Genomic structure and sequence of the gilthead seabream (Sparus aurata) growth hormone-encoding gene: identification of minisatellite polymorphism in intron I.

The growth hormone (GH) gene of the gilthead seabream (Sparus aurata) (saGH) has been cloned, sequenced, and characterized. The saGH gene spans approximately 4.3 kb and consists of six exons and five introns, as found for all cloned teleost GH genes with the exception of carps and catfish. The first and third introns contain long stretches of repetitive tandem repeats. The second intron, which is unusually long compared with that in other teleosts (and other vertebrates) spans 1747 nucleotides (nt) and contains several inverted repeats. Intron-targeted polymerase chain reaction (PCR) analysis identified length polymorphism of the first intron. Sequence analysis of four variants (405, 424, 636, and 720 nt) out of many variants found revealed that the variation in length is due to differences in the number of repeat monomers (17-mer or 15-mer) as well as minor changes in their length. This repeat unit contains the consensus half-site motif of the thyroid hormone response element (TRE) and estrogen response element (ERE). Polymorphism was found also in the third intron. This is the first report of such high polymorphism of the first intron of GH gene in a vertebrate.

Isolation and characterization of medaka ribosomal protein S3a (fte-1) cDNA and gene.

Many ribosomal protein genes were cloned from different organisms. We describe here, for the first time, the isolation of the ribosomal protein S3a cDNA and gene from a teleost - the medaka (Oryzias latipes). The cDNA sequence is 863bp long and encodes an open reading frame of 266 amino acids. The gene is 2927bp long and contains six introns and five introns. The levels of the S3a mRNA are elevated during embryonal development. Transcription of the gene was also detected in different tissues of adult medaka. At the 5' untranslated region of the cDNA, the terminal pyrimidine tract, a common feature with all ribosomal protein genes, was found. snoRNA sequences were found in introns 3 and 5, similar to human and mouse U73b and U73a.

The high copper tolerance of Candida albicans is mediated by a P-type ATPase.

The pathogenic yeast Candida albicans has higher resistance than the baker's yeast Saccharomyces cerevisiae to elevated concentrations of copper. To understand the basis of this differential resistance, we performed a functional screen for C. albicans genes involved in copper detoxification. Here, we report the isolation of two such genes: a metallothionein, CaCUP1, and a copper-transporting P-type ATPase, CaCRP1. Both genes are induced by extracellular copper. Gene disruptions indicated that the copper extrusion pump is responsible for the unusual resistance of C. albicans to copper, whereas the metallothionein is responsible for the residual copper resistance of the Cacrp1Delta mutant. We show further that under acidic and anaerobic conditions, such as prevail in the natural niche of C. albicans, the digestive tract of animals, CaCRP1 function becomes essential for survival in the presence of even very low copper concentrations. These observations suggest that copper in the gastrointestinal tract may present a toxic challenge to which enteric organisms had to adapt.

Preparation of recombinant gilthead seabream (Sparus aurata) growth hormone and its use for stimulation of larvae growth by oral administration.

Gilthead seabream (Sparus aurata) growth hormone (gsGH) cDNA coding for the mature protein was cloned in a pGEM-T vector and then transferred into prokaryotic expression vector pET-8 and expressed in E. coli BL21 (DE3) cells upon induction with IPTG. The expressed protein, contained within the inclusion-body pellet, was solubilized in 4.5 M urea, refolded at pH 11.3 in the presence of catalytic amounts of cysteine, and purified to over 98% purity, as evidenced by SDS-PAGE. Gel-filtration on a Superdex column under nondenaturing conditions and partial amino acid N-terminal sequence showed the purified protein to be a monomeric alanyl-gsGH. Over 90% pure bacterial beta-lactamase was copurified as a by-product. Binding assays of the [125I]gsGH to gs liver microsomal fraction resulted in high specific binding characterized by a Kd = 1.93 nM. Recombinant gsGH, like ovine placental lactogen, exhibited growth-stimulating activity when applied orally to S. aurata larvae or intraperitoneally to juvenile fish.

Somatolactin, a novel pituitary protein: isolation and characterization from Sparus aurata.

