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Vikrant K. Bhosle (08-04-2013)

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Promoter proximal elements vs. Enhancers Genomic imprinting Position effect variegation Bioinformatics Genome annotation, BLAST, functional genomics/proteomics Forward vs. Reverse Genetics Summary of transfection methods from the lecture Developmental genetics- Why drosophila? Maternal effect vs. Zygotic genes

What are HOX genes? Two hybrid assay (yeast) RNA interference: summary

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All of them are cis-acting, DNA elements in eukaryotes Either one of them can be bound by transcription factors

Promoter proximal elements (PPE) are always present upstream of the gene andwithin few hundred bases (

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Last week (week-10 conference), I mentioned that effect of histonemethylation depends on the location.

Histone methylation can lead to either euchromatin orheterochromatin formation. This is correct. HOWEVER,

We, after discussion with Prof. Hipfner, have decided to simplifythe situation for Biol-202 students.

For the purposes of this course only, histone methylation leads toheterochromatin spreading (e.g. H3K9me3) -> Leading torepression of the gene transcription

The enzymes for histone methylation -> Histone methyltransferase (HMT)

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Mating type in yeast- Not covered Position effect variegation (PEV) in

drosophila (lect-30) X- chromosome inactivation in mammals

(non-coding RNA)

What is genomic imprinting?

Only one allele of autonomous gene isactive. Other allele is silenced byepigenetic mechanisms

e.g. Igf2 is active on paternal allele andH19 is active on maternal allele of

chromosome 11 in humans.

Role of CTCF in H19/Igf2 imprinting.CTCF binds to insulator (ICR) only if lateris unmethylated

Imprinting refers to the silenced gene

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1. Heterochromatin protein-1 or HP1 (Normal)- Promoteheterochromatin formation and spreading

2. Histone methyl transferase or HMT (Normal)- Promotespreading of heterochromatin

3. Histone acetyl transferase or HAT (Normal)- Prevent spreadingof heterochromatin

Loss of function mutations

HP1 and HMT (mutated) suppress heterochromatin spreadingi.e. Su(var)-> More gene expression -> More red color

HAT (mutated) promote heterochromatin spread i.e. E(var) ->Less gene expression -> More white color

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Bioinformaticsis the field of science in which 1. biology, 2. computerscience, and 3. information technology merge into a single discipline. (NCBI;2001)

Some useful websites- OMIM- http://www.ncbi.nlm.nih.gov/omim NCBI BLAST- http://blast.ncbi.nlm.nih.gov/Blast.cgi UCSC Genome browser- http://genome.ucsc.edu/ NCBI primer (PCR)- http://www.ncbi.nlm.nih.gov/tools/primer-blast/

Genome annotation- There is huge amount of genomic information (3 Gb of DNA in human

genome) but protein-coding genes are minimum (

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Pseudogenes (non-functional duplicated copies of genes almost asnumerous as functional genes) They have accumulated many mutationsand stop codons during years of evolutions.

Information obtained from cDNAs

Identify what are likely genes Identify what are exons & what are introns Identify patterns of splicing (alternative splicing)

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An Open Reading Frame (ORF) is a contiguous stretch of DNA that codes foramino acids without being interrupted by Stop Codons(s).

Computer programs scan 6 possible reading frames (3 per strand) & markthose with large ORFs (say, greater than 50 amino acids)

Introns (avg. size- 5 kb) in eukaryotes can interrupt reading frames. Only way to definitively confirm presence of gene is alignment with cDNA The problem is cDNA library might not detect mRNAs with very low

expression levels

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Comparative genomics is the study of genome structure, including, numbers,types and location of genes between chromosomes, or, more usually, betweenorganisms.

Within an organism (species) Gene families created by segmental duplication Genes with sequence homology in same organism are called paralogs

Between organisms Types of genes & proteins (sequence homology) Genes with sequence homology in different species are called orthologs Beware of Codon Bias Different organisms make more use of some amino acids codons less/more

than others

Remember more homologies you find between two organisms at genetic level,stronger the evolutionary relationship between the two (recent commonancestor)

Most commonly used program for comparing sequences is called BLAST.BLAST stands for Basic Local Alignment Search Tool.

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Gene family- Genes within same or closely related organism(s) which showsequence homology. They might encode for proteins with similar function.

Synteny- It is defined as conservation of gene order in blocks of chromosomesfrom distant organisms with a common ancestor.

