Norman Maclean - Academia.edu (original) (raw)

Papers by Norman Maclean

Journal of Cell Science, 1975

The mass ratio of nuclear non-histone protein: DNA in the immature circulating erythroid cells of... more The mass ratio of nuclear non-histone protein: DNA in the immature circulating erythroid cells of phenylhydrazine-induced anaemic Xenopus is approximately threefold higher than in mature erythrocytes. This is largely due to the presence of increased amounts of low and intermediate molecular weight proteins in the nuclei of the immature cells. There are a few qualitative differences in the components of this class of proteins between the mature and immature cells, the most notable of which is the presence of a protein of molecular weight approximately 115000 in the former which is not detectable in the latter. These changes are discussed in relation to the changing synthetic capacities of cells and to certain generalizations about the function of the nuclear non-histone protein based on studies of other differentiating systems.

Preface Notes on use Appendix 1. Common names and latin names of some key organisms in cell biolo... more Preface Notes on use Appendix 1. Common names and latin names of some key organisms in cell biology and genetics. Appendix 2. Chromosome numbers in various species. Contents Appendix 3. DNA content of haploid genomes. Appendix 4. The greek alphabet. Appendix 5.

Molecular Ecology Resources, 2008

We present primers and amplification conditions for 15 microsatellite loci developed for the Cope... more We present primers and amplification conditions for 15 microsatellite loci developed for the Cope's giant salamander (Dicamptodon copei), 14 of which are tetranucleotide repeats. Cross-species amplification revealed 10 of these loci to also be polymorphic in the Pacific giant salamander (Dicamptodon tenebrosus). Several loci produced nonoverlapping allelic ranges between the two species and may be useful in species identification. These polymorphic microsatellite loci are potentially useful for future studies of population genetics in dicamptodontid salamanders.

Human HOX homeobox genes, D.Acampora et al the growth hormone/prolactin receptor gene family, P.K... more Human HOX homeobox genes, D.Acampora et al the growth hormone/prolactin receptor gene family, P.Kelly et al organization and evolution of satellite, minisatellite and microsatellite DNAs in teleost fishes, J.Franck et al nucleosome positioning and regulated gene expression, B.Pina et al two distinct tumour necrosis factor receptors - members of a new cytokine receptor gene family, H.Loetscher et al the CTLA-1 gene - a member of the granzyme multigene family in human and murine cytotoxic T cells, P.Haddad.

New Developments in Marine Biotechnology, 1998

Since the first introduction of novel genes in fish (Maclean and Talwar, 1984; Zhu et al., 1985),... more Since the first introduction of novel genes in fish (Maclean and Talwar, 1984; Zhu et al., 1985), a wide range of fish species have been used in transgenic research either for commercial or academic purposes. For commercial exploitation this technology has been applied in several commercially important fish species to improve desirable genetic traits such as growth (Rokkones et al., 1989; Zhang et al., 1990; Brem et al., 1988; Penman et al., 1990; Gross et al., 1992), cold tolerance (Fletcher et al., 1988), or disease resistance (Anderson et al., 1996) for aquaculture development. Most of these gene transfer experiment have concentrated on growth enhancement using regulatory sequences and coding sequences of distantly related species (Rokkones et al., 1989; Penman et al., 1990; Brem et al., 1988; Zhang et al., 1990). Although successful gene transfer in these fish was observed in only a few cases has germline transmission and expression of transgenes in progeny been convincingly demonstrated (see reviews by Maclean and Rahman, 1994; Gong and Hew, 1995). In studies where the regulatory sequences used were of mammalian origin, low or nil expression was observed (Guyomard et al., 1989; Alam et al., 1996). In other studies, enhanced growth was observed in transgenic carp (Zhang et al., 1990) and in tilapia (Martinez et al., 1996) when growth hormone sequences were driven by a viral promoter. However, the main objective of the production of transgenic fish with GH gene is to generate novel strain of growth enhanced fish which could subsequently be used in aquaculture. It is, therefore, important that the gene constructs used for producing trans-genic fish be of fish origin, and not from mammalian or viral origin. Moreover, it was also observed that regulatory sequences from fish are found to be more effective than those of mammalian origin in respect to expression of transgenes (Alam et al., 1996) in fish.

