Genetic identification of an endoproteinase encoded by the adenovirus genome.

The temperature-sensitive adenovirus type 2 mutant H2ts1 is defective for polypeptide processing at the non-permissive temperature. We have in the present study mapped the mutation by marker rescue and DNA sequencing techniques: the mutation is a C/T transition located at map co-ordinate 61.1. Previous sequencing studies have identified an uninterrupted translational reading frame in this part of the adenovirus genome, encoding a hypothetical 23 X 10(3) Mr polypeptide. The mutation leads to a proline/leucine substitution in the 23 X 10(3) Mr polypeptide.

A common sequence in the inverted terminal repetitions of human and avian adenoviruses.

The termini of the avian chick embryo lethal orphan (CELO) virus DNA have been sequenced. The results revealed a 63-bp-long inverted terminal repetition (ITR) which shared the sequence ATAATA with all adenovirus termini, thus far analyzed. The CELO virus ITR differed from those of the mammalian adenoviruses in two major aspects: (i) it is not a perfect duplication; (ii) it begins with a 5'-guanylic acid residue instead of the cytidylic acid normally observed in adenoviruses.

Efficient secretion of human parathyroid hormone by Saccharomyces cerevisiae.

A cDNA encoding mature human parathyroid hormone (hPTH) was expressed in Saccharomyces cerevisiae, after fusion to the prepro region of yeast mating factor alpha (MF alpha). Radioimmunoassay showed high levels of hPTH immunoreactive material in the growth medium (up to 10 micrograms/ml). More than 95% of the immunoreactive material was found extracellularly as multiple forms of hormone peptides. Three internal cleavage sites were identified in the hPTH molecule. The major cleavage site, after a pair of basic amino acids (aa) (Arg25Lys26 decreases Lys27), resembles that recognized by the KEX2 gene product on which the MF alpha expression-secretion system depends. The use of a protease-deficient yeast strain and the addition of high concentrations of aa to the growth medium, however, not only changed the peptide pattern, but also resulted in a significant increase in the yield of intact hPTH (1-84) (more than 20% of the total amount of immunoreactive material). The secreted hPTH (1-84) migrates like a hPTH standard in two different gel-electrophoretic systems, co-elutes with standard hPTH on reverse-phase high-performance liquid chromatography, reacts with two hPTH antibodies raised against different parts of the peptide, has a correct N-terminal aa sequence, and has full biological activity in a hormone-sensitive osteoblast adenylate cyclase assay.

Localization of salmon gonadotropin-releasing hormone mRNA and peptide in the brain of Atlantic salmon and rainbow trout.

The decapeptide gonadotropin-releasing hormone (GnRH) is a key hormone for the central regulation of reproduction. The distribution of salmon GnRH (sGnRH), which is the major form in salmonids, has been studied in different fish species by immunocytochemistry. Discrepancies in data concerning the distribution of sGnRH perikarya led us to investigate this problem in two species, the Atlantic salmon and the rainbow trout, with in situ hybridizaiton of sGnRH messenger, a highly specific molecular tool. By Northern blot analysis, the rainbow trout sGnRH messenger appears to be about 500 bases in length, which is close to those isolated from Atlantic salmon or masu salmon and characterized previously. In situ hybridization with riboprobes generated with Atlantic salmon sGnRH cDNA demonstrated that sGnRH perikarya are restricted to the ventral part of olfactory bulbs, telencephalon, and preoptic area. They are distributed on a nearly continuous line extending from the olfactory bulbs to the preoptic area in both salmonid species studied. Despite the presence of GnRH-like immunoreactivity in the preoptic magnocellular nucleus (NPOm) and in the tegmentum of the midbrain (MT), the sGnRH mRNA is not present in these two structures. Stained cells in NPOm could be target cells for GnRH and immunoreactive neurons in MT are likely to be chicken GnRH-II containing cells. Our study not only gives a precise distribution of the sGnRH system in two salmonids, Atlantic salmon and rainbow trout, but also clarifies the ambiguous data published up to now in rainbow trout.

Sequence conservation in kringle IV-type 2 repeats of the LPA gene.