The complete amino acid sequence of somatolactin, a new pituitary protein belonging to the growth hormone/prolactin family, from the gilthead sea bream Sparus aurata has been determined. Somatolactin was isolated from the pituitary by alkaline extraction, gel filtration on a Sephadex G-100 column, and reversed-phase high-performance liquid chromatography (rpHPLC) on a TSK gel ODS-120T column. The purified protein was confirmed to be somatolactin by immunoblotting using chum salmon somatolactin antisera. It was found that Sparus aurata somatolactin consists of two forms; one form (28 kD) is probably a glycosylated form, while the other (25 kD) is a simple protein form, as was found also in Atlantic cod. The somatolactin consists of 207 amino acids that show remarkable conservation among fish somatolactins.

Molecular identification of Candida albicans.

The benomyl resistant gene (BenR) found in Candida albicans, but not in other species of Candida, was used as a probe for the identification of C. albicans in clinical specimens. The utility of this probe to detect this species was demonstrated by Southern and dot-blot analysis, and by PCR. The possible use of this gene in C. albicans typing by the RFLP method is also demonstrated.

Isolation of growth hormone and in vitro translation of mRNA isolated from pituitaries of the gilthead sea bream Sparus aurata.

Growth hormone (GH) polypeptide was purified from pituitary glands of the gilthead sea bream (Sparus aurata) by a two-step procedure involving gel filtration on Sephadex G-100 and reverse-phase high-performance liquid chromatography (rpHPLC). At each stage of purification, fractions were monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting using anti-bonito GH antiserum. The molecular weight of the sea bream GH was estimated by SDS-PAGE to be 21 kDa when electrophoresed in the absence of beta-mercaptoethanol (nonreduced conditions) and 22 kDa when electrophoresed under reduced conditions (in the presence of 1% beta-mercaptoethanol). Pituitary RNA was used to direct cell-free translation. When specific immunoisolation from 35S-labeled proteins was conducted, using antisera against Sparus or tilapia GH, a larger prehormone was immunoprecipitated. The size of the pre-GH was estimated to be 27-28 kDa under reduced conditions and 26-27 kDa under nonreduced conditions, in agreement with the calculated molecular weight of Sparus pre-GH of 26,296 based on the deduced amino acid sequence of Sparus GH cDNA. The specificity of the immunoprecipitation reaction was demonstrated by the ability of recombinant tilapia GH to compete with the radioactively labeled translation product. No such competition was found after the addition of BSA. Our results demonstrate that the sea bream GH is similar in its size to other purified fish GHs and provide direct evidence for the synthesis of GH as a prepeptide, thus supporting the conclusions presented earlier by GH cDNA cloning.

All-fish gene constructs for growth hormone gene transfer in fish.

In order to develop all-fish expression vectors for microinjection into fertilized fish eggs, we have prepared the following constructs: rainbow trout metallothionein a/b and the gilthead seabream growth hormone cDNA (ptMTa-gbsGHcDNA, ptMTb-gsbGHcDNA), carp ß-actin gilthead seabream GH cDNA (pcAß-gsbGHcDNA). 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 ligated 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 to be 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.

Developmental expression of the growth hormone gene in the gilthead sea bream Sparus aurata.

Expression of growth hormone (GH) gene during early stages of larval development of the teleost Sparus aurata was determined by Northern blot analysis. Poly(A+) RNA was prepared from a pool of larvae collected on different days after hatching. When hybridized to Sparus aurata GH cDNA, GH specific mRNA was first seen on day 6 post-hatching. In contrast, the levels of beta-actin mRNA, which was used to normalize for RNA amounts, were already high on the day of hatching. Our results suggest that expression of the GH gene is very low immediately after hatching, and increases dramatically within 6 days.

Cloning and sequencing of the gilthead seabream (Sparus aurata) growth hormone-encoding cDNA.

