Search

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. This protocol describes a straightforward but crude isolation strategy for genomic DNA that has been effective on all tested choanoflagellate species. The resulting genomic DNA provides a suitable template for a variety of polymerase chain reaction (PCR) techniques, but is too impure for procedures that require very high-quality DNA such as Southern blotting.

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. However, the preparation of genomic DNA from cultures of Monosiga brevicollis or other choanoflagellates can be complicated by the presence of abundant bacterial DNA. Indeed, bacterial DNA can represent as much as 90% of the total yield from a standard genomic DNA preparation. This protocol describes the use of a CsCl gradient to separate choanoflagellate genomic DNA from the DNA of its bacterial prey. This strategy works only when the G+C content of the choanoflagellate genomic DNA is significantly different from that of its bacterial food. The final DNA sample from this protocol is expected to be 90%-95% enriched for choanoflagellate genomic DNA and to provide an adequate template for genome sequencing projects.

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. Most, if not all, choanoflagellates are heterotrophs that prey on bacteria. Thus, all choanoflagellate media provide nutrition for bacteria, which are in turn consumed by the choanoflagellates. If a culture is axenic or has low bacterial content, it can be supplemented with a specific bacterial strain that has been grown separately. On the other hand, if a culture already contains a well-established flora of bacteria, it can be cocultured with the choanoflagellate isolate. In either case, it is common to enrich the medium with an organic extract, such as liver extract, cereal grass infusion, or a mixture of proteose peptone and yeast extract. This protocol describes how to start cultures of the marine choanoflagellate species, Monosiga brevicollis, from frozen stocks. These cultures can then be maintained and expanded in preparation for DNA or RNA isolation or cell biological assays.

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. Choanoflagellates can be stored under stable conditions by freezing them with liquid nitrogen, as described here. To prevent cross-contamination among vials stored in the liquid phase of a liquid nitrogen freezer, the use of CryoFlex tubing to fully seal the vials is encouraged.

INTRODUCTIONChoanoflagellates are the closest living relatives of the metazoa, and the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. These heterotrophic flagellates are a cosmopolitan group of small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Monosiga brevicollis, a marine species, has emerged as a representative of the group because it is easily grown and manipulated in the laboratory and was the subject of a recently completed genome project. Because of the similarity in morphology and ecology shown across the order Choanoflagellida, the protocols mentioned in this article should be transferable to most choanoflagellate species.

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. They occur as spherical to ovoid cells exhibiting morphological similarities to the choanocytes (feeding cells) of sponges. Specifically, they are characterized by a single anterior flagellum surrounded by a funnel-shaped collar composed of 30 or more actin-based tentacles or microvilli. These structures can be visualized by staining for actin and β-tubulin. In this protocol, Monosiga brevicollis cells are fixed in dilute formaldehyde, attached to coverslips, and then exposed to antibodies against β-tubulin and an actin-specific stain; a simple DNA stain highlights the nucleus. The central challenge of this protocol is to treat the cells gently so that they retain their ultrastructure and remain attached to the coverslips during processing.

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. This protocol describes how to enrich freshly collected field samples and isolate single choanoflagellate cells. Two methods are commonly used: isolation by micropipetting single cells and isolation by dilution. The two methods are complementary and each has its advantages and disadvantages. The micropipette technique requires considerable experience. However, provided a single cell has been isolated, any culture arising from it will be clonal. The dilution technique does not require the same level of skill, but single-cell isolation is not ensured. For the latter technique, repeating the isolation procedure using higher dilutions is advised to ensure that only one cell is the basis of an established culture.

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. This protocol describes a simple genomic DNA isolation strategy that should be effective for almost all choanoflagellate species and will provide genomic templates suitable for polymerase chain reactions and Southern blot analysis.

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. The preparation of RNA is an important prerequisite for characterizing the gene expression profile of choanoflagellates under different conditions. Although most standard protocols (e.g., those using TRIzol) are sufficient for many purposes, the approach described here minimizes RNA degradation and provides a high-quality template for downstream protocols, including real-time polymerase chain reaction.