Trypsin and trypsinogen from an Antarctic fish: molecular basis of cold adaptation.

Trypsin from Antarctic fish Paranotothenia magellanica displays molecular and kinetic properties typical of enzymes produced by psychrophilic organisms. The enzyme has a high catalytic efficiency at low and moderate temperatures and is rapidly inactivated at temperatures higher than 30 degrees C. The nucleotide sequence was determined after mRNA extraction and cDNA synthesis. The cDNA encodes a pretrypsinogen which includes a seven residue activation peptide containing only three acidic residues preceeding the 222 amino-acid mature enzyme. A three-dimensional model of the enzyme was built. Structural parameters possibly involved in the adaptation to cold have been derived from comparison with the three-dimensional structure of the bovine enzyme. Among them are the lack of Tyr-151 in the substrate binding pocket, an overall decrease in the number of salt bridges and hydrophobicity and the increase in the surface hydrophilicity.

Comparative study of the milk fat globule membrane and the mouse mammary tumour virus prepared from the milk of an infected strain of Swiss albino mice.

Milk fat globule membranes and mammary tumour virus particles (d=1.17 g/cm3) have been obtained from the milk of a Swiss albino mice strain. Comparative biochemistry shows that these two structures differ significantly in the phospholipid, polypeptide and glycopolypeptide patterns and enzymatic activities. However, the lipid profile and the morphology of both structures suggest a filiation with the plasma membrane. Density fractions obtained from the crude virus preparation have been thoroughly investigated. The results suggest that most of these fractions represent degraded virus and/or atypical virus assembly.

Cloning and overexpression of the triosephosphate isomerase genes from psychrophilic and thermophilic bacteria. Structural comparison of the predicted protein sequences.

We focused on the temperature adaptation of triosephosphate isomerase (TIM; E.C. 5.3.1.1.) by comparing the structure of TIMs isolated from bacterial organisms living in either cold or hot environments. The TIM gene from psychrophilic bacteria Moraxella sp. TA137 was cloned and its nucleotide sequence determined. Its deduced amino acid sequence revealed 34% identity with the thermophilic bacteria Bacillus stearothermophilus TIM. Expression vectors were constructed and recombinant Moraxella TA137 and Bacillus stearothermophilus TIMs were overproduced and purified to homogeneity. Recombinant TIM inactivation constants (Ki), measured at various temperatures, compared to those of the mesophilic Escherichia coli recombinant TIM clearly show that Moraxella TA137 and B. stearothermophilus TIMs have respectively psychrophilic and thermophilic characteristics. To try to elucidate the structure-thermolability and structure-thermostability relationship, factors affecting the overall stability of these two TIMs were examined, based on the alignment with the mesophilic chicken TIM, the three-dimensional structure of which is already known. From this comparison, it appears that the adaptability of TIM to high temperature is favored by better stabilizing residues for the helix dipole as well as better helix-forming residues whereas the adaptability of TIM to low temperature seems to reside in the nature of helix-capping residues.

APH-1, a POU homeobox gene expressed in the salt gland of the crustacean Artemia franciscana.

We characterized the first POU-homeoprotein in a crustacean (designated APH-1 for Artemia POU-Homeoprotein, EMBL Y15070). The amino acid sequence of the APH-1 POU-domain is identical, except for two residues, to that of the two class III POU proteins Cf1-a (Drosophila) and POU-M1 (Bombyx mori). Southern blot analysis suggests that crustaceans have only one class III POU gene. RT-PCR and whole-mount in situ hybridization show that APH-1 mRNA is present in larvae specifically in the salt gland, an organ which is involved in osmoregulation, and disappears in the adult.

Evidence of pre-prosomatostatin mRNA in human normal and tumoral anterior pituitary gland.

Expression of the SRIH gene was investigated in six human normal anterior pituitaries, six GH-, three PRL-, three mixed GH/PRL-secreting and four nonsecreting adenomas. Total cellular RNA and poly(A+) mRNAs were analyzed by dot and Northern blot hybridization to a 3'-end labeled oligonucleotide probe specific for the human pre-proSRIH mRNA. A weak but detectable pre-proSRIH hybridization signal was present in human normal anterior pituitaries and in the four groups of adenomas. The size of this pre-proSRIH mRNA was indistinguishable from that found in our hypothalamic samples and close to that described in the literature. The wide variation of the signal intensity from one case to the other in each group of the different types of normal and tumoral antehypophyseal samples prevented establishment of any correlation between the level of pre-proSRIH mRNA and the nature of the pituitary tissue. The presence of SRIH mRNA in human normal and tumoral anterior pituitary tissues provides a sound basis to substantiate the hypothesis of a SRIH biosynthesis in the human anterior pituitary gland.

