540 Chemie und zugeordnete Wissenschaften
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Xiphophorus meyeri n. sp. is described as an endemic to Muzquiz, Coahuila, Mexico. It appears to be the northernmost species of the genus. The new species is related to X. couchianus and X. gordoni, but differs morphologically from those by dorsal fin ray number, by the expression of some gonopodial features and most markedly by the appearance of macromelanophores or tr-melanophores.
In order to develop all-fish expression vectors for microinjection into fertilized fish eggs, we have prepared the following cunstructs: rainbow trout metallothionein a/b and the gilthead seabream growth hormone cDNA (ptMTa-gbsGHcDNA, ptMTb-gsbGHcDNA), carp ß-actin gilthead seabream GH cDNA (pcAßgsbGHcDNA). The inducible metallothionein promoters a and b were cloned from rainbow trout, and the constitutive promoter ß-actin was isolated from carp. The metallothionein promoters were cloned by using the PCR technique. The tMTa contains 430 bp, while the tMTb contains 260 bp (Hong et al. 1992). These two promoters were introduced to pGEM-3Z containing the GH cDNA of Sparus aurata to form ptMTa-gsbGH and ptMTb-gsbGH, respectively. The carp cytoplasmic ß-actin gene was chosen as a source for isolating strong constitutive regulatory sequences. One of these regulatory sequences in pUC118 was Iigated to GH cDNA of S. aurata to form the pcAß-gsbGHcDNA. Expression of the constructs containing the metallothionein promoters was tested in fish cell culture and was found tobe induced effectively by zinc. The ptMTa gsb-GH cDNA construct was microinjected into fertilized carp eggs, and integration in the genome of carp was detected in the DNA isolated from fins at the age of two months.
Melanotic melanoma (MM) of Xiphophorus (Teleostei: Poeciliidae) was studied by conventional preparations and freeze-etch preparations for electron microscopy. MM of Xiphophorus exhibits tightly packed pigment cells with prominent dendritic processes and interdigitations of their plasma membranes. The most impressive feature of MM cells is the occurrence of Iarge lobulated nuclei with numerous nuclear pores and some nuclear pockets. Abundant spheroidal or ellipsoidal melanosomes (diameter 200-650 nm) and vesicular structures are distributed throughout the cellular dendrites, whereas the perinucJear cytoplasm is free of melanosomes.
A further characteristic feature of melanoma cells in fish is the occurrence of melanosome complexes (i.e., "compound melanosomes"). These melanosome complexes consist of a few to numerous melanosomes, which are enveloped by a separate rnembrane. Pinocytotic vesicles couJd be demonstrated with distinct differences in frequency and distribution patterns, indicating differences in the metabolic activities of the cells in the same melanoma. Intercellular junctions are lacking in the MM cells.
The conventional TEM technique showed clear advantages in the demonstration of intemal architecture of organelles, whereas FE bad considerable potential in respect to the visualization of membrane surface specializations.
Efficient expression systems are required for analysis of gene regulation and function in teleost fish. To develop such systems, a nurober of inducible or constitutive promoter and enhancer sequences of fish or higher vertebrate origin were tested for activity in a variety of fish celllines andin embryos of the Japanese medaka fish (Oryzias latipes) and Xiphophorus. The activity of the different promoterenhancer combinations were quantitated. Considerable differences were found for some constructs if tested in vitro or in vivo. From the data obtained, a set of expression vectors for basic research as weH as for aquaculture purposes were established.
The melanoma·inducing gene of Xiphophorus fish encodes the Xmrk receptor tyrosine kinase. U sing a highly specific antiserum p~oduced against the recombinant receptor expressed with a baculovirus, it is shown that Xmrk is the most abundant phosphotyrosine protein in fish melanoma and thus highly activated in the tumors. Studies on a melanoma cellline revealed that these cells produce an activity that considerably stimulates receptor autophosphorylation. The stimulating activity induces receptor down-regulation and can be depleted from the melanoma cellsupernatant by the immobilized recombinant receptor protein. The fish melanoma cells can thus be considered autocrine tumor cells providing a source for future purification and characterization of the Xmrk ligand.
Malignant melanomas (MM) in the fish Xiphophorus and in humans were studied both by transmission electron microscopy (TEM) and freeze-etching (FE). In both fish and human melanomas the cells show interdigitations of the,plasma membranes. The nuclei are large and lobulated and have many nuclear pores. Melanosomes are abundant and melanosome complexes ("compound melanosomes") occur regularly. Pinocytotic vesicles could be demonstrated in fish and human melanomas showing iocal differences in frequency and distribution patterns in the tumor. lntercellular junctions are lacking in MM cells from fish and humans. The FE technique showed considerable advantages in demonstrating membrane-surface peculiarities such as nuclear pores or pinocytotic vesicles. The FE replicas of fish melanomas are like those of humans. These findings may support the hypothesis that melanoma in fish and humans reflect the same biological phenomenon.