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Question 8.6. According to the Holliday model for genetic recombination, what factor determines the length of the heteroduplex in the recombination intermediate?
Question 8.7. Holliday junctions can be resolved in two different ways. What are the consequences of the strand choice used in resolution?
Question 8.8. Why do models for recombination include the generation of heteroduplexes in the products?
Question 8.9. Consider two DNA duplexes that undergo recombination by the double-strand break mechanism. The parental duplex indicated by thin lines has dominant alleles for genes M, N, O, P, and Q, and the parental duplex shown in thick lines has recessive alleles, indicated by the lower case letters. The recombination intermediate with two Holliday structures is also shown.
- a) What duplexes result from resolution of the left Holliday junction vertically and the right junction horizontally?
- b) After the vertical-horizontal resolution, what will the genotype be of the recombination products with respect to the flanking markers M and Q? In answering, use a slash to separate the designation for the 2 chromosomes, each of which is indicated by a line (i.e. the parental arrangement is M___Q / m___q).
- c) If the products of the vertical-horizontal resolution were separated by meiosis, and then replicated by mitosis to generate 8 spores in an ordered array (as in the Ascomycetefungi), what would be the phenotype of the spores with respect to alleles of gene O? Assume that the sister chromatids of these chromosomes did not undergo recombination in this region (i.e. one parental duplex from each homologous chromosome remains from the 4n stage).
For the next 3 problems, consider two DNA duplexes that undergo recombination by the double-strand break mechanism. The parental duplex denoted by thin black lines has dominant alleles (capital letters) for genes (or loci) K, L, and M, and the parental duplex denoted by thick gray lines has recessive alleles, indicated by k, l, m. The genes are shown as boxes with gray outlines. In the diagram on the right, the double strand break has been made in the L gene in the black duplex and expanded by the action of exonucleases.
Question 8.10. When recombination proceeds by the double-strand break mechanism, what is the structure of the intermediate with Holliday junctions, prior to branch migration? Please draw the structure, and distinguish between the DNA chains from the parental duplexes.
Question 8.11. If the recombination intermediates are resolved to generate a chromosome with the dominant K allele of the K gene and the recessive m allele of the M gene on the same chromosome (K___m), which allele (dominant L or recessive l) will be be at the L, or middle, gene?
Question 8.12. If the left Holliday junction slid leftward by branch migration all the way through the K gene (K allele on the black duplex, k allele on the gray duplex), what will the structure of the product be, prior to resolution?
Question 8.13. According to the original Holliday model and the double-strand break model for recombination, what are the predicted outcomes of recombination between a linear duplex chromosome and a (formerly) circular duplex carrying a gap in the region of homology? The homology is denoted by the boxes labeled ABC on the linear duplex and ac on the gapped circle. The regions flanking the homology (P and Q versus X and Y) are not homologous.
The results of an experiment like this are reported in Orr-Weaver, T. L., Szostak, J. W. and Rothstein, R. J. (1981) Yeast transformation: a model system for the study of recombination. Proc. Natl. Acad. Sci. USA 78: 6354-6358. These data were instrumental in formulating the double-strand-break model for recombination.
Question 8.14.A variety of in vitro assays have been developed for strand exchange catalyzed by RecA. For each of the substrates shown below, what are the expected products when incubated with RecA and ATP (and SSB to facilitate removal of secondary structures from single-stranded DNA)? In practice, the reactions proceed in stages and one can see intermediates, but answer in terms of the final products after the reaction has gone to completion.
In each case, the molecule with at least partical single stranded region is shown with thick blue strands, and the duplex that will be invaded is shown with thin red lines. The DNA substrates are as follows.
- A. Single-stranded circle and duplex linear. The two substrates are the same length and are homologous throughout.
- B. Single-stranded short linear fragments and duplex circle. The short fragments are homologous to the circle.
- C. Single-stranded linear and duplex linear. The two substrates are the same length and are homologous throughout.
- D. Gapped circle and duplex linear. The intact strand of the circle is the same length as the linear and is homologous throughout. The gapped strand of the circle is complementary to the intact strand, of course, but is just shorter.
DNA cloning by homologous recombination in Escherichia coli
The cloning of foreign DNA in Escherichia coli episomes is a cornerstone of molecular biology. The pioneering work in the early 1970s, using DNA ligases to paste DNA into episomal vectors, is still the most widely used approach. Here we describe a different principle, using ET recombination 1,2 , for directed cloning and subcloning, which offers a variety of advantages. Most prominently, a chosen DNA region can be cloned from a complex mixture without prior isolation. Hence cloning by ET recombination resembles PCR in that both involve the amplification of a DNA region between two chosen points. We apply the strategy to subclone chosen DNA regions from several target molecules resident in E. coli hosts, and to clone chosen DNA regions from genomic DNA preparations. Here we analyze basic aspects of the approach and present several examples that illustrate its simplicity, flexibility, and remarkable efficiency.