At the local level, genes often can have the same neighbors in related organisms(microsynteny)

~3% of the human genome consists of non-repetitive, non- coding DNAsequences that are highly conserved in related species (e.g. mouse) ULTRACONSERVED SEQUENCES

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Functional Genomics (done in the lab)- Large scale, high throughputprojects to study functions of many genes at once.

Transcriptomics, also referred to as expression profiling, examines theexpression level of mRNAs in a given cell population at a given time, oftenusing high-throughput techniques .

Common methods- microarray, RNA-seq. The proteome is the entire set of proteins expressed by a genome, cell, tissue

or organism at a certain time.

The interactome- 1. Protein-protein interaction- studied by co-immunoprecipitation 2. Protein-DNA interaction- studied by chromatin immunoprecipitation

(CHIP)

CHIP-seq - It identifies all possible binding sites for one transcription factorin entire genome

In situ hybridization- Hybridization to tissue sections or whole organisms todetect cells making mRNA of gene of interest

Northern blots on tissue using RNA probe complementary to mRNA(antimRNA)

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You start with an unique phenotype. Ask following questions Q.1) How many genes control this phenotype? Q.2) What are those genes? Q.3) If more than one, do these genes interact with each other?

Forward genetics- Going from phenotype to gene

This approach is also known as Classical genetics (e.g. Mendelian genetics)

One approach- Mutagenesis (e.g.- radiation) followed by forward geneticscreen

Genetic Selection- Individuals lacking a phenotype are killed (e.g.- antibioticresistance). This is easier to do on large scale.

Genetic screen Analyzes all individuals and then identify unexpectedphenotypes.

Screening is more time-consuming and costly but provides more information.

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Now you are going from gene to phenotype.

Common approaches- Targeted mutagenesis, fusion gene/protein The new version of gene is called Transgene (Transformed Gene) Approaches to introduce a transgene into an organism vary. Another approach- specific gene knockout or RNAi knockdown Phenocopy- It (phenotypically) resembles a mutant but is produced

by non-genetic mechanisms (e.g.- environmental influences)

Genetic organism Technique

1. Arabidopsis (plant) T-DNA from Agarobacterium (Non-mobile,random insertion, stable)

2 Drosophila (fruit-fly) P-element mediated gene transfer in embryos

(Transposable element)

3. C. elegans (roundworm) Gonadal injection, vector commonly remainextra-chromosomal, rarely integrated into hostgenome

4. Mouse (mammal) Using embryonic stem cells for transformationand injecting them into normal embryo

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Targeted knockout (insertion) means at specific location (NOT random location) Two selection markers are used- 1. One within (inside) flanking regions- Neomycin resistance (NeoR) gene 2. Onejustoutside flanking region Thymidine Kinase (TK) gene (Note- Before transfection, cells dont have NeoR or TK gene) Targeted insertion ALWAYS takes place by homologous recombination The CORRECT targeted knockout will always have ONLY NeoR gene BUT NOT TK gene

Random insertion (non-homologous methods) will always have BOTH NeoR

& TK genes

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Why use two selection makers?

1. Neomycin analog (G418) Selects for cells with NeoR gene

If used alone, it will select bothtargeted and random insertions

2. Ganciclovir- It selects cellswithout TK geneIf used alone, it will select

targeted insertion as well as NO

insertion

3. When you use both (1) and (2), you

are selecting ONLY for cells withtargeted insertion (NeoR present and

TK absent)

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Transient transfection

Advantages- 1. Temporary andquick (48-72 hours)

2. No selection needed Disadvantages- 1. Gene is not

integrated into the genome

2. Only some cells take up thevector (plasmid)

Stable transfection Advantages- 1. Permanent

(gene is integrated intochromosome)

2. Large quantities of proteinproduced

Disadvantages- 1. Slow (4-6weeks),

2. Selection (G418) needed

Transcriptional fusion

It is to study transcriptionalregulation of a specific gene

1. Cut a regulatory sequence incl.promoter from a gene of interest.

2. Paste the regulatory sequencefrom (1) in front (upstream) of a

promoterless reporter gene (e.g.Lac Z or GFP gene)

This is promoter/reporter fusion Translational fusion It is to study protein production/

localization etc. It can be done by attaching a tag

to a protein-coding gene (GFP)

Tag gets transcribed & translatedalong with the gene (N-terminalor C-terminal)

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Why do Geneticists like to study drosophila for developmental genetics? The adult fruit-fly body can be divided into 3 regions viz. head, thorax,

abdomen (common for many higher animals incl. humans)

Drosophila has segmented development (14 segments)- Head- Segments 1,2 Thorax- Segments 3, 4, 5 Abdomen- Remaining 9 segments Each gene controls development of specific segment(s) Trivia- The drosophila larva has a noncellular exoskeleton made up of

polysaccharide which makes detection of mutant phenotypes easier.