Crystal structure of recombinant triosephosphate isomerase from Bacillus stearothermophilus. An analysis of potential thermostability factors in six isomerases with known three-dimensional structures points to the importance of hydrophobic interactions.

The structure of the thermostable triosephosphate isomerase (TIM) from Bacillus stearothermophilus complexed with the competitive inhibitor 2-phosphoglycolate was determined by X-ray crystallography to a resolution of 2.8 A. The structure was solved by molecular replacement using XPLOR. Twofold averaging and solvent flattening was applied to improve the quality of the map. Active sites in both the subunits are occupied by the inhibitor and the flexible loop adopts the "closed" conformation in either subunit. The crystallographic R-factor is 17.6% with good geometry. The two subunits have an RMS deviation of 0.29 A for 248 C alpha atoms and have average temperature factors of 18.9 and 15.9 A2, respectively. In both subunits, the active site Lys 10 adopts an unusual phi, psi combination. A comparison between the six known thermophilic and mesophilic TIM structures was conducted in order to understand the higher stability of B. stearothermophilus TIM. Although the ratio Arg/(Arg+Lys) is higher in B. stearothermophilus TIM, the structure comparisons do not directly correlate this higher ratio to the better stability of the B. stearothermophilus enzyme. A higher number of prolines contributes to the higher stability of B. stearothermophilus TIM. Analysis of the known TIM sequences points out that the replacement of a structurally crucial asparagine by a histidine at the interface of monomers, thus avoiding the risk of deamidation and thereby introducing a negative charge at the interface, may be one of the factors for adaptability at higher temperatures in the TIM family. Analysis of buried cavities and the areas lining these cavities also contributes to the greater thermal stability of the B. stearothermophilus enzyme. However, the most outstanding result of the structure comparisons appears to point to the hydrophobic stabilization of dimer formation by burying the largest amount of hydrophobic surface area in B. stearothermophilus TIM compared to all five other known TIM structures.

Expression of the growth hormone variant gene in human placenta.

Besides the hGH-N gene, which codes for the pituitary 22 and 20K GH variants, the human genome contains a second GH gene, namely the GH-V, which has been thought to be silent. We recently discovered a placental variant of human growth hormone (hPGH), which appears in maternal serum at mid-pregnancy and which rises in concentration thereafter to term. As hPGH and GH-V proteins display very similar characteristics, including a high affinity for hepatic GH receptors, they could be identical. To verify this hypothesis, we sought hGH-V mRNA in placenta. Hybridization experiments were performed between dot-blotted mRNA originating either from placenta or from one pituitary hGH secreting adenoma and synthetic polynucleotide probes corresponding to specific portions of the hGH-V or hGH-N gene sequences. The results indicate that the V gene is indeed expressed in the placenta and, at a very low level, in the pituitary adenoma. Therefore hPGH is most likely the expression product of the hGH-V gene.

Sequence of the triosephosphate isomerase-encoding gene isolated from the thermophile Bacillus stearothermophilus.

By analysis of genomic clones, we have determined the complete nucleotide sequence of the gene encoding triosephosphate isomerase (TIM; EC 5.3.1.1) in the thermophilic bacterium, Bacillus stearothermophilus. The gene encodes a 253-amino-acid TIM which is 39% identical to that of the mesophile, Escherichia coli.

Evidence that two tilapia (Oreochromis niloticus) prolactins have different osmoregulatory functions during adaptation to a hyperosmotic environment.