Molecular Genetics is a broad survey course in which students must acquire a firm conceptual understanding of the molecular mechanisms that govern basic genetic principles. This book provides the necessary foundation for most of the upper division courses in biology, and it is on par with similar molecular genetic courses at major research universities in the United States.
This manual serves as a companion to the molecular genetics textbooks and was adapted from lectures presented from the Lewin-inspired Genes textbooks. It is not intended to cover all of the material presented in those lectures, but instead offers undergraduate students an opportunity to focus on topics that are more challenging in small, active learning environments. Often these problem-based concepts cannot be mastered until the student invests the time to work through them in their entirety. The exercises presented in Molecular Genetics are designed to satisfy this important learning objective of the course.
Students are reminded that answers to the questions at the end of each discussion can sometimes be found within the written explanations in the manual. Other times, it will be necessary to refer to class notes, the textbook, and/or other resources to answer these questions correctly.
Genes Are Composed of DNA
Genes Can Be Identified By Analyzing Mutant Phenotypes
Whole Genome Analyses
Genome Evolution and Genome Packaging
DNA Recombination and DNA Repair
Mobile Genetic Elements
Prokaryotic Gene Regulatory Circuits
Regulation of Gene Expression in Eukaryotes
Stanley Cohen and Herbert Boyer's historic experiment used techniques to cut and paste DNA to create the first custom-made organism containing recombined or "recombinant" DNA. Cohen and Boyer inserted the recombinant DNA molecule they created into E. coli bacteria by means of a plasmid, thereby inducing the uptake and expression of a foreign DNA sequence known as "transformation."
This animation is also available as VIDEO .
Stanley Cohen and Herbert Boyer's historic experiment used techniques to cut and paste DNA to create the first custom-made organism containing recombined or "recombinant" DNA. Cohen and Boyer inserted the recombinant DNA molecule they created into E. coli bacteria by means of a plasmid, thereby inducing the uptake and expression of a foreign DNA sequence known as "transformation."
e coli bacteria,dna transformation,dna molecule,herbert boyer,stanley cohen,recombinant dna,dna sequence,e coli,plasmid,expression
Prior to the discovery of EcoR1, the recombination of genetic material was, as Paul Berg has conceded, “cumbersome, technically challenging, and perhaps not replicable.” EcoR1 changed all of that.
EcoR1 is a restriction endonuclease, an enzyme that cuts DNA molecules into pieces. Scientists believe that restriction enzymes evolved in bacteria to defend against infiltrating viral genomes. The first molecular specimens were identified in the late 1960s. Biochemists undertook a broad search to find others. No one knew how many awaited discovery. EcoR1 turned up in San Francisco in 1971, and it was a boon to DNA recombination projects.
As a type II restrictor, EcoR1 reliably severs DNA at a specific recognition site, a definite DNA sequence, and always makes the same precise cut within it. As Boyer learned on a Saturday morning in the spring of 1972, EcoR1’s recognition sequence is:
The enzyme cleaves between the G and A nucleotides on both strands to produce a signature staggered cut:
Both pieces of the severed molecule are left with an overhang of four nucleotides at one end. The sequences are palindromic complements: AATT and TTAA. Mertz and Davis called the ends ‘sticky’ because, in contrast to restricted DNA molecules with blunt ends, the overhangs on the EcoR1-restricted fragments will readily combine with complementary sequences. And because the enzyme makes identical cuts in any piece of DNA from any organism, fragments produced by EcoR1 restriction are naturally complimentary, and can be joined without further biochemical treatment.
EcoR1’s ‘sticky ends’ permitted researchers to cut and paste DNA with far greater ease, flexibility, and precision than prior methods and materials. It served as an important enabling tool in the development of recombinant DNA technology.
Stan Cohen heard about the enzyme from a colleague in San Diego, molecular biologist Don Helinski. Cohen and Helinski were organizing an international conference on plasmids to be held in November, in Hawaii. Helinski suggested that Cohen extend an invitation to Boyer to talk about the significance of EcoR1 in plasmid genetics.
Stanford and UCSF authors simultaneously published four papers on EcoR1 in the November 1972 issue of the Proceedings of the National Academy of Sciences (PNAS): Morrow and Berg, Mertz and Davis, Sgaramella, and Hedgpeth, Goodman, and Boyer.
Cloning Vectors used in Recombinant DNA Technology: 3 Cloning Vectors
The plasmid based vectors used for cloning DNA molecules generally carry up to 10 kb of inserted DNA. However, for the formation of library, it is often helpful to be able to maintain larger pieces of DNA. For this reason, E. coli virus (Bacteriophage, phage) lambda (λ) has been developed as a cloning vehicle. In its life cycle, bacteriophage λ infects E. coli and after injection of the viral DNA, two possibilities exist.