Transgenic Research, 2000

Three lines of transgenic tilapia (Oreochromisniloticus) fish were generated with a constructcont... more Three lines of transgenic tilapia (Oreochromisniloticus) fish were generated with a constructcontaining a lacZ reporter gene spliced to a 4.7?kb 5' regulatory region of a carp beta actin gene. All these three lines contain different copy numbers oftransgenes and the levels of lacZ expressionwere found to be related to transgene copy number.Mosaic patterns of somatic lacZ expression wereobserved in these

Transgenic Research, 2007

Several lines of growth hormone (GH)overexpressing fish have been produced and analysed for growt... more Several lines of growth hormone (GH)overexpressing fish have been produced and analysed for growth and fertility parameters. However, only few data are available on the growth-promoting hormone insulin-like growth factor I (IGF-I) that mediates most effects of GH, and these are contradictory. Using quantitative real-time RT-PCR, radioimmunoassay, in situ hybridization, immunohistochemistry, and radiochromatography we investigated IGF-I and IGF binding proteins (IGFBPs) in an adult (17 months old) transgenic (GH-overexpressing) tilapia (Oreochromis niloticus). The transgenics showed an around 1.5-fold increase in length and an approximately 2.3-fold higher weight than the non-transgenics. Using radioimmunoassay, the serum IGF-I levels were lower (6.22 ± 0.75 ng/ml) in transgenic than in wild-type (15.01 ± 1.49 ng/ml) individuals (P = 0.0012). Radioimmunoassayable IGF-I in transgenic liver was 4.2times higher than in wild-type (16.0 ± 2.21 vs. 3.83 ± 0.71 ng/g, P = 0.0017). No hepatocytes in wild-type but numerous hepatocytes in transgenic liver contained IGF-I-immunoreactivity. RT-PCR revealed a 1.4-times higher IGF-I mRNA expression in the liver of the transgenics (10.51 ± 0.82 vs. 7.3 ± 0.49 pg/mg total RNA, P = 0.0032). In correspondence, in situ hybridization showed more IGF-I mRNA containing hepatocytes in the transgenics. A twofold elevated IGF-I mRNA expression was determined in the skeletal muscle of transgenics (0.33 ± 0.02 vs. 0.16 ± 0.01 pg/mg total RNA, P < 0.0001). Both liver and serum of transgenics showed increased IGF-I binding. The increased IGFBP content in the liver may lead to retention of IGF-I, and/or the release of IGF-I into the circulation may be slower resulting in accumulation of IGF-I in the hepatocytes. Our results indicate that the enhanced growth of the transgenics likely is due to enhanced autocrine/paracrine action of IGF-I in extrahepatic sites, as shown here for skeletal muscle.

Transgenic Research, 2009

Several lines of GH-overexpressing fish have been produced and characterized concerning organ int... more Several lines of GH-overexpressing fish have been produced and characterized concerning organ integrity, growth, fertility and health but few and contradictory data are available on IGF-I that mediates most effects of GH. Furthermore, nothing is known on IGF-II. Thus, the expression of both IGFs in liver and various extrahepatic sites of adult transgenic (GH-overexpressing) tilapia and agematched wild-type fish was determined by real-time PCR. Both IGF-I and IGF-II mRNA were found in all organs investigated and were increased in gills, kidney, intestine, heart, testes, skeletal muscle and brain of the transgenics (IGF-I: 1.4-4-fold; IGF-II: 1.7-4.2-fold). Except for liver, brain and testis the increase in IGF-I mRNA was higher than that in IGF-II mRNA. In pituitary, no significant change in IGF-I or IGF-II mRNA was detected. In spleen, however, IGF-I and IGF-II mRNA were both decreased in the transgenics, IGF-I mRNA even by the 19-fold. In agreement, in situ hybridisation revealed a largely reduced number of IGF-I mRNA-containing leukocytes and macrophages when compared to wild-type. These observations may contribute to better understanding the reported impaired health of GH-transgenic fish. Growth enhancement of the transgenics may be due to the increased expression of both IGF-I and IGF-II in extrahepatic sites. It is also reasonable that the markedly enhanced expression of liver IGF-II mRNA that may mimick an early developmental stage is a further reason for increased growth.

