9.E: Transposition of DNA (Exercises) - Biology

9.E: Transposition of DNA (Exercises) - Biology

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Question 9.5. Suppose you are studying a gene that is contained within a 5 kb EcoRI fragment for the wild type allele. When analyzing mutations in that gene, you found one that converted the 5 kb fragment to an 8 kb EcoRI fragment. Recombinant plasmids carrying the 8 kb EcoRI fragment conferred resistance to the antibiotic kanamycin in the host bacteria, whereas neither the parental cloning vector nor a recombinant plasmid carrying the 5 kb EcoRI fragment did. What do you conclude is the basis for this mutation? What other enzyme activities might you expect to be encoded in the additional DNA?

Use the following diagram to answer the next two questions. Transposase encoded by a transposable element (TE) has nicked on each side of the TE in the donor (black) replicon and made a staggered break in the recipient (gray) replicon, and the ends of the TE have been joined to the target (T) site in the recipient replicon. The strands of the replicons have been designated top (t) or bottom (b). The open triangles with 1 or 2 in them just refer to locations in the figure; they are not part of the structure.

Question 9.6. The action of DNA polymerase plus dNTPs, primed at positions 1, followed by ligase (with ATP or NAD) leads to what product or result? (In this scenario, nothing occurs at positions 2).

Question 9.7. The action of an endonuclease at the positions labeled 2 followed by DNA polymerase and dNTPs to fill in the gaps (from positions 1 to the next 5' ends of DNA fragments), and finally DNA ligase (with ATP or NAD) leads to what product or result?

Question 9.8. Refer to the model for a crossover intermediate in replicative transposition in Fig. 9.13. If the transposon moved to a second site on the same DNA molecule by replicative transposition (not to a different molecule as shown in the Figure), what are the consequences for the DNA between the donor and recipient sites?

Question 9.9.The technique of transposon tagging uses the integration of transposons to mutate a large numbers of genes while leaving a "tag" in the mutated gene to allow subsequent isolation of the gene using molecular probes (such as hybridization probes for the transposon). What is a good candidate for transposon tagging in mammalian cells?

9.E: Transposition of DNA (Exercises) - Biology

Some good sources of additional microbial genetics problems include:

  • Freifelder, D. 1983. Problems for Molecular Biology. Jones and Bartlett Publishers, Boston.
  • Maloy, S., J. Cronan, and D. Freifelder. 1994. Microbial Genetics (Second edition). Jones and Bartlett Publishers, Boston.
  • Schleif, R. 1993. Genetics and Molecular Biology (Second edition). Johns Hopkins University Press, Baltimore.
  • Smith-Keary, P. 1989. Molecular Genetics of Escherichia coli . Guilford Press, NY.

For more background on many of these topics, see the Microbial Genetics supplement.

Problem solving plays an integral role in teaching microbial genetics, but developing a large bank of problems is very time consuming. Thus, this web site was designed as a Co-op to provide a continually evolving collection of microbial genetics problems that could be freely used by anyone interested.

Please use any of these problems or contribute problems you think others may find useful. The only restriction is that the questions must not be copywrited. If you have any comments or would like to submit problems (send both the question and your solution) please send them via email to the address shown at the bottom of this page. Unless you indicate that you wish to remain anonymous, I will indicate your name in the problem.

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Essentials of Genetics

Known for its focus on conceptual understanding, problem solving, and practical applications, this bestseller strengthens problem-solving skills and explores the essential genetics topics that today’s students need to understand. The Ninth Edition maintains the text’s brief, less-detailed coverage of core concepts and has been extensively updated with relevant, cutting-edge coverage of emerging topics in genetics.

MasteringGenetics™ is not included. Students, if MasteringGenetics is a recommended/mandatory component of the course, please ask your instructor for the correct ISBN. MasteringGenetics should only be purchased when required by an instructor. Instructors, contact your Pearson representative for more information.