The cDNA clones encoding gilthead seabream (gsb) (Sparus aurata) growth hormone (GH) have been isolated from a cDNA library prepared from seabream pituitary gland poly(A)+ RNA. The cDNA library was screened using red seabream and rainbow trout GH cDNAs. The complete nucleotide (nt) sequence of gsbGH has been determined. The cDNA sequence codes for a polypeptide of 204 amino acids (aa), including a putative signal peptide of 17 aa. The 5'- and 3'-untranslated regions of the message are 55 and 236 nt long, respectively. The predicted aa sequence of gsbGH revealed 97% homology with red seabream GH, 95% with tuna GH, 85% with yellowtail GH, and 65% with rainbow trout GH.

Growth hormone increases plasma levels of insulin-like growth factor (IGF-I) in a teleost, the gilthead seabream (sparus aurata).

A heterologous radioimmunoassay (RIA) was applied for the determination of immunoreactive (IR)--insulin-like growth factor (IGF-I) in a teleost, the gilthead seabream (Sparus aurata). Serial dilutions of the fish plasma gave a linear curve when added to constant amounts of 125I-labelled human IGF-I(53-70) and antiserum prepared against this fragment. The RIA was used to study the effect of GH on plasma levels of IR-IGF-I in S. aurata. A single injection of human recombinant GH (1 micrograms/g) resulted in a significant increase in IR-IGF-I at 29, 48 and 72 h, when compared with saline-injected fish. This novel observation suggests that in fish, as in mammals, circulating IGF-I levels are modulated by GH.

Ability of Daphnia Cell-Free Extract to Damage Escherichia coli Cells.

A cell-free extract of Daphnia magna was found to lyse Escherichia coli cells as shown by leakage of the enzymes alkaline phosphatase and beta-galactosidase from the bacteria. The cell-free extract was separated on Sephadex G-200, and the fractions showing an ability to lyse E. coli cels were isolated. The factor which was responsible for the lysis of the bacterial cells was probably a protein with a molecular weight of several thousands. Mg and Ca ions augmented the activity of the Daphnia extract on E. coli cells.

Effect of temperature on heterotrophic glucose uptake, mineralization, and turnover rates in lake sediments.

The V(max) and turnover rates (TR) of [U-C]glucose uptake and mineralization of Lake Kinneret (Israel) sediment are temperature dependent. The following activation energies were determined: glucose uptake, approximately 15,000 cal (62,760 J); TR of glucose uptake, approximately 10,000 cal (41,840 J); glucose mineralization, 7,500 to 15,000 cal (31,380 to 62,760 J); and TR of glucose mineralization, approximately 15,000 cal. Q(10) values varied as follows: glucose uptake, approximately 2.3; TR of glucose uptake, approximately 1.8; and glucose mineralization, approximately 2.5. K + S(n) values increased slightly with temperature and might reflect an increased K with increased temperatures. Glucose respiration/uptake ratios were low (9.5 to 12%) and were apparently not greatly influenced by the presence or absence of oxygen or by different assay temperatures. Aerobic or anaerobic sediments assayed under either aerobic or anaerobic conditions did not exhibit greatly different V(max), TR, or K + S(n) values.

Effect of temperature on growth and activity of Aeromonas spp. and mixed bacterial populations in the Anacostia River.

During the winter months, total bacterial counts in the water column and in the sediment in the Anacostia River were two- to eightfold higher than at other times of the year, whereas Aeromonas spp. decreased in number of several orders of magnitude. This significant decrease in number in the Anacostia River during the cold months of the year can be explained by the low metabolic activity of Aeromonas at low temperatures.

Effects of pharmaceutical wastes on microbial populations in surface waters at the puerto rico dump site in the atlantic ocean.

