyme activity suggests no gene action

but, what about pre-existing inactive enzymes converting to --> active forms

ZYMOGENS - pepsinogen -----> pepsin

- trypsinogen -----> trypsin

thus, we have 2 possibilities: 1) pre-existing inactive

enzyme --> active 2) de novo (new) enzyme

synthesis (gene action)

Mechanism of Gene Action (turning on/off genes) in PROCARYOTES...

model: LACTOSE OPERON - Jacob & Monod

[glossary]*

E. coli (grown on) glucose lactose

NO beta-galactosidase beta-galacotsidase

OPERON* = series of mapable-linked genes controlling synthesis of protein

p Rg crp p O Sg1 Sg2 Sg3 Rg (i gene) regulator - makes repressor protein what if regulator

binds lactose* p promoter - binds RNA polymerase figure O operator - binds repressor protein figure

S structural - make enzyme proteins figure*

Sumanas, Inc. animation - Lac Operon* Catabolic Repression

Control of Gene Expression - in EUKARYOTES

Mechanism of Gene Action (turning on/off genes) is more complex

much more DNA & it's inside a compartment (nucleus) and, there are no operons present

have many more promoters - sites where RNA polymerase binds

enhancer sequence - sites where enhancers/transcription factors bind

transcription factors - proteins that help transcription but, individual genes are not contiguous, thus no operons

3 levels for eukaryotic controls* - transcriptional, translational, post-translational

multiple places for control* - of whether a gene make a protein or not

McGraw-Hill higher Ed movie on control of

gene expression

Some examples for Eukaryotic gene expression controls:

Differential Gene Activity... is the selective expression of genes

i.e., different cell types express different genes [liver vs. lens cell]

1. role of activators in selective gene expression

(Differential Gene Activity*)

ex: Steroid Hormones (figure*)

2. Molecular turnover - ½ life mRNA's* & longevity of some proteins*

3. Processing of RNA transcript (figure*) cut/spliced in nucleus and capped for

transport intron - pieces cut out (non gene-

proteins) exons - pieces transported to cytoplasm alternative splicing = figure C17.11* and

some examples*

ex. cont. Eukaryotic gene expression controls:

5. cancer often results from gene changes affecting cell cycle control.

cancer genes, such as adenomatous polyposis coli, which cause 15% of

colorectal cancers is a tumor suppressor gene, a type of Oncogenesg*

2 kinds of human cancer genes:

Ras (proto-oncogene) causes 30% human cancers: is a G-protein that promotes other cell division

proteins a Ras mutation --> hyperactive Ras protein -->

cell division fig 19.12a

p53 (tumor suppressor geneg = 50% human cancers) fig 19.12b*

p53 is a transcription factor that promotes the synthesis of cell cycle

inhibiting proteins [DNA damage --> active p53 --> p51 gene --> protein binds to

cyclin dependent kinase stops cell division]

thus a p53 mutation --> leads to excess cell division (cancer)

- other cancer genes can lead to new gene actions resulting in

cancer BRCA1 and BRCA2 (tumor suppressor genes) are involved in

50% of breast cancers in humans

Organization of the Genome* - the structural organization of genome

in eukaryotes influence its expression. Size of Human genome:

3million+ base pairs, or some 500,000 pages of journal Nature.

reading a 5 bases/sec it would take you about 60 year @ 8h/d 7d/w

A definition of a GENE*.

Definition of a Gene Mendel's Particles... unit of heredity responsible for phenotype

Morgan's Loci... he placed genes on a chromosome, i.e., it's a cellular entity, that is part of chromosome & is mapable Watson & Crick... it's a sequence of specific nucleotides along the

length of a double helical DNA molecule Molecular Definition...

length: 1 nucleotide = 0.34nm thus tRNA = 81n x 0.34 = 27.5nm

mass: 1 nucleotide = 340amu thus tRNA = 81n x 340 = 27,540amu

Modern functional definition... a DNA sequence coding for a specific

polypeptide: but, also must include... Split Genes... presence of Introns &

Exons : eukaryotic genes contain

non-coding segments (introns) and coding segments (exons

- that make proteins)

Others DNA pieces... any definition must also include: segments that code for rRNA, tRNA, & snRNP's also promoters, enhancer segments, regulator

genes, operators ? BEST ≈ "a GENE is a region of DNA that CODES for an RNA" end.

MST II restriction cuts of normal sickle beta-gene

( pink is DNA sequence & blue = 4 gel fragments) _________|__________________|CCTNAGG GAA

_____________|____________

a b c d

In 1978, Yuet Wai Kan and Andrees Dozy of the University of California-San Francisco showed that the restriction enzyme Mst II,

which cuts normal b globin DNA at a particular site, but will not recognize and therefore will not cut DNA that contains the sickle cell mutation. Mst II recognized the sequence CCTNAGG (where N = any

nucleotide). Sickle cell disease is due to a single point mutation in the beta globin gene on chromosme 11 that changes CCTGAGG to

CCTGTGG. MST II restriction cuts of recessive sickle beta-gene (blue = 3 gel

fragments) _________|___________________CCTGTGG ________________|

____________

¥ Sickle Cell disease occurs when the DNA sequence for

glutamic acid is converted to valine. This results from a change in the nucleotide T to A. This change eliminates a

site recognized by the restriction enzyme DdeI.