Bacteriophage λ can enter a lytic cycle, which after 20 minutes lead to the lysis of host cells and the release of about 100 phage particles. Alternatively, the injected bacteriophage λ DNA can be integrated into the E. coli chromosome (DNA) as a prophage and can be maintained more or less indefinitely (Lysogeny stage).
However, under conditions of nutritional or environmental stress, the integrated bacteriophage λ DNA can be excised and enter a lytic cycle. The bacteriophage λ DNA is about 50 kb in length, of which approximately 20 kb is essential for the integration excision (I/E) events. For forming genomic libraries, 20 kb of DNA can be replaced with 20kb of cloned DNA.
Cloning Vectors based on the Bacteriophage Lambda (λ):
Derivatives of the genome of bacterioplage lambda have been constructed to serve as cloning vectors. Transfection or transduction is used to introduce such vectors into E. coli.
Two properties of the lambda genome make it suitable for use as a vector:
1. Only about 50% of the 50 genes of lambda are essential for its replication and for lysis of the host cell. Most of these non essential genes are located together in a cluster around the middle of the genome.
2. Lambda genome is packaged inside the phage head by what is known as the ‘head-full mechanism’. This means that not only there is an upper limit of the amount of DNA that is packaged inside the phage head, but there is a lower limit also. Effective packaging takes place only when a minimum amount of DNA is present, i.e., 35 kb (kilo = thousand base).
An infective bacteriophage λ consists of a tubular protein tail with a few tail fibres and a protein head. The production and assembly of heads and tails, and packaging of DNA are highly coordinated sequence of events. The DNA within head of a λ phage is a 50 kb linear molecule with a 12-base, single stranded extension at the 5′ end. These extensions are called cohesive (cos) ends, because they contain sequences that are complementary to each other.
In E. coli, these cos ends base pair to form a circular DNA. DNA replication from the circular DNA creates a linear form of λ DNA that is composed of several contiguous lengths of 50 kb units. Each new assembly is filled with 50 kb DNA (Fig. 14.3).
Inserting Type Lambda (λ) Vectors:
A lambda cloning vector can therefore be constructed by deletion (in vivo or in vitro by restriction deletion) of a part of the non-essential region such that the remainder is not less than 35kb. Other mutations are also introduced such that restriction sites in the enemies regions are eliminated. A segment of foreign DNA can be cloned in an unique restriction site in the non-essential region, the only condition being that the vector and the insert together would not to be more than 53 kb long. Such vectors are termed “insertion vectors”.
Some of the Charon vectors are examples of this type of vectors. Bacteriophage X cloning vector has two Bam HI sites that flank the I/E region. When this DNA is cut with Bam HI, three segments are produced. The middle segment I/E region, which is replaced by cloned DNA of 20 kb size. The source is cut with Bam HI, and DNA pieces that are 15 to 20 kb in length are isolated. The two DNA samples (phage and source) are combined and incubated with T4 DNA ligase.
Then empty bacteriophage heads and tail parts are added. Under these conditions 50 kb unit of DNA are packed in to the heads, and infective phages are produced. Other products from ligation reaction cannot be packed, because they are either too large (>52 kb) or too small (>38 kb). Recombinant bacteriophage λ can undergo lytic cycle only in an E. coli strain that does not allow reconstituted phage λ (non-recombinant) with intact I/E regions to grow.
Recombinant phage is maintained by lytic cycles in fresh E. coli cultures. Bacteriophage libraries can be screened by using either DNA probes or immunological assays. For this purpose, individual lytic zones are tested (compared to bacterial colonies in plasmid cloning vactors).
Substitution Type Lambda (λ) Vector:
The second type of lambda vector is of the substitution type, the example being the lambda gt vectors and the EMBL vectors. These vectors have two Eco R1 sites or two BamHI sites in the non-essential region.
On digestion with Eco Rl, at least three piece(s) are produced, two terminal ones containing the essential regions and the central piece(s) containing the non-essential genes.
The central piece (s) is separated out by sucrose density gradient sedimentation and replacing by the foreign segment to be cloned. The limits of the size of foreign DNA that can be cloned in the lambda gt vector is 1-14 kb and in the Charon 4 vector is 8.2-22.2 kb.
Such a replacement cloning vector has an advantage over the insertion vectors. The terminal pieces by themselves if joined by DNA ligase, do not make up 35 kb and hence cannot be packaged. Packaging occurs only when a segment of foreign DNA gets cloned between the two terminal pieces of the vector and hence no separate selection for recombination molecules to is necessary.
