Plasmids Small circular autonomously replicating extrachromosomal DNA Modified plasmids, called cloning vectors Are used by molecular biologists to isolate Large quantities of a given DNA fragment Plasmids used for cloning share three properties Unique restriction site Antibiotic resistance Origin of replication Bacterial genome (5000kb) Plasmid DNA (3kb)

Plasmid elements Origin of replication: This is a DNA element that allows the plasmid to be replicated and duplicated in bacteria. Each time the bacterium divides, the plasmid also needs to divide and go with the daughter cells. If a plasmid cannot replicate in bacteria, then it will be lost.

Plasmid elements Antibiotic resistance: This allows for the presence of the plasmid to be selectively maintained in a given strain of bacteria Lab bacterial strains are sensitive to antibiotics. When grown on plates with antibiotics, they die. The presence of a plasmid with the antibiotics resistance gene allows these lab strains to grow on plates with the antibiotic. You are therefore selecting for bacterial colonies with the Plasmid

Plasmid elements Unique restriction sites: For cloning the plasmid needs too be linearized. Most cloning vectors have unique restriction sites. If the plasmid contains more than one site for a given restriction enzyme, this results in fragmentation of the plasmid Why does this matter?

pUC18 pUC18 is a commonly used plasmid: pUC= plasmid University of California Plasmid replicon copy No pBR322 pMB1 15 pUC18 pMB1 500 pACYC p15A 10 pSC101 pSC101 5

Why are plasmids important? Most genes are present as two copies in the entire genome. Plasmids allow us to obtain 1000’s of copies of a gene in a pure form

How is a specific gene isolated (CLONED)? Its like going to the library and looking for a specific book. It involves screening through a genomic library. A genomic library is a large collection of plasmids containing pieces of DNA from a specific species. The set of cloned fragments is so comprehensive that virtually the entire genome is represented in the library. The fragments that make up the library are initially generated by digesting genomic DNA (e.g. human) with a restriction enzyme- say EcoRI The EcoRI sites are randomly distributed in the genome- fragments of varying lengths will be generated. Some fragments will contain one gene, others two genes or cut genes in half. Gene1 Gene2 Gene3 A B C D E F

The library is random! Each fragment is cloned into the plasmid, each plasmid is put (transformed) into E.coli C D A B Gene1 Gene2 Gene3 A B C D E F

Fragments,bookmark title The library is not bookmarked or even titled and is in fragments! There is no organization to the library. It is simply a populations of cloned fragments representing the entire genome. The equivalent of this would be if you went to the University Library to find all the books in a large heap, the books had no title, and in addition instead of entire books you often found parts of books. How do you use such a library? How do you find the book you are interested in. Lets work our way through this problem with a simple example Organism has EIGHT genes in its genome A B C D E F G H EcoRI

Genomic library If we wanted to study gene C- Create a restriction map of gene C Determine it sequence Study proteinC What do we need to do We need to initially clone the gene and make many copies of gene C Creating a genomic library provides a means of obtaining many copies of gene C To generate a genomic library: Total genomic DNA is isolated from the species of interest The DNA is cut with EcoRI A B C D E F G H A b d E F G h h b C d

Genomic library These genomic DNA fragments are mixed with a plasmid that has been linearized at a single EcoRI site (say pUC18) A b d E F G h h b C d Ori Ampr Ori Ampr Ori Ampr Ori Ampr Both the plasmid and genomic DNA have been cut with EcoRI, they have complementary sticky ends | G A A T T C C T T A A G

Recombinant plasmid This process where foreign DNA is joined to plasmid DNA is called ligation It results in recombinant plasmid (foreign DNA+plasmid) Each plasmid has one foreign EcoRI fragment Each foreign fragment is still present as only one copy! This is not useful.