A series of cruises during 1979 and 1980 to the pharmaceutical dump site located 64 km north of Arecibo, Puerto Rico, in the Atlantic Ocean, was carried out to evaluate effects of wastes on the ecology of the microflora of surface waters of the dump site. In addition to bacteriological monitoring of the waste plume created by the release of wastes from the disposal barge, stations along a series of transects, extending north from coastal waters through and beyond the dump site, were sampled. Largest numbers of culturable bacteria on marine agar were found at stations closest to shore and in the vicinity of the dump site. Bacteria recovered on marine agar were predominantly Vibrio and Aeromonas spp., with the relative abundance of these organisms decreasing as gram-positive organisms (staphylococci, micrococci, and bacilli) became dominant in areas immediately affected by waste dumping. Total numbers of bacteria (determined by acridine orange direct counts [AODC]), which were relatively stable throughout the region, and a direct estimate of viable cells (DVC), i.e., those cells responsive to additions of yeast extract and nalidixic acid, were determined by acridine orange staining and epifluorescence microscopy. Heterotrophic bacterial activity, measured by the uptake (V(max)) of C-labeled amino acids, declined relative to distance from land. Increases in specific activity indices (DVC/AODC and V(max)/AODC) were observed near the dump site. The composite results of this study, i.e., increased specific activities (determined by two methods), increased numbers of culturable marine bacteria, and marked alteration of the taxonomic composition of the culturable bacterial community in waters within and surrounding the Puerto Rico dump site, indicate demonstrable changes in the marine microbial community in the region used for waste disposal.

Nitrogen Fixation by the Photosynthetic Sulfur Bacterium Chlorobium phaeobacteroides from Lake Kinneret.

N(2) fixation by Chlorobium phaeobacteroides from Lake Kinneret was dependent on ammonia concentration and light intensity. In the thermocline of Lake Kinneret, N(2) fixation and photosynthesis were low. It was concluded that the bacteria do not contribute significantly to the organic nitrogen load of the lake.

Investigations on the photosynthetic sulfur bacterium Chlorobium phaeobacteroides causing seasonal blooms in Lake Kinneret.

Between May and December, the annual stratification period in Lake Kinneret, sulfide is formed and accumulates in the hypolimnion. In July-August a large population (up to 10(6) cells/mL) of green, photosynthetic, sulfur bacteria develops at the boundary of the oxidative and reductive zones of the water column lasting for 3--8 weeks. These bacteria were isolated from the lake and identified as Chlorobium phaeobacteroides. Optimal growth conditions included 160 mg S=L-1 and light intensities of 5--30 micron Einstein (micron E) m-2s-1. Glucose and acetate augmented growth when added to the mineral medium. The lowest light intensity which still supported growth was 0.3 micron E m-2s-1 when acetate was present and 1.0 micron E m-2s-1 when no organic substrate was present. Under complete darkness, either with or without organic substrate, the bacteria die. Photosynthetic activity was higher when no organic compound was added to the medium. Uptake of acetate was light-dependent. In the lake the photosynthetic activity of the bacteria is low because of the limited light intensity (0.3 micron E m-2s-1) at the bloom layer. It is suggested that the appearance and the disappearance of the bloom are caused by the influence of the daily internal seiche.

Seasonal distribution of vitamin B12 in Lake Kinneret.

Vitamin B12 is formed in Lake Kinneret in the hypolimnion and in the sediment. The highest value of B12 recorded in the lake water was about 100 ng/liter in November and December of 1975 at a 40-m depth. The vitamin was liberated from the hypolimnion during the turnover period. This supply of the vitamin to the photic zone was accompanied by increasing biomass of Dinoflagellates, Bacillariophyta, and Chlorophyta. The decrease in the vitamin concentration, followed by an increase, is correlated with a decline and subsequent rise in the algal biomass, respectively. Cyanophyta biomass, on the other hand, increased when the vitamin concentration in the photic zone was at its lowest level.

Sensitive enzymatic assay for glucose determination in natural waters.

A new enzymatic method for glucose determination is described. It allows measurement of glucose concentration as low as 10 M. Such sensitivity makes this method particularly appropriate for estimation of glucose in natural-water bodies, generally without prior concentration or extraction. The method is based on the reaction between glucose and adenosine 5'-triphosphate, catalyzed by hexokinase to form glucose-6-phosphate. The amount of adenosine 5'-triphosphate consumed in this reaction, which is directly proportional to the amount of glucose present in the sample, is measured by the luciferin-luciferase assay. The optimal conditions for glucose determination by this method have been defined as follows: 20 min of incubation at 30 degrees C, magnesium concentration of 10 M, and pH in the range of 7.5 to 10.5. The specificity of the assay to different carbohydrates has also been studied. Recovery of known amounts of glucose added to Lake Kinneret water was in the range of 80 to 114%. Application of this method is demonstrated in eight monthly profiles of the glucose content in Lake Kinneret.