Prolactin cDNA Research Articles

Molecular cloning of cDNA for porcine prolactin precursor

Porcine prolactin cDNA clones were screened using antiserum against ovine prolactin from a cDNA library of porcine anterior pituitary, and their nucleotide sequences were determined by the chain-termination method. The nucleotide sequence of the 5' untranslated region and part of the signal peptide region were determined by direct RNA sequencing with reverse transcriptase. The composite sequence of 957 nucleotides showed a signal sequence of 30 amino acids and a further 199 amino acids corresponding to the mature prolactin molecule. The predicted sequence confirmed the amino acid sequence determined previously by direct protein analysis, except for one amide form at residue 122 (Gln instead of the reported Glu). Northern blot analysis showed that the length of the porcine prolactin mRNA was about 1.1 kb. The porcine prolactin amino acid sequence showed 81, 80, 64, 62, 80 and 31% homology with human, bovine, rat, mouse, chick and salmon forms respectively. The identical amino acid residues showed marked clustering in four domains, two of which are highly conserved throughout a wide range of species. The hydropathy and secondary structure of porcine prolactin were analysed and compared with those of porcine GH, which shares the same ancestral gene. The two highly conserved regions of both hormones showed similar hydrophilicity, and the predicted secondary structures indicated that these regions in each hormone form different structures with differences in extension of the hydrophilic residues outside the molecule.

Expression of Biologically Active Rat Prolactin in Mammalian COS-1 Cells.

Rat prolactin (PRL) cDNA was constructed in mammalian expression vector, pSVL. Transient expression of rat PRL was performed in COS-1 cells by the DEAE-dextran method. The production of recombinant rat PRL started within 48 h from the cells and reached the level of 1.0-1.5 micrograms/ml/5 x 10(5) cells. The molecular size of recombinant rat PRL was the same as that of standard rat PRL (Mw: 23,000), suggesting successful removal of the signal peptide. The radioimmunoassay and isoelectric focusing analysis showed that recombinant rat PRL has almost the same immunological and biochemical characteristics as those of standard rat PRL. As biological tests, receptor-binding activity, Nb 2 node lymphoma cell growth activity, and mammary gland stimulating activity were examined. The radioreceptor assay showed that recombinant rat PRL has binding activity to mammary microsomal membrane similar to that of standard rat PRL. Recombinant rat PRL also stimulated the growth of Nb 2 lymphoma cells as standard rat PRL. Finally it was shown that recombinant rat PRL promotes the synthesis of the secretory materials in the lumen of mouse mammary gland with the same potency as that of standard rat PRL. In conclusion, recombinant rat PRL, which was produced in mammalian cells in the present experiment, has immunological, biochemical and biological characteristics similar to those of standard PRL, and has full bioactivity.

Characterization of a human cDNA encoding a widely expressed and highly conserved cysteine-rich protein with an unusual zinc-finger motif

A human term placental cDNA library was screened at low stringency with a human prolactin cDNA probe. One of the cDNAs isolated hybridizes to a 1.8 kb mRNA present in all four tissues of the placenta as well as to every nucleated tissue and cell line tested. The sequence of the full-length cDNA was determined. An extended open reading frame predicted an encoded protein product of 20.5 kDa. This was directly confirmed by the in vitro translation of a synthetic mRNA transcript. Based upon the characteristic placement of cysteine (C) and histidine (H) residues in the predicted protein structure, this molecule contains four putative zinc fingers. The first and third fingers are of the C4 class while the second and fourth are of the C2HC class. Based upon sequence similarities between the first two and last two zinc fingers and sequence similarities to a related rodent protein, cysteine-rich intestinal protein (CRIP), these four finger domains appear to have evolved by duplication of a preexisting two finger unit. Southern blot analyses indicate that this human cysteine-rich protein (hCRP) gene has been highly conserved over the span of evolution from yeast to man. The characteristics of this protein suggest that it serves a fundamental role in cellular function.

Метод быстрого клонирования специфических кДНК. клонирование кДНК пролактина человека

Метод быстрого клонирования специфических кДНК. клонирование кДНК пролактина человека

Ontogeny of prolactin mRNA in the rat pituitary gland as evaluated by in situ hybridization

We have studied the ontogeny of prolactin (PRL) messenger RNA (mRNA) in male and female rats. Quantitative in situ hybridization was performed on sections of fixed pituitaries using a 32S-labeled PRL cDNA probe. With this technique, hybridization signal was first detected on day 19 of gestation. The PRL mRNA levels were very low in foetuses and newborn animals. Higher PRL mRNA levels were found in 5-day-old animals. Thereafter, mRNA concentrations regularly increased to reach a plateau at 60 and 90 days of age in males and females, respectively. Sexual dimorphism was first observed in 20-day-old animals, the PRL mRNA levels being higher in the female than in the male. This difference in PRL mRNA became more marked after puberty such that in 90-day-old animals the amounts of PRL mRNA in females were 2.7 times those observed in males. Since sexual dimorphism in PRL mRNA levels occurs well before sexual dimorphism in PRL secretion, which takes place first during puberty, it is suggested that during sexual maturation PRL secretion is regulated translationally as well as transcriptionally.