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  • Three new Special Topics in Modern Genetics mini-chapters explore cutting-edge topics, including Emerging Roles of RNA, Genetically Modified Foods, and Gene Therapy.
  • Review Questions and Discussion Questions have been added to the end of each of the six Special Topic chapters to help reinforce key ideas and to facilitate class discussions. The questions are also assignable in MasteringGenetics.
  • Evolving Concept of the Gene sections are integrated in key chapters and describe how scientists’ understanding of the gene has changed over time, helping students better understand how the science of genetics has developed.
  • Genetics, Technology, and Society boxes discuss advances in genetics that have societal importance. Your Turn questions direct students to related information for further exploration.
  • Exploring Genomics exercises appear in selected chapters and help students connect genetics with genomics, bioinformatics, and proteomics. Exercises direct students to explore a wide variety of online resources.


1. Introduction to Genetics
2. Mitosis and Meiosis
3. Mendelian Genetics
4. Modifications of Mendelian Ratios
5. Sex Determination and Sex Chromosomes
6. Chromosome Mutations: Variation in Number and Arrangement
7. Linkage and Chromosome Mapping in Eukaryotes
8. Genetic Analysis and Mapping in Bacteria and Bactierophages
9. DNA Structure and Analysis
10. DNA Replication and Recombination
11. Chromosome Structure and DNA Sequence Organization
12. The Genetic Code and Transcription
13. Translation and Proteins
14. Gene Mutation, DNA Repair, and Transposition
15. Regulation of Gene Expression
16. The Genetics of Cancer
17. Recombinant DNA Technology
18. Genomics and Proteomics
19. Applications and Ethics of Genetic Engineering and Biotechnology
20. Developmental Genetics
21. Quantitative Genetics and Multifactorial Traits
22. Population and Evolutionary Genetics

Special Topics in Modern Genetics 1: Epigenetics
Special Topics in Modern Genetics 2: Emerging Roles of RNA
Special Topics in Modern Genetics 3: DNA Forensics
Special Topics in Modern Genetics 4: Genomics and Personalized Medicine
Special Topics in Modern Genetics 5: Genetically Modified Foods
Special Topics in Modern Genetics 6: Gene Therapy


In the first session, we will mutagenize the pGLO (alias pBAD-GFPuv) plasmid with the EZ-Tn5 transposon. pGLO DNA (Fig. 1), the complete sequence of which can be found on the Bio-Rad website ( or searched as pBAD-GFPuv against Entrez, The Life Sciences Search Engine (, can be purchased from Bio-Rad. This plasmid has been engineered to contain three core genes: the bla gene which encodes the enzyme β-lactamase, responsible for resistance toward the antibiotic ampicillin (Amp R ) the green fluorescent protein (GFP) gene, originally derived from jellyfish (Aequorea victoria), which encodes the GFP and, the arabinose operon repressor (araC) gene which encodes a repressor protein that ensures that the GFP gene of pGLO is expressed only when glucose in the growth medium is replaced by arabinose [ 3 ].

The pGLO plasmid showing positions of genes of interest. The approximate positions of restriction sites for the three restriction enzymes NdeI, PsiI, and EcoRI used in Session 5 are shown by arrows. [Color figure can be viewed in the online issue, which is available at]

Several genes under the control of one or two regulatory proteins are organized into the ara operon and contribute to arabinose metabolism. The ara operon, which is regulated by the araC gene product, is expressed when glucose in the growth medium is replaced by arabinose [ 4 , 5 ]. In the genetically engineered pGLO plasmid, the genes involved in arabinose metabolism have been replaced by the GFP (aequorin) gene [ 3 ], thus placing expression of this fluorescent protein under the control of the araC gene [ 6 ]. When bacteria transformed with pGLO containing a wild-type copy of the GFP gene are grown in the presence of arabinose, they fluoresce a brilliant green color under UV light.

The Amp R antibiotic resistance marker enables the selection of E. coli transformed with pGLO DNA by plating them on growth media containing ampicillin. The GFP gene, whose expression is activated when the sugar arabinose is added to the growth medium, enables the direct visualization of transformants. Transformed colonies appear white under UV light on agar plates lacking arabinose but fluoresce green on plates containing arabinose. An intact araC gene is required for the expression of the GFP gene.