Transgenic Research, 1996

For the purpose of studying the factors that cause wide variation in transient transgene expressi... more For the purpose of studying the factors that cause wide variation in transient transgene expression in individual fish, a lacZ reporter gene linked to a carp [3-actin regulatory sequence was introduced into zebrafish embryos. As a general trend, a correlation between the number of transgene copies injected and the level of transgene expression was found. However, a substantial variation in the level of expression still occurred that could not be attributed to teclmical factors such as the difference in injected volume of the transgene. Co-injection of 32p-dCTP and transgene into the same embryo followed by detection of 13-galactosidase activity, has shown that the volume used for transgene injection, which was determined in terms of radioactivity, is not closely related to the level and location of transgene expression. Injection into the animal pole at zygote stage and the yolk cytoplasmic layer (YCL) at the 64-cell stage followed by determination of transgene expression in terms of unit injection volume, revealed that there are marked differences among tissues with regard to their capacity for transgene expression, and that the yolk syncytial layer is higher in this capacity. This high activity is assumed to be due to the high transcriptional activity or enhanced transgene replication in the syncytial layer, which is known to contain giant polyploid nuclei. The high levels of expression in the YSL may influence transient expression studies using quantitative comparative analyses and should be taken into consideration when expression data are derived from homogenates of yolk sac embryos.

Transgenic Research, 2005

We have previously produced transgenic fish from crosses between a wild-type female tilapia (Oreo... more We have previously produced transgenic fish from crosses between a wild-type female tilapia (Oreochromis niloticus) and a G1 transgenic male. This line of growth-enhanced tilapia carries a single copy of a chinook salmon (s) growth hormone (GH) gene spliced to an ocean pout antifreeze promoter (OPA-FPcsGH) co-ligated to a carp b-actin/lacZ reporter gene construct, integrated into the tilapia genome. Because little is known about the expression sites of transgenes, we have characterised the gene expression patterns of sGH and tilapia (t)GH in transgenic tilapia using a newly established real-time PCR to measure the absolute mRNA amounts of both hormones. The sGH gene, which was expected to be expressed mainly in liver, was also found to be expressed in other organs, such as gills, heart, brain, skeletal muscle, kidney, spleen, intestine and testes. However, in pituitary no sGH mRNA but only tGH mRNA was found. Tilapia GH mRNA in wild-type pituitary amounted to 226 ± 30 pg/lg total RNA but in transgenics only to 187 ± 43 pg/lg total RNA. Liver exhibited the highest level of sGH mRNA (8.3 ± 2.5 pg/lg total RNA) but the extrahepatic sites expressed considerable amounts of sGH mRNA ranging from 4.1 ± 2.0 pg/lg total RNA in gills to 0.2 ± 0.08 pg/lg total RNA in kidney. The widespread expression of the sGH gene is assumed to be due to the tissue specificity of the type III AFP gene promoter. It is assumed that our transgenic experiments, which in contrast to some other approaches caused no obvious organ abnormalities, mimick the GH expression during ontogeny. Because sGH mRNA is expressed both in liver and in extrahepatic sites it may not only promote secretion and release of liver-derived (endocrine) IGF-I leading to an overall growth enhancement but also stimulate IGF-I expression within the different organs in a paracrine/autocrine manner and, thus, further promote organ growth.

Transgenic Research, 1996

Transgenic fish, owing to a number of advantages which they offer over other species, are proving... more Transgenic fish, owing to a number of advantages which they offer over other species, are proving to be valuable model systems for the study of gene regulation and developmental genetics in addition to being useful targets for the genetic manipulation of commercially important traits. Despite having begun only a decade ago, the production of transgenic fish has become commonplace in a number of laboratories worldwide and considerable progress has been made. In this review, we initially consider the various regulatory elements and coding genes which have been used in fish, and subsequently discuss and compare both the transient and long-term fate and expression patterns of injected DNA sequences in the context of the different factors which are likely to have an effect on the expression of transgenes.

