Pause-melting misalignment: a novel model for the birth and motif indel of tandem repeats in the mitochondrial genome.

BACKGROUND: Tandem repeats (TRs) in the mitochondrial (mt) genome control region have been documented in a wide variety of vertebrate species. The mechanism by which repeated tracts originate and undergo duplication and deletion, however, remains unclear. RESULTS: We analyzed DNA sequences of mt genome TRs (mtTRs) in the ridged-eye flounder (Pleuronichthys cornutus), and characterized DNA sequences of mtTRs from other vertebrates using the data available in GenBank. Tandem repeats are concentrated in the control regions; however, we found approximately 16.6% of the TRs elsewhere in the mt genome. The flounder mtTRs possess three motif types with hypervariable characteristics at the 3' end of the control region (CR). CONCLUSION: Based on our analysis of this larger dataset of mtTR sequences, we propose a novel model of Pause Melting Misalignment (PMM) to describe the birth and motif indel of tandem repeats. PMM is activated during a pause event in mitochondrial replication in which a dynamic competition between the nascent (N) heavy strand and the displaced (D) heavy strand may lead to the melting of the N-strand from the template (T) light strand. When mispairing occurs during rebinding of the N-strand, one or several motifs can be inserted or deleted in both strands during the next round of mt-replication or repair. This model can explain the characteristics of TRs in available vertebrate mt genomes.

Reinventing the ames test as a quantitative lab that connects classical and molecular genetics.

While many institutions use a version of the Ames test in the undergraduate genetics laboratory, students typically are not exposed to techniques or procedures beyond qualitative analysis of phenotypic reversion, thereby seriously limiting the scope of learning. We have extended the Ames test to include both quantitative analysis of reversion frequency and molecular analysis of revertant gene sequences. By giving students a role in designing their quantitative methods and analyses, students practice and apply quantitative skills. To help students connect classical and molecular genetic concepts and techniques, we report here procedures for characterizing the molecular lesions that confer a revertant phenotype. We suggest undertaking reversion of both missense and frameshift mutants to allow a more sophisticated molecular genetic analysis. These modifications and additions broaden the educational content of the traditional Ames test teaching laboratory, while simultaneously enhancing students' skills in experimental design, quantitative analysis, and data interpretation.

An Expanded Role for the RFX Transcription Factor DAF-19, with Dual Functions in Ciliated and Nonciliated Neurons.

Regulatory Factor X (RFX) transcription factors (TFs) are best known for activating genes required for ciliogenesis in both vertebrates and invertebrates. In humans, eight RFX TFs have a variety of tissue-specific functions, while in the worm Caenorhabditis elegans, the sole RFX gene, daf-19, encodes a set of nested isoforms. Null alleles of daf-19 confer pleiotropic effects including altered development with a dauer constitutive phenotype, complete absence of cilia and ciliary proteins, and defects in synaptic protein maintenance. We sought to identify RFX/daf-19 target genes associated with neuronal functions other than ciliogenesis using comparative transcriptome analyses at different life stages of the worm. Subsequent characterization of gene expression patterns revealed one set of genes activated in the presence of DAF-19 in ciliated sensory neurons, whose activation requires the daf-19c isoform, also required for ciliogenesis. A second set of genes is downregulated in the presence of DAF-19, primarily in nonsensory neurons. The human orthologs of some of these neuronal genes are associated with human diseases. We report the novel finding that daf-19a is directly or indirectly responsible for downregulation of these neuronal genes in C. elegans by characterizing a new mutation affecting the daf-19a isoform (tm5562) and not associated with ciliogenesis, but which confers synaptic and behavioral defects. Thus, we have identified a new regulatory role for RFX TFs in the nervous system. The new daf-19 candidate target genes we have identified by transcriptomics will serve to uncover the molecular underpinnings of the pleiotropic effects that daf-19 exerts on nervous system function.

Using restriction mapping to teach basic skills in the molecular biology lab.

Digestion of DNA with restriction enzymes, calculation of volumes and concentrations of reagents for reactions, and the separation of DNA fragments by agarose gel electrophoresis are common molecular biology techniques that are best taught through repetition. The following open-ended, investigative laboratory exercise in plasmid restriction mapping allows students to gain technical expertise while simultaneously exploring the utility of gel electrophoresis and restriction mapping. Because of its interpretive nature, this project also provides data suitable for a written report, and can thus be used to reinforce lessons on figure presentation and science writing skills.

Isolation of Caenorhabditis elegans genomic DNA and detection of deletions in the unc-93 gene using PCR.

PCR, genomic DNA isolation, and agarose gel electrophoresis are common molecular biology techniques with a wide range of applications. Therefore, we have developed a series of exercises employing these techniques for an intermediate level undergraduate molecular biology laboratory course. In these exercises, students isolate genomic DNA from the nematode Caenorhabditis elegans and use PCR to detect deletions in the C. elegans unc-93 gene. In advance of the exercises, wild-type and three different unc-93 deletion mutant strains are grown, harvested, and frozen by the instructor. In one approach, students isolate genomic DNA from each strain using a genomic DNA isolation kit and use agarose gel electrophoresis to analyze the DNA and to estimate its concentration. PCRs using primers directed to two different regions of the unc-93 gene are carried out on the genomic DNA from wild-type and mutant strains, and the PCR products are analyzed by agarose gel electrophoresis. Students analyze the gel to determine the approximate location and size of deletions in the three mutant strains. Alternatively, students may lyse single nematodes and carry out PCR in one laboratory session. These exercises should be easily adaptable to detection of well characterized deletions in any organism.

Suppressors, screens, and genes: an educational primer for use with "A network of genes antagonistic to the LIN-35 retinoblastoma protein of Caenorhabditis elegans".

An article by Polley and Fay in this issue of GENETICS provides an excellent opportunity to introduce or reinforce concepts of reverse genetics and RNA interference, suppressor screens, synthetic phenotypes, and phenocopy. Necessary background, explanations of these concepts, and a sample approach to classroom use of the original article, including discussion questions, are provided.

RFLP analysis and allelic discrimination with real-time PCR using the human lactase persistence trait: A pair of molecular genetic investigations.

We describe here two open-ended laboratory investigations for an undergraduate laboratory course that uses students' DNA as templates for quantitative real-time PCR and for traditional PCR followed by RFLP analysis. Students are captivated by the immediacy of the application and the relevance of the genotypes and traits, lactase persistence or nonpersistence, under study. Concepts ranging from nucleotide polymorphisms to natural selection can be demonstrated and reinforced by these laboratories. Students have a high degree of success with the laboratories and both objective and self-assessments of student learning indicate that concepts and techniques are better understood following implementation of these laboratory investigations.