PubMed Health⌕ Search

Biomedical subjects

N Maclean

Publications and source records attributed to N Maclean.

At least 37 records · Page 2Linked to original sources
PubMed1998

Isolation and functional analysis of the histone H3 promoter from atlantic salmon (Salmo salar L.).

The histone H3 (sH3) promoter of Atlantic salmon (Salmo salar) was cloned via polymerase chain reaction using primers designed from the rainbow trout (Oncorhynchus mykiss) promoter sequence. A comparison of the nucleotide sequence with the equivalent sequences from rainbow trout and sockeye salmon (Oncorhynchus nerka) revealed a high degree of conservation. In vivo expression analysis of the sH3 promoter was carried out in both rainbow trout and zebrafish (Danio rerio) embryos. A direct comparison of the sH3 promoter with the viral RSV promoter in rainbow trout resulted in stronger expression of the sH3 promoter. Furthermore, lacZ expression directed by the sH3 promoter was ubiquitous in several different cell types in developing zebrafish embryos. These results suggest that the sH3 promoter will be useful in transgenic studies in Atlantic salmon.

Animals↗
PubMed1997

Activator effect of coinjected enhancers on the muscle-specific expression of promoters in zebrafish embryos.

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 beta-actin regulatory region together with the MyHC promoter were compared by accumulating the type, number and spatial distribution of beta-galactosidase-expressing cells on an expression map. beta-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 beta-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.

Actins↗
PubMed1997

Genetic structure of an aphid studied using microsatellites: cyclic parthenogenesis, differentiated lineages and host specialization.

In a previous study, samples of the grain aphid Sitobion avenae (F.) were collected from wheat and adjacent cocksfoot hosts in a population thought to be primarily parthenogenetic, and DNA from individual aphids was analysed with a multilocus technique. Here we have applied single-locus microsatellites and a mitochondrial DNA marker to a subset of the same DNA extracts, and have made several additional inferences about important genetic and population processes in S. avenae. Microsatellite analysis indicated very high levels of genic and genotypic variation. S. avenae fell into three genotypic groups inferred to be almost noninterbreeding, while analysis of linkage and Hardy-Weinberg equilibria suggested high levels of sexual recombination within each genotypic group. Host specialization was evident: one lineage was found only on wheat, and one (bearing many alleles inferred to be introgressed from the blackberry-grass aphid S. fragariae (Walker)) was found only on cocksfoot. The third group of interrelated genotypes was found commonly on both hosts. Although most genotypes were found only once, some were much more numerous in the sample than expected from the frequency of the alleles they contained. This, and rapid temporal changes in genotypic composition of samples, indicates strong selective differences between genotypes and lineages. In the major genotypic group, the commonest genotypes were significantly more homozygous than were rare ones: thus these data may help to explain the frequent observation of homozygous excess in aphid allozymes. The genotype group showing S. avenae-like as well as S. fragariae-like alleles also carried S. fragariae-like mitochondrial DNA in at least 25/31 cases, indicating gender-asymmetrical hybridization.

Animals↗
PubMed1996

Comparison of the activity of carp and rat beta-actin gene regulatory sequences in tilapia and rainbow trout embryos.

A comparative study on the level of expression of lacZ reporter constructs driven by equivalent carp and rat beta-actin regulatory sequences was carried out in embryos of tilapia and rainbow trout. DNA was microinjected into fertilised tilapia and rainbow trout eggs and the embryos/fry were assayed at various developmental stages for beta-galactosidase expression. We provide evidence to demonstrate that the carp beta-actin promoter/ lacZ reporter gene is expressed at higher levels than the equivalent rat beta-actin construct in both species.

Actins↗
PubMed1996

Regulation and expression of transgenes in fish -- a review.

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 development 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 world-wide 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.

Animals↗
PubMed1996

High transgene activity in the yolk syncytial layer affects quantitative transient expression assays in zebrafish Danio rerio) embryos.

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 beta-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 technical 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 beta-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.

Actins↗
PubMed1994

An analysis of secondary spread by putative clones of Sitobion avenae within a Hampshire wheat field using the multilocus (GATA)4 probe.

Grain aphids (Sitobion avenae (F.)) were collected from forty-four wheat ears in a Hampshire field at three times during the growing season. On each occasion, individual aphids were profiled using the multilocus (GATA)4 probe. During the full head emergence and full anthesis growth stages of wheat, each ear generally supported a genetically distinguishable aphid colony which consisted of genetically indistinguishable individuals (putative clones). This information strongly suggests that individual ears were colonized by single immigration events. By the late milky ripe stage, most ears supported two or more such clones. The total number of clones declined and the spatial separation of identical clones increased markedly over the duration of the study, which strongly suggests that secondary spread rather than increased immigration was responsible for the increased clonal diversity of ears. In addition, the profiles of individual S. avenae became more alike as the season progressed and samples became dominated by particular clones indicating either differential survival or reproduction among clones.

