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Lab Report: Caenorhabditis Elegans Report


Background

Caenorhabditis elegans is a nonparasitic, translucent roundworm that is commonly found in temperate soil ecosystems (Husson et al., 2009). Its length is approximately 1 millimeter even though adult worms can grow to a length of 1.5 millimeters. The worm is unsegmented and has a pseudocoelom. It does not have a respiratory and a circulatory system. The gut of C. elegans contains gut pellets that emanate a bright blue fluorescence, which is predominant when the animal dies. Most of these roundworms are hermaphrodites. However, the male worms possess dedicated tails for mating, which have spicules.

C. elegans is an extensively investigated experimental prototypical organism that has led to great discoveries in the field of genetic analysis. Some of the features that contribute to its applicability in genetic studies include a short, three-day life cycle, small size, and an uncomplicated laboratory cultivation process (Andersen, Krichevsky, Leheste, & Moloney, 2008). Hundreds of these roundworms can be cultivated on one Petri dish containing a lawn of Escherichia coli as the source of food. In addition, the worm has been used to study molecular biology techniques such as RNA interference. Also, the worm is often used as a model to study physiological processes that occur in higher organisms (Benian & Epstein, 2011).

The unc-22 gene codes for the production of a protein called twitchin, which is a gigantic intracellular protein with numerous fibronectin- and immunoglobulin-like realms. The protein also contains one protein kinase domain that resembles titin. The unc-22 gene is fundamental in tissues where it controls the actinomyosin contraction-relaxation cycle. The gene is also responsible for the sustenance of standard muscle morphology. Unc-22 interacts with the muscle protein myosin and is confined to A-bands of the worm’s striated muscles that make up the wall of the animal’s body. The unc-22 proteins can add phosphate groups to the light chain peptides that make up myosin and go through autophosphorylation outside a living organism. Twitchin influences muscle synchronization in C. elegans and controls muscular contraction. Interference with the unc-22 gene in C. elegans affects its function leading to compromised movement and the characteristic “twitcher” phenotype.

Studies involving the manipulation of the unc-22 gene including the introduction of mutations and silencing various alleles of the gene have helped elucidate the structure and function of the gene, which is beneficial to the field of muscle physiology.

Research Question

Which alleles of the unc-22 gene elicit the strongest response in muscle movement when mutated?

The unc-22 gene is fascinating because of its rare phenotype. Distorted alleles of the locus bring about different degrees of compromised movement and muscle incompetence. Weak alleles show a minimal effect on the motion or organization of the muscle. When strong alleles are affected, the result is almost complete paralysis of adult worms. Moreover, there may be aberrant muscle structure where the thick and thin filaments are available in the usual amounts, but their distribution is altered. However, all alleles result in a continuous, subcellular jerk in all the cells making up the muscles of the body wall. It has been reported that the alleles of the unc-54 myosin heavy-chain gene and one of lev-ll alleles also contribute to this observation further indicating the interaction of unc-22 with other alleles. The twitch is aggravated by choline agonists.

Hypothesis

If the strong alleles of the unc-22 gene are knocked down, the strongest muscle movement will be evident.

Specific Aims

To test for the interference of the strong alleles of the unc-22 gene, different alleles of the unc-22 genes will be introduced with mutations and the strength of the mutations on muscle function will be evaluated.

Genetic investigations have provided a number of intuitions about the nature of the gene and its function. The unc-22 gene is an abnormally large mutational target such that the intragenic recombination rates for alleles at the far ends of the gene are equal or higher than rates observed for the most secluded allelic pairs of the unc-54 gene that encodes the myosin heavy chain. Most alleles of the unc-22 gene are conditionally dominant. Therefore, animals with heterozygous mutations in the unc-22 gene exhibit normal motion as well as conventional muscle structure. Nevertheless, they can be provoked to jerk forcefully in solutions containing choline agonists. This dominance implies that the unc-22 gene most likely encodes a protein that is needed stoichiometrically to exert its effects. An interesting observation is that the reversion analysis of unc-22 alleles shows that specific missense alleles of the myosin heavy-chain gene unc-54 can overpower the unc-22 phenotype. Several changes occur from this procedure including repression of the muscle twitch, partial restitution of motility and reorganization of the muscle structure. Sequence evaluation of the unc-54 repressors reveals that the transmutations are present in the region that encodes the myosin head, a few are situated around the nucleotide binding domain while others are located close to the preserved thiol domain.

The 1st Specific Aim: Forward genetic screen

A study by Chu et al. (2014) has shown that the “twitcher” phenotype can be provoked by putting worms without the unc-22 gene on 1% nicotine resulting in violent twitching by the mutant worms for several hours. The wild-type worms become rigid and restrained. Nicotinic agonists such as levamisole can also be used for this test. Therefore, placing the worms on 1% nicotine solution will enable the screening of mutant worms from the wild-type worms by observing the twitching thus visually separating the mutant worms from the wild-type worms.

The 2nd Specific Aim: Reverse genetic screen of mutation

Muscular twitching is a consequence of defective unc-22 gene. A deletion of the unc-22 gene should produce abnormal muscle twitching in C. elegans. Unc-22 RNAi will be introduced to the worm and observe the resultant phenotypes. Thereafter, the gene loci responsible for the mutation will be isolated and mapped. The unc-22 gene has multiple loci and alleles, and only a few of these loci will produce the twitching phenotype. It is reported that the large protein is responsible for muscular contraction because this protein is often found localized to the Actin A of myosin. Therefore, the absence of this protein in the isolated loci will indicate the particular loci that are greatly affected by this mutation.