Nucleic Acids Research, 1984

The methylation sensitive restriction enzymes Hha I and Hpa II were used to analyse the methylati... more The methylation sensitive restriction enzymes Hha I and Hpa II were used to analyse the methylation pattern of a tRNA gene cluster in germ line and somatic DNA of Xenopus laevis. A single tDNA repeat containing 8 tRNA genes was studied and all copies were found to be fully modified in sperm DNA. In the DNA from erythroid cells, hepatocytes, kidney and brain, most tDNA repeats were found to be fully r,uoaified. However, in a fraction of the repeats, specific demethylated sites can be detected, giving rise to a pattern which does not vary significantly frorii one tissue to another. Although our results do not allow a straightforward correlation between hypomethylation and tRNA gene transcription, they are in agreement with the observation that hypomethylation accompanies differentiation and development. The tRNA genes of X. laevis are reiterated 8,000 times in toto in the haploid genome (12) and due to their clustered repetitive organization, they

Molecular Reproduction and Development, 1996

Molecular Reproduction and Development, 1997

The transient expression of reporter gene constructs in embryos provides a powerful tool to chara... more The transient expression of reporter gene constructs in embryos provides a powerful tool to characterise cis-acting transcriptional elements of the genes involved in development. In the present study, we have analysed the expression pattern of several muscle-specific and ubiquitous regulatory sequences in microinjected zebrafish embryos. By using a fast and reproducible coinjection strategy, the mosaic expression of lacZ reporter gene was monitored in wholemount embryos injected with sequences containing putative enhancer elements and a carp myosin heavy chain promoter/lacZ reporter construct. We have found that a 0.9-kb myosin heavy chain (MyHC) proximal promoter containing several putative myogenic regulatory factors (MRF) binding sites is sufficient to restrict lacZ expression to the skeletal muscle fibres of prim-6 stage zebrafish embryos. Expression of a rat-derived foetal myosin light chain enhancer (MyLC) and different fragments of a carp b-actin regulatory region together with the MyHC promoter were compared by accumulating the type, number and spatial distribution of b-galactosidase-expressing cells on an expression map. b-galactosidase activity increased similarly whether the MyLC enhancer was ligated to the promoter/ reporter construct directly or when coinjected as a separate fragment whilst skeletal muscle specificity was retained. The coinjection of two different forms of the b-actin regulatory elements also showed a marked effect on the MyHC promoter activity. The coinjection of putative enhancers with minimal promoter constructs and subsequent analysis of the transient expression pattern in the developing embryos provides a rapid and simple technique to identify cis acting activator elements of genes expressed in the vertebrate embryo.

Marine Biotechnology, 2005

Atlantic spiny lobsters support major fisheries in northeastern Brazilian waters and in the Carib... more Atlantic spiny lobsters support major fisheries in northeastern Brazilian waters and in the Caribbean Sea. To avoid reduction in diversity and elimination of distinct stocks, understanding their population dynamics, including structuring of populations and genetic diversity, is critical. We here explore the potential of using the hypervariable domain in the control region of the mitochondrial DNA as a genetic marker to characterize population subdivision in spiny lobsters, using Panulirus argus as the species model. The primers designed on the neighboring conserved genes have amplified the entire control region (approx. 780 bases) of P. argus and other closely related species. Average nucleotide and haplotype diversity within P. argus were found to be high, and population structuring was hypothesized. The data suggest a division of P. argus into genetically different phylogeographic groups. The hypervariable domain seems to be useful for determining genetic differentiation of geographically distinct stocks of P. argus and other Atlantic spiny lobsters.