We have studied the homology of repeating kringle IV-type 2 (K IV-type 2) elements of the LPA gene. Two K IV-type 2 genomic polymerase chain reaction (PCR) fragment libraries were constructed, one from an individual with high and one from an individual with low Lp(a) lipoprotein level. Only minor K IV-type 2 repeat length heterogeneity was observed. Sequence analysis data from the cloned K IV-type 2 repeats revealed a high degree of LPA sequence conservation in exons as well as in introns both within and between the two libraries. This sequence conservation of the IV-type 2 kringles is in agreement with our previously reported results of simultaneous 'batch' DNA sequence analyses of all the K IV-type 2 repeats from single individuals. Sequence data from the clones, combined with genomic DNA sequencing, revealed that the K IV-type 2 reading frame of exons 1 and 2 are extended into the conserved flanking introns by 519 base pairs (bp) and 312 bp, respectively. The theoretical coding capacity of the exon 1 extended open reading frame (ORF I) is three times larger (173 amino acids, aa) than the translated exon 1, and that of the extended open reading frame of exon 2 (ORF II) is about twice (104 aa) the length of exon 2. A central portion of the intron separating exons 1 and 2 also exhibited a high degree of sequence conservation, with the exception of a polymorphic CA repeat. Within the 61 K IV repeat clones analysed, 19 different CA repeat patterns with 12-18 CA dinucleotide repeats were observed. A comparison between the 37 clones from the individual with high Lp(a) lipoprotein level and the 24 clones from the individual with low Lp(a) lipoprotein level, revealed that seven of the CA repeat variants were present in both clone libraries. The observed high level of sequence conservation in K IV-type 2 exons and introns matches relevant areas of the plasminogen gene, and our findings fit with recent K IV-type 2 duplications and evolutionary selection pressure theories, although gene conversion events could also explain the findings. DNA sequences within K IV-type 2 appeared to have no influence on Lp(a) lipoprotein level.

Histopathology of arterial lesions in LPA transgenic mice on cholesterol-enriched chow.

The aortic root from 21 LPA transgenic mice and 18 control litter mates on cholesterol enriched chow were studied histologically for the presence of atherosclerotic lesions. Serial sections were cut and the total area of the lesions was measured by use of computerised image analysis. Lipid staining lesions were found in 17 aortas of the transgenic mice and were five times more common than in the controls. Foam cell lesions were the only type of lesion in 12 of the aortas from transgenic animals, while five animals had developed fibrofatty lesions. Immunostaining revealed monocytes/macrophages on the endothelial surface, and in the subendothelial space of foam cell lesions. In fibrofatty lesions, spindle shaped cells formed a cap around the lipid core. This study supports the view that transgenic mice expressing human apolipoprotein (a) on a high fat and cholesterol diet, are more susceptible to aortic lesions than control mice and develop early atherosclerotic lesions comparable to lesions in man. Aminoguanidin in the drinking water had no effect on the aortic lesions, but lesion size was significantly, negatively correlated with plasma glucose concentration.

Transgenic rainbow trout expressed sGnRH-antisense RNA under the control of sGnRH promoter of Atlantic salmon.

A recombinant vector containing antisense DNA complementary to Atlantic salmon (Salmo salar) sGnRH cDNA driven by specific promoter Pab derived from a corresponding sGnRH gene was introduced into rainbow trout (Oncorhynchus mykiss) eggs. This resulted in transgenic animals that had integrated one copy of the transgene into their genome and transmitted it through the germline. Antisense-sGnRH mRNA (AS) was expressed mainly in the brain of transgenic AS(+) fish. Levels of sGnRH endogenous mRNA in the brain were lower in 11-month-old AS(+) fish compared with nontransgenic AS(-) individuals from the same F2 progeny. sGnRH levels significantly decreased in the pituitary of transgenic males and females around the maturation period and in the brain of AS(+) immature females compared with controls. No reliable statistical difference was found in the levels of FSH and LH between AS(+) and AS(-) groups either in immature or mature fish. The majority of transgenic fish reached maturity at the same time as did nontransgenic individuals, although the maturation of AS(+) animals seemed to be more asynchronous. For the first time, the influence of antisense messengers on endogenous mRNA in transgenic fish and the corresponding protein is described.