Type # 2. Phagemids as Cloning Vectors:
A phagemids is a hybrid of a plasmid and a filamentous coliphage that can be propagated in either form. The coliphage could be either of the three virtually identical phages, M13 fd or f1. These are male specific phages that contain single stranded circular DNA as their genome. Upon infection of E. coli by the bacteriophage, double stranded DNA is first formed as the replicative intermediate.
Finally single stranded DNA is packaged into the virion. Both the replication origins of the plasmid and the coliphage are incorporated in the phagemid. The auxiliary replication functions necessary in trans for the coliphage replication are not, however, incorporate in the phagemid. Hence, replication from the coliphage origin can take place only in the presence of a helper phage.
Otherwise, replication takes place from the plasmid replication origin. The pBLUESCRIPT phagemids have both Col E, (pMB 9 like) origin and the filamentous phage f1 origin. The cloning is done in any of the multiple cloning sites in the double stranded circular DNA of the plasmid form of the vector. This is introduced in to E.coli by transformation and the synthesis of single stranded DNA from the phage fl origin is induced by superinfection with a helper phage.
The single stranded DNA formed is packaged in to the phage rods because of the presence of the phage packaging signals as well in the phagemid. Direct base sequencing can be undertaken using the single stranded DNA isolated from the virions secreted from the E. coli cells.
Depending on the orientation of the f1 replication origin in the phagemid, either the (+) strand or the (-) strand of the phagemid is replicated in presence of the helper phage. The pEMBL phagemids are similar to the pBLUESCRIPT phagemids.
Phagemids that have replication origin of pUC plasmids and of the M13 bacteriophage have also been developed and are available under the trade name of LITMUS vectors. Replication in the circular double stranded plasmid form uses the pUC replication origin.
Cloning is done in the multiple cloning sites located in the lacZ gene and the usual blue/white selection is available. Here also replication of the single stranded phage DNA form is induced by super infecting with the M13 helper phage.
Type # 3. Cosmids as Cloning Vectors:
Plasmid vectors are not suitable for cloning DNA fragments very much larger than their own size, as the transformation frequency fall beyond acceptable limits and cloned fragments or their parts very often get deleted. Takagi and co-workers observed as early as 1976 that the presence of the cohesive end site cos λ from the bacteriophage lambda DNA in a plasmid allows it to be packaged in vivo into virus particles. The interesting finding was that the in vivo packaging mechanism would be select DNA molecules of the full size of the lambda genome (
Making use of this finding, cosmid vectors were first developed in 1978 by J. Collins and coworkers to facilitate cloning of larger DNA fragments in plasmids. Extracts of lambda lysogens have been successfully used for in vitro packaging of the lambda capsids. An example of a commonly used cosmid is pHV79 which is nothing but pBR322 containing the cohesive end site cos λ and which can accommodate up to 45 kb sized inserts.
A great advantage of such a cosmid vector is that:
(1) Gene libraries consisting of a smaller number of clone members can span the whole genome of an organism. For example, the genome of Escherichia coli can be accommodated in just 120 cosmids.
(2) Other advantages are that large gene can be studied intact and genetic linkage studies can be carried out at the molecular level.
(3) An important practical advantage of a cosmid is that background molecules which do not have the intact and genetics linkage studies can be carried out at the molecular level.
(4) An important practical advantage of a cosmid is that background molecules which do not have inserts or have smaller inserts are eliminated during packaging. This is not possible to achieve with plasmid cloning vectors.
(5) Besides, the frequency of transformation of the lambda capsids with an in vitro packaging extract is much higher than the transformation frequency of plasmids.
Cosmid cloning vectors can carry 40 kb of cloned DNA and can be maintained as plasmids in E. coli. Cosmids combine the properties of plasmids and bacteriophage λ vectors. The commonly used cosmid pLFR-5 (6kb size) has two cos sites (cos ends) from bacteriophage λ separated by a Sea I restriction endonuclease site, a multiple cloning sequence with six unique sites (Hind III, PstI, Sail, BamHI, SmaI, and EcoRI), an origin of DNA replication (ori) and a tetracycline resistance (Jet) gene. This cosmid carry about 40 kb of cloned DNA.
For this vector, pieces of DNA that are approximately 40 kb in length are purified by sucrose density gradient configuration from a partial digestion of source DNA with Bam HI. The pFLR-5 DNA is cleaved first with Seal and then with Bam HI. The two DNA samples are mixed and ligated. Some of the ligand products will have a 40kb DNA piece inserted between the two fragments that are derived from the digestion of the pLFR-5 DNA.
The molecules formed by joining will be about 50 kb long, with cos sequences that are about 50 kb apart. Therefore, these DNA constructs can be successfully packaged into bacteriophage λ heads in vitro (as described above, phage λ head accommodate only 50kb DNA). After formation of complete phage, the DNA is delivered by infection into E. coli.