Transposon Insertion Reaction

The transposon insertion reaction is made by incubating equimolar concentrations of the target pGLO DNA with EZ-Tn5 <KAN-2> transposon as described in the EZ-Tn5 Transposon Tools Technical Manual (EPICENTRE Biotechnologies). Students prepare the transposon insertion reaction mixture by the sequential addition of 1 μL EZ-Tn5 10X reaction buffer, 6.5 μL distilled water, 1 μL of a 0.2 μg/μL solution of pGLO DNA in water, 0.5 μL of a molar equivalent of EZ-Tn5 <KAN-2> transposon, and 1 μL EZ-Tn5 transposase to give a final volume of 10 μL. After incubation for 2 h at 37 °C the reaction is stopped by adding 1 μL of EZ-Tn5 10X stop solution and heating the mixture at 70 °C for 10 min. The mixture is stored at –20 °C for use in Session 2.

Supplies and Equipment for Session 1

Epicentre's Transposome Kit (EZ1982K contains Tn5, 10X reaction buffer, distilled water, transposase, and 10X stop solution), pGLO DNA (0.2 μg/μL), heat block (at 70 °C), water bath (at 37 °C) and floating water bath racks, Eppendorf tubes, beaker, P-2 and P-20 micropipetters and tips, microfuge, and adapter for small Eppendorfs.

Assignment for Students for Session 1

Consult text books, articles, manuals, and the Internet to write a one-page single-spaced description of transposons (their characteristics, types, composition, etc.). Concentrate on Tn transposons as much as possible.

––1–– μL EZ-Tn5 10X reaction buffer

Use the formula: μM target DNA = μg target DNA/[(base pairs in target DNA) × 660 as detailed in EZ-Tn5 <KAN-2> Insertion Kit from EPICENTRE Biotechnologies (]. Show your calculations.

Chromatin is regulated epigenetically and changes in senescence and aging

It has long been appreciated that an important aspect of aging may be biological structures that are inherently difficult to maintain 23, 24 . Chromatin is one such structure, and it is rapidly becoming clear that it is subject to extensive age-associated remodeling 10 . Changes in the organization of the genome into euchromatin and heterochromatin, as well as in DNA methylation patterns during aging and cellular senescence have been documented in several species 25, 26 . For example, a decrease in histone expression has been noted in senescent cells 27, 28 , and histone overexpression can extend the lifespan of yeast 29 . In the nematode, preventing excess accumulation of histone activating marks (H3K4Me3) extended lifespan 30 . Mammalian cells undergoing senescence reorganize their genomes into prominent senescence-associated heterochromatin foci (SAHF) 31 . Historically, changes in the methylation of CpG residues in DNA were the first noted effects of aging on chromatin 32, 33 , and have been widely studied 34, 35 . While overall levels of DNA methylation decrease, methylation of CpG islands associated with promoters (and hence transcriptional regulation of genes) changes in complex and tissue-specific patterns, showing an overall increase in many cases.

Using high throughput sequencing approaches, we recently examined genome-wide changes in chromatin accessibility that occur during replicative cellular senescence 36 . We found that the fundamental architecture of the genome undergoes profound alterations: an overall closing of chromatin in euchromatic gene-rich regions, which is opposed by a somewhat paradoxical opening of heterochromatic gene-poor regions. The former was associated with a dampening of gene expression, and the latter with increased transcription of retrotransposable elements (RTEs, below), which are normally heavily heterochromatinized. Quite remarkably, this culminated in active transposition, as evidenced by increases in copy number. Heterochromatin in centromeric and pericentromeric regions also became more open, and transcription of satellite sequences increased.


Immune system Edit

IL-6 is secreted by macrophages in response to specific microbial molecules, referred to as pathogen-associated molecular patterns (PAMPs). These PAMPs bind to an important group of detection molecules of the innate immune system, called pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). These are present on the cell surface and intracellular compartments and induce intracellular signaling cascades that give rise to inflammatory cytokine production. IL-6 is an important mediator of fever and of the acute phase response.

IL-6 is responsible for stimulating acute phase protein synthesis, as well as the production of neutrophils in the bone marrow. It supports the growth of B cells and is antagonistic to regulatory T cells.