Signal transduction alterations in GH(1)2C1 rat pituitary tumour cells following treatment with 5-azacytidine.

In GH(1)2C1 rat pituitary cells treated with 5-azacytidine, the stimulatory effects exerted by vasoactive intestinal peptide (VIP), the GTP analogue guanyl-5'-yl imidodiphosphate (Gpp(NH)p), 12-O-tetradecanoyl phorbol 13-acetate, cholera toxin and pertussis toxin on the membrane-bound adenylyl cyclase were almost completely abolished. The corresponding inhibitory effect of somatostatin was increased. Alterations in adenylyl cyclase responsiveness began at the end of the drug treatment, and were most pronounced on day 5 after removal of 5-azacytidine. The cells subsequently and completely recovered after 10 days in the absence of the drug. Measurements of cholera toxin- and VIP-enhanced cyclic AMP levels in intact cells confirmed these results, and VIP appeared to have no stimulatory effect on GH secretion after 5-azacytidine treatment. Down-regulation of G alpha s RNA also occurred on day 5 after cessation of drug treatment. ADP-ribosylation subsequent to stimulation with pertussis toxin was markedly increased, indicating an enhancement of G alpha i and/or G alpha o. Furthermore, both basal and Gpp(NH)p-stimulated phospholipase C activities were augmented by pre-exposure to 5-azacytidine. Treatment of GH(1)2C1 rat pituitary tumour cells with 5-azacytidine therefore causes a marked but temporary increase in the ratio of G alpha i/G alpha s protein levels.

Estrogen receptor binds to the salmon GnRH gene in a region with long palindromic sequences.

Footprinting and gel shift assays demonstrated that the human estrogen receptor (hER) specifically binds to two estrogen response element (ERE)-like motifs in the gonadotropin releasing hormone (GnRH) gene promoter region of Atlantic salmon (Salmo salar). The two ER binding sites are situated approximately 1.5 kb upstream of the transcriptional start site of the GnRH gene and are localized 49 bp from each other. Each ERE-like motif is composed of two palindromic ERE half-sites interspaced by 8 and 9 nucleotides, respectively. The salmon GnRH gene promoter region contains an almost perfect 426-bp-long palindromic sequence that might form a cruciform structure.

The Atlantic salmon prepro-gonadotropin releasing hormone gene and mRNA.

Screening for the gene encoding salmon gonadotropin releasing hormone (sGnRH) in an Atlantic salmon (Salmo salar) genomic library resulted in isolation of a positive clone designated lambda sGnRH-1. An anchor polymerase chain reaction (PCR) technique was used to amplify GnRH cDNA derived from salmon hypothalamic mRNA. The cDNA sequence was aligned to the 7607 base pair genomic sequence which was shown to encode the entire prepro-GnRH gene. The cDNA proved that the cloned gene is expressed in the hypothalamus of mature salmon. The coding domain of sGnRH differs from the mammalian GnRH by six nucleotide changes which allow the two amino acid differences between the two GnRH variants. Salmon GnRH associated peptide (GAP) differs extensively in sequence and size from the mammalian counterpart. Compared to the GnRH cDNA of a cichlid species the similarity is 69.3% in the protein coding sequence.

Gene-Gun-Mediated Transfer of Reporter Genes to Somatic Zebrafish (Danio rerio) Tissues.

We describe the use of gene-gun-mediated transfer of luciferase and green fluorescent protein (GFP) reporter genes in zebrafish (Danio rerio). Optimization of DNA transfer parameters indicated highest overall luciferase expression in epidermis and dermis using 1-µm microcarriers and 1 µg of pCMVL plasmid DNA at a delivery pressure of 200 psi. Time course studies revealed luciferase activity peaking at 18 hours and decreasing to 30% of the maximum at day 8 after DNA transfer. Onset of reporter gene (GFP) expression was detected at 13 minutes after DNA delivery, and by 65 minutes approximately 100% of the cells in the target area exhibited GFP expression. No germline association or integration events were detected in a screen of approximately 250,000 zebrafish sperm cells by fluorescence in situ hybridization at 15 or 30 days after delivery of 1 µg of pCMVL DNA, suggesting incidental male germline integration should not be considered as a risk factor when using the biolistic DNA delivery parameters and target tissues described.