Transformation The entire collection of these plasmids bearing genomic DNA inserts is called a Genomic Library! These plasmids are added back into bacteria by a process called transformation Ori Ampr Gene The bacteria are selected for the presence of the Plasmid by growth on media containing antibiotics Petri dish d E F G h b C A Each colony of E. coli will harbor one plasmid with one piece of genomic DNA We at this point don’t know which colony has which piece of foreign DNA

How are genomic libraries used? If we are interested in studying gene C, you need the plasmid containing gene C Having a genomic library means you have gene C, but where is it? Which colony on the Petri dish contains gene C? Genomic libraries are much more complex than the one described for our hypothetical 8 gene organism You need to identify one recombinant plasmid out of 100,000’s present in a library. Identifying and isolating a specific plasmid is called screening a library. This requires a probe A probe is a sequence complementary to part of the sequence one wishes to pull out You radiolabel the probe and once labeled the probe is used to identify the plasmid containing E. coli colony How do we get the probe?

PROBES Probes are obtained in a number of ways RNA as a source The probe for hemoglobin can be obtained from mRNA of immature red blood cells. The major transcript of these cells is from the hemoglobin gene. So isolating RNA from these cells, we can obtain a relatively pure probe for the hemoglobin gene Protein If you have a purified protein, the amino acid sequence can be determined. From the amino acid sequence, using the genetic code a corresponding DNA sequence can be synthesized and this small DNA piece can be used as a probe Homology Probes from conserved genes-Many genes are conserved from one species to another Chimpanzee and human DNA are 97% identical. If you know the sequence of a gene in chimps, then you will be able to know the sequence for the gene in humans! The histone genes are highly conserved across phyla. Histone proteins have three Amino acid differences between humans and peas Histone genes have been isolated in yeast, they can serve as probes for screening a Human genomic library- cloning by phone The computer databases

How is the probe used to screen a genomic library? The E.coli containing the genomic inserts are plated onto a series of petridishes. The media on the petridishes contain ampicillin so only E.coli with the plasmid containing the ampicillin resistance gene will grow and form colonies Petri dish Colonies are imprinted onto A paper filter Filter The bacteria on the filter are lysed The DNA in the bacteria (plasmid and chromosomal) is denatured (converted from ds to ss) Then you take the filter and to it add radiolabeled probe (small part of Gene C). The probe will bind the single stranded plasmid that has a complementary sequence. The bacterial colony containing the plasmid with gene C will bind the probe.

Autoradiography d E F G h Filter autoradiogram b C d Petri dish A b h Now you have the complete GeneC!!!

Isolate the plasmid To isolate the gene C fragment, we grow up a large population of E. coli containing the plasmid with the gene C insert. A simple procedure allows us to isolate the plasmid (which is smaller than Chromosomal DNA) Once we have purified the plasmid we have 1000’s of copies of Gene C in a plasmid We can take the plasmid and cut it with EcoRI. When the digest is run on an agarose gel, we get two bands- one corresponding to the plasmid and one to the insert. The DNA present in the band corresponding to the insert can be isolated from the gel PURE GENE C!!!!! Marker Uncut EcoRI Gene C plasmid

The genomic library and a specific probe enabled us to achieve two goals Out of the billions of base pairs in a large genome, we have been able to identify a few 1000 base pairs that correspond to a specific gene of interest. In addition we were able to isolate this sequence on a specifically engineered plasmid That allows us to make large quantities of this rare sequence. Genomic libraries are described in terms of average fragment size and the number of plasmids that must be screened to have the entire genome represented To have a good probability (>99%) of identifying a given DNA sequence (gene) present in the collection of plasmids (library). The number of plasmids (colonies) that must be screened is a function of the size of the genome of the species from which the Library was constructed.

Genomic libraries Species Genome size average #plasmids insert size screened E. Coli 5000kb 16 kb 1300 Drosophila 150,000kb 16 kb 46,000 Human 3000,000kb 16 kb >100,000 The larger the genome, the more difficult the task of isolating a plasmid with a given gene from a library At present, genomic libraries exist for a large number of organisms including Yeast, C.elegans, Drosophila, Zebrafish, Xenopus, Chickens, Mouse, Humans etc