Transgenic Research, 1998

Several lines of transgenic G1 and G2 tilapia fish (Oreochromis niloticus) have been produced fol... more Several lines of transgenic G1 and G2 tilapia fish (Oreochromis niloticus) have been produced following egg injection with gene constructs carrying growth hormone coding sequences of fish origin. Using a construct in which an ocean pout antifreeze promoter drives a chinook salmon growth hormone gene, dramatic growth enhancement has been demonstrated, in which the mean weight of the 7 month

Journal of Cell Science, 1975

The mass ratio of nuclear non-histone protein: DNA in the immature circulating erythroid cells of... more The mass ratio of nuclear non-histone protein: DNA in the immature circulating erythroid cells of phenylhydrazine-induced anaemic Xenopus is approximately threefold higher than in mature erythrocytes. This is largely due to the presence of increased amounts of low and intermediate molecular weight proteins in the nuclei of the immature cells. There are a few qualitative differences in the components of this class of proteins between the mature and immature cells, the most notable of which is the presence of a protein of molecular weight approximately 115000 in the former which is not detectable in the latter. These changes are discussed in relation to the changing synthetic capacities of cells and to certain generalizations about the function of the nuclear non-histone protein based on studies of other differentiating systems.

Preface Notes on use Appendix 1. Common names and latin names of some key organisms in cell biolo... more Preface Notes on use Appendix 1. Common names and latin names of some key organisms in cell biology and genetics. Appendix 2. Chromosome numbers in various species. Contents Appendix 3. DNA content of haploid genomes. Appendix 4. The greek alphabet. Appendix 5.

Molecular Ecology Resources, 2008

We present primers and amplification conditions for 15 microsatellite loci developed for the Cope... more We present primers and amplification conditions for 15 microsatellite loci developed for the Cope's giant salamander (Dicamptodon copei), 14 of which are tetranucleotide repeats. Cross-species amplification revealed 10 of these loci to also be polymorphic in the Pacific giant salamander (Dicamptodon tenebrosus). Several loci produced nonoverlapping allelic ranges between the two species and may be useful in species identification. These polymorphic microsatellite loci are potentially useful for future studies of population genetics in dicamptodontid salamanders.

Human HOX homeobox genes, D.Acampora et al the growth hormone/prolactin receptor gene family, P.K... more Human HOX homeobox genes, D.Acampora et al the growth hormone/prolactin receptor gene family, P.Kelly et al organization and evolution of satellite, minisatellite and microsatellite DNAs in teleost fishes, J.Franck et al nucleosome positioning and regulated gene expression, B.Pina et al two distinct tumour necrosis factor receptors - members of a new cytokine receptor gene family, H.Loetscher et al the CTLA-1 gene - a member of the granzyme multigene family in human and murine cytotoxic T cells, P.Haddad.

New Developments in Marine Biotechnology, 1998

Since the first introduction of novel genes in fish (Maclean and Talwar, 1984; Zhu et al., 1985),... more Since the first introduction of novel genes in fish (Maclean and Talwar, 1984; Zhu et al., 1985), a wide range of fish species have been used in transgenic research either for commercial or academic purposes. For commercial exploitation this technology has been applied in several commercially important fish species to improve desirable genetic traits such as growth (Rokkones et al., 1989; Zhang et al., 1990; Brem et al., 1988; Penman et al., 1990; Gross et al., 1992), cold tolerance (Fletcher et al., 1988), or disease resistance (Anderson et al., 1996) for aquaculture development. Most of these gene transfer experiment have concentrated on growth enhancement using regulatory sequences and coding sequences of distantly related species (Rokkones et al., 1989; Penman et al., 1990; Brem et al., 1988; Zhang et al., 1990). Although successful gene transfer in these fish was observed in only a few cases has germline transmission and expression of transgenes in progeny been convincingly demonstrated (see reviews by Maclean and Rahman, 1994; Gong and Hew, 1995). In studies where the regulatory sequences used were of mammalian origin, low or nil expression was observed (Guyomard et al., 1989; Alam et al., 1996). In other studies, enhanced growth was observed in transgenic carp (Zhang et al., 1990) and in tilapia (Martinez et al., 1996) when growth hormone sequences were driven by a viral promoter. However, the main objective of the production of transgenic fish with GH gene is to generate novel strain of growth enhanced fish which could subsequently be used in aquaculture. It is, therefore, important that the gene constructs used for producing trans-genic fish be of fish origin, and not from mammalian or viral origin. Moreover, it was also observed that regulatory sequences from fish are found to be more effective than those of mammalian origin in respect to expression of transgenes (Alam et al., 1996) in fish.

