Question: The image below s.......... Edit
Answer: Two methods commonly used are to either insert large genomic DNA fragments into cosmid vectors, which are then packaged into Phage lambda heads and propagated in Escherichia coli,1 or into yeast artificial chromosome (YAC) vectors propagated in Saccharomyces cerevisiae.2 The use of cosmids results in high transformation efficiencies, but insert size is limited by the size of the Phage lambda head to 45-48 kilibase pairs (kbp). The advantage of using YAC vectors is their ability to accommodate DNA as large as 200-800 kbp. Disadvantages are the low transformation efficiency, which decreases with insert size; the need to process transformants individually prior to screening; and the difficulty in obtaining large amounts of recombinant DNA from transformed cells.
To overcome some of the problems associated with using cosmid or YAC systems, a novel method for cloning and packaging DNA fragments using a Bacteriophage P1 system has been developed3 that offers the ability to clone large genomic DNA fragments of between 70-95 kb in size with efficiencies approaching those of cosmids. In addition, the P1 DNA Packaging System uses host E. coli strains and in vitro packaging extracts obtained from strains that are deficient in restricition and recombination abilities. These prevent the degradation and recombination of methylated genomic DNA.
Principle of the Method
Using a strategy analogous to Phage lambda packaging, partially digested and size selected genomic DNA between 70 and 95 kb is ligated onto linearized plasmid vector arms. The SacBII vector used contains a Phage P1 pac-cleavage site and two Phage P1 loxP recombination sites in addition to replication origins and an antibiotic resistance gene (Fig. 1) The recombinant vector is cleaved at the pac-cleavage site in a "Pacase" extract and the resulting DNA is then inserted into an empty P1 Phage head using a second extract containing phage packaging proteins. The attachment of P1 phage tails to the heads results in the formation of infectious recombinant phage particles that are then used to infect a restriction minus Escherichia coli host strain containing an expressed cre gene.
After injection into the host strain, the recombinant DNA is circularized between the two Phage P1 loxP sites by Cre recombinase. DNA which does not circularize is degraded by host nucleases. The circular DNA molecule now replicates and is maintained stably at one copy per host cell by the P1 Plasmid replicon.
Prior to alkaline lysis plasmid isolation,5 the recombinant plasmid copy number is increased more than 25-fold by isopropyl B-Dthiogalactopyranoside (IPTG) induction of the lac promoter
controlled high-copy P1 lytic replicon. A schematic detailing the P1 DNA packaging strategy is shown in Figure 2.
Figure 2. The P1 DNA Packaging Strategy.
The SacBII vector is digested with restriction endonucleases Scal and BamHI and the ends dephosphorylated. This generates two vector "arms," one consisting of the "short" Scal to BamHI fragment and the other the "long" Scal to BamHI fragment. Genomic DNA is partially digested with BamHI or other compatible end restriction endonucleases and size-selected on a sucrose gradient. Fragments between 70 kb and 95 kb in length are isolated and ligated to the vector arms, generating a series of linear molecules. If ligation occurs between two "short" arms, the resulting molecule will neither contain the origins of replication nor the kanR gene, and will be nonviable. If both arms are "long," there will be no pac site, and no packaging into the phage heads will occur. The only viable recombinant will be one consisting of the insert sequence flanked by both a short and long arm. Phage P1 uses a headful packaging strategy and can accommodate a total DNA length of approximately 110-115 kb. Any inserts longer than 95-100 kb will result in truncation of the packaged DNA before the distal loxP site is inserted, and the molecule will be unable to circularize upon injection into the host. Once injected into the cre+ host cell, the cre protein circularizes the injected DNA at the loxP sites, and DNA now replicates using the plasmid origin of replication. Propagation of cells on sucrose containing media only permits growth of colonies with genomic DNA inserts (positive selection). Plasmid copy number is increased by induction with IPTG. The recombinant DNAs are then isolated as plasmids using traditional methods.
Phage P1 uses a headful packaging strategy. Once the phage head is filled with DNA (about 110-115 kb), a "headful cut" occurs, cleaving any remaining DNA away from the head before it is packaged. This packaging mode suggests that if the insert DNA is too large (>95 kb), it will be packaged but not recovered in bacteria because the "headful" packaging process will terminate before the distal loxP is incorporated into the phage head. It also suggests that DNA too small to generate a headful when inserted into the vector should not be packaged into phage particles.