Differential gene expression and biomarkers in zebrafish (Danio rerio) following exposure to produced water components.

The main effluent from oil and gas production is produced water (PW), a waste that contains low to moderate concentrations of oil-derived substances such as polycyclic aromatic hydrocarbons (PAHs) and alkylphenols (APs). PW components may be present in seawater at low concentrations over large areas in the vicinity of oil and gas production facilities. In this study, zebrafish (Danio rerio) were exposed to control and three treatments (high-, pulsed-, low-dose) of a synthetic PW mixture for 1, 7 and 13 weeks. The aim was to investigate the development of transcriptome and biomarker responses as well as relationships between early responses and population-relevant effects. The synthetic PW contained a mixture of low-molecular-weight PAHs (<5 ring) and short-chain APs (C1-C4). The water-borne exposure levels (sum PAH) ranged from 0.54 ppb (low dose) to 5.4 ppb (high dose). Bile pyrene metabolites ranged from 17-133 ng g(-1) bile in the control group to 23-1081 ng g(-1) bile in the high exposure group. Similar levels have been observed in wild fish, confirming an environmentally relevant exposure. The expression of mRNAs of hepatic genes was investigated in the high exposure group using the Zebrafish OligoLibrary from Compugen. Functional clustering analysis revealed effects in the reproductive system, the nervous system, the respiratory system, the immune system, lipid metabolism, connective tissue and in a range of functional categories related to cell cycle and cancer. The majority of differentially expressed mRNAs of genes were down-regulated, suggesting reduction in gene transcription to be as relevant as up-regulation or induction when assessing biological responses to PW exposure. Biomarkers for effects of PAHs (cytochrome P450 1A) and environmental estrogens (vitellogenin) did not appear to be affected by the chronic exposure to low concentration of PW components. Effects at the population level included a reduction in condition factor in male fish from all exposed groups and spinal column deformations in the F1 generation of exposed groups. The different exposure regimes did not produce any significant differences in reproduction or recruitment. The results from this study demonstrate that environmentally relevant concentrations of PW affect gene expression and population-relevant endpoints in zebrafish, although links between the two were not obvious.

Nuclear localization signal of SV40 T antigen directs import of plasmid DNA into sea urchin male pronuclei in vitro.

Nuclear import of plasmid DNA mediated by a nuclear localization signal (NLS) derived from SV40 T antigen was investigated in a cell-free extract. In vitro assembled sea urchin male pronuclei were incubated in a 100,000g supernatant of a zebrafish fertilized egg lysate, together with fluorescently labeled plasmid DNA bound to NLS or nuclear import deficient reverse NLS (revNLS) peptides. After 3 hr, DNA-NLS, but not DNA-revNLS, complexes were bound around the nuclear periphery. We demonstrate that nuclear import of DNA-NLS complexes is a two-step process involving binding to, and translocation across, the nuclear envelope. Binding is ATP-independent, occurs at 0 degree C and is Ca(2+)-independent. By contrast, translocation requires ATP hydrolysis, Ca2+, is temperature dependent and is blocked by the lectin wheat germ agglutinin. Both binding and translocation are competitively inhibited by albumin-NLS conjugates, require heat-labile cytosolic factors, and are inhibited by N-ethylmaleimide treatment of the cytosol. Binding and translocation are differentially affected by cytosol dilutions, suggesting that at least two distinct soluble fractions are required for nuclear import. The requirements for NLS-mediated nuclear import of plasmid DNA are similar to those for nuclear import of protein-NLS conjugates in permeabilized cells.

Nuclear localization signals enhance germline transmission of a transgene in zebrafish.