cDNA Often we have RNA rather than DNA as the starting material For instance in the case of the human hemoglobin gene, we started with globin mRNA RNA is difficult to work with. In contrast to DNA, RNA breaks down and degrades very easily. There are no restriction enzymes that cut RNA at specific sites. RNA cannot be cloned. It cannot be inserted into a plasmid and amplified since all Plasmids are DNA. The enzyme reverse transcriptase has proven very useful to molecular biologists. This enzyme catalyzes the synthesis of DNA from a RNA template. It is normally found in a large class of viruses. The genome of these viruses is RNA!! These viruses are called retroviruses.They infect eukaryotic cells and use these cells to grow/replicate Retroviruses carry an RNA genome. Interestingly they will integrate into the DNA of the host. For RNA to integrate into DNA, first the RNA has to be converted to DNA Remember the central dogma of molecular biology Information flows from DNA to RNA to protein! DNA---->RNA---->protein Reverse Transcriptase reverses this dogma (partially)

cDNA synthesis Protein coat RNA genome Reverse transcriptase

cDNA/splicing So from globin mRNA, a complementary DNA molecule can be created using reverse Transcriptase. This complementary DNA is called cDNA. This DNA can now be inserted into a plasmid and cloned. What is the relationship between a cDNA clone and a genomic clone? Splicing In eukaryotes, the coding sequences are interrupted by introns Gene 7700 nt 1 2 3 4 5 6 7 Ovalbumin Primary transcript Splicing mRNA 1872 nt Genomic clones represent the organization of the DNA in the nucleus!

Genomic clones represent the organization of the DNA in the nucleus! cDNA represents the organization of mRNA sequences after the gene has been transcribed, processed and exported to the cytoplasm. cDNA clones contain the sequence of nucleotides that code for the protein! cDNA clones do not contain the sequence of the promoter of the gene or the intron. cDNA libraries can be made. They are analogous to genomic libraries discussed earlier The starting material is different. Organism, tissue or cell type. First mRNA is isolated, then this is converted to to dsDNA using reverse transcriptase These DNA fragments are inserted into plasmids, transformed into E. coli to produce a cDNA library.

Properties of genomic and cDNA libraries Genomic library cDNA library Source Nucleii cytoplasmic RNA (any cell) (specific cell type) Frequency Depends on copy Depends on abundance of a gene number, gene & stability of RNA in library size, genome size Use Studies on gene Studies directed organization & towards coding regions structure

Southern blotting Rapid method of identifying specific DNA fragments in a large mixture of fragments plasmid EcoRI Insert GeneC Marker Uncut EcoRI How do you determine which band corresponds to insert and which to the plasmid

Southern blotting allows us to answer this question This method is very similar to the method we used to screen a genomic library TTTTTTT AAAAAAA The probe AAAAAAA specifically hybridizes with the insert (geneC) A probe with this specific sequence is generated and made radioactive The plasmid is cut with restriction enzyme and run on an agarose gel The DNA is transferred to a membrane Incubate the filter with the radio-labeled probe Marker Uncut EcoRI

What about a genome? What if Gene C was in a large genome. Could we identify the fragment by this method? EcoRI GeneC Marker Marker Marker Transfer to membrane Hybridize with Probe

GeneC E Mapping deletions.

You can build a genomic restriction map using this method. If we digest the DNA with HindIII instead of EcoRI what will happen? E E E E GeneC H H H H Marker Marker

Rapid low resolution analysis of hemoglobin gene MstII 0.2kb 1.1kb Exon1 Exon2 0.3kb deletion Marker WT Del 1.1 0.2 1

Northern blot This is a rapid method that allows you to determine the cell type in which a specific gene is active (MAKING RNA) and being transcribed. Brain Bone Blood Embryo Lung liver These tissues differ because each is transcribing a unique Subset of genes. Each tissue contains a unique and distinct mRNA population Presence of RNA is a reflection of gene activity

Microarrays These are glorified northern blots These are a recent development Allows us to examine gene expression of all of the genes in the genome! They are a reverse northern blot Each spot is DNA for one defined gene Each gene DNA is spotted in a grid So extensive that they cover the entire genome. Make total RNA from normal and mutant cell Label each total RNA differently Add labeled RNA from normal and mutant to array and let hybridize Measure label and determine change WT Mut 1 2 Ratio of WT/mut 3 4 5