Transgenic Research, 2000

Three lines of transgenic tilapia (Oreochromisniloticus) fish were generated with a constructcont... more Three lines of transgenic tilapia (Oreochromisniloticus) fish were generated with a constructcontaining a lacZ reporter gene spliced to a 4.7?kb 5' regulatory region of a carp beta actin gene. All these three lines contain different copy numbers oftransgenes and the levels of lacZ expressionwere found to be related to transgene copy number.Mosaic patterns of somatic lacZ expression wereobserved in these

Transgenic Research, 2007

Several lines of growth hormone (GH)overexpressing fish have been produced and analysed for growt... more Several lines of growth hormone (GH)overexpressing fish have been produced and analysed for growth and fertility parameters. However, only few data are available on the growth-promoting hormone insulin-like growth factor I (IGF-I) that mediates most effects of GH, and these are contradictory. Using quantitative real-time RT-PCR, radioimmunoassay, in situ hybridization, immunohistochemistry, and radiochromatography we investigated IGF-I and IGF binding proteins (IGFBPs) in an adult (17 months old) transgenic (GH-overexpressing) tilapia (Oreochromis niloticus). The transgenics showed an around 1.5-fold increase in length and an approximately 2.3-fold higher weight than the non-transgenics. Using radioimmunoassay, the serum IGF-I levels were lower (6.22 ± 0.75 ng/ml) in transgenic than in wild-type (15.01 ± 1.49 ng/ml) individuals (P = 0.0012). Radioimmunoassayable IGF-I in transgenic liver was 4.2times higher than in wild-type (16.0 ± 2.21 vs. 3.83 ± 0.71 ng/g, P = 0.0017). No hepatocytes in wild-type but numerous hepatocytes in transgenic liver contained IGF-I-immunoreactivity. RT-PCR revealed a 1.4-times higher IGF-I mRNA expression in the liver of the transgenics (10.51 ± 0.82 vs. 7.3 ± 0.49 pg/mg total RNA, P = 0.0032). In correspondence, in situ hybridization showed more IGF-I mRNA containing hepatocytes in the transgenics. A twofold elevated IGF-I mRNA expression was determined in the skeletal muscle of transgenics (0.33 ± 0.02 vs. 0.16 ± 0.01 pg/mg total RNA, P < 0.0001). Both liver and serum of transgenics showed increased IGF-I binding. The increased IGFBP content in the liver may lead to retention of IGF-I, and/or the release of IGF-I into the circulation may be slower resulting in accumulation of IGF-I in the hepatocytes. Our results indicate that the enhanced growth of the transgenics likely is due to enhanced autocrine/paracrine action of IGF-I in extrahepatic sites, as shown here for skeletal muscle.

Transgenic Research, 2009

Several lines of GH-overexpressing fish have been produced and characterized concerning organ int... more Several lines of GH-overexpressing fish have been produced and characterized concerning organ integrity, growth, fertility and health but few and contradictory data are available on IGF-I that mediates most effects of GH. Furthermore, nothing is known on IGF-II. Thus, the expression of both IGFs in liver and various extrahepatic sites of adult transgenic (GH-overexpressing) tilapia and agematched wild-type fish was determined by real-time PCR. Both IGF-I and IGF-II mRNA were found in all organs investigated and were increased in gills, kidney, intestine, heart, testes, skeletal muscle and brain of the transgenics (IGF-I: 1.4-4-fold; IGF-II: 1.7-4.2-fold). Except for liver, brain and testis the increase in IGF-I mRNA was higher than that in IGF-II mRNA. In pituitary, no significant change in IGF-I or IGF-II mRNA was detected. In spleen, however, IGF-I and IGF-II mRNA were both decreased in the transgenics, IGF-I mRNA even by the 19-fold. In agreement, in situ hybridisation revealed a largely reduced number of IGF-I mRNA-containing leukocytes and macrophages when compared to wild-type. These observations may contribute to better understanding the reported impaired health of GH-transgenic fish. Growth enhancement of the transgenics may be due to the increased expression of both IGF-I and IGF-II in extrahepatic sites. It is also reasonable that the markedly enhanced expression of liver IGF-II mRNA that may mimick an early developmental stage is a further reason for increased growth.