Expression of biologically active recombinant-derived chicken prolactin in Escherichia coli.

The putative chicken prolactin (chPRL) cDNA clone PRL101 was manipulated in vitro and cloned into the Escherichia coli expression vector pKK2332 to produce a plasmid coding for recombinant-derived mature chPRL (R-chPRL). Expression of this manipulated cDNA sequence in E. coli resulted in the production of a 23 kDa protein which cross-reacted with specific chPRL antisera in Western blots. The partially purified protein stimulated ring dove crop sac mucosa to proliferate in a PRL bioassay, demonstrating that the R-chPRL was biologically active. R-chPRL was expressed at a level of approximately 1.5% of total cell protein.

Isolation of a novel prolactin-like cDNA clone from bovine placenta: Occurrence of new family members

A novel cDNA clone that hybridized to bovine prolactin cDNA was isolated from a bovine ( Bos taurus) placental cDNA library and the nucleotide sequence was analyzed. The cDNA clone, named bPLP-III, contained one open reading frame encoding a protein consisting of 239 amino acids. The amino-acid sequence of bPLP-III is 43 and 57% homologous to bovine preprolactin and a bovine prolactin-related protein, bPRC-I, respectively, but only 23% homologous to bovine pregrowth hormone. The predicted mature protein of bPLP-III is distinct from bovine placental lactogens in amino-acid composition, suggesting that bPLP-III is the clone for a new member of prolactin-related mRNA expressed in bovine placenta.

Cloning and nucleotide sequence of ovine prolactin cDNA

A cDNA expression library was constructed in the λgt11 phage vector using ovine (o) pituitary mRNA. The clone, pOP1, carrying a 934-bp insert contains an open reading frame beginning with the first nucleotide (nt) and ending with the stop codon TAA at nt position 781. Two potential translation start codons (ATGs) are present in the 5′ region of this cDNA. Translation initiation could occur at the 5′ proximal ATG at nt position 61. The nucleotide sequence around this ATG (TCCATGG), resembles the optimum sequence context for translation initiation by the eukaryotic ribosomes, as defined by mutational analysis [Kozak, Cell 44 (1986) 283–292)], with its substitution of the A at −3 of the consensus sequence by a T residue in this clone. Translation initiated at this codon could potentially code for the entire pre-prolactin (pre-PRL) molecule. The 3′ -untranslated region is 154 nt long and contains a polyadenylation signal AATAAA. The deduced amino acid sequence agrees in totality with the published amino acid sequence of the mature hormone. The present study reports on the nucleotide sequence of o-PRL mRNA and the deduced amino acid sequence in the signal peptide of the hormone.

Cloning and nucleotide sequence of an ovine prolactin cDNA.

Cloning and nucleotide sequence of an ovine prolactin cDNA.

Molecular cloning and expression of salmon prolactin cDNA.

Prolactin was purified from chum salmon pituitaries. It was resolved into two variants by reverse-phase high-performance liquid chromatography. A cDNA library was prepared from Pacific chinook salmon pituitaries. Salmon prolactin gene was screened using a synthetic oligonucleotide based on partial protein sequence. A positive clone (PRL-10) was identified and sequenced. It is a full-size clone containing 1.1 kb and coding for a preprolactin of 211 amino acids. A modified prolactin plasmid (PRL-10A), in which the 5' untranslated sequence and the nucleotide sequence coding for the signal peptide of prolactin were deleted, was reconstructed into an expression vector using the heat-inducible lambda pL promotor. Mature prolactin, a single polypeptide of 22 kDa, was efficiently expressed in the bacteria at an elevated temperature.

Cloning and expression of cDNA for salmon prolactin in Escherichia coli.

cDNA clones which include the coding sequences of chum salmon prolactin (sPRL) have been isolated from a cDNA library prepared from chum salmon pituitary gland poly(A) + RNA. A synthetic oligonucleotide probe based on amino acid residues 122~127 of sPRL was used as a hybridization probe to select recombinant plasmids carrying the sPRL coding sequences. One of the cDNA clones, which includes the longest cDNA insert, has been sequenced. The cDNA sequence codes for a polypeptide of 210 amino acids, including a putative signal sequence of 23 amino acids. The deduced amino acid sequence of the mature region matched that of the sPRL polypeptide isolated from chum salmon pituitaries (ref. 1). sPRL cDNA was expressed in Escherichia coli under the control of the E. coli trp promoter. sPRL was identified by Western blotting using rabbit antiserum to authentic sPRL.