Restriction enzyme: DdeI (recognition sequence: 5'-C^TNAG-3')

Southern blotting probe: fragment of ß-globin coding sequence

Pattern result: normal cell = 3 fragments (1 large, a 201bp piece, and a 175bp piece

sickle cell = 2 fragments (1 large, and a 376bp piece)

fig 20.9* Thus the number of RFLP piece can indicate presence of defective alleles.

reading frame (1 codon) = CAT point mutations at hot spots - [fig]

C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-

1st point insertion or deletion

C-A-T-X-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A = mutant

2nd insertion or deletion C-A-T-X-Y-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-

C-A-T-C-A-T-C = mutant3rd insertion or deletion

C-A-T-X-Y-Z-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T = norma

DO NOT study the material below Gene expressions in pharmacogenomics &

toxicogenomics via microarrays 1 cM = about 1 Mb

TRANSPOSONS - pieces of DNA prone to moving & creating repeat sequences

LINE - long interspersed nuclear element holds promoter & 2 genes: RT & integrase

an anomaly - RNA Recoding*

Simple Tandem Repeats (short- 5n to 6n) or trinucleotide (3n) repeats can undergo an increase in copy

number by a process of dynamic mutation; # of tandem repeats is unique to a genetic indiv.

Variation in the length of these repeats is polymorphic. figure* individual A has ACA repeated 65 times @ loci 121, 118, and 129

individual B has a different repeat pattern at these loci STR'sa can cause genetic diseases as well:

CCG trinucleotide occur in fragile sites on human chromosomes (folate-sensitive group).

fragile X (FRAXA) is responsible for familial mental retardation. another FRAXE is responsible for a rarer mild form of mental

retardation. mutations of AGC repeats give rise to a number of neurological

disorders

3. Forensics - DNA fingerprinting is the vogue judicial modus operandi

a murder case* & a rape case* + DNA prints in Health & Society & DNA Forensic Science

DNA fingerprinting usually looks a 5 RFLP markers and blood is tested via

Southern Blotting (20.10) using probes for these alleles

4. Environmental Clean-up... bacteria can extract heavy metals (Cu, Pb,

Ni) from the environment & convert them into non-toxic compounds

genetically modified bacteria may be the "miner's" of the future

5. Franken Food... genetically modified (GM) animals & agricultural crops

Transgenics - organisms with inserted foreign DNA in their genomes

Animals* - GFP novelties* + Dolly - animal cloning companies --->

mammalian cloning success? - "pharm" animals (20.18*) --->

transgenic animal movie sheep carry human blood

protein gene that inhibits enzymes

fibrosis; artificially insemination, microinjection

of human gene, fertilized ova are put into a surrogate sheep:

chimeras mated to produce homozygote- Milk tested for active protein.

Plants - genetically modified crop plants - fig 20.19* - to get Ti plasmids in = a DNA gun* Purdue

University Gene Gun movie - Frankenfood & Edible Vaccines - National Plant Genome Initiative

Plan update future

6. Synthetic Biology... artificially manufactured biological systems

- virus models* (synthetic Biology)

ð An overview of biotechnology History of Biotechnology Human Genome Project &

Biotech Companies HHMI funded DNA Interactive

tutorial

What are Introns? and What is the Role of Intron DNA?

don't really know, but Percentage of non-coding DNA during evolution* goes up.

INTRONS - DNA Junk or sophisticated Genetic Control Elements?

Current dogma of Molecular Biology

DNA --> RNA --> Proteins, (proteins supposedly regulate gene expression) figure*

in 1977 Phillip Sharp & Richard Roberts discovered DNA contains

introns intervening DNA segments that do NOT code for proteins

a primary RNA transcript is processed by splicing to assemble protein coding exons

Presence of Introns: Absent in prokaryotes: they have few non-coding DNA sequences

as eukaryotic complexity grows so does non-coding DNA [figure]

makes up greater than 95% of the DNA less than 1.5% of human genome encodes proteins, but all of

DNA is transcribed 40% of human genome is Transposons & repeat genetic

elements.

Evolutionary Origins? may have been self-splicing mobile genetic elements

that inserted themselves into host genomes Advent of Spliceosomes: catalytic RNA/protein

complexes that snip RNAs out of mRNAs,

would encourage introns to proliferate, mutate, evolve

that inserted themselves into host genomes

Advent of Spliceosomes: catalytic RNA/protein complexes

that snip RNAs out of mRNAs,

would encourage introns to proliferate, mutate, evolve

Role of Introns? Not Junk, but rather Genetic Control Elements [figure*]

Micro RNAs - derived from introns? - occur in plants, animals, & fungi

a) help control timing of developmental processes as cell proliferation,

apoptosis, and stem cell maintenance b) help tag chromatin with methyl and acetyl

groups c) may help in alternative splicing mechanisms

COMPLEXITY: to build a complex structure one must have bricks & mortar,

as well as an architectural plan. DNA, therefore should contain both - the materials and the plan:

a) component molecules - proteins, carbs, lipids, and nucleic acids:

all known living organism use the same bricks and mortar

b) the difference between Man & Monkey is the architectural plan

Where is the Architectural Information? we've always assumed in the regulatory proteins

Maybe it's in the non-coding mirco-RNAs (intronic elements)

Thus the greater proportion of the genome of complex organisms, the introns, isn't junk,

but rather, it is functional RNA that regulates time dependent complexity?