Transgenic Research, 1996

For the purpose of studying the factors that cause wide variation in transient transgene expressi... more For the purpose of studying the factors that cause wide variation in transient transgene expression in individual fish, a lacZ reporter gene linked to a carp [3-actin regulatory sequence was introduced into zebrafish embryos. As a general trend, a correlation between the number of transgene copies injected and the level of transgene expression was found. However, a substantial variation in the level of expression still occurred that could not be attributed to teclmical factors such as the difference in injected volume of the transgene. Co-injection of 32p-dCTP and transgene into the same embryo followed by detection of 13-galactosidase activity, has shown that the volume used for transgene injection, which was determined in terms of radioactivity, is not closely related to the level and location of transgene expression. Injection into the animal pole at zygote stage and the yolk cytoplasmic layer (YCL) at the 64-cell stage followed by determination of transgene expression in terms of unit injection volume, revealed that there are marked differences among tissues with regard to their capacity for transgene expression, and that the yolk syncytial layer is higher in this capacity. This high activity is assumed to be due to the high transcriptional activity or enhanced transgene replication in the syncytial layer, which is known to contain giant polyploid nuclei. The high levels of expression in the YSL may influence transient expression studies using quantitative comparative analyses and should be taken into consideration when expression data are derived from homogenates of yolk sac embryos.

Transgenic Research, 2005

We have previously produced transgenic fish from crosses between a wild-type female tilapia (Oreo... more We have previously produced transgenic fish from crosses between a wild-type female tilapia (Oreochromis niloticus) and a G1 transgenic male. This line of growth-enhanced tilapia carries a single copy of a chinook salmon (s) growth hormone (GH) gene spliced to an ocean pout antifreeze promoter (OPA-FPcsGH) co-ligated to a carp b-actin/lacZ reporter gene construct, integrated into the tilapia genome. Because little is known about the expression sites of transgenes, we have characterised the gene expression patterns of sGH and tilapia (t)GH in transgenic tilapia using a newly established real-time PCR to measure the absolute mRNA amounts of both hormones. The sGH gene, which was expected to be expressed mainly in liver, was also found to be expressed in other organs, such as gills, heart, brain, skeletal muscle, kidney, spleen, intestine and testes. However, in pituitary no sGH mRNA but only tGH mRNA was found. Tilapia GH mRNA in wild-type pituitary amounted to 226 ± 30 pg/lg total RNA but in transgenics only to 187 ± 43 pg/lg total RNA. Liver exhibited the highest level of sGH mRNA (8.3 ± 2.5 pg/lg total RNA) but the extrahepatic sites expressed considerable amounts of sGH mRNA ranging from 4.1 ± 2.0 pg/lg total RNA in gills to 0.2 ± 0.08 pg/lg total RNA in kidney. The widespread expression of the sGH gene is assumed to be due to the tissue specificity of the type III AFP gene promoter. It is assumed that our transgenic experiments, which in contrast to some other approaches caused no obvious organ abnormalities, mimick the GH expression during ontogeny. Because sGH mRNA is expressed both in liver and in extrahepatic sites it may not only promote secretion and release of liver-derived (endocrine) IGF-I leading to an overall growth enhancement but also stimulate IGF-I expression within the different organs in a paracrine/autocrine manner and, thus, further promote organ growth.

Transgenic Research, 1996

Transgenic fish, owing to a number of advantages which they offer over other species, are proving... more Transgenic fish, owing to a number of advantages which they offer over other species, are proving to be valuable model systems for the study of gene regulation and developmental genetics in addition to being useful targets for the genetic manipulation of commercially important traits. Despite having begun only a decade ago, the production of transgenic fish has become commonplace in a number of laboratories worldwide and considerable progress has been made. In this review, we initially consider the various regulatory elements and coding genes which have been used in fish, and subsequently discuss and compare both the transient and long-term fate and expression patterns of injected DNA sequences in the context of the different factors which are likely to have an effect on the expression of transgenes.