During phage packaging, cos ends are cleaved. Once inside the bacteria, the cos ends base pair and form a circular DNA molecules (Fig. 14.4). This circular form is stable, so the cloned DNA can be maintained as a plasmid-insert DNA construct because the vector contains a complete set of plasmid functions. Moreover, the tetracycline resistance gene allows colonies that carry the cosmid to grow in presence of tetracycline non-transformed cells are sensitive to tetracycline and die.
The following are the steps for construction of a cosmid library:
(i) Cleavage of the genome by partial digestion with restriction endonuclease,
(ii) Sizing of the fragments by gel electrophoresis or velocity centrifugation
(iii) Cleavage of the cosmid vector and treatment with phosphate to minimize polycomid formation
(iv) Ligation of the genomic DNA and the cosmid DNA
(v) Packaging the ligated DNA into infectious phage particles
(vi) Transduction into E. coli.
Therefore, it is evident that different vectors have different capacity of carry foreign DNA (Table 14.2).
Recombinant DNA technology is used to produce hormones for women with fertility issues. Recombinant human follicle stimulating hormone (r-hFSH), recombinant luteinizing hormone (r-hLH) and recombinant human chorionic gonadotropin (r-hCG) are all hormones that facilitate the proper functioning of ovulation and follicular maturation necessary for fertilization to become a success. As opposed to earlier methods of hormone production, recombinant DNA technology will bring about a higher efficacy, easier access and safer, less invasive infertility treatments.
When it comes to DNA repair, it's not one tool fits all
Our cells are constantly dividing, and as they do, the DNA molecule -- our genetic code -- sometimes gets broken. DNA has twin strands, and a break in both is considered especially dangerous. This kind of double-strand break can lead to genome rearrangements that are hallmarks of cancer cells, said James Daley, PhD, of the Long School of Medicine at The University of Texas Health Science Center at San Antonio.
Dr. Daley is first author of research, published June 18 in the journal Nature Communications, that sheds light on a double-strand break repair process called homologous recombination. Joined by senior authors Patrick Sung, DPhil, and Sandeep Burma, PhD, and other collaborators, Dr. Daley found that among an array of mechanisms that initiate homologous recombination, each one is quite different. Homologous recombination is initiated by a process called DNA end resection where one of the two strands of DNA at a break is chewed back by resection enzymes.
"What's exciting about this work is that it answers a long-held mystery among scientists," Dr. Daley said. "For a decade we have known that resection enzymes are at the forefront of homologous recombination. What we didn't know is why so many of these enzymes are involved, and why we need three or four different enzymes that seem to accomplish the same task in repairing double-strand breaks."
An array of tools, each one finely tuned
"On the surface of it, there seems to be quite a bit of redundancy," said Dr. Sung, who holds the Robert A. Welch Distinguished Chair in Chemistry at UT Health San Antonio. "Our study is significant in showing that the perceived redundancy is really a very naïve notion."
DNA resection pathways actually are highly specific, the findings show.
"It's like an engine mechanic who has a set of tools at his disposal," Dr. Sung said. "The tool he uses depends on the issue that needs to be repaired. In like fashion, each DNA repair tool in our cells is designed to repair a distinctive type of break in our DNA."
The research team studied complex breaks that featured double-strand breaks with other kinds of DNA damage nearby -- such complex breaks are more relevant physiologically, Dr. Daley said. Studies in the field of DNA repair usually tend to look at simpler versions of double-strand breaks, he said. Dr. Daley found that each resection enzyme is tailored to deal with a specific type of complex break, which explains why a diverse toolkit of resection enzymes has evolved over millennia.
Dr. Burma, the Mays Family Foundation Distinguished Chair in Oncology at UT Health San Antonio MD Anderson Cancer Center, said the fundamental understandings gleaned from this research could one day lead to improved cancer treatments.
"The cancer therapeutic implications are immense," Dr. Burma said. "This research by our team is timely because a new type of radiation therapy, called carbon ion therapy, is now being considered in the U.S. While being much more precisely aimed at tumors, this therapy is likely to induce exactly the sort of complex DNA damage that we studied. Understanding how specific enzymes repair complex damage could lead to strategies to dramatically increase the efficacy of cancer therapy."
Part of the research is funded by NASA. "These kinds of complex DNA breaks are also induced by space radiation," Dr. Burma said. "Therefore, the research is relevant not just to cancer therapy, but also to cancer risks inherent to space exploration."
Role of Restriction Endonuclease Enzyme | Genetics
Restriction endonuclease enzymes occur naturally in bacteria as a chemical weapon against the invading viruses. They cut both strands of DNA when certain foreign nucleotides are introduced in the cell. Endonucleases break strands of DNA at internal positions in random manner.
Types of Restriction Enzymes:
1. Restriction enzyme Type I:
These enzymes interact with an unmodified recognition sequence in double-stranded DNA and then attach to long DNA molecule. After travelling for distance between 1000 to 5000 nucleotides the enzymes cleaves only one strand of the DNA at an apparently random site, and creates a gap of about 75 nucleotides in length.
