Mary S. Tyler

Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica

The sea slug Elysia chlorotica acquires plastids by ingestion of its algal food source Vaucheria litorea. Organelles are sequestered in the molluscs digestive epithelium, where they photosynthesize for months in the absence of algal nucleocytoplasm. This is perplexing because plastid metabolism depends on the nuclear genome for >90% of the needed proteins. Two possible explanations for the persistence of photosynthesis in the sea slug are (i) the ability of V. litorea plastids to retain genetic autonomy and/or (ii) more likely, the mollusc provides the essential plastid proteins. Under the latter scenario, genes supporting photosynthesis have been acquired by the animal via horizontal gene transfer and the encoded proteins are retargeted to the plastid. We sequenced the plastid genome and confirmed that it lacks the full complement of genes required for photosynthesis. In support of the second scenario, we demonstrated that a nuclear gene of oxygenic photosynthesis, psbO, is expressed in the sea slug and has integrated into the germline. The source of psbO in the sea slug is V. litorea because this sequence is identical from the predator and prey genomes. Evidence that the transferred gene has integrated into sea slug nuclear DNA comes from the finding of a highly diverged psbO 3′ flanking sequence in the algal and mollusc nuclear homologues and gene absence from the mitochondrial genome of E. chlorotica. We demonstrate that foreign organelle retention generates metabolic novelty (“green animals”) and is explained by anastomosis of distinct branches of the tree of life driven by predation and horizontal gene transfer.

Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

Phosphoribulokinase (PRK), a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction (Calvin) cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc (sea slug) Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclear-encoded proteins that are directed to complex (i.e. four membrane-bound) algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V. litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V. litorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.

The Teaching Demonstration: What Faculty Expect and How to Prepare for This Aspect of the Job Interview

To help job candidates understand faculty expectations of the teaching demonstration portion of an interview for a tenure-track faculty position, we canvassed biology faculty from a variety of institutions. We asked faculty to identify the elements of an effective teaching demonstration and to give advice on how candidates can best prepare for this aspect of the interview.

Development of osteogenic and chondrogenic potentials along the mediolateral axis of the embryonic chick mandible

Various regions of the mandibular process were tested for these potentials to determine whether regional differences exist and vary with embryonic age. Mandibular processes from HH stages 17-21.5 were cultured and grafted intact, or were subdivided into medial, mediolateral and lateral fragments and the separate regions cultured or grafted. The intact mandible from all these stages can form cartilage and membrane bone, but the 3 regions are not equally osteogenic and chondrogenic. The lateral region from all stages could form cartilage and membrane bone; the mediolateral region could form cartilage and membrane bone but, in mediolateral fragments from HH stage 17, membrane bone was formed only in scant amounts. The medial region from HH stages 17 and 18 formed cartilage in only 50 per cent of cases and never formed membrane bone. By HH stage 20, the medial region could form membrane bone, but only in scant amounts. Medial fragments from HH stage 21.5 formed extensive membrane bone and cartilage. The acquisition of these potentials, therefore, proceeds in a lateral-to-medial sequence, and the acquisition of an osteogenic potential lags slightly behind that of a chondrogenic potential. These findings do not indicate the mechanisms by which the two subpopulations of chondrogenic and osteogenic cells are distinguished from one another, but they give the temporal and spatial sequence in which this determination must occur.

Tissue interactions promoting osteogenesis in chorioallantoic-grown explants of secondary palatal shelves of the embryonic chick

Abstract Intramembranous osteogenesis in the neural crest-derived maxilla and mandible depends upon interactions between the bone-forming mesenchyme and the overlying epithelium. Whether or not an epithelium is required for the formation of the neural crest-derived palatine bone was examined. Palatal shelf mesenchyme, removed from embryonic chicks (5–7 days of incubation) prior to in-vivo palatal osteogenesis (10 days of incubation), was grafted with and without its epithelium to the chorioallantoic membrane of host chick embryos. During early secondary palatal development, removal of the palatal epithelium resulted in the failure of osteogenesis within the palatal mesenchyme. Removal of the palatal epithelium at subsequent developmental stages did not interfere with palatal osteogenesis. Epithelial influences, therefore, are required during early palatal development to promote palatal osteogenesis but cease to be required several days prior to the actual onset of ossification.

Effects of dibutyryl cyclic AMP and theophylline on in-vitro development of the secondary palate in the embryonic chick

The avian secondary palate is normally cleft and does not show a rise in intracellular cyclic AMP like that of the fused mammalian palate. The secondary palate of the embryonic chick (HH stages 25-34) was exposed in vitro to dibutyryl cyclic AMP and theophylline to determine whether raising intracellular cyclic-AMP levels would alter medial-epithelial development. Differentiation of the medial epithelium was not altered but there was mucous-cell hyperplasia in nasal epithelium and inhibition of membrane-bone formation in the mesenchyme. Thus the developmental factors that cause the avian palate to be cleft and the mammalian palate to be fused are more complex than differences in intracellular cyclic-AMP levels.

Effects of elevated levels of cyclic AMP on the embryonic chick mandible in vitro

Mandibles were cultured in the presence of dibutyryl cyclic AMP (1 mM) and theophylline (1 mM) and in control medium. Controls differentiated normally but the timing of events differed from that in ovo and feathers did not form. With elevated intracellular cyclic-AMP levels, membrane bone did not form in the earlier stages tested and was inhibited or reduced in older ones; chondrogenesis was inhibited only in young explants (HH stage 20) and, in certain instances, it was enhanced. There was precocious and hyperplastic differentiation of mucous cells within oral epithelium but the aboral epithelium was unchanged.

Promotion of osteogenesis by extra-embryonic epithelia in maxillary mesenchyme of the embryonic chick

Maxillary mesenchyme (HH stages 19-21), separated from its epithelium and grown as a graft on the chorioallantoic membranes of host chick embryos, did not form membrane bone. However, when isolated maxillary mesenchyme was recombined directly with chorioallantoic endoderm or chorioallantoic ectoderm and then grafted, it did form membrane bone in 87.5 and 70% of cases, respectively. This novel finding of extra-embryonic epithelia eliciting an osteogenic response from membrane bone-forming mesenchyme indicates that the germ-layer origin of an epithelium does not necessarily restrict its potential to promote membrane bone formation.