The 3rd Specific Aim: To determine strong and weak unc-22 alleles

To determine the alleles that lead to strong phenotypes of unc-22, a mutation analysis will be done by placing Tc1 insertions in the unc-22 region. The first step will be isolating genomic DNA from the wild-type and extracting the unc-22 regions. Approximately 12 different Tc1 insertions will then be placed into a minimum of 10 different sites in the unc-22 alleles, which have already been identified (Thompson et al., 2013). These recombinant fragments will then be used to transform different C. elegans samples using E. coli cloning vectors. The process of transformation will be relatively simple because C. elegans feeds on E. coli. The ingested bacteria cells containing the mutant alleles will then be incorporated into the host cell genome leading to the manifestation of the unc-22 phenotype. The transformed animals will be labeled accordingly based on the specific allele that was mutated. The impact of the insertions in the phenotype will be determined by observing the movement of the animals in the presence and the absence of choline inhibitors. The strength of the muscular twitching will be scored to determine which mutations have the strongest impact on the muscular contraction of C. elegans. Minimal twitching will be indicative of weak unc-22 alleles while aggressive and prolonged twitching will show strong unc-22 alleles.

Research Objectives

Broader Impact

Understanding the mutation of the unc-22 genes and the particular alleles that lead to strong muscle impairment will improve the understanding of structure and muscle function. The unc-22 gene is similar to the TTN and OBSCN genes in humans that function in muscle contraction (Matsunaga, Qadota, Furukawa, Choe, & Benian, 2015). TTN gene codes for a large predominant protein of the striated muscle. Mutations in this gene are linked to human diseases such as familial hypertrophic cardiomyopathy 9, hereditary myopathy and muscular dystrophy. Also, patients with the autoimmune disease scleroderma have been reported to produce antibodies against titin.

The obscurin gene is responsible for the production of a protein that belongs to the category of colossal sarcomeric signaling proteins such as titin and nebulin. It may play a significant role in the arrangement of myofibrils in the course of assembly and may facilitate associations between the sarcoplasmic reticulum and myofibrils. The obscurin gene is thought to take part in various physiological processes such as apoptosis, initiation of programmed cell death via extracellular indications, cell differentiation, the development of multicellular organisms, the addition of phosphate groups to amino acids in proteins and the control of Rho protein signal transduction.

Knowledge of the strongest mutations can help in the development of appropriate gene therapies to remedy the illnesses caused by the mutation of these genes.

Future Directions

Following the identification of the strong alleles in unc-22 mutations, future studies could look into ways of protecting against these mutations.

References

Andersen, J., Krichevsky, A., Leheste, J. R., & Moloney, D. J. (2008). Caenorhabditis elegans as an undergraduate educational tool for teaching RNAi. Biochemistry and Molecular Biology Education, 36(6), 417-427.

Benian, G. M., & Epstein, H. F. (2011). Caenorhabditis elegans muscle: A genetic and molecular model for protein interactions in the heart. Circulation Research, 109(9), 1082-1095.

Chu, J. S. C., Chua, S. Y., Wong, K., Davison, A. M., Johnsen, R., Baillie, D. L., & Rose, A. M. (2014). High-throughput capturing and characterization of mutations in essential genes of Caenorhabditis elegans. BMC Genomics, 15(1), 361.

Husson, S. J., Landuyt, B., Nys, T., Baggerman, G., Boonen, K., Clynen, E.,… & Schoofs, L. (2009). Comparative peptidomics of Caenorhabditis elegans versus C. briggsae by LC–MALDI-TOF MS. Peptides, 30(3), 449-457.

Matsunaga, Y., Qadota, H., Furukawa, M., Choe, H. H., & Benian, G. M. (2015). Twitchin kinase interacts with MAPKAP kinase 2 in Caenorhabditis elegans striated muscle. Molecular Biology of the Cell, 26(11), 2096-2111.

Thompson, O., Edgley, M., Strasbourger, P., Flibotte, S., Ewing, B., Adair, R.,… & Kieffer, A. (2013). The million mutation project: A new approach to genetics in Caenorhabditis elegans. Genome Research, 23(10), 1749-1762.

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IvyPanda. (2020, July 28). Lab Report: Caenorhabditis Elegans. Retrieved from https://ivypanda.com/essays/lab-report-caenorhabditis-elegans/

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"Lab Report: Caenorhabditis Elegans." IvyPanda, 28 July 2020, ivypanda.com/essays/lab-report-caenorhabditis-elegans/.

1. IvyPanda. "Lab Report: Caenorhabditis Elegans." July 28, 2020. https://ivypanda.com/essays/lab-report-caenorhabditis-elegans/.


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IvyPanda. "Lab Report: Caenorhabditis Elegans." July 28, 2020. https://ivypanda.com/essays/lab-report-caenorhabditis-elegans/.

References

IvyPanda. 2020. "Lab Report: Caenorhabditis Elegans." July 28, 2020. https://ivypanda.com/essays/lab-report-caenorhabditis-elegans/.

References

IvyPanda. (2020) 'Lab Report: Caenorhabditis Elegans'. 28 July.

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