Two forms of prolactin (tiPRLI and tiPRLII), with only 69% sequence identity, have been previously described in the cichlid fish tilapia (Oreochromis species). In the present study we have attempted to investigate the biological activity of these two prolactin forms during adaptation to a hyperosmotic environment. For this purpose, we have developed two highly sensitive (sensitivity: 0.05 ng/ml) and specific (cross-reactivity < 0.04%) radioimmunoassays for tiPRLI and tiPRLII, using recombinant hormones. When fish were directly transferred from fresh to brackish water, the measured levels of plasma tiPRLI and tiPRLII dropped abruptly until 12 h after transfer. Thereafter, plasma tiPRLII remained stable (around 0.5 ng/ml) until the end of the experiment, whereas plasma tiPRLI continued to decrease to undetectable levels. These different patterns of change are reflected in the calculated ratio of plasma tiPRLII to tiPRLI, which increased from 2-3 in fresh water-adapted fish to over 10 in fish which had spent 3 days or more in brackish water. The pituitary contents of tiPRLI and tiPRLII varied in a qualitatively similar fashion after transfer to brackish water. The tiPRLI content dropped continuously after 12 h, reaching one-twelfth of its initial level after 2 weeks. The pituitary tiPRLII content, on the other hand, did not decrease significantly until day 7, and after a 2-week exposure to brackish water it had only decreased by 50%. When injected into tilapia adapted to brackish water, both ovine prolactin and recombinant tiPRLI induced a clear dose-dependent ion-retaining effect. In contrast, the effect induced by tiPRLII treatment was markedly smaller and not dose-dependent. Northern blot analysis of tiPRL mRNAs using either a tiPRLI or a tiPRLII cDNA probe indicated the presence of two mRNAs differing in size: a 1.7 kb mRNA coding for tiPRLI and a 1.3 kb mRNA coding for tiPRLII. After transfer to brackish water, levels of the two mRNAs decreased similarly. The present study indicates that, in O. niloticus, the two forms of prolactin have different osmoregulatory roles during adaptation to brackish water. Accordingly, their synthesis are differentially regulated after transfer to a hyperosmotic environment, presumably at a post-transcriptional level.

Characterization of a single prolactin (PRL) receptor in tilapia (Oreochromis niloticus) which binds both PRLI and PRLII.

In tilapia, there are two forms of prolactin (PRL) whose effects on sodium and chloride movements differ and depend on the living environment of the fish. To see whether different receptors or the same receptor mediates these different effects, we have characterized the specific binding of both forms of tilapia (ti)PRL in two osmoregulatory organs, the gill and kidney. Two recombinant tiPRLs were used for this analysis. The recombinant hormones had the same properties as the native hormones in a tilapia gill radioreceptor assay. Specific binding to gill and kidney membranes was increased by optimizing the quality of the tissue preparations (physiological state of fish, membrane preparation) and the incubation conditions (pH, salt concentrations, temperature, time). Under these optimized conditions, we detected only one class of high affinity PRL receptor in gill and kidney. Its binding affinity was higher for tiPRLI than for tiPRLII in both gill and kidney (for tiPRLI the respective affinity values were 2.9 and 2.3 x 10(10) per M, for tiPRLII they were 1.9 and 0.5 x 10(10) per M). In competition studies, tiPRLI was more potent, followed by tiPRLII and ovine (o)PRL. tiGH and oGH did not significantly displace either tiPRL. The receptor we have characterized thus recognizes quite specifically both tiPRLs.

A simple method for the preparation of the covalently closed circular form of plasmid DNA.

We describe a simple, rapid, inexpensive method for isolation of covalently closed circular plasmid DNA. The method involves the electrophoresis of crude DNA preparations in an agarose gel, electrotransfer onto a dialysis membrane and elution of the highly purified circular covalently closed plasmid DNA. Native and recombinant plasmid DNA have been purified by this method and shown to be suitable for restriction enzyme digestion and transformation of bacteria. The yield of this rapid purification procedure makes it a good alternative method to standard centrifugation in cesium chloride ethidium bromide gradients.

Regulation of gill prolactin receptors in tilapia (Oreochromis niloticus) after a change in salinity or hypophysectomy.

Prolactin (PRL) receptors in gill tissue have been analyzed in tilapia (Oreochromis niloticus) after transfer from fresh water (FW) to brackish water (BW). This study has indicated the presence of only one class of tilapia PRL (tiPRL) receptor whatever the salinity. After transfer, however, the percentage of specific binding of the two forms of tiPRL (tiPRLI and tiPRLII) increased significantly. Scatchard analysis of tiPRLI binding indicated an increase in receptor affinity, an effect which was not accompanied by any change in receptor specificity. Transfer to BW also caused the number of tiPRL receptors to increase rapidly, remaining high in fish adapted to BW for 28 days. Based on the sharp reduction in plasma tiPRLI and tiPRLII levels after transfer to BW, one possible explanation may be that tiPRL itself is an important factor regulating the number of free receptors. This hypothesis finds support in the fact that the number of tiPRL receptors also increased in hypophysectomized fish reared in FW. However, the absence of change in receptor affinity after hypophysectomy suggested that yet other factors are involved in tiPRL receptor regulation during the transfer from FW to BW. The paradoxically high numbers of tiPRL receptors in the gills of BW-adapted tilapia, even though PRL is known to be a FW-adapting hormone, is discussed with regard to the environment in which tilapia live.

