New Role for an Old Protein: An Educational Primer for Use with "The Identification of a Novel Mutant Allele of topoisomerase II in Caenorhabditis elegans Reveals a Unique Role in Chromosome Segregation During Spermatogenesis"

Abstract

Modern experimental techniques, such as whole-genome sequencing and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 endogenous genome editing, are enabling researchers to identify and further characterize the roles of proteins that were previously thought of as well defined. In the December 2016 issue of GENETICS, an article by Jaramillo-Lambert et al. identified a new role for the enzyme topoisomerase II in Caenorhabditis elegans male meiosis. This Primer article is designed to provide essential background information on C. elegans spermatogenesis and the relevant scientific techniques that will assist students and instructors in their understanding and discussion of the related article.Related article in GENETICS: Jaramillo-Lambert, A., A. S. Fabritius A. S., T. J. Hansen T. J., H. E. Smith H. E., and A. Golden A., 2016 The identification of a novel mutant allele of topoisomerase II in Caenorhabditis elegans reveals a unique role in chromosome segregation during spermatogenesis. Genetics204: 1407-1422.

Keywords: Caenorhabditis elegans; meiosis; oogenesis; spermatogenesis; topoisomerase.

Figure 1

The hermaphrodite and male C. elegans reproductive systems. (A) A schematic of the hermaphrodite bilobed, U-shaped gonad. The distal end contains the mitotically dividing germ cells, and germ cells progress toward the spermatheca (sperm storage organ) undergoing the various stages of prophase I of meiosis. The uterus contains embryos undergoing early development, which will ultimately be laid through the vulva to the external environment. (B) A schematic of the male gonad with germ cells undergoing mitosis at the distal end and progressing through the various stages of meiosis as they move toward the vas deferens. For both (A) and (B), the chromosome configurations can be utilized to identify specific stages of meiosis.

Figure 2

Chromosome dynamics during C. elegans spermatogenesis and oogenesis. Following chromosome appearance can help determine which stage of meiosis a germ cell is in. In this figure, the chromosomes appear as dark lines. Karyosome formation is only observed during spermatogenesis. Note that spermatids complete meiosis before fertilization, while oocyte meiosis completion is triggered upon fertilization in the +1 embryo. n/a, not applicable.

Figure 3

Strategy for one-step Hawaiian single-nucleotide polymorphisms (SNP) mapping and whole-genome sequencing (WGS). The red asterisks indicate the mutation of interest in an N2 background. Step 1 involves mating the strain carrying the mutation of interest to Hawaiian males. Step 2 involves allowing the F1 progeny to self-fertilize, thereby generating F2 progeny that have undergone recombination events between the original N2 and Hawaiian chromosomes. Step 3 involves isolating F2 animals exhibiting the mutation phenotype of interest and that represent a unique recombination event between the N2 (Bristol) and Hawaiian chromosomes. The progeny of the F2 hermaphrodites are then pooled, the DNA isolated, and WGS performed. Step 4 involves computationally analyzing the results of the WGS and the Hawaiian/N2 SNP ratios to determine the genomic loci, which has little to no Hawaiian SNPs, and thus is comprised of N2 genomic sequence. The researcher would then have a narrowed down genomic region to look at for the causative mutation (the boxed region in Step 3 and red arrow in Step 4).

Figure 4

Overview of CRISPR/CAS-9 endogenous genome editing. The DNA sequence to be edited is recognized by the crRNA portion of the sgRNA. The tracrRNA portion of sgRNA brings the Cas9 endonuclease enzyme. Cas9 must bind to a PAM site before initiating a DSB upstream of the PAM site. The DSB can then be repaired by error-prone NHEJ or by error-free HDR utilizing a specifically designed repair template. CRISPR, clustered regularly interspaced short palindromic repeats; crRNA, CRISPR RNA; DSB, double-strand break; HDR, homology-directed repair; NHEJ, nonhomologous end joining; PAM, protospacer-adjacent motif; sgRNA, single-guide RNA; tracrRNA, trans-activating CRISPR RNA.