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Institute
- Theodor-Boveri-Institut für Biowissenschaften (118) (remove)
Hierarchical structures among male indlviduals in a population are frequently reflected ln differences in aggressive and reproductive behavior and access to the females. In general, sodal dominance requires the Investments, which in turn then may have to be compensated for by high reproductive success. However, this hypothesls has so far only been sufficiently tested in small mating groups (one or two males with one or two females) due to the difficulties of determining paternity by conventional methods. DNA fingerprinting overcomes these problems by offering the possibility to determine genetic relationships and mating patterns within larger groups [Borke, T. (1989) Trends Ecol. Evol. 4, 139-144]. We show here that in the poecUiid fish Limia perugitu, in small matlng groups the dominant male has 8 mating success of 100%, whereas ln larger groups lts contribution to the offspring unexpectedly drops to zero.
The demonstration ofthe chromosomal mode ofsex determinationvia genetic experiments as well as the absence of heteromorphic sex chromosomes affirm poeciliid fishes as a unique group among vertebrates that are endowed with the mostprimitive form of sex chromosornes. In many different taxa the evolutionary process involved in the differentiation ofadvanced sex chromosomes is outlined through sex specifically organized repetitive sequences. In this investigation hydridization of synthetic probes specific to genomic simple repeat motifs uncovers a sex-specific hybridization pattern in certain viviparaus fishes ofthe family Poeciliidae. The hybridization pattern together with specific staining ofthe constitutive heterochromatin by C-banding reveals heterogamety in males (Poecilia reticulata) as weil as in females (P. sphenops). In P. velifera, however, C-banding alone fails to unravel the heterogametic status. The female specific W-chromosome can be detected by simple repetitive sequence probes. Therefore, the principal significance of heterochromatization as a means of generating differentiated sex chromosomes is evident.
The promoter of the rainbow trout metallothionein B gene ( tMTb) was isolated from genomic DNA by the polymerase chain reaction (PCR ), fused to the bacterial chloramphenicol acetyltransferase (CAT) genein an expression vector, and functionally analyzed in one human cellline and four fish celllines. This promoter exhibited an extremely low basal expression in all celllines and was zincand cadmium-inducible except in the fish melanoma cell line where the promoter was completely inactive. The metal-induced expression patterns were cellline-specific. In general the fish promoter was more species- and cell type-specific than its human counterpart. In a transient assay it was functional in developing embryos of the medaka ( Oryzias /atipes). These properties make this promoter suitable for inducible, tissue-specific expression of transgenes and for in vivo studies of gene function and regulation.
Hereditary melanoma in Xiphophorus hybrids canying the melanoma·induclng Tu-Sd locus is caused by transcriptional activation of the Xmrk gene that resides at the Tu·Sd locus and encodes a novel member of receptor tyrosine kinases (RTK). ln this study, a total of 17 hereditary melanomas from various hybrid genotypes harbouring 7 different Tu alleles were also found to aver-express the correspondlng Xmrlc alleles. The Ievei of over-expression correlated with the degree of malignancy of the melanoma. ln addition, Xsrc expression was high ln many malignant melanomas. Expression pattems and Ieveis of the Xiphophorus EGF-receptor gene (Xerb B), the c-myc (Xmyc), and the PDGF (Xsls) gene(s) were not intriguing. Transcription of the ras gene(s) may be correlated to secondary events of melanoma progression. Expression pattems of Xfms, the Xiphophorus CSF-1 receptor homologue, can be explained by different contents of infiltrating macrophages in the tumors. ln carcinogen-induced tumors includlng one melanoma no significant expression of the Xmrk oncogene could be detected. Xsrc expression, however, was strikingly high. This indicates that activation of oncogenes other than Xmrk ls instrumental in tumorigenesls of neoplasia of non-hereditary origin.
Which home for coelacanth?
(1993)
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.
When bovine or human growth hormones (GH) were injected into 6 months old (about 10 g) gilthead seabream, the growth rate of the fish, as measured by changes in their weight, increased by only about 15% compared with the saline-injected control. No effect or even slight inhibition of the growth rate was obtained when chicken or porcine GHs were injected. In a preliminary experiment, it was found that injection ofthe native GH increased the growth rate ofthe fish by about 20% after treatment for only 2 weeks. An expression vector, using the pRE1 plasmid and transformation into MZl cells, produced the gilthead seabream GH, providing a supply for further experiments on the effect of the homologaus GH on growth. Two reporter genes, ß-galactosidase (lacZ) and melanoma oncogene of Xiphophorus (mrk YY), were microinjected into fertilized eggs of S. aurata. Expression of these two genes could be demonstrated in 2-day-old embryos, the lacZ gene by staining of its enzymatic product, and the mrk YY gene by its phenotypic expression.
In Xiphophorus melanoma formation has been attributed by classical genetic findings to the overexpression of a cellular oncogene (Tu) due to elimination of the corresponding regulatory gene locus in hybrids. We have attempted to elucidate this phenomenon on the molecular biological level. Studies on the structure and expression of known proto-oncogenes revealed that several of these genes, especially the c-src gene of Xiphophorus, may act as effectors in establishing the neoplastic phenotype of the melanoma cells . However, these genes appear more to participate in secondary steps of tumorigenesis. Another gene, being termed Xmrk, which represents obviously a so far unknown proto-oncogene but with a cons iderably high similarity to the epidermal growth-factorreceptor gene, was mapped to the Tu-containing region of the chromosome. This gene shows features with respect to its structure and expression that seem to justify it to be regarded as a candidate for a gene involved in the primary processes leading to neoplastic transformation of pigment cells in Xiphophorus.