Douglas L. Chalker, in Methods in Cell Biology, 2012
Tetrahymena ThermophilaVI The Use of Heterokaryon Strains for the Study of Essential Genes
Essential genes can be difficult to study. If an essential gene is disrupted in the macronucleus, it is unlikely that complete replacement of the wild-type copies with the selectable marker will ever be achieved. In some cases, one can detect phenotypes in the cells that have the fewest copies of the essential gene remaining, but this takes very careful observation. Generation of germline knockout heterokaryons offers a potentially powerful means to characterize such difficult to study genes. These strains are homozygous for the knockout allele in their silent micronuclei, but are wild type in their macronucleus. When cells homozygous for the gene knockout in their micronuclei are mated, all progeny cells will be complete knockouts. If the gene is essential, then no viable progeny will be recovered. Nevertheless, it is possible to examine the phenotype of the mutant cells as they deplete their maternal load of protein in the cells as they undergo the first few rounds of postconjugative cell division (e.g., Cervantes et al., 2006; Malone et al., 2008).
Germline heterokaryon strains can also be used for mutational analysis. If the germline is homozygous for the knockout, copies of the gene can be introduced during postzygotic development, either on replicating vectors or integrating constructs to assess whether an introduced copy can rescue the knockout phenotype. Epitope-tagged versions of the gene can be introduced to determine whether or not the tag disrupts function. Truncated versions of the gene can be tested to map essential domains of a protein of interest. The major advantage over simply trying to replace the macronuclear gene by assortment is that one can be confident that the only version of the protein expressed is the one introduced during conjugation.
A rapid means of creating two homozygous germline knockout heterokaryons is to mate an existing germline transformant with “star” strains, which have defective micronuclei (e.g., B*VI and B*VII). This genomic exclusion cross-transfers the micronucleus from the knockout line to the star strain. The exconjugant derived from the star parent will have a homozygous micronucleus of the knockout, but retain its wild-type macronucleus. As this was an abortive mating, the strains are mature and ready to mate; therefore, as soon as these cell lines are expanded by growth, they can be used in phenotypic studies.
Nucleic Acids Res. 2004 Jan 1; 32(Database issue): D271–D272.
PMID: 14681410
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Abstract
Essential genes are genes that are indispensable to support cellular life. These genes constitute a minimal gene set required for a living cell. We have constructed a Database of Essential Genes (DEG), which contains all the essential genes that are currently available. The functions encoded by essential genes are considered a foundation of life and therefore are likely to be common to all cells. Users can BLAST the query sequences against DEG. If homologous genes are found, it is possible that the queried genes are also essential. Users can search for essential genes by their function or name. Users can also browse and extract all the records in DEG. Essential gene products comprise excellent targets for antibacterial drugs. Analysis of essential genes could help to answer the question of what are the basic functions necessary to support cellular life. DEG is freely accessible from the website http://tubic.tju.edu.cn/deg/.
INTRODUCTION
Essential genes are genes that are indispensable to support cellular life. These genes constitute a minimal gene set required for a living cell. Therefore, the functions encoded by this gene set are essential and could be considered as a foundation of life itself (,). The definition of the minimal gene set needed to sustain a living cell is of considerable interest not only because it represents a fundamental question in biology, but also because it has much significance in practical use. For example, since most antibiotics target essential cellular processes, essential gene products of microbial cells are promising new targets for antibacterial drugs ().
DATABASE DESCRIPTION
The determination of the minimal gene set for bacteria has only been possible with the advent of the completion of many whole genome sequencing projects and the genome-scale gene inactivation technology. Consequently, essential genes have been determined in a number of different organisms. Essential genes have been determined in Staphylococcus aureus by an antisense RNA technique (), in Mycoplasma genitalium by transposon mutagenesis (), in Haemophilus influenzae by high-density transposon mutagenesis (), in Vibrio cholerae by a mariner-based transposon (), in yeast by genetic footprinting (), and in M.genitalium and H.influenzae by comparative genomics ().
We have constructed a Database of Essential Genes (DEG) that contains all the essential genes currently available. These genes include the essential genes identified in the genomes of M.genitalium (), H.influenzae (), V.cholerae (), S.aureus (), Escherichia coli and Saccharomyces cerevisiae. The essential genes in the E.coli genome were extracted from the web site http://magpie.genome.wisc.edu/~chris/essential.html, in which the essential genes are collected from a large number of related references. The essential genes in yeast genome were extracted from the yeast genome database (http://www.mips.biochem.mpg.de/proj/yeast), which is maintained by the Munich Information Center for Protein Sequences ().
Each entry of essential genes has a unique DEG identification number, gene reference number, gene function and sequence. All information is stored and operated by using an open-source database management system, MySQL. Users can browse and extract all the records of these entries. In addition, users can also search DEG by gene function or name. Furthermore, we have installed the BLAST program locally. Therefore, users can BLAST the query sequences against all the essential gene sequences in DEG.
Pubmed
One of the applications is the prediction of essential genes based on homologous sequence search against DEG. The functions encoded by essential genes are considered to be generally essential for all cells (). It is even believed that some basic functions and principles are common to all cellular life on this planet (). Therefore, if the query sequences compared using BLAST have homologous genes in DEG, it is likely that the queried genes are also essential. In addition, by performing the BLAST search against DEG for all the protein-coding genes in a genome, it is possible to define the putative essential genes for the proteomes of newly sequenced genomes. However, caution must be taken in interpreting the BLAST results, since many essential genes are essential only in given growth conditions, such as in rich or minimal medium.
Another application is that by analyzing all the essential genes in DEG, some principles or regulations could be found to answer the question of what are the basic functions necessary to support cellular life. Those principles could lead to the development of new algorithms to predict essential genes. Some functions encoded by essential genes are expected, such as DNA replication, gene transcription, protein synthesis, energy production and cell division. Some essential genes, however, are somewhat unexpected, such as Embden–Meyerhof–Parnas pathway genes and a purine biosynthesis gene (). Analysis of DEG, which has all essential genes among different organisms, could help to classify those ‘unexpected’ essential genes.
Currently some essential gene projects are still ongoing and the identification of more essential genes is expected. DEG will be updated periodically to include more entries upon the availability of newly identified essential genes. We plan to integrate more information about experimental methods for each entry. In the next version of DEG, we also plan to include the essential genes of vertebrates, such as mouse. We welcome users’ comments, corrections and new information, which will be used for updating.
DEG is freely available at the web site http://tubic.tju.edu.cn/deg/, and should be cited with the present publication as reference.
ACKNOWLEDGEMENTS
We are indebted to Professor Jingchu Luo for advice on database construction. The present study was supported in part by the 973 Project of China (grant 1999075606).
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Science. Author manuscript; available in PMC 2015 Nov 28.
Published in final edited form as:
Published online 2015 Oct 15. doi: 10.1126/science.aac7041
NIHMSID: NIHMS732683
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