Improved tissue preparation for in situ localization of specific mRNA using non-radioactive DNA probes: Effects of protease digestion and probe size on signal detection in frozen and paraffin sections of rat pituitary glands.

To better describe the physiological state of cells, detection of specific mRNA by in situ hybridization at the cellular level is often demanded. In order to accomplish this reliably, various factors such as morphological preservation, retention of mRNA, accessibility of the labeled probe to the target mRNA and efficiency of the hybridization should be considered during the tissue processing. In this paper, using non-radioactive probes, we investigated the effects of protease treatment and the size of probe on the efficiency of in situ hybridization with mRNAs in frozen and paraffin-embedded tissue sections of the rat pituitary glands. As non-radioactive haptenic DNA probes, we used dinitrophenyl (DNP) -labeled pro-opiomelanocortin (POMC) DNA and thymine-thymine (T-T) dimerized prolactin cDNA. The signals were visualized by the indirect enzyme-immunohis-tochemistry. The best results were obtained when frozen sections of tissues fixed by 4% paraformaldehyde in phosphate buffered saline were mildly digested with protease and hybridized with the haptenized DNAs of about 200-400 base pairs.

Nucleotide sequence of carp prolactin cDNA

Nucleotide sequence of carp prolactin cDNA

Molecular cloning of a prolactin-related mRNA expressed in bovine placenta.

Bovine (Bos taurus) prolactin-related cDNA I (bPRC-I), distinct from the isolated bovine placental lactogen, was derived from bovine fetal placental mRNA by molecular cloning. The nucleotide sequence is 63% homologous to bovine prolactin cDNA and only 45% to bovine growth hormone. The region of bPRC-I corresponding to the 5' portion of the signal peptide and 5' untranslated region of bovine prolactin mRNA is markedly different from prolactin. The predicted protein is 39% homologous to bovine prolactin and about 30% to the related placental hormones in rodents. This identification of a prolactin-related gene in the cow in addition to those reported in rodents suggests that multiple prolactin-related genes expressed in the placenta may be a general phenomenon in nonprimates. The role of these related hormones during gestation remains to be investigated.

Cloning and sequence analysis of cDNA for mouse prolactin

The present study was undertaken to find out whether or not sexual dimorphism in biological activities and amino acid compositions of mouse prolactin might be due to heterogeneity in mRNA for mouse prolactin. Cloned cDNAs for mouse prolactin were first isolated from a mouse pituitary cDNA library by hybridization with a rat prolactin cDNA. Then, one clone of about 140 positive clones obtained from 2000 transformants was subjected to nucleotide sequence analysis and verified to contain a nearly full length of cDNA sequence coding for mouse prolactin precursor. The deduced complete amino acid sequence indicates that the precursor molecule consists of 31 amino acids as the signal peptide and 197 amino acids of prolactin, in which two amino acids were found to be diferent from the amino acid sequence previously published elsewhere. S 1 nuclease mapping analysis using male and female pituitary RNAs indicates that mouse preprolactin is encoded by two mRNAs in both sexes. The two mRNAs differ from each other based upon the deletion of three nucleotides in the coding region for the signal peptide determined by the nucleotide sequence analysis in other cDNA clones. In the present study, no sexual difference was revealed in murine prolactin mRNA.

Novel prolactin related mRNAs in rat pituitary cells

From cytoplasm of rat pituitary GH4C1 tumour cells, anti prolactin anti-serum precipitates a polypeptide with apparent molecular weight of 75.000 in addition to prolactin. In vitro translation of size fractionated RNA shows that a 82.000 molecular weight PRL-like polypeptide is encoded by a mRNA larger than the 1 kb prolactin mRNA. Northern blot analysis shows that a rat prolactin cDNA probe hybridize to a 3.2 kb RNA and a 1.5 kb RNA in addition to the 1 kb PRL mRNA. The 82.000 molecular weight translation product and the 3.2 kb mRNA is also detected in rat anterior pituitary cytoplasm. We conclude that at least one high molecular weight mRNA which code for a prolactin-like polypeptide, is present in normal rat anterior pituitary gland and in GH4C1 cells.

Synthesis of bovine prolactin in Escherichia coli.