Nucleic Acids Research, 1984

The methylation sensitive restriction enzymes Hha I and Hpa II were used to analyse the methylati... more The methylation sensitive restriction enzymes Hha I and Hpa II were used to analyse the methylation pattern of a tRNA gene cluster in germ line and somatic DNA of Xenopus laevis. A single tDNA repeat containing 8 tRNA genes was studied and all copies were found to be fully modified in sperm DNA. In the DNA from erythroid cells, hepatocytes, kidney and brain, most tDNA repeats were found to be fully r,uoaified. However, in a fraction of the repeats, specific demethylated sites can be detected, giving rise to a pattern which does not vary significantly frorii one tissue to another. Although our results do not allow a straightforward correlation between hypomethylation and tRNA gene transcription, they are in agreement with the observation that hypomethylation accompanies differentiation and development. The tRNA genes of X. laevis are reiterated 8,000 times in toto in the haploid genome (12) and due to their clustered repetitive organization, they

Molecular Reproduction and Development, 1996

Molecular Reproduction and Development, 1997

The transient expression of reporter gene constructs in embryos provides a powerful tool to chara... more The transient expression of reporter gene constructs in embryos provides a powerful tool to characterise cis-acting transcriptional elements of the genes involved in development. In the present study, we have analysed the expression pattern of several muscle-specific and ubiquitous regulatory sequences in microinjected zebrafish embryos. By using a fast and reproducible coinjection strategy, the mosaic expression of lacZ reporter gene was monitored in wholemount embryos injected with sequences containing putative enhancer elements and a carp myosin heavy chain promoter/lacZ reporter construct. We have found that a 0.9-kb myosin heavy chain (MyHC) proximal promoter containing several putative myogenic regulatory factors (MRF) binding sites is sufficient to restrict lacZ expression to the skeletal muscle fibres of prim-6 stage zebrafish embryos. Expression of a rat-derived foetal myosin light chain enhancer (MyLC) and different fragments of a carp b-actin regulatory region together with the MyHC promoter were compared by accumulating the type, number and spatial distribution of b-galactosidase-expressing cells on an expression map. b-galactosidase activity increased similarly whether the MyLC enhancer was ligated to the promoter/ reporter construct directly or when coinjected as a separate fragment whilst skeletal muscle specificity was retained. The coinjection of two different forms of the b-actin regulatory elements also showed a marked effect on the MyHC promoter activity. The coinjection of putative enhancers with minimal promoter constructs and subsequent analysis of the transient expression pattern in the developing embryos provides a rapid and simple technique to identify cis acting activator elements of genes expressed in the vertebrate embryo.

Marine Biotechnology, 2005

Atlantic spiny lobsters support major fisheries in northeastern Brazilian waters and in the Carib... more Atlantic spiny lobsters support major fisheries in northeastern Brazilian waters and in the Caribbean Sea. To avoid reduction in diversity and elimination of distinct stocks, understanding their population dynamics, including structuring of populations and genetic diversity, is critical. We here explore the potential of using the hypervariable domain in the control region of the mitochondrial DNA as a genetic marker to characterize population subdivision in spiny lobsters, using Panulirus argus as the species model. The primers designed on the neighboring conserved genes have amplified the entire control region (approx. 780 bases) of P. argus and other closely related species. Average nucleotide and haplotype diversity within P. argus were found to be high, and population structuring was hypothesized. The data suggest a division of P. argus into genetically different phylogeographic groups. The hypervariable domain seems to be useful for determining genetic differentiation of geographically distinct stocks of P. argus and other Atlantic spiny lobsters.

Transgenic Research, 1998

Several lines of transgenic G1 and G2 tilapia fish (Oreochromis niloticus) have been produced fol... more Several lines of transgenic G1 and G2 tilapia fish (Oreochromis niloticus) have been produced following egg injection with gene constructs carrying growth hormone coding sequences of fish origin. Using a construct in which an ocean pout antifreeze promoter drives a chinook salmon growth hormone gene, dramatic growth enhancement has been demonstrated, in which the mean weight of the 7 month