However, it has been observed that DNA less than a P1 "headful" is packaged in vitro in viable phage particles with an efficiency of about 10-15 percent that of "headful" DNA (Pierce & Sternberg, manuscript in preparation). While it is not completely clear why this should occur, it points out the need to size-select the insert DNA on sucrose gradient before attempting to ligate it to vector in order to maximize the recovery of large inserts. In particular, size fractions that maximize DNA fragments in the 70-95 kb range, and minimize the presence of shorter fragments, should be used. The presence of small fragments is undesirable also because it increases the possibility that they will be ligated together and then recovered in one clone. This would significantly complicate subsequent screening and gene localization processes.
Finally, as the second stage packaging extract contains about 5-10 percent small heads (headful size 47 kb), small DNA fragments can be recovered by a headful packaging process in phage particles containing these heads.
An 11 kb "stuffer" region of Adenovirus type 2 DNA has been engineered into the pAd10-SacBII cloning vector. It is designed to provide a segment of DNA in which the "headful" cut can be made. Using the P1 DNA Packaging System, genomic DNA from 70-95 kb can be readily cloned and manipulated. The major advantages of the P1 DNA packaging method over other genomic cloning methods are: 1) the ability to clone inserts two to three times the size of those used with cosmids and lambda vectors, 2) no rearrangement or deletion of methylated DNA occurs because of the use of restriction-minus host strains, and 3) recombinant DNA is easily recovered as plasmids for further screening and minipulations.
C.1 Regulation of transcription controls when transcription occurs and how much RNA is created. Transcription of a gene by RNA polymerase can be regulated by at least six mechanisms:
• Specificity factors alter the specificity of RNA polymerase for a given promoter or set of promoters, making it more or less likely to bind to them (i.e. sigma factors used in prokaryotic transcription).
• Repressors bind to non-coding sequences on the DNA strand that are close to or overlapping the promoter region, impeding RNA polymerase's progress along the strand, thus impeding the expression of the gene.
• General transcription factors These transcription factors position RNA polymerase at the start of a protein-coding sequence and then release the polymerase to transcribe the mRNA.
• Activators enhance the interaction between RNA polymerase and a particular promoter, encouraging the expression of the gene. Activators do this by increasing the attraction of RNA polymerase for the promoter, through interactions with subunits of the RNA polymerase or indirectly by changing the structure of the DNA.
• Enhancers are sites on the DNA helix that are bound to by activators in order to loop the DNA bringing a specific promoter to the initiation complex.
• Silencers are regions of DNA that are bound by transcription factors in order to silence gene expression. The mechanism is very similar to that of enhancers.
In eukaryotes, transcriptional regulation tends to involve combinatorial interactions between several transcription factors, which allow for a sophisticated response to multiple conditions in the environment. This permits spatial and temporal differences in gene expression. Eukaryotes also make use of enhancers, distant regions of DNA that can loop back to the promoter. A major difference between eukaryotes and prokaryotes is the fact the eukaryotes have a nuclear envelope, which prevents simultaneous transcription and translation. RNA interference also regulates gene expression in most eukaryotes, both by epigenetic modification of promoters and by breaking down mRNA.
Regulation of transcription machinery
In order for a gene to be expressed, several things must happen. First, there needs to be an initiating signal. This is achieved through the binding of some ligand to a receptor. Activation of g-protein-coupled receptors can have this effect; as can the binding of hormones to intra- or extracellular receptors.
This signal gives rise to the activation of a protein called a transcription factor, and recruits other members of the "transcription machine". Transcription factors generally simultaneously bind DNA as well as an RNA polymerase, as well as other agents necessary for the transcription process (HATs, scaffolding proteins, etc.). Transcription factors, and their cofactors, can be regulated through reversible structural alterations such as phosphorylation or inactivated through such mechanisms as proteolysis.
Transcription is initiated at the promoter site, as an increase in the amount of an active transcription factor binds a target DNA sequence. Other proteins, known as "scaffolding proteins" bind other cofactors and hold them in place. DNA sequences far from the point of initiation, known as enhancers, can aid in the assembly of this "transcription machinery." Histone arms are acetylated, and DNA is transcribed into RNA.
Frequently, extracellular signals induce the expression of immediate early genes (IEGs) such as c-fos, c-jun, or AP-1. These are in and of themselves transcription factors or components thereof, and can further influence gene expression.
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