Animals↗
PubMed1992

Response of Xenopus laevis to cadmium administration.

1. Levels of copper, zinc and cadmium have been determined in the plasma, liver and kidneys of Xenopus laevis. 2. Administration of cadmium by subcutaneous injection induces metallothionein synthesis in the liver of Xenopus laevis. Cadmium is found to accumulate in both liver and kidneys following injection. 3. Cadmium uptake, following injection, is biphasic, suggesting a saturable first-phase uptake mechanism, followed by a linear second phase. 4. Zinc is displaced from the liver by administration of cadmium in a dose-dependent manner.

Animals↗
PubMed1991

Patterns of transgene inheritance in rainbow trout (Oncorhynchus mykiss).

There have been very few studies of the inheritance of introduced genes (transgenes) in fish. We have followed the inheritance of the mammalian fusion gene MTrGH from founder generation transgenics (originating from eggs microinjected with the MTrGH DNA) to offspring in crosses with control fish. Initial screening of the founder generation transgenics was by analysing DNA from blood samples. Only three out of six fish which carried the novel gene in blood DNA transmitted it to their offspring, despite the presence of the gene in DNA extracted from the sperm of all four male fish in this group. The frequency of transgenics in the progeny groups from the three fish which transmitted the gene varied widely: in one of these groups more than one type of MTrGH restriction pattern was found. These results suggest widespread mosaicism in founder generation transgenics.

Animals↗
PubMed1990

Effects of 5-azacytidine and 5-aza-2-deoxycytidine on alphafetoprotein levels in mice.

1. Production of alphafetoprotein in adult C3H mice was monitored by radial immunodiffusion both in controls, and in animals treated with carbon tetrachloride, 5-azacytidine, or 5-aza-2-deoxycytidine, either alone or in combination. 2. Carbon tetrachloride routinely induced alphafetoprotein synthesis in our experiments, but neither of the cytidine analogues showed any effects on the serum levels of this protein when administered alone. 3. Treatment of mice with either cytidine analogue prior to carbon tetrachloride injection markedly reduced the consequent production of alphafetoprotein, whereas if carbon tetrachloride injection was followed by a subsequent injection with either cytidine analogue, a markedly enhanced level of serum alphafetoprotein was detected. 4. It is suggested that carbon tetrachloride induces alphafetoprotein production in adult mice by inducing liver damage, followed by synthesis of the protein in the dividing and differentiating cells during recovery. We also propose that the cytidine analogues ablate this response by a cytotoxic effect on the liver cells when they are administered prior to the CCl4, but enhance the alphafetoprotein levels when administered after the CCl4 because they inhibit the methylation of cytidine residues in the recovery cell population in the liver and thus prevent early cessation of synthesis of the protein.

Animals↗
PubMed1989

Detection of retrovirus in patients with myeloproliferative disease.

RNA-directed DNA polymerase (reverse transcriptase) activity was detected in platelets from 4/4 patients with primary proliferative polycythaemia (PPP), 7/7 patients with essential thrombocythaemia (ET), 1/4 patients with relative "stress" polycythaemia, 0/2 patients with secondary polycythaemia, and 0/3 normal subjects. The activity appeared to be particle-associated and was detected under conditions not appropriate for any known cellular enzymes. Active particulate fractions from 1 patient with PPP and 1 patient with ET were examined by electron microscopy and revealed objects with the features of retroviruses. Similar retrovirus-like particles were observed in 2/2 further patients with PPP and 2/2 further patients with ET but in 0/3 controls.

Blood Platelets↗
PubMed1988

Responses of trout fry (Salmo gairdneri) and Xenopus laevis tadpoles to cadmium and zinc.

1. Toxicity of cadmium chloride and zinc sulphate in solution has been determined for Xenopus tadpoles and rainbow trout fry in recirculated water systems. 2. Both animals show approximately equal tolerance of zinc but Xenopus tadpoles tolerate cadmium at approximately 10 times the lethal concentration for trout fry. 3. In the case of Xenopus tadpoles, pre-treatment with sub-lethal concentrations of cadmium or zinc protects against subsequent exposure to either metal. For trout fry, cadmium gives little protection against later exposure to either cadmium or zinc, while zinc gives moderate protection against either metal. 4. Protection is attributed to metallothionein [MT] synthesis.

Animals↗
PubMed1987

Injection of the mouse MT-1 gene into rainbow trout eggs and assay of trout fry for resistance to cadmium and zinc toxicity.

The mouse MT-1 gene, cloned into the plasmid pBR 322, has been injected into fertilised eggs of the rainbow trout, Salmo gairdneri. About 10(6) copies of the construct, either circular or linearized, have been injected into 1 cell stage eggs within 4 hours of fertilisation, and fry hatched from such eggs are being assayed for evidence of integration of the injected sequence. In addition, with a view to the development of an assay which can be used to monitor for expression of the injected gene, uninjected fry have been exposed to various concentrations of cadmium chloride or zinc sulphate, with or without prior exposure to inducing doses of these compounds. Whereas a distinct protection is evident following induction with zinc, no such protection has been detected with cadmium induction.

Animals↗
PubMed1984

The methylation pattern of tRNA genes in Xenopus laevis.

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 modified. However, in a fraction of the repeats, specific demethylated sites can be detected, giving rise to a pattern which does not vary significantly from 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.

Animals↗
PubMed1984

Hydrocarbon transport in chylomicrons and high-density lipoproteins in rat.

A lipoprotein system is described that transports gut hydrocarbons of low polarity in chylomicrons of intestinal lymph and plasma to plasma high density lipoproteins (HDL) in rat. Four highly lipophilic aryl and alkyl hydrocarbons [benzo(alpha)pyrene; 1,1,1-trichloro-2,2-bis(p-chlorophenol)ethane (DDT), hexadecane and octadecane] were selected to give a graded range of polarity. Chylomicrons were labeled doubly with radioisotopes in triacylglycerol and a single hydrocarbon by feeding [3H]-glycerol and [14C]hydrocarbon. All hydrocarbons were transported in the triacylglycerol oil phase of chylomicrons. Injected chylomicron triacylglycerol and 3 of 4 hydrocarbons were cleared simultaneously from plasma consistent with lipoprotein-lipase dependent hydrocarbon clearance but DDT was cleared more rapidly. HDL was the major plasma acceptor of all labelled hydrocarbons. Plasma chemical fluxes were measured for octadecane and DDT and both showed net fluxes from chylomicrons to HDL. HDL selectively concentrated chylomicron hydrocarbons from chylomicron triacylglycerol. Lipoprotein lipase stimulation by intravenous heparin significantly increased transfer of alkanes from chylomicrons to HDL. These results indicate that (a) chylomicrons transport gut-derived hydrocarbons with a wide range of structure and polarity as triacylglycerol solutes; (b) HDL are a major plasma acceptor of all these hydrocarbons, demonstrating both selective solute uptake from triacylglycerol and net chemical uptake for the 2 hydrocarbons studied and (c) efflux of these chylomicron hydrocarbons from plasma and into HDL is regulated partly by hydrolysis of chylomicron triacylglycerol.

Alkanes↗
PubMed1983

A calcium-activated nuclease endogenous to Xenopus erythrocytes.

An endogenous nuclease is found in purified Xenopus erythrocyte nuclei. This enzyme is activated specifically by Ca2+ in the presence of which chromatin is digested into discrete fragments. Both 10 bp and 200 bp ladders are detected upon electrophoresis of DNA. This endogenous enzyme therefore resembles mammalian and chicken liver endonucleases.

Animals↗
PubMed1981

Structural transitions of chromatin in isolated Xenopus erythrocyte nuclei. I. The effects of ions.

Nuclei from Xenopus erythrocytes have been isolated under varying ionic conditions and their morphology studied by light and electron microscopy. The 200-A beaded chromatin fibre observed in vivo is maintained in media containing MgCl2 or CaCl2, but not in those containing Mn2+ as the sole divalent cation. In the absence of these alkaline-earth metal ions, 100-A filaments predominate. This suggests that the native 200-A fibre in isolated nuclei is also stabilized by specific divalent cations. Gross chromatin condensation within the nucleus occurs independently of whether the chromatin is in the form of 100-A or 200-A fibres, but requires the presence of divalent cations. Lower concentrations of MgCl2 or CaCl2 are necessary as compared with MnCl2. In the presence of sufficient divalent cations, decreasing KCl concentration causes peripheral condensation, the condensation of chromatin towards the nuclear membrane. Monovalent cation alone does not appear capable of inducing peripheral condensation. The transcriptional capacities of the different morphological types identified have been studied using E. coli RNA polymerase as a probe. Neither the degree of chromatin condensation nor the state of higher-order coiling have a significant effect on the rate of transcription of the DNA. These results are discussed with regard to further in vitro studies on eukaryotic gene activity.

Animals↗