Metabolic Edit

It is capable of crossing the blood-brain barrier [7] and initiating synthesis of PGE2 in the hypothalamus, thereby changing the body's temperature setpoint. In muscle and fatty tissue, IL-6 stimulates energy mobilization that leads to increased body temperature. At 4 degrees C, both the oxygen consumption and core temperature were lower in IL-6-/- compared with wild-type mice, suggesting a lower cold-induced thermogenesis in IL-6-/- mice. [8]

In the absence of inflammation 10–35% of circulating IL-6 may come from adipose tissue. [9] IL-6 is produced by adipocytes and is thought to be a reason why obese individuals have higher endogeneous levels of CRP. [10] IL-6 may exert a tonic suppression of body fat in mature mice, given that IL-6 gene knockout causes mature onset obesity. [11] [12] [13] Moreover, IL-6 can suppress body fat mass via effects at the level of the CNS. [11] The antiobesity effect of IL-6 in rodents is exerted at the level of the brain, presumably the hypothalamus and the hindbrain. [14] [15] [16] ). On the other hand, enhanced central IL-6 trans-signaling may improve energy and glucose homeostasis in obesity [17] Trans-signaling implicates that a soluble form of IL-6R (sIL-6R) comprising the extracellular portion of the receptor can bind IL-6 with a similar affinity as the membrane bound IL-6R. The complex of IL-6 and sIL-6R can bind to gp130 on cells, which do not express the IL-6R, and which are unresponsive to IL-6. [17]

Studies in experimental animals indicate that IL-6 in the CNS partly mediates the suppression of food intake and body weight exerted by glucagon-like peptide-1 (GLP-1) receptor stimulation. [18]

Outside the CNS, it seems that IL-6 stimulates the production of GLP-1 in the endocrine pancreas and the gut. [19] Amylin is another substance that can reduce body weight, and that may interact with IL-6. Amylin-induced IL-6 production in the ventromedial hypothalamus (VMH) is a possible mechanism by which amylin treatment could interact with VMH leptin signaling to increase its effect on weight loss. [20]

It is assumed that interleukin 6 in the liver activates the homologue of the human longevity gene mINDY expression via binding to its IL-6-receptor, which is associated with activation of the transcription factor STAT3 (which binds to the binding site in the mIndy promoter) and thereby rise of citrate uptake and hepatic lipogenesis. [21] [22]

Central nervous system Edit

Intranasally administered IL-6 has been shown to improve sleep-associated consolidation of emotional memories. [23]

There are indications of interactions between GLP-1 and IL-6 in several parts of the brain. One example is the parabrachial nuclei of the pons, where GLP-1 increases IL-6 levels [24] [25] and where IL-6 exerts a marked anti-obesity effect. [26]

IL-6 is also considered a myokine, a cytokine produced from muscle, which is elevated in response to muscle contraction. [27] It is significantly elevated with exercise, and precedes the appearance of other cytokines in the circulation. During exercise, it is thought to act in a hormone-like manner to mobilize extracellular substrates and/or augment substrate delivery. [28]

Like in humans, there seems to be an increase in IL-6 expression in working muscle and plasma IL-6 concentration during exercise in rodents. [29] [30] Studies in mice with IL-6 gene knockout indicate that lack of IL-6 in mice affect exercise function. [9]

It has been shown that the reduction of abdominal obesity by exercise in human adults can be reversed by the IL-6 receptor blocking antibody tocilizumab. Together with the findings that IL-6 prevents obesity, stimulates lipolysis and is released from skeletal muscle during exercise, the tocilizumab finding indicates that IL-6 is required for exercise to reduce visceral adipose tissue mass. [31] Bone may be another organ affected by exercise induced IL-6, given that muscle-derived interleukin 6 has been reported to increase exercise capacity by signaling in osteoblasts. [32]

IL-6 has extensive anti-inflammatory functions in its role as a myokine. IL-6 was the first myokine that was found to be secreted into the blood stream in response to muscle contractions. [33] Aerobic exercise provokes a systemic cytokine response, including, for example, IL-6, IL-1 receptor antagonist (IL-1ra), and IL-10. IL-6 was serendipitously discovered as a myokine because of the observation that it increased in an exponential fashion proportional to the length of exercise and the amount of muscle mass engaged in the exercise. It has been consistently demonstrated that the plasma concentration of IL-6 increases during muscular exercise. This increase is followed by the appearance of IL-1ra and the anti-inflammatory cytokine IL-10. In general, the cytokine response to exercise and sepsis differs with regard to TNF-α. Thus, the cytokine response to exercise is not preceded by an increase in plasma-TNF-α. Following exercise, the basal plasma IL-6 concentration may increase up to 100-fold, but less dramatic increases are more frequent. The exercise-induced increase of plasma IL-6 occurs in an exponential manner and the peak IL-6 level is reached at the end of the exercise or shortly thereafter. It is the combination of mode, intensity, and duration of the exercise that determines the magnitude of the exercise-induced increase of plasma IL-6. [34]