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Genes with products provided byfemale to the egg.

In drosophila, developing egg cellsare surrounded by maternal cellscalled nurse cells.

Maternal effect genes are first

expressed in nurse cells. ThemRNA and/or protein product istransported to egg cells throughconnecting tubular structures.

Maternal effect genes control post-fertilization development of egg.

The Phenotypes of offspring forthese genes depend only onmaternal genotype

Therefore, they affect very earlystages of zygote formation.

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Gap genes- They control formation of contiguous block of segments. Mutation ingap genes leads to large gaps in segmentation.

Pair-rule genes- The mutants are missing alternate segments (e.g.- odd skipped oreven skipped)

Segment Polarity gene- They affect patterning of each segment. Mutants displaysegment polarity (i.e. part of each segment missing) and number.

Remember gap, pair-rule & segment polarity genes control formation of segmentsin drosophila embryo.

Hox genes (*on next slide*) then control fate (identity) of each segment i.e. alreadyformed.

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HOX (Homeotic) genes- They were first discovered in drosophila but are alsopresent in other organisms (e.g.- Mouse, humans etc)

Properties of HOX genes- They are present in gene clusters. For e.g.- In drosophila, there are 8 HOX genes

divided into 2 complexes (Bithorax- 3 genes & Antennapedia- 5 genes) on

chromosome 3. They share DNA sequence homology (partial); the region is called

Homeobox. In line with DNA similarity, HOX proteins also have commonmotif.

HOX genes are expressed in spatially restricted domains. In other words, eachHOX gene is only expressed in specific segments as detected by in-situhybridization.

Single point mutation in HOX genes & can result in loss of function or gain offunction (Homeotic mutations)

Homeotic mutation- The mutant phenotype looks normal but is present at thewrong location. For example, one type of homeotic mutation in the fruit fly,causes legs to develop where antennae normally form

Expression of HOX genes in controlled by combinatorial mechanism

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Molecular biology technique developed in 1980s to study protein-protein interaction What are the major steps? 1. Choose a reporter gene and its unique transcription factor. One example of yeast reporter gene is His gene. His gene is required for synthesis of

essential amino acid histidine

Cells will survive in media lacking histidine only if they make His mRNA (& protein) Any transcription factor (TF) has two distinct domains viz. DNA binding domain and

Activation domain 2. Construct two plasmids by Plasmid-1 By attaching DNA binding domain to bait (ONE protein of interest) Plasmid-2 By attaching Activation domain to predicted interacting partners with bait

(MANY fish)

3. Transform yeast cells with both plasmids; followed by selection of cells which took bothplasmids

4. ONLY if two proteins interact, they will activate transcription ofHis (reporter)mRNA Drawback- Proteins (bait or fish) on their own should not activate reporter transcription Animation-

http://www.sumanasinc.com/webcontent/animations/content/yeasttwohybrid.html

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dsRNA (exogenous or endogenous) is processed by DICER like enzymes toproduce small double stranded RNA (avg. 21 bp; precursor of siRNA)

Pre-miRNA (always endogenous) is also processed by DICER like enzymesto produce precursor of miRNA

RNAi-induced silencing complex (RISC) produces final single strandedRNA (antisense)

Finally, single stranded RNA (antisense) binds to its complimentarymessenger RNA (sense)

This binding might be perfect complimentarity (for siRNA) or imperfect(endogenous miRNA)

The downstream effects of are diverse 1. mRNA cleavage by cellular enzymes (typical for siRNA) 2. Inhibition of translational elongation and/or polyadenylation of mRNA

(typical for miRNA)

3. Recruiting chromatin modifying enzymes (e.g. centromere silencing) RNAi animation- http://www.youtube.com/watch?v=cK-OGB1_ELE

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Thank You Its been a great time teaching both Biol-200 and Biol-202 Good Luck for Biol-202 finals Good luck for rest of the courses and I wish all of you

bright academic future.