Acid soluble oligonucleotides removed from the gap are released. A second enzyme molecule is needed to cleave the remaining strand of DNA. The cofactors for the enzyme are Mg 2+ ions, ATP and S-adenosyl- methionine. This kind of enzyme is not useful for genetic engineering, because its cleavage sites are non-specific.
Type I restriction enzyme can simultaneously hold two different sites on DNA creating a loop in nucleic acid. This enzyme consists of three types of subunits. The Eco K enzyme, for example, has the structure R2M2S.
The R subunit is responsible for restriction and the M subunit for methylation. The binding of enzyme to DNA may be succeeded by either restriction or modification and this property is characterized by S subunit.
2. Restriction enzyme Type II:
These enzymes recognise a particular target sequence in a double-stranded DNA molecule. They cleave the polynucleotide chain within or near that sequence to give rise to distinct DNA fragments of defined length and sequence. They require Mg 2+ ions for the action (i.e., restriction). Type II enzymes are used for gene manipulation studies.
3. Restriction enzyme Type III:
These enzymes cleave double-stranded DNA at well-defined sites. They require ATP, Mg 2+ ions and have very partial requirement for S-adenosyl-methionine for restriction. They have intermediate properties between Type I and Type II REs.
Naming of Restriction Endonuclease Enzymes:
About 350 types of restriction endonucleases have been isolated from more than 200 bacterial strains. Large number of these enzymes require a system of uniform nomenclature. A system based on the proposals of Smith and Nathans (1973) has been followed for the most part.
Naming exercise of RE enzymes is based on following rules:
1. Each RE enzyme is named by a three-letter code.
2. The first letter of this code is derived from the first epithet (first letter of name) of the genus name. It is printed in italics.
3. The second and third letters are from the first two letters of its species name. They are also printed in italics.
4. This is followed by the strain number. If a particular strain has more than one restriction enzyme, these will be identified by Roman numerals as I, II, III, etc.
For example, the enzyme Eco RI was isolated from the bacterium Escherichia (E) coli (co) strain RY13 (R) and it was the first endonuclease (I). R also indicates antibiotic resistant plasmid of the bacterium. Likewise, Hind II from Haemophilus influenzae strain Rd and Bgl I from Bacillus globigii. A few restriction endonuclease enzymes and their sources are given in Table 55.3.
Target Sites of Restriction Endonuclease Enzymes:
A restriction endonuclease enzyme of type H recognises a specific recognition site (base sequence) on the DNA and makes a cut at this site only. These target sites are 4 to 6 nucleotides long (Fig. 55.3).
They exhibit palindromic symmetry, i.e., nucleotide pair sequences are same reading forward or backward from a central axis of symmetry, like the nonsense phrase-AND ‘MADAM DNA’.
The term palindromic has also been applied to sequences such as:
both of which are palindromic strands.
X-ray crystallography of RE enzyme-DNA complex indicate that endonuciease acts as a dimer of identical subunits and that the palindromic nature of target sequence reflects the two fold rotational symmetry of the dimeric protein.
Nature of Cut Ends:
Two types of cut ends of DNA, namely blunt or flush ends and sticky or cohesive ends, are produced by the restriction endonuclease s. The nature of these cut ends generated by the REs are very important in designing the gene cloning experiments.
In case of the blunt cut end, the enzyme (e.g., Haelll, Smal) makes a simple double-stranded cut in the middle of the recognition sequence. Thus the blunt ends or flush ends are formed. The RE Hae III makes a cut in the 5′-GGCC-3′ target site as shown in Fig. 55.3.
The utility of generation of blunt end cuts during the joining of DNA fragments is that any pair of ends may be joined together irrespective of sequence. This is especially useful for those researchers who are interested to join two defined sequences without introducing any additional material between them. Table 55.4 shows certain blunt-end restriction sites.
2. Sticky-or Cohesive ends:
Many restriction enzymes (e.g., Eco RI, Bam HI, and Hind III) make staggered, single-stranded cuts, producing short single-stranded projections at each end of the cleaved DNA, called sticky ends.
Since the restriction sites are symmetrical, so that both strands have the same sequence when read in the 5′ to 3′ direction. Thus, such staggered cuts will generate identical single-stranded projections on the either site of the cut (Fig. 55.4).
These ends are not only identical, but complementary, and will base pair with each other they are therefore, known as cohesive or sticky ends. Because of specificity of restriction enzymes, every copy of a given DNA molecule will give the same set of fragments when cleaved with a particular enzyme.