IL-6 had previously been classified as a proinflammatory cytokine. Therefore, it was first thought that the exercise-induced IL-6 response was related to muscle damage. [35] However, it has become evident that eccentric exercise is not associated with a larger increase in plasma IL-6 than exercise involving concentric "nondamaging" muscle contractions. This finding clearly demonstrates that muscle damage is not required to provoke an increase in plasma IL-6 during exercise. As a matter of fact, eccentric exercise may result in a delayed peak and a much slower decrease of plasma IL-6 during recovery. [34]

Recent work has shown that both upstream and downstream signalling pathways for IL-6 differ markedly between myocytes and macrophages. It appears that unlike IL-6 signalling in macrophages, which is dependent upon activation of the NFκB signalling pathway, intramuscular IL-6 expression is regulated by a network of signalling cascades, including the Ca2+/NFAT and glycogen/p38 MAPK pathways. Thus, when IL-6 is signalling in monocytes or macrophages, it creates a pro-inflammatory response, whereas IL-6 activation and signalling in muscle is totally independent of a preceding TNF-response or NFκB activation, and is anti-inflammatory. [36]

IL-6, among an increasing number of other recently identified myokines, thus remains an important topic in myokine research. It appears in muscle tissue and in the circulation during exercise at levels up to one hundred times basal rates, as noted, and is seen as having a beneficial impact on health and bodily functioning when elevated in response to physical exercise. [37]

IL-6 signals through a cell-surface type I cytokine receptor complex consisting of the ligand-binding IL-6Rα chain (CD126), and the signal-transducing component gp130 (also called CD130). CD130 is the common signal transducer for several cytokines including leukemia inhibitory factor (LIF), ciliary neurotropic factor, oncostatin M, IL-11 and cardiotrophin-1, and is almost ubiquitously expressed in most tissues. In contrast, the expression of CD126 is restricted to certain tissues. As IL-6 interacts with its receptor, it triggers the gp130 and IL-6R proteins to form a complex, thus activating the receptor. These complexes bring together the intracellular regions of gp130 to initiate a signal transduction cascade through certain transcription factors, Janus kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs). [38]

IL-6 is probably the best-studied of the cytokines that use gp130, also known as IL-6 signal transducer (IL6ST), in their signalling complexes. Other cytokines that signal through receptors containing gp130 are Interleukin 11 (IL-11), Interleukin 27 (IL-27), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC), leukemia inhibitory factor (LIF), oncostatin M (OSM), Kaposi's sarcoma-associated herpesvirus interleukin 6-like protein (KSHV-IL6). [39] These cytokines are commonly referred to as the IL-6 like or gp130 utilising cytokines [40]

In addition to the membrane-bound receptor, a soluble form of IL-6R (sIL-6R) has been purified from human serum and urine. Many neuronal cells are unresponsive to stimulation by IL-6 alone, but differentiation and survival of neuronal cells can be mediated through the action of sIL-6R. The sIL-6R/IL-6 complex can stimulate neurites outgrowth and promote survival of neurons and, hence, may be important in nerve regeneration through remyelination.

There is considerable functional overlap and interaction between Substance P (SP), the natural ligand for the neurokinin type 1 receptor (NK1R, a mediator of immunomodulatory activity) and IL-6.

IL-6 stimulates the inflammatory and auto-immune processes in many diseases such as Multiple sclerosis, [46] Neuromyelitis Optica Spectrum Disorder (NMOSD), [46] diabetes, [47] atherosclerosis, [48] depression, [49] Alzheimer's Disease, [50] systemic lupus erythematosus, [51] multiple myeloma, [52] prostate cancer, [53] Behçet's disease, [54] rheumatoid arthritis, [55] and intracerebral hemorrhage. [56]

Hence, there is an interest in developing anti-IL-6 agents as therapy against many of these diseases. [57] [58] The first such is tocilizumab, which has been approved for rheumatoid arthritis, [59] Castleman's disease [60] and systemic juvenile idiopathic arthritis. [61] Others are in clinical trials. [62]

Rheumatoid arthritis Edit

The first FDA approved anti-IL-6 treatment was for rheumatoid arthritis.