Production and purification of biologically active recombinant tilapia (Oreochromis niloticus) prolactins.

Recombinant expression vectors carrying tilapia prolactin-I or -II (tiPRL-I or tiPRL-II) cDNA were constructed and the tiPRL-I and II proteins were produced in E. coli as inclusion bodies. These inclusion bodies were dissolved in 6 mol urea/l. Refolding of the proteins was followed by SDS-PAGE under non-reducing conditions so as to visualize the oxidized state of the molecules. Proteins tiPRL-I and tiPRL-II were purified by gel filtration and ion-exchange chromatography. The N-terminal sequence and bioactivities of both purified proteins were then analysed. Recombinant tiPRL-I and tiPRL-II induced a significant rise in plasma calcium levels as well as in mucocyte density in the abdominal skin epithelium. When tested on kidney membrane, both proteins exhibited potency in competing with 125I-labelled tiPRL-I for binding sites, but tiPRL-I seemed to be more potent than tiPRL-II in competing for these sites. The results obtained for the biological activities tested suggest that both recombinant prolactins were correctly refolded and had retained the full biological activity previously observed with the natural hormone preparations extracted from the animals.

Evidence of rainbow trout prolactin interaction with its receptor through unstable homodimerisation.

This study aims to characterise Prolactin receptor (PRLR) in rainbow trout for which no information is available despite the availability of Salmonid PRL preparations. By screening a freshwater rainbow trout intestine cDNA library with a probe corresponding to the extracellular domain (ECD) of tilapia PRLR, we have cloned a 2.5 kb insert coding for the PRLR. The mature protein of 614 amino acid residues is similar to PRLR isolated in tilapia and also the long form of mammalian PRLR. Analysis of PRLR gene expression in osmoregulatory organs revealed the presence of a unique transcript, thus confirming the involvement of this hormone in the control of osmoregulation in this fish species. By using surface plasmon resonance (SPR) technology, kinetic measurement of interaction between trout PRL and its receptor ECD was studied. This approach allowed us to demonstrate the formation of a transient, unstable homodimeric complex. This unstability could explain the inability to perform binding experiments using homologous PRL. In contrast, heterologous lactogenic ligands were able to interact through a more stable complex. Whether these characteristics of PRL-receptor interaction in rainbow trout are different to what occurs in tilapia where a homologous radioreceptor assay was developed would require further studies.

Structure of the tilapia (Oreochromis mossambicus) prolactin I gene.

The tilapia (Oreochromis mossambicus) prolactin-I (PRL-I) gene has been cloned and sequenced. Its transcript (3,677 bases long) begins with a guanine and is organized in five exons and four introns like the other known prolactin genes. Analysis of the 1,555-bp 5'-flanking region suggests that pituitary-specific expression of the gene could be regulated through a trans-factor related to the mammalian pituitary-specific factor Pit-1. Two potential binding sites for such a factor were found in the first intron, suggesting a possible regulatory role for this region. Moreover, two potential Z-DNA regions are located at positions -837 to -812 and -246 to -179 from the transcription start site. These two regions could play an important role in the regulation of PRL gene expression.

Analysis of JC virus DNA purified directly from human progressive multifocal leukoencephalopathy brains.

Human polyomavirus JC DNA was purified directly from the diseased brain tissue of two patients with progressive multifocal leukoencephalopathy (PML) by a method employing differential salt precipitation (B. Hirt, J. Mol. Biol. 26:365-369, 1967). Each of the viral genomes (JC-NIH-1 and JC-NIH-2) was molecularly cloned intact in Escherichia coli, using pBR322, at their unique EcoRI (0.00 map unit) and BamHI (0.51 map unit) sites. The JC-NIH-1 genome was approximately 50 base pairs larger and the JC-NIH-2 genome was approximately 50 base pairs smaller than the prototype human polyomavirus JC (Mad-1) DNA. Analysis of the restriction endonuclease cleavage fragments of these two DNAs and the human polyomavirus JC (Mad-1) DNA revealed only slight differences which mapped in a region of the genome extending from 0.67 to 0.74 map unit. From previous homology studies, this region of variance corresponds to the noncoding region to the late side of the origin of DNA replication.