We report that cytoplasmic injection into zebrafish eggs of 10(4) copies of plasmid DNA complexed to nuclear localization signal (NLS) peptides, as compared to 10(6) copies of naked DNA, increased nuclear uptake of transgene DNA early during embryo development and enhanced transgene integration frequency into the germline of founders. Monitoring the dynamics of nuclear uptake of DNA-NLS complexes by fluorescence in situ hybridization (FISH) of interphase nuclei indicates that NLS enhances both the proportion of nuclei importing DNA during early embryo development, and the amount of DNA imported by individual nuclei. The use of NLS increases the proportion of germline transgenic founders from 14 to 43% (P < 0.01) as assessed by polymerase chain reaction analysis of F1s. From germline transgenic DNA-NLS-injected founders, 47% transgenic F1s are obtained in wild-type crosses, as opposed to 6% from naked DNA-injected founders (P < 0.01). In both cases, the transgene is transmitted to the F2 generation. In addition, high-resolution FISH analysis of transgenic F1s reveals that the use of NLS increases the number of distinct transgene integration sites along chromatin fibres.

Rapid targeting of plasmid DNA to zebrafish embryo nuclei by the nuclear localization signal of SV40 T antigen.

Binding SV40 T antigen nuclear localization signals (NLSs) to plasmid DNA promotes transgene expression following injection of DNA-NLS complexes into the cytoplasm of zebrafish eggs. We now demonstrate that NLS peptides mediate import of DNA from the cytoplasm into embryo nuclei, under conditions in which naked DNA is not imported. Plasmid DNA was localized by polymerase chain reaction (PCR) in isolated nuclei, and relative amounts were quantified by densitometry. Binding DNA to NLSs, but not to nuclear-import-deficient peptides, promoted rapid targeting of DNA-NLS complexes to nuclei, and transport across the nuclear envelope. Import of DNA-NLS complexes was competed by co-injected albumin-NLS conjugates. NLS, but not reverse NLS, was detected on blots of nuclei probed with 32P-labeled DNA. The results suggest that NLS-mediated DNA transfer into nuclei may constitute a valuable tool for several gene transfer applications.

Nuclear localization signals: a driving force for nuclear transport of plasmid DNA in zebrafish.

Nuclear localization signals (NLSs) are short peptides required for nuclear transport of karyophilic proteins. We review in this paper how the nuclear targeting property of NLS peptides has been taken advantage of to enhance the efficiency of nuclear uptake of transgene DNA in zebrafish and how it may improve the efficiency of transgenesis in this species. Synthetic NLS peptides can bind to plasmid DNA by ionic interactions. Cytoplasmic injection of DNA-NLS complexes in zebrafish eggs enhances the rate and the amount of plasmid DNA taken up by embryonic nuclei. Nuclear import of DNA-NLS complexes has been duplicated in vitro and exhibits energetic and cytosolic requirements similar to those for nuclear protein import. Furthermore, binding NLSs to DNA increases expression frequency of the transgene. We suggest that NLS peptides may constitute a valuable tool to improve the efficiency of transgenesis in zebrafish and other species.

The nuclear localization sequence of the SV40 T antigen promotes transgene uptake and expression in zebrafish embryo nuclei.

We report luciferase expression in zebrafish embryos after cytoplasmic injection of low copy numbers of plasmid DNA coupled to the SV40 T antigen nuclear localization sequence (NLS). Binding of NLS to plasmid DNA (pCMVL) occurs at room temperature in 0.25 M KCl, as assayed by gel retardation at molar ratios of NLS:pCMVL of at least 100:1. Luciferase expression is induced in 35% of embryos with as low as 10(3) NLS-bound pCMVL copies. With 10(4) copies, the proportion of expression increases from 6% at 0:1 to 70% 100:1 NLS:pCMVL (p < 0.01). The beneficial effect of NLS is abolished at DNA concentrations promoting high frequencies of transgene expression without NLS. Regardless of the DNA concentration, the use of NLS does not affect embryo viability for at least up to 10 days. The specificity of NLS on luciferase expression was tested by using a nuclear import deficient reverse NLS peptide (revNLS). revNLS binds to pCMVL, causing gel retardation similarly to NLS, but does not promote transgene expression. Binding of equimolar amounts of revNLS and NLS to DNA reduces by 50% the beneficial effect of NLS on transgene expression. The results suggest efficient targeting of MLS-bound plasmid DNA to the nucleus, and subsequent enhanced uptake of DNA by the nucleus. The data suggest that the use of NLS may reduce the need for using elevated DNA copy numbers in some gene transfer applications.

