Polyketide synthase genes from marine dinoflagellates.

Rapidly developing techniques for manipulating the pathways of polyketide biosynthesis at the genomic level have created the demand for new pathways with novel biosynthetic capability. Polyketides derived from dinoflagellates are among the most complex and unique structures identified thus far, yet no studies of the biosynthesis of dinoflagellate-derived polyketides at the genomic level have been reported. Nine strains representing 7 different species of dinoflagellates were screened for the presence of type I and type II polyketide synthases (PKSs) by polymerase chain reaction (PCR) and reverse transcriptase PCR. Seven of the 9 strains yielded products that were homologous with known and putative type I PKSs. Unexpectedly, a PKS gene was amplified from cultures of the dinoflagellate Gymnodinium catenatum, a saxitoxin producer, which is not known to produce a polyketide. In each case the presence of a PKS gene was correlated with the presence of bacteria in the cultures as identified by amplification of the bacterial 16S ribosomal RNA gene. However, amplification from polyadenylated RNA, the lack of PKS expression in light-deprived cultures, residual phylogenetic signals, resistance to methylation-sensitive restriction enzymes, and the lack of hybridization to bacterial isolates support a dinoflagellate origin for most of these genes.

A virus-like agent associated with neurofibromatosis in damselfish.

Damselfish neurofibromatosis (DNF) is a transmissible disease involving neurofibromas and chromatophoromas affecting bicolor damselfish Stegastes partitus on Florida reefs. Analysis of genomic DNA by Southern blotting techniques demonstrated the presence of a group of extrachromosomal DNAs in tumors from fish affected with DNF but not in healthy individuals. Cell lines obtained from tumors contained identical DNAs and were shown to be tumorigenic in vivo, while lines established from healthy fish did not contain such DNA and were not tumorigenic. These DNA patterns were also observed in experimentally induced tumors. A DNase resistant component of this DNA was isolated from both tumor cells and conditioned media of tumor cell lines suggesting that these sequences were encapsulated in viral particles. These data support the hypothesis that one or more of these extrachromosomal DNA forms is the genome of an unusual virus and that this virus is the etiologic agent of DNF. We have tentatively termed this agent the damselfish virus-like agent (DVLA).

Progression of infection and tumor development in damselfish.

The bicolor damselfish (Stegastes partitus) is a tropical marine teleost naturally affected by multiple neurofibromas and chromatophoromas on South Florida reefs. Damselfish neurofibromatosis is a transmissible disease caused by a subcellular agent. Development of tumors is associated with the appearance of a series of extrachromosomal DNAs ranging in size from 1.2 to 7 kb that appear to be the genome of a small virus-like agent which we termed the damselfish virus-like agent (DVLA). This DNA was found at high copy number in most spontaneous and experimentally induced tumors. An essentially identical pattern of DNA, but with lower copy numbers, was observed in non-tumor-bearing tissue from diseased fish. Copy numbers of DVLA DNA in tumors and nontumorous tissues increased as the disease progressed from early to late stages. In healthy fish in which DVLA DNA was detected, the quantities were much lower than those in diseased fish. Healthy fish from populations with a high prevalence of disease exhibited more infected tissues than fish from populations with low levels.

Structure and function of the axillary organ of the gulf toadfish, Opsanus beta (Goode and Bean).

The structure of the axillary organ of a batrachoidid species, the gulf toadfish (Opsanus beta Goode and Bean 1879), has been examined and several simple experiments designed to elucidate its function performed. Electron microscopy (EM) studies revealed cells and structures suggesting secretory and iono regulatory roles (e.g., abundant intracytoplasmic secretory particles, rough endoplasmic reticulum, sparse Golgi bodies, indented epithelial cells with microvilli, numerous endocytotic vesicles, etc.). Our physiological experiments allowed us to reach several conclusions: the organs do not excrete significant quantities of urea relative to other areas of the fish (head and gills), the organs do not secrete a substance that is toxic to a teleost test fish (Gambusia affinis), the secretions do not induce short-term modifications in locomotory activity of other gulf toadfish (e.g., by pheromonal means) and the secretions do not inhibit the growth of several species of microorganisms in culture. The function of the organ and its secretions remains unknown, representing a fertile area for research on structure and function in comparative physiology.

An in vivo screen for the luciferase transgene in zebrafish.

A simple and economical large-scale in vivo screen for firefly luciferase expression in transgenic zebrafish is described. The screen is a film assay of luminescence during embryogenesis. Either luciferin substrate can be microinjected into the embryo, or the embryo can be raised in a luciferin solution. In a test of transient expression in the G0 (microinjected) generation, a construct with the human cytomegalovirus (CMV) promoter gave higher levels of expression than three other constructs. Using the CMV promoter, injection of supercoiled or linear DNA led to approximately equivalent amounts of expression. Although G0 transient luciferase expression is high enough to be reliably screened, G1 integrated expression is either low or nonexistent, and therefore unscreenable. In the G1 and G2 generations, low-level expression was increased with application of 5-azacytidine. The fact that both transgene methylation and 5-azacytidine activation of expression occurred suggests that methylation is involved in either reducing or eliminating integrated luciferase expression. This in vivo luciferase screen may be useful for insertional mutagenesis, promoter, gene, or enhancer traps, promoter analysis, and optimization of conditions for gene transfer.

Inheritance of P element and reporter gene sequences in zebrafish.

We have established 15 separate lines of zebrafish transgenic for two plasmid DNAs containing Drosophila P element sequences and either the reporter genes chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), and beta-galactosidase (BGAL) or the reporter gene hygromycin phosphotransferase (HPT). Transient expression of CAT, but not NPT or BGAL, could be measured in the G0 (microinjected) generation, while expression in subsequent generations was below the limit of detection for CAT and NPT. All 15 lines contain stable low copy number integrations of between one and five transgene copies per cell with characteristic Southern blot "junction fragments" and have shown Mendelian inheritance after the G1 generation. The transgenes in 13 of 15 of the lines were originally inherited by about 10% or less of the G1 fish, suggesting a mosaic integration into the germline. Of 14 tested lines, none is associated with an embryonic recessive lethal phenotype as scored in the G3 generation.

Developmental control of transduced dopa decarboxylase genes in D. melanogaster.

Seventeen new euchromatic integration sites of the dopa-decarboxylase gene (Ddc) have been generated using p-mediated transduction. The developmental expression of the integrated genes was examined by monitoring the embryonic induction of dopa decarboxylase enzyme activity (DDC) and by monitoring the developmental pattern of DDC activity from late third instar to eclosion. The majority of inserts are regulated correctly within about 30% of controls. Several cases of multiple insertion events were recovered and these show correspondingly elevated levels of activity and are regulated normally. The pattern of expression of one insert (15C) falls outside the normal range. Multiple copies of transduced Ddc genes are used to test for effects of elevated gene dose on levels of expression. One insert on the X chromosome shows little or no dosage compensation. Possible reasons for the differences between the regulation of transduced genes in Drosophila and the regulation of transformed genes in mammalian systems are discussed.