Cancer Edit

Anti-IL-6 therapy was initially developed for treatment of autoimmune diseases, but due to the role of IL-6 in chronic inflammation, IL-6 blockade was also evaluated for cancer treatment. [63] [64] IL-6 was seen to have roles in tumor microenvironment regulation, [65] production of breast cancer stem cell-like cells, [66] metastasis through down-regulation of E-cadherin, [67] and alteration of DNA methylation in oral cancer. [68]

Advanced/metastatic cancer patients have higher levels of IL-6 in their blood. [69] One example of this is pancreatic cancer, with noted elevation of IL-6 present in patients correlating with poor survival rates. [70]

Diseases Edit

Enterovirus 71 Edit

High IL-6 levels are associated with the development of encephalitis in children and immunodeficient mouse models infected with Enterovirus 71 this highly contagious virus normally causes a milder illness called Hand, foot, and mouth disease but can cause life-threatening encephalitis in some cases. EV71 patients with a certain gene polymorphism in IL-6 also appear to be more susceptible to developing encephalitis.

Epigenetic modifications Edit

IL-6 has been shown to lead to several neurological diseases through its impact on epigenetic modification within the brain. [71] [72] IL-6 activates the Phosphoinositide 3-kinase (PI3K) pathway, and a downstream target of this pathway is the protein kinase B (PKB) (Hodge et al., 2007). IL-6 activated PKB can phosphorylate the nuclear localization signal on DNA methyltransferase-1 (DNMT1). [73] This phosphorylation causes movement of DNMT1 to the nucleus, where it can be transcribed. [73] DNMT1 recruits other DNMTs, including DNMT3A and DNMT3B, which, as a complex, recruit HDAC1. [72] This complex adds methyl groups to CpG islands on gene promoters, repressing the chromatin structure surrounding the DNA sequence and inhibiting transcriptional machinery from accessing the gene to induce transcription. [72] Increased IL-6, therefore, can hypermethylate DNA sequences and subsequently decrease gene expression through its effects on DNMT1 expression. [74]

Schizophrenia Edit

The induction of epigenetic modification by IL-6 has been proposed as a mechanism in the pathology of schizophrenia through the hypermethylation and repression of the GAD67 promoter. [72] This hypermethylation may potentially lead to the decreased GAD67 levels seen in the brains of people with schizophrenia. [75] GAD67 may be involved in the pathology of schizophrenia through its effect on GABA levels and on neural oscillations. [76] Neural oscillations occur when inhibitory GABAergic neurons fire synchronously and cause inhibition of a multitude of target excitatory neurons at the same time, leading to a cycle of inhibition and disinhibition. [76] These neural oscillations are impaired in schizophrenia, and these alterations may be responsible for both positive and negative symptoms of schizophrenia. [77]

Aging Edit

IL-6 is commonly found in the senescence-associated secretory phenotype (SASP) factors secreted by senescent cells (a toxic cell-type that increases with aging). [78] [79] Cancer (a disease that increases with age) invasiveness is promoted primarily though the actions of the SASP factors metalloproteinase, chemokine, IL-6, and interleukin 8 (IL-8). [80] [78] IL-6 and IL-8 are the most conserved and robust features of SASP. [81]

Depression and major depressive disorder Edit

The epigenetic effects IL-6 have also been implicated in the pathology of depression. The effects of IL-6 on depression are mediated through the repression of brain-derived neurotrophic factor (BDNF) expression in the brain DNMT1 hypermethylates the BDNF promoter and reduces BDNF levels. [82] Altered BDNF function has been implicated in depression, [83] which is likely due to epigenetic modification following IL-6 upregulation. [82] BDNF is a neurotrophic factor implicated in spine formation, density, and morphology on neurons. [84] Downregulation of BDNF, therefore, may cause decreased connectivity in the brain. Depression is marked by altered connectivity, in particular between the anterior cingulate cortex and several other limbic areas, such as the hippocampus. [85] The anterior cingulate cortex is responsible for detecting incongruences between expectation and perceived experience. [86] Altered connectivity of the anterior cingulate cortex in depression, therefore, may cause altered emotions following certain experiences, leading to depressive reactions. [86] This altered connectivity is mediated by IL-6 and its effect on epigenetic regulation of BDNF. [82]