Different DNA molecules will in general, give different sets of fragments when treated with the same enzyme. The table 55.5 shows sticky or cohesive-end restriction enzymes and sites:
Host Controlled Restriction and Modification:
Certain strains of bacteria are immune to bacteriophages. This phenomenon is called host controlled restriction. This restriction is due to these restriction endonuclease enzymes (e.g., Eco RI) which could recognise and split specific loci in the foreign DNA. Thus these enzymes prevent or restrict the survival of foreign DNA in the host. This is analogous to an immune system.
All restriction sites in host chromosome of a bacterium are protected from its own restriction endonuclease enzyme due to a modification system. This system helps in preventing suicidal self-degradation.
Such modification occurs by methylation of specific bases in the recognition sequence of the endonuclease. The enzymes involved in such modification are called methyltransferases.
These enzymes methylate adenine (i.e., adds a methyl group to the base) in the N6 position and cytosine either in N5 or W position and produce 6 methyl adenine and 5 or 4 methyl cytosine respectively.
Unmodified foreign DNA entering the cell is degraded by the host restriction system. As both the enzymes, i.e., methyltransferases and endonucleases recognise the restriction site, they are together called as restriction and modification system.
Various REs show , the star activity when they exhibit relaxation in specificity of sequence under non-optimal conditions. In such condition, endonuclease enzymes even recognise other alternative base instead of a specific base.
The following factors are known to alter the DNA recognition sequence for several to alter the DNA recognition sequence for several endonuclease enzymes: non-ideal strength buffers, high glycerol concentration (more than 5% vv) and high enzyme concentration.
Isoschizomers are restriction endonuclease enzymes which are isolated from different organisms but recognize identical base sequences in the DNA. For example, Asp 718 and Kpn I have identical recognition sites-
Source of Asp718 is Achromobacter species 718 source of Kpn I is Klebsiella pneumoniae OK 8. Some pairs of isochizomers cut their target at different places (e.g., Sma I, Xma I).
Use of Restriction Endonuclease Enzymes in Genetic Engineering:
In gene cloning experiments, DNA molecules have to be cut in a very precise and reproducible manner. Restriction endonuclease enzymes play an important role in cutting the desired gene as well as cleaving the vector.
The required DNA fragment from a large DNA molecule should be cleaved in a precise manner for further genetic manipulations. A particular restriction endonuclease enzyme can recognize and bind to specific base sequence of the DNA and then will cleave it. It is highly reproducible and can be programmed according to DNA sequences of required gene and particular endonuclease enzymes identifying and cleaving it.
2. Cutting the vectors:
The function of a vector DNA molecule is to carry a gene of interest to a second organism where it can express it (i.e., can produce a gene specific product). During this technique the DNA to be cloned is integrated with the plasmid.
Hence each vector molecule should be cleaved with same restriction site at a single position to open the circular form so that the new DNA fragment can be inserted at these complementary sites.
If foreign DNA is introduced into E. coli host, it may be attacked by restriction endonucleases active in a host cell. Because restriction phenomenon provides a natural defence against invasion by foreign DNA, it is usual to employ a K restriction deficient E. coli K12 strain as a host in transformation with newly created recombinant DNA molecules. This will eliminate the chance that the incoming sequence will be restricted.
Were Ancient Humans Healthier Than Us?
A curious thing happened when researchers at Georgia Tech used modern human genome sequences to look back at the possible health of our long-ago ancestors &ndash they found that while the Neanderthals and Denisovans of 30,000 to 50,000 years ago seemed to have been genetically sicker than us, &ldquorecent ancients&rdquo from a few thousand years ago may actually have been healthier. Their paper, &ldquoThe Genomic Health of Ancient Hominins,&rdquo is published in Human Biology.
How could that be? Perhaps drugs and procedures that enable us to live with certain conditions also perpetuate gene variants that would otherwise sicken us enough to not reproduce. We pass on those genes and inexorably weaken our global gene pool.
A cheetah with hereditary visual impairment would be weeded out, outrun and not likely to leave offspring. But we humans just use corrective lenses and have kids.
We&rsquove come a long way from imagining what life must have been like for ancient humans from just rare teeth and bits of bone. Now sequenced genomes from a sliver of a Denisovan pinkie, from Neanderthal skeletons, and from several sampled tissues of Ötzi the Tyrolean ice man, offer glimpses of past health. Had arrows and wounds not done in Ötzi 5300 years ago, his inherited heart disease might have. He also had brown eyes and type O blood, and would have been lactose intolerant if he&rsquod eaten dairy. Lactose intolerance is the wild type ancestral condition, which didn&rsquot begin to be selected against until adults started consuming nonhuman milk products and suffering cramps.