Calcitropic effects of recombinant prolactins in Oreochromis mossambicus.

Homologous recombinant tilapia prolactin-188 (PRL-I) and tilapia prolactin-177 (PRL-II) were tested for calcitropic activity in tilapia, Oreochromis mossambicus. Injection of PRL-I and PRL-II (4 injections, 12.5 pmol/g, over an 8-day period) induced hypercalcemia that resulted from an enhanced calcium influx via the gills and a decreased calcium efflux. Both PRLs increased the density of the Ca(2+)-transporting Ca(2+)-adenosinetriphosphatase in a plasma membrane preparation of the branchial epithelium. Dose-response studies (doses tested: 0.75-12.5 pmol/g) demonstrated that PRL-I was roughly twofold more potent than PRL-II in inducing hypercalcemia, in enhancing basal levels of cortisol, and in stimulating opercular ionocyte density. PRL-I and PRL-II were equipotent in stimulating the dermal mucocyte frequency. We conclude that in this species PRL-I and PRL-II have calcitropic effects, and that PRL-I is more potent than PRL-II in this respect. We postulate that PRL has corticotrophic activity in this fish.

Detection of mRNA encoding an antibacterial-metalloprotein (MPII) by in situ hybridization with a cDNA probe generated by polymerase chain reaction in the worm Nereis diversicolor.

Based on partial amino acid sequence of an antibacterial-metalloprotein (MPII) consisting of a N-terminal fragment (1-33th) and an 43 amino acids long internal fragment, two oligonucleotides primers were synthesized and used to generate a cDNA fragment by the PCR method. The specificity of the PCR synthesized 220 bp fragment was verified by hybrid-arrest translation and sequencing. In situ hybridization performed with this cDNA fragment on Nereis diversicolor whole body defined two specific sites of recognition: a cluster of cells floating in the coelom and two types of muscles (perineural and oblique). Finally, the chronological expression of the MPII was postulated.

Molecular cloning and characterization of two forms of trout growth hormone cDNA: expression and secretion of tGH-II by Escherichia coli.

We constructed a cDNA library using mRNA isolated from rainbow trout pituitaries. Two types of cDNA clones encoding growth hormone (GH) were isolated and their complete nucleotide sequences determined. Twenty seven nucleotide substitutions in the coding region and 108 in the noncoding region distinguish the cDNAs of trout GH-I and II. Both cDNAs encode polypeptides of 210 amino acids, including a putative signal peptide of 22 amino acids, which differ by 12 residues. In both trout and salmon, GH-I mRNA is predominant, which suggests that the variation in the amount of secreted GH originates from a transcriptional event. Moreover, comparison of rainbow trout and chum salmon GH reveals that, in both cases, the predominant GH-I has mutated less than its GH-II counterpart. Mature tGH-II was expressed in Escherichia coli using the pIN-III-ompA-Hind secretion vector.

Tilapia prolactin: molecular cloning of two cDNAs and expression in Escherichia coli.

We have isolated cDNA clones encoding tilapia prolactin (tiPRL) from a cDNA library prepared from tilapia (Oreochromis niloticus) anterior pituitary glands. A trout PRL cDNA fragment was used as hybridization probe to select the recombinant plasmids carrying the tiPRL coding sequence. Two types of PRL cDNA were isolated and their complete nucleotide sequence determined. The larger cDNA (tiPRL-I) codes for a polypeptide of 212 amino acids, including a putative signal sequence of 24 amino acids, and contains a 3' untranslated region of 787 bp. The second prolactin cDNA (tiPRL-II) encodes a polypeptide of 200 amino acids, including a presumptive signal peptide of 23 amino acids, and contains a noncoding region of 512 bp. tiPRL-I and tiPRL-II cDNA sequences are 81% similar, whereas the encoded proteins share 69% amino acid identity at optimal alignment. Mature tiPRL-I was efficiently expressed in Escherichia coli carrying a plasmid in which the tiPRL-I cDNA was under the control of the phi 10 promoter of T7 bacteriophage. The new recombinant protein representing about 45% of the total cellular proteins was found in inclusion bodies and cross-reacted with salmon PRL antiserum.