Transformation of Escherichia coli cells with a recombinant plasmid (pESP4) containing a modified bovine prolactin cDNA clone in a pEMBL vector resulted in efficient expression of prolactin. The cDNA was modified by removal of a 5' nontranslated sequence as well as the sequence that specified the signal peptide of preprolactin. To achieve a high level of synthesis, a sequence of 30 nucleotides in the cDNA, which included the ATG initiation codon and the first 7 codons of mature bovine prolactin, was replaced by a chemically synthesized oligonucleotide duplex. The sequence of this duplex was chosen from the consensus sequence around the initiation codon of E. coli genes and by the amino acid sequence of the protein. Prolactin, a single-chain polypeptide of molecular weight 24,000, was identified by Coomassie Blue staining of NaDodSO4-polyacrylamide gels of total protein from transformed E. coli cells, and by reaction with specific antibody. Increased levels of expression of the hormone, corresponding to the form secreted from the pituitary, were observed in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG).

Estrogen induced prolactin mRNA accumulation in adult male rat pituitary as revealed by in situ hybridization.

Adult male rats were injected with different doses (1, 10, and 100 micrograms) of 17 beta-estradiol daily for 5 days, and the changes in prolactin (PRL) mRNA levels were examined by in situ hybridization and cytoplasmic dot blot hybridization using cloned cDNA for rat prolactin mRNA. An increase in cytoplasmic PRL mRNA content was evident in all the animals treated with estrogen as revealed with cytoplasmic dot blot analysis. There were however, no significant differences in PRL mRNA content among the three estradiol treated groups. Cytoplasmic PRL mRNA was also demonstrated by in situ hybridization on the frozen pituitary sections using a 3H-labeled PRL cDNA probe. The number of grains per cell was increased after estrogen treatment. 3H-thymidine uptake into pituitary cells was also examined in vivo using combined techniques of immunocytochemistry and autoradiography. Although the percentage of immunoreactive PRL cells which took up thymidine in their nuclei increased to more than double after estrogen treatment, the increase in the total number of immunoreactive PRL cells was small. These results suggest that the major effect of estrogen on PRL cells is an increase in the accumulation of PRL mRNA in the individual PRL cells. The number of grains per cell was found to vary from cell to cell, both in control and estrogen treated animals. This variability is discussed in relation to the functional heterogeneity within the PRL cell population.

Nucleotide sequence of a growth-related mRNA encoding a member of the prolactin-growth hormone family.

As part of the proliferative response to serum, mouse 3T3 cells produce a set of growth-related mRNAs identified by hybridization to cloned cDNAs. One of these mRNAs, which is about 1 kilobase long, appears within a few hours after stimulation of resting cells with serum or platelet-derived growth factor and reaches a high level during the transition from the G1 to the S phase of growth. This mRNA is translated in vitro into a protein of approximately 25 kilodaltons. The corresponding cloned cDNA of 791 base pairs has been sequenced; it contains a single open reading frame that encodes a protein of 224 amino acids with extensive sequence homology to mammalian prolactins. The initial 29-amino acid segment of the encoded protein resembles the signal sequences of prehormones. That the growth-related protein is not mouse prolactin is indicated by comparison of its predicted amino acid composition with that of mouse prolactin and by the distinct fragment patterns seen when restricted mouse DNA is probed with the cloned cDNA or rat prolactin cDNA. Therefore, the growth-related protein appears to be a new member of the prolactin-growth hormone family. Because of its relationship to prolactin and growth hormone and its association with cell proliferation, the protein has been called "proliferin."

Molecular cloning and nucleotide sequence of DNA complementary to human decidual prolactin mRNA.

Three human decidual prolactin (PRL) cDNA clones (pdPL-1:349 base pairs, pdPL-2:584 base pairs, pdPL-3:807 base pairs without poly(A) tract) were prepared and sequenced. The insert DNA of the largest clone pdPL-3 contained the coding region corresponding to 217 amino acid residues including 18 amino acid residues of the signal peptide, and 157 nucleotides of the 3'-untranslated region. A comparison of the pdPL-3 cDNA sequence with that of human pituitary PRL cDNA revealed 4 silent nucleotide differences. Two of the base changes occurred in the third position of the amino acid codons, and the other two occurred in the 3'-untranslated region. Therefore, the amino acid sequence of decidual PRL deduced from the nucleotide sequence of pdPL-3 was identical with that of pituitary PRL. Minor changes were also observed among the three PRL mRNAs: a silent change in the third codon in the translated region, and another change in the 3'-untranslated region. Southern blot hybridization of human cellular DNA with the pdPL-3 probe indicates that PRL gene may occur once per haploid genome. Therefore, these minor changes may reflect microheterogeneity of PRL gene. The existence of multiple poly(A)-adjacent sequences was shown in the three species of human decidual PRL mRNA and in human pituitary PRL mRNA. This variation may be due to heterogeneity in the processing at the 3' terminus of mRNA or the termination sites of the transcription.