Joe Lachance, Ali J. Berens, and Taylor L. Cooper of Georgia Tech started out with 449 genome sequences from individuals who&rsquod lived at various times from 430 to 50,000 years ago. The trio compared the ancient genome sequences to a compendium of 3,180 gene variants associated with specific diseases today, ignoring trivial traits and genes carried on the X or Y chromosome. Then they selected the 147 genomes that had more than 50% of the risk gene variants to further analyze. They focused mostly on individuals who lived 2,000 to 9,000 years ago, and categorized each as from a hunter-gatherer, a farmer, or a pastoralist (someone who raises sheep or cattle).
A similar study, one of my favorites, compared Neanderthal DNA sequences to electronic health records from 28,000 modern Europeans. It added to the list of predispositions that Neanderthals might have faced: depression, skin lesions, urinary tract infections, and tobacco addiction.
The Georgia Tech researchers used an overall genetic risk score (GRS) as well as 9 specific disease categories.
The GRS included all 3,180 gene variants weighted for the magnitude of their effects. In determining cholesterol level in the blood, for example, some gene variants have enormous effects, such as the mutation that causes familial hypercholesterolemia, and others not so much. The GRS paints a portrait of risk, not necessarily actual diseases.
A high GRS is bad. A value of 82, for example, means the person would have been at greater risk to get sick from risk genes than 82% of modern humans. &ldquoHow this percentile maps to actual disease risks is likely to be trait-specific. For example, you might only be high risk for a disease if you are above the 90th percentile (think of a liability threshold), with only minimal differences in risk between individuals who are at the 25th and 50th percentiles,&rdquo said Dr. Lachance.
The 9 disease categories were allergy/autoimmune, cancer, cardiovascular disease, dental/periodontal, gastrointestinal/liver, metabolic/weight, miscellaneous, morphological/muscle, and neurological/psychological.
The analysis revealed several apparent trends:
&bull Pastoralists had &ldquoextremely healthy genomes&rdquo for allergy/autoimmunity, cancer, gastrointestinal ills, and dental health. Farmers and hunter-gatherers were less healthy but about even. Dr. Lachance can&rsquot explain this finding, but suggests that the better health of those who work with cattle and sheep may have been due to exposure to different environments, perhaps like today farm kids have fewer allergies than kids raised in cities.
&bull Ancient people who lived in the north were healthier. They had better teeth and less cancer.
&bull The most ancient individuals were less likely to have been predisposed to cancer and neurological/psychological conditions. Maybe they didn&rsquot live long enough to develop cancer or neurological disorders, and therapist records from thousands of years ago are scarce.
&bull Farmers had the worst teeth. Was it due to the residents of the dental plaque microbiome that formed in response to mashing all those new carbs from grains?
A particularly decrepit specimen was the famed Altai Neanderthal, who lived 50,000 to 100,000 years ago in the Altai mountains of Siberia. Her genome indicates that her parents were siblings, which must have been common when humanity struggled to survive in isolated caves. She had &ldquopoor genomic health&rdquo and a GRS of a whopping 97%, with genetic signposts of cancer, digestive and metabolic issues, muscle problems, and immune problems. But she had lower risk of cardiovascular disease.
The new work also fleshes out Ötzi: he had gastrointestinal problems and was prone to infection, but was neurologically intact.
A huge caveat to the study is the use of modern genomes as a point of comparison, which was necessary because ancient ones tend to be fragmented in different places. But incomplete evidence was a challenge even when all we had was fossil evidence. Remember the old view of Neanderthals as brown-haired brutes? Thanks to DNA analysis we now know that some of them were redheads.
The range of genetic risk scores among ancient and modern genomes is about the same, but overall genomic health seems to have improved. That could be due to context.
&ldquoIt&rsquos important to consider how well an individual&rsquos genome is suited for the present environment,&rdquo Dr. Lachance told me, like the effects of a dairy diet on whether or not lactose intolerance becomes noticeable enough to affect reproduction, the currency of evolution. Imagine using a time machine to send someone today back to the distant past, where vision goes uncorrected, influenza is deadly, and tendency to bleed too easily or not fast enough is life-threatening. Gene function always filters through the lens of the environment.
Maybe the blame can&rsquot be placed on our treating diseases that have genetic components to explain why recent ancients may have been healthier. Study design may be a simpler explanation for why the genomes from people who lived a few thousand years ago have a median GRS below 50%, and were therefore seemingly healthier than us. The study looked only at those above 50% &ndash 302 of the 449 ancient genomes fell below that mark.
Consider this: Gene variants that elevated risks of certain diseases, or mutations that actually caused them, might have been stripped from the ancient populations as affected people were too sick to have children, so they wouldn&rsquot even be in the modern genomes to which the older ones were compared. And the healthy pastoralists might have been a sampling error &ndash they contributed only 19 of the 147 genomes.
Dr. Lachance thinks the second possibility &ndash inability to compare full genomes from thousands of years ago to modern ones &ndash the more likely explanation. The truth is out there, but it&rsquoll take more experiments and data to illuminate it.