Additional preclinical and clinical data, suggest that Substance P [SP] and IL-6 may act in concert to promote major depression. SP, a hybrid neurotransmitter-cytokine, is co-transmitted with BDNF through paleo-spinothalamic circuitry from the periphery with collaterals into key areas of the limbic system. However, both IL6 and SP mitigate expression of BDNF in brain regions associated with negative affect and memory. SP and IL6 both relax tight junctions of the blood brain barrier, such that effects seen in fMRI experiments with these molecules may be a bidirectional mix of neuronal, glial, capillary, synaptic, paracrine, or endocrine-like effects. At the cellular level, SP is noted to increase expression of interleukin-6 (IL-6) through PI-3K, p42/44 and p38 MAP kinase pathways. Data suggest that nuclear translocation of NF-κB regulates IL-6 overexpression in SP-stimulated cells. [87] This is of key interest as: 1) a meta-analysis indicates an association of major depressive disorder, C-reactive protein and IL6 plasma concentrations, [88] 2) NK1R antagonists [five molecules] studied by 3 independent groups in over 2000 patients from 1998 to 2013 validate the mechanism as dose-related, fully effective antidepressant, with a unique safety profile. [89] [90] (see Summary of NK1RAs in Major Depression), 3) the preliminary observation that plasma concentrations of IL6 are elevated in depressed patients with cancer, [91] and 4) selective NK1RAs may eliminate endogenous SP stress-induced augmentation of IL-6 secretion pre-clinically. [92] These and many other reports suggest that a clinical study of a neutralizing IL-6 biological or drug based antagonist is likely warranted in patients with major depressive disorder, with or without co-morbid chronic inflammatory based illnesses that the combination of NK1RAs and IL6 blockers may represent a new, potentially biomarkable approach to major depression, and possibly bipolar disorder.

The IL-6 antibody sirukumab is now undergoing clinical trials against major depressive disorder. [93]

Asthma Edit

Obesity is a known risk factor in the development of severe asthma. Recent data suggests that the inflammation associated with obesity, potentially mediated by IL-6, plays a role in causing poor lung function and increased risk for developing asthma exacerbations. [94]

Interleukin is the main member of the IL-6 superfamily (Pfam PF00489), which also includes G-CSF, IL23A, and CLCF1. A viral version of IL6 is found in Kaposi's sarcoma-associated herpesvirus. [95]

Principles of microbiology, including the study of major microbial groups, cultivation, physiology and genetics, destruction, and control of microorganisms in nature and disease. For students in programs requiring one semester of microbiology (not premedical or medical technology students). Includes laboratory (Fall, Spring, Summer I)

BIOL-M 200 must be taken concurrently. Introduction to basic techniques and procedures of microbiology laboratories. Emphasis on aspects useful to nursing students. Growth and transfer of living microorganisms, aseptic techniques, and the staining of and identification of bacteria. (Fall, Spring, Summer I)

Is the DNA insertion problem solved?

Both research groups showed that it is possible to precisely insert DNA sequences in a desired chromosome location, but only in bacteria. As CRISPR is a bacterial system, there shouldn’t be CRISPR-associated transposable elements in eukaryotes. It will be interesting to see whether INTEGRATE or CAST systems can function as they are in other organisms, or if the eukaryotic transposons can be engineered to associate with a non-cutting CRISPR protein.

There are several genetic engineering applications that may require chromosome insertions of long DNA sequences, such as gene therapy and crop improvement. Developing a cut-free insertion system is definitely a step towards more versatile, safer, and less laborious CRISPR applications.

Kostas Vavitsas

Kostas Vavitsas is a Research Associate at the University of Athens, Greece. He is also community editor for PLOS Synbio, member of the steering committee of EUSynBioS, and communications editor for Omic Engine and EFB-EBBS. Find him on Twitter or LinkedIn

Watch the video: DNA cut-and-paste transposition (January 2023).