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Biomedical subjects

M Lion

Publications and source records attributed to M Lion.

7 recordsLinked to original sources
PubMed1991

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.

Animals↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

Rainbow trout prolactin cDNA cloning in Escherichia coli.

We describe the isolation and characterization of a cDNA for trout prolactin (tPrl). An extensive analysis of tPrl recombinant clones by restriction analysis and sequencing revealed the presence of only one form of tPrl mRNA. The deduced protein sequence consists of 210 amino acids, including a signal peptide of 23 amino acids. The amino acid sequence of the mature protein is compared among teleosts and mammals, showing two domains of strong similarity that may be involved in biological activity.

Amino Acid Sequence↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

Tilapia growth hormone: molecular cloning of cDNA and expression in Escherichia coli.

A cDNA library was prepared from poly(A)+RNA extracted from tilapia Oreochromis niloticus anterior pituitaries. The recombinant clones carrying the cDNA sequence of tilapia growth hormone (tiGH) were selected using a fragment of the trout growth hormone (tGH) cDNA as hybridization probe. The nucleotide sequence of the full-length tiGH cDNA was determined. This cDNA encodes a protein of 204 amino acids, including the putative signal peptide of 17 amino acids. Mature tiGH cDNA was inserted in an Escherichia coli expression vector which led to the production of tiGH protein with a yield estimated to be 20% of the total bacterial proteins.

Amino Acid Sequence↗
PubMed1987

Identification and characterization of the functional alpha origin of DNA replication of the R6K plasmid and its relatedness to the R6K beta and gamma origins.

The functional R6K alpha origin is composed of two DNA elements, one of 580 bp carrying the alpha origin sequences and the other of 277 bp containing the seven 22 bp direct repeats previously identified as also required for gamma and beta origin activity. These two genetic elements are separated by approximately 3,000 bp of R6K sequences which are dispensable for alpha origin activity. The function of the alpha origin depends on the presence in cis of the 580 bp and the 277 bp fragments and requires that they be oriented as in the intact R6K. Activation of the alpha origin depends on the R6K replication initiation protein pi. Within the 580 bp of the alpha origin, there is a sequence of 98 bp which appears as an inverted repeat of 96 bp in the beta replicon. Deletion of the 96 bp or 98 bp results in inactivation of the alpha and the beta origins respectively. These long repeats are palindromic and it is suggested that these may serve as the recognition signals for initiation of DNA replication in the alpha and the beta origins of R6K. DNA homology analysis performed on alpha, beta and gamma origin sequences, also reveals 10-23 bp sequences in the alpha and the beta origins that are related to the family of 22 bp direct repeats in the gamma origin which were shown previously to be binding sites for the pi protein.

Base Sequence↗