Sequence of inverted terminal repetitions from different adenoviruses: demonstration of conserved sequences and homology between SA7 termini and SV40 DNA.

We have established the nucleotide sequence for the inverted terminal repetition of human adenovirus type 3, a subgroup B adenovirus. The repetition, which is 136 bp long, shows a high degree of homology with the known sequence for the inverted repetition of adenovirus type 5 (Steenbergh et al., 1977) a subgroup C adenovirus. Partial sequence information convering 120 bp of the inverted terminal repetitions of human serotype 12, a subgroup A member, and of simian adenovirus type 7 has also been obtained. A comparison of the established sequences shows that the terminal repetitions, in particular the first 50 bp from the ends, contain sequences that have been well conserved in adenovirus evolution. For instance, only six mismatched base pairs were detected among the first 50 bp in the repetitions of simian adenovirus type 7 and human adenovirus type 5, although the homology between simian adenovirus 7 and human subgroup C adenoviruses was estimated to be only 30%. A 14 bp sequence located 9-22 nucleotides from the ends is present in DNAs from all the human serotypes examined as well as in simian adenovirus 7 DNA. Furthermore, the simian adenovirus 7 repetition contains a 21 bp sequence which is present in SV40 DNA, close to the origin of DNA replication.

A functional study of the salmon GnRH promoter.

The Pa and Pb promoters of the Atlantic salmon (Salmo salar) GnRH gene were fused together or individually to the Escherichia coli lacZ gene, and their transcriptional activities were measured in transient expression assays in zebrafish (Danio rerio). In 48-hour embryos, both promoters were preferentially expressed in the brain, whereas a cytomegalovirus (CMV) promoter-lacZ fusion gene displayed high levels of activity in nonbrain tissues. Pa and Pb exhibited different cell specificity in the forebrain. Pb was active in large neuron-like cells exclusive in the olfactory placode region, whereas Pa appeared active in nonneuron-like cells in the forebrain. In Atlantic salmon forebrain tissue, both Pa and Pb exhibited endogenous activity, as assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. However, only the Pb transcript contained the prepro-GnRH exon II-IV sequences, suggesting that Pa activity may not be related to GnRH production in this species.

High-degree sequence conservation in LPA kringle IV-type 2 exons and introns.

In the search for factors contributing to the regulation of the Lp(a) lipoprotein concentration, we have sequenced the kringle IV-type 2 encoding exons 1 and 2 together with the flanking intron sequences of the LPA gene in individuals with different serum concentrations of Lp(a) lipoprotein. The high degree of sequence identity between the kringle IV-type 2 repeats made it possible to analyse all the 3-42 kringles simultaneously by polymerase chain reaction and direct DNA sequencing. The strategy used allowed us to determine approximately 700 bp from each kringle IV-type 2 repeat, resulting in a rapid screen of on average 28,000 bp of the LPA gene from each individual. Comparing these bipartite kringle IV-type 2 repeat sequences from 12 individuals with high and 11 individuals with low Lp(a) lipoprotein level revealed that: 1. no sequence polymorphism could be detected in the exons examined; 2. no sequence polymorphism could be detected in the consensus GT/AG splicing signals of exon/intron junctions; and 3. the proximal intron sequences seemed almost completely conserved in the 76-135 bp analysed. Only one position in the intron sequences exhibited the pattern of a G/A polymorphism. We observed no differences between the group with high and the group with low Lp(a) lipoprotein level. The very high conservation of intron sequences could support the hypothesis that the LPA gene evolved relatively recently. The contradictory finding of a corresponding sequence conservation between the human LPA and the plasminogen gene suggests that an evolutionary pressure has preserved these intron